Impaired antibody synthesis after spinal cord injury is level dependent and is due to sympathetic nervous system dysregulation

Individuals with spinal cord injury (SCI) are highly susceptible to infection. This post-traumatic immune suppression is thought to occur via alterations in sympathetic nervous system (SNS) or hypothalamic-pituitary-adrenal (HPA) axis function. Normally, the HPA axis and SNS help coordinate proper immune function. After SCI, the HPA axis becomes activated and descending input to sympathetic preganglionic neurons (SPNs) is impaired. Because lymphoid organs are innervated by SPNs distributed throughout the thoracolumbar spinal cord, we predicted level-dependent immune suppression after SCI due to activation of the HPA axis and loss of descending input to SPNs. We tested this hypothesis by measuring indices of HPA (circulating corticosterone; CORT) and SNS function (norepinephrine (NE) in spleen) as well as antigen-specific antibody synthesis against an exogenous non-self protein following high- or low-level SCI. Using a mid-thoracic (T9) spinal contusion injury model, we found that CORT was elevated after SCI with aberrant patterns of diurnal CORT synthesis evident through at least the first 24 h post-injury. However, splenic NE and antibody synthesis were similar to uninjured controls. Injury severity did not change these parameters. Indeed, CORT, NE and antibody synthesis were similar after T9 contusion or transection SCI. In contrast, high-level SCI (T3) caused sustained increases in CORT and splenic NE along with impaired antibody synthesis and elevated splenocyte apoptosis. The immunosuppressive effects of T3 SCI were caused by NE acting at beta2-adrenergic receptors (beta2AR) and could be reversed using beta2AR blockers. Interestingly, impaired antibody after T3 SCI could be mimicked after T9 SCI with a beta2AR agonist. These data illustrate the immunosuppressive effects of the SNS after high-level SCI and indicate that immune deficits may be overcome using beta-blockers.     

Immunopathological reactions in viral infections of the central nervous system

Viral infections of the central nervous system (CNS), as of any other organ, evoke humoral and cellular immune responses which enable the host to eliminate the pathogen. However, effective responses may themselves produce tissue damage sometimes exceeding that caused by the virus itself. The relative contribution of the various immunopathological mechanisms in human viral encephalitides remains mostly ill defined. Most of our understanding of the immunopathogenesis in viral CNS infections comes from studies on experimentally infected animals. The prototype model of a virus-induced, cell-mediated, immunopathological CNS disease is the neurological illness of mice that follows intracerebral inoculation with lymphocytic choriomeningitis virus. Virus-specific cytotoxic T cells are crucial to the pathogenesis but death of the animals only results when these cells are targeted into functionally essential brain structures like the circumventricular organs or the medulla oblongata and cervical spinal cord.     

Neuroprotective effects of melanocortins in experimental spinal cord injury. An experimental study in the rat using topical application of compounds with varying affinity to melanocortin receptors

Summary. The possibility that local administration of low molecular weight non-peptide compounds with varying affinities at melanocortin receptors in the spinal cord will influence pathophysiological outcome of spinal cord injury (SCI) was examined in a rat model. Five new Melacure compounds ME10092, ME10354, ME10393, ME10431 and ME10501 were used in this investigation. Each compound was dissolved in saline and tested at 3 different doses, i.e. 1 μg, 5 μg and 10 μg total dose in 10 μl applied topically 5 min after SCI. The animals were allowed to survive 5 h and trauma induced edema formation, breakdown of the blood–spinal cord barrier (BSCB) and cell injuries were examined and compared with untreated injured rats. A focal SCI inflicted by an incision into the right dorsal horn of the T10–11 segments resulted in marked edema formation, breakdown of the BSCB to Evans blue albumin and caused profound nerve cell injury in the T9 and the T12 segments. Topical application of ME10501 (a compound with high affinity at melanocortin, MC-4 receptors) in high doses (10 μg) resulted in most marked neuroprotection in the perifocal spinal cord (T9 and T12) segments. On the other hand, only a mild or no effect on spinal cord pathology was observed in the traumatized animals that received ME10092, ME10354, ME10393 and ME10431 at 3 different doses. These observations suggest that non-peptide compounds with varying affinity to melanocortin receptors are able to influence the pathophysiology of SCI. Furthermore, compounds acting at melanocortin, MCR4 receptors are capable to induce neuroprotection in spinal cord following trauma. Present addresses: AstraZeneca R&D, S?dert?lje, Sweden Present addresses: Department of Organic Pharmaceutical Chemistry, Uppsala University, Uppsala, Sweden     

