Publications

Systemic immunity supports lifelong brain function. Obesity posits a chronic burden on systemic immunity. Independently, obesity was shown as a risk factor for Alzheimer’s disease (AD). Here we show that high-fat obesogenic diet accelerated recognition-memory impairment in an AD mouse model (5xFAD). In obese 5xFAD mice, hippocampal cells displayed only minor diet-related transcriptional changes, whereas the splenic immune landscape exhibited aging-like CD4+ T-cell deregulation. Following plasma metabolite profiling, we identified free N-acetylneuraminic acid (NANA), the predominant sialic acid, as the metabolite linking recognition-memory impairment to increased splenic immune-suppressive cells in mice. Single-nucleus RNA-sequencing revealed mouse visceral adipose macrophages as a potential source of NANA. In vitro, NANA reduced CD4+ T-cell proliferation, tested in both mouse and human. In vivo, NANA administration to standard diet-fed mice recapitulated high-fat diet effects on CD4+ T cells and accelerated recognition-memory impairment in 5xFAD mice. We suggest that obesity accelerates disease manifestation in a mouse model of AD via systemic immune exhaustion.

Recent functional and anatomical discoveries of brain-immune relationships have overturned previous beliefs regarding the brain's immune privilege. Here, we propose that the brain and immune cells at its borders operate as an "ecosystem" to support the brain's robustness and resilience. Modulation of this ecosystem can be harnessed in the clinic.

For decades, neurodegenerative diseases were thought to be caused by the accumulation of toxic compounds, exacerbated by local inflammation, which together lead to neuronal loss and cognitive impairment . An additional factor that was long overlooked , is the role of the systemic immune system, which provides a defense mechanism against internal and external intruders in all bodily tissues. The evolving understanding of the life-long cross-talk between the CNS and the immune system led to an awareness of the function of systemic adaptive immunity in containing emerging destructive factors within the brain. . This includes harnessing of circulating myeloid cells to help the brain. However, as damage accumulates within the brain, the systemic immune system loses its protective capacity. Under such conditions, the dysregulated immune system becomes an escalating factor itself, thereby driving a vicious cycle that must be arrested.
•The brain's privilege does not imply immune isolation, but rather, a unique mode of brain–immune communication.•Both adaptive and innate immune support are needed for brain maintenance and repair.•Neurodegenerative diseases have an essential peripheral component, which is often manifested by immune dysfunction.•While not a primary cause, the immune dysfunction develops at an early stage in neurodegenerative disease, presumably before symptoms appear, resulting in a vicious cycle that further escalates disease progression.•Breaking the vicious cycle by activating the systemic immune system can harness bone marrow-derived macrophages and regulatory T cells to the brain to help defeat disease.

The incidence of age-related dementia is growing with increased longevity, yet there are currently no disease-modifying therapies for these devastating disorders. Studies over the last several years have led to an evolving awareness of the role of the immune system in supporting brain maintenance and repair, displaying a diverse repertoire of functions while orchestrating the crosstalk between the periphery and the brain. Here, we provide insights into the current understanding of therapeutic targets that could be adopted to modulate the immune system, either systemically or locally, to defeat brain aging and neurodegeneration.

For decades, it was commonly accepted that the brain is secluded from peripheral immune activity and is self-sufficient for its maintenance and repair. This simplistic perception was based on the presence of resident immune cells, the microglia, and barrier systems within the brain, and the assumption that the central nervous system (CNS) lacks lymphatic drainage. This view was revised with the discoveries that higher functions of the CNS, homeostasis and repair are supported by peripheral innate and adaptive immune cells. The findings of bone marrow-derived immune cells in specialized niches, and the renewed observation that a lymphatic drainage system exists within the brain, further contributed to this revised model. In this Review, we describe the immune niches within the brain, the contribution of professional immune cells to brain functions, the bidirectional relationships between the CNS and the immune system and the relevance of immune components to brain aging and neurodegenerative diseases.

The role of non-neuronal cells in Alzheimer's disease progression has not been fully elucidated. Using single-nucleus RNA sequencing, we identified a population of disease-associated astrocytes in an Alzheimer's disease mouse model. These disease-associated astrocytes appeared at early disease stages and increased in abundance with disease progression. We discovered that similar astrocytes appeared in aged wild-type mice and in aging human brains, suggesting their linkage to genetic and age-related factors.

The immune system supports brain plasticity and homeostasis, yet it is prone to changes following psychological stress. Thus, it remains unclear whether and how stress-induced immune alterations contribute to the development of mental pathologies. Here, we show that following severe stress in mice, leukocyte trafficking through the choroid plexus (CP), a compartment that mediates physiological immune-brain communication, is impaired. Blocking glucocorticoid receptor signaling, either systemically or locally through its genetic knockdown at the CP, facilitated the recruitment of Gata3- and Foxp3-expressing T cells to the brain and attenuated post-traumatic behavioral deficits. These findings functionally link post-traumatic stress behavior with elevated stress-related corticosteroid signaling at the brain-immune interface and suggest a novel therapeutic target to attenuate the consequences of severe psychological stress.

Microglia differentiate from progenitors that infiltrate the nascent CNS during early embryonic development. They then remain in this unique immune-privileged environment throughout life. Multiple immune mechanisms, which we collectively refer to as microglial checkpoints, ensure efficient and tightly regulated microglial responses to perturbations in the CNS milieu. Such mechanisms are essential for proper CNS development and optimal physiological function. However, in chronic disease or aging, when a robust immune response is required, such checkpoint mechanisms may limit the ability of microglia to protect the CNS. Here we survey microglial checkpoint mechanisms and their roles in controlling microglial function throughout life and in disease, and discuss how they may be targeted therapeutically.

Hypoxia occurs physiologically in the developing body, and changing oxygen tensions are known to direct tissue differentiation; however, in the context of pathology, the same hypoxia-activated mechanisms may negatively affect tissue function. In this issue of The EMBO Journal, Esteban-Martinez et al (2017) report that programmed mitophagy, dependent on hypoxia-induced NIP-3-like protein X (BNIP3L, best known as NIX), is an essential step in differentiation of both retinal neurons and inflammatory macrophages.

For decades, the brain was considered to be a tissue behind barriers, and thus autonomous with respect to its maintenance needs. Several years ago, our group suggested that the ability of the brain to operate optimally, with relatively little deterioration throughout life, is dependent on continuous support from circulating immune cells. We envisioned that such immune support is provided from specific permissive sites that lie between the brain and the immune system, enabling immune-brain dialogue without the risk of an inflammatory autoimmune response. The brain's choroid plexus has been identified as an active neuroimmunological interface that is continuously exposed to signals from the brain and from the periphery. The interplay between these bidirectional signals shapes brain function in health and disease, including in multiple sclerosis. Accumulating evidence suggests that autoimmune T cells that recognize brain antigens are involved in this dialogue. Here, we discuss the fate of this dialogue under neurodegenerative conditions, and how boosting autoimmunity, rather than suppressing it, by either active vaccination with central nervous system-derived antigens, or by breaking peripheral immune suppression, could be harnessed for mitigating neurodegenerative diseases.

Hippocampal neurogenesis is affected throughout life by factors external to the brain, including what we eat, our gut microbiota, and the immune system. However, the mechanisms that link microbiota to neurogenesis are still puzzling. Now in Cell Reports, Mohle et al. (2016) attribute a role to Ly6C(hi) monocytes in this gut-immune-brain axis.

Hemorrhagic stroke, primarily caused by rupture of blood vessels in the brain, is a leading cause of death and disability in adults. In this issue of Immunity, Liu et al. (2016) demonstrate that repair of cerebrovascular ruptures can be directly mediated by myeloid cells.

Recent findings have revealed distinct roles for type I and II interferons (IFN-I and IFN-gamma) in the recruitment of immune cells to the central nervous system (CNS) and highlighted the importance of this process for brain maintenance and protection/repair. Furthermore, manipulation of IFN-I and IFN-gamma pathways in pathological contexts has yielded conflicting results. We discuss these findings, focusing on two distinct conditions; relapsing remitting multiple sclerosis (RRMS) and brain aging. Using these examples, we propose that regulation of immune cell entry to the CNS is a mechanism through which interaction between IFN-I and -II can affect brain function from its anatomical borders. Deviation from homeostatic IFN-I/-II balance may contribute to distinct brain pathologies, resulting from either insufficient immune surveillance of the CNS and loss of immune-dependent protection, or overwhelming leukocyte entry and immune-mediated destruction.

Systemic immune suppression may curtail the ability to mount the protective, cell-mediated immune responses that are needed for brain repair. By using mouse models of Alzheimer's disease (AD), we show that immune checkpoint blockade directed against the programmed death-1 (PD-1) pathway evokes an interferon (IFN)-gamma-dependent systemic immune response, which is followed by the recruitment of monocyte-derived macrophages to the brain. When induced in mice with established pathology, this immunological response leads to clearance of cerebral amyloid-beta (A beta) plaques and improved cognitive performance. Repeated treatment sessions were required to maintain a long-lasting beneficial effect on disease pathology. These findings suggest that immune checkpoints may be targeted therapeutically in AD.

Chronic neuroinflammation is evident in brain aging and neurodegenerative disorders and is often associated with excessive nitric oxide (NO) production within the central nervous system (CNS). Under such conditions, increased NO levels are observed at the choroid plexus (CP), an epithelial layer that forms the blood-cerebrospinal fluid barrier (BCSFB) and serves as a selective gateway for leukocyte entry to the CNS in homeostasis and following injury. Here, we hypothesized that elevated cerebral NO levels interfere with CP gateway activity. We found that induction of leukocyte trafficking determinants by the CP and sequential leukocyte entry to the CSF are dependent on the CP epithelial NFκB/p65 signaling pathway, which was inhibited upon exposure to NO. Examining the CP in 5XFAD transgenic mouse model of Alzheimer's disease (AD-Tg) revealed impaired ability to mount an NFκB/p65-dependent response. Systemic administration of an NO scavenger in AD-Tg mice alleviated NFκB/p65 suppression at the CP and augmented its gateway activity. Together, our findings identify cerebral NO as a negative regulator of CP gateway activity for immune cell trafficking to the CNS.

Immune cell infiltration to the brain's territory was considered for decades to reflect a pathological process in which immune cells attack the central nervous system (CNS); such a process is observed in the inflammatory autoimmune disease, multiple sclerosis (MS). As neuroinflammatory processes within the CNS parenchyma are also common to other CNS pathologies, regardless of their etiology, including neurodegenerative disorders such as Alzheimer's disease (AD) and Amyotrophic lateral sclerosis (ALS), these pathologies have often been compared to MS, a disease that benefits from immunosuppressive therapy. Yet, over the last decade, it became clear that autoimmunity has a bright side, and that it plays a pivotal role in CNS repair following damage. Specifically, autoimmune T cells were found to facilitate CNS healing processes, such as in the case of sterile mechanical injuries to the brain or the spinal cord, mental stress, or biochemical insults. Even more intriguingly, autoimmune T cells were found to be involved in supporting fundamental processes of brain functional integrity, such as in the maintenance of life-long brain plasticity, including spatial learning and memory, and neurogenesis. Importantly, autoimmune T cells are part of a cellular network which, to operate efficiently and safely, requires tight regulation by other immune cell populations, such as regulatory T cells, which are indispensable for maintenance of immunological self-tolerance and homeostasis. Here, we suggest that dysregulation of the balance between peripheral immune suppression, on one hand, and protective autoimmunity, on the other, is an underlying mechanism in the emergence and progression of the neuroinflammatory response associated with chronic neurodegenerative diseases and brain aging. Mitigating chronic neuroinflammation under these conditions necessitates activation, rather than suppression, of the peripheral immune response directed against self. Accordingly, we propose tha

Keywords: Immunology; Neurosciences

Keywords: Immunology; Neurosciences

Keywords: Immunology; Neurosciences

Immune activity in the CNS parenchyma under various acute and chronic neurodegenerative conditions has been often interpreted as a sign of pathological inflammation. The apparent resemblance of the local neuroinflammatory processes to autoimmune diseases, such as multiple sclerosis (MS), generated the view that, despite differences in etiology and pathology, neurodegenerative disorders with a local inflammatory component can benefit from systemic anti-inflammatory therapy. In addition, as CNS self-reactive T cells are associated with the etiology of MS, autoimmunity was assumed to solely reflect pathology, and therefore, was universally linked to autoimmune disease. Yet, it is becoming increasingly clear that CNS-specific T cells, along with circulating and local innate immune cells, can enhance CNS healing processes following non-infectious injuries, or any deviation from homeostasis, including chronic pathological conditions. Here, we discuss the theory of "protective autoimmunity," which describes the activity of an immune cell network encompassing effector and regulatory T cells with specificity for CNS antigens, in CNS maintenance and repair. Such an immune network, evoked in response to external and internal threats, functions in a tightly regulated way, ensuring restoration of the brain's equilibrium and return to homeostasis.

Adaptive immunity was repeatedly shown to play a role in maintaining lifelong brain function. Under physiological conditions, this activity was associated with CD4(+) T cells specific for brain self-antigens. Nevertheless, direct interactions of T cells with the healthy neuronal parenchyma are hardly detectable. Recent studies have identified the brain's choroid plexus (CP) as an active neuro-immunological interface, enriched with CNS-specific CD4(+) T cells. Strategically positioned for receiving signals from both the central nervous system (CNS) through the cerebrospinal fluid (CSF), and from the circulation through epithelium-immune cell interactions, the CP has recently been recognized as an important immunological compartment in maintaining and restoring brain homeostasis/allostasis. Here, we propose that CNS-specific T cells shape brain function via the CP, and suggest this immunological control to be lost as part of aging, in general, and immune senescence, in particular. Accordingly, the CP may serve as a novel target for immunomodulation to restore brain equilibrium. (C) 2013 Elsevier Inc. All rights reserved.

Functional macrophage heterogeneity is recognized outside the central nervous system (CNS), where alternatively activated macrophages can perform immune-resolving functions. Such functional heterogeneity was largely ignored in the CNS, with respect to the resident microglia and the myeloid-derived cells recruited from the blood following injury or disease, previously defined as blood-derived microglia; both were indistinguishably perceived detrimental. Our studies have led us to view the myeloid-derived infiltrating cells as functionally distinct from the resident microglia, and accordingly, to name them monocyte-derived macrophages (mo-M Phi). Although microglia perform various maintenance and protective roles, under certain conditions when they can no longer provide protection, mo-M Phi are recruited to the damaged CNS; there, they act not as microglial replacements but rather assistant cells, providing activities that cannot be timely performed by the resident cells. Here, we focus on the functional heterogeneity of microglia/mo-M Phi, emphasizing that, as opposed to the mo-M Phi, microglia often fail to timely acquire the phenotype essential for CNS repair.

