metallothionein and Encephalitis

Neurotoxic effects of the environmentally abundant mycotoxin Ochratoxin A (OTA) were studied in histotypic 3D rat brain cell cultures, comprising all brain cell types. Cultures were exposed to nanomolar OTA concentrations and samples were collected 48h after a single exposure, or after 10 days of repeated administration. OTA-induced changes in gene- and protein expression, as well as alterations in cell morphology were assessed. Forty-eight-hour OTA exposure resulted in a disruption of the neuronal cytoskeleton and reduced expression of several oligodendrocyte-specific markers indicative of demyelination. Astrocyte disturbances were revealed by a decrease in two astrocytic proteins involved in regulation of inflammatory responses, metallothioneins I and II. Repeated OTA administration induced a neuroinflammatory response, as visualized by an increase of isolectin B4 labelled cells, increased expression of pro-inflammatory cytokines, and detection of macrophagic ED1/CD68 positive cells, as well as an upregulation of neurodegenerative M1 microglial phenotype markers. Partial recovery from OTA-induced deleterious effects on oligodendrocytes and astrocytes was achieved by co-treatment with sonic hedgehog (SHH). In addition, metallothionein I and II co-treatment partially restored OTA-induced effects on oligodendrocytes after 48h, and modulated microglial reactivity after 10 days. These results suggest that OTA-exposure affects Shh-signalling, which in turn may influence both oligodendrocytes and astrocytes. Furthermore, the primarily astrocytic proteins MTI/MTII may affect microglial activation. Thus the neuroinflammatory response appears to be downstream of OTA-induced effects on demyelination, axonal instabilities and astrocytes disturbances. In conclusion, repeated OTA-exposure induced a secondary neuroinflammatory response characterized by neurodegenerative M1 microglial activation and pro-inflammatory response that could exacerbate the neurodegenerative process.

Topics: Animals; Brain; Cells, Cultured; Encephalitis; Metallothionein; Microglia; Neuroglia; Ochratoxins; Phenotype; Rats; Rats, Sprague-Dawley

The distribution of metallothioneins I/II in the mouse brain and their specific area distribution upon physical stress were studied. To induce physical stress, groups of mice were subjected to total darkness for different periods (2 weeks, 1 month, and 2 months). The concentration of metallothioneins, evaluated by immunohistochemistry, as well as area-specific protein expression, were found in the following quantitative order: corpus striatum, cerebellum, mesencephalon, hippocampus with fornix, parts of thalamus, and pons. All other brain areas were marginally affected, or even unaffected, in terms of immunopositive metallothionein reaction. Metallothionein I/II expression was compared with the immunopositivity of glial fibrillary acidic protein (GFAP). It is noteworthy that metallothioneins and GFAP are expressed in different types of astrocytes.

Topics: Animals; Astrocytes; Brain; Circadian Rhythm; Copper; Darkness; Encephalitis; Glial Fibrillary Acidic Protein; Gliosis; Male; Metallothionein; Mice; Reaction Time; Stress, Physiological; Up-Regulation; Zinc

Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-IL6 mice) induces significant inflammation and neurodegeneration but also affords neuroprotection against acute traumatic brain injury. This neuroprotection is likely mediated by the IL-6-induced protective factors metallothioneins-I and -II (MT-I+II). Here we evaluate the neuroprotective roles of IL-6 vs. MT-I+II during 6-aminonicotinamide (6-AN)-induced neurotoxicity, by using GFAP-IL6 mice and transgenic mice overexpressing MT-I (TgMT) as well as GFAP-IL6 mice crossed with TgMT mice (GFAP-IL6 x TgMT). 6-AN caused acute damage of brainstem gray matter areas identified by necrosis of astrocytes, followed by inflammatory responses. After 6-AN-induced toxicity, secondary damage was observed, consisting of oxidative stress, neurodegeneration, and apoptotic cell death. We hereby show that the primary injury caused by 6-AN was comparable in wild-type and GFAP-IL6 mice, but MT-I overexpression could significantly protect the brain tissue. As expected, GFAP-IL6 mice showed increased CNS inflammation with more gliosis, macrophages, and lymphocytes, including increased cytokine expression, relative to the other mice. However, GFAP-IL6 mice showed reduced oxidative stress (judged from nitrotyrosine, malondialdehyde, and 8-oxoguanine stainings), neurodegeneration (accumulation of neurofibrillary tangles), and apoptosis (determined from TUNEL and caspase-3). MT-I+II expression was significantly higher in GFAP-IL6 mice than in wild types, which may contribute to the IL-6-induced neuroprotection. In support of this, overexpression of MT-I in GFAP-IL6 x TgMT as well as TgMT mice protected the brainstem tissue significantly from 6-AN-induced toxicity and secondary brain tissue damage. Overall, the results demonstrate that brain MT-I+II proteins are fundamental neuroprotective factors, which in the future may become therapeutic agents.

