Magnetic resonance imaging features of COVID-19-related cranial nerve lesions.

The complete features of the neurological complications of coronavirus disease 2019 (COVID-19) still need to be elucidated, including associated cranial nerve involvement. In the present study we describe cranial nerve lesions seen in magnetic resonance imaging (MRI) of six cases of confirmed COVID-19, involving the olfactory bulb, optic nerve, abducens nerve, and facial nerve. Cranial nerve involvement was associated with COVID-19, but whether by direct viral invasion or autoimmunity needs to be clarified. The development of neurological symptoms after initial respiratory symptoms and the absence of the virus in the cerebrospinal fluid (CSF) suggest the possibility of autoimmunity.

Impact of peripheral immune status on central molecular responses to facial nerve axotomy.

When facial nerve axotomy (FNA) is performed on immunodeficient recombinase activating gene-2 knockout (RAG-2-/-) mice, there is greater facial motoneuron (FMN) death relative to wild type (WT) mice. Reconstituting RAG-2-/- mice with whole splenocytes rescues FMN survival after FNA, and CD4+ T cells specifically drive immune-mediated neuroprotection. Evidence suggests that immunodysregulation may contribute to motoneuron death in amyotrophic lateral sclerosis (ALS). Immunoreconstitution of RAG-2-/- mice with lymphocytes from the mutant superoxide dismutase (mSOD1) mouse model of ALS revealed that the mSOD1 whole splenocyte environment suppresses mSOD1 CD4+ T cell-mediated neuroprotection after FNA. The objective of the current study was to characterize the effect of CD4+ T cells on the central molecular response to FNA and then identify if mSOD1 whole splenocytes blocked these regulatory pathways. Gene expression profiles of the axotomized facial motor nucleus were assessed from RAG-2-/- mice immunoreconstituted with either CD4+ T cells or whole splenocytes from WT or mSOD1 donors. The findings indicate that immunodeficient mice have suppressed glial activation after axotomy, and cell transfer of WT CD4+ T cells rescues microenvironment responses. Additionally, mSOD1 whole splenocyte recipients exhibit an increased astrocyte activation response to FNA. In RAG-2-/- + mSOD1 whole splenocyte mice, an elevation of motoneuron-specific Fas cell death pathways is also observed. Altogether, these findings suggest that mSOD1 whole splenocytes do not suppress mSOD1 CD4+ T cell regulation of the microenvironment, and instead, mSOD1 whole splenocytes may promote motoneuron death by either promoting a neurotoxic astrocyte phenotype or inducing Fas-mediated cell death pathways. This study demonstrates that peripheral immune status significantly affects central responses to nerve injury. Future studies will elucidate the mechanisms by which mSOD1 whole splenocytes promote cell death and if inhibiting this mechanism can preserve motoneuron survival in injury and disease.

Neutrophil-lymphocyte ratio: a new predictive and prognostic factor in patients with Bell palsy.

OBJECTIVE: The aim of this study was to investigate whether neutrophil-lymphocyte ratio (NLR) levels are elevated in patients with Bell palsy (BP). Moreover, we aimed to find out whether there is a correlation between NLR levels and the severity and prognosis of BP. MATERIALS AND METHODS: The study group consisted of 25 subjects who presented with BP and 25 control subjects with no evidence of facial nerve pathology. The subjects underwent a general physical examination; an assessment of laboratory blood parameters; and a cranial magnetic resonance imaging, using gadolinium as a contrast medium. RESULTS: The mean (SD) NLR values were 2.16 (0.80) in the patients with BP and 1.36 (0.48) in the control group. The mean NLR values in the patients with BP were significantly higher than in the control group (P = 0.0001). There was a positive correlation between NLR values and grade of facial paralysis (r = 0.661, P = 0.0001). The mean (SD) NLR values in the grades III, IV, V, and VI BP groups were 1.40 (0.54), 1.78 (0.44), 3.00 (0.63), and 2.60 (0.54), respectively. The mean NLR values in the grade V BP group were significantly higher than in the other groups (P = 0.0001). In addition, there was a positive correlation between NLR values and prognosis of facial paralysis (r = 0.239, P = 0.251). CONCLUSIONS: There is no previous study that investigated the association between NLR and BP in the literature. Higher NLR values in patients with BP may be a predictor of worse prognosis.