Central nervous system demyelination in Venezuelan equine encephalomyelitis infection: An experimental model of virus-induced myelin injury

Arboviruses are important pathogens for both animals and humans. Venezuelan equine encephalomyelitis virus (VEEV) is an arbovirus whose pathogenicity for grey matter structures has been previously studied. To our knowledge, the present study is the first to describe extensive inflammation and demyelination in spinal cord white matter of mice infected with VEEV. To probe a possible immunepathogenesis of white matter alterations in this infection, nude mice and heterozygous controls were similarly infected. Whereas controls still showed inflammatory demyelination, nude mice showed no white matter changes in the absence of a mononuclear inflammatory response. These results suggest that white matter changes in VEEV infection are dependent upon the host immune-response, rather than produced by primary viral cytolytic activity. Such findings are similar to those we and others obtained in a number of different viral infections and support the possibility that the host immune response may be the common denominator leading to myelin injury in a variety of viral diseases. The hypothesis of “by-stander killing” of myelin is discussed as a possible host-mediated mechanism of demyelination in viral infections.     

MMP9 deficiency does not decrease blood-brain barrier disruption, but increases astrocyte MMP3 expression during viral encephalomyelitis

Expression of matrix metalloproteinases (MMPs), especially MMP9 correlates with blood-brain barrier (BBB) disruption during many neuroinflammatory diseases. During neurotropic coronavirus virus (JHMV) induced encephalomyelitis, MMP9 activity is restricted to neutrophils. Furthermore, myeloid cell depletion implicated MMP9 in facilitating leukocyte central nervous system (CNS) infiltration via loss of BBB integrity. The requirement of MMP9 in BBB disruption was thus assessed in JHMV infected MMP9 deficient (MMP9(-/-)) mice. Depletion of neutrophils reduced CNS accumulation of monocytes and T cells, albeit without affecting overall pathogenesis. By contrast, infected MMP9(-/-) mice revealed no differences in CNS leukocyte infiltration, composition or localization, consistent with BBB disruption similar to wild-type (WT) mice. Unimpaired T cell mediated virus control supported an unexpectedly redundant role of MMP9 in promoting leukocyte access to the brain parenchyma. Although MMP9 deficiency did not expand the overall limited pattern of MMP expression during JHMV infection, it coincided with MMP3 upregulation. MMP3 expression remained largely confined to astrocytes, similar to WT mice. These data demonstrate that neutrophil-derived MMP9 is not the sole mediator facilitating parenchymal leukocyte entry via BBB disruption during viral encephalomyelitis. Moreover, significantly enhanced MMP3 expression by astrocytes in infected MMP9(-/-) mice suggests an active role of resident cells in participating and potentially collaborating with infiltrating cells in regulating BBB permeability. Overall, these results highlight the complexity of targeting individual MMPs as a strategy to regulate inflammation.     

