metallothionein and Brain-Injuries

Metallothionein (MT) is a kind of metal binding protein. As an important member in metallothionein family, MT-I/II regulates metabolism and detoxication of brain metal ion and scavenges free radicals. It is capable of anti-inflammatory response and anti-oxidative stress so as to protect the brain tissue. During the repair process of brain injury, the latest study showed that MT-I/II could stimulate brain anti-inflammatory factors, growth factors, neurotrophic factors and the expression of the receptor, and promote the extension of axon of neuron, which makes contribution to the regeneration of neuron and has important effect on the recovery of brain injury. Based on the findings, this article reviews the structure, expression, distribution, adjustion, function, mechanism in the repair of brain injury of MT-I/II and its application prospect in forensic medicine. It could provide a new approach for the design and manufacture of brain injury drugs as well as for age estimation of the brain injury.

Topics: Animals; Astrocytes; Brain; Brain Injuries; Cytokines; Forensic Medicine; Gene Expression Regulation; Humans; Metallothionein; Neurons; Neuroprotective Agents; Oxidative Stress

In traumatic brain injury (TBI), the primary, irreversible damage associated with the moment of impact consists of cells dying from necrosis. This contributes to fuelling a chronic central nervous system (CNS) inflammation with increased formation of proinflammatory cytokines, enzymes and reactive oxygen species (ROS). ROS promote oxidative stress, which leads to neurodegeneration and ultimately results in programmed cell death (secondary injury). Since this delayed, secondary tissue loss occurs days to months following the primary injury it provides a therapeutic window where potential neuroprotective treatment could alleviate ongoing neurodegeneration, cell death and neurological impairment following TBI. Various neuroprotective drug candidates have been described, tested and proven effective in pre-clinical studies, including glutamate receptor antagonists, calcium-channel blockers, and caspase inhibitors. However, most of the scientific efforts have failed in translating the experimental results into clinical trials. Despite intensive research, effective neuroprotective therapies are lacking in the clinic, and TBI continues to be a major cause of morbidity and mortality. This paper provides an overview of the TBI pathophysiology leading to cell death and neurological impairment. We also discuss endogenously expressed neuroprotectants and drug candidates, which at this stage may still hold the potential for treating brain injured patients.

Topics: Brain Injuries; Cell Death; Humans; Metallothionein; Neuroprotective Agents

There is a large body of evidence demonstrating that metallothioneins (MTs) expressed in astrocytes following CNS injury, exhibit both neuroprotective and neuroregenerative properties and are critical for recovery outcomes. As these proteins lack signal peptides, and have well characterized free radical scavenging and heavy metal binding properties, the neuroprotective functions of MTs have been attributed to these intracellular roles. However, there is an increasing realization that the neuroprotective functions of MTs may also involve an extracellular component. In this issue of Journal of Neurochemistry, Ambjørn et al. reveal considerable insight into this novel function of MTs. In this review, we examine the seminal work of Ambjørn et al. in the context of our current understanding of the role of MT in astrocyte-neuron interactions in the injured brain, and also discuss the significant therapeutic potential of their work.

Topics: Animals; Astrocytes; Brain Injuries; Humans; Metallothionein; Models, Biological; Neuroprotective Agents; Signal Transduction

Topics: 6-Aminonicotinamide; Animals; Brain; Brain Diseases; Brain Injuries; Central Nervous System; Central Nervous System Diseases; DNA Fragmentation; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Inflammation Mediators; Lymphocyte Activation; Macrophages; Metallothionein; Necrosis; Neovascularization, Pathologic; Neurofibrillary Tangles; Oxidative Stress; Tumor Necrosis Factor-alpha

For many years, research focus on metallothioneins, small zinc binding proteins found predominantly within astrocytes in the brain, has centred on their ability to indirectly protect neurons from oxygen free radicals and heavy metal-induced neurotoxicity. However, in recent years it has been demonstrated that these proteins have previously unsuspected roles within the cellular response to brain injury. The aim of this commentary is to provide an overview of the exciting recent experimental evidence from several laboratories including our own suggesting a possible extracellular role for these proteins, and to present a hypothetical model explaining the newly identified function of extracellular metallothioneins in CNS injury and repair.

Topics: Animals; Brain Injuries; Extracellular Fluid; Humans; Metallothionein

Metallothioneins (MTs) are small cysteine-rich proteins which are found widely throughout the mammalian body, including the CNS. There are extensive data on the structure and expression of MTs, and many basic properties pertinent to MT biology in the CNS appear to be well established. As discussed in this review, one isoform class (MT-I/II) is rapidly induced following many types of CNS insult, and is strongly neuroprotective, whilst another isoform class (MT-III) shows major differences in its expression profile and physiological properties. As in other tissues, there is no clear consensus on the mechanism of MT action in the CNS and how it exerts its protective role, despite a number of excellent animal and cell culture models of MT expression in the brain, and a large literature on the physico-chemical properties of MTs extending over several decades. This review is therefore an attempt to summarise the recent literature on the expression of MTs in the adult mammalian brain and how MTs possibly act to protect the brain following physical or chemical insult. One exciting finding from recent work is that perturbing the levels of MT in the brain has an effect that extends beyond cells which normally express MT to other cell types including neurons, microglia and cells of the immune system. These observations were made mainly using animal models in which MT action can be observed in its normal cellular context, and this review focuses particularly on work conducted in animal models of physical and chemical injury in the brain.

