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Abstract: Delayed neuronal death following transient cerebral ischemia is mixed with apoptosis and necrosis, and the activation of microglia are activated after the ischemic insult. In the present study, we examined the long-term changes in neuronal degeneration and microglial activation in the gerbil hippocampal CA1 region after 5min of transient cerebral ischemia using specific markers for neuronal damage and microliosis. Transient ischemia-induced neuronal death was shown in CA1 pyramidal cells 4days after ischemia/reperfusion (I/R). However, neuronal degeneration of the pyramidal cells were observed up to 45days in the CA1 region after I/R. Microglial activation was also observed in the CA1 region after I/R. Isolectin B4- (IB4) immunoreactive (+) microglia appeared in the CA1 region 4days after I/R. On the other hand, ionized calcium-binding adapter molecule 1 (Iba-1)+ microglia was markedly increased after I/R, and peaked at 15days after I/R. Thereafter, Iba-1 immunoreactivity was decreased with time-dependant manner in the ischemic CA1 region. These results indicate that neuronal degeneration of CA1 pyramidal cells may last about 45days in the CA1 region after ischemic damage, and microglial activation may be diverse according to their function, such as phagocytosis, after I/R. [Copyright &y& Elsevier]

Abstract: Background context: Polyetheretherketone (PEEK) is gaining favor as a spinal implant material for interbody and corpectomy cages as well as stabilizing rods. However, there has been little correlation to a relevant and reproducible clinical model. Biomechanical data on PEEK rod constructs have not been reported. Purpose: To quantify the stabilizing effects of PEEK versus titanium (Ti) instrumentation in a thoracolumbar corpectomy model. Study design: Corpectomy and randomized instrumentation with an all-Ti, all-PEEK, and hybrid cage/rod construct were performed on cadaveric spines to assess biomechanical differences. Methods: Pure unconstrained bending moments were applied to the intact spine and subsequent test constructs in the three physiologic planes using a load control protocol. Motion tracking and analysis were carried out to quantify and compare the range of motion (ROM) between different test constructs in each plane. Results: Flexion ROM did not show significant changes compared with intact, whereas the all-Ti and hybrid construct reduced ROM significantly in extension. Lateral bending was significantly reduced in all the treatment groups. Rotational stability of the construct was significantly compromised by an all-PEEK spinal construct. Conclusion: The rigidity of the corpectomy construct increased as the amount of Ti in the construct increased. A hybrid construct incorporating a PEEK corpectomy cage and Ti rods may provide adequate stability for an anterior thoracolumbar reconstruction in the sagittal and coronal planes. An all-PEEK construct may provide adequate stability in the coronal and sagittal planes but may compromise the stability significantly in axial rotation. Consideration should be given for supplemental posterior instrumentation if an all-PEEK construct is used in an anterior thoracolumbar spinal reconstruction procedure. [Copyright &y& Elsevier]

CCT2 is a eukaryotic chaperonin TCP-1 ring complex subunit that mediates protein folding, autophagosome incorporation, and protein aggregation. In this study, we investigated the effects of CCT on oxidative and ischemic damage using in vitro and in vivo experimental models. The Tat-CCT2 fusion protein was efficiently delivered into HT22 cells in a concentration- and time-dependent manner, and the delivered protein was gradually degraded in HT22 cells. Incubation with Tat-CCT2 significantly ameliorated the 200 µM hydrogen peroxide (H2O2)-induced reduction in cell viability in a concentration-dependent manner, and 8 µM Tat-CCT2 treatment significantly alleviated H2O2-induced DNA fragmentation and reactive oxygen species formation in HT22 cells. In gerbils, CCT2 protein was efficiently delivered into pyramidal cells in CA1 region by intraperitoneally injecting 0.5 mg/kg Tat-CCT2, as opposed to control CCT2. In addition, treatment with 0.2 or 0.5 mg/kg Tat-CCT2 mitigated ischemia-induced hyperlocomotive activity 1 d after ischemia and confirmed the neuroprotective effects by NeuN immunohistochemistry in the hippocampal CA1 region 4 d after ischemia. Tat-CCT2 treatment significantly reduced the ischemia-induced activation of astrocytes and microglia in the hippocampal CA1 region 4 d after ischemia. Furthermore, treatment with 0.2 or 0.5 mg/kg Tat-CCT2 facilitated ischemia-induced autophagic activity and ameliorated ischemia-induced autophagic initiation in the hippocampus 1 d after ischemia based on western blotting for LC3B and Beclin-1, respectively. Levels of p62, an autophagic substrate, significantly increased in the hippocampus following treatment with Tat-CCT2. These results suggested that Tat-CCT2 exerts neuroprotective effects against oxidative stress and ischemic damage by promoting the autophagic removal of damaged proteins or organelles. [ABSTRACT FROM AUTHOR]