The poor recovery of the central nervous system (CNS) after injury, coupled with its complex and immunologically-privileged nature, led to the belief that CNS repair is different from the repair of other tissues. Here, we consider CNS repair from a novel perspective, suggesting that CNS responses to injury resemble wound healing. Extrapolating the classical wound healing model suggests that poor CNS recovery is an outcome of insufficient resolution of interim reparative events that precede tissue regeneration and renewal, a state reminiscent of chronic/unresolved wounds. This comparison requires reevaluation of the inflammatory response, glial scarring, and barrier permeability, traditionally considered obstacles to CNS repair. Understanding the similarity to wound healing suggests new research directions and therapeutic avenues for CNS injuries.

Monocyte-derived macrophages are essential for recovery after spinal cord injury, but their homing mechanism is poorly understood. Here, we show that although of common origin, the homing of proinflammatory (M1) and the "alternatively activated'' anti-inflammatory (M2) macrophages to traumatized spinal cord (SC) was distinctly regulated, neither being through breached blood-brain barrier. The M1 macrophages (Ly6c(hi)CX3CR1(lo)) derived from monocytes homed in a CCL2 chemokine-dependent manner through the adjacent SC leptomeninges. The resolving M2 macrophages (Ly6c(lo)CX3CR1(hi)) derived from monocytes trafficked through a remote blood-cerebrospinal-fluid (CSF) barrier, the brain-ventricular choroid plexus (CP), via VCAM-1-VLA-4 adhesion molecules and epithelial CD73 enzyme for extravasation and epithelial transmigration. Blockage of these determinants, or mechanical CSF flow obstruction, inhibited M2 macrophage recruitment and impaired motor-function recovery. The CP, along with the CSF and the central canal, provided an anti-inflammatory supporting milieu, potentially priming the trafficking monocytes. Overall, our finding demonstrates that the route of monocyte entry to central nervous system provides an instructional environment to shape their function.

The adaptive arm of the immune system has been suggested as an important factor in brain function. However, given the fact that interactions of neurons or glial cells with T lymphocytes rarely occur within the healthy CNS parenchyma, the underlying mechanism is still a mystery. Here we found that at the interface between the brain and blood circulation, the epithelial layers of the choroid plexus (CP) are constitutively populated with CD4(+) effector memory cells with a T-cell receptor repertoire specific to CNS antigens. With age, whereas CNS specificity in this compartment was largely maintained, the cytokine balance shifted in favor of the T helper type 2 (Th2) response; the Th2-derived cytokine IL-4 was elevated in the CP of old mice, relative to IFN-gamma, which decreased. We found this local cytokine shift to critically affect the CP epithelium, triggering it to produce the chemokine CCL11 shown to be associated with cognitive dysfunction. Partial restoration of cognitive ability in aged mice, by lymphopenia-induced homeostasis-driven proliferation of memory T cells, was correlated with restoration of the IL-4:IFN-gamma ratio at the CP and modulated the expression of plasticity-related genes at the hippocampus. Our data indicate that the cytokine milieu at the CP epithelium is affected by peripheral immunosenescence, with detrimental consequences to the aged brain. Amenable to immunomodulation, this interface is a unique target for arresting age-related cognitive decline.

Philosophers defined the eye as a window to the soul long before scientists addressed this cliche to determine its scientific basis and clinical relevance. Anatomically and developmentally, the retina is known as an extension of the CNS; it consists of retinal ganglion cells, the axons of which form the optic nerve, whose fibres are, in effect, CNS axons. The eye has unique physical structures and a local array of surface molecules and cytokines, and is host to specialized immune responses similar to those in the brain and spinal cord. Several well-defined neurodegenerative conditions that affect the brain and spinal cord have manifestations in the eye, and ocular symptoms often precede conventional diagnosis of such CNS disorders. Furthermore, various eye-specific pathologies share characteristics of other CNS pathologies. In this Review, we summarize data that support examination of the eye as a noninvasive approach to the diagnosis of select CNS diseases, and the use of the eye as a valuable model to study the CNS. Translation of eye research to CNS disease, and deciphering the role of immune cells in these two systems, could improve our understanding and, potentially, the treatment of neurodegenerative disorders. London, A. et al. Nat. Rev. Neurol. 9, 44-53 (2013); published online 20 November 2012; doi:10.1038/nrneurol.2012.227

Understanding of ocular diseases and the search for their cure have been based on the common assumption that the eye is an immune privileged site, and the consequent conclusion that entry of immune cells to this organ is forbidden. Accordingly, it was assumed that when immune cell entry does occur, this reflects an undesired outcome of breached barriers. However, studies spanning more than a decade have demonstrated that acute insults to the retina, or chronic conditions resulting in retinal ganglion cell loss, such as in glaucoma, result in an inferior outcome in immunocompromised mice; likewise, steroidal treatment was found to be detrimental under these conditions. Moreover, even conditions that are associated with inflammation, such as age-related macular degeneration, are not currently believed to require immune suppression for treatment, but rather, are thought to benefit from immune modulation. Here, we propose that the immune privilege of the eye is its ability to enable, upon need, the entry of selected immune cells for its repair and healing, rather than to altogether prevent immune cell entry. The implications for acute and chronic degenerative diseases, as well as for infection and inflammatory diseases, are discussed.

The brain has been commonly regarded as a "tissue behind walls." Appearance of immune cells in the brain has been taken as a sign of pathology. Moreover, since infiltrating monocyte-derived macrophages and activated resident microglia were indistinguishable by conventional means, both populations were considered together as inflammatory cells that should be mitigated. Yet, because the microglia permanently reside in the brain, attributing to them negative properties evoked an ongoing debate; why cells that are supposed to be the brain guardians acquire only destructive potential? Studies over the last two decades in the immune arena in general, and in the context of central nervous system pathology in particular, have resulted in a paradigm shift toward a more balanced appreciation of the contributions of immune cells in the context of brain maintenance and repair, and toward the recognition of distinct roles of resident microglia and infiltrating monocyte-derived macrophages.

Background: Circulating immune cells including autoreactive T cells and monocytes have been documented as key players in maintaining, protecting and repairing the central nervous system (CNS) in health and disease. Here, we hypothesized that neurodegenerative diseases might be associated, similarly to tumors, with increased levels of circulating peripheral myeloid derived suppressor cells (MDSCs), representing a subset of suppressor cells that often expand under pathological conditions and inhibit possible recruitment of helper T cells needed for fighting off the disease. Methods and Findings: We tested this working hypothesis in amyotrophic lateral sclerosis (ALS) and its mouse model, which are characterized by a rapid progression once clinical symptoms are evident. Adaptive transfer of alternatively activated myeloid (M2) cells, which homed to the spleen and exhibited immune suppressive activity in G93A mutant superoxide dismutase-1 (mSOD1) mice at a stage before emergence of disease symptoms, resulted in earlier appearance of disease symptoms and shorter life expectancy. The same protocol mitigated the inflammation-induced disease model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), which requires circulating T cells for disease induction. Analysis of whole peripheral blood samples obtained from 28 patients suffering from sporadic ALS (sALS), revealed a two-fold increase in the percentage of circulating MDSCs (LIN(-/Low)HLA-DR(-)CD33(+)) compared to controls. Conclusions: Taken together, these results emphasize the distinct requirements for fighting the inflammatory neurodegenerative disease, multiple sclerosis, and the neurodegenerative disease, ALS, though both share a local inflammatory component. Moreover, the increased levels of circulating MDSCs in ALS patients indicates the operation of systemic mechanisms that might lead to an impairment of T cell reactivity needed to overcome the disease conditions within the CNS. This high le

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing fatal neurodegenerative disorder characterized by the selective death of motor neurons (MN) in the spinal cord, and is associated with local neuroinflammation. Circulating CD4(+) T cells are required for controlling the local detrimental inflammation in neurodegenerative diseases, and for supporting neuronal survival, including that of MN. T-cell deficiency increases neuronal loss, while boosting T cell levels reduces it. Here, we show that in the mutant superoxide dismutase 1 G93A (mSOD1) mouse model of ALS, the levels of natural killer T (NKT) cells increased dramatically, and T-cell distribution was altered both in lymphoid organs and in the spinal cord relative to wildtype mice. The most significant elevation of NKT cells was observed in the liver, concomitant with organ atrophy. Hepatic expression levels of insulin-like growth factor (IGF)-1 decreased, while the expression of IGF binding protein (IGFBP)-1 was augmented by more than 20-fold in mSOD1 mice relative to wild-type animals. Moreover, hepatic lymphocytes of pre-symptomatic mSOD1 mice were found to secrete significantly higher levels of cytokines when stimulated with an NKT ligand, ex-vivo. Immunomodulation of NKT cells using an analogue of alpha-galactosyl ceramide (alpha-GalCer), in a specific regimen, diminished the number of these cells in the periphery, and induced recruitment of T cells into the affected spinal cord, leading to a modest but significant prolongation of life span of mSOD1 mice. These results identify NKT cells as potential players in ALS, and the liver as an additional site of major pathology in this disease, thereby emphasizing that ALS is not only a non-cell autonomous, but a non-tissue autonomous disease, as well. Moreover, the results suggest potential new therapeutic targets such as the liver for immunomodulatory intervention for modifying the disease, in addition to MN-based neuroprotection and systemic treatments

An organism's behavior is determined by the way it senses and perceives the surrounding environment, and by its responses to these stimuli. The major factors known to affect the behavioral response to an event are genetic background, environmental factors, and past experiences, and their imprinting on the relevant brain circuits. Recently, circulating immune cells were introduced as novel players into this system. It was proposed that the brain and circulating immune cells engage in a continuous dialogue that takes place within the brain's territory, though outside the parenchyma (occurring within the brain's borders - the choroid plexi, the brain meninges and the cerebrospinal fluid (CSF)). The cytokines secreted by activated leukocytes residing at the borders were shown to affect neurotrophic factors production within the parenchyma. Here, we suggest that such a dialogue is stimulated at the brain's borders, upon need, by a "danger" signal that originates in the parenchyma in response to any destabilizing event, and discuss the potential role of reactive oxygen species (ROS) in transmitting this signal. Accordingly, a failure to restore balance is likely to lead to aberrant responses to subsequent events. This view thus supports the contention that circulating immune cells are required to maintain the brain's balanced activity and suggests a novel mechanism whereby the surveying immune cells are sensing the brain's status and needs. (C) 2010 Elsevier Inc. All rights reserved.

Noninvasive monitoring of beta-amyloid (A beta) plaques, the neuropathological hallmarks of Alzheimer's disease (AD), is critical for AD diagnosis and prognosis. Current visualization of A beta plaques in brains of live patients and animal models is limited in specificity and resolution. The retina as an extension of the brain presents an appealing target for a live, noninvasive optical imaging of AD if disease pathology is manifested there. We identified retinal A beta plaques in postmortem eyes from AD patients (n = 8) and in suspected early stage cases (n=5), consistent with brain pathology and clinical reports; plaques were undetectable in age-matched non-AD individuals (n = 5). In APP(SWE)/PS1(Delta E9) transgenic mice (AD-Tg; n = 18) but not in non-Tg wt mice (n = 10), retinal A beta plaques were detected following systemic administration of curcumin, a safe plaque-labeling fluorochrome. Moreover, retinal plaques were detectable earlier than in the brain and accumulated with disease progression. An immune-based therapy effective in reducing brain plaques, significantly reduced retinal A beta plaque burden in immunized versus non-immunized AD mice (n = 4 mice per group). In live AD-Tg mice (n = 24), systemic administration of curcumin allowed noninvasive optical imaging of retinal A beta plaques in vivo with high resolution and specificity; plaques were undetectable in non-Tg wt mice (n = 11). Our discovery of A beta specific plaques in retinas from AD patients, and the ability to noninvasively detect individual retinal plaques in live AD mice establish the basis for developing high-resolution optical imaging for early AD diagnosis, prognosis assessment and response to therapies. (C) 2010 Elsevier Inc. All rights reserved.

Until recently, the local inflammation that occurs in response to spinal cord injury has received a negative reputation; overall, it was assumed to be one of the major causes of a vicious neurotoxic cycle that leads to impaired recovery following injury. This local inflammation involves both the activated tissue-resident microglia and monocyte-derived macrophages infiltrating from the blood. Ten years ago, we proposed that the blood-derived macrophages, reminiscent of "alternatively activated" macrophages (also known as tissue repairing, M2), are not spontaneously recruited in sufficient numbers to sites of injured central nervous system (CNS). We further demonstrated that their exogenous administration to the margins of injured spinal cord improved functional outcome. However, our suggestions evoked criticism, claiming that we were adding macrophages to a site that is already overwhelmed with inflammatory cells. Using experimental paradigms that enabled functional distinction between the resident and infiltrating cells, our most recent studies further corroborated our repair perception, showing that (a) infiltrating monocyte-derived macrophages are recruited following injury and localize to the margins of the lesion, unlike the activated resident microglia that are not compartmentalized, and (b) activated resident microglia and infiltrating monocyte-derived macrophages perform distinct roles; recruited blood-derived macrophages display an (IL-10-dependent) anti-inflammatory phenotype when they become co-localized with the glial scar. We further found that post-injury recruitment of blood monocytes is indeed suboptimal. Augmentation of the levels of naive blood monocytes leads to their increased recruitment to the same zones that are the targets of the infiltrated endogenous monocytes, and they acquire the same anti-inflammatory activity, leading to improved recovery. Thus, boosting the levels of the relevant blood monocytes reinforces the body's own repair mecha

Neuropsychological syndromes including schizophrenia often do not manifest until late adolescence or early adulthood. Studies attributing a role in brain maintenance to the immune system led us to propose that malfunction of immune-dependent regulation of brain functions at adolescence underlies the late onset of such diseases/syndromes. One such function is sensorimotor gating, the ability to segregate a continuous stream of sensory and cognitive information, and to selectively allocate attention to a significant event by silencing the background (measured by prepulse inhibition; PPI). This activity is impaired in schizophrenia, as well as in several other neuropsychological diseases. Using a model of prenatal immune activation (maternal polyriboinosinic-polyribocytidylic acid (poly I: C) injection), often used as a model for schizophrenia, and in which abnormal PPI has a delayed appearance, we demonstrated a form of immune deficit in the adult offspring. Similar abnormal PPI with a delayed appearance was found in congenitally immune-deficient mice (severe combined immune deficient, SCID), and could be reversed by immune reconstitution. This functional deficit correlated with impairment of both hippocampal neurogenesis and expression of the gene encoding kisspeptin (Kiss1) that manifested at adulthood. Moreover, exogenous administration of a kisspeptin-derived peptide partially reversed the gating deficits in the SCID mice. Our results suggest that a form of congenital immune deficiency may be a key factor that determines manifestation of developmental neuropsychological disorders with onset only at early adulthood. Molecular Psychiatry (2010) 15, 415-425; doi: 10.1038/mp.2009.66; published online 28 July 2009