Topics: Animals; Brain Injuries; Humans; Mammals; Metallothionein; Neuroprotective Agents

Parkinson's disease (PD) is the second most common neurodegenerative disease caused by selective degeneration of dopaminergic neurons in the substantia nigra. Metallothionein has been shown to act as a neuroprotectant in various brain injury. Thus, this study aims to identify the effects of full-length human metallothionein 2 peptide (hMT2) in paraquat-induced brain injury in the zebrafish.. A total of 80 adult zebrafish were divided into 4 groups namely control, paraquat-treated, pre-hMT2-treated, and post-hMT2-treated groups. Fish were treated with paraquat intraperitoneally every 3 days for 15 days. hMT2 were injected intracranially on day 0 (pre-treated group) and day 16 (post-treated group). Fish were sacrificed on day 22 and the brains were collected for qPCR, ELISA and immunohistochemistry analysis.. qPCR analysis showed that paraquat treatment down-regulated the expression of genes related to dopamine activity and biosynthesis (. Paraquat has been identified as one of the pesticides that can cause the death of dopaminergic neurons and affect dopamine biosynthesis. Treatment with exogenous hMT2 could reverse the effects of paraquat in the zebrafish brain.

Topics: Animals; Brain Injuries; Dopamine; Dopaminergic Neurons; Humans; Metallothionein; Mice; Mice, Inbred C57BL; Neurodegenerative Diseases; Neuroprotective Agents; Paraquat; Parkinson Disease; Substantia Nigra; Zebrafish

The divalent cation zinc is associated with cortical plasticity. However, the mechanism of zinc in the pathophysiology of cortical injury-associated neurobehavioral damage following neonatal seizures is uncertain. We have previously shown upregulated expression of ZnT-3; MT-3 in hippocampus of neonatal rats submitted to flurothyl-induced recurrent seizures, which was restored by pretreatment with ketogenic diet (KD). In this study, utilizing a novel "twist" seizure model by coupling early-life flurothyl-induced seizures with later exposure to penicillin, we further investigated the long-term effects of KD on cortical expression of zinc homeostasis-related genes in a systemic scale. Ten Sprague-Dawley rats were assigned each averagely into the non-seizure plus normal diet (NS + ND), non-seizure plus KD (NS + KD), recurrent seizures plus normal diet (RS + ND) and recurrent seizures plus KD (RS + KD) group. Recurrent seizures were induced by volatile flurothyl during P9-P21. During P23-P53, rats in NS + KD and RS + KD groups were dieted with KD. Neurological behavioral parameters of brain damage (plane righting reflex, cliff avoidance reflex, and open field test) were observed at P43. At P63, we examined seizure threshold using penicillin, then the cerebral cortex were evaluated for real-time RT-PCR and western blot study. The RS + ND group showed worse performances in neurological reflex tests and reduced latencies to myoclonic seizures induced by penicillin compared with the control, which was concomitant with altered expressions of ZnT-7, MT-1, MT-2, and ZIP7. Specifically, there was long-term elevated expression of ZIP7 in RS + ND group compared with that in NS + ND that was restored by chronic ketogenic diet (KD) treatment in RS + KD group, which was quite in parallel with the above neurobehavioral changes. Taken together, these findings indicate that the long-term altered expression of the metal transporter ZIP7 in adult cerebral cortex might correlate with neurobehavioral damage and reduced seizure threshold following recurrent neonate seizures and further highlights ZIP7 as a candidate for therapeutic target of KD for the treatment of neonatal seizure-induced long-term brain damage.

Topics: Animals; Brain Injuries; Cation Transport Proteins; Cerebral Cortex; Diet, Ketogenic; Female; Flurothyl; Gene Expression Regulation; Hippocampus; Male; Metallothionein; Nerve Tissue Proteins; Rats; Rats, Sprague-Dawley; Seizures

We recently reported that administration of the non-selective cyclic GMP-phosphodiesterase (cGMP-PDE) inhibitor zaprinast to cortically cryoinjured rats results three days post-lesion in reduced neuronal cell death that was associated to decreased macrophage/microglial activation and oxidative stress and increased astrogliosis and angiogenesis. Similar effects have been observed in cryoinjured animals overexpressing metallothioneins I/II (MT-I/II), metal-binding cysteine-rich proteins that are up-regulated in response to injury. In this work we have examined the effect of administration of the selective PDE5 inhibitor sildenafil (10mg/kg, sc) 2h before and 24 and 48h after induction of cortical cryolesion in wild-type and MT-I/II-deficient mice. Our results show that in wild-type animals sildenafil induces similar changes in glial reactivity, angiogenesis and antioxidant and antiapoptotic effects in the cryolesioned cortex as those observed in rats with zaprinast, indicating that inhibition of PDE5 is responsible for the neuroprotective actions. However, these effects were not observed in mice deficient in MT-I/II. We further show that sildenafil significantly increases MT-I/II protein levels in homogenates of lesioned cortex and MT-I/II immunostaining in glial cells around the lesion. Taken together these results indicate that cGMP-mediated pathways regulate expression of MT-I/II and support the involvement of these proteins in the neuroprotective effects of sildenafil in focal brain lesion.

