Alpha Tocopherol Loaded Polymeric Nanoparticles: Preparation, Characterizations, and In Vitro Assessments Against Oxidative Stress in Spinal Cord Injury Treatment.

Spinal cord injury (SCI) is characterized by mechanical injury or trauma to the spinal cord. Currently, SCI treatment requires extremely high doses of neuroprotective agents, which in turn, causes several adverse effects. To overcome these limitations, the present study focuses on delivery of a low but effective dose of a naturally occurring antioxidant, α-tocopherol (α-TP). Calcium alginate nanoparticles (CA-NP) and poly D,L-lactic-co-glycolic acid nanoparticles (PLGA-NP) prepared by ionotropic gelation and solvent evaporation technique had particle size of 21.9 ± 11.19 and 152.4 ± 10.6 nm, respectively. Surface morphology, surface charge, as well as particle size distribution of both nanoparticles were evaluated. Entrapment of α-TP into CA-NP and PLGA-NP quantified by UPLC showed entrapment efficiency of 4.00 ± 1.63% and 76.6 ± 11.4%, respectively. In vitro cytotoxicity profiles on human astrocyte-spinal cord (HA-sp) showed that blank CA-NP at high concentrations reduced the cell viability whereas blank PLGA-NP showed relatively safer cytotoxic profiles. In addition, PLGA nanoparticles encapsulated with α-TP (α-TP-PLGA-NP) in comparison to α-TP alone at high concentrations were less toxic. Pretreatment of HA-sp cells with α-TP-PLGA-NP showed two-fold higher anti-oxidative protection as compared to α-TP alone, when oxidative stress was induced by H2O2. In conclusion, CA-NP were found to be unsuitable for treatment of SCI due to their cytotoxicity. Comparatively, α-TP-PLGA-NP were safer and showed high degree of protection against oxidative stress than α-TP alone.

Regulation of Prdx6 by Nrf2 Mediated Through aiPLA2 in White Matter Reperfusion Injury.

Hypoxia and reperfusion produces overproduction of ROS (reactive oxygen species), which may lead to mitochondrial dysfunction leading to cell death and apoptosis. Here, we explore the hypothesis that Prdx6 protects the spinal cord white matter from hypoxia-reperfusion injury and elucidate the possible mechanism by which Prdx6 elicits its protective effects. Briefly, rats were deeply anesthetized with isoflurane. A 30-mm section of the spinal cord was rapidly removed and placed in cold Ringer's solution (2-4 °C). The dissected dorsal column was exposed to hypoxia with 95% N2 and 5% CO2 and reperfusion with 95% O2 and 5% CO2. The expression of Prdx6 significantly upregulated in white matter after hypoxia compared to the sham group, whereas reperfusion caused a gradual decrease in Prdx6 expression after reperfusion injury. For the first time, our study revealed the novel expression and localized expression of Prdx6 in astrocytes after hypoxia, and possible communication of astrocytes and axons through Prdx6. The gradual increase in Nrf2 expression suggests a negative regulation of Prdx6 through Nrf2 signaling. Furthermore, inhibition of aiPLA2 activity of Prdx6 by MJ33 shows that the regulation of Prdx6 by Nrf2 is mediated through aiPLA2 activity. The present study uncovers a differential distribution of Prdx6 in axons and astrocytes and regulation of Prdx6 in hypoxia-reperfusion injury. The low levels of Prdx6 in reperfusion injury lead to increased inflammation and apoptosis in the white matter; therefore, the results of this study suggest that Prdx6 has a protective role in spinal hypoxia-reperfusion injury.

Curcumin Protects against White Matter Injury through NF-κB and Nrf2 Cross Talk.