Circulating immune cells support hippocampal neurogenesis, spatial memory, expression of brain-derived neurotrophic factor, and resilience to stress. Nevertheless, considering the immune privileged status of the central nervous system (CNS), such cells were assumed to be excluded from the healthy brain. It is evident, however, that the CNS is continuously surveyed by leukocytes, though their function is still a mystery. Coupling this routine leukocyte trafficking with the function attributed to circulating T cells in brain plasticity led us to propose here that CNS immunosurveillance is an integral part of the functioning brain. Anatomical restriction of selected self-recognizing leukocytes to the brain's borders and fluids (cerebrospinal fluid) not only supports the brain's activity, but also controls the potential aggressiveness of such cells. Accordingly, the brain's 'privilege' is its acquisition of a private peripheral immunological niche under its own control, which supports brain function. Immune malfunction may comprise a missing link between a healthy and diseased mind. Molecular Psychiatry (2010) 15, 342-354; doi: 10.1038/mp.2010.31

Background: Although macrophages (M Phi) are known as essential players in wound healing, their contribution to recovery from spinal cord injury (SCI) is a subject of debate. The difficulties in distinguishing between different M Phi subpopulations at the lesion site have further contributed to the controversy and led to the common view of M Phi as functionally homogenous. Given the massive accumulation in the injured spinal cord of activated resident microglia, which are the native immune occupants of the central nervous system (CNS), the recruitment of additional infiltrating monocytes from the peripheral blood seems puzzling. A key question that remains is whether the infiltrating monocyte-derived M Phi contribute to repair, or represent an unavoidable detrimental response. The hypothesis of the current study is that a specific population of infiltrating monocyte-derived M Phi is functionally distinct from the inflammatory resident microglia and is essential for recovery from SCI. Methods and Findings: We inflicted SCI in adult mice, and tested the effect of infiltrating monocyte-derived M Phi on the recovery process. Adoptive transfer experiments and bone marrow chimeras were used to functionally distinguish between the resident microglia and the infiltrating monocyte-derived M Phi. We followed the infiltration of the monocyte-derived M Phi to the injured site and characterized their spatial distribution and phenotype. Increasing the naive monocyte pool by either adoptive transfer or CNS-specific vaccination resulted in a higher number of spontaneously recruited cells and improved recovery. Selective ablation of infiltrating monocyte-derived M Phi following SCI while sparing the resident microglia, using either antibody-mediated depletion or conditional ablation by diphtheria toxin, impaired recovery. Reconstitution of the peripheral blood with monocytes resistant to ablation restored the lost motor functions. Importantly, the infiltrating monocyte-derived M

The psychobiological mechanisms that contribute to the development of stress resilience are not fully elucidated. One potential approach for enhancing resilience is the exposure to mild challenges. According to this approach, a mildly stressful episode may immunize the individual, thereby strengthening resistance to subsequent stressors. This phenomenon is often viewed as a form of behavioral immunization. Although, the term 'behavioral immunization' was borrowed from the field of immunology, the involvement of the adaptive immune system in stress resilience was never investigated. However, based on accumulated new data, we suggest that the immunological memory does have a significant role in developing coping responses to stress. Although, immune deficiency results in an impaired ability to cope with stress, boosting immunological memory can increase stress resilience. Therefore, we propose that defense against mental challenge, similarly to defense against intruders, involves an immunological mechanism, which establishes stress resilience to a later challenge. Here, we review the involvement of the adaptive immune system in coping mechanisms in response to psychological stress, and discuss the connection between cognitive memory and immunological memory in establishing ability to efficiently cope with stressful episodes.

Our research group has been working for more than a decade on the cross-talk between the immune and the nervous systems. Due to the unique nature of the central nervous system (CNS) as an immune privileged site, it was commonly believed that the nervous system functions optimally without any immune intervention, and that any immune cell infiltration to the CNS is a sign of pathology. However, since the immune system constitutes the body's major defense and repair mechanism, it seemed unreasonable that the CNS would have completely lost the need for assistance from this system. This insight prompted us to revisit the entire question of whether immune cells are required for recovery from CNS injuries. We subsequently made numerous fundamental observations that led us to formulate a unified theory linking all neurodegenerative conditions; thus, we suggested that "T-cell immunity to self maintains the self," at least in the CNS. According to this view, immunity to self ("protective autoimmunity") provides a pivotal role in maintenance, protection, and repair of the healthy and diseased CNS. We further showed that the T cells mediate their effect, at least under pathological conditions, by controlling the recruitment of blood-borne monocytes, which play a crucial local role that cannot be replaced by the resident microglia. Boosting of such a T cell response specific for brain proteins, while carefully choosing the antigen, the carrier, timing, dosing, and regimen should be considered as a way of augmenting a physiological repair mechanism needed to ameliorate disease conditions while restoring equilibrium needed for protection, repair and renewal; such therapy is not intended to modify a single mediator of a single disease, but rather, would serve as an approach for adjusting the levels of the immune response needed to restore a lost balance. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Retinal neurogenesis ceases by the early postnatal period, although retinal progenitor cells (RPCs) persist throughout life. In this study, we show that in the mammalian eye, the function of Toll-like receptor 4 (TLR4) extends beyond regulation of the innate immune response; it restricts RPC proliferation. In TLR4-deficient mice, enhanced proliferation of cells reminiscent of RPCs is evident during the early postnatal period. In vitro experiments demonstrate that TLR4 acts as an intrinsic regulator of RPC fate decision. Increased TLR4 expression in the eye correlates with the postnatal cessation of cell proliferation. However, deficient TLR4 expression is not sufficient to extend the proliferative period but rather contributes to resumption of proliferation in combination with growth factors. Proliferation in vivo is inhibited by both MyD88-dependent and -independent pathways, similar to the mechanisms activated by TLR4 in immune cells. Thus, our study attributes a novel role to TLR4 as a negative regulator of RPC proliferation.

Aging is often associated with a decline in hippocampus-dependent spatial memory. Here, we show that functional cell-mediated immunity is required for the maintenance of hippocampus-dependent spatial memory. Sudden imposition of immune compromise in young mice caused spatial memory impairment, whereas immune reconstitution reversed memory deficit in immune-deficient mice. Analysis of hippocampal gene expression suggested that immune-dependent spatial memory performance was associated with the expression of insulin-like growth factor (Igf1) and of genes encoding proteins related to presynaptic activity (Syt10, Cplx2). We further showed that memory loss in aged mice could be attributed to age-related attenuation of the immune response and could be reversed by immune system activation. Homeostatic-driven proliferation of lymphocytes, which expands the existing T cell repertoire, restored spatial memory deficits in aged mice. Thus, our results identify a novel function of the immune system in the maintenance of spatial memory and suggest an original approach for arresting or reversing age-associated memory loss.

Background Chondroitin sulfate proteoglycan (CSPG) is a major component of the glial scar. It is considered to be a major obstacle for central nervous system (CNS) recovery after injury, especially in light of its well-known activity in limiting axonal growth. Therefore, its degradation has become a key therapeutic goal in the field of CNS regeneration. Yet, the abundant de novo synthesis of CSPG in response to CNS injury is puzzling. This apparent dichotomy led us to hypothesize that CSPG plays a beneficial role in the repair process, which might have been previously overlooked because of nonoptimal regulation of its levels. This hypothesis is tested in the present study. Methods and Findings We inflicted spinal cord injury in adult mice and examined the effects of CSPG on the recovery process. We used xyloside to inhibit CSPG formation at different time points after the injury and analyzed the phenotype acquired by the microglia/macrophages in the lesion site. To distinguish between the resident microglia and infiltrating monocytes, we used chimeric mice whose bone marrow-derived myeloid cells expressed GFP. We found that CSPG plays a key role during the acute recovery stage after spinal cord injury in mice. Inhibition of CSPG synthesis immediately after injury impaired functional motor recovery and increased tissue loss. Using the chimeric mice we found that the immediate inhibition of CSPG production caused a dramatic effect on the spatial organization of the infiltrating myeloid cells around the lesion site, decreased insulin-like growth factor 1 (IGF-1) production by microglia/macrophages, and increased tumor necrosis factor alpha (TNF-alpha) levels. In contrast, delayed inhibition, allowing CSPG synthesis during the first 2 d following injury, with subsequent inhibition, improved recovery. Using in vitro studies, we showed that CSPG directly activated microglia/macrophages via the CD44 receptor and modulated neurotrophic factor secretion by these cells. Co

Although there is a growing acceptance that immune cells could play a protective role under various injurious or pathological conditions of the central nervous system (CNS), the possibility that the immune system is constantly involved in day-to-day maintenance of CNS functional integrity has not been acknowledged. Here, we propose a unifying hypothesis, based on a recent collection of experimental results, suggesting that the loss of immunity to certain self-antigens or its insufficiency when encountering increased levels of risk factors is an important underlying factor in the onset or escalation of neurodegenerative processes, age-related dementia or mental dysfunction. We further suggest a model that explains how immunity to self exerts its roles in the special immune-privileged context of the CNS.

Neurogenesis, the formation of new neurons from stem/progenitor cells, occurs in the hippocampal dentate gyrus throughout life. Although the exact function of adult hippocampal neurogenesis is currently unknown, recent studies suggest that the newly formed neuronal population plays an important role in hippocampal-dependent cognitive abilities, including declarative memory. The process of adult neurogenesis is greatly influenced by the interaction between cells of the adaptive immune system and CNS-resident immune cells. Our laboratory has recently demonstrated that immune cells contribute to maintaining life-long hippocampal neurogenesis. The regulation of such immune-cell activity is crucial: too little immune activity (as in immune deficiency syndromes) or too much immune activity (as in severe inflammatory diseases) can lead to impaired hippocampal neurogenesis, which could then result in impaired hippocampal-dependent cognitive abilities. From these converging discoveries arise a mechanism that can explain one route by which our body affects our mind. (C) 2007 Elsevier Inc. All rights reserved.

Glaucoma, once thought as a single disease, is actually a group of diseases of the optic nerve involving loss of retinal ganglion cells. The process of cell death occurs in a characteristic pattern of optic neuropathy, a broad term for a certain pattern of damage to the optic nerve (the bundle of nerve fibers that carries information from the eye to the brain). Untreated glaucoma leads to permanent damage of the optic nerve and resultant visual field loss, which can progress to blindness. Worldwide, it is estimated that about 66.8 million people have visual impairment as a result of glaucoma, with 6.7 million suffering from blindness.

Neurogenesis-the formation of new neurons in the adult brain - is considered to be one of the mechanisms by which the brain maintains its lifelong plasticity in response to extrinsic and intrinsic changes(1). The mechanisms underlying the regulation of neurogenesis are largely unknown. Here, we show that Toll-like receptors (TLRs), a family of highly conserved pattern-recognizing receptors involved in neural system development in Drosophila(2) and innate immune activity in mammals(3,4), regulate adult hippocampal neurogenesis. We show that TLR2 and TLR4 are found on adult neural stem/progenitor cells (NPCs) and have distinct and opposing functions in NPC proliferation and differentiation both in vitro and in vivo. TLR2 deficiency in mice impaired hippocampal neurogenesis, whereas the absence of TLR4 resulted in enhanced proliferation and neuronal differentiation. In vitro studies further indicated that TLR2 and TLR4 directly modulated self-renewal and the cell-fate decision of NPCs. The activation of TLRs on the NPCs was mediated via MyD88 and induced PKC alpha/beta-dependent activation of the NF-kappa B signalling pathway. Thus, our study identified TLRs as players in adult neurogenesis and emphasizes their specified and diverse role in cell renewal.

Microglia are resident cells in the central nervous system (CNS), of hematopoietic origin with a high plasticity. In this study, we examined whether adaptive immune system, involving in CNS maintenance and repair, can induce microglia to express markers of neural cells. We show that long exposure (above 10 days) of microglia to low doses (10 ng/ml) of the 'proinflammatory' T-cell derived cytokine, IFN-gamma, induced them to express neuronal markers including gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD-67). In contrast, exposure of microglia to low doses (10 ng/ml) of the 'antiinflammatory' T-cell derived cytokine, IL-4, induced the expression of oligodendrocyte markers and dendritic cell (DC) marker, CD11c. The microglial origin of the neural-like cells was confirmed using microglia from transgenic mice expressing GFP under promoter of the chemokine fractalkine receptor CX(3)CR1, and diphtheria toxin receptor, under CD11c promoter. This study emphasizes that microglial plasticity includes their ability to give rise to neural-like cells and shows that cytokines produced by the adaptive immune system are involved in these processes. (c) 2007 Published by Elsevier Inc.

Glaucomatous neuropathy, like other neurodegenerative diseases, is a multi-dimensional disease in which various molecular and cellular factors contribute to the pathological process. These factors, while not initially causative, are key elements in disease progression and may continue to contribute even after the primary pathology is alleviated. An entire field of research of neuroprotection and restoration has opened up as part of the search for additional ways of slowing disease progression. We have proposed, on the basis of experimental evidence, that a vaccine could be a means of recruiting the immune system to help eliminate many of the factors associated with glaucomatous neurodegeneration and thus prevent disease progression, though not its onset. This immune defence involves lymphocytes, resident and infiltrating innate immune cells, the microglia, and macrophages. The antigens of choice are synthetic antigens, such as glatiramer acetate, that weakly cross-react with self-antigens in the retina and optic nerves. The vaccine induces a beneficial immune response that recruits immune effector cells to counteract or neutralize many of the compounds and factors that contribute to ongoing destruction, and in addition supports cell renewal and repair.

The ability of the central nervous system to cope with stressful conditions was shown to be dependent on proper T-cell-mediated immune response. Because the therapeutic window for neuroprotection after acute insults such as stroke is relatively narrow, we searched for a procedure that would allow the relevant T cells to be recruited rapidly. Permanent middle cerebral artery occlusion was induced in adult rats. To facilitate a rapid poststroke T cell activity, rats were treated with poly-YE using different regimens. Control and poly-YE-treated rats were assessed for functional recovery using neurological severity score and Morris water maze. Neuroprotection, neurogenesis, growth factor expression, and microglial phenotype were assessed using histological and immunofluorescence methods. Administration of poly-YE as late as 24 hours after middle cerebral artery occlusion yielded a beneficial effect manifested by better neurological performance, reduced neuronal loss, attenuation of behavioral deficits, and increased hippocampal and cortical neurogenesis. This compound affected the subacute phase by modulating microglial response and by increasing local production of insulin-like growth factor-I, known to be a key player in neuronal survival and neurogenesis. The relative wide therapeutic window, coupled with its efficacy in attenuating further degeneration and enhancing restoration, makes poly-YE a promising immune-based candidate for stroke therapy. (Stroke. 2007;38[part 2]:774-782.)