Topics: Animals; Apoptosis; Blotting, Western; Brain Injuries; Cerebral Cortex; Cold Temperature; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme-Linked Immunosorbent Assay; Gliosis; Immunohistochemistry; Macrophage Activation; Metallothionein; Mice; Mice, Knockout; Microglia; Neovascularization, Physiologic; Neuroprotective Agents; Oxidative Stress; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Sildenafil Citrate; Sulfones; Up-Regulation

Experiments with transgenic over-expressing, and null mutant mice have determined that metallothionein-I and -II (MT-I/II) are protective after brain injury. MT-I/II is primarily a zinc-binding protein and it is not known how it provides neuroprotection to the injured brain or where MT-I/II acts to have its effects. MT-I/II is often expressed in the liver under stressful conditions but to date, measurement of MT-I/II expression after brain injury has focused primarily on the injured brain itself. In the present study we measured MT-I/II expression in the liver of mice after cryolesion brain injury by quantitative reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) with the UC1MT antibody. Displacement curves constructed using MT-I/II knockout (MT-I/II(-/-)) mouse tissues were used to validate the ELISA. Hepatic MT-I and MT-II mRNA levels were significantly increased within 24 hours of brain injury but hepatic MT-I/II protein levels were not significantly increased until 3 days post injury (DPI) and were maximal at the end of the experimental period, 7 DPI. Hepatic zinc content was measured by atomic absorption spectroscopy and was found to decrease at 1 and 3 DPI but returned to normal by 7DPI. Zinc in the livers of MT-I/II(-/-) mice did not show a return to normal at 7 DPI which suggests that after brain injury, MT-I/II is responsible for sequestering elevated levels of zinc to the liver.. MT-I/II is up-regulated in the liver after brain injury and modulates the amount of zinc that is sequestered to the liver.

Topics: Animals; Antibodies; Brain; Brain Injuries; Cold Temperature; Corticosterone; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Liver; Logistic Models; Metallothionein; Mice; Radioimmunoassay; Reference Standards; Reproducibility of Results; RNA, Messenger; Spectrophotometry, Atomic; Tissue Extracts; Zinc

N-Methyl-D-aspartate (NMDA) receptor overactivation-mediated oxidative stress has been proposed to contribute to brain injury. Metallothionein-I/II (MT-I/II), a member of cysteine-rich metalloproteins, has been found to express in the central nervous system primarily in cortical tissues and be upregulated following brain injury. To address the role of MT-I/II on NMDA-mediated oxidative injury, we established primary cortical neuron/astrocyte cultures from neonatal MT-I/II deficient (MT⁻/⁻) and wild type (MT+/+) mice to test whether basal MT-I/II protects cortical cultures against NMDA-mediated injury. We found that MT-I/II expression was increased by NMDA in MT+/+ cultures but was not detectable in MT⁻/⁻ cultures. NMDA concentration-dependently induced oxidative injury in both MT+/+ and MT⁻/⁻ cultures as evidenced by decrease of cell viability, increases of lipid peroxidation and DNA damage. However, these toxic effects were greater in MT⁻/⁻ than MT+/+ cultures. NMDA significantly increased reactive oxygen species (ROS) generation and disrupted mitochondrial membrane potential in neurons in MT+/+ cultures, and these effects were exaggerated in MT⁻/⁻ cultures. Our findings clearly show that basal MT-I/II provides protection against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures, and suggest that the protective effects are possibly associated with inhibition of ROS generation and preservation of mitochondrial membrane potential.

Topics: Animals; Animals, Newborn; Astrocytes; Brain Injuries; Cells, Cultured; Cerebral Cortex; Down-Regulation; Membrane Potential, Mitochondrial; Metallothionein; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Transgenic; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Oxidative Stress; Reactive Oxygen Species; Up-Regulation