Immune cell-mediated neuroprotection is independent of estrogen action through estrogen receptor-alpha.

It has been well documented that both estrogen and immune cells (CD4+ T cells) mediate neuroprotection in the mouse facial nerve axotomy model. Estrogen has been shown to play an important role in regulating the immune response. However, it is unclear whether immune cell-mediated neuroprotection is dependent on estrogen signaling. In this study, using FACS staining, we confirmed that the majority of CD4+ T cells express high levels of estrogen receptor-alpha (ERα), suggesting that CD4+ T cell-mediated neuroprotection may be modulated by estrogen signaling. We previously found that immunodeficient Rag-2KO mice showed a significant increase in axotomy-induced facial motoneuron death compared to immunocompetent wild-type mice. Therefore, we investigated axotomy-induced facial motoneuron loss in immunodeficient Rag-2KO mice that received 17ß-estradiol treatment or adoptive transfer of immune cells from mice lacking functional ERα. Our results indicate that while estradiol treatment failed to rescue facial motoneurons from axotomy-induced cell death in Rag-2KO mice, immune cells lacking ERα successfully restored facial motoneuron survival in Rag-2 KO mice to a wild-type level. Collectively, we concluded that CD4+ T cell-mediated neuroprotection is independent of estrogen action through ERα.

The presence of herpes simplex-1 and varicella zoster viruses is not related with clinical outcome of Bell's Palsy.

Bell's Palsy is the most frequent acute neuropathy of cranial nerves; it has been associated in various reports to herpes viruses. In a prospective study we searched the presence of DNA from five herpes viruses (HSV-1 and 2, VZV, EBV and HHV-6) in 79 patients at the acute phase of Bell's Palsy. Results were related with various parameters; age, gender and clinical outcome. We found the significant presence (p˂0.001) of HSV-1 and VZV in 39% and 42% of patients. However, a large percentage of cases were negative. When comparisons were made between subgroups according to gender and age no differences were found with viral findings nor with clinical outcome of palsy, which was of clinical remission in most cases (78%). Our results suggest that herpes viruses might participate in the complex mechanisms of autoimmunity of Bell's Palsy but not as determinant etiological element.

Mechanisms of the Immunomodulation Effects of Bone Marrow-Derived Mesenchymal Stem Cells on Facial Nerve Injury in Sprague-Dawley Rats.

Normal facial nerve (FN) function is very important for human being. However, if injured, FN function is difficult to restore completely. Recently, many studies reported the immune regulation function of stem cells (SCs). However, the immunomodulation function of SCs on FN injury is still unclear. Our study aims to explore the mechanism of immunomodulation effect of Sprague-Dawley rat bone marrow-derived SCs (BMSCs) on FN injury and specially focus on the regulation of Th17 and the protection effects of BMSCs on central facial motor neurons (FMNs). First, rat FNs were harvested. FN and BMSCs were cultured together or separately and levels of transforming growth factor (TGF)-ß1, interleukin (IL)-6, hepatocyte growth factor (HGF), inducible nitric oxide synthase (iNOS), and prostaglandin E2 (PGE2) in supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Then, after treating with or without local BMSCs injection, the proportion of Th17 in neck lymph nodes (LNs) was investigated in rat FN injury models. Furthermore, the apoptotic index of FMNs was studied in rat FN injury models that were treated with or without BMSCs. We found that BMSCs could secrete high levels of IL-6, HGF, PGE2, iNOS, and TGF-ß1 in culture. The percentage of Th17 of neck LNs in BMSCs-treated group was significantly lower than that in the control group. The apoptotic index of FMNs in BMSCs-treated group was significantly lower than that in the control group. In conclusion, our research indicates BMSCs could independently secrete cytokines IL-6, HGF, PGE2, iNOS, and TGF-ß1, and these cytokines could regulate the balance among subsets of CD4+ T cells and could protect FMNs by inhibiting neuron apoptosis.

Phenotype of CD4+ T cell subsets that develop following mouse facial nerve axotomy.