Malate dehydrogenase (MDH) plays an important role in the conversion of malate to oxaloacetate during the tricarboxylic acid cycle. In this study, we examined the role of cytoplasmic MDH (MDH1) in hydrogen peroxide (H2O2)-induced oxidative stress in HT22 cells and ischemia-induced neuronal damage in the gerbil hippocampus. The Tat-MDH1 fusion protein was constructed to enable the delivery of MDH1 into the intracellular space and penetration of the blood–brain barrier. Tat-MDH1, but not MDH1 control protein, showed significant cellular delivery in HT22 cells in a concentration- and time-dependent manner and gradual intracellular degradation in HT22 cells. Treatment with 4 μM Tat-MDH1 significantly ameliorated 200 μM H2O2-induced cell death, DNA fragmentation, and reactive oxygen species formation in HT22 cells. Transient increases in MDH1 immunoreactivity were detected in the hippocampal CA1 region 6–12 h after ischemia, but MDH1 activity significantly decreased 2 days after ischemia. Supplementation of Tat-MDH1 immediately after ischemia alleviated ischemia-induced hyperlocomotion and neuronal damage 1 and 4 days after ischemia. In addition, treatment with Tat-MDH1 significantly ameliorated the increases in hydroperoxides, lipid peroxidation, and reactive oxygen species 2 days after ischemia. Tat-MDH1 treatment maintained the redox status of the glutathione system in the hippocampus 2 days after ischemia. These results suggest that Tat-MDH1 exerts neuroprotective effects by reducing oxidative stress and maintaining glutathione redox system in the hippocampus. [ABSTRACT FROM AUTHOR]

p27Kip1 (p27) regulates the cell cycle by inhibiting G1 progression in cells. Several studies have shown conflicting results on the effects of p27 against cell death in various insults. In the present study, we examined the neuroprotective effects of p27 against H2O2-induced oxidative stress in NSC34 cells and against spinal cord ischemia-induced neuronal damage in rabbits. To promote delivery into NSC34 cells and motor neurons in the spinal cord, Tat-p27 fusion protein and its control protein (Control-p27) were synthesized with or without Tat peptide, respectively. Tat-p27, but not Control-27, was efficiently introduced into NSC34 cells in a concentration- and time-dependent manner, and the protein was detected in the cytoplasm. Tat-p27 showed neuroprotective effects against oxidative stress induced by H2O2 treatment and reduced the formation of reactive oxygen species, DNA fragmentation, and lipid peroxidation in NSC34 cells. Tat-p27, but not Control-p27, ameliorated ischemia-induced neurological deficits and cell damage in the rabbit spinal cord. In addition, Tat-p27 treatment reduced the expression of α-synuclein, activation of microglia, and release of pro-inflammatory cytokines such as interleukin-1β and tumor necrosis factor-α in the spinal cord. Taken together, these results suggest that Tat-p27 inhibits neuronal damage by decreasing oxidative stress, α-synuclein expression, and inflammatory responses after ischemia. [ABSTRACT FROM AUTHOR]

