The effect of herpes simplex virus-1 on nitric oxide synthase in the rat brain: the role of glucocorticoids.

OBJECTIVE: Herpes simplex virus-1 (HSV-1) is a common cause of viral encephalitis manifested by activation of the adrenocortical axis, fever and behavioral changes. We investigated the early effects of HSV-1 on constitutive (c) and inducible (i) nitric oxide synthase (NOS) activity in rat brain and in mixed glial cell culture. The effect of glucocorticoids (GCs) on NOS responses to HSV-1 was also determined. METHODS: NOS activity was evaluated by the conversion of ³H-arginine to ³H-citrulline. Nitrites were measured in supernatants of activated glial cells. RESULTS: Under basal conditions, the highest cNOS activity was found in the cerebellum, while activity was much lower in the pons and negligible in the hypothalamus and hippocampus. Forty-eight hours after intracerebral injection of HSV-1, serum corticosterone was increased and NOS activity in the cerebellum and pons was inhibited. Adrenalectomy had no effect on the basal NOS activity but completely abrogated the inhibitory effect of HSV-1. Administration of the iNOS inhibitor aminoguanidine did not significantly change NOS activity, suggesting that the activity found in the cerebellum and pons can be attributed to the cNOS isoform. In mixed glial cell culture infected with HSV-1 and then activated with lipopolysaccharide, NOS activity and nitrite production were inhibited by 77 and 53%, respectively. CONCLUSIONS: These results suggest that brain NOS activity is inhibited in the early stages of HSV-1 infection and requires the presence of circulating GCs. HSV-1-induced brain NOS inhibition may play a role in neuronal viral invasion and in the activation of the adrenocortical axis.

Neuroprotection and immunomodulation with mesenchymal stem cells in chronic experimental autoimmune encephalomyelitis.

OBJECTIVE: To investigate the therapeutic potential of mesenchymal stromal cells (MSCs) in the chronic model of experimental autoimmune encephalomyelitis (EAE). DESIGN: Mesenchymal stromal cells were obtained from the bone marrow of naïve C57BL and green fluorescent protein-transgenic mice and cultured with Eagle minimum essential medium/alpha medium after removal of adhering cells. Following 2 to 3 passages, MSCs were injected intraventricularly or intravenously into mice in which chronic EAE had been induced with myelin oligodendrocyte glycoprotein 35-55 peptide. RESULTS: In 8 separate experiments, the intravenously and intraventricularly injected green fluorescent protein-positive MSCs were attracted to the areas of central nervous system inflammation and expressed galactocerebroside, O4, glial fibrillary acidic protein, and beta-tubulin type III. The clinical course of chronic EAE was ameliorated in MSC-treated animals (0% mortality; mean [SE] maximal EAE score, 1.76 [1.01] and 1.8 [0.46] in the intraventricular and intravenous groups, respectively, vs 13% and 21% mortality and 2.80 [0.79] and 3.42 [0.54] mean maximal score in the controls). A strong reduction in central nervous system inflammation, accompanied by significant protection of the axons (86%-95% intact axons vs 45% in the controls) was observed in the animals injected with MSCs (especially following intraventricular administration). Mesenchymal stromal cells injected intravenously were detected in the lymph nodes and exhibited systemic immunomodulatory effects, downregulating proliferation of lymphocytes in response to myelin antigens and mitogens. Mesenchymal stromal cells cultured with fibroblast growth factor and brain-derived neurotrophic factor in vitro acquired neuronal-lineage cell morphology and expressed beta-tubulin type III, nestin glial fibrillary acidic protein, and O4. CONCLUSIONS: Our results indicate that stem cells derived from bone marrow may provide a feasible and practical way for neuroprotection, immunomodulation, and possibly remyelination and neuroregeneration in diseases such as multiple sclerosis.

Increased KPI containing amyloid precursor protein in experimental autoimmune encephalomyelitis brains.