We have previously shown that CD4(+) T helper (Th) 2 cells, but not Th1 cells, participate in the rescue of mouse facial motoneurons (FMN) from axotomy-induced cell death. Recently, a number of other CD4(+) T cell subsets have been identified in addition to the Th1 and Th2 effector subsets, including Th17, inducible T regulatory type 1 (Tr1), and naturally thymus-born Foxp3(+) regulatory (Foxp3(+) Treg) cells. These subsets regulate the nature of a T cell-mediated immune response. Th1 and Th17 cells are pro-inflammatory subsets, while Th2, Tr1, and Foxp3(+) Treg cells are anti-inflammatory subsets. Pro-inflammatory responses in the central nervous system are thought to be neurodestructive, while anti-inflammatory responses are considered neuroprotective. However, it remains to be determined if another CD4(+) T cell subset, other than the Th2 cell, develops after peripheral nerve injury and participates in FMN survival. In the present study, we used FACS analysis to determine the temporal frequency of Th1, Th17, Th2, Tr1 and Foxp3(+) Treg CD4(+) T cell subset development in C57BL/6 wild type mice after facial nerve transection at the stylomastoid foramen in the mouse. The results indicate that all of the known CD4(+) T cell subsets develop and expand in number within the draining lymph node, with a peak in number primarily at 7 days postoperative (dpo), followed by a decline at 9 dpo. In addition to the increase in subset frequency over time, FACS analysis of individual cells showed that the level of cytokine expressed per cell also increased for interferon-gamma (IFN-gamma), interleukin (IL)-10 and IL-17, but not IL-4. Additional control double-cytokine labeling experiments were done which indicate that, at 7dpo, the majority of cells indeed have committed to a specific phenotype and express only 1 cytokine. Collectively, our findings indicate for the first time that there is no preferential activation and expansion of any single CD4(+) T cell subset after peripheral nerve injury but, rather, that both pro-inflammatory and anti-inflammatory CD4(+) T cells develop.

Neuroprotective effect of acute prior inflammation with lipopolysaccharide for adult male rat facial motoneurones.

Increases in inflammatory cytokines are reported to have both neuroprotective and neurotoxic effects depending on the type and age of neurones studied. This study aimed to determine the effect of experimental inflammation induced by Lipopolysaccharide (LPS) on the survival of injured male adult rat facial motoneurones. Time- and dose-response studies were done to optimise the LPS administration time and dose, to best correlate with inflammatory levels previously reported for aged rats. 12 cytokines were assayed through multiplex analysis. 24 h after intraperitoneal injection of 0.5 mg/kg Lipopolysaccharide in rats, IL-1ß, IL-5 and IL-12p70 levels were elevated, with no observed LPS-associated sickness behaviour. In other groups of 5-6 adult rats, the facial nerve was either crushed (as mild injury) or avulsed (as severe injury) after the LPS priming injection. Stereology revealed that most motoneurones survived 28 days after nerve crush only and LPS- or saline-priming preceding nerve crush. Most motoneurones died following nerve avulsion only, whereas over half survived when LPS-priming preceded nerve avulsion. We suggest that elevated levels of experimental inflammation are neuroprotective for severely injured adult male rat facial motoneurones.

IL-15 and IL-15R alpha gene deletion: effects on T lymphocyte trafficking and the microglial and neuronal responses to facial nerve axotomy.

IL-15 is a potent T cell chemoattractant, and this cytokine and its unique alpha subunits, IL-15R alpha, can modify immune cell expression of several T cell chemokines and their receptors. Facial nerve axotomy in mice leads to T cell migration across an intact blood-brain-barrier (BBB), and under certain conditions T cells can provide neuroprotection to injured neurons in the facial motor nucleus (FMN). Although chemokines and chemoattractant cytokines are thought to be responsible for T cell migration to the injured cell bodies, data addressing this question are lacking. This study tested the hypothesis that T cell homing to the axotomized FMN would be impaired in knockout (KO) mice with the IL-15 and IL-15R alpha genes deleted, and sought to determine if microglial responsiveness and motoneuron death are affected. Both IL-15KO and IL-15R alpha KO mice exhibited a marked reduction in CD3(+) T cells and had fewer MHC2(+) activated microglia in the injured FMN than their respective WT controls at day 14 post-axotomy. Although there was a relative absence of T cell recruitment into the axotomized FMN in both knockout strains, IL-15R alpha KO mice had five times more motoneuron death (characterized by perineuronal microglial clusters engulfing dead motoneurons) than their WT controls, whereas dead neurons in IL-15KO did not differ from their WT controls. Further studies are needed to dissect the mechanisms that underlie these observations (e.g., central vs. peripheral immune contributions).