CD8+-dependent CNS demyelination following ocular infection of mice with a recombinant HSV-1 expressing murine IL-2

Demyelinating diseases comprise a spectrum of immunopathologic syndromes in which myelin, the fatty covering of nerve cell fibers in the brain and spinal cord, is destroyed. In this study, we have shown for the first time that ocular infection of BALB/c mice with a recombinant herpes simplex virus type 1 (HSV-1) expressing IL-2 (HSV-IL-2) results in CNS demyelination as determined by light microscopy and EM. The demyelinated lesions involve periventricular white matter, brain stem, and spinal cord white matter. Demyelination was detected in the CNS of infected mice up to 75 days (the longest time point tested) post HSV-IL-2 infection. In contrast, mice infected with HSV-IFN-gamma or HSV-IL-4, which are identical to HSV-IL-2 but express IFN-gamma or IL-4 instead of IL-2, did not exhibit demyelination. Control mice infected with wild-type HSV-1 or parental virus also remained free of these symptoms. During early times (days 3-7), post-infection with HSV-IL-2 virus, a T(H)1 + T(H)2 pattern of cytokines was produced by lymphocytes of infected mice while mice infected with HSV-IFN-gamma or control viruses produced a T(H)1 pattern of cytokine. By day 21 post-infection, all infected groups exhibited a T(H)1 pattern of response. Immunohistochemistry and FACS analyses of infiltrates in the brains and spinal cords of HSV-IL-2-infected mice showed elevations in CD4+ and CD8+ T cells and macrophages. However, T cell depletion studies suggest that only central memory CD8+ T cells are directly involved in the demyelination process, with macrophages being involved through a bystander effect.     

A comparative study of acute and chronic diseases induced by two subgroups of Theiler’s murine encephalomyelitis virus

Theiler’s murine encephalomyelitis viruses (TMEV) are divided into two subgroups on the basis of their different biological activities. The GDVII strain produces acute polioencephalomyelitis in mice, whereas the DA strain produces demyelination with virus persistence in the spinal cord. A comparative study of GDVII and DA strains suggested that low host immune responses are responsible for the development of acute GDVII infection and that the persistence of infected macrophages plays a crucial role in the development of chronic white matter lesions in DA infection. All 78 mice infected with GDVII died or became moribund by day 13, while none of 54 mice infected with DA died. In the acute stage, the distribution of viral antigens in the central nervous system (CNS) tissue was similar in both GDVII and DA infections, although the virus titer was higher in GDVII infection. In DA infection, a substantial number of T cells were recruited to the CNS on day 6 when they were virtually absent in GDVII infection. The titer of neutralizing antibody was already high on day 6 in DA infection but was negligible in GDVII infection. Development of chronic paralytic disease from day 35 of the DA infection was accompanied by focal accumulation of viral antigen-positive macrophages in the spinal white matter. In addition, white matter lesions comparable to those in chronic DA infection were induced in the spinal cord within 7 days after intracerebral injection of DA-infected murine macrophages. Received: 26 June 1995 / Revised, accepted: 27 December 1995     

Effects of regional spinal X-irradiation on demyelinating disease caused by Theiler's virus, mouse hepatitis virus or experimental allergic encephalomyelitis

The effects of X-irradiation on the course of chronic demyelinating disease were examined in mice with experimental allergic encephalitis (EAE), mouse hepatitis virus (MHV) or Theiler's virus (DAV) infection. One month after the induction of EAE or 2-16 months after inoculation of DAV, exposure of the cervical spinal cord to 20 Gy X-rays caused local exacerbation of disease activity but spinal irradiation did not affect MHV-induced demyelination. In EAE, there was a significant increase in the number of inflammatory cells in the irradiated part of the cord. Mice infected with DAV showed locally increased demyelination and axonal degeneration but no change in the titer of infectious virus within the cord. Thus in DAV infection, as in EAE, the exacerbation of disease seemed to be due to vascular or immunological factors rather than viral reactivation.     

Endogenous gamma interferon produced in central nervous system by systemic infection infection with Theiler's virus in mice

Theiler's virus GD VII strain causes acute encephalomyelitis by intracerebral inoculation. We established acute encephalomyelitis in mice by the intravenous (i.v.) inoculation of Theiler's virus GD VII strain. Replication of Theiler's virus injected i.v. could be observed in both the brain and spinal cord of mice, and interferon (IFN)-γ could be detected in the extracts of brain and spinal cord in parallel with viral replication. Furthermore, by the injection of anti-IFN-γ monoclonal antibody (mAb) on Day 1 post-infection (p.i.), mortality and virus titres in the spinal cord increased compared with the control mice treated with normal rat globulin. The histological exacerbation of inflammation was observed in spinal cord of anti-IFN-γ mAb-treated mice. These results indicate that endogenous IFN-γ, produced locally in the brain and spinal cord of mice through both antiviral action and anti-inflammatory action of IFN-γ in central nervous system, plays an important role in Theiler's virus infection.     