Metallothionein-I and -II (MT-I/II) is produced by reactive astrocytes in the injured brain and has been shown to have neuroprotective effects. The neuroprotective effects of MT-I/II can be replicated in vitro which suggests that MT-I/II may act directly on injured neurons. However, MT-I/II is also known to modulate the immune system and inflammatory processes mediated by the immune system can exacerbate brain injury. The present study tests the hypothesis that MT-I/II may have an indirect neuroprotective action via modulation of the immune system.. Wild type and MT-I/II(-/-) mice were administered cryolesion brain injury and the progression of brain injury was compared by immunohistochemistry and quantitative reverse-transcriptase PCR. The levels of circulating leukocytes in the two strains were compared by flow cytometry and plasma cytokines were assayed by immunoassay.. Comparison of MT-I/II(-/-) mice with wild type controls following cryolesion brain injury revealed that the MT-I/II(-/-) mice only showed increased rates of neuron death after 7 days post-injury (DPI). This coincided with increases in numbers of T cells in the injury site, increased IL-2 levels in plasma and increased circulating leukocyte numbers in MT-I/II(-/-) mice which were only significant at 7 DPI relative to wild type mice. Examination of mRNA for the marker of alternatively activated macrophages, Ym1, revealed a decreased expression level in circulating monocytes and brain of MT-I/II(-/-) mice that was independent of brain injury.. These results contribute to the evidence that MT-I/II(-/-) mice have altered immune system function and provide a new hypothesis that this alteration is partly responsible for the differences observed in MT-I/II(-/-) mice after brain injury relative to wild type mice.

Topics: Animals; Astrocytes; Brain Injuries; Chemokines; Cytokines; Leukocyte Count; Leukocytes; Macrophages; Male; Metallothionein; Mice; Mice, Knockout; Neurons; Neuroprotective Agents

The discovery of neural stem cells (NSCs) provides new therapeutic strategies for brain injury by means of endogenous cell renewal. In the injured mouse brain, bio-liberated gold ions from gold implants mediate anti-inflammatory and antiapoptotic effects and activation of NSCs. This paper investigates the neuroprotective effects of gold following brain injury in mice. We show for the first time that endogenous NSCs express macrophage colony-stimulating factor (M-CSF) as part of their post-injury activation and that gold implants increase this response. Also, gold increases expression of neurotrophin (NT)-4, transforming growth factor-beta 3 (TGF-beta 3), leukemia inhibitory factor (LIF) and metallothionein I+II (MT-I+II) post-injury. This paper shows that gold ions modulate neurotrophic factors after injury and that hematopoietic factor M-CSF is expressed in activated NSCs.

Topics: Adult Stem Cells; Animals; Brain Injuries; Disease Models, Animal; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Gold; Lateral Ventricles; Macrophage Colony-Stimulating Factor; Metallothionein; Mice; Mice, Inbred C57BL; Nerve Growth Factors; Time Factors

Traumatic injury to the brain is one of the leading causes of injury-related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock-out mice. We herewith report the results comparing full mouse MT-1 with the independent alpha and beta domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota-rod tests; all the proteins showed a modest effect in the former test, while only full MT-1 improved the performance of animals in the rota-rod, and the alpha domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host-response genes such as GFAP, Mac1 and ICAM, in some cases being the beta domain more effective than the alpha domain or even the full MT-1. A MT-1-to-MT-3 mutation blunted some but not all the effects caused by the normal MT-1, and in some cases increased its potency. Thus, splitting the two MT-1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions.

Topics: Animals; Body Weight; Brain Injuries; Disease Models, Animal; Gene Expression Regulation; Metallothionein; Metallothionein 3; Mice; Mice, Knockout; Motor Activity; Mutation; Nerve Tissue Proteins; Protein Structure, Tertiary; Psychomotor Performance

A major obstacle that hampers the design of drug therapy for traumatic brain injury is the incomplete understanding of the biochemical pathways that lead to secondary cellular injury and contribute to cell death. One such pathway involves reactive species that generate potentially cytotoxic zinc ion fluctuations as a major executor of neuronal, and possibly glial, cell death. Whether zinc ions released during traumatic brain injury are toxic or protective is controversial but can be approached by investigating the exact concentrations of free zinc ions, the thresholds of compromised zinc buffering capacity, and the mechanism of cellular homeostatic control of zinc. Rapidly stretch-injured rat pheochromocytoma (PC12) cells express cellular zinc ion fluctuations that depend on the production of nitric oxide. Chelation of cellular zinc ions after rapid stretch injury, however, increases cellular reactive oxygen species. In a rat model of traumatic brain injury, parasagittal fluid percussion, analysis of the metal load of metallothionein was used as an indicator of changes in cellular zinc ion concentrations. The combined results from the cellular and in vivo investigations caution against interpreting zinc ion fluctuations in the early phase (24h) after injury as a primarily cytotoxic event.