Neurogenesis is known to continuously take place in certain ‘neurogenic’ areas of the adult central nervous system (CNS) and can be induced in ‘non-neurogenic’ areas under traumatic or degenerative conditions. Here we introduce T cells and CNS-resident microglia as important players in the regulation of adult neurogenesis. Under normal conditions, immune deficient mice (SCID and nude) and transgenic mice that most of their T-cell pool is specific for an irrelevant antigen (ovalbumin) exhibited impaired hippocampal neurogenesis. In contrast, mice in which the majority of T cells specifically recognize the CNSabundant antigen myelin basic protein showed normal neurogenesis. CNSspecific T cells were also found to be important for spatial learning abilities and for brain-derived neurotrophic factor expression in the dentate gyrus. Environmental enrichment did not evoke enhanced neurogenesis in immune-deficient animals, whereas in wild-type animals it led to enhanced hippocampal neurogenesis coupled with recruitment of T cells and activation of microglia. The clinical implications of these findings were tested using a rat model of cerebral ischemic insult. We demonstrate that Tcell based immune activation following stroke induces a robust elevation of neurogenesis in the hippocampus as well as in the cerebral cortex. Our results suggest that T cells, acting via resident antigen presenting cells, are important regulators of adult neurogenesis under both physiological and pathological conditions.

Alzheimer's disease (AD) is characterized by plaque formation, neuronal loss, and cognitive decline. The functions of the local and systemic immune response in this disease are still controversial. Using AD double-transgenic (APP/PS1) mice, we show that a T cell-based vaccination with glatiramer acetate, given according to a specific regimen, resulted in decreased plaque formation and induction of neurogenesis. It also reduced cognitive decline, assessed by performance in a Morris water maze. The vaccination apparently exerted its effect by causing a phenotype switch in brain microglia to dendritic-like (CD11c) cells producing insulin-like growth factor 1. In vitro findings showed that microglia activated by aggregated beta-amyloid, and characterized as CD11b(+)/CD11c(-)/MHC class II-/TNF-alpha(+) cells, impeded neurogenesis from adult neural stem/progenitor cells, whereas CD11b(+)/CD11c(+)/MHC class II+/TNF-alpha(-) microglia, a phenotype induced by IL-4, counteracted the adverse beta-amyloid-induced effect. These results suggest that dendritic-like microglia, by facilitating the necessary adjustment, might contribute significantly to the brain's resistance to AD and argue against the use of antiinflammatory drugs.

The ability to cope with ongoing neurodegeneration after injury to the central nervous system of mammals differs among strains and depends in part on the animal's ability to manifest a T-cell-mediated protective response. After CNS injury, strain-related differences were observed. Moreover, the post-injury effect of naturally occurring regulatory CD4+CD25+ T cells was found to differ in different strains. In this study, using partially injured optic nerves of Balb/c/OLA and C57BL/6J mice as models, we observed strain-related differences in the T-cell-mediated protection obtained by antigens administered via the nasal route. Active immunization with myelin-related antigens emulsified in complete Freund's adjuvant had a beneficial effect on both strains, whereas mucosal administration of the same antigens was destructive in mice of the Balb/c/OLA strain but protective in C57BL/6J mice. (c) 2006 Elsevier B.V. All rights reserved.

The role of activated microglia (MG) in demyelinating neurodegenerative diseases such as multiple sclerosis is controversial. Here we show that high, but not low, levels of IFN-gamma (a cytokine associated with inflammatory autoimmune diseases) conferred on rodent MG a phenotype that impeded oligodendrogenesis from adult neural stem/progenitor cells. IL-4 reversed the impediment, attenuated TNF-alpha. production, and overcame blockage of IGF-I production caused by IFN-gamma. In rodents with acute or chronic EAE, injection of IL-4-activated MG into the cerebrospinal fluid resulted in increased oligodendrogenesis in the spinal cord and improved clinical symptoms. The newly formed oligodendrocytes were spatially associated with MG expressing MHC class II proteins and IGF-I. These results point to what we believe to be a novel role for MG in oligodendrogenesis from the endogenous stem cell pool.

T-cell-mediated autoimmunity participates in physiological defense, maintenance and repair of the adult brain. However, unless such autoimmune responses to insults are rigorously controlled, they might lead to an autoimmune disease or other immune-related defects, where destructive activity outweighs the beneficial effect. Here, we discuss these apparently contradictory effects of autoimmunity in schizophrenic patients, whose typical immune aberrations have prompted recent speculation about an autoimmune-related etiology. We found that, although schizophrenic patients have active immune systems, they often lack autoimmune clones specifically reactive to a major myelin protein, myelin basic protein (MBP). This, in conjunction with our discovery in rodents that T cells that recognize brain-resident proteins are needed for normal cognitive functioning, led us to propose an immune-based neurodevelopmental hypothesis, in which autoimmune-T-cell deficiency is suggested to cause onset or progression of schizophrenia.

Microglia, the standby cells for immune defense in the CNS, have a reputation for exacerbating the neural damage that occurs in neurodegenerative diseases. However, research over the past few years has established that microglia do not constitute a single, uniform cell population, but rather comprise a family of cells with diverse phenotypes - some that are beneficial and others that the CNS can barely tolerate and that are therefore destructive. This finding raised several questions. What instructs microglia to acquire a particular phenotype, and how do these phenotypes differ? How committed are microglia to a specific phenotype? Can destructive microglia become protective, and can protective microglia retain their beneficial phenotype even when they encounter a destructive environment? Here, we address these questions, and the background of research that elicited them.

Chondroitin sulfate proteoglycan (CSPG), a matrix protein that occurs naturally in the central nervous system (CNS), is considered to be a major inhibitor of axonal regeneration and is known to participate in activation of the inflammatory response. The degradation of CSPG by a specific enzyme, chondroitinase ABC, promotes repair. We postulated that a disaccharidic degradation product of this glycoprotein (CSPG-DS), generated following such degradation, participates in the modulation of the inflammatory responses and can, therefore, promote recovery in immune-induced neuropathologies of the CNS, such as experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU). In these pathologies, the dramatic increase in T cells infiltrating the CNS is far in excess of the numbers needed for regular maintenance. Here, we show that CSPG-DS markedly alleviated the clinical symptoms of EAE and protected against the neuronal loss in EAU. The last effect was associated with a reduction in the numbers of infiltrating T cells and marked microglia activation. This is further supported by our in vitro results indicating that CSPG-DS attenuated T cell motility and decreased secretion of the cytokines interferon-gamma and tumor necrosis factor-alpha. Mechanistically, these effects are associated with an increase in SOCS-3 levels and a decrease in NF-kappa B. Our results point to a potential therapeutic modality, in which a compound derived from an endogenous CNS-resident molecule, known for its destructive role in CNS recovery, might be helpful in overcoming inflammation-induced neurodegenerative conditions.

Nerve agents are highly toxic organophosphates (OPs) that can cause severe damage to the central and peripheral nervous systems. The central nervous system insult results in seizures and neuronal death. The glutamatergic system apparently contributes to the neuropathology. Using a model of OP intoxication causing death of retinal ganglion cells in the mouse eye, we show here that intoxication is exacerbated if the mice are devoid of mature T cells. The retinal neurons could be protected from these effects by vaccination, 7 days before or immediately after intoxication, with the copolymer glatiramer acetate (Cop-1), recently found to limit the usual consequences of an acute glutamate insult to the eye. These findings underlie a new therapeutic approach to protection against OP intoxication, based on the rationale that boosting of the adaptive immunity recruited at the site of intoxication helps the local cellular machinery such as resident microglia to withstand the neurotoxic effects.

With the increasing use of the rat as an animal model for glaucoma and for the evaluation of neuroprotective treatments, there is a need for a sensitive test of retinal ganglion cell (RGC) function in this species. The aims of this study were to detect functional abnormalities of the inner retina in a rat model of high intraocular pressure (IOP) using the pattern electroretinogram (PERG), and to correlate them with morphometric analysis of RGC survival and the functional integrity of the inner retina. Unilateral ocular hypertension was induced in 17 Lewis rats through laser photocoagulation. Pattern ERGs were recorded prior to lasering and 3 weeks later, using a series of shifting patterns of decreasing spatial frequency projected directly onto the animals' fundus. IOP was measured at the same intervals, and the number of surviving RGCs estimated. Low amplitude PERG signals could be recorded in response to a narrow grating of 0.368 cycles per degree (cpd), and increased with stimulus size. Lasering caused mean (+/- S.D.) IOP to increase significantly from 18.3 +/- 4.5 (baseline) to 29.8 8.8 mmHg within 3 weeks (p

Protective e autoimmunity refers to a well-controlled anti-self response that helps the body resist neurodegeneration. The response is mediated by autoimmune T cells, which produce cytokines and growth factors. Using an in vitro assay of hippocampal slices, we show that the cytokines interferon-gamma and (especially) interleukin-4, characteristic of pro-inflammatory and anti-inflarnmatory T cells, respectively, can make microglia neuroprotective. Aggregated beta-amyloid, like bacteria cell wall-derived lipopolysaccharide, rendered the microglia cytotoxic. Cytotoxicity was correlated with a signal transduction pathway that down-regulates expression of class-II major histocompatibility proteins (MHC-II) through the MHC-II-transactivator and the invariant chain. Protection by interleukin-4 was attributed to down-regulation of tumor necrosis factor-alpha, and up-regulation of insulin-like growth factor I. These findings suggest that beneficial or harmful expression of the local immune response in the damaged CNS depends on how microglia interpret the threat, and that a well-regulated T-cell-mediated response enables microglia to alleviate rather than exacerbate stressful situations in the CNS. (c) 2005 Published by Elsevier Inc.

The trigger that leads to the pathogenesis of type 1 diabetes is currently unknown. It is well established that the pathophysiology of the disease is biphasic. In the first stage, leukocytes infiltrate the pancreatic islets in a response that does not cause damage. In the second phase, which occurs only in diabetes-prone individuals and strains, autoreactive T cells acquire aggressive potential and destroy the majority of the pancreatic islets. Rodents and humans exhibit a physiological ripple of apoptotic beta-cell death shortly after birth, which induces an adaptive autoimmune response towards islet-antigens, both in diabetes-prone non-obese diabetic (NOD) mice and in mice that do not develop diabetes. Here, we propose that the early T cell-mediated autoimmune response towards islet-antigens is physiological, purposeful and beneficial.

The failure of the spinal cord to recover after injury has been associated with the immune privilege mechanism that suppresses immune activity throughout the central nervous system. Primed macrophages and dendritic cells were shown to promote neurological recovery in preclinical models of spinal cord injury. A cell therapy consisting of autologous incubated macrophages is now being tested on spinal cord injury patients in clinical trials.

Chondroitin sulphate proteoglycan (CSPG) inhibits axonal regeneration in the central nervous system (CNS) and its local degradation promotes repair. We postulated that the enzymatic degradation of CSPG generates reparative products. Here we show that an enzymatic degradation product of CSPG, a specific disaccharide (CSPG-DS), promoted CNS recovery by modulating both neuronal and microglial behaviour. In neurons, acting via a mechanism that involves the PKCalpha and PYK2 intracellular signalling pathways, CSPG-DS induced neurite outgrowth and protected against neuronal toxicity and axonal collapse in vitro. In microglia, via a mechanism that involves ERK1/2 and PYK2, CSPG-DS evoked a response that allowed these cells to manifest a neuroprotective phenotype ex vivo. In vivo, systemically or locally injected CSPG-DS protected neurons in mice subjected to glutamate or aggregated beta-amyloid intoxication. Our results suggest that treatment with CSPG-DS might provide a way to promote post-traumatic recovery, via multiple cellular targets.

Autoimmune T cells have been viewed for decades as an outcome of immune system malfunction, and specifically as a failure to distinguish between components of self and non-self. The need for discrimination between self and non-self as a way to avoid autoimmunity has been repeatedly debated over the years. Recent studies suggest that autoimmunity, at least in the nervous system, is the body's defense mechanism against deviations from the normal. The ability to harness neuroprotective autoimmunity upon need is evidently allowed by naturally occurring CD4+CD25+ regulatory T cells, which are themselves controlled by brain-derived compounds. These findings challenge widely accepted concepts of the need for discrimination between self and non-self, as they suggest that while such discrimination is indeed required, it is needed not as away to avoid an anti-self response but to ensure its proper regulation. Whereas a response to non-self can be self-limited by a decreased presence of the relevant antigen, the response to self needs a mechanism for strict control, such as that provided by the naturally occurring regulatory T cells.

Fighting off neuronal degeneration requires a well controlled T-cell response against self-antigens residing in sites of the CNS damage. The ability to evoke this response is normally suppressed by naturally occurring CD4(+) CD25(+) regulatory T-cells (Treg). No physiological compound that controls Treg activity has yet been identified. Here, we show that dopamine, acting via type 1 dopamine receptors ( found here to be preferentially expressed by Treg), reduces the suppressive activity and the adhesive and migratory abilities of Treg. Treg activity was correlated with activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) signaling pathway. Systemic injection of dopamine or an agonist of its type 1 receptors significantly enhanced, via a T-cell-dependent mechanism, protection against neuronal death after CNS mechanical and biochemical injury. These findings shed light on the physiological mechanisms controlling Treg and might open the way to novel therapeutic strategies for downregulating Treg activity (e.g., in neuronal degeneration) or for strengthening it (in autoimmune diseases).

The effects of the adaptive immune system on the cognitive performance and abnormal behaviors seen in mental disorders such as schizophrenia have never been documented. Here, we show that mice deprived of mature T cells manifested cognitive deficits and behavioral abnormalities, which were remediable by T cell restoration. T cell-based vaccination, using glatiramer acetate (copolymer-1, a weak agonist of numerous self-reactive T cells), can overcome the behavioral and cognitive abnormalities that accompany neurotransmitter imbalance induced by (+)dizocilpine maleate (MK-801) or amphetamine. The results, by suggesting that peripheral T cell deficit can lead to cognitive and behavioral impairment, highlight the importance of properly functioning adaptive immunity in the maintenance of mental activity and in coping with conditions leading to cognitive deficits. These findings point to critical factors likely to contribute to age- and AIDS-related dementias and might herald the development of a therapeutic vaccination for fighting off cognitive dysfunction and psychiatric conditions.

Protective autoimmunity was only recently recognized as a mechanism for attenuating the progression of neurodegeneration. Using a rat model of optic nerve crush or contusive spinal cord injury, and a mouse model of neurodegenerative conditions caused by injection of a toxic dose of intraocular glutamate, we show that a single low dose of whole-body or lymphoid-organ gamma-irradiation significantly improved the spontaneous recovery. Animals with severe immune deficiency or deprived of mature T cells were unable to benefit from this treatment, suggesting that the irradiation-induced neuroprotection is immune mediated. This suggestion received further support from the findings that irradiation was accompanied by an increased incidence of activated T cells in the lymphoid organs and peripheral blood and an increase in mRNA encoding for the pro-inflammatory cytokines interleukin-12 and interferon-gamma, and that after irradiation, passive transfer of a subpopulation of suppressive T cells (naturally occurring regulatory CD4(+)CD25(+) T cells) wiped out the irradiation-induced protection. These results suggest that homeostasis-driven proliferation of T cells, induced by a single low-dose irradiation, leads to boosting of T cell-mediated neuroprotection and can be utilized clinically to fight off neurodegeneration and the threat of other diseases in which defense against toxic self-compounds is needed.