Inflammation and oxidative stress play a central role in the pathogenesis of white matter injury (WMI). Curcumin (Cur), a polyphenolic compound, exhibits anti-inflammatory and anti-oxidant effects on several conditions. The objective of this study was to investigate neuroprotective effects of Cur on WMI and explore its underlying mechanisms of action. Sprague-Dawley rats were subjected to the removal of white matter from the dorsal column of the spinal cord. Dorsal columns were randomly divided into three groups: Sham (Ringer's solution bubbled with 95% O2 and 5% CO2), hypoxia (Hyp; Ringer's solution bubbled with 95% N2 and 5% CO2 for 1 h), and Cur-treated (Hyp+Cur; Ringer's solution bubbled with 95% N2 and 5% CO2 for 1 h in the presence of 50 µM Cur). For NF-κB inhibition experiments, dorsal columns were incubated with 50 µM BAY 11-7082 (BAY) for 30 min in 95% O2 and 5% CO2 prior to 1-h incubation with 50 µM Cur in 95% N2 and 5% CO2. Our data show that Cur inhibited hypoxia-induced HIF1-α expression and tissue damage by demonstrating the improved morphology of astrocytes and remarkable reduction in vacuolation. Cur also inhibited the hypoxia-induced upregulation of glial fibrillary acidic protein (GFAP) and neurofilament-H (NF-H) after hypoxia and downregulated the expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 1 (IL-1). Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-assay analysis showed that Cur effectively attenuated apoptosis in white matter. In addition, we demonstrated that Cur exerted its neuroprotective effect through cross talk between nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. In conclusion, our results indicate that treatment with Cur inhibited the hypoxia, inflammation and apoptosis associated with WMI. Further, the Nrf-2 pathway inhibits NF-κB activation by preventing IkB degradation and increasing HO-1 expression, which in turn reduces reactive oxygen species (ROS) and as a result NF-κB activation is suppressed. Similarly, NF-κB-mediated transcription reduces Nrf2 activation by reducing anti-oxidant response element (ARE) gene and free CREB binding protein by competing with Nrf2 for CBP thus inhibiting the Nrf-2 activation.

Neuroprotective effects of Riluzole and Curcumin in human astrocytes and spinal cord white matter hypoxia.

Damage to the spinal cord (SC) can result in irreversible impairments or complete loss of motor, sensory, and autonomic functions. Riluzole, a sodium channel-blocker and glutamate inhibitor, is in preclinical use for SC injury (SCI), and curcumin is an intracellular calcium inhibitor that attenuates glutamate-induced neurotoxicity. As riluzole and curcumin have different mechanisms to protect against SCI, we aimed to investigate the neuroprotective effects of a combination of riluzole and curcumin in human astrocytes and white matter injury (WMI) model of SCI. Our data show that a combination of riluzole (1 µM) and curcumin (1 µM) was effective in inhibiting hydrogen peroxide (H2O2)-induced oxidative dress in astrocytes derived from human SC, however, curcumin alone showed a significant inhibition. In addition, our results demonstrated that curcumin alone downregulates the hypoxia-induced expression of HIF-1, GFAP, and NF-H proteins in WMI, whereas riluzole alone and in combination with curcumin remained ineffective in changing the expression of these proteins. Contrarily, after inhibiting Ca2+ influx with EGTA, riluzole alone and in combination with curcumin significantly downregulated hypoxia-induced expression of GFAP and NF-H. After analysis of caspase 9 and cleaved caspase 9, we observed that curcumin and riluzole both inhibit apoptosis significantly, whereas their combination remains ineffective. Furthermore, we observed that neuroprotective effects of curcumin and riluzole are mediated through Nrf2/HO-1 signaling. In conclusion, our results demonstrate that curcumin and riluzole protect astrocytes from oxidative stress and white matter from hypoxia. However, their combination is not beneficial to reduce hypoxia-induced astrocytosis, axonal damage, and apoptosis. From our results, it is evident that curcumin is more effective in reducing WMI than riluzole.

Review of psychiatric comorbidities and their associations with opioid use in elective lumbar spine surgery.

