metallothionein and Spinal-Cord-Injuries

After spinal cord injury (SCI), some self-destructive mechanisms start leading to irreversible neurological deficits. It is known that oxidative stress and apoptosis play a major role in increasing damage after SCI. Metallothioneins I and II (MT) are endogenous peptides with known antioxidant, neuroprotective capacities. Taking advantage of those capacities, we administered exogenous MT to rats after SCI in order to evaluate the protective effects of MT on the production of reactive oxygen species (ROS) and lipid peroxidation (LP), as markers of oxidative stress. The activities of caspases-9 and -3 and the number of annexin V and TUNEL-positive cells in the spinal cord tissue were also measured as markers of apoptosis. Rats were subjected to either sham surgery or SCI and received vehicle or two doses of MT (10 

Topics: Animals; Antioxidants; Apoptosis; Female; Lipid Peroxidation; Metallothionein; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Recovery of Function; Spinal Cord Injuries

The aim of this study is to investigate the effect of ketogenic metabolism, induced by different diet interventions, on histone acetylation and its potential antioxidant capacity to injured spinal cord tissue in rats. 72 male Sprague-Dawley rats were randomly divided into 4 groups, fed with ketogenic diet (KD), every other day fasting (EODF), every other day ketogenic diet (EODKD) and standard diet (SD) respectively for 2 weeks. β-Hydroxybutyrate (βOHB) concentration was measured both in serum and cerebrospinal fluid (CSF). C5 spinal cord tissue was harvested before, at 3 h and 24 h after injury for analysis of HDAC activity, histone acetylation and oxidative makers. All three dietary interventions resulted in a significant increase of βOHB level in both serum and CSF, and inhibited HDAC activity by 31-43% in spinal cord. Moreover, the expressions of acetylated histone AcH3K9 and AcH3K14 were significantly increased. Anti-oxidative stress genes Foxo3a and Mt2 and related proteins, such as mitochondrial superoxide dismutase (SOD), FOXO3a, catalase were increased in dietary intervention groups. After SCI, high ketogenic metabolism demonstrated significant reduction of the expression of lipid peroxidation factors malondialdehyde (MDA), and this might contribute to the reported neuroprotection of the spinal cord from oxidative damage possibly mediated by increasing SOD. The result of this study suggested that by inhibiting HDAC activity and modifying related gene transcription, ketogenic metabolism, induced by KD, EODF or EODKD, might reduce oxidative damage in the spinal cord tissue after acute injury.

Topics: 3-Hydroxybutyric Acid; Animals; Diet, Ketogenic; Forkhead Box Protein O3; Histone Deacetylases; Male; Metallothionein; Oxidative Stress; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries

After spinal cord injury (SCI), a complex cascade of pathophysiological processes rapidly damages the nervous tissue. The initial damage spreads to the surrounding tissue by different mechanisms, including oxidative stress. We have recently reported that the induction of metallothionein (MT) protein is an endogenous rapid-response mechanism after SCI. Since the participation of MT in neuroprotective processes after SCI is still unknown, the aim of the present study was to evaluate the possible neuroprotective effect of exogenously administered MT-II during the acute phase after SCI in rats. Female Wistar rats weighing 200-250g were submitted to spinal cord contusion by means of a computer-controlled device (NYU impactor). Rats received several doses of MT-II (3.2, 10 and 100μg) at 2 and 8h after SCI. Results of the BBB scale were statistically analysed using an ANOVA of repeated-measures, followed by Tukey's test. Among the three doses tested, only 10 and 100μg were able to significantly increase (p<0.05) BBB scale scores eight weeks after SCI from a mean of 7.88 in the control group, to means of 12.63 and 10.88 for the 10 and 100μg doses of MT-II, respectively. The amount of spared tissue was also higher in the groups treated with 10 and 100μg, as compared to the control group values. Results from the present study demonstrate a significant neuroprotective effect of exogenously administered MT-II. Further studies are needed in order to characterize the mechanisms involved in this neuroprotective action.