Topics: Animals; Brain; Brain Injuries; Cell Survival; Disease Models, Animal; Intracellular Space; Ions; Male; Metallothionein; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; PC12 Cells; Random Allocation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Time Factors; Zinc

Compared with adults, immature metallothionein I and II knockout (MT(-/-)) mice incur greater neuronal loss and a more rapid rate of microglia accumulation after target deprivation-induced injury. Because minocycline has been proposed to inhibit microglial activation and associated production of neuroinflammatory factors, we investigated its ability to promote neuronal survival in the immature, metallothionein-deficient brain. After ablation of the visual cortex, 10-day-old MT(-/-) mice were treated with minocycline or saline and killed 24 or 48 hr after injury. By means of stereological methods, the number of microglia and neurons were estimated in the ipsilateral dorsal lateral geniculate nucleus (dLGN) by an investigator blinded to the treatment. No effect on neuronal survival was observed at 24 hr, but 48 hr after injury, an unanticipated but significant minocycline-mediated increase in neuronal loss was detected. Further, while failing to inhibit microglial accumulation, minocycline treatment increased the proportion of amoeboid microglia in the ipsilateral dLGN. To understand the molecular mechanisms underlying this neurotoxic response, we identified minocycline-mediated changes in the expression of three potentially proapoptotic/inflammatory genes: growth arrest- and DNA damage-inducible gene 45gamma (GADD45gamma); interferon-inducible protein 1 (IFI1), and cytokine-induced growth factor. We also observed increased mitogen-activated protein kinase p38 phosphorylation with minocycline treatment. Although minocycline inhibited calpain activity at 12 hr after injury, this effect was not sustained at 24 hr. Together, these results help to explain how minocycline has a deleterious effect on neuronal survival in this injury model.

Topics: Animals; Brain; Brain Injuries; Calpain; Cell Survival; Cerebral Cortex; GADD45 Proteins; Gene Expression; Geniculate Bodies; GTP-Binding Proteins; Intracellular Signaling Peptides and Proteins; Metallothionein; Mice; Mice, Knockout; Microglia; Minocycline; Neural Pathways; Neurons; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Thalamus

The kynurenine pathway has been implicated as a major component of the neuroinflammatory response to brain injury and neurodegeneration. We found that the neurotoxic kynurenine pathway intermediate quinolinic acid (QUIN) is rapidly expressed, within 24 h, by reactive microglia following traumatic injury to the rodent neocortex. Furthermore, administration of the astrocytic protein metallothionein attenuated this neuroinflammatory response by reducing microglial activation (by approximately 30%) and QUIN expression. The suppressive effect of MT was confirmed upon cultured cortical microglia, with 1 mug/ml MT almost completely blocking interferon-gamma induced activation of microglia and QUIN expression. These results demonstrate the neuroimmunomodulatory properties of MT, which may have therapeutic applications for the treatment of traumatic brain injury.

Topics: Analysis of Variance; Animals; Brain Injuries; Cell Count; Cells, Cultured; Cerebral Cortex; Culture Media, Conditioned; Dose-Response Relationship, Drug; Ferritins; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Interferon-gamma; Metallothionein; Microglia; Neocortex; Neurons; Quinolinic Acid; Rats; Rats, Wistar

Metallothioneins (MTs) are metal-binding proteins that can be upregulated in the brain after injury and are associated with neuroprotection. A recent genomics study has shown that brain MT-1 and MT-2 mRNA levels are upregulated following intracerebral hemorrhage (ICH) in rats. Our study examines whether brain MT-1 and MT-2 protein levels are increased after ICH. We also investigated the effect of exogenous MT-1 in perihematomal edema formation in vivo and iron-induced cell death in vitro. We found that MT-1/-2 immunoreactivity in ipsilateral basal ganglia was significantly increased after ICH and exogenous MT-1 attenuated perihematomal edema formation. In addition, MT-1 also reduced cell death induced by iron in cultured astrocytes. These results suggest a role for MT in ICH-induced brain injury, and MT could be a therapeutic target for ICH.

Topics: Analysis of Variance; Animals; Animals, Newborn; Astrocytes; Basal Ganglia; Brain Injuries; Cells, Cultured; Cerebral Cortex; Cerebral Hemorrhage; Disease Models, Animal; Dose-Response Relationship, Drug; L-Lactate Dehydrogenase; Male; Metallothionein; Rats; Rats, Sprague-Dawley; Up-Regulation

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.

Topics: Animals; Astrocytes; Axons; Brain Injuries; Cells, Cultured; Free Radical Scavengers; Metallothionein; Optic Nerve; Protein Transport; Rats; Regeneration; Retinal Ganglion Cells

Cytokines, such as tumour necrosis factor (TNF)-alpha and lymphotoxin-alpha, 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 approximately 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.

Topics: Analysis of Variance; Animals; Behavior, Animal; Brain; Brain Injuries; Exploratory Behavior; Gene Expression Regulation; In Situ Hybridization; Maze Learning; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Oligonucleotide Array Sequence Analysis; Psychomotor Performance; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Recovery of Function