The opioid epidemic is an ongoing concern in the United States and efforts to ameliorate this crisis are underway on multiple fronts. Opiate use is an important consideration for patients undergoing lumbar spine surgery with concurrent psychiatric diagnoses and more information is needed regarding the factors involved in these patients. That information may help guide opioid prescribing practices for individual patients with certain psychiatric conditions that are undergoing these procedures. This study was done to identify psychiatric conditions that are associated with preoperative and postoperative opioid use in this cohort of veteran patients undergoing elective lumbar spine surgery.A 3 month preoperative and 3 month postoperative chart review was conducted on 25 patients per year who underwent elective lumbar spine surgery over a 16-year period at the Veterans Affairs Nebraska-Western Iowa Healthcare Center (n = 376 after exclusion criteria applied). The association between psychiatric comorbidities and use of opioids during the 90-day period after surgery was assessed using a linear model that adjusted for surgical type, opioid use prior to surgery, and other relevant comorbidities.Patients are more likely to use opioids preoperatively if they have major depression (P = .02), hepatitis C (P = .01), or musculoskeletal disorders (P = .04). PTSD (P = .02) and lumbar fusion surgery (P < .0001) are associated with increased postoperative use, after adjusting for preoperative use and other comorbidities.Certain psychiatric comorbidities are significantly correlated with opioid use for this cohort of lumbar spine surgery patients in the preoperative and postoperative periods. Awareness of an individual's psychiatric comorbidity burden may help guide opioid prescription use.

Pre and post treatment with curcumin and resveratrol protects astrocytes after oxidative stress.

The two most studied polyphenolic compounds, curcumin (Cur) and resveratrol (Res), have been reported to protect oxidative damage of astrocytes. The present study is designed to examine the comparative anti-oxidative effect of Cur and Res on astrocytes by studying their potential to protect H2O2 induced oxidative stress at 4 h and 24 h time exposure. The effect of Cur and Res on cell viability, ROS production, inflammation and astrogliosis was compared. The effect of these two on Nrf2 expression and its translocation to nuclear compartment was investigated. The results showed that both Cur and Res significantly increase astrocytes survival after oxidative stress at both time points, however, Res demonstrated better effect on cell viability than the Cur. Res, showing significant inhibition of ROS production at both time points. Cur displayed significant inhibition of ROS production at 4 h, suggesting that Cur is more active on ROS inhibition in the earlier phase of insult. Comparing the expression of NF-κB, Cur showed better anti-inflammatory action on NF-κB while Res did not have any effect of NF-κB expression at 4 h. Interestingly, Cur showed an upregulation of nuclear Nrf2 expression at 24 h whereas Res displayed no effect after 24 h incubation. Both Cur and Res inhibited the H2O2 induced translocation of Nrf2 into nucleus. In conclusion, based on our observation, we found that Cur and Res both protected astrocytes from oxidative stress. In addition, we observed that Cur is most effective in early hours of insult while Res is effective in late hours suggesting that Res may or may not have immediate effect on astrocytes.

Curcumin alleviates oxidative stress and mitochondrial dysfunction in astrocytes.

Oxidative stress plays a critical role in various neurodegenerative diseases, thus alleviating oxidative stress is a potential strategy for therapeutic intervention and/or prevention of neurodegenerative diseases. In the present study, alleviation of oxidative stress through curcumin is investigated in A172 (human glioblastoma cell line) and HA-sp (human astrocytes cell line derived from the spinal cord) astrocytes. H2O2 was used to induce oxidative stress in astrocytes (A172 and HA-sp). Data show that H2O2 induces activation of astrocytes in dose- and time-dependent manner as evident by increased expression of GFAP in A172 and HA-sp cells after 24 and 12h respectively. An upregulation of Prdx6 was also observed in A172 and HA-sp cells after 24h of H2O2 treatment as compared to untreated control. Our data also showed that curcumin inhibits oxidative stress-induced cytoskeleton disarrangement, and impedes the activation of astrocytes by inhibiting upregulation of GFAP, vimentin and Prdx6. In addition, we observed an inhibition of oxidative stress-induced inflammation, apoptosis and mitochondria fragmentation after curcumin treatment. Therefore, our results suggest that curcumin not only protects astrocytes from H2O2-induced oxidative stress but also reverses the mitochondrial damage and dysfunction induced by oxidative stress. This study also provides evidence for protective role of curcumin on astrocytes by showing its effects on attenuating reactive astrogliosis and inhibiting apoptosis.