Introduction: We examined the effects of exogenous protein disulfide isomerase A3 (PDIA3) on hippocampal neurogenesis in gerbils under control and ischemic damage. Methods: To facilitate the delivery of PDIA3 to the brain, we constructed Tat‐PDIA3 protein and administered vehicle (10% glycerol) or Tat‐PDIA3 protein once a day for 28 days. On day 24 of vehicle or Tat‐PDIA3 treatment, ischemia was transiently induced by occlusion of both common carotid arteries for 5 min. Results: Administration of Tat‐PDIA3 significantly reduced ischemia‐induced spontaneous motor activity, and the number of NeuN‐positive nuclei in the Tat‐PDIA3‐treated ischemic group was significantly increased in the CA1 region compared to that in the vehicle‐treated ischemic group. Ki67‐ and DCX‐immunoreactive cells were significantly higher in the Tat‐PDIA3‐treated group compared to the vehicle‐treated control group. In vehicle‐ and Tat‐PDIA3‐treated ischemic groups, the number of Ki67‐ and DCX‐immunoreactive cells was significantly higher as compared to those in the vehicle‐ and Tat‐PDIA3‐treated control groups, respectively. In the dentate gyrus, the numbers of Ki67‐immunoreactive cells were comparable between vehicle‐ and Tat‐PDIA3‐treated ischemic groups, while more DCX‐immunoreactive cells were observed in the Tat‐PDIA3‐treated group. Transient forebrain ischemia increased the expression of phosphorylated cAMP‐response element‐binding protein (pCREB) in the dentate gyrus, but the administration of Tat‐PDIA3 robustly increased pCREB‐positive nuclei in the normal gerbils, but not in the ischemic gerbils. Brain‐derived neurotrophic factor (BDNF) mRNA expression was significantly increased in the Tat‐PDIA3‐treated group compared to that in the vehicle‐treated group. Transient forebrain ischemic increased BDNF mRNA levels in both vehicle‐ and Tat‐PDIA3‐treated groups, and there were no significant differences between groups. Conclusions: These results suggest that Tat‐PDIA3 enhances cell proliferation and neuroblast numbers in the dentate gyrus in normal, but not in ischemic gerbils, by increasing BDNF mRNA and phosphorylation of pCREB. [ABSTRACT FROM AUTHOR]

Abstract Ischemia causes oxidative stress in the endoplasmic reticulum (ER), accelerates the accumulation of unfolded and misfolded proteins, and may ultimately lead to neuronal cell apoptosis. In the present study, we investigated the effects of protein disulfide-isomerase A3 (PDIA3), an ER-resident chaperone that catalyzes disulfide-bond formation in a subset of glycoproteins, against oxidative damage in the hypoxic HT22 cell line and against ischemic damage in the gerbil hippocampus. We also confirmed the neuroprotective effects of PDIA3 by using PDIA3-knockout HAP1 cells. The HT22 and HAP1 cell lines showed effective (dose-dependent and time-dependent) penetration and stable expression of the Tat-PDIA3 fusion protein 24 h after Tat-PDIA3 treatment compared to that in the control-PDIA3-treated group. We observed that the fluorescence for both 2′,7′-dichlorofluorescein diacetate (DCF-DA) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), which are markers for the formation of hydrogen peroxide (H 2 O 2)-induced reactive oxygen species and apoptosis, respectively, was higher in HAP1 cells than in HT22 cells. The administration of Tat-PDIA3 significantly reduced the (1) DCF-DA and TUNEL fluorescence in HT22 and HAP1 cells, (2) ischemia-induced hyperactivity that was observed 1 day after ischemia/reperfusion, (3) ischemia-induced neuronal damage and glial (astrocytes and microglia) activation that was observed in the hippocampal CA1 region 4 days after ischemia/reperfusion, and (4) lipid peroxidation and nitric oxide generation in the hippocampal homogenates 3–12 h after ischemia/reperfusion. Transient forebrain ischemia significantly elevated the immunoglobulin-binding protein (BiP) and C/EBP-homologous protein (CHOP) mRNA levels in the hippocampus at 12 h and 4 days after ischemia, relative to those in the time-matched sham-operated group. Administration of Tat-PDIA3 ameliorated the ischemia-induced upregulation of BiP mRNA levels versus the Tat peptide- or control-PDIA3-treated groups, and significantly reduced the induction of CHOP mRNA levels, at 12 h or 4 days after ischemia. Collectively, these results suggest that Tat-PDIA3 acts as a neuroprotective agent against ischemia by attenuating oxidative damage and blocking the apoptotic pathway that is related to the unfolded protein response in the ER. Highlights • Tat-PDIA3 can efficiently transduce into the hippocampal cells and is stably expressed within the cells. • Tat-PDIA3 strongly ameliorates H 2 O 2 -induced neuronal death and reactive oxygen species formation in the hippocampal cells. • Tat-PDIA3 protects neurons from ischemic damage in the gerbil hippocampus by reducing the lipid peroxidation, nitric oxide generation, and CHOP expression. [ABSTRACT FROM AUTHOR]