The clinical manifestations of inflicted traumatic brain injury in infancy most commonly result from intracranial hemorrhage, axonal stretch and disruption, and cerebral edema. Often hypoxia ischemia is superimposed, leading to early forebrain and later thalamic neurodegeneration. Such acute and delayed cellular injury activates microglia in the CNS. Although activated microglia provide important benefits in response to injury, microglial release of reactive oxygen species can be harmful to axotomized neurons. We have previously shown that the antioxidants metallothionein I and II (MT I & II) promote geniculocortical neuronal survival after visual cortex lesioning. The purpose of this investigation was to determine the influence of MT I & II on the density and rate of thalamic microglial activation and accumulation following in vivo axotomy. We ablated the visual cortex of 10-day-old and adult MT I & II knock out (MT(-/-)) and wild-type mice and then determined the density of microglia in the dorsal lateral geniculate nucleus (dLGN) over time. Compared to the wild-type strain, microglial activation occurred earlier in both young and adult MT(-/-) mice. Similarly, microglial density was significantly greater in young MT(-/-) mice 30, 36, and 48 hours after injury, and 3, 4, and 5 days after injury in MT(-/-) adults. In both younger and older mice, time and MT I & II deficiency each contributed significantly to greater microglial density. Only in younger mice did MT I & II expression significantly slow the rate (density x time) of microglial accumulation. These results suggest that augmentation of MT I & II expression may provide therapeutic benefits to infants with inflicted brain injury.

Topics: Aging; Animals; Axotomy; Brain Injuries; Cell Count; Cell Death; Immunohistochemistry; Metallothionein; Mice; Mice, Knockout; Microglia; Nerve Degeneration; Neurons; Thalamus; Visual Cortex

Metallothionein (MT) expression is rapidly up-regulated following CNS injury, and there is a strong correlation between the presence or absence of MTand improved or impaired (respectively) recovery from such trauma.We now report that a distinct subset of NG2-positive, GFAP-negative glial cells bordering the injury tract express MT following focal injury to the adult rat neocortex. To confirm the ability of these NG2 glial cells to express MT, we have isolated and cultured them and identified that they can express MT following stimulation with zinc. To investigate the functional importance of MT expression by NG2 glial cells, we plated cortical neurons onto these cells and found that expression of MT enhanced the permissivity of NG2 glial cells to neurite outgrowth. Our data suggest that expression of MT by NG2 glial cells may contribute to the overall permissiveness of these cells to axon regeneration.

Topics: Animals; Brain Injuries; Cells, Cultured; Coculture Techniques; Gene Expression Regulation; Metallothionein; Neocortex; Nerve Regeneration; Neurites; Neuroglia; Rats; Rats, Wistar; Zinc

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.

Topics: Analysis of Variance; Animals; Antigens, CD; Apoptosis; Brain Injuries; Cell Count; Drug Interactions; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Inflammation; Macrophages; Male; Metallothionein; Mice; Mice, Knockout; Neurobiology; Neuronal Plasticity; Rabbits; Recombinant Proteins; Sensitivity and Specificity

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.

Topics: Analysis of Variance; Animals; Aquaporin 2; Brain Injuries; Catalase; Cluster Analysis; Gene Expression Profiling; Gene Expression Regulation; Immunohistochemistry; Metallothionein; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Oligonucleotide Array Sequence Analysis; Stem Cells; Time Factors

The aim of this study was to evaluate the correlation of neuron specific enolase (NSE), protein S100B and time-profile of Glasgow Coma Score (GCS) development with metallothionein (MT) blood levels in patients with traumatic brain injury (TBI) during 10 days of hospitalization. Patients were divided into 2 groups with respect to NSE and S100B levels - with (group I) and without (group II) GCS improvement.. Serum NSE and S100B concentrations were measured by immunochemical methods; serum metallothionein concentration by electrochemical technique. Cortical biopsies were investigated immunohistochemically and by electron microscope. A cDNA microarray containing 700 gene probes was used to study the changes in gene expression in the ipsilateral cortex.. Values of MT in the blood of group I showed a non-significant decrease compared to group II during 1-3 days after admission. There was an increase of MT during 4-8 days in comparison with values of 1-3 days. The highest value of MT during hospitalization was found in a patient with diffuse axonal injury (group II). The data of cDNA microarray suggested an increase in expression of gene transcripts for oxygen free radical scavenger proteins corresponding with the increase of MT during 4-8 days in both groups.. The experimental data indicate that monitoring the content of MT in patients with trauma brain injury would be a suitable approach to evaluate the degree of injury or duration of prolonging unconsciousness, particularly in diagnosis of diffuse axonal injury.

Topics: Brain Injuries; Gene Expression Profiling; Humans; Magnetic Resonance Imaging; Metallothionein; Nerve Growth Factors; Phosphopyruvate Hydratase; Radiography; S100 Calcium Binding Protein beta Subunit; S100 Proteins