Abrogation of resistance to Theiler's virus-induced demyelination in C57BL mice by total body irradiation

Theiler's murine encephalitis virus (TMEV) produces an unusual biphasic disease in susceptible mice characterized by poliomyelitis with early viral replication in neurons, followed by chronic demyelination with viral antigen expression in spinal cord white matter. In addition, infectious virus persists in the central nervous system (CNS) throughout the chronic phase of disease. Previous studies have indicated an important role for major histocompatibility complex (MHC)-gene products in determining resistance/susceptibility to disease. In particular, certain class I gene products of the D region of the H-2 gene complex render mice of the C57BL lineage resistant to induction of demyelination. Intracerebral infection of B10.S(DS) mice results in demyelination in the spinal cord while infection of C57BL/10(Db) or B10.S(9R)(Dd) fails to produce white matter destruction. In this study we showed that immunosuppression with gamma irradiation renders normally resistant B10.S(9R) and C57BL/10 mice susceptible to TMEV-induced demyelination and allowed for increased viral replication. In addition, the majority of irradiated C57BL/10 mice infected with virus showed extensive areas of CNS remyelination by oligodendrocytes beginning at 63 days post-infection. In contrast, immunosuppression of normally susceptible B10.S mice resulted in acute disease and high mortality accompanied by overwhelming destruction of neurons. The study supports the hypothesis that MHC-conferred resistance in C57BL mice is associated with MHC D region products and indicate an important active role for the immune system early in infection in limiting vital infection during disease induction in nonimmunosuppressed mice.     

The dual role of CD8+ T lymphocytes in the development of stress-induced herpes simplex encephalitis

Despite the generally restrictive nature of the blood–brain barrier (BBB), circulating lymphocytes can infiltrate into the central nervous system (CNS) during a variety of disease states. Although the contributions of these lymphocytes to CNS-associated disease have been identified in some viral models, the factors which govern this infiltration following herpes simplex virus (HSV) infection remain to be elucidated. We have developed a murine model of HSV encephalitis (HSE) to define the relationship among psychological stress, the recruitment of HSV-specific T cells into the CNS, and the development of HSE. Naive mice, as well as mice that had been vaccinated with a recombinant vaccinia virus (rVVESgB498–505) that elicits the generation of HSV-1 gB498–505-specific CD8⁺ T cells, were infected intranasally (i.n.) with HSV-1 McIntyre. Beginning one day prior to HSV-1 infection and continuing for a total of 9 days, naive and vaccinated mice were exposed to a well-established stressor, restraint stress. Naive, stressed mice exhibited increased symptoms of HSE and HSE-associated mortality as compared to non-stressed controls. A concomitant increase in CD4⁺ and CD8⁺ T cells in the brain was observed throughout the infection, with CD8⁺ T cells outnumbering CD4⁺ T cells. The development of HSE in these naive, stressed mice was accompanied by a delayed infiltration of gB498–505-specific CD8⁺ T cells after HSV spread into the brain. In contrast, both stressed and non-stressed rVVESgB498–505-vaccinated mice possessed gB498–505-specific CD8⁺ T cells prior to HSV challenge and were protected against HSE despite having detectable HSV-1 DNA in the brain. Together, these findings suggest that a delayed infiltration of CD8⁺ T cells into the brain may promote HSE in naive mice, while the presence of HSV-specific CD8⁺ T cells in the brain prior to HSV challenge is protective, possibly by limiting HSV replication and spread within the CNS.     