Amyloid precursor protein can be translated from three alternatively spliced mRNAs. We measured levels of amyloid precursor protein isoforms containing the Kunitz protease inhibitor domain (KPIAPP), and amyloid precursor protein without the Kunitz protease inhibitor domain (KPIAPP) in brain homogenates of acute experimental autoimmune encephalomyelitis mice. At the preclinical phase of the disease, both KPIAPP and KPIAPP levels were significantly higher in homogenates from brains of autoimmune encephalomyelitis mice, whereas at the acute phase of the disease only KPIAPP remained significantly elevated compared with controls. At the recovery phase, no differences were observed between the groups. The early and isoform-specific elevation of KPIAPP in autoimmune encephalomyelitis mice suggests a possible role for amyloid precursor protein in the immune response mediating the disease.

Neural precursors attenuate autoimmune encephalomyelitis by peripheral immunosuppression.

OBJECTIVE: Intracerebroventricular or intravenous (IV) injection of neural precursor cells (NPCs) attenuates experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Although stem cell therapy was introduced initially for cell replacement, we examine here whether NPCs possess immunomodulatory effects. METHODS: We examined the effects of systemic administration of NPCs on central nervous system (CNS) inflammation in EAE and the interactions between NPCs and T cells in vitro and in vivo. RESULTS: IV NPC therapy decreased significantly CNS inflammation and tissue injury and attenuated the clinical severity of EAE. IV-injected NPCs could not be found in the CNS but were detected in lymphoid organs. Coculture experiments showed that NPCs inhibited the activation and proliferation of lymph node-derived T cells in response to CNS-derived antigens and to nonspecific polyclonal stimuli. The relevance of NPC/lymph node cell interactions in vivo was further demonstrated when lymph node cells obtained from IV NPC-treated mice exhibited poor encephalitogenicity on transfer to naive mice and caused a markedly milder EAE compared with those obtained from nontreated mice. INTERPRETATION: IV administration of neural precursors inhibits EAE by a peripheral immunosuppressive effect. Our findings suggest a profound bystander inhibitory effect of NPCs on T-cell activation and proliferation in the lymph nodes, leading to amelioration of EAE.

Cytokine-mediated modulation of MMPs and TIMPs in multipotential neural precursor cells.

Recent studies have implicated the inflammatory process during experimental allergic encephalomyelitis (EAE) in triggering migration and differentiation of transplanted neural precursors cells (NPCs) into the inflamed white matter. The pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma are key factors in the pathogenesis of brain inflammation in EAE and were shown to enhance NPCs migration in vitro. As cell migration is dependent on extracellular matrix remodeling, involving proteolytic enzyme members of the matrix metalloproteinase (MMPs) family, we characterized the profile of expression of MMPs and their endogenous inhibitors (TIMPs) in rat NPCs, and evaluated the effects of TNF-alpha, IFN-gamma and IFN-beta, a clinically proven modulator of brain inflammation, on the expression of these molecules. Newborn rat striatal NPCs were expanded in spheres as nestin+, PSA-NCAM+ and NG2(-) cells, which can differentiate into astrocytes, oligodendrocytes and neurons. NPCs' gelatinase activities of MMP-2 and MMP-9, as determined by zymography, were increased by TNF-alpha, and to a lesser extent by IFN-gamma. Semi-quantitative RT-PCR indicated that TNF-alpha also upregulated MMP-9 mRNA levels. IFN-beta suppressed the TNF-alpha-induced levels of secreted MMP-9 and MMP-2, while enhancing the expression of TIMP-1 and TIMP-2 mRNA. These results suggest that MMPs activity is induced in NPCs by pro-inflammatory cytokines to mobilize them for promoting reparative processes. IFN-beta, on the other hand, appears to have an anti-proteolytic influence that may attenuate such NPC-mediated repair processes.

Transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis.