Oxygen free radicals and nitric oxide (NO) participate in the pathogenesis of acute central nervous system (CNS) injury by forming peroxynitrite, which promotes oxidative damage and tyrosine nitration. Neuronal nitration is associated with cell death, but little is known of the characteristics and cell fate of nitrated astrocytes. In this study, we have used a postnatal excitotoxic lesion model (intracortical NMDA injection) and our aims were (i) to evaluate the temporal and spatial pattern of astroglial nitration in correlation with the neuropathological process and the sources of NO; and (ii) to establish, if any, the correlation among astrocyte nitration and other events such as expression of cytoskeletal proteins, antioxidant enzymes, and cell death markers to cope with nitration and/or undergo cell death. Our results show that after postnatal excitotoxic damage two distinct waves of nitration were observed in relation to astrocytes. At 24 h post-lesion, early-nitrated astrocytes were found within the neurodegenerating area, coinciding with the time of maximal cell death. These early-nitrated astrocytes are highly ramified protoplasmic cells, showing diffuse glial fibrillary acidic protein (GFAP) content and expressing inducible NOS. At later time-points, when astrogliosis is morphologically evident, nitrated hypertrophied reactive astrocytes are observed in the penumbra and the neurodegenerated area, displaying increased expression of GFAP and vimentin cytoskeletal proteins and of metallothionein I-II and Cu/Zn superoxide dismutase antioxidant proteins. Moreover, despite revealing activated caspase-3, they do not show TUNEL labeling. In summary, we show that nitrated astrocytes in vivo constitute a subpopulation of highly reactive astrocytes which display high resistance towards oxidative stress induced cell death.

Topics: Animals; Animals, Newborn; Astrocytes; Brain Injuries; Cytoskeletal Proteins; Disease Models, Animal; Female; Male; Metallothionein; Nerve Tissue Proteins; Neurotoxins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Rats, Long-Evans; Superoxide Dismutase; Tyrosine

Recent data suggests that metallothioneins (MTs) are major neuroprotective proteins within the CNS. In this regard, we have recently demonstrated that MT-IIA (the major human MT-I/-II isoform) promotes neural recovery following focal cortical brain injury. To further investigate the role of MTs in cortical brain injury, MT-I/-II expression was examined in several different experimental models of cortical neuron injury. While MT-I/-II immunoreactivity was not detectable in the uninjured rat neocortex, by 4 days, following a focal cortical brain injury, MT-I/-II was found in astrocytes aligned along the injury site. At latter time points, astrocytes, at a distance up to several hundred microns from the original injury tract, were MT-I/-II immunoreactive. Induced MT-I/-II was found both within the cell body and processes. Using a cortical neuron/astrocyte co-culture model, we observed a similar MT-I/-II response following in vitro injury. Intriguingly, scratch wound injury in pure astrocyte cultures resulted in no change in MT-I/-II expression. This suggests that MT induction was specifically elicited by neuronal injury. Based upon recent reports indicating that MT-I/-II are major neuroprotective proteins within the brain, our results provide further evidence that MT-I/-II plays an important role in the cellular response to neuronal injury.

Topics: Animals; Astrocytes; Brain Injuries; Cell Communication; Gene Expression Regulation; Male; Metallothionein; Neuroglia; Neurons; Rats; Up-Regulation

Both the immediate insult and delayed apoptosis contribute to functional deficits after brain injury. Secondary, delayed apoptotic death is more rapid in immature than in adult CNS neurons, suggesting the presence of age-dependent protective factors. To understand the molecular pathobiology of secondary injury in the context of brain development, we identified changes in expression of oxidative stress response genes during postnatal development and target deprivation-induced neurodegeneration. The antioxidants metallothionein I and II (MT I/II) were increased markedly in the thalamus of adult C57BL/6 mice compared to mice <15 days old. Target deprivation generates reactive oxygen species that mediate neuronal apoptosis in the central nervous system; thus the more rapid apoptosis observed in the immature brain might be due to lower levels of MT I/II. We tested this hypothesis by documenting neuronal loss after target-deprivation injury. MT I/II-deficient adult mice experienced greater thalamic neuron loss at 96 hr after cortical injury compared to that in controls (80 +/- 2% vs. 57 +/- 4%, P < 0.01), but not greater overall neuronal loss (84 +/- 4% vs. 79 +/- 3%, MT I/II-deficient vs. controls). Ten-day-old MT I/II-deficient mice, however, experienced both faster onset of secondary neuronal death (30 vs. 48 hr) and greater overall neuronal loss (88 +/- 2% vs. 69 +/- 4%, P = 0.02). MT I/II are thus inhibitors of age-dependent secondary brain injury, and the low levels of MT I/II in immature brains explains, in part, the enhanced susceptibility of the young brain to neuronal loss after injury. These findings have implications for the development of age-specific therapeutic strategies to enhance recovery after brain injury.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Apoptosis; Brain Injuries; Cell Count; Decerebrate State; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Functional Laterality; Gene Expression Profiling; Gene Expression Regulation, Developmental; Geniculate Bodies; Immunoblotting; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Degeneration; Neurons; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Thalamus; Time Factors

In this study, we investigated the expression of metallothionein (MT)-I and MT-II in the rat brain following traumatic brain injury (TBI). In the early stage, significant induction of MT-I and MT-II were observed in various regions including ventricle walls, pia mater, and dentate gyrus. At 12-24 h after TBI, strong induction of MT-I mRNA was observed in cerebral cortical layer II/III, amygdala, and piriform cortex where neurons reside. On the other hand, MT-II appeared to be expressed mainly in glial cells localized in the cerebral cortex and hippocampal formation. Three days after TBI, MTs were observed in the vimentin-positive astrocytes in the penumbra as revealed by double immunohistochemistry. The differences in expression of MT-I and MT-II in different brain regions and cell types (neuron vs. glial cells) suggests that multiple regulatory mechanisms are involved in the control of MT expression following brain injury.