Up-regulation of Fas ligand (FasL) in the central nervous system: A mechanism of immune evasion by rabies virus

Following its injection into the hindlimbs of mice, CVS, a highly pathogenic strain of rabies virus, invades the spinal cord and brain resulting in the death of the animal. In contrast, central nervous system (CNS) invasion by PV, a strain of attenuated pathogenicity, is restricted to the spinal cord and mice infected with this virus survive. Lymphocytes display transient migration into the infected CNS in fatal rabies and sustained migration in nonfatal rabies. The transient migration of T cells in fatal rabies is associated with an increase in T-cell apoptosis. We found that the early production of Fas ligand (FasL) mRNAs was up-regulated only in fatal rabies. FasL is produced by several neuronal cells and mainly in infected neurons. In mice lacking FasL (gld), infection with the neuroinvasive rabies virus strain was less severe, and the number of CD3 T cells undergoing apoptosis was smaller than that in normal mice. These data provide strong evidence that fatal rabies virus infection involves the early triggering of FasL production leading to the destruction of migratory T cells by the Fas/FasL apoptosis pathway. This mechanism could be in part responsible for the fact that T cells cannot control neuroinvasive rabies infection. Thus, rabies virus seems to use an immunosubversive strategy that takes advantage of the immune privilege status of the CNS.     

Abortive rabies virus central nervous infection is controlled by T lymphocyte local recruitment and induction of apoptosis

Nonfatal paralysis, induced by the attenuated Pasteur strain of rabies virus, is characterised by local and irreversible flaccid paralysis of the inoculated limbs. We characterised the spread and localisation of virus in the CNS of infected mice, determined the nature of cell injury and examined the role of the immune response. Data indicate that infection of BALB/c mice induced paralysis in 60% of infected mice, the others recovering without sequelae. In both groups of mice, virus was detected in restricted sub-populations of neurons from the brain and spinal cord, and intensity of the neuropathology correlated with levels of rabies RNA and apoptotic infected neurons. However, apoptosis of neurons and paralysis were not due to a direct deleterious effect of the virus, but induced by a T-dependent immune response, as evidenced by their absence in nude mice. Paralysed and asymptomatic mice developed a similar rabies virus-specific IgG2a antibody response, thus excluding the role of any modification of the humoral immune response. In contrast, three events were critically associated with the development of neurological symptoms: the amount of virus in the CNS, the level of apoptosis in both infected neurons and uninfected surrounding cells and the progressive parenchymal infiltration of CD4+ and CD8+ T cells at the site of infection. These data suggest that during nonfatal rabies infection, the levels of viral replication and primary degeneration of infected neurons by apoptosis could be responsible for the infiltration of T lymphocytes capable of inducing secondary degeneration of neural cells.     

Castration of male C57L/J mice increases susceptibility and estrogen treatment restores resistance to Theiler's virus-induced demyelinating disease

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination in selective mouse strains. We have previously demonstrated that the males of C57L mice are significantly more susceptible to TMEV-induced demyelinating disease. To assess further the hormonal influence for this gender-associated differential susceptibility, estrogen-treated, castrated C57L mice were infected with TMEV and compared with sham-operated and/or placebo-treated mice. Interestingly, castration further elevated the susceptibility to virally induced demyelinating disease compared with sham-castrated control mice, and prolonged treatment of castrated mice with estrogen restored the resistance to the level of control mice. These results strongly suggest that sex hormone levels contribute to the gender-biased susceptibility to TMEV-induced demyelinating disease. Mice treated with estrogen showed a significantly decreased level of virus-specific Th1 responses both in the periphery and in the CNS. In addition, in vitro estrogen treatment was able to inhibit viral replication directly in macrophages, consistent with the lower level of viral RNA in microglia/macrophages in the CNS from castrated estrogen-treated mice compared with controls. Also, estrogen treatment inhibited VCAM-1 expression induced by tumor necrosis factor-alpha in cerebral vascular endothelial (CVE) cells via inhibition of nuclear factor-kappaB (NFkappaB), which is produced in various glial cells upon TMEV infection. Overall, estrogen treatment appears to exert its effects on viral replication, induction of immune responses, as well as infiltration of activated immune cells into the CNS via inhibition of NFkappaB function.     