Topics: 6-Aminonicotinamide; Animals; Apoptosis; Brain Stem; Caspase 3; Caspases; Crosses, Genetic; Disease Models, Animal; Down-Regulation; Encephalitis; Free Radicals; Glial Fibrillary Acidic Protein; Gliosis; Interleukin-6; Metallothionein; Mice; Mice, Transgenic; Nerve Degeneration; Neurofibrillary Tangles; Neuroglia; Neuroprotective Agents; Neurotoxins; Oxidative Stress; Promoter Regions, Genetic

Transgenic expression of IL-6 in the CNS under the control of the GFAP gene promoter, glial fibrillary acidic protein-interleukin-6 (GFAP-IL-6) mice, raises an inflammatory response and causes significant brain damage. However, the results obtained in the GFAP-IL-6 mice after a traumatic brain injury, such as a cryolesion, demonstrate a neuroprotective role of IL-6. Thus, the GFAP-IL-6 mice showed faster tissue repair and decreased oxidative stress and apoptosis compared with control litter-mate mice. The neuroprotective factors metallothionein-I+II (MT-I+II) were upregulated by the cryolesion to a higher extent in the GFAP-IL-6 mice, suggesting that they could be related to the neuroprotection afforded by the transgenic expression of IL-6. To examine this possibility, we have crossed GFAP-IL-6 mice with transgenic mice overexpressing MT-I (TgMT), producing double transgenic GFAP-IL-6 TgMT mice. The results obtained after cryolesion in GFAP-IL-6 TgMT mice, as well as in TgMT mice, consistently supported the idea that the increased MT-I+II levels observed in GFAP-IL-6 mice are a fundamental and important mechanism for coping with brain damage. Accordingly, MT-I overexpression regulated the inflammatory response, decreased oxidative stress and apoptosis significantly, and increased brain tissue repair in comparison with either GFAP-IL-6 or control litter-mate mice. Overall, the results demonstrate that brain MT-I+II proteins are fundamental neuroprotective factors.

Topics: Animals; Apoptosis; Astrocytes; Brain; Brain Injuries; Down-Regulation; Encephalitis; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Gliosis; Immunohistochemistry; Interleukin-6; Male; Metallothionein; Mice; Mice, Transgenic; Nerve Regeneration; Oxidative Stress; Promoter Regions, Genetic; Tumor Necrosis Factor-alpha

6-Aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray-matter astrocytes followed by a vigorous inflammatory response. Macrophage colony stimulating factor (M-CSF) is important during inflammation, and in order to further clarify the roles for M-CSF in neurodegeneration and brain cell death, we have examined the effect of 6-AN on osteopetrotic mice with genetic M-CSF deficiency (op/op mice). The 6-AN-induced degeneration of gray-matter areas was comparable in control and op/op mice, but the numbers of reactive astrocytes, macrophages, and lymphocytes in the damaged areas were significantly decreased in op/op mice relative to controls. The levels of oxidative stress (as determined by using immunoreactivity for inducible nitric oxide synthase, nitrotyrosine, and malondialdehyde) and apoptotic cell death (as determined by using TUNEL and immunoreactivity for caspases and cytochrome c) were significantly increased in 6-AN-injected op/op mice relative to controls. From a number of antioxidant factors assayed, only metallothioneins I and II (MT-I+II) were decreased in op/op mice in comparison to controls. Thus, the present results indicate that M-CSF is an important growth factor for coping with 6-AN-induced central nervous system damage and suggest that MT-I+II are likely to have a significant role.