Topics: Animals; Brain; Brain Injuries; Cytokines; Gene Expression Regulation; Immunohistochemistry; In Situ Hybridization; Male; Metallothionein; Microscopy, Fluorescence; Neuroglia; Neurons; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors

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

Metallothioneins (MTs) are small, cysteine-rich, metal binding proteins. Their function has often been considered as stress-related proteins capable of protecting cells from heavy metal toxicity and oxidative free radicals. However, recent interest has focused on the brain-specific MT-III isoform, which has neurite-inhibitory properties. To investigate the effect of another MT isoform, human MT-IIA, on neurite growth, we used rat cortical neuron cultures. MT-IIA promoted a significant increase in the rate of initial neurite elongation of individually plated neurons. We also investigated the effect of MT-IIA on the neuronal response to axonal transection in vitro. MT-IIA promoted reactive axonal growth after injury, and, by 18 hr after transection, MT-IIA had promoted axonal growth across the injury tract. Exogenous application of MT-IIA after cortical brain injury promoted wound healing, as observed by a significant decrease in cellular degradation at 4 d after injury. Furthermore, MT-IIA-treated rats exhibited numerous SMI-312-immunoreactive axonal processes within the injury tract. This was in contrast to vehicle-treated animals, in which few axonal sprouts were observed. By 7 d after injury, MT-IIA treatment resulted in a total closing over of the injury tract by microglia, astrocytes, and reactive axonal processes. However, although some reactive axonal processes were observed within the injury tract of vehicle-treated rats, the tract itself was almost never entirely enclosed. These results are discussed in relation to a possible physiological role of metallothioneins in the brain, as well as in a therapeutic context.

Topics: Animals; Astrocytes; Axons; Brain Injuries; Cell Division; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Humans; Immunohistochemistry; Male; Metallothionein; Microglia; Neurites; Neurofilament Proteins; Neurons; Rats; Rats, Wistar; Wound Healing

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-alpha, interleukin (IL)-1 alpha/beta, 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.

Topics: Animals; Apoptosis; Brain Injuries; Cell Count; Cerebral Cortex; Cytokines; Freezing; GAP-43 Protein; Gene Expression Regulation; Gliosis; Metallothionein; Metallothionein 3; Mice; Mice, Inbred Strains; Mice, Knockout; Nerve Growth Factors; Nerve Tissue Proteins; Oxidative Stress; T-Lymphocytes

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

Topics: Animals; Brain; Brain Injuries; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression; Humans; Inflammation Mediators; Metallothionein; Mice; Mice, Knockout; Multiple Sclerosis; Neurodegenerative Diseases; Oxidative Stress; Rats; RNA, Messenger

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

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

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.

Topics: Animals; Astrocytes; Blood-Brain Barrier; Brain Injuries; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Inhibitors; In Situ Hybridization; Interleukin-6; Ki-67 Antigen; Macrophages; Metallothionein; Metallothionein 3; Mice; Mice, Inbred C57BL; Microglia; Nerve Tissue Proteins; Neurons

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

Exposure of the adult rat brain parenchyma to zinc induces an increase in the intracerebral expression of the metal-binding protein, metallothionein, which is normally confined to astrocytes, ependymal cells, choroid plexus epithelial cells, and brain endothelial cells. Metallothionein is expressed only in diminutive amounts in astrocytes of the neonatal rat brain, which could imply that neonatal rats are devoid of the capacity to detoxify free metals released from a brain wound. In order to examine the influence of a brain injury on the expression of metallothionein in the neonatal brain, PO rats were subjected to a localized freeze lesion of the neocortex of the right temporal cortex. This lesion results in a disrupted blood-brain interface, leading to extravasation of plasma proteins. From 16 h, reactive astrocytosis, defined as an increase in the number and size of cells expressing GFAP and vimentin, was observed surrounding the neocortical lesion site. Astrocytes and pial cells situated adjacent to the area of injury also became positively stained for metallothionein. At 3-6 days post-lesion, the highest level of reactive astrocytes expressing metallothionein was observed. Neo-Timm staining revealed that histochemically reactive zinc had disappeared from the lesion site. Extracellular albumin and metallothionein-positive astrocytes were absent approximately 2 weeks after the lesion, whereas reactive astrocytosis was still observed. These results show that a lesion of the neonatal rat brain induces a transient expression of metallothionein in reactive astrocytes, probably as a response to metals released from the site of the brain injury.

Topics: Animals; Animals, Newborn; Astrocytes; Blood Proteins; Blood-Brain Barrier; Brain Injuries; Cell Count; Cerebral Cortex; Immunohistochemistry; Metallothionein; Rats; Rats, Wistar; Time Factors; Zinc