Effect of Protein Concentration on the Flow of Cerebrospinal Fluid Through Shunt Tubing.

BACKGROUND: Protein levels in cerebrospinal fluid (CSF) are commonly thought to be related to sterile shunt malfunction. OBJECTIVE: To investigate the relationship between protein concentration and flow through CSF shunt tubing and a shunt valve. METHODS: New and explanted shunt catheters were tested with and without a shunt valve attached at various protein concentrations. The protein concentrations used were 0.5, 2, 5, and 10 g/L. A flask with artificial CSF attached to the proximal end provided flow. The flow was allowed to stabilize over 1 hour, and then the change in pressure between the proximal and distal end of the catheter was measured and recorded. The resistance to flow was calculated for new and explanted catheters for adult shunt systems, as well as with the addition of a programmable siphon control valve. The resistance was examined after the addition of various protein concentrations to a normal CSF solution. RESULTS: Both new and explanted catheters exhibited a decrease in the resistance to flow with higher concentrations of protein. CONCLUSION: In our laboratory setting, there was decreased resistance of adult CSF shunt catheters with and without a valve as the concentration of protein in the CSF increased. The decrease in the resistance of CSF shunt catheters with the addition of protein to the CSF may be related to the lowering of surface tension. This is the first study to examine the effects of varying protein concentrations across different lengths of shunt tubing for both new and explanted catheters.

Patch-clamp recordings from white matter glia in thin longitudinal slices of adult rat spinal cord.

We developed a technique of whole cell patch-clamp recordings from white matter oligodendrocytes and astrocytes in 200-250 microm-thick horizontal slices of adult (>2 months, 240-260 g) rat thoracic spinal cord. The viability of the white matter, sectioned in Na(+)-free, low Ca(2+) media, and the function of axons were preserved for >8 h, as demonstrated by the propagation of TTX-sensitive compound action potentials (CAPs) and the sensitivity of their refractory period to K(+) channel blocker 4-aminopyridine (1 microM). Glial cells were visually identified within the slices with a 40 x water immersion objective using infra-red differential interference contrast (IR-DIC) video microscopy, and the details of their morphology were further elucidated after filling the cells with Lucifer Yellow or Alexa 350 fluorescent dyes during whole-cell recording. Using voltage steps and ramps, we revealed pronounced non-linearity of I-V relationships in both oligodendrocytes and astrocytes. Both types of cells expressed TEA-sensitive outward delayed rectifier-type currents activated at positive voltages but showed little, if any, signs of inward rectification at voltages up to -140 mV. At -70 mV holding voltage, bath-applied kainic acid (100 microM) activated inward currents in both types of cells. This novel horizontal slice preparation of adult rat thoracic cord will facilitate the examination of mature glial cell physiology, glial-axonal signaling and the pathophysiology of spinal cord trauma and ischemia.

Role of Na(+)-Ca(2+) exchanger after traumatic or hypoxic/ischemic injury to spinal cord white matter.