Role of the inflammasome-related cytokines Il-1 and Il-18 during infection with murine coronavirus

The inflammasome, a cytosolic protein complex that mediates the processing and secretion of pro-inflammatory cytokines, is one of the first responders during viral infection. The cytokines secreted following inflammasome activation, which include IL-1 and IL-18, regulate cells of both the innate and adaptive immune system, guiding the subsequent immune responses. In this study, we used murine coronavirus, mouse hepatitis virus (MHV), infection of the central nervous system and liver to assess of the role of the inflammasome and its related cytokines on pathogenesis and host defense during viral infection. Mice lacking all inflammasome signaling due to the absence of caspase-1 and -11 were more vulnerable to infection, with poor survival and elevated viral replication compared to wild-type mice. Mice lacking IL-1 signaling experienced elevated viral replication but similar survival compared to wild-type controls. In the absence of IL-18, mice had elevated viral replication and poor survival, and this protective effect of IL-18 was found to be due to promotion of interferon gamma production in αβ T cells. These data suggest that inflammasome signaling is largely protective during murine coronavirus infection, in large part due to the pro-inflammatory effects of IL-18.     

Effect of cyclosporin A on an experimental chronic viral infection of the central nervous system

The effects of cyclosporin A (CsA) on neuropathological lesions induced by a chronic viral infection have been tested in the experimental model of the mouse hepatitis virus 3 (MHV3) infection. Daily injections of CsA (50 mg/kg) inhibited the expression of the MHV3-induced ependymitis, meningitis, hydrocephalus and vasculitis. The effect was preserved even if CsA treatment was initiated 15 days after virus infection but was lost if CsA treatment was given later on or for a shorter period of time. Viral titers in brains of chronically infected mice were not affected by CsA treatment. During the first week following MHV3 infection, CsA treatment increased both the percentage of acute death (31 vs. 10%) and the viral titers in brain and liver of infected mice. In this model, the timing of CsA treatment appeared critical for the balance between its beneficial effect on CNS lesions and the risk of increased acute mortality.     

ICAM-1 is crucial for protection from TMEV-induced neuronal damage but not demyelination

Previous work has suggested that the factors protecting mice from Theiler's murine encephalomyelitis virus (TMEV)-induced spinal cord demyelination are distinct from those involved in protection of the brain during the acute encephalitic phase. In this study, we examined the requirement for intercellular adhesion molecule-1 (ICAM-1) in both of these processes. During the acute phase of infection (days 7 to 10 after intracerebral infection with TMEV), no differences in brain or spinal cord pathology or virus burdens were observed between ICAM-1-knockout mice and the infected immunocompetent control mice of a similar background. Examination of brain pathology later in infection (that is, day 45 post infection [p.i.]) revealed that ICAM-1-deficient mice experienced increased levels of pathology in gray matter regions of the brain. We observed an increase in striatal damage and meningeal inflammation in the brains of TMEV-infected ICAM-1-knockout mice compared to C57BL/6J mice. Despite the increase in brain pathology, no immunoreactivity to viral antigens was detected, suggesting that the virus had been cleared by this time. Resistance to demyelination was similar in both groups, indicating that the resulting immune response was sufficient for protection of the spinal cord white matter.     

Macrophage-mediated optic neuritis induced by retrograde axonal transport of spike gene recombinant mouse hepatitis virus