CD4(+) T cell-mediated facial motoneuron survival after injury: Distribution pattern of cell death and rescue throughout the extent of the facial motor nucleus.

We have previously demonstrated that CD4(+) T cells transiently rescue facial motoneurons (FMN) from axotomy-induced death in immunodeficient mice. Three subpopulations of motoneurons have been observed within the facial motor nucleus following axotomy: one that always survives axotomy (50%), one that is amenable to rescue from axotomy-induced death through the addition of neurotrophic factors or CD4(+) T cells (30-40%), and one that always dies after axotomy (10-15%). The objective of this study was to anatomically map the extent of axotomy-induced cell death and immune cell rescue in the facial nucleus to study the differential survival capabilities of each subpopulation. Wild-type (WT) mice, recombinase activating gene 2 knockout (RAG-2 KO) mice, and RAG-2 KO mice reconstituted with CD4(+) T cells were subjected to right facial nerve axotomy. At 4 weeks post-axotomy, topographical mapping of axotomy-induced cell death throughout the rostro-caudal extent of the facial nucleus was accomplished in accordance with previously published maps of the subnuclear arrangement of the facial neurons. The results indicate that all 3 subpopulations of FMN can be found in each of the subnuclear groups throughout the entire rostro-caudal extent of the facial nucleus. These data are discussed in context of recent work in amyotrophic lateral sclerosis, a fatal motoneuron disease.

Exacerbation of facial motoneuron loss after facial nerve transection in severe combined immunodeficient (scid) mice.

The immune system functions to protect an organism against microbial infections and may be involved in the reparative response to nerve injury. The goal of this study was to determine whether the immune system plays a role in regulating motoneuron survival after a peripheral nerve injury. After a right facial nerve axotomy, facial motoneuron (FMN) survival in C.B-17 (+/+) wild-type mice was found to be 87 +/- 3.0% of the unaxotomized left side control. In contrast, facial nerve axotomy in C.B-17 (-/-) severe combined immunodeficient (scid) mice, lacking functional T and B lymphocytes, resulted in an average FMN survival of 55 +/- 3.5% relative to the unaxotomized left side control. This represented an approximately 40% decrease in FMN survival compared with wild-type controls. The reconstitution of scid mice with wild-type splenocytes containing T and B lymphocytes restored FMN survival in these mice to the level of the wild-type controls. These results suggest that immune cells associated with acquired immunity play a role in regulating motoneuron survival after a peripheral nerve injury.

Peripheral nerve lesion produces increased levels of major histocompatibility complex antigens in the central nervous system.

Proliferation of central nervous system (CNS) glia in response to peripheral nerve injury occurs without apparent participation of cells of the immune system. It is shown here that following transection of the rat facial nerve there is strongly elevated expression of class I, and to a lesser extent, class II antigens of the major histocompatibility complex (MHC) in the facial nucleus. It is demonstrated by double-immunofluorescence studies that the cells responsible for increased levels of MHC class I antigens are endogenous brain microglia. These findings emphasize the thought that microglia are immunocompetent cells, but, at the same time, raise the possibility for a non-immunological function of MHC antigens under conditions of neural regeneration.

Inflammation and proinflammatory cytokine production, but no demyelination of facial nerves, in experimental autoimmune neuritis in Lewis rats.

Experimental autoimmune neuritis (EAN) is a CD4(+) T cell-mediated, inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for Guillain-Barré syndrome (GBS) in humans. The facial nerve paralysis is relatively commonly found in GBS patients. Here, EAN was established in Lewis rats by immunization with P2 peptide 57-81, a purified component of peripheral nerve myelin, and Freund's complete adjuvant (FCA). To study whether the facial nerves are involved in the pathogenic process during the EAN course, we observed the clinical and pathological changes as well as cytokine production in facial nerves on Day 14 postimmunization (p.i.), i.e. at height of clinical EAN. As a result, all rats immunized with P2 peptide 57-81 developed severe EAN on Day 14 p.i., but none of the rats manifested clinical signs of facial nerve paralysis. Additionally, only mild inflammatory cell infiltration and proinflammatory cytokine, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF-alpha) production as well as devoid demyelination were seen in facial nerves of the EAN rats. On the contrary, severe inflammation and demyelination as well as upregulated IFN-gamma and TNF-alpha production were observed in sciatic nerves of the same EAN rats. The underlying mechanism for the difference of the local manifestation of the disease process of EAN remains to be resolved.