Purpurin, an anthraquinone, has potent anti-oxidant and anti-inflammatory effects in various types of brain damage. In a previous study, we showed that purpurin exerts neuroprotective effects against oxidative and ischemic damage by reducing pro-inflammatory cytokines. In the present study, we investigated the effects of purpurin against D-galactose-induced aging phenotypes in mice. Exposure to 100 mM D-galactose significantly decreased cell viability in HT22 cells, and purpurin treatment significantly ameliorated the reduction of cell viability, formation of reactive oxygen species, and lipid peroxidation in a concentration-dependent manner. Treatment with 6 mg/kg purpurin significantly improved D-galactose-induced memory impairment in the Morris water maze test in C57BL/6 mice and alleviated the reduction of proliferating cells and neuroblasts in the subgranular zone of the dentate gyrus. In addition, purpurin treatment significantly mitigated D-galactose-induced changes of microglial morphology in the mouse hippocampus and the release of pro-inflammatory cytokines such as interleukin-1β, interleukin-6, and tumor necrosis factor-α. In addition, purpurin treatment significantly ameliorated D-galactose-induced phosphorylation of c-Jun N-terminal kinase and cleavage of caspase-3 in HT22 cells. These results suggest that purpurin can delay aging by reducing the inflammatory cascade and phosphorylation of the c-Jun N-terminal in the hippocampus. • Purpurin ameliorates D-galactose-induced cell death, ROS formation, and lipid peroxidation in HT22 cells. • Purpurin alleviates impairments in memory formation and hippocampal neurogenesis induced by D-galactose treatment. • Purpurin decreases pro-inflammatory cytokine releases and JNK phosphorylation in D-galactose-treated mice. [ABSTRACT FROM AUTHOR]

Phosphoglycerate mutase 5 (PGAM5), a glycolytic enzyme, plays an important role in cell death and regulation of mitochondrial dynamics. In this study, we investigated the effects of PGAM5 on oxidative stress in HT22 hippocampal cells and ischemic damage in the gerbil hippocampus to elucidate the role of PGAM5 in oxidative and ischemic stress. Constructs were designed with a PEP-1 expression vector to facilitate the intracellular delivery of PGAM5 proteins. We observed time- and concentration-dependent increases in the intracellular delivery of the PEP-1-PGAM5 protein, but not its control protein (PGAM5), in HT22 cells, and morphologically demonstrated the localization of the transduced protein, which was stably expressed in the cytoplasm after 12 h of PEP-1-PGAM5 treatment. PEP-1-PGAM5 treatment significantly ameliorated cell death, reactive oxygen species formation, DNA fragmentation, and the reduction of cell proliferation induced by H 2 O 2 treatment in HT22 cells. In addition, PEP-1-PGAM5 was effectively delivered to the gerbil hippocampus 8 h after treatment, and ischemia-induced hyperlocomotion and neuronal death in the hippocampal CA1 region were significantly alleviated 1 and 4 days after ischemia, respectively. Ischemia-induced microglial activation was also mitigated by treatment with 1.0 mg/kg PEP-1-PGAM5. At 3 h after ischemia, PEP-1-PGAM5 treatment significantly ameliorated the increase in lipid peroxidation, as assessed by malondialdehyde and hydroperoxide levels, and decreased glutathione levels (increases in glutathione disulfide, the oxidized form of glutathione) in the hippocampus. Two days after ischemia, treatment with PEP-1-PGAM5 significantly alleviated the ischemia-induced reduction in glutathione peroxidase activity and further increased superoxide dismutase activity in the hippocampus. The neuroprotective effects of PEP-1-PGAM5 are partially mediated by a reduction in oxidative stress, such as the formation of reactive oxygen species, and increases in the activity of antioxidants such as glutathione peroxidase and superoxide dismutase. • PEP-1-PGAM5 is delivered into HT22 cells with time- and concentration-dependent manner. • PEP-1-PGAM5 alleviates H 2 O 2 -induced ROS formation, DNA fragmentation, and cell death in HT22 cells. • PEP-1-PGAM5 ameliorates ischemic neuronal death in the hippocampus by reducing lipid peroxidation and increasing glutathione. [ABSTRACT FROM AUTHOR]