In neurodegenerative disorders, as well as in acute central nervous system (CNS) injuries, the initial impairment triggers a cascade of destructive events, collectively termed secondary degeneration, which eventually cause much more extensive damage. To investigate the process of secondary degeneration and ways to prevent it, we designed a well-calibrated model of optic nerve crush injury. Until recently, the main purpose of the immune system was thought to be protection of the body against alien pathogens. Since mechanical or biochemical insults do not involve exogenous pathogens, recruitment of the adaptive immune system was not considered relevant in such cases. We recently demonstrated, however, that a T-cell-mediated immune response directed against self-antigens residing in the site of damage can be beneficial for the injured optic nerve or spinal cord. This protective autoimmune response was found to be spontaneously evoked in some individuals, but not strongly enough to significantly affect recovery. Our aim was to boost this protective response in those individuals capable of spontaneously manifesting it, and to induce it in those incapable of manifesting it spontaneously. Optimal functional recovery requires the application of a proper combination of neuroprotection and neuroregeneration. (C) 2003 Published by Elsevier Inc.

Injury-induced self-destructive processes cause significant functional loss after incomplete spinal cord injury (SCI). Cellular elements of both the innate (macrophage) and the adaptive (T-cell) immune response can, if properly activated and controlled, promote post-traumatic regrowth and protection after SCI. Dendritic cells (DCs) trigger activation of effector and regulatory T-cells, providing a link between the functions of the innate and the adaptive immune systems. They also initiate and control the body's response to pathogenic agents and regulate immune responses to both foreign and self-antigens. Here we show that post-injury injection of bone marrow-derived DCs pulsed with encephalitogenic or nonencephalitogenic peptides derived from myelin basic protein, when administered (either systemically or locally by injection into the lesion site) up to 12 d after the injury, led to significant and pronounced recovery from severe incomplete SCI. No significant protection was seen in DC recipients deprived of mature T-cells. Flow cytometry, RT-PCR, and proliferation assays indicated that the DCs prepared and used here were mature and immunogenic. Taken together, the results suggest that the DC-mediated neuroprotection was achieved via the induction of a systemic T-cell-dependent immune response. Better preservation of neural tissue and diminished formation of cysts and scar tissue accompanied the improved functional recovery in DC-treated rats. The use of antigen-specific DCs may represent an effective way to obtain, via transient induction of an autoimmune response, the maximal benefit of immune-mediated repair and maintenance as well as protection against self-destructive compounds.

Glutamate, a key neurotransmitter, is pivotal to CNS function. Alterations in its concentration can be dangerous, as seen for example in acute injuries of the CNS, chronic neurodegenerative disorders and mental disorders. Its homeostasis is attributed to the efficient removal of glutamate from the extracellular milieu by reuptake via local transport mechanisms. Our recent studies suggest that glutamate, either directly or indirectly, elicits a purposeful systemic T-cell-mediated immune response directed against immunodominant self-antigens that reside at the site of glutamate-induced damage. We suggest that the harnessed autoimmunity (which we have termed 'protective autoimmunity') helps the resident microglia in their dual function as antigen-presenting cells (serving the immune system) and as cells that clear the damaged site of potentially harmful material (serving the nervous system). The interplay between glutamate and an adaptive immune response illustrates the bidirectional dialog between the immune and nervous systems, under both physiological and pathological conditions. These results point to the possible development of a therapeutic vaccination with self-antigens, or with antigens cross-reactive with self-antigens, as a way to augment autoimmunity without inducing an autoimmune disease, thus providing a safe method of limiting degeneration. This approach, which boosts a physiological mechanism for the regulation of glutamate, and possibly also that of other self-compounds, might prove to be a feasible strategy for therapeutic protection against glutamate-associated neurodegenerative or mental disorders.

Primary open-angle glaucoma is a chronic, progressive optic neuropathy associated with a gradual decline in visual function, which may lead to blindness. In most cases, the optic neuropathy is associated with increased intraocular pressure. It is now generally accepted, however, that normalization of pressure, although necessary, is often not-sufficient as a remedial measure. This is because of the existence of additional factors, some of which emerge as a consequence of the initial damage. This situation is reminiscent of the response to a traumatic axonal insult, in which some of the damage is immediate and is caused by the insult itself, and some is secondary and is caused by a deficiency of growth-supportive factors as well as by toxic factors derived from the damaged tissue. Accordingly, the author has suggested that glaucoma may be viewed as a neurodegenerative disease and consequently amenable to any therapeutic intervention applicable to neurodegenerative diseases. There is evidence that neuroprotection can be achieved both pharmacologically and immunologically. Pharmacologic intervention neutralizes some of the effects of the nerve-derived toxic factors and possibly increases the ability of the remaining healthy neurons, at any given time, to cope with the stressful conditions. Immunologic intervention boosts the body's repair mechanisms for counteracting the toxicity of physiologic compounds acting as stress signals.

Therapeutic vaccination with Copaxone (glatiramer acetate, Cop-1) protects motor neurons against acute and chronic degenerative conditions. In acute degeneration after facial nerve axotomy, the number of surviving motor neurons was almost two times higher in Cop-1-vaccinated mice than in nonvaccinated mice, or in mice injected with PBS emulsified in complete Freund's adjuvant (P

The function of the adaptive immune response against exogenous (non-self) agents is to help the innate arm of the immune system (represented by phagocytic cells) to fight and eliminate these agents. We suggest that the body also protects itself against potentially harmful self components using mechanisms similar to those used for fighting and eliminating non-self agents, and that the protective immune activity against self-components competes with the activity of self-destructive compounds. Tolerance to self is thus not a lack of response to self, but the ability to tolerate an active defense response to self without developing an autoimmune disease.

Purpose: To evaluate the neuroprotective effect of memantine, an NMDA receptor channel blocker, in two retinal ganglion cell (RGC) injury models in rats. Methods: Neuroprotective effect of memantine was tested in partial optic nerve injury and chronic ocular hypertensive models. In the optic nerve injury model, memantine (0.1 - 30 mg/kg) was injected intraperitoneally immediately after injury. Two weeks later, optic nerve function was determined by measuring compound action potential and surviving RGC was determined by retrograde labeling with dextran tetramethyl rhodamine. Chronic ocular hypertension was attained by laser photocoagulation of episcleral and limbal veins. Memantine (5 or 10 mg/kg) was administered continuously each day with an osmotic pump, either immediately after or 10 days after first laser photocoagulation, for 3 weeks, after which RGC survival was determined. Results: Two weeks after partial optic nerve injury, there was approximate to80% reduction in RGC number. Memantine (5 mg/kg) caused a twofold increase in compound action potential amplitude and a 1.7-fold increase in survival of RGCs, respectively. In the chronic ocular hypertension model there was 37% decrease in RGCS after 3 weeks of elevated intraocular pressure. Memantine (10 mg/kg daily) reduced ganglion cell loss to 12% when applied immediately after first laser photocoagulation, and prevented any further loss when applied 10 days after first laser photocoagulation. Conclusion: The protective effect of memantine suggests that excessive stimulation of NMDA receptors by glutamate is involved in causing cell damage in these RGC injury models.

Autoimmunity, at least in the central nervous system (CNS), is not only an outcome of immune system malfunction, but is the body's own protective mechanism against destructive self-compounds. Likewise, the naturally occurring regulatory CD4(+)CD25(+) T cells have a physiological function, and are not merely an evolutionary adaptation to suppress self-reactive T-cell clones that escaped deletion in the thymus. We postulate that the regulatory T (Tr) cells are the product of an evolutionary compromise between the need for autoimmunity on alert for tissue maintenance and the need to control autoimmunity to avoid autoimmune disease. In the event of an insult to the CNS, the balance between self-reactive (effector) T cells and Tr cells determines the time of onset, the intensity and the duration of the autoimmune response. This response might thus represent an adaptive mechanism, which is optimal for day-to-day maintenance, but insufficient in extreme cases of CNS damage or failure of regulation. Downregulation or upregulation of CD4(+)CD25(+) Tir cells might be a way to achieve better protection from neurodegenerative conditions induced by self-destruction or avoid autoimmune inflammatory disease development, respectively.

Immune cells have been shown to contribute to spontaneous recovery from central nervous system (CNS) injury. Here we show that adult female rats and mice recover significantly better than their male littermates from incomplete spinal cord injury (ISCI). This sexual dimorphism is wiped out and recovery is worse in adult mice deprived of mature T cells. After spinal cord contusion in adult rats, functional recovery ( measured by locomotor scores in an open field) was significantly worse in females treated with dihydrotestosterone prior to the injury than in placebo-treated controls, and significantly better in castrated males than in their noncastrated male littermates. Post-traumatic administration of the testosterone receptor antagonist flutamide promoted the functional recovery in adult male rats. These results, in line with the known inhibitory effect of testosterone on cell-mediated immunity, suggest that androgen-mediated immunosuppression plays a role in ISCI-related immune dysfunction and can therefore partly explain the worse outcome of ISCI in males than in female. We suggest that females, which are more prone to develop autoimmune response than males, benefit from this response in cases of CNS insults.

The resistance of rats or mice to glutamate-induced toxicity depends on their ability to spontaneously manifest a T cell-dependent response to the insult. Survival of retinal ganglion cells (RGCs) exposed to glutamate in BALB/c SCID mice (a strain relatively resistant to glutamate toxicity) was significantly worse than in the wild type. In the susceptible C57BL/6J mouse strain, however, significantly more RGCs survived among SCID mutants than in the matched wild type. RGC survival in the SCID mutants of the two strains was similar. These results suggest 1) that immunodeficiency might be an advantage in strains incapable of spontaneously manifesting protective T cell-dependent immunity and 2) that B cells might be destructive in such cases. After exposure of RGCs to toxic glutamate concentrations in three variants of B cell-deficient C57BL/6J mice, namely muMT(-/-) (B cell knockout mice) and Ii(-/-) mice reconstituted with transgenically expressed low levels of Ii p31 isoforms (p31 mice) or Ii p41. isoforms (p41 mice), significantly more RGCs survived in these mice than in the wild type. The improved survival was diminished by replenishment of the B cell-deficient mice with B cells derived from the wild type. It thus seems that B cells have an adverse effect on neuronal recovery after injury, at least in a strain that is unable to spontaneously manifest a T cell-dependent protective mechanism. These findings have clear implications for the design of immune-based therapies for CNS injury.

PURPOSE. Glaucoma is widely accepted as a neurodegenerative disease in which retinal ganglion cell (RGC) loss is initiated by a primary insult to the optic nerve head, caused, for example, by increased intraocular pressure (IOP). In some cases, the surviving RGCs, despite adequate IOP control, may continue to degenerate as a result of their heightened susceptibility to self-destructive processes evoked by the initial damage. In animal models of mechanical or biochemical injury to the optic nerve or retina, a T-cell-mediated immune response evoked by the insult helps to reduce this ongoing loss. The current study was conducted to find out whether the ability to resist the IOP-induced loss of RGCs in a rat model is affected by the immune system. METHODS. The ocular veins and limbal plexus of rats of two strains differing in their resistance to experimental autoimmune encephalomyelitis (EAE) and in their ability to manifest a beneficial autoimmune response were laser irradiated twice to induce an increase in IOP. The pressure was measured weekly, and RGC losses were assessed 3 and 6 weeks after the first irradiation. To verify the existence of a relationship between the immune system and RGC survival, we assessed neuronal survival in Sprague-Dawley (SPD) rats devoid of mature T cells as well as after transferring splenocytes from Fisher rats, an EA,E-resistant rat strain capable of manifesting T-cell-mediated neuroprotection, to rats of a major histocompatibility complex (MHC)-matched EAE-susceptible strain (Lewis), in which the ability to manifest such protective immunity is limited. RESULTS. Both 3 and 6 weeks after the increase in IOP was initiated, the number of surviving RGCs in SPD rats, a strain in which a beneficial autoimmune response can be evoked spontaneously, was significantly higher than in Lewis rats. Moreover, in SPD rats that were thymectomized at birth, the number of surviving RGCs after an increase in IOP as adults was significantly diminished. Pa

Protective autoimmunity is the body's defense mechanism against destructive self-compounds such as those commonly associated with neurodegenerative disorders. Autoimmune disease and neurodegenerative disorders can thus be viewed as two extreme manifestations of the same process. Therefore, when designing therapy, it is important to avoid an approach that will cure the one by invoking the other. One way to stop, or at least slow down, the progression of neurodegeneration without risking development of an autoimmune disease is by boosting protective autoimmunity in a well-controlled way. Copolymer 1 (Cop-1), an approved drug for the treatment of multiple sclerosis, can be used as a treatment for autoimmune diseases and as a therapeutic vaccine for neurodegenerative diseases. We propose that the protective effect of Cop-1 vaccination is obtained through a well-controlled inflammatory reaction, and that the activity of Cop-1 in driving this reaction derives from its ability to serve as a 'universal antigen' by weakly activating a wide spectrum of self-reactive T cells.

Neurodegenerative diseases, whatever their primary causes, are characterized by certain common features, one of which is their self-perpetuating nature. The ongoing progression of the disorder is due to the effects of destructive self-compounds, whose presence in the tissues is an outcome of the early phase of the disease and which gradually destroy remaining functional neurons. Studies in our laboratory have led to the recent formulation of a novel concept of protective autoimmunity as the body's mechanism of defense against these destructive self-compounds. This autoimmune response to central nervous system (CNS) insults is mediated by T-cells and presumably operates by activating and regulating local microglia and infiltrating macrophages (inflammatory response) to carry out their function of clearing destructive material from the tissue at risk. We suggest that a well-controlled autoimmunity counteracts and overcomes the destructive effects of the potentially harmful self-compounds, at the cost of some loss of tissue. An additional risk to the individual is the induction of an autoimmune disease, which is likely to occur if the autoimmune response is malfunctioning. An optimal balance of the various factors will lead to an outcome of maximal benefit at minimal cost to the tissue. A procedure for safely boosting the autoimmune response, by vaccination with a weak self-crossreactive antigen such as glatiramer acetate (also known as Cop-1) was found to protect rats from glutamate toxicity, a major mediator of the spread of damage and a well-known causative factor in neurodegenerative disorders. Cop-1, when administered according to a different regimen, is an FDA-approved drug for the treatment of multiple sclerosis. Different formulations of the same drug can therefore be used to treat two extreme manifestations of chronic degenerative diseases of the CNS. (C) 2002 Wiley-Liss, Inc.