Topics: Animals; Female; Metallothionein; Motor Activity; Neuroprotective Agents; Rats; Rats, Wistar; Recovery of Function; Spinal Cord; Spinal Cord Injuries

Spinal cord injury (SCI) is a world-wide health problem. After traumatic injury, spinal cord tissue starts a series of self-destructive mechanisms, known as the secondary lesion. The leading mechanisms of damage after SCI are excitotoxicity, free radicals' overproduction, inflammation and apoptosis. Metallothionein (MT) and reduced glutathione (GSH) are low-molecular-weight, cysteine-rich peptides able to scavenge free radicals. MT and GSH participation as neuroprotective molecules after SCI is unknown. The aim of the present study is to describe the changes of MT and GSH contents and GSH peroxidase (GPx) activity in the acute phase after SCI in rats. Female Wistar rats weighing 200-250g were submitted to spinal cord contusion model, by means of a computer-controlled device (NYU impactor). Rats receiving laminectomy were used as a control group. Animals were killed 2, 4, 12 and 24h after surgery. MT was quantified by the silver-saturation method, using atomic absorption spectrophotometry. GSH and GPx were assayed by spectrophotometry. Results indicate an increased MT content by effect of SCI, only at 4 and 24h, as compared to sham group values. Meanwhile, GSH was found decreased at 4, 12 and 24h after SCI. Interestingly, GPx activity was raised at all time points, indicating that this enzymatic defense is activated soon after SCI. Results suggest that thiol-based defenses, MT and GSH, are differentially expressed by spinal cord tissue to cope with the various processes of damage after lesion.

Topics: Animals; Antioxidants; Disease Models, Animal; Female; Gene Expression Regulation; Glutathione; Metallothionein; Oxidoreductases; Rats; Rats, Wistar; Spinal Cord Injuries; Time Factors

Metallothionein (MT) is a small molecular weight protein possessing metal binding and free radical scavenging properties. We hypothesized that MT-1/MT-2 null (MT(-/-)) mice would display exacerbated soleus muscle atrophy, oxidative injury, and contractile dysfunction compared with the response of wild-type (WT) mice following acute spinal cord transection (SCT). Four groups of mice were studied: WT laminectomy, WT transection, MT(-/-) laminectomy (MT(-/-) lami), and MT(-/-) transection (MT(-/-) trans). Laminectomy animals served as surgical controls. Mice in SCT groups experienced similar percent body mass (BM) losses at 7 days postinjury. Soleus muscle mass (MM) and MM-to-BM ratio were lower at 7 days postinjury in SCT vs. laminectomy mice, with no differences observed between strains. However, soleus muscles from MT(-/-) trans mice showed reduced maximal specific tension compared with MT(-/-) lami animals. Mean cross-sectional area (microm(2)) of type I and type IIa fibers decreased similarly in SCT groups compared with laminectomy controls, and no difference in fiber distribution was observed. Lipid peroxidation (4-hydroxynoneal) was greater in MT(-/-) trans vs. MT(-/-) lami mice, but protein oxidation (protein carbonyls) was not altered by MT deficiency or SCT. Expression of key antioxidant proteins (catalase, manganese, and copper-zinc superoxide dismutase) was similar between the groups. In summary, MT deficiency did not impact soleus MM loss, but resulted in contractile dysfunction and increased lipid peroxidation following acute SCT. These findings suggest a role of MT in mediating protective adaptations in skeletal muscle following disuse mediated by spinal cord injury.

Topics: Acute Disease; Animals; Antioxidants; Catalase; Disease Models, Animal; Laminectomy; Lipid Peroxidation; Male; Metallothionein; Mice; Mice, Knockout; Muscle Contraction; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle Strength; Muscle, Skeletal; Muscular Atrophy; Oxidative Stress; Protein Carbonylation; Spinal Cord Injuries; Superoxide Dismutase; Thoracic Vertebrae