Complement activation and CD59 expression in the motor facial nucleus following intracranial transection of the facial nerve in the adult rat.

Intracranial transection of the facial nerve has been shown to cause a massive neuronal cell death in the motor facial nucleus. Complement activation has been proposed to contribute to neuronal degeneration following axotomy. Using immunocytochemistry and in situ hybridization we show in the present study that there is complement activation in the facial nucleus after intracranial facial nerve transection as well as increase of the complement regulators CD59 and clusterin. We propose a neuroprotective role for the complement regulators CD59 and clusterin against homologous attack of complement to facial motor neurons.

IL-2 gene knockout affects T lymphocyte trafficking and the microglial response to regenerating facial motor neurons.

Following facial nerve axotomy in mice, T cells cross the intact blood-brain barrier (BBB), home to nerve cell bodies in the facial motor nucleus (FMN), and augment neuroregenerative processes. The pivotal T cell immunoregulatory cytokine, IL-2, appears to have bidirectional effects on neuronal and microglial cell function, suggesting rival hypotheses that IL-2 could either enhance or disrupt processes associated with regeneration of axotomized facial motor neurons. We tested these competing hypotheses by comparing the effect of facial nerve axotomy on C57BL/6-IL-2(-/-) knockout and C57BL/6-IL-2(+/+) wild-type littermates. Since IL-2 may also be produced endogenously in the brain, we also sought to determine whether differences between the knockout and wild-type mice were attributable to loss of IL-2 gene expression in the CNS, loss of peripheral sources of IL-2 and the associated effects on T cell function, or a combination of these factors. To address this question, we bred novel congenic mice with the SCID mutation (mice lacking T cell derived IL-2) that were homozygous for either the IL-2 knockout or wild-type gene alleles (C57BL/6scid-IL-2(-/-) and C57BL/6scid-IL-2(+/+) littermates, respectively). Groups were assessed for differences in (1) T lymphocytes entering the axotomized FMN; (2) perineuronal CD11b(+) microglial phagocytic clusters, a measure of motor neuron death; and (3) activated microglial cells as measured by MHC-II positivity. C57BL/6-IL-2(-/-) knockout mice had significantly higher numbers of T cells and lower numbers of activated MHC-II-positive microglial cells in the regenerating FMN than wild-type littermates, although the number of CD11b(+) phagocytic microglia clusters did not differ. Thus, despite the significant impairment of T cell function known to be associated with loss of peripheral IL-2, the increased number of T cells entering the axotomized FMN appears to have sufficient activity to support neuroregenerative processes. Congenic C57BL/6scid-IL-2(-/-) knockout mice had lower numbers of CD11b(+) microglial phagocytic clusters than congenic C57BL/6scid-IL-2(+/+) wild-type littermates, suggesting that loss of the IL-2 gene in the CNS (and possibly the loss of other unknown sources of the gene) enhanced neuronal regeneration. Further study of IL-2's complex actions in neuronal injury may provide greater understanding of key variables that determine whether or not immunological processes in the brain are proregenerative.

C-terminals on motoneurons: electron microscope localization of cholinergic markers in adult rats and antibody-induced depletion in neonates.