Topics: 6-Aminonicotinamide; Animals; Antioxidants; Apoptosis; Astrocytes; Brain Stem; Caspases; Encephalitis; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Homozygote; In Situ Nick-End Labeling; Lymphocytes; Macrophage Colony-Stimulating Factor; Macrophages; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microglia; Niacin; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress

We have evaluated the physiological relevance of metallothionein-1+2 (MT-1+2) in the CNS following damage caused by a focal cryolesion onto the cortex. In comparison to normal mice, transgenic mice overexpressing the MT-1 isoform (TgMTI* mice) showed a significant decrease of the number of activated microglia/macrophage and of CD3+ T lymphocytes in the area surrounding the lesion, while astrocytosis was increased. The TgMTI* mice showed a diminished peripheral macrophage but not CD3 T cell response to the cryolesion. This altered inflammatory response produced a decreased expression of the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha and an increased expression of the growth factors bFGF, TGFbeta1, and VEGF in the TgMTI* mice relative to control mice, which might be related to the increased angiogenesis and regeneration of the parenchyma of the former mice. The overexpression of MT-1 dramatically reduced the cryolesion-induced oxidative stress and neuronal apoptosis. Remarkably, these effects were also obtained by the intraperitoneal administration of MT-2 to both normal and MT-1+2 knock-out mice. These results fully support the notion that MT-1+2 are essential in the CNS for coping with focal brain injury and suggest a potential therapeutic use of these proteins.

Topics: Animals; Apoptosis; Astrocytes; Brain; Brain Injuries; Cytokines; Cytoprotection; Disease Progression; Encephalitis; Freezing; Gene Expression Regulation; Growth Substances; Immunohistochemistry; In Situ Hybridization; Injections, Intraperitoneal; Macrophages; Metallothionein; Mice; Mice, Knockout; Mice, Transgenic; Microglia; Neovascularization, Physiologic; Oxidative Stress; Protein Isoforms; T-Lymphocytes

The role of interleukin-6 in hippocampal tissue damage after injection with kainic acid, a rigid glutamate analogue inducing epileptic seizures, has been studied by means of interleukin-6 null mice. At 35mg/kg, kainic acid induced convulsions in both control (75%) and interleukin-6 null (100%) mice, and caused a significant mortality (62%) only in the latter mice, indicating that interleukin-6 deficiency increased the susceptibility to kainic acid-induced brain damage. To compare the histopathological damage caused to the brain, control and interleukin-6 null mice were administered 8.75mg/kg kainic acid and were killed six days later. Morphological damage to the hippocampal field CA1-CA3 was seen after kainic acid treatment. Reactive astrogliosis and microgliosis were prominent in kainic acid-injected normal mice hippocampus, and clear signs of increased oxidative stress were evident. Thus, the immunoreactivity for inducible nitric oxide synthase, peroxynitrite-induced nitration of proteins and byproducts of fatty acid peroxidation were dramatically increased, as was that for metallothionein I+II, Mn-superoxide dismutase and Cu/Zn-superoxide dismutase. In accordance, a significant neuronal apoptosis was caused by kainic acid, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling and interleukin-1beta converting enzyme/Caspase-1 stainings. In kainic acid-injected interleukin-6 null mice, reactive astrogliosis and microgliosis were reduced, while morphological hippocampal damage, oxidative stress and apoptotic neuronal death were increased. Since metallothionein-I+II levels were lower, and those of inducible nitric oxide synthase higher, these concomitant changes are likely to contribute to the observed increased oxidative stress and neuronal death in the interleukin-6 null mice. The present results demonstrate that interleukin-6 deficiency increases neuronal injury and impairs the inflammatory response after kainic acid-induced seizures.

Topics: Animals; Apoptosis; Blood-Brain Barrier; Encephalitis; Epilepsy; Excitatory Amino Acid Agonists; Gene Expression; Gliosis; Hippocampus; In Situ Nick-End Labeling; Interleukin-6; Kainic Acid; Macrophages; Metallothionein; Mice; Mice, Knockout; Microglia; Nerve Degeneration; Oxidative Stress; Seizures; Superoxide Dismutase

The role of zinc- and copper-deficient diets on the inflammatory response to traumatic brain injury (TBI) has been evaluated in adult rats. As expected, zinc deficiency decreased food intake and body weight gain, and the latter effect was higher than that observed in pair-fed rats. In noninjured brains, zinc deficiency only affected significantly lectin (increasing) and glial fibrillary acidic protein (GFAP) and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (decreasing) immunoreactivities (irs). In injured brains, a profound gliosis was observed in the area surrounding the lesion, along with severe damage to neurons as indicated by neuron specific enolase (NSE) ir, and the number of cells undergoing apoptosis (measured by TUNEL) was dramatically increased. Zinc deficiency significantly altered brain response to TBI, potentiating the microgliosis and reducing the astrogliosis, while increasing the number of apoptotic cells. Metallothioneins (MTs) are important zinc- and copper-binding proteins in the CNS, which could influence significantly the brain response to TBI because of their putative roles in metal homeostasis and antioxidant defenses. MT-I+II expression was dramatically increased by TBI, and this response was significantly blunted by zinc deficiency. The MT-III isoform was moderately increased by both TBI and zinc deficiency. TBI strongly increased oxidative stress levels, as demonstrated by malondialdehyde (MDA), protein tyrosine nitration (NITT), and nuclear factor kappaB (NF-kappaB) levels irs, all of which were potentiated by zinc deficiency. Further analysis revealed unbalanced expression of prooxidant and antioxidant proteins besides MT, since the levels of inducible nitric oxide synthase (iNOS) and Cu,Zn-SOD were increased and decreased, respectively, by zinc deficiency. All these effects were attributable to zinc deficiency, since pair-fed rats did not differ from normally fed rats. In general, copper deficiency caused a similar pattern of responses, albeit more moderate. Results obtained in mice with a null mutation for the MT-I+II isoforms strongly suggest that most of the effects observed in the rat brain after zinc and copper deficiencies are attributable to the concomitant changes in the MT expression.