Abstract: Response to cerebral ischemia in young animals was very different from that in the adult. The aim of this study was to investigate differences in neuronal death and gliosis in the hippocampal CA1 region (CA1) between adult and young gerbils following 5 and 15min of transient cerebral ischemia. Delayed neuronal death (DND) of pyramidal cells occurred in the CA1 was similar in all the adult gerbils after 5 and 15min of ischemia: the DND occurred 4days after ischemia. In the young groups, DND of pyramidal cells in the CA1 region occurred 7 and 3days after 5 and 15min of ischemia, respectively. On the other hand, the activation of GFAP-immunoreactive (+) astrocytes and Iba-1+ microglia was different in the young groups from the adult groups after ischemia. The change pattern of GFAP immunoreactivity in the adult groups was similar in both the adult groups after ischemia; in the young groups, the activation of GFAP+ astrocytes after 5min of ischemia was much delayed than that after 15min of ischemia. Activated Iba-1+ microglia were aggregated in the stratum pyramidale 4days after ischemia in all the adult ischemia-operated groups; in the young groups, activated Iba-1+ microglia were aggregated in the stratum pyramidale 7days after 5min of ischemia and 3days after 15min of ischemia. These observations indicate that DND in young animals is very different from the adult according to different duration of transient cerebral ischemia and glial activation is very different in young animals after different duration of transient ischemia. [Copyright &y& Elsevier]

Abstract: Objective: Because of atlantoaxial complex has a unique and complicated anatomy and instability of this complex is very dangerous. We investigated the clinical results of posterior C1–C2 fixation with a polyaxial screw–rod system. Methods: Between July 2001 and December 2007, the authors treated 17 patients suffering from atlantoaxial deformity and instability. Atlantoaxial fusion was employed in 9 patients with upper cervical fracture and dislocation, in 6 patients with atlantoaxial subluxation, in 1 patient with pure transverse ligament injury, and in 1 patient with basilar invagination. The mean age at the time of surgery was 40.4 years (range, 15–68 years). Results: Operative times ranged from 165 to 420min (average 306min), and the postoperative mean VAS score was 2.4. The mean follow-up period was 26 months. Solid fusion was achieved in 15 patients at the last follow up; no injury of the vertebral artery or spinal cord and no operative mortality occurred in these cases. Conclusions: We suggest that posterior atlantoaxial fixation using the polyaxial screw–rod system is an effective and relatively safe technique. The navigation guidance system employed during the surgical procedure was helpful methods. Future studies of the feasibility of navigation system-guided surgical procedures will be required. [Copyright &y& Elsevier]