C-terminals on motoneurons are defined as those accompanied by characteristic postsynaptic specializations termed subsurface cisterns. We have previously shown, by light microscope immunolabelling methods, that subsurface cisterns occur regularly beneath choline acetyltransferase- and acetylcholinesterase-containing boutons on motoneurons. In the present study, the cholinergic nature of C-terminals suggested by these results was further investigated by immunohistochemistry and electron microscopy in adult rats and in neonates treated with a murine monoclonal acetylcholinesterase antibody which was previously shown to cause immunological lesions of central cholinergic systems. In both the facial nucleus and lumbar segment of spinal cord of adult rats, C-terminals were seen intensely immunostained for the cholinergic markers choline acetyltransferase and acetylcholinesterase. Immunolabelled terminals made contact with either neuronal somata or large calibre dendrites, which were positive for the cholinergic markers, and exhibited club-shaped or thin elongated morphologies suggestive of terminal or en passant type synaptic interactions. The close relationship found between cholinergic markers and immunolabelled subsurface cisterns in adults was maintained on motoneurons of eight-day-old rats. While subcutaneous treatment of newborn rat with acetylcholinesterase antibody appeared to have no effect on the distribution of immunopositive subsurface cisterns in motoneurons when examined on postnatal day 8, the density of labelling for the two cholinergic markers around these neurons was reduced. Areas of neuropil immediately surrounding motoneurons in treated animals often showed signs of extensive swelling and deterioration indicative of a lesion event, and these motoneurons frequently displayed subsurface cisterns unapposed to C-terminals. These results support our earlier conclusion, based on light microscope investigation, that the majority if not all C-terminals are cholinergic in the areas investigated and demonstrate the potential utility of immunolesion methods in the study of C-terminal function.

Induction of neuropeptide gene expression and blockade of retrograde transport in facial motor neurons following local peripheral nerve inflammation in severe combined immunodeficiency and BALB/C mice.

Peripheral nerve inflammation is a common clinical problem that accompanies nerve injury and several diseases including Guillain-Barre syndrome and acute and chronic inflammatory demyelinating polyneuropathy. To determine if neuropeptides are induced in motor neurons after inflammation and to study the mechanisms involved, a nerve cuff soaked in complete Freund's adjuvant (CFA) was applied locally to the facial nerve of Balb/C mice. This procedure resulted in an influx of lymphocytes and macrophages to the affected area and a blockade of retrograde axonal transport distal, but not proximal, to the site of application. The same treatment resulted in a strong ipsilateral induction of pituitary adenylyl cyclase activating peptide (PACAP) gene expression in motor neurons in the facial motor nucleus. Because the changes could have occurred due to the loss of target-derived factors or to the production of new factors by immune cells, we studied the effect of the inflammatory stimulus on PACAP mRNA in mice with severe combined immunodeficiency (SCID). As expected, SCID mice showed a severely reduced influx of T-lymphocytes but not macrophages to the peripheral nerve. Moreover, although retrograde transport distal to the inflammation site was blocked similarly in control and SCID mice, the number of motor neurons expressing PACAP mRNA after CFA application was significantly reduced in SCID mice. The data indicate that the induction of PACAP mRNA during nerve inflammation requires the involvement of lymphocytes. However, because the induction of PACAP gene expression was only partially blocked in SCID mice, macrophages, loss of target-derived factors, or other mechanisms may also contribute to the upregulation of PACAP gene expression in motor neurons after nerve inflammation.

Immune rejection of a facial nerve xenograft does not prevent regeneration and the return of function: an experimental study.

Nerve grafts may be used to repair damaged peripheral nerves and also to facilitate spinal cord regeneration after experimental trauma. Little is known, however, about the possible use of xenografts and the role of immune rejection in the outcome of repair. In rats, excision of a short (7-8 mm) segment of facial nerve at its exit point from the skull base results in a permanent deficit in eye closure in the blink reflex. This deficit can be repaired by transplantation of a segment of either syngeneic rat facial nerve or xenogeneic Balb-C mouse sciatic nerve either with or without cyclosporine immunosuppression. With longer (15-20 mm) transplants, however, restoration of eye closure becomes dependent on cyclosporine administration. Thus, in a situation where nerve repair does not occur without a graft, a host immune attack has an attritional effect which is not sufficient to prevent repair over short distances, but becomes obvious when the regenerating fibres have to cross longer segments of transplanted tissue.

Facial nerve transection causes expansion of myelin autoreactive T cells in regional lymph nodes and T cell homing to the facial nucleus.