Topics: Animals; Brain; Brain Injuries; Copper; Diet; Eating; Encephalitis; Male; Metallothionein; Neuroglia; Neurons; Protein Isoforms; Rats; Rats, Sprague-Dawley; Weight Gain; Zinc

We examined the effects of interleukin-6 (IL-6) deficiency on brain inflammation and the accompanying bone marrow (BM) leukopoiesis and spleen immune reaction after systemic administration of a niacin antagonist, 6-aminonicotinamide (6-AN), which causes both astroglial degeneration/cell death in brain stem gray matter areas and BM toxicity. In both normal and genetically IL-6-deficient mice (IL-6 knockout (IL-6KO) mice), the extent of astroglial degeneration/cell death in the brain stem was similar as determined from disappearance of GFAP immunoreactivity. In 6-AN-injected normal mice reactive astrocytosis encircled gray matter areas containing astroglial degeneration/cell death, which were infiltrated by several macrophages and some T-lymphocytes. Reactive astrocytes and a few macrophages increased significantly the antioxidants metallothionein-I+II (MT-I+II) and moderately the MT-III isoform. In 6-AN-injected IL-6KO mice reactive astrocytosis and recruitment of macrophages and T-lymphocytes were clearly reduced, as were BM leukopoiesis and spleen immune reaction. Expression of MT-I+II was significantly reduced while MT-III was increased. Oxidative stress, as determined by measuring nitrated tyrosine and malondialdehyde, was increased by 6-AN to a greater extent in IL-6KO mice. The blood-brain barrier to albumin was only disrupted in 6-AN-injected normal mice, which likely is due to the substantial migration of blood-derived inflammatory cells into the CNS. The present results demonstrate that inflammation in CNS is clearly reduced during IL-6 deficiency and this effect is likely due to significant inhibition of BM leukopoiesis. We also show that IL-6 deficiency reduces the levels of neuroprotective antioxidants MT-I+II followed by an increased oxidative stress during CNS inflammation.

Topics: 6-Aminonicotinamide; Animals; Antigens, Differentiation; Astrocytes; Blood-Brain Barrier; Bone Marrow Cells; Brain Stem; Encephalitis; Glial Fibrillary Acidic Protein; Hematopoiesis; Immunohistochemistry; Interleukin-6; Macrophages; Malondialdehyde; Metallothionein; Metallothionein 3; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Nerve Tissue Proteins; Oxidative Stress; Protein Isoforms; Serum Albumin; T-Lymphocytes; Tyrosine

To study the importance of metallothionein-I and -II (MT-I+II) for brain inflammation and regeneration, the authors examined normal and MT-I+II knock-out (MT-KO) mice subjected to a cortical freeze injury. Normal mice showed profound neurodegeneration, inflammation, and gliosis around the injury, which was repaired by 20 days postlesion (dpl). However, in MT-KO mice the lesion-associated inflammation was still present as late as 90 dpl. Scanning electron microscopy demonstrated that the number of capillaries was lower, and ultrastructural preservation of the lesioned parenchyma was poorer in MT-KO mice, suggesting an altered angiogenesis. To gain insight into the mechanisms involved, a number of cytokines and growth factors were evaluated. The number of cells expressing the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha was higher in MT-KO mice than in normal mice, which was confirmed by RNase protection analysis, whereas the number of cells expressing the growth factors bFGF, TGFbeta1, VEGF, and NT-3 was lower. Increased expression of proinflammatory cytokines could be involved in the sustained recruitment of CD-14+ and CD-34+ inflammatory cells and their altered functions observed in MT-KO mice. Decreases in trophic factors bFGF, TGFbeta1, and VEGF could mediate the decreased angiogenesis and regeneration observed in MT-KO mice after the freeze lesion. A role for MT-I+II in angiogenesis was also observed in transgenic mice expressing IL-6 under the control of the promoter of glial fibrillary acidic protein gene (GFAP-IL6 mice) because MT-I+II deficiency dramatically decreased the IL-6-induced angiogenesis of the GFAP-IL6 mice. In situ hybridization analysis indicated that the MT-III expression was not altered by MT-I+II deficiency. These results suggest that the MT-I+II isoforms have major regulatory functions in the brain inflammatory response to injury, especially in the angiogenesis process.