Stem cell transplantation was introduced as a mean of cell replacement therapy, but the mechanism by which it confers clinical improvement in experimental models of neurological diseases is not clear. Here, we transplanted neural precursor cells (NPCs) into the ventricles of mice at day 6 after induction of chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Transplanted cells migrated into white matter tracts and attenuated the clinical course of disease. NPC transplantation down-regulated the inflammatory brain process at the acute phase of disease, as indicated by a reduction in the number of perivascular infiltrates and of brain CD3+ T cells, an increase in the number and proportion of regulatory T cells and a reduction in the expression of ICAM-1 and LFA-1 in the brain. Demyelination and acute axonal injury in this model are considered to result mainly from the acute inflammatory process and correlate well with the chronic neurological residua. In consequence to inhibition of brain inflammation, precursor cell transplantation attenuated the primary demyelinating process and reduced the acute axonal injury. As a result, the size of demyelinated areas and extent of chronic axonal pathology were reduced in the transplanted brains. We suggest that the beneficial effect of transplanted NPCs in chronic EAE is mediated, in part, by decreasing brain inflammation and reducing tissue injury.

Survival of neural precursor cells in growth factor-poor environment: implications for transplantation in chronic disease.

A key issue for therapeutic neural stem cell transplantation in chronic diseases is the long-term survival of transplanted cells in the brain. The normal adult central nervous system does not support the survival of transplanted cells. Presumably, the limited availability of trophic factors maintains the survival of resident cells but is insufficient for supporting the survival of transplanted cells. Specifically, in multiple sclerosis, a chronic relapsing disease, it would be necessary to maintain long-term survival of transplanted cells through phases of relapses and remissions. It may be beneficial to transplant cells as early as possible, in a form that will keep their survival independent of tissue support and ready for immediate mobilization upon tissue demand during disease relapse. In the present study, we examined whether, in the form of neurospheres, multipotential neural precursor cells (NPCs) survive in a growth factor-poor environment while maintaining their potential to respond to environmental cues. We found that after removal of growth factors from the culture medium of neurospheres in vitro, NPC proliferation decreased significantly, but most cells survived for a prolonged time and maintained their stem cell characteristics. After re-exposure to growth factors, neurosphere cells resumed proliferation and could differentiate along neural lineages. Furthermore, neurospheres, but not single NPCs, that were transplanted into the brain ventricles of intact animals survived within the ventricles for at least a month and responded to induction of experimental autoimmune encephalomyelitis and brain inflammation by extensive migration into the brain white matter and differentiated into glial lineage cells.

Increased thrombin inhibition in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory diseases of the central nervous system (CNS). Activated coagulation factors are associated with inflammation and are elevated in the plasma of animals with EAE. Thrombin is a key coagulation factor and its major endogenous inhibitors are antithrombin III (ATIII) in the plasma and protease nexin 1 (PN-1) in the brain. We measured the capacity of brain homogenates to inhibit exogenous thrombin and the CNS levels of ATIII and PN-1 during the course of EAE. Acute EAE was induced in SJL/J mice by immunization with mouse spinal cord homogenates. On Days 8, 13, and 22 post-immunization, inhibition of exogenous thrombin activity was measured by a recently developed fluorimetric assay. PN-1 and ATIII were assayed both by immunohistochemistry and by immunoblots in the brain and spinal cord. Total brain thrombin inhibitory activity increased (32%) in EAE mice at the peak of clinical disease (Day 13, P=0.04 compared to controls). Brain ATIII also increased at the peak of disease (2.5-fold higher than controls, P=0.0001), and correlated significantly with clinical scores at all stages of disease (r=0.72, P=0.0068). In contrast, PN-1 elevations were more pronounced at the preclinical stage on Day 8 (3-fold higher than controls, P=0.01) than on Day 13 (1.4-fold higher, P=0.005). Increased brain thrombin inhibition at the clinical peak of EAE probably reflects increased influx of plasma thrombin inhibitors. Early PN-1 changes represent a potential target for thrombin modulating drugs in EAE and MS.

Intraventricular transplantation of neural precursor cell spheres attenuates acute experimental allergic encephalomyelitis.