BACKGROUND CONTEXT: Spinal cord injury is a devastating condition in which clinical disability results from demyelination of white matter tracts. Changes in glial-axonal signaling, and enhanced Ca(2+) channel activity with excessive accumulation of intracellular Ca(2+), is a common phenomenon after hypoxia/ischemia or mechanical trauma to spinal cord dorsal column white matter tracts leading to irreversible injury. PURPOSE: In the present study we examined the role of Na(+)-Ca(2+) exchanger (NCX) at physiological temperatures after hypoxia/ischemia and compressive injury to spinal cord dorsal column white matter in vitro. STUDY DESIGN: A 30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber (maintained at 37 degrees C) and injured by exposure to a hypoxic atmosphere for 60 minutes or compressed with a modified aneurysm clip (2-gm closing force) for 15 seconds. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential (CAP) with glass microelectrodes. RESULTS: The mean CAP decreased to 49.5 +/- 5.7% and 49.4 +/- 2.6% of control (p<.05) after hypoxia/ischemia and compressive injury, respectively. KB-R7943, a potent, selective NCX reverse mode inhibitor, significantly promoted greater recovery of CAP amplitude to 82.0 +/- 10.0% and 70.8 +/- 10.7% of control (p<.05) after hypoxic/ischemic or compressive injury to dorsal column white matter, respectively, when applied at 10 microM concentration. Bepridil (Research Biochemical Inc., Natick, MA, USA) (a less selective NCX inhibitor), when applied at 10 microM and 50 microM concentration promoted CAP amplitude recovery only to 46.8 +/- 7.8% and 29.9 +/- 3.3% of control, respectively, after hypoxic/ischemic injury to dorsal column white matter. Western blot analysis identified NCX presence with positive immunolabeling of 160 kD and 120 kD NCX proteins in the spinal cord white matter. CONCLUSION: In conclusion, at physiological temperature NCX activation plays an important role in intracellular calcium overload after hypoxic/ischemic and compressive injury to spinal cord dorsal column white matter in vitro.

Role of calcineurin in calcium-mediated hypoxic injury to white matter.

BACKGROUND CONTEXT: Calcium influx into cells is responsible for initiating the "final pathway" to cell death in neuronal tissue after traumatic or hypoxic injury. The specific pathways in this cascade are myriad and the importance each one plays is controversial. It is clear, though, that blocking individual pathways confers protection to these tissues. PURPOSE: In the present study we examined the role of Cyclosporin A (CsA), FK-506 and rapamycin in modulating the effects of Ca(2+) influx through their interactions with immunophilins and specifically the end result of calcineurin modulation. METHODS: Dorsal columns were isolated from the spinal cord of adult rats and injured by exposure to hypoxic conditions for 60 minutes. The samples were monitored electrophysiologically in an in vitro recording chamber (maintained at 37 C degrees ) during injury, and the compound action potential (CAP) was monitored with glass microelectrodes. The dorsal column was exposed to hypoxic Ringers solution alone or with the different immunosuppressants and compared with baseline readings. Functional recovery of the dorsal column was then assessed by recovery of the CAP. RESULTS: The mean CAP decreased to about 20% of baseline control levels during hypoxia and returned 53.8+/-7.6% of baseline (p<.05) after reoxygenation. CsA, an immunosuppressant known to inhibit calcineurin, promoted a significantly greater recovery of CAP amplitude to 76.8+/-5.2% and 72.1+/-13.2% of control (p<.05) after hypoxic injury and reoxygenation of dorsal column white matter when applied at concentrations of 1 microM and 10 microM, respectively. FK-506, which also inhibits calcineurin, was applied at a concentration of 0.1 microM, and promoted CAP amplitude recovery to 82.6+/-5.0% of control after hypoxic injury and reoxygenation of dorsal column white matter. The addition of rapamycin (1 microM), which binds to the same immunophilin as FK-506, to the FK-506 (0.1 microM) solution during hypoxic injury showed recovery of CAP amplitudes to only 56.9+/-6.7% of control. Electron microscopy revealed remarkable protection of axons and prevention of organelle disruption in segments treated with CsA and FK-506 during hypoxia when compared with hypoxic controls. CONCLUSION: In conclusion, both CsA and FK-506 confer in vitro protection to dorsal columns during hypoxic injury at physiological temperatures, and rapamycin blocks the protective effect of FK-506. Thus, calcineurin may play an important role in the physiology of neuronal injury.

Fasudil reduces GFAP expression after hypoxic injury.