Abstract: Transplantation of adipose-derived stem cells (ASCs) is one of the possible therapeutic tools for ischemic damage. In this study, we observed the effects of ASCs against ischemic damage in the ventral horn of L5–6 levels in the rabbit spinal cord. ASCs were isolated from rabbits, and cell type was confirmed by flow cytometry analysis, labeling with CM-DiI dye and differentiation into adipocytes in adipogenesis differentiation medium. ASCs were administered intrathecally into recipient rabbits (2×105) immediately after reperfusion following a 15-min aortic artery occlusion in the subrenal region. Transplantation of ASCs significantly improved functions of the hindlimb and morphology of the ventral horn of spinal cord although CM-DiI-labeled ASCs were not observed in the spinal cord parenchyma. In addition, transplantation of ASCs significantly increased brain-derived neurotrophic factor (BDNF) levels at 72h after ischemia/reperfusion. These results suggest that transplantation of ASCs prevents motor neurons from spinal ischemic damage and reactive gliosis by increasing neurotrophic factors such as BDNF in the spinal cord. [Copyright &y& Elsevier]

Abstract: Cyclooxygenase (COX) is a rate-limiting enzyme in synthesis of prostaglandins from arachidonic acid. In this study, we observed the effects of a physical exercise on COX-2 immunoreactivity in the hippocampus using immunohistochemistry in rats. In addition, we examined effects of administration of a COX-2 inhibitor, celecoxib, on neuroblast differentiation. At 6weeks of age, Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats were put on a treadmill with or without running for 1h/session/day for 5weeks. The running speed was gradually increased from 16 to 22m/min with 2m/min per 2weeks. In the ZLC and ZDF rats, COX-2 immunoreaction was detected in the granule cell layer of the dentate gyrus and in the stratum pyramidale of the CA2/3 region; COX-2 immunoreaction in the CA1 region was hardly detected. In the exercised-ZLC and ZDF rats, COX-2 immunoreactivity was significantly increased compared to that in the ZLC and ZDF rats, showing that COX-2 immunoreactivity in the exercised-ZDF rats was slightly low than that in the exercised-ZDF rats. In addition, weak COX-2 immunoreactivity was shown in the CA1 region by exercise. On the other hand, the repeated oral administration of celecoxib to 4-week-old ZDF rats significantly decreased the neuroblasts in the subgranular zone of the dentate gyrus. These results suggest that COX-2 may be associated with the increase of synaptic plasticity or contacts in the hippocampus. [Copyright &y& Elsevier]

Abstract: In this study, we examined transient ischemia-induced changes in transcription factor E2F1 and c-myb expressions in the gerbil hippocampus after 5 min of transient forebrain ischemia. E2F1 immunoreactivity significantly increased in the CA1 region 6–12 h after ischemia/reperfusion. c-myb immunoreactivity increased mainly in CA1 pyramidal cells with time by 12 h after ischemia. Thereafter, E2F1 and c-myb immunoreactivities significantly decreased compared to those in the 12 h post-ischemic group. Four days after ischemia/reperfusion, E2F1 and c-myb immunoreactivities were detected in non-pyramidal cells. Ten days after ischemia, c-myb immunoreactivity increased again: at this time, astrocytes as well as non-pyramidal cells showed E2F1 and c-myb immunoreactivities. In the CA2/3 region, E2F1 and c-myb immunoreactivities mainly changed in non-pyramidal cells, and 10 days after ischemia, c-myb immunoreactivity was not expressed in astrocytes. In conclusion, E2F1 and c-myb significantly alter in pyramidal cells and express in astrocytes in the gerbil hippocampal CA1 region after transient ischemia. These results indicate that E2F1 and c-myb in the CA1 region after ischemic damage may be associated with delayed neuronal death. [Copyright &y& Elsevier]

Abstract: Much evidence has been accumulated that the increased expression of calbindin D-28k (CB) is involved in the blockade of calcium-evoked excitotoxicity in cerebral ischemia. We investigated the expression of CB in the basal lamina of microvessels in the ventral horn of the rabbit spinal cord after transient spinal cord ischemia. Spinal cord sections at the level of L7 were immunostained using monoclonal antibody raised against CB at light and electron microscopic levels. CB immunoreactivity was detected in the basal lamina of microvessels at 30 min after ischemic insult. By 3 h after ischemia, CB immunoreactivity was increased in the basal lamina of the microvessels. CB immunoreactivity began to decrease at 6 h after ischemia and nearly disappeared at 48 h after ischemic insult. For calcium detection in the blood vessels of spinal cord, we conducted an alizarin red staining. Alizarin red reactivity was detected in some microvessels at 3 h after ischemic insult. Our results suggest that the ectopic expression of CB in the microvascular basal laminae may be associated with the buffering of calcium in the endothelial cells of microvessels after ischemic damage. [Copyright &y& Elsevier]