Brain transplantation of neural precursor cells (NPCs) has been proposed to enhance CNS regeneration. As the pathogenesis of most acute CNS diseases involves an inflammatory component, we studied whether NPC transplantation affects brain inflammation. Newborn rat multipotential NPCs were transplanted intraventriculary into acute experimental allergic encephalomyelitis (EAE) rats, a model for disseminated brain inflammation. Cells migrated into inflamed white matter and differentiated into glial cells. NPC transplantation attenuated the clinical severity of EAE and the brain inflammation, indicated by reduction in perivascular infiltrates and decreased expression of ICAM-1 and LFA-1. NPCs inhibited basal proliferation and proliferative responses to Concavalin-A and to MOG peptide of EAE rat-derived lymphocytes in vitro. Purified astrocytes inhibited lymphocyte proliferation in vitro, but did not migrate into EAE brains in vivo, and did not reduce EAE severity or brain inflammation. Thus, transplanted NPCs attenuate acute EAE via an anti-inflammatory mechanism which depends on cell ability to migrate into inflamed brain tissue.

Effects of proinflammatory cytokines on the growth, fate, and motility of multipotential neural precursor cells.

We have recently shown that the inflammatory process during experimental allergic encephalomyelitis (EAE), the animal model of MS, attracts transplanted NPC migration into the inflamed white matter. Here we studied how the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) affect NPC growth, survival, differentiation, and migration. Newborn rat striatal NPCs were expanded in spheres as nestin+, PSA-NCAM+, NG2(-) cells, which differentiated into astrocytes, oligodendrocytes, and neurons. NPCs expressed receptors of TNFalpha and IFNgamma but not interleukin-1. TNFalpha and IFNgamma inhibited sphere cell proliferation, determined by [(3)H]thymidine and BrdU incorporation. IFNgamma increased apoptotic cell death (determined by TUNEL stains); this effect partially blocked by TNFalpha. Neither cytokine affected NPC lineage fate, determined by percentage of GFAP+, neurofilament+, and GalC+ cells after differentiation. TNFalpha and IFNgamma increased outward migration of cells from spheres in vitro. Thus, TNFalpha and IFNgamma, key players in MS and EAE, inhibit NPC proliferation and induce their migration.

MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain.

Stem cell transplantation is being explored as a new paradigm for the treatment of demyelinating diseases. Magnetically labeled multipotential neural precursor cells were transplanted into the ventricles of rats with acute experimental allergic encephalomyelitis (EAE) and high-resolution (microscopic) MR images were obtained ex vivo. Migration patterns of live cells into periventricular white matter structures could be easily visualized, with a good correlation of the corresponding histopathology. The present results confirm that MR cell tracking can be used to guide the development of successful transplantation protocols.

Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis.

Transplanted neural precursor cells remyelinate efficiently acutely demyelinated focal lesions. However, the clinical value of cell transplantation in a chronic, multifocal disease like multiple sclerosis will depend on the ability of transplanted cells to migrate to the multiple disease foci in the brain. Here, we expanded newborn rat neural precursor cells in spheres and transplanted them intracerebroventricularly or intrathecally in rats. The cells were labeled by the nuclear fluorescent dye Hoechst or by incubation with BrdU to enable their identification at 2 days and 2 weeks after transplantation, respectively. Spheres consisted of PSA-NCAM(+), nestin(+), NG2(-) undifferentiated precursor cells that differentiated in vitro into astrocytes, oligodendrocytes, and neurons. Spheres that were transplanted into intact rats remained mostly in the ventricles or in the spinal subarachnoid space. Following transplantation at peak of experimental autoimmune encephalomyelitis, cells migrated into the brain or spinal cord parenchyma, exclusively into inflamed white matter but not into adjacent gray matter regions. After 2 weeks, many transplanted cells had migrated into distant white matter tracts and acquired specific markers of the astroglial and oligodendroglial lineages. Thus, the inflammatory process may attract targeted migration of transplanted precursor cells into the brain parenchyma.