Fasudil (HA-1077), a specific Rho kinase II (ROCKII) inhibitor, is in clinical trials for recovery from spinal cord injury (SCI). The primary role of Fasudil is in axonal regeneration, as it inhibits ROCKII, the key signaling molecule involved in collapse of axon growth cone. Astrogliosis, due to the activation of astrocytes is an indicator of CNS injury. In early stages of injury, GFAP expression increases, helping to restore the integrity of the CNS. An increase in GFAP expression is also a marker of astrogliosis. Thus, reducing GFAP and hence astrogliosis at later stages of SCI is important for neuroregeneration and functional recovery. CoCl2 was used to induce hypoxic injury in astrocytic cell lines A172 (24h) and in spinal cord dorsal column white matter (8h). Several different techniques were used to study the changes in GFAP expression such as real-time PCR, western blotting and immunofluorescence staining with confocal microscopy. Hypoxia increased the expression of GFAP in A172 cells and in the spinal cord dorsal column after CoCl2 (100µM) treatment for 24h and 8h, respectively. We observed 11 folds increase in protein expression in A172 cells (24h) and 4.5 folds in spinal cord dorsal column (8h). The RNA expression was increased 3 folds in A172 cells after 24h of treatment and 4 folds in spinal cord dorsal column after 8h of treatment with 100µM CoCl2. Treatment with fasudil (20µM) significantly reduces the expression of GFAP in A172 cells and in spinal cord dorsal column. Fasudil also decreased activation of NF-κB in A172 cells after hypoxic injury. In the present study, we observed that fasudil reduces the expression of GFAP (consequently, astrogliosis) after hypoxic injury to A172 cells and spinal cord dorsal column. Our studies demonstrate that fasudil also plays a role in GFAP expression by reducing NF-κB activation at the injury site which could further help in axonal regeneration.

Aneurysm embolization with biologically active coils: an animal study.

OBJECTIVES: Cellular adhesion on the surface of Guglielmi detachable coils (GDC) promotes accelerated occlusion of aneurysms. We hypothesized that coils coated with transforming growth factor beta (TGF-beta) and vascular endothelial growth factor (VEGF) will promote clot organization and endothelial or cellular proliferation to facilitate closure of large experimental aneurysms relative to uncoated GDC coils. METHODS: GDC were inserted either uncoated, coated with albumin, coated with TGF-beta (500 µg/ml) or with VEGF (500 µg/ml) into the ligated common carotid artery of adult male wistar rats for 14 days. Twenty-four adult male Wistar rats (280-300 g) were divided into four groups (n = 6 per group). Subjects were killed 2 weeks after implantation and common carotid artery (CCA) segments were harvested and coils were removed. Arterial tissue was evaluated histopathologically. RESULTS: Significant differences in the proportion of aneurysm luminal area (mm(2)) was shown among Group i (0·22±0·14 SD) and Group ii (0·16±0·8 SD) compared to Group iii (0·04±0·03 SD; P<0·05), and Group iv (0·02±0·02 SD; P<0·05) analyzed 2 weeks postoperatively. Light microscopy showed well organized fibrous tissue formation of epithelial cells and intimal hyperplasia around the coils when using the coated GDC's (Group iii and Group iv). CONCLUSION: The result of this study suggests that the coated GDC with TGF-beta or VEGF appears beneficial in promoting endothelization, clot organization, and cellular tissue integration of the coils.

Upregulation of RyR2 in hypoxic/reperfusion injury.

Calcium influx into cells is responsible for initiating the cell death in neuronal tissue after hypoxic injury. Changes in intracellular calcium with subsequent increased expression of ryanodine receptor 2 (RyR2) are hypothesized to cause cell death after hypoxic injury. In the present study we have examined the time-dependent changes of RyR2 expression in hypoxic/reperfusion injury of spinal cord dorsal column. In this study we used western blotting, real time PCR (RT-PCR) and immunohistochemistry to examine changes in protein and gene expression of RyR2 after spinal cord injury (SCI) in the rat. Quantitative immunoblotting showed increase in the expression of RyR2 at 4 h during hypoxic/reperfusion injury of dorsal column. Moreover, RT-PCR showed 36-fold increases in mRNA of RyR2 after 4 h of hypoxic injury of white matter. By double immunofluorescence staining, RyR2 was localized on axons and astrocytes in the white matter of the spinal cord. After treatment with KN-62; (inhibitor of CaMKII) and SP600125 (inhibitor of JNK), there is a significant reduction in the expression of RyR2, indicating the role of these molecules in RyR2 regulation. Further removal of extracellular calcium does not have significant effect on RyR2 expression and phosphorylation of CaMKII, which was further confirmed by treatment with intracellular Ca(++) chelator BAPTA-AM. Finally, bioassay with quantitative analysis showed that treatment with inhibitor significantly reduced the cellular oxidative stress suggesting RyR2 is responsible for increased cellular oxidative load. In summary, we provide evidence that RyR2 gene and protein expression in astrocyte and axons is markedly increased after hypoxic injury. Further CaMKII/JNK pathway upregulates RyR2 expression after hypoxic injury. Therefore we propose that inhibitors of CaMKII/JNK pathway would reduce the cellular oxidative load and thereby have a neuroprotective role.