We examined ischemia-related changes of calbindin D-28k (CB) immunoreactivity in L7 of the spinal ventral horn after transient spinal cord ischemia in rabbits. In the sham-operated group, CB immunoreactivity was not present in the spinal ventral horn, but CB immunoreactivity was detectable in the dorsal horn. CB immunoreactivity was detectable in the ventral horn at 30min after ischemia: the CB immunoreactivity was found in glial cells identified as astrocytes. At 1h after ischemia, CB immunoreactivity was highest and present at a few somata located in the lamina VII as well as many glial cells. CB immunoreactivity was lower in the lamina VII at 3h after ischemia compared to 1h post-ischemic group. By 2 days after ischemia, CB immunoreactivity was decreased in this region. In addition, the result of Western blot result showed the pattern of CB expression similar to that of immunohistochemistry. In conclusion, the ischemia-related changes of CB immunoreactivity in neurons and glial cells in the ischemic spinal ventral horn in rabbits may be related to modulation of intracellular calcium following transient ischemia. [Copyright &y& Elsevier]

The present study explored the effects of endophilin A1 (SH3GL2) against oxidative damage brought about by H2O2 in HT22 cells and ischemic damage induced upon transient forebrain ischemia in gerbils. Tat-SH3GL2 and its control protein (Control-SH3GL2) were synthesized to deliver it to the cells by penetrating the cell membrane and blood–brain barrier. Tat-SH3GL2, but not Control-SH3GL2, could be delivered into HT22 cells in a concentration- and time-dependent manner and the hippocampus 8 h after treatment in gerbils. Tat-SH3GL2 was stably present in HT22 cells and degraded with time, by 36 h post treatment. Pre-incubation with Tat-SH3GL2, but not Control-SH3GL2, significantly ameliorated H2O2-induced cell death, DNA fragmentation, and reactive oxygen species formation. SH3GL2 immunoreactivity was decreased in the gerbil hippocampal CA1 region with time after ischemia, but it was maintained in the other regions after ischemia. Tat-SH3GL2 treatment in gerbils appreciably improved ischemia-induced hyperactivity 1 day after ischemia and the percentage of NeuN-immunoreactive surviving cells increased 4 days after ischemia. In addition, Tat-SH3GL2 treatment in gerbils alleviated the increase in lipid peroxidation as assessed by the levels of malondialdehyde and 8-iso-prostaglandin F2α and in pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and interleukin-6; while the reduction of protein levels in markers for synaptic plasticity, such as postsynaptic density 95, synaptophysin, and synaptosome associated protein 25 after transient forebrain ischemia was also observed. These results suggest that Tat-SH3GL2 protects neurons from oxidative and ischemic damage by reducing lipid peroxidation and inflammation and improving synaptic plasticity after ischemia. [ABSTRACT FROM AUTHOR]

Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that increases glycolytic flux in the brain. In the present study, we examined the effects of PGAM1 in conditions of oxidative stress and ischemic damage in motor neuron-like (NSC34) cells and the rabbit spinal cord. A Tat-PGAM1 fusion protein was prepared to allow easy crossing of the blood-brain barrier, and Control-PGAM1 was synthesized without the Tat peptide protein transduction domain. Intracellular delivery of Tat-PGAM1, not Control-PGAM1, was achieved in a time- and concentration-dependent manner. Immunofluorescent staining confirmed the intracellular expression of Tat-PGAM1 in NSC34 cells. Tat-PGAM1, but not Control-PGAM1, significantly alleviated H2O2-induced oxidative stress, neuronal death, mitogen-activated protein kinase, and apoptosis-inducing factor expression in NSC34 cells. After ischemia induction in the spinal cord, Tat-PGAM1 treatment significantly improved ischemia-induced neurological impairments and ameliorated neuronal cell death in the ventral horn of the spinal cord 72 h after ischemia. Tat-PGAM1 treatment significantly mitigated the ischemia-induced increase in malondialdehyde and 8-iso-prostaglandin F2α production in the spinal cord. In addition, Tat-PGAM1, but not Control-PGAM1, significantly decreased microglial activation and secretion of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α induced by ischemia in the ventral horn of the spinal cord. These results suggest that Tat-PGAM1 can be used as a therapeutic agent to reduce spinal cord ischemia-induced neuronal damage by lowering the oxidative stress, microglial activation, and secretion of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α. [ABSTRACT FROM AUTHOR]