Functional loss after injury to the mammalian central nervous system (CNS) has been attributed not only to the immediate loss of neurons but also to secondary neuronal degeneration caused by the toxicity of physiological Compounds present in abnormally high amounts as a result of the injury. One such compound appears to be the protease thrombin. Here we show that the beneficial effect of T cells directed against myelin self-antigens can be attributed, at least in part, to the ability of these 'autoimmune' T cells to produce antithrombin III. Using transgenic mice lacking the thrombin receptor PAR-1, we also present molecular evidence indicating that down-regulation of PAR-I by genetic manipulation leads to increased post-traumatic survival of CNS neurons. We further show that the ability of autoimmnune T cells to produce thrombin inhibitors and to exert post-traumatic neuroprotection are both independent of their PAR-1 expression. These findings suggest that thrombin plays a key role in post-injury neuronal survival, and that its post-traumatic toxicity can be down-regulated by appropriate alteration of the amounts of PAR-1 receptors or of antithrombin III, the latter achieved by T cell-mediated autoimmunity. (C) 2001 Elsevier Science B.V. All rights reserved.

The myelin-associated protein Nogo-A has received more research attention than any other inhibitor of axonal regeneration in the injured central nervous system (CNS). Circumvention of its inhibitory effect, by using antibodies specific to Nogo-A, has been shown to promote axonal regrowth. Studies in our laboratory have demonstrated that active or passive immunization of CNS-injured rats or mice with myelin-associated peptides induces a T-cell-mediated protective autoimmune response, which promotes recovery by reducing posttraumatic degeneration. Here, we show that neuronal degeneration after incomplete spinal-cord contusion in rats was substantially reduced, and hence recovery was significantly promoted, by posttraumatic immunization with p472, a peptide derived from Nogo-A. The observed effect seemed to be mediated by T cells and could be reproduced by passive transfer of a T cell line directed against the Nogo-A peptide. Thus, it seems that after incomplete spinal-cord injury, immunization with a variety of myelin-associated peptides, including those derived from Nogo-A, can be used to evoke a T cell-mediated response that promotes recovery. The choice of peptide(s) for clinical treatment of spinal-cord injuries should be based on safety considerations; in particular, the likelihood that the chosen peptide will not cause an autoimmune disease or interfere with essential functions of this peptide or other proteins. From a therapeutic point of view, the fact that the active cellular agents are T cells rather than antibodies is an advantage, as T cell production commences within the time window required for a protective effect after spinal-cord injury, whereas antibody production takes longer.

Axonal injury initiates a process of neuronal degeneration, with resulting death of neuronal cell bodies. We show here that in C57BL/6J mice, previously shown to have a limited ability to manifest a post-traumatic protective immunity, the rate of neuronal survival is increased if IL-6 is deficient during the first 24 hours after optic nerve injury. Immunocytochemical staining preformed 7 days after the injury revealed an increased number of activated microglia in the IL-6-deficient mice compared to the wild-type mice. In addition, IL-6-deficient mice showed an increased resistance to glutamate toxicity. These findings suggest that the presence of IL-6 during the early post-traumatic phase, at least in mice that are susceptible to autoimmune disease development, has a negative effect on neuronal survival. This further substantiates the contention that whether immune-derived factors are beneficial or harmful for nerve recovery after injury depends on the phenotype of the immune cells and the timing and nature of their dialog with the damaged neural tissue. (C) 2001 Elsevier Science B.V. All rights reserved.

PURPOSE. Primary open-angle glaucoma is a chronic, progressive optic neuropathy associated with a gradual decline in visual functions, which may lead to blindness. METHODS. In most cases, the optic neuropathy is associated with increased intraocular pressure. However, it is now generally accepted, that normalization of pressure, although necessary, is often not sufficient as a remedial measure. This is because of the existence of additional risk factors, some of which emerge as a consequence of the initial damage. This situation is reminiscent of the response to a traumatic axonal insult: some of the damage is immediate and is caused by the insult itself, while some is secondary and is caused by a deficiency of growth-supportive factors, as well as by toxic factors derived from the damaged tissue. Accordingly, we have suggested that glaucoma may be viewed as a neurodegenerative disease and consequently is amenable to any therapeutic intervention applicable to these diseases. CONCLUSIONS. There is evidence that neuroprotection can be achieved both pharmacologically and immunologically. Pharmacological intervention (e.g. by using selective alpha-2 adrenergic receptor agonists) neutralizes some of the effects of the nerve-derived toxic factors and possibly increases the ability of the remaining healthy neurons, at any given time, to cope with the stressful conditions. Immunological intervention boosts the body's own repair mechanisms for counteracting the toxicity of physiological compounds acting as stress signals.

Primary damage caused by injury to the CNS is often followed by delayed degeneration of initially spared neurons. Studies in our laboratory have shown that active or passive immunization with CNS myelin-associated self-antigens can reduce this secondary loss. Here we show, using four experimental paradigms in rodents, that CNS trauma spontaneously evokes a beneficial T cell-dependent immune response, which reduces neuronal loss. (1) Survival of retinal ganglion cells in rats was significantly higher when optic nerve injury was preceded by an unrelated CNS (spinal cord) injury. (2) Locomotor activity of rat hindlimbs (measured in an open field using a locomotor rating scale) after contusive injury of the spinal cord (T8) was significantly better (by three to four score grades) after passive transfer of myelin basic protein (MBP)-activated splenocytes derived from spinally injured rats than in untreated injured control rats or rats similarly treated with splenocytes from naive animals or with splenocytes from spinally injured rats activated ex vivo with ovalbumin or without any ex vivo activation. (3) Neuronal survival after optic nerve injury was 40% lower in adult rats devoid of mature T cells (caused by thymectomy at birth) than in normal rats. (4) Retinal ganglion cell survival after optic nerve injury was higher (119 +/- 3.7%) in transgenic mice overexpressing a T cell receptor (TcR) for MBP and lower (85 +/- 1.3%) in mice overexpressing a T cell receptor for the non-self antigen ovalbumin than in matched wild types. Taken together, the results imply that CNS injury evokes a T cell-dependent neuroprotective response.

In glaucoma, as in other degenerative diseases of the central nervous system (CNS), there are some neurons which, although susceptible to degeneration, are amenable to neuroprotection. Until very recently, attempts: to attenuate the spread of damage after CNS trauma or in neurodegenerative diseases did not include recruitment of the immune system, because it was assumed that ill the C:NS any immune activity, particularly autoimmune activity, would be harmful. Using the injured optic nerve of the rat as a model, we recently showed, however, that this 'secondary degeneration' call be slowed down by a well-controlled adaptive immune response, which is mediated by T cells: against C:NS sells antigens, such as myelin basic protein (MBP), and can be achieved either by passive transfer of MBP-activated T cells or by active immunization with MBP. Accordingly, we suggested that;autoimmune T cells can be neuroprotective. We further showed that the neuroprotective autoimmunity is a physiological response to the injury, perhaps insufficient in its natural state, but amenable to boosting. The neuroprotective activity of these T cells probably depends on their reactivation by their specific antigen after they are targeted to the injured nerve. As nerve degeneration is initiated and sustained by many. factors, it would probably best be counteracted by a comprehensive type of therapy rather than treatment that addresses only some aspects of nerve damage. T cell therapy, being physiological rather than pharmaceutical in nature, may provide a global answer. However, since the neuroprotective immune response is directed against die self, it must be rigorously regulated to avoid inducing an autoimmune disease. We showed that the synthetic copolymer Cop-1 can passively or actively evoke T cell-mediated neuroprotection, probably by cross-reacting with MBP. Safe synthetic peptides that resemble self-antigens and are cross-activated by CNS-associated self antigens may be a useful starti

The increasing ability to augment antitumor immunity in model:systems has led to increased numbers of clinical trials. However, progress in detecting immune responses by patients against autologous tumors has been slow, Although a considerable number of tumor antigens, as well as peptides derived from them, and the MHC determinants together with which they are presented have been identified for melanoma, this is not so for the majority of solid tumors. Furthermore, tumor cells themselves are poor stimulators of immunity. Thus, approaches that do not depend upon defined antigens or using tumor cells as stimulators would be desirable. To attempt to measure immune responses in these situations, we tested whether total peptides, prepared from autologous tumor tissue, stimulated cytokine release by T cells. Peripheral blood mononuclear cells (PBMCs) were mixed with antigen-presenting cells (APCs), pulsed with tumor peptides, and tested in the ELISPOT assay for IFN-gamma secretion. Few spots were obtained when PBMCs were cultured with unpulsed APCs or in wells with peptide-pulsed APC alone. In contrast, a strong response was seen when PBMCs were cultured with APCs that had been pulsed with autologous total tumor peptides, This system should help to identify those immunotherapeutic approaches that induce responses against tumor cells in vivo. Because different cytokine profiles are associated with distinct arms of the immune response, testing in the ELISPOT assay may also help us understand the mechanisms responsible.

The functional loss that often follows injury of the mammalian CNS has been attributed not only to the immediate neural loss, but also to secondary neuronal degeneration caused by toxic biochemical mediators in the environment of the injured nerve. We report here that a high thrombin content, produced as a result of injury-induced activation of prothrombin, appears to be an important mediator of secondary damage. Measurement of post-traumatic neuronal survival in vivo revealed that post-traumatic local application of the thrombin inhibitor N-alpha-(2-naphthylsulphonylglycyl)-4-(D,L)-amidinophenylalanine piperidide acetate in the rat optic nerve subjected to mild partial crush injury left twice as many retinal ganglion cells with functioning axons as in controls. Thus, by readjusting thrombin activity, thereby possibly obtaining a moderate post-traumatic increase and thus gaining the benefit of thrombin without its toxic effects, it may be possible to create an environment that is more favourable towards post-traumatic survival.

Spinal cord injury and its devastating consequences are the subject of intensive research aimed at reversing or at least minimizing functional loss, Research efforts focus on either attenuating the post-injury spread of damage (secondary degeneration) or inducing some regeneration. In most of these studies, as well as in clinical situations, evaluation of the state of the injured spinal cord poses a serious difficulty. To address this problem, we carried out a diffusion-weighted MRI experiment and developed an objective routine for quantifying anisotropy in injured rat spinal cords. Rats were subjected to a contusive injury of the spinal cord caused by a controlled weight drop. Untreated control rats were compared with rats treated with T cells specific to the central nervous system self-antigen myelin basic protein, a form of therapy recently shown to be neuroprotective, After the rats were killed their excised spinal cords were fixed in formalin and imaged by multislice spin echo MRI, using two orthogonal diffusion gradients. Apparent diffusion coefficient (ADC) values and anisotropy ratio (Al) maps were extracted on a pixel-by-pixel basis. The calculated sum of Al values (SAl) for each slice was defined as a parameter representing the total amount of anisotropy. The mean-Al and SAI values increased gradually with the distance from the site of the lesion. At the site itself, the mean-Al and SAI values were significantly higher in the spinal cords of the treated animals than in the controls (P = 0.047, P = 0.028, respectively). These values were consistent with the score of functional locomotion. The difference was also manifested in the Al maps, which revealed well-organized neural structure in the treated rats but not in the controls. The SAI values, Al histograms, and Al maps proved to be useful parameters for quantifying injury and recovery in an injured spinal cord. These results encourage the development of diffusion anisotropy MRI as a helpful approach fo

The innate and adaptive arms of the immune system, represented principally by macrophages and by T and B cells, respectively, provide body tissues with mechanisms of defence, protection and repair. In the central nervous system (CNS), probably because of its status of 'immune privilege', any immune activity has long been viewed as detrimental. Recent studies have provided evidence, however, that immune activity after traumatic CNS injury may have a beneficial effect, manifested by promotion of regeneration and reduction in the secondary degeneration of neurons that escaped direct injury. Rigorous regulation of immune system activity allows the individual to derive the benefit of such neuroprotection without the risk of detrimental side effects. Recently, our research group found a way to boost the T-cell-mediated autoimmune protection while avoiding the risk of autoimmune disease.

Neurotrophins (NTs) promote neuronal survival and maintenance during development and after injury. However, their role in the communication between the nervous system and the immune system is not yet clear. We observed recently that passively transferred activated T cells of various antigen specificities home to the injured central nervous system (CNS), yet only autoimmune T cells specific to a CNS antigen, myelin basic protein (MBP), protect neurons from secondary degeneration after crush injury of the rat optic nerve. Here we examined the involvement of NTs in T-cell-mediated neuroprotection, and the possible significance of the antigen specificity of the T cells in this activity. Analysis of cytokine and NT expression in various rat T cell lines showed that the T cells express mRNA for cytokines of Th1, Th2, and Th3 phenotypes. in addition, the T cells express mRNA and protein specific to nerve growth factor, brain-derived neurotrophic factor, NT-3, and NT-4/5. Antigen activation significantly increased NT secretion. Thus, reactivation of CNS autoimmune T cells by locally presented antigens to which they are specific can lead to enhanced secretion of NTs and possibly also of other factors in injured optic nerves. mRNA for TrkA, TrkB and p75 receptors was expressed in the injured nerve, suggesting that these specific receptors can mediate the effects of the T-cell-derived NTs. The neuroprotective effect of the passively transferred autoimmune anti-MBP T cells in injured optic nerves was significantly decreased after local applicaiton of a tyrosine kinase inhibitor known to be associated with NT-receptor activity. These results suggest that the neuroprotective effect of autoimmune T cells involves the secretion of factors such as NTs by the T cells reactivated by their specific antigen in the injured CNS. T cell intervention in the injured CNS might prove to be a useful means of promoting post-injury CNS maintenance and recovery, possibly via supply of NTs and o

We recently demonstrated that autoimmune T cells protect neurons from secondary degeneration after central nervous system (CNS) axotomy in rats. Here we show, using both morphological and electrophysiological analyses, that the neuroprotection is long-lasting and is manifested functionally. After partial crush injury of the rat optic nerve, systemic injection of autoimmune T cells specific to myelin basic protein significantly diminished the loss of retinal ganglion cells and conducting axons, and significantly retarded the loss of the visual response evoked by light stimulation. These results support our challenge to the traditional concept of autoimmunity as always harmful, and suggest that in certain situations T cell autoimmunity may actually be beneficial. It might be possible to employ T cell intervention to slow down functional loss in the injured CNS. (C) 2000 Published by Elsevier Science B.V. All rights reserved.

Several issues remain to be resolved before the efficacy of various approaches to elicit anti-tumor immunity in patients can be evaluated. First, in vitro assays able to detect responses by T cells primed in vivo are needed. Second, a source of tumor antigen to stimulate patients' lymphocytes in vitro is required. The ELISPOT assay is attractive, because it can be performed with a small numbers of cells and requires only short-term culture in vitro. A source of tumor antigen is more problematic, since for most tumors, tumor-associated antigens (TAA) have not been identified and/or cloned. In this report we demonstrate that autologous antigen-presenting cells (APC) pulsed with total tumor peptides from autologous tumor tissue can stimulate IFN gamma release by patients' lymphocytes in the ELISPOT assay. Thus, this approach should be considered for monitoring immune responses in clinical immunotherapy trials. (C) 2000 Elsevier Science B.V. All rights reserved.

Autoimmunity is usually considered only as a cause of disease; nevertheless, human T-cell repertoires are filled naturally with autoimmune lymphocytes. Here, we review evidence that autoimmune T cells can help heal damaged tissues, indicating that natural autoimmunity could also be a cause of health.

