We examined metallothionein (MT)-induced neuroprotection during kainic acid (KA)-induced excitotoxicity by studying transgenic mice with MT-I overexpression (TgMT mice). KA induces epileptic seizures and hippocampal excitotoxicity, followed by inflammation and delayed brain damage. We show for the first time that even though TgMT mice were more susceptible to KA, the cerebral MT-I overexpression decreases the hippocampal inflammation and delayed neuronal degeneration and cell death as measured 3 days after KA administration. Hence, the proinflammatory responses of microglia/macrophages and lymphocytes and their expression of interleukin (IL)-1, IL-6, IL-12, tumor necrosis factor-alpha and matrix metalloproteinases (MMP-3, MMP-9) were significantly reduced in hippocampi of TgMT mice relative to wild-type mice. Also by 3 days after KA, the TgMT mice showed significantly less delayed damage, such as oxidative stress (formation of nitrotyrosine, malondialdehyde, and 8-oxoguanine), neurodegeneration (neuronal accumulation of abnormal proteins), and apoptotic cell death (judged by TUNEL and activated caspase-3). This reduced bystander damage in TgMT mice could be due to antiinflammatory and antioxidant actions of MT-I but also to direct MT-I effects on the neurons, in that significant extracellular MT presence was detected. Furthermore, MT-I overexpression stimulated astroglia and increased immunostaining of antiinflammatory IL-10, growth factors, and neurotrophins (basic fibroblastic growth factor, transforming growth factor-beta, nerve growth factor, brain-derived neurotrophic factor, glial-derived neurotrophic factor) in hippocampus. Accordingly, MT-I has different functions that likely contribute to the increased neuron survival and improved CNS condition of TgMT mice. The data presented here add new insight into MT-induced neuroprotection and indicate that MT-I therapy could be used against neurological disorders.
Metallothioneins (MTs) are major zinc binding proteins in the CNS that could be involved in the control of zinc metabolism as well as in protection against oxidative stress. Mice lacking MT-I and MT-II (MT-I + II deficient) because of targeted gene inactivation were injected with kainic acid (KA), a potent convulsive agent, to examine the neurobiological importance of these MT isoforms. At 35 mg/kg KA, MT-I + II deficient male mice showed a higher number of convulsions and a longer convulsion time than control mice. Three days later, KA-injected mice showed gliosis and neuronal injury in the hippocampus. MT-I + II deficiency decreased both astrogliosis and microgliosis and potentiated neuronal injury and apoptosis as shown by terminal deoxynucleotidyl transferase-mediated in situ end labelling (TUNEL), detection of single stranded DNA (ssDNA) and by increased interleukin-1beta-converting enzyme (ICE) and caspase-3 levels. Histochemically reactive zinc in the hippocampus was increased by KA to a greater extent in MT-I + II-deficient compared with control mice. KA-induced seizures also caused increased oxidative stress, as suggested by the malondialdehyde (MDA) and protein tyrosine nitration (NITT) levels and by the expression of MT-I + II, nuclear factor-kappaB (NF-kappaB), and Cu/Zn-superoxide dismutase (Cu/Zn-SOD). MT-I + II deficiency potentiated the oxidative stress caused by KA. Both KA and MT-I + II deficiency significantly affected the expression of MT-III, granulocyte-macrophage colony stimulating factor (GM-CSF) and its receptor (GM-CSFr). The present results indicate MT-I + II as important for neuron survival during KA-induced seizures, and suggest that both impaired zinc regulation and compromised antioxidant activity contribute to the observed neuropathology of the MT-I + II-deficient mice.
Summary: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-1, IL-6, and TNF-␣ 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, TGF1, 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, TGF1, 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. Key Words: MT-I+II-MT-IIIInflammation-Angiogenesis-Regeneration-Cytokines.Metallothioneins are a family of cysteine-rich, ubiquitous intracellular proteins. In the central nervous system (CNS), MT-I+II are expressed coordinately and are increased in macrophages/microglia and astrocytes during various inflammatory and pathologic conditions, including human neurodegenerative disorders, experimentally induced brain injury, epileptic seizures, brain ischemia, astroglial cell death, metal exposure, stress, and myelin deficiency
Injury to the central nervous system (CNS) elicits an inflammatory response involving activation of microglia, brain macrophages, and astrocytes, processes likely mediated by the release of proinflammatory cytokines. In order to determine the role of interleukin‐6 (IL‐6) during the inflammatory response in the brain following disruption of the blood–brain barrier (BBB), we examined the effects of a focal cryo injury to the fronto‐parietal cortex in interleukin‐6‐deficient (IL‐6−/−) and normal (IL‐6+/+) mice. In IL‐6+/+ mice, brain injury resulted in the appearance of brain macrophages and reactive astrocytes surrounding the lesion site. In addition, expression of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and metallothionein‐I+II (MT‐I+II) were increased in these cells, while the brain‐specific MT‐III was only moderately upregulated. In IL‐6−/− mice, however, the response of brain macrophages and reactive astrocytes was markedly depressed and the number of NSE positive neurons was reduced. Brain damage‐induced GM‐CSF and MT‐I+II expression were also markedly depressed compared to IL‐6+/+ mice. In contrast, MT‐III immunoreactivity was markedly increased in brain macrophages and astrocytes. In situ hybridization analysis indicates that MT‐I+II but not MT‐III immunoreactivity reflect changes in the messenger levels. The number of cell divisions was similar in IL‐6+/+ and IL‐6−/− mice. The present results demonstrate that IL‐6 is crucial for the recruitment of myelo‐monocytes and activation of glial cells following brain injury with disrupted BBB. Furthermore, our results suggest IL‐6 is important for neuroprotection and the induction of GM‐CSF and MT expression. The opposing effect of IL‐6 on MT‐I+II and MT‐III levels in the damaged brain suggests MT isoform‐specific functions. GLIA 25:343–357, 1999. © 1999 Wiley‐Liss, Inc.
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