Nervous tissue expression of immunological signal and recognition molecules, as well as lymphoid tissue immune responses after facial nerve trauma was studied in male rats of the Lewis and Brown Norway (BN) strains. In both rat strains nerve transection caused within four days the appearance of IFN-gamma-like immunoreactivity in the cytoplasm of axotomized motor neurons and an induction of MHC class I and II, and CD4 molecules on surrounding glial cells to a similar extent. T lymphocytes also infiltrated the facial nuclei ipsilateral to the axotomy in all animals. The number of autoreactive T cells in superficial cervical lymph nodes, which in response to whole myelin or peptides of myelin basic protein (MBP) secreted IFN-gamma increased markedly after axotomy. This response was more conspicuous in Lewis rats, which are susceptible to experimental allergic encephalomyelitis (EAE), than in BN rats, which are EAE resistant. A proportion of the axotomized Lewis rats also developed widespread perivascular infiltration of mononuclear cells in the CNS, reminiscent of EAE. Hypothetically, a strong expansion of myelin autoreactive IFN-gamma producing T cells secondary to nerve trauma may have immunopathological consequences in genetically predisposed individuals. It is also possible that myelin reactive T cells, whether recruited to the lesioned nerve, could have impact on macrophage function during Wallerian degeneration in the distal stump.

The cerebral perivascular cells.

This monograph reviews the literature and presents experimental data on the intracerebral presentation of antigen(s) to the immune system as a consequence of neuronal cell death. "Which cells are the antigen presenting cells (APC) of the brain?" is the main question of this book. The immune surveillance of the CNS occurs through specialized resident cells, which present (auto)antigen(s) to the immune system and thus initiate an (auto)immune response. There are four established prerequisites necessary to identify resident APC of the brain. First, the APC must be capable to phagocytose dead neurons. Second, in order to be recognized by T lymphocytes, these neuronophages must express Major Histocompatibility Complex (MHC) cells II glycoproteins on their surface. Third, in order to present (auto)antigen, the MHC class II-positive neuronophages must also be able to contact T lymphocytes. Fourth, in order to exert a stimulatory effect on T lymphocytes, the APC should be able to produce the cytokine interleukin-1 beta (IL-128 Mb). Three main tools were used to identify and characterize the APC of the brain. First, a lesion model was employed that yields a slowly progressing neuronal cell loss without disruption of the blood-brain barrier. This model consisted of resection of 10 mm of the facial nerve, which caused a slowly occurring neuronal death so that one year after resection the amount of facial neurons was about 44% of the control value. Second, neuronophages were labeled in vivo in situ via phagocytosis of the permanent fluorescent marker Fluoro-Gold (FG) from decaying pre-loaded facial motoneurons. Third, the FG-labeled neuronophages were immunocytochemically characterized with the new method "immunoquenching of fluorescence". Sections of the brainstem containing FG-labeled, i.e. fluorescent, neuronophages were incubated with a variety of primary antibodies, followed by avidin-HRP and DAB-nickel as a dark brown reaction product for bright-field microscopy. In the fluorescent mode this DAB reaction product selectively quenches the fluorescence of all immunopositive cells, i.e. only those neuronophages that do not bind to the primary antibody remain fluorescent. Combining FG-labeling of neuronophages with immunoquenching, a population of small round fluorescent cells was discovered, localized in the immediate vicinity of the motoneurons long after the neuronofugal migration of microglia. As the fluorescence of these cells was not quenched after a triple immunostaining with anti-neuronal-specific enolase, anti-GFAP and OX-42 (quenching all fluorescence from neurons, astroglia, and microglia), they seem to represent a new, immunologically unidentified neuronophage. Following this triple immunostaining, a broad panel of antibodies was tested to stain, quench fluorescence, and thus immunotype these enigmatic phagocytes. Only the monoclonal antibody ED2, the classical marker for perivascular cells, specifically stained the small round neuronophages. Although the perivascular cells are in the vicinity of the basal lamina of the cerebral vasculature, they must not be confused with the pericytes, which are not able to perform phagocytosis. In contrast, the perivascular cells are macrophages-ED2 recognizes an established macrophage membrane antigen. In addition, after neuronal injury a subset of the perivascular cells starts to synthesize MHC class II glycoproteins and IL-1 beta. Hence this population of cells seems to possess the complete machinery required for antigen presentation: They are macrophages, upregulate MHC class II molecules and IL-1 beta, and due to their anatomical location, have access to circulating T lymphocytes. What was still lacking, however, was a direct proof of neuronophagia. Our experiments provided this proof. (ABSTRACT TRUNCATED)