Autoimmune T cells against central nervous system myelin associated peptide reduce the spread of damage and promote recovery in injured rat spinal cord, findings that might lead to neuroprotective cell therapy without risk of autoimmune disease.

Injuries of the central nervous system (CNS) lead to an inevitable and irreversible loss of function because of the lack of neurogenesis, poor regeneration, and the spread of degeneration. In most tissues, protection and repair are the function of the immune system. It has long been thought that this does not apply to the CNS, where because of its immune-privileged character-any immune activity was assumed to be detrimental. We have recently proposed, however, that provided care is taken to avoid the attendant risks, both repair and protection of injured CNS neurons can benefit from immune intervention. In the following I will summarize the data that led to this concept and describe the evidence supporting it.

Neurodegenerative diseases are characterized by a relentless loss of specific groups of neuronal subtypes. Many of these diseases share similar molecular mechanisms and extracellular mediators of neuronal loss. We now suggest that neurodegeneration originating in the neuronal cell bodies (e.g. in Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis) should be distinguished from that originating in the axons (e.g. in glaucoma, certain peripheral neuropathies and spinal stenosis). We propose that the former group of diseases be defined as "somagenic" and the latter as 'axogenic'. Although axogenic disorders may share common symptoms and mediators of toxicity with somagenic disorders, they have distinct temporal, subcellular and signal-transduction features. We further suggest that, by adopting this classification of disorders based on pathophysiological processes, we will come go recognize additional diseases (in particular, those defined as axogenic) as being neurodegenerative and therefore possibly amenable to neuroprotective therapy.

The central nervous system (CNS), unlike the peripheral nervous system (PNS), is an immune-privileged site in which local immune responses are restricted. Whereas immune privilege in the intact CNS has been studied intensively, Little is known about its effects after trauma. In this study, we examined the influence of CNS immune privilege on T cell response to central nerve injury. Immunocytochemistry revealed a significantly greater accumulation of endogenous T cells in the injured rat sciatic nerve than in the injured rat optic nerve (representing PNS and CNS white matter trauma, respectively). Use of the in situ terminal deoxytransferase-catalyzed DNA nick end labeling (TUNEL) procedure revealed extensive death of accumulating T cells in injured CNS nerves as well as in CNS nerves of rats with acute experimental autoimmune encephalomyelitis, but not in injured PNS nerves, Although Fas ligand (FasL) protein was expressed in white matter tissue of both systems, it was more pronounced in the CNS, Expression of major histocompatibility complex (MHC) class II antigens was found to be constitutive in the PNS, but in the CNS was induced only after injury, Our findings suggest that the T cell response to central nerve injury is restricted by the reduced expression of MHC class II antigens, the pronounced Fast expression, and the elimination of infiltrating lymphocytes through cell death.

It is now widely accepted that injured nerves, like any other injured tissue, need assistance from their extracellular milieu in order to heal. We compared the postinjury activities of thrombin and gelatinases, two types of proteolytic activities known to be critically involved in tissue healing, in nonregenerative (rat optic nerve) and regenerative (fish optic nerve and rat sciatic nerve) neural tissue. Unlike gelatinases, whose induction pattern was comparable in all three nerves, thrombin-like activity differed clearly between regenerating and nonregenerating nervous systems. Postinjury levels of this latter activity seem to dictate whether it will display beneficial or detrimental effects on the capacity of the tissue for repair. The results of this study further highlight the fact that tissue repair and nerve regeneration are closely linked and that substances that are not unique to the nervous system, but participate in wound healing in general, are also crucial for regeneration or its failure in the nervous system.

Autoimmunity to antigens of the central nervous system is usually considered detrimental. T cells specific to a central nervous system self antigen, such as myelin basic protein, can indeed induce experimental autoimmune encephalomyelitis, but such T cells may nevertheless appear in the blood of healthy individuals. We show here that autoimmune T cells specific to myelin basic protein can protect injured central nervous system neurons from secondary degeneration. After a partial crush injury of the optic nerve, rats injected with activated anti-myelin basic protein T cells retained approximately 300% more retinal ganglion cells with functionally intact axons than did rats injected with activated T cells specific for other antigens. Electrophysiological analysis confirmed this finding and suggested that the neuroprotection could result from a transient reduction in energy requirements owing to a transient reduction in nerve activity. These findings indicate that T-cell autoimmunity in the central nervous system, under certain circumstances, can exert a beneficial effect by protecting injured neurons from the spread of damage.

The adult mammalian central nervous system (CNS) fails to regenerate its axons following injury. A comparison between its postinjury response and that of axons of nervous systems capable of regeneration reveals major differences with respect to inflammation. In regenerative systems, a large number of macrophages rapidly invade the injured site during the first few hours arid days after the injury. Following their activation/differentiation through interaction with the host tissue, they play a central role in tissue healing through phagocytosis of cell debris and communication with cellular and molecular elements of the damaged tissue. Relative to the peripheral nervous System (PNS), macrophage recruitment in the adult mammalian CNS is delayed and is restricted in amount and activity. It was recently proposed that in injured mammalian CNS tissue, implantation of macrophages stimulated by prior co-culture with segments of peripheral (sciatic) nerves can compensate, at least in part, for the restricted postinjury inflammatory reaction. In the present study, this experimental paradigm is further explored and shows that there is no conflict between the systemic use of anti-inflammatory compounds and treatment with stimulated macrophages to promote regrowth of neuronal tissue. (C) 1998 Elsevier Science Ltd. All rights reserved.

GTP and Ca2+, two well-known modulators of intracellular signaling pathways, control a structural/functional switch between two vital and mutually exclusive activities, cross-linking and G(alpha) activity, in the same enzyme. The enzyme, a brain-derived tissue-type transglutaminase (TGase), was recently cloned by us in two forms, one of which (s-TGN) lacks a C-terminal region that is present in the other (1-TGN). Immunoreaction with antibodies directed against a peptide present in the C-terminus of 1-TGN but missing in s-TGN suggested that this site, which is located in the C-terminal fourth domain, undergoes conformational changes as a result of interaction between 1-TGN and GTP. Site-directed mutagenesis suggested that the third domain is involved in mediating the inhibition of the cross-linking activity. These results were supported by molecular modeling, which further suggested that domains III and IV both participate in conformational changes leading to the functional switch between the Ca2+-dependent cross-linking activity and the G(alpha) activity. (C) 1998 Academic Press.

Neuroprotective therapy is a relatively new development in the approach to the treatment of acute and chronic brain damage. Though initially viewed in the framework of acute CNS injuries, the concept was recently extended to include chronic injuries, in which at any given time there are some neurons in an acute phase of degeneration coexisting with others that are healthy, marginally damaged, or dead. The healthy neurons and those that are only marginally damaged are the potential targets for neuroprotection. For the development of neuroprotective therapies, it is essential to employ an animal model in which the damage resulting from secondary degeneration can be quantitatively distinguished from primary degeneration. This is of particular relevance when the site of the damage is in the white matter (nerve fibers) rather than in the gray matter (cell bodies). In the present work we reexamine the concepts of secondary degeneration and neuroprotection in white matter lesions. Using a partial crush injury of the adult rat optic nerve as a model, we were able to assess both primary and secondary nerve damage. We show that neurons whose axons were not damaged or only marginally damaged after an acute insult will eventually degenerate as a consequence of their existence in the degenerative environment produced by the injury. This secondary degeneration does not occur in all of the neurons at once, but affects them in a stepwise fashion related to the severity of the damage inflicted. These findings, which may be applicable to the progression of acute or chronic neuropathy, imply that neuroprotective therapy may have a beneficial effect even if there is a time lag between injury and treatment. (C) 1998 Academic Press.

Axons in the central nervous system (CNS) of adult mammals do not regenerate after injury. Mammalian CNS differs In this respect from other mammalian tissues, Including the peripheral nervous system (PNS), and from the CNS of lower vertebrates. In most parts of the body, including the nervous system, injury triggers an Inflammatory reaction involving macrophages. This reaction Is needed for tissue healing; when it Is delayed or insufficient, healing Is incomplete. The CNS, although needing an efficient inflammatory reaction resembling that In the periphery for tissue healing, appears to have lost the ability to supply it. We suggest that restricted CNS recruitment and activation of macrophages are linked to regeneration failure and might reflect the Immune privilege that characterizes the mammalian CNS. As macrophages play a critical role In tissue restoration, and because their recruitment and activation are among the most upstream of the events leading to tissue healing, overcoming the deficiencies In these steps might trigger a self-repair process leading to recovery after CNS Injury.

Background: Acute partial lesion of the rat optic nerve, although not a model for glaucoma, mimics some of the features of the disease. Objective: To learn whether degeneration of rat optic nerve fibers and death of their retinal ganglion cells induced by an acute partial lesion are associated with elevated levels of glutamate, known to occur in the eyes of humans and monkeys with glaucoma. Materials and Methods: Rat optic nerve was subjected to a partial crush injury. Aqueous humor samples were aspirated from the anterior and posterior aqueous chambers at specified times and their amino acid contents were determined by means of highperformance liquid chromatography. Results: Three and 7 days after injury, intraocular glutamate and aspartate levels were found to be significantly higher than in normal or sham-operated-on eyes, and returned to normal by day 14, Conclusions: Degeneration of the optic nerve, induced by a mechanical injury of the axons, leads to intraocular elevation of glutamate and aspartate levels. These results illustrate that the model of the partial optic nerve lesion exhibits another feature typical of a long-term optic neuropathy, such as glaucoma.

Tissue-type transglutaminases (TGases) were recently shown to exert dual enzymatic activities; they catalyze the posttranslational modification of proteins by transamidation, and they also act as guanosine triphosphatase (GTPase). Here we show that a tissue-type TGase is expressed in rat brain astrocytes in vitro, and is induced by the inflammation-associated cytokines interleukin-1 beta and to a lesser extent by tumor necrosis factor-alpha. Induction is accompanied by overexpression and appearance of an additional shorter clone, which does not contain the long 3'-untranslated region and encodes for a novel TGase enzyme whose C terminus lacks a site that affects the enzyme's interaction with guanosine triphosphate (GTP), Expression of two clones revealed that the long form is inhibited noncompetitively by GTP, but the short form significantly less so, The different affinities for GTP may account for the difference in physiological function between these two enzymes.

In this study we present a method to achieve a complete transection of optic nerve axons in adult rat, while preserving the vasculature and retaining the continuity of the meninges. Under deep anesthesia, the optic nerve of adult rat is exposed. Using specially designed instruments built from disposable glass microsampling pipets, a small opening is created in the meninges of the optic nerve, 2-3 mm behind the eye globe. A glass dissector is introduced through the opening and is used to cut all the axons through the whole width of the nerve. Complete transfection of the optic nerve axons was achieved, while retaining the continuity of the meninges and avoiding damage to the nerve's vascular supply, Transection was confirmed by transillumination showing a complete gap in the continuity of the nerve axons, and by both morphological and electrophysiological criteria. Nerve transection performed by the conventional technique leads to neuroma formation and hampers regeneration. Crush injury may cause nerve ischemia, which is detrimental to axonal recovery. Both of these disadvantages are avoided by the method of transection presented here. The opening created in the 'meningeal tube' can be used to inject substances that may be of benefit in recovery, rescue and/or regeneration of the injured axons. The model is particularly suitable for in vivo studies on nerve regeneration, and especially for screening of putative therapeutic agents.

Macrophages have long been known to play a key role in the healing processes of tissues that regenerate after injury; however, the nature of their involvement in healing of the injured central nervous system (CNS) is still a subject of controversy, Here we show that the absence of regrowth in transected rat optic nerve (which, like all other CNS nerves in mammals, cannot regenerate after injury) can be overcome by local transplantation of macrophages preincubated ex vivo with segments of a nerve (e.g., sciatic nerve) that can regenerate after injury, The observed effect of the transplanted macrophages was found to be an outcome of their stimulated activity, as indicated by phagocytosis, Thus, macrophage phagocytic activity was stimulated by their preincubation with sciatic nerve segments but inhibited by their preincubation with optic nerve segments. We conclude that the inability of nerves of the mammalian CNS to regenerate is related to the failure of their macrophages recruited after injury to acquire growth-supportive activity. We attribute this failure to the presence of a CNS resident macrophage inhibitory activity, which may be the biochemical basis underlying the immune privilege of the CNS, The transplantation of suitably activated macrophages into injured nerves may overcome multiple malfunctioning aspects of the CNS response to trauma, and thus may be developed into a novel, practical, and multipotent therapy for CNS injuries.

Migration of astrocytes is thought to play a role in nerve regeneration and to be mediated, at least in part, by inflammation-associated cytokines, Plasminogen activators are secreted proteases that function in fibrinolysis and participate in cellular migration and invasion and, in some cases, are modulated by cytokines, Here, we show that two cytokines, tumor necrosis factor-alpha and interleukin-1 beta, can modulate plasminogen activation in astrocytes, each causing 90% reduction of total plasminogen activator activity. Direct and reverse zymography indicated that this reduction resulted from two simultaneous events, a pronounced decrease in tissue-type plasminogen activator activity and an induction of plasminogen activator inhibitor-1. Northern hybridization analysis indicated a 30-fold increase of the steady-state level of plasminogen activator inhibitor-1 mRNA following treatment with each of the two cytokines. Both of the cytokine-induced effects could be blocked by cycloheximide or actinomycin D. When signal transduction pathways were blocked, the results indicated the involvement of reduction in cyclic AMP levels, protein kinase activity, and arachidonic metabolites of the lipoxygenase pathway. The results thus show that the two cytokines reduce the ability of astrocytes to conduct fibrinolysis and extracellular proteolysis, and suggest that the effect of these cytokines on members of the plasminogen activation system is through a common signal transduction pathway.

Axons of the mammalian central nervous system do not regenerate spontaneously after axonal injury, unlike the central nervous system axons of fish and amphibians and the peripheral nervous system of mammals, which possess a good regenerative ability (Grafstein: The Retina: A Model for Cell Biology Studies, Part II, 1986; Kiernan: Biol Rev 54:155-197, 1979; Murray: J Comp Neurol 168:175-196, 1976; Ramon y Cajal: Degeneration and Regeneration of the Nervous System, 1928; Reier and Webster: J Neurocytol 3:591-618, 1974; Sperry: Physiol Zool 23:351-361, 1948). It was previously believed that intrinsic differences between the central nervous system neurons of mammals and fish account for their differences in regenerative ability. The past decade, however, has seen an accumulation of evidence, indicating that mammalian central nervous system neurons are able to regenerate injured axons, at least to some extent. This was first demonstrated by Aguayo and colleagues (David and Aguayo: Science 214:931-933, 1981; Kierstead et al: Science 246:255-257, 1989), who showed that injured mammalian central nervous system axons can grow for a considerable distance into an autograft of a peripheral nerve. It was also demonstrated that injured rabbit optic axons can regenerate into their own environment (i.e., into the distal part of the injured optic nerve), if the injured nerve is treated so as to make it conducive for growth (Lavie et al: J Comp Neurol 298:293-314, 1990; Eitan et al: Science 264:1764-1768, 1994). It is generally believed today that the glial cells surrounding the axons play a crucial role in determining the regenerative capacity of any central nervous system. In this review, we discuss various aspects of glial cells in the context of regeneration, with special emphasis on similarities and differences between the glial cells of rat and fish optic nerves, representing a nonregenerative and a regenerative system, respectively. (C) 1995 Wiley-Liss, Inc.