After intracranial inoculation, neurovirulent mouse hepatitis virus (MHV) strains induce acute inflammation, demyelination, and axonal loss in the central nervous system. Prior studies using recombinant MHV strains that differ only in the spike gene, which encodes a glycoprotein involved in virus-host cell attachment, demonstrated that spike mediates anterograde axonal transport of virus to the spinal cord. A demyelinating MHV strain induces optic neuritis, but whether this is due to the retrograde axonal transport of viral particles to the retina or due to traumatic disruption of retinal ganglion cell axons during intracranial inoculation is not known. Using recombinant isogenic MHV strains, we examined the ability of recombinant MHV to induce optic neuritis by retrograde spread from the brain through the optic nerve into the eye after intracranial inoculation. Recombinant demyelinating MHV induced macrophage infiltration of optic nerves, demyelination, and axonal loss, whereas optic neuritis and axonal injury were minimal in mice infected with the nondemyelinating MHV strain that differs in the spike gene. Thus, optic neuritis was dependent on a spike glycoprotein-mediated mechanism of viral antigen transport along retinal ganglion cell axons. These data indicate that MHV spreads by retrograde axonal transport to the eye and that targeting spike protein interactions with axonal transport machinery is a potential therapeutic strategy for central nervous system viral infections and associated diseases.     

Characterization of the central nervous system innervation of the rat spleen using viral transneuronal tracing

Splenic immune function is modulated by sympathetic innervation, which in turn is controlled by inputs from supraspinal regions. In the present study, the characterization of central circuits involved in the control of splenic function was accomplished by injecting pseudorabies virus (PRV), a retrograde transynaptic tracer, into the spleen and conducting a temporal analysis of the progression of the infection from 60 hours to 110 hours postinoculation. In addition, central noradrenergic cell groups involved in splenic innervation were characterized by dual immunohistochemical detection of dopamine-beta-hydroxylase and PRV. Infection in the CNS first appeared in the spinal cord. Splenic sympathetic preganglionic neurons, identified in rats injected with Fluoro-Gold i.p. prior to PRV inoculation of the spleen, were located in T(3)-T(12) bilaterally; numerous infected interneurons were also found in the thoracic spinal cord (T(1)-T(13)). Infected neurons in the brain were first observed in the A5 region, ventromedial medulla, rostral ventrolateral medulla, paraventricular hypothalamic nucleus, Barrington's nucleus, and caudal raphe. At intermediate survival times, the number of infected cells increased in previously infected areas, and infected neurons also appeared in lateral hypothalamus, A7 region, locus coeruleus, subcoeruleus region, nucleus of the solitary tract, and C3 cell group. At longer postinoculation intervals, infected neurons were found in additional hypothalamic areas, Edinger-Westphal nucleus, periaqueductal gray, pedunculopontine tegmental nucleus, caudal ventrolateral medulla, and area postrema. These results demonstrate that the sympathetic outflow to the spleen is controlled by a complex multisynaptic pathway that involves several brainstem and forebrain nuclei.     

Differential induction of apoptosis in demyelinating and nondemyelinating infection by mouse hepatitis virus

The role of apoptosis in mouse hepatitis virus (MHV) infection is still controversial. To better assess the role of apoptosis in MHV infection, we used three different biologic phenotypes of MHV to examine their differential effect on the induction of apoptosis. MHV-A59 produces acute hepatitis, meningoencephalitis, and chronic demyelination. MHV-2 causes only acute hepatitis and meningitis, whereas Penn98-1 produces acute hepatitis and meningoencephalitis without demyelination. We detected TdT-mediated dUTP nick-end labeling (TUNEL) staining in the livers and meninges of MHV-A59-, MHV-2-, and Penn98-1-infected mice. TUNEL staining in brain parenchyma was only detected in MHV-A59- and Penn98-1-infected mice. We detected apoptosis by electronmicroscopy in olfactory neurons during acute infection with MHV-A59. The kinetics and distribution of TUNEL staining correlated with the pathologic damage and colocalized with viral antigen in some cells. At 1 month, TUNEL staining was found exclusively in areas of demyelination in the spinal cord of MHV-A59-infected mice; however, it was not found in nondemyelinated mice infected with MHV-2 or Penn98-1, or in mock-infected mice. TUNEL-positive cells were identified as macrophage/microglial cells, some astrocytes, and some oligodendrocytes, by colabeling with cell-specific markers. The presence of TUNEL staining in oligodendrocytes suggests that apoptosis may play an important role in MHV-induced demyelination.