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Cryolesion of the frontoparietal cortex in mice is a well-described brain injury paradigm that results in increased astrogliosis surrounding the lesion site and is accompanied by a prominent increase in the MAO-B levels in astrocytes. Whether these increased MAO-B levels contribute to cellular damage or modulate reactive astrocytosis remains unclear. MAO-B activity may contribute to cellular damage, since its metabolism products are highly toxic to the cells. Additionally, it has been suggested that MAO-B inhibition may regulate astrocytic reaction. In this study, we have determined the relative contribution of MAO-B activity to the outcome following freeze injury. Freeze injury induced a prominent increase of several inflammatory markers, including ICAM, Mac-1, EB22, and GFAP. Inhibition of MAO-B activity using the selective inhibitor PF9601N did not reduce this cryolesion-induced inflammatory response. Additional data revealed that the expression of several cryolesion-induced cell death genes, such as Fas, Rip, p53, and ICE, was not reduced in PF9601N-treated mice, evidencing that MAO-B activity did not contribute to cryolesion-induced cell death. Definitive functional analysis of the mice using the ladder beam task revealed that MAO-B inhibition did not improve the cryolesion-induced motor impairment. These data strongly suggest that, although MAO-B is highly expressed in the area surrounding the lesion site, its activity does not contribute to the cellular damage or play any role in regulating astrocytic reactivity.

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.

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.

Cytokines, such as tumour necrosis factor (TNF)-α and lymphotoxin-α, have been described widely to play important roles in the brain in physiologic conditions and after traumatic injury. However, the exact mechanisms involved in their function have not been fully elucidated. We give some insight on their role by using animals lacking either Type 1 receptor (TNFR1KO) or Type 2 (TNFR2KO) and their controls (C57Bl/6). Both TNFR1KO and to a greater extent TNFR2KO mice showed increased exploration/activity neurobehavioral traits in the hole board test, such as rearings, head dippings, and ambulations, compared with wild-type mice, suggesting an inhibitory role of TNFR1/TNFR2 signaling. In contrast, no significant differences were observed in the elevated plus maze test, ruling out a major role of these receptors in the control of anxiety. We next evaluated the response to a freeze injury to the somatosensorial cortex. The effect of the cryolesion on motor function was evaluated with the horizontal ladder beam test, and the results showed that both TNFR1KO and TNFR2KO mice made fewer errors, suggesting a detrimental role for TNFR1/TNFR2 signaling for coping with brain damage. Expression of ∼22600 genes was analyzed using an Affymetrix chip (MOE430A) at 0 (unlesioned), 1, or 4 days post-lesion in the three strains. The results show a unique and major role of both TNF receptors on the pattern of gene expression elicited by the injury but also in normal conditions, and suggest that blocking of TNFR1/TNFR2 receptors may be beneficial after a traumatic brain injury. © 2006 Wiley-Liss, Inc.

Traumatic injury to the brain is one of the leading causes of injury-related death or disability, especially among young people. Inflammatory processes and oxidative stress likely underlie much of the damage elicited by injury, but the full repertoire of responses involved is not well known. A genomic approach, such as the use of microarrays, provides much insight in this regard, especially if combined with the use of gene-targeted animals. We report here the results of one of these studies comparing wild-type and metallothionein-I + II knockout mice subjected to a cryolesion of the somatosensorial cortex and killed at 0, 1, 4, 8, and 16 days postlesion (dpl) using Affymetrix genechips/oligonucleotide arrays interrogating approximately 10,000 different murine genes (MG_U74Av2). Hierarchical clustering analysis of these genes readily shows an orderly pattern of gene responses at specific times consistent with the processes involved in the initial tissue injury and later regeneration of the parenchyma, as well as a prominent effect of MT-I + II deficiency. The results thoroughly confirmed the importance of the antioxidant proteins MT-I + II in the response of the brain to injury and opened new avenues that were confirmed by immunohistochemistry. Data in KO, MT-I-overexpressing, and MT-II-injected mice strongly suggest a role of these proteins in postlesional activation of neural stem cells.

Brain injury and neuroinflammation are pathophysiologic contributors to acute and chronic neurologic disorders, which are progressive diseases not fully understood. Mammalian metallothioneins I and II (MT-I&II) have significant neuroprotective functions, but the precise mechanisms underlying these effects are still unknown. To gain insight in this regard, we have evaluated whether a distant, most likely single-domain MT (Drosophila MTN) functions similarly to mammalian MT-I&II (recombinant mouse MT-I and human MT-IIa and native rabbit MT-II) after cryogenic injury to the cortex in Mt1&2 KO mice. All the recombinant proteins showed similar neuroprotective properties to native MT-II, significantly reducing brain inflammation (macrophages, T cells, and pro-inflammatory cytokines), oxidative stress, neurodegeneration, and apoptosis. These results in principle do not support specific protein–protein interactions as the mechanism underlying the neuroprotective effects of these proteins because a non-homologous and structurally unrelated MT such as Drosophila MTN functions similarly to mammalian MTs. We have also evaluated for the first time the neurobiologic effects of exogenous MT-III, a major CNS MT isoform. Human rMT-III, in contrast to human nMT-IIa, did not affect inflammation, oxidative stress, and apoptosis, and showed opposite effects on several growth factors, neurotrophins, and markers of synaptic growth and plasticity. Our data thus highlight specific and divergent roles of exogenous MT-III vs. the MT-I&II isoforms that are consistent with those attributed to the endogenous proteins, and confirm the suitability of recombinant synthesis for future therapeutic use that may become relevant to clinical neurology. © 2006 Wiley-Liss, Inc.

Tumor necrosis factor-alpha (TNF-alpha) is one of the mediators dramatically increased after traumatic brain injury that leads to the activation, proliferation, and hypertrophy of mononuclear, phagocytic cells and gliosis. Eventually, TNF-alpha can induce both apoptosis and necrosis via intracellular signaling. This cytokine exerts its functions via interaction with two receptors: type-1 receptor (TNFR1) and type-2 receptor (TNFR2). In this work, the inflammatory response after a freeze injury (cryolesion) in the cortex was studied in wild-type (WT) animals and in mice lacking TNFR1 (TNFR1 KO) or TNFR2 (TNFR2 KO). Lack of TNFR1, but not of TNFR2, significantly decreased the inflammatory response and tissue damage elicited by the cryolesion at both 3 and 7 days postlesion, with decreased gliosis, lower IL-1beta immunostaining, and a reduction of apoptosis markers. Cryolesion produced a clear induction of the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6, and TNF-alpha; this induction was significantly lower in the TNFR1 KO mice. Host response genes (ICAM-1, A20, EB22/5, and GFAP) were also induced by the cryolesion, but to a lesser extent in TNFR1 KO mice. Lack of TNFR1 signaling also affected the expression of apoptosis/cell death-related genes (Fas, Rip, p53), matrix metalloproteinases (MMP3, MMP9, MMP12), and their inhibitors (TIMP1), suggesting a role of TNFR1 in extracellular matrix remodeling after injury. However, GDNF, NGF, and BDNF expression were not affected by TNFR1 deficiency. Overall, these results suggest that TNFR1 is involved in the early establishment of the inflammatory response and that its deficiency causes a decreased inflammatory response and tissue damage following brain injury.

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.