In a previous study, we utilized a proteomic approach and found a significant reduction in phosphatidylethanolamine-binding protein 1 (PEBP1) protein level in the spinal cord at 3 h after ischemia. In the present study, we investigated the role of PEBP1 against oxidative stress in NSC34 cells in vitro, and ischemic damage in the rabbit spinal cord in vivo. We generated a PEP-1-PEBP1 fusion protein to facilitate the penetration of blood-brain barrier and intracellular delivery of PEBP1 protein. Treatment with PEP-1-PEBP1 significantly decreased cell death and the induction of oxidative stress in NSC34 cells. Furthermore, administering PEP-1-PEBP1 did not show any significant side effects immediately before and after ischemia/reperfusion. Administration of PEP-PEBP1 improved the Tarlov's neurological score at 24 and 72 h after ischemia, and significantly improved neuronal survival at 72 h after ischemia based on neuronal nuclei (NeuN) immunohistochemistry, Flouro-Jade B staining, and western blot study for cleaved caspase 3. PEP-1-PEBP1 administration decreased oxidative stress based on malondialdehyde level, advanced oxidation protein products, and 8-iso-prostaglandin F2α in the spinal cord. In addition, inflammation based on myeloperoxidase level, tumor necrosis factor-α level, and high mobility group box 1 level was decreased by PEP-1-PEBP1 treatment at 72 h after ischemia. Thus, PEP-1-PEBP1 treatment, which decreases oxidative stress, inflammatory cytokines, and neuronal death, may be an effective therapeutic strategy for spinal cord ischemia. [ABSTRACT FROM AUTHOR]

Heat shock protein 70 (HSP70) is an ATP-dependent molecular chaperone, and it has been shown that its levels increase after exposure to various types of stress, including ischemia. In the present study, we investigated the effects of HSP70 against H 2 O 2 -induced neuronal stress in NSC34 cells and against spinal cord ischemia in rabbits. Tat-HSP70 proteins facilitated the intracellular delivery of HSP70 into the NSC34 cells and enabled them to cross the blood-brain barrier in the rabbit spinal cord. Tat-HSP70 was effectively transduced into NSC34 cells in a concentration- and time-dependent manner, while control-HSP70 protein could not be delivered intracellularly at any concentration or time after treatment. Treatment with Tat-HSP70 reduced the generation of reactive oxygen species and cell death induced by H 2 O 2 , while the control-HSP70 did not show any significant effect on the NSC34 cells exposed to H 2 O 2. In rabbit spinal cord, the administration of Tat-HSP70 showed significant amelioration of neurological defects and neuronal death in the ventral horn of spinal cord. In addition, Tat-HSP70 treatment significantly reduced lipid peroxidation and increased Cu, Zn-superoxide dismutase activities in the spinal cord, but glutathione peroxidase and Mn-superoxide dismutase activities remained unchanged. These results suggest that Tat-HSP70, not control-HSP70, decreases cell damage by reducing oxidative stress in NSC34 cells and rabbit spinal cord, and it can be employed for the reduction of neuronal damage caused after spinal cord ischemia. • Tat-HSP70 enables to efficiently transduce into the NSC34 cells. • Tat-HSP70 reduces the H 2 O 2 -induced neuronal death and oxidative damage in the NSC34 cells. • Tat-HSP70 protects motor neurons from spinal cord ischemia by reducing the lipid peroxidation. [ABSTRACT FROM AUTHOR]