We examined the effects of spinal cord injury (SCI) on alterations in gene expression and respective protein products in human skeletal muscle 2 days and 5 days post-SCI. Biopsies were taken from skeletal muscle of 9 men and 1 woman (n = 10) (43.9 +/- 6.7 years) 2 days and 5 days post-SCI and from 5 healthy young men who served as controls (20.4 +/- 0.5 years). Global changes in gene expression were analysed using Affymetrix GeneChips on a subsample of subjects (n = 3). Candidate genes were then pursued via qRT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes. Immunohistochemistry (IHC) was used to localize proteins. Groups of transcripts showing the greatest percentage of altered expression, the most robust fold-changes, and indicative of involvement of an entire pathway using the GeneChip included genes involved in the ubiquitin proteasome pathway (UPP), metallothionein function, and protease inhibition. qRT-PCR analysis confirmed increases in gene expression for UPP components (UBE3C, Atrogin-1, MURF1, and PSMD11), the metallothioneins (MT1A, MT1F, MT1H), and the protease inhibitor, SLPI (P < 0.05) at 2 days and 5 days post-SCI. Protein levels of the proteasome subunit (PSMD11) and the metallothioneins were increased 5 days post-SCI. Protein levels of UBE3C, Atrogin-1, MURF1 and SLPI were unchanged (P > 0.05). IHC showed increased staining for PSMD11 and the metallothioneins 5 days post-SCI, along the peripheral region of the cells. IHC also showed altered staining for Atrogin-1 at 5 days post-SCI along the membrane region. Thus, there was a profound increase in gene expression of UPP components, the metallothioneins, and the protease inhibitor, SLPI, within 5 days of SCI. Increased protein levels for PSMD11 and the metallothioneins 5 days post-SCI, specifically along the cell periphery, indicate that proteins in this region may be early targets for degradation post-SCI.

Topics: Adult; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Male; Metallothionein; Middle Aged; Muscle Fibers, Skeletal; Muscle, Skeletal; Oligonucleotide Array Sequence Analysis; Proteasome Endopeptidase Complex; RNA, Messenger; Secretory Leukocyte Peptidase Inhibitor; Spinal Cord Injuries; Ubiquitin

Topics: Animals; Antioxidants; Calpain; Disease Models, Animal; Humans; Immobilization; Metallothionein; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Oxidative Stress; Peptide Hydrolases; Proteasome Endopeptidase Complex; Spinal Cord Injuries; Time Factors; Ubiquitin; Ubiquitin-Protein Ligases

The purpose of this study was to use a cDNA microarray to identify new genes involved in healing of spinal cord injury. C57BL/6 mice (7-8 weeks, male) were subjected to spinal cord compression injury (SCI) at the T7/8 level (20 g, 5 min; SCI group). For the control group, mice underwent only laminectomy. Mice were killed at 1, 3 and 7 days. cDNA transcribed from mRNA was hybridized to NIA mice 15K microarrays at each time point. We found 84 genes showing significant expressional changes, including higher and lower expression levels in the SCI groups than in the control [more than 1.0 or less than -1.0 using log ratio (base 2)]. Five genes were selected for further quantitative gene expression analysis by real-time reverse transcription (RT)-PCR. For histological examination, we applied in situ hybridization and fluorescence immunohistochemistry. Cathepsin D, metallothionein-1 (MT-1), metallothionein-2 (MT-2), osteopontin (OPN), and tenascin-C were selected for quantitative and histological analysis. Microarray analysis revealed that SCI led to the up-regulation of OPN and cathepsin D expression at 7 days and also of MT-1, MT-2, and tenascin-C expression at 1 day. Tenascin-C was re-up-regulated at 7 days. These values agreed with those of real-time RT-PCR analysis. By double labeling with in situ hybridization and fluorescence immunohistochemistry, MT-1, MT-2 and tenascin-C expression was observed in neurons and glial cells at 1 day, whereas at 7 days the main MT-2 and tenascin-C expression was found in fibronectin-positive fibroblasts. The main cathepsin D and OPN expression was observed in activated macrophages/microglia at 3 and 7 days. The five genes picked up by microarray gene expression profiling were shown to exhibit temporal and spatial changes of expression after SCI. This system is potentially useful for identifying genes that are involved in the response to SCI.

Topics: Animals; Behavior, Animal; Cathepsin D; Cluster Analysis; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Expression Regulation; In Situ Hybridization; Male; Metallothionein; Mice; Mice, Inbred C57BL; Motor Activity; Neuroglia; Neurons; Oligonucleotide Array Sequence Analysis; Osteopontin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Spinal Cord; Spinal Cord Injuries; Tenascin; Time Factors