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6-aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray matter astrocytes mainly in the brainstem. We have examined the role of interleukin-6 (IL-6) in this degenerative process by using transgenic mice with astrocyte-targeted IL-6 expression (GFAP-IL6 mice). This study demonstrates that transgenic IL-6 expression significantly increases the 6-AN-induced inflammatory response of reactive astrocytes, microglia/macrophages, and lymphocytes in the brainstem. Also, IL-6 induced significant increases in proinflammatory cytokines IL-1, IL-12, and tumor necrosis factor-alpha as well as growth factors basic fibroblast growth factor (bFGF), transforming growth factor-beta, neurotrophin-3, angiopoietin, vascular endothelial growth factor, and the receptor for bFGF. In accordance, angiogenesis was increased in GFAP-IL6 mice relative to controls after 6-AN. Moreover, oxidative stress and apoptotic cell death were significantly reduced by transgenic IL-6 expression. IL-6 is also a major inducer in the CNS of metallothionein I and II (MT-I+II), which were significantly increased in the GFAP-IL6 mice. MT-I+II are antioxidants and neuroregenerative factors in the CNS, so increased MT-I+II levels in GFAP-IL6 mice could contribute to the reduction of oxidative stress and cell death in these mice.

The effect of CNS-targeted IL-6 gene expression has been thoroughly investigated in the otherwise nonperturbed brain but not following brain injury. Here we examined the impact of astrocyte-targeted IL-6 production in a traumatic brain injury (cryolesion) model using GFAP-IL6 transgenic mice. This study demonstrated that transgenic IL-6 production significantly increased wound healing following the cryolesion. Thus, at 20 days postlesion (dpl) the GFAP-IL6 mice showed almost complete wound healing compared to litter mate nontransgenic controls. It seems likely that a reduced inflammatory response in the long term could be responsible for this IL-6-related effect. Thus, while in the acute phase following cryolesion (1-6 dpl) the recruitment of macrophages and T lymphocytes was higher in GFAP-IL6 mice, at 10-20 dpl it was significantly reduced compared to controls. Reactive astrogliosis was also significantly increased up to but not including 20 dpl in the GFAP-IL6 mice. Oxidative stress as well as apoptotic cell death was significantly decreased throughout the time period studied in the GFAP-IL6 mice compared to controls. This could be linked to the altered inflammatory response as well as to the transgenic IL-6-induced increase of the antioxidant, neuroprotective proteins metallothionein-I + II. These results indicate that although in the brain the chronic astrocyte-targeted expression of IL-6 spontaneously induces an inflammatory response causing significant damage, during an acute neuropathological insult such as following traumatic injury, a clear neuroprotective role is evident.

We evaluated the physiological relevance of metallothionein-III (MT-III) in the central nervous system following damage caused by a focal cryolesion onto the cortex by studying Mt3 -null mice. In normal mice, dramatic astrogliosis and microgliosis and T-cell infiltration were observed in the area surrounding the lesioned tissue, along with signs of increased oxidative stress and apoptosis. There was also significant upregulation of cytokines/growth factors such as tumor necrosis factor-α, interleukin (IL)-1 α/β, and IL-6 as measured by ribonuclease protection assay. Mt3 -null mice did not differ from control mice in these responses, in sharp contrast to results obtained in Mt1- Mt2 -null mice. In contrast, Mt3 -null mice showed increased expression of several neurotrophins as well as of the neuronal sprouting factor GAP-43. Thus, unlike MT-I and MT-II, MT-III does not affect the inflammatory response elicited in the central nervous system by a cryoinjury, nor does it serve an important antioxidant role, but it may influence neuronal regeneration during the recovery process.

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.

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) causes significant damage and alters the expression of many genes, including a dramatic upregulation of metallothionein-I (MT-I). The findings in this report support the idea that the upregulation of MT-I observed in GFAP-IL6 mice is an important mechanism for coping with brain damage. Thus, GFAP-IL6 mice that were crossed with TgMTI transgenic mice (GFAP-IL6xTgMTI) and overexpressed MT-I in the brain showed a decreased upregulation of cytokines such as IL-6 and a diminished recruitment and activation of macrophages and T cells throughout the CNS but mainly in the cerebellum. The GFAP-IL6 mice showed clear evidence of increased oxidative stress, which was significantly decreased by MT-I overexpression. Interestingly, MT-I overexpression increased angiogenesis in GFAP-IL6 mice but not in control littermates. Overall, the results strongly suggest that MT-I+II proteins are valuable factors that protect against cytokine-induced CNS injury.

6-Aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray matter astrocytes. Metallothionein 1+2 (MT-1+2) are neuroprotective factors in the central nervous system (CNS), and to determine the roles for MT after 6-AN, we have examined transgenic mice overexpressing MT-1 (TgMTI* mice) after an i.p. injection with 6-AN. In control mice injected with 6-AN, astrocytes in specific gray matter areas of the brainstem showed degeneration. Reactive astrocytes surrounded the degenerated areas, which were heavily infiltrated by macrophages and T lymphocytes. MT-1+2 expression was significantly decreased in the damaged brainstem areas, but it increased in reactive astrocytes surrounding these areas and also in infiltrating macrophages. The levels of oxidative stress, as determined by immunoreactivity for inducible nitric-oxide synthase (iNOS), malondialdehyde (MDA), and nitrotyrosine (NITT), and the number of terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate [dUTP]-digoxigenin nick end labeling–positive (TUNEL+), caspase-3+ apoptotic cells were significantly increased in the brainstem of normal mice after 6-AN. In the TgMTI* mice, the 6-AN–induced tissue damage was decreased in comparison to control mice, and they showed significantly reduced numbers of recruited macrophages and T lymphocytes, and a drastic reduction of oxidative stress and apoptotic cell death. In addition, the accompanying reactive astrogliosis was increased in the transgenic mice. To further study the potential protective role of MT, we administered intraperitoneally Zn-MT-2 to 6-AN–injected normal mice and found essentially the same results as those obtained in TgMTI* mice. Thus, we hereby report that endogenous MT-1 overexpression and exogenous MT-2 treatment have significant neuroprotective roles during CNS pathological conditions. J. Comp. Neurol. 444:174–189, 2002. © 2002 Wiley-Liss, Inc.