Regulation of inositol 1,4,5-triphosphate receptor, type 1 (IP3R1) in hypoxic/reperfusion injury of white matter.

OBJECTIVE: Calcium overloading is responsible for initiating the cell death in neuronal tissue after hypoxic injury. Inositol 1,4,5-triphosphate receptors (IP3Rs) is an important calcium channel which regulates cellular calcium homeostasis. IP3R1 is widely expressed in brain and spinal tissue. In the present study, we have studied the regulation of IP3R1 in hypoxic/reperfusion injury of spinal cord dorsal column in vitro. METHODS: Dorsal columns were isolated from the spinal cord of adult rats and injury was induced by exposing to hypoxic condition for 1 hour. After injury, reperfusion was carried out for 0, 2, 4, and 8 hours. Tissues were collected and processed for western blotting, immunohistochemistry and real-time PCR. RESULTS: In the present study, we have found increased expression of IP3R1 after hypoxic/reperfusion injury of spinal cord dorsal column in vitro. Maximum expression of IP3R1 has been seen at 4 hours after hypoxia. Double immunofluorescence studies show the localization of IP3R1 in axons and astrocytes. Further identifying the signaling pathway involved in the regulation, we found Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-62 and c-Jun N-terminal kinase (JNK) inhibitor SP600125 reduced the expression of IP3R1 suggesting the role of CaMKII and JNK in the regulation of IP3R1 expression. We did not find role of ERK and p38 in the regulation IP3R1 expression in hypoxic/reperfusion injury of dorsal column in vitro. DISCUSSION: The result presented in this study showed that IP3R1 expression is increased in hypoxic/reperfusion injury of spinal cord white matter and it is regulated by the CaMKII-JNK pathway.

Flow characteristics of cerebrospinal fluid shunt tubing.

OBJECT: Cerebrospinal fluid shunt systems malfunction for a multitude of reasons, including malpostitioning, obstruction of the ventricular or distal catheter, obstruction of the shunt valve, and catheter disruptions or disconnections. The goal of this study was to examine the hydrodynamic resistance and flow in new and explanted catheters and also in catheters with 1 or 2 straight connectors. METHODS: Explanted catheters of multiple lengths, 2-piece catheters, 3-piece catheters, and new catheters were attached to a proximal and distal manometer. A flask with artificial CSF attached to the proximal end provided flow. The flow was allowed to stabilize over 1 hour; then the change in pressure between the proximal and distal end of the catheter was measured. RESULTS: The resistance to flow was calculated for new, never-implanted catheters and compared with the resistance of explanted distal shunt catheters. The resistance of the new catheters was examined after the addition of 1 and 2 straight connectors. Explanted catheters exhibited a slight increase in the resistance to flow of artificial CSF compared with new catheters. Two-piece and 3-piece catheters had a significant increase in resistance to flow compared with new catheters. For all catheters, resistance to flow increased as length increased (new, p = 0.01; explanted, p = 0.009; 1 connector, p = 0.01; 2 connectors, p = 0.03). In this paper, effective diameter is defined as the available cross-sectional area of catheter contacted by the artificial CSF. For new and explanted catheters, a decrease in the effective diameter of the catheter was associated with an increase in the resistance to flow of artificial CSF (new, p = 0.1083; explanted, p = 0.0091). However, after the addition of 1 or 2 connectors, an inverse trend was observed: resistance to flow increased with effective diameter. CONCLUSIONS: There appears to be some increase in resistance of CSF shunt catheters as they age, altering flow dynamics. In addition, the use of straight connectors within a CSF shunt system increases the resistance to flow of artificial CSF within the shunt system. The increase in resistance appears to be related to the duration of implantation and the length of the catheter and inversely related to the diameter of the catheter. This increase in resistance may be related to sterile shunt malfunction. The addition of straight connectors is associated with a significant increase in resistance in comparison with catheters without connectors (p = 0.005).