Topics: Animals; Brain; Capillaries; Cerebrovascular Circulation; Cytokines; Encephalitis; Freezing; Growth Substances; Macrophages; Metallothionein; Mice; Mice, Knockout; Microscopy, Electron; Neovascularization, Physiologic; Protein Isoforms; Reference Values; Stem Cells

In order to determine the role of the neuropoietic cytokine interleukin-6 (IL-6) during the first 3 weeks after a focal brain injury, we examined the inflammatory response, oxidative stress and neuronal survival in normal and interleukin-6-deficient (knockout, IL-6KO) mice subjected to a cortical freeze lesion. In normal mice, the brain injury was followed by reactive astrogliosis and recruitment of macrophages from 1 day postlesion (dpl), peaking at 3-10 dpl, and by 20 dpl the transient immunoreactions were decreased, and a glial scar was present. In IL-6KO mice, the reactive astrogliosis and recruitment of macrophages were decreased throughout the experimental period. The expression of the antioxidant and anti-apoptotic factors metallothionein I+II (MT-I+II) was increased prominently by the freeze lesion, but this response was significantly reduced in the IL-6 KO mice. By contrast, the expression of the antioxidants Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-SOD, and catalase remained unaffected by the IL-6 deficiency. The lesioned mice showed increased oxidative stress, as judged by malondialdehyde (MDA) and nitrotyrosine (NITT) levels and by formation of inducible nitric oxide synthase (iNOS). IL-6KO mice showed higher levels of MDA, NITT, and iNOS than did normal mice. Concomitantly, in IL-6KO mice the number of apoptotic neurons was significantly increased as judged by TUNEL staining, and regeneration of the tissue was delayed relative to normal mice. The changes in neuronal tissue damage and in brain regeneration observed in IL-6KO mice are likely caused by the IL-6-dependent decrease in MT-I+II expression, indicating IL-6 and MT-I+II as neuroprotective factors during brain injury.

Topics: Animals; Apoptosis; Astrocytes; Brain; Brain Injuries; Catalase; Encephalitis; In Situ Nick-End Labeling; Interleukin-6; Macrophages; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Degeneration; Nerve Regeneration; Neurons; Oxidative Stress; Superoxide Dismutase

Metallothioneins (MTs) are a family of proteins which in mammals is comprised of four isoforms (MT-I-IV). MT-I and MT-II are expressed in many tissues, whereas MT-III is expressed exclusively in the central nervous system (CNS). In contrast to the liver, the knowledge of the regulation of the different MT isoforms in the brain is scarce. A number of cytokines have been shown to be important regulators of MT synthesis in vivo and in vitro. In accordance with this concept, the i.p. administration of endotoxin, which elicits the release of cytokines not only in peripheral tissues but also in the brain, caused an overall increase of MT-I + II levels in the rat brain which was very significant in medulla + pons and cerebellum. Among the putative cytokines involved in endotoxin-elicited brain MT-I+II induction, interleukin-1 (IL-1) and interleukin-6 (IL-6) are likely candidates. These cytokines have a variety of effects in the brain, and they are major regulators of MT-I+II synthesis in tissues such as the liver. Here we show the administration of IL-1 and IL-6 into the third ventricle increased MT-I+II protein levels in specific brain areas in the rat. IL-1 tended to increase MT-I+II levels in all brain areas studied, but significantly in the striatum, hypothalamus, medulla + pons and cerebellum. The effect of IL-6 was more restricted, but a significant increase of MT-I+II levels was still observed in frontal cortex, hypothalamus and cerebellum. The results suggest that IL-1 and IL-6 are important regulators of brain MT-I+II and that these cytokines could mediate MT-I+II induction after an immunological insult.

Topics: Animals; Brain; Brain Injuries; Encephalitis; Endotoxins; Gene Expression Regulation; Injections, Intraventricular; Interleukin-1; Interleukin-6; Male; Metallothionein; Rats; Rats, Sprague-Dawley