The central nervous system has long been regarded as an immunologically privileged site. Accumulating evidence suggests, however, that the privilege is not total, and that certain immune functions involving immune components and resident glial cells can operate in the central nervous system. The nervous and immune systems interact during normal development, but in the mature brain their interaction is restricted mainly to cases of pathogenic infections and traumatic lesions. The focus of this review is on bidirectional interactions between immune and neuroglial components in response to nerve injury. The macrophage is the most ubiquitous of the immune-derived cell types associated with injury. Its role, as in any other organ, is tissue remodeling and promotion of healing. Macrophage activities include removal of dead tissue and debris by phagocytosis, lipid recycling, and secretion of a wide spectrum of cytokines possessing trophic, mitogenic, and chemotactic properties. These activities affect the behavior of resident cells in the vicinity of the wound. We discuss the possible association of these cytokines with the ability of injured nerves to regenerate. Finally, we consider the apparently conflicting effects of posttraumatic inflammation on the recovery of function.

Neu differentiation factor (NDF, also called heregulin) was isolated from mesenchymal cells on the basis of its ability to elevate phosphorylation of ErbB proteins. Earlier in situ hybridization analysis showed that NDF was transcribed predominantly in the central nervous system during embryonic development. To gain insights into the role of NDF in brain we analyzed its distribution by inmunohistochemistry and in situ hybridization. Late-gestation (day 17) rat embryos displayed high NDF immunoreactivity in both motor (e.g., putamen) and limbic (e.g., septum) regions. Lower levels of the factor were exhibited by adult brain, except for the cerebellum, where NDF expression was increased postnatally. Both neurons and glial cells were identified by immunohistochemistry as NDF-producing cells (e.g., pyramidal neurons in the cerebral cortex and glial cells in the corpus callosum). By establishment of primary cultures of rat brain cells we confirmed that NDF was expressed in neurons as well as in astrocytes. In addition, by using such primary cultures we observed that NDF treatment exerted only a limited mitogenic effect, which was accompanied by significant acceleration of astrocyte maturation. Furthermore, long-term incubation with the factor specifically protected astrocytes from apoptosis, implying that NDF functions in brain as a survival and maturation factor for astrocytes.

Recent results shed new light on how some nervous systems can regenerate after injury while others cannot. Until recently it was widely believed that the main difference between systems that regenerate and those that do not lies in the normal state of their permissiveness to the regenerating axons, Thus, while nonregenerative systems such as the rat optic nerve, were shown to contain myelin-associated growth inhibitors, regenerative systems such as the fish optic nerve, were thought to have no such inhibitors. However, it has now been demonstrated that spontaneously regenerating systems do contain growth inhibitors, though their levels seem to be lower than in nonregenerative systems. The main difference, however, appears to reside in the system's response to injury. This article discusses the involvement of myelin-associated growth inhibitors in the spontaneously regenerating nervous system of fish, traces the apparent discrepancy, and shows how it has been resolved recently.

This study shows that the fish optic nerve, which is able to regenerate after injury, contains myelin-associated growth inhibitors similar to the growth inhibitors present in mammalian central nervous system (CNS) myelin. The ability of nerves to regenerate was previously correlated with the ability of sections from these nerves to support neuronal attachment and axonal growth in vitro. Thus neuroblastoma cells or embryonic neurons became attached to and grew axons on sections of rat sciatic nerve or fish optic nerve, which are spontaneously regenerating systems, but not on sections of rat optic nerve, a nonregenerating system. Failure of the latter to support axonal growth has been attributed, at least in part, to growth inhibitors. Recently it was shown that adult neurons, which differ in their growth requirement from embryonic neurons, are unable to extend neurites on sections of normal sciatic nerve but are able to extend neurites on sections of sciatic nerve that was injured prior to its excision. We found a similar situation in the fish optic nerve, i.e., that the nerve is normally not permissive to growth of adult retinal axons but becomes growth permissive after injury. The nonpermissiveness of the normal fish optic nerve was found to correlate with the presence of myelin-associated growth-inhibitory molecules. This inhibitory activity of fish myelin was neutralized by IN-1 antibodies, known to neutralize rat myelin growth inhibitors. The results thus demonstrate that fish optic nerve myelin contains inhibitors apparently similar or even identical to those of rat, but possibly present in lower amounts than in the rat. Results are discussed with respect to the possibility that fish optic nerve, like the rat sciatic nerve and unlike the rat optic nerve, undergoes certain changes after injury that support regeneration of adult neurons. Such changes might include elimination or neutralization of growth inhibitors. (C) 1994 Wiley-Liss, Inc.

The expression of intermediate filaments is developmentally regulated. In the mammalian embryo keratins are the first to appear, followed by vimentin, while the principal intermediate filament of the adult brain is glial fibrillary acidic protein. The intermediate filaments expressed by a cell thus reflect its state of differentiation. The differentiation state of cells, and especially of glial cells, in turn determines their ability to support axonal growth. In this study we used three new antibodies directed against three fish intermediate filaments (glial fibrillary acidic protein, keratin 8 and vimentin), in order to determine the identity and level of expression of intermediate filaments present in fish glial cells in culture. We found that fish astrocytes and oligodendrocytes are both able to express keratin 8 and vimentin. We further demonstrate that under proliferative conditions astrocytes express high keratin 8 levels and most oligodendrocytes also express keratin 8, whereas under nonproliferative conditions the astrocytes express only low keratin 8 levels and most oligodendrocytes do not express keratin 8 at all. These results suggest that the fish glial cells retain characteristics of immature cells. The findings are also discussed in relation to the fish glial lineage.

The poor regenerative ability of neurons of the central nervous system in mammals, as compared with their counterpart in fish or amphibians, is thought to stem from differences in their immediate nonneuronal environment and its response to axonal injury. We describe one aspect of the environmental response to axonal injury in a spontaneously regenerating system-the fish optic nerve. The aspect under investigation was the reaction of glial cells at the injury site. This was examined by the use of antibodies that specifically recognize vimentin in fish glial cells. In the present study, affinity-purified vimentin antibodies were raised against a nonconserved N-terminal 14-amino acid peptide, which was predicted from the nucleotide sequence of vimentin. These antibodies were found to react specifically with glial cells in vitro. Moreover, the antivimentin antibodies stained both the optic nerve and the optic tract, but with different patterns. Specificity of the antibodies was verified by protein immunoblotting, tissue distribution, and labeling patterns. After injury, vimentin immunoreactivity initially disappeared from the site of the lesion due to cell death. Early signs of glial cell migration toward the injury site were evident a few days later. It is suggested that the reappearance of vimentin-positive glial cells at the site of injury is associated with axonal elongation across it, and that they contribute to the regenerative ability of the fish optic nerve. (C) 1994 Wiley-Liss, Inc.

The elements that control neuronal proliferation are largely unknown. Proliferating neurons in cultures of goldfish brain were studied in an attempt to identify the cell types involved. Neuronal proliferation was found to occur only when the neuronal stem cells were in direct contact with astrocytes, and never directly on the substrate. The regulation of neuronal proliferation thus appears to be mediated, at least in part, by contact with astrocytes. In addition, neurite extension was inhibited by medium conditioned by fish astrocytes. Since neurite extension and neuronal proliferation are mutually exclusive processes, inhibition of neurite extension by soluble substances derived from the astrocytes is probably one of the mechanisms controlling neuronal proliferation. The complex reciprocal relationship between neurons and astrocytes is also demonstrated by an observed inhibition of astrocytic proliferation by medium conditioned by differentiating fish neurons. This inhibition of astrocytic proliferation might be part of a mechanism through which interference with neuronal differentiation by astrocytes is avoided. The results of this study thus suggest that astrocytes, in addition to their known roles in controlling neuronal migration, neuronal differentiation and neurite elongation, may also play a role in the control of neuronal proliferation.

Regenerating optic nerves from fish produce a factor that is cytotoxic to oligodendrocytes. The cytotoxic factor is recognized by antibodies to interleukin-2 (IL-2) and has the apparent molecular size of a dimer of IL-2. An enzyme, identified as a nerve transglutaminase, was purified from regenerating optic nerves of fish and was found to catalyze dimerization of human IL-2. The dimerized IL-2, unlike monomeric IL-2, is cytotoxic to oligodendrocytes from rat brain in culture. The results suggest that posttranslational modification of a cytokine can alter its activity. Under conditions in which oligodendrocytes inhibit neuronal regeneration, dimerization of IL-2 might provide a mechanism to permit nerve growth.

It has long been known that while damaged nerves of the peripheral nervous system in mammals are able to regenerate, this does not occur in the central nervous system. Recent studies have shown that a chemical factor present in regenerating fish optic nerves facilitates regeneration of nerve axons in rodents. Further research encourages the hope that this may ultimately lead to the successful treatment of nerve damage in humans.

Axons of the central nervous system in adult mammals do not regenerate spontaneously after injury, partly because of the presence of oligodendrocytes that inhibit axonal growth. This is not the case in lower vertebrates (e.g., in fish), where regeneration of the optic nerve does occur spontaneously and has been correlated with the presence of factors cytotoxic to oligodendrocytes. The present study provides evidence that the substance originating from the fish optic nerves, which is cytotoxic to oligodendrocytes, is an interleukin 2-like substance.

Previous studies have suggested that L1, the cell adhesion molecule, is present in the regenerating fish optic nerve. The present study was undertaken in order to determine whether L1 is expressed by fish neurons and specifically by non-myelinated axons, using fish retinal explants in vitro and the developing fish visual system in vivo. In vitro, the nonmyelinated axons emerging from retinal explants showed L1 immunoreactivity and in vivo, L1 immunoreactive sites were found to be associated with areas rich in non-myelinated axons. At embryonic stage 23, as the eye developed and optic nerve axons began to elongate towards the tectum, L1-like immunoreactivity was seen both in optic nerve and in plexiform layers of the retina. At this stage and until hatching, the cellular layers within the retina showed little or no staining relative to the layers containing axons or dendrites. After hatching, L1 immunoreactivity was also observed in the ganglion cell layer, but upon maturation both the retina and the optic nerve lost most of their L1 immunoreactivity. We therefore suggest that non-myelinated axons of the fish visual system express L1 during development, which is lost after myelination and presumably reappears during regeneration.

Regeneration of injured central nervous system axons is largely dependent on the response of the associated nonneuronal glial cells to injury. Glial cells of the mammalian central nervous system, unlike those of fish, are apparently not conducive to axonal regeneration. While the lineage of rat glial cells is well characterized and its role in the support or inhibition of regenerative growth is beginning to be understood, little is known about fish glial cells. Accordingly, glial cells in cultures of adult goldfish brain and of newly hatched goldfish larvae were studied in an attempt to establish their lineage. The cells were identified by means of indirect immunofluorescence, using antibodies against fish astrocytes and oligodendrocytes. The cell count in the cultures increased from a small number of cells at 24 h after plating to a large number of both astrocytes and oligodendrocytes after 1 week in culture. Both of these cell types had originated from proliferating cells, as shown by their uptake of tritiated thymidine and by the inhibition of cell proliferation by 5-fluoro-2'-deoxyuridine. Both astrocytes, i.e., glial fibrillary acidic protein-positive cells, and oligodendrocytes, i.e., 6D2-positive cells, were positively labeled also by A2B5 antibodies, which are known to label progenitors of type-2 astrocytes and oligodendrocytes in the rat optic nerve. The results suggest that A2B5 positive progenitor cells in the goldfish central nervous system, as in the rat optic nerve, might be a common progenitor of astrocytes and oligodendrocytes.

The high post-traumatic regenerative ability of fish central nervous system has been partially attributed to the hospitable nature of the surrounding non-neuronal cells and their appropriate response to injury. Uncovering the correlation between fish non-neuronal cell structure and behavior might yield a better understanding of what makes them supportive to axonal growth. Towards this goal, structural proteins expressed by fish non-neuronal cells need to be characterized. In the present study we isolated cDNA clones encoding fish intermediate filaments which are prominent structural proteins in astrocytes. Among the isolated clones, one was identified as fish vimentin and another was found identical to the cloned fish keratin 8. Results are discussed with respect to the use of these cDNAs for further understanding of fish non-neuronal cell plasticity.

Mammalian central nervous system (CNS) axons are virtually incapable of regenerating after injury. However, CNS neurons of lower vertebrates, such as fish and amphibians, are endowed with a high regenerative capacity. Lately, the glial cells have been credited with the regenerative ability of any specific CNS. We have previously demonstrated that many oligodendrocytes are recovered in cultures of injured rat optic nerve, while only a few oligodendrocytes are recovered from injured fish optic nerve in culture. We further demonstrated that medium conditioned by regenerating fish optic nerves (CM), which has been shown to cause axonal elongation in injured rabbit optic nerves, causes a decrease in the number of oligodendrocytes in rat glial cultures. In the present study, we demonstrate that soluble factors in the CM are capable of reducing the number of fish oligodendrocytes in fish optic nerve cultures. In addition, an inverse relationship was found between the number of macrophages and the number of oligodendrocytes. These results thus suggest that macrophages and/or activated resident microglial cells are directly or indirectly responsible for the presence of these soluble factor(s) that regulate the postinjury number of oligodendrocytes in the fish optic nerves.

In the present study, we have developed an animal model for central nervous system (CNS) damage using the graded crush injury of the adult rat optic nerve. This injury model has the following characteristics: (a) the injuries are reproducible; (b) the injuries can be created with controlled severity; and (c) variations in the severity of the injuries correlate with definable electrophysiological and behavioral outcomes. Self-closing forceps, modified by attaching a screw to the handle, were used as a lesion-causing device. Rat optic nerves were crushed in vitro. The severity of the injury, calibrated by a measured applied force, was determined, electrophysiologically, in vitro. Compound action potential was assessed and recorded by suction electrodes connected to both sides of the nerve. In vivo studies correlated the calibrated crush injuries with behavioral outcome. After severe crush (applied force: 205 g or higher), the rats were unable to orient towards visual stimuli presented in the visual field of the damaged nerve and only minor transient recovery was observed. After moderate crush (applied force: 120 g), however, there was a significant recovery of orienting performance up to the 6th postoperative day, followed by a gradual, secondary loss of function. In the mildly injured group (applied force lower than 30 g), functional improvement continued for up to 10 days and remained stable thereafter, with only minor secondary loss. This CNS graded injury model may be valuable to study the molecular and anatomical mechanisms underlying secondary degeneration and the potency of various posttraumatic treatments in leading to recovery of function.