Restraint stress-induced oxidative damage and its amelioration with selenium.

Stress is a state of threatened cellular homeostasis which results in free radical generations and subsequent oxidative damage. The aim of this study was to evaluate the effect of selenium on restraint stress-induced oxidative damage in hippocampus, striatum and frontal cortex. Rats were pre-treated with sodium selenite (0.3 mg/kg; intraperitoneally) for 15 days and divided into six groups (n=8). Rats were then subjected to restraint stress for 1 h and 4 h. Lipid peroxidation, glutathione (GSH) and activities of antioxidant enzymes viz. selenium-dependent glutathione peroxidase (Se-GPx), glutathione reductase (GR), glutathione S-transferase (GST) and catalase were evaluated in the frontal cortex, striatum and hippocampus. Restraint stress-induced for 1 h and 4 h caused a significant decrease (P<0.001) in intracellular GSH content and the activity of Se-GPx, GR, GST and catalase with a significant increase (P<0.001) in the level of lipid peroxidation in all 3 regions of the brain. Selenium pre-treatment exhibited restoration of antioxidant enzymes activity, GSH content and decrease in the level of lipid peroxidation in hippocampus, striatum and frontal cortex in both 1 h and 4 h restraint stress groups. Protective effect of selenium pre-treatment was found to be more pronounced in 4 h restraint stress group as compared to 1 h restraint stress group. Selenium per se had no effect on GSH, lipid peroxidation level or activities of antioxidant enzymes in hippocampus, striatum and frontal cortex. In conclusion, selenium pre-treatment protected the brain against restraint stress-induced oxidative damage at 4 h in hippocampus, striatum and frontal cortex.

Confocal imaging of changes in glial calcium dynamics and homeostasis after mechanical injury in rat spinal cord white matter.

Periaxonal glia play an important role in maintaining axonal function in white matter. However, little is known about the changes that occur in glial cells in situ immediately after traumatic injury. We used fluo-3 and confocal microscopy to examine the effects of localized (<0.5 mm) mechanical trauma on intracellular calcium (Ca(i)(2+)) levels in glial cells in a mature rat spinal cord white matter preparation in vitro. At the injury site, the glial Ca(i)(2+) signal increased by 300-400% within 5 min and then irreversibly declined indicating cell lysis and death. In glial cells at sites adjacent to the injury (1.5-2 mm from epicenter), Ca(i)(2+) levels peaked at 10-15 min, and thereafter declined but remained significantly above rest levels. At distal sites (6-9 mm), Ca(i)(2+) levels rose and declined even slower, peaking at 80-90 min. Injury in zero calcium dampened Ca(i)(2+) responses, indicating a role for calcium influx in the generation and propagation of the injury-induced Ca(i)(2+) signal. By 50-80 min post-injury, surviving glial cells demonstrated an enhanced ability to withstand supraphysiological Ca(i)(2+) loads induced by the calcium ionophore A-23187. Glial fibrillary acidic protein (GFAP) and CNPase immunolabeling determined that the glial cells imaged with fluo-3 included both astrocytes and oligodendrocytes. These data provide the first direct evidence that the effects of localized mechanical trauma include a glial calcium signal that can spread along white matter tracts for up to 9 mm within less than 3 h. The results further show that trauma can enhance calcium regulation in surviving glial cells in the acute post-injury period.