Protective effects of dantrolene and methylprednisolone against spinal cord injury-induced early oxidative damage in rabbit bladder: A comparative experimental study

Conclusions. Dantrolene and MP may have potential benefits against oxidative damage in the bladder after SCIs because of their anti-inflammatory and antioxidant effects. In particular, the combined use of DNT and MP at different doses can be considered a treatment strategy

Sustained Na⁺/H⁺ exchanger activation promotes gliotransmitter release from reactive hippocampal astrocytes following oxygen-glucose deprivation

Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na +/H+ exchanger isoform 1 (NHE1) protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX). 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H+ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1-5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na+ and Ca2+ overload. The latter was mediated by reversal of Na+/Ca2+ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1-24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na+ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H + homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na+ and Ca2+ homeostasis, which reduces Na+-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI. © 2014 Cengiz et al

TrkB Receptor Agonist 7, 8 Dihydroxyflavone Triggers Profound Gender-Dependent Neuroprotection in Mice After Perinatal Hypoxia and Ischemia

In this study, we investigated the effects of a bioactive high-affinity TrkB receptor agonist 7,8-dihydroxyflavone (7,8 DHF) on neonatal brain injury in female and male mice after hypoxia ischemia (HI). HI was induced by exposure of postnatal day 9 (P9) mice to 10% O-2 for 50 minutes at 37 degrees C after unilateral ligation of the left common carotid artery. Animals were randomly assigned to HI-vehicle control group [phosphate buffered saline (PBS), intraperitoneally (i.p.)] or HI + 7,8 DHF-treated groups (5 mg/kg in PBS, i.p at 10 min, 24 h, or with subsequent daily injections up to 7 days after HI). The HI-vehicle control mice exhibited neuronal degeneration in the ipsilateral hippocampus and cortex with increased Fluoro-Jade C positive staining and loss of microtubule associated protein 2 expression. In contrast, the 7,8 DHF-treated mice showed less hippocampal neurodegeneration and astrogliosis, with more profound effects in female than in male mice. Moreover, 7,8 DHF-treated mice improved motor learning and spatial learning at P30-60 compared to the HI-vehicle control mice. Diffusion tensor imaging of ex vivo brain tissues at P90 after HI revealed less reduction of fractional anisotropy values in the ipsilateral corpus callosum of 7,8 DHF-treated brains, which was accompanied with better preserved myelin basic protein expression and CA1 hippocampal structure. Taken together, these findings strongly suggest that TrkB agonist 7,8 DHF is protective against HI-mediated hippocampal neuronal death, white matter injury, and improves neurological function, with a more profound response in female than in male mice

Metabolic and Structural Imaging at 7 Tesla After Repetitive Mild Traumatic Brain Injury in Immature Rats

Mild traumatic brain injury (mTBI) in children is a common and serious public health problem. Traditional neuroimaging findings in children who sustain mTBI are often normal, putting them at risk for repeated mTBI (rmTBI). There is a need for more sensitive imaging techniques capable of detecting subtle neurophysiological alterations after injury. We examined neurochemical and white matter changes using diffusion tensor imaging of the whole brain and proton magnetic resonance spectroscopy of the hippocampi at 7 Tesla in 18-day-old male rats at 7 days after mTBI and rmTBI. Traumatic axonal injury was assessed by beta-amyloid precursor protein accumulation using immunohistochemistry. A significant decrease in fractional anisotropy and increase in axial and radial diffusivity were observed in several brain regions, especially in white matter regions, after a single mTBI versus sham and more prominently after rmTBI. In addition, we observed accumulation of beta-amyloid precursor protein in the external capsule after mTBI and rmTBI. mTBI and rmTBI reduced the N-acetylaspartate/creatine ratio (NAA/Cr) and increased the myoinositol/creatine ratio (Ins/Cr) versus sham. rmTBI exacerbated the reduction in NAA/Cr versus mTBI. The choline/creatine (Cho/Cr) and (lipid/Macro Molecule 1)/creatine (Lip/Cr) ratios were also decreased after rmTBI versus sham. Diffusion tensor imaging findings along with the decrease in Cho and Lip after rmTBI may reflect damage to axonal membrane. NAA and Ins are altered at 7 days after mTBI and rmTBI likely reflecting neuro-axonal damage and glial response, respectively. These findings may be relevant to understanding the extent of disability following mTBI and rmTBI in the immature brain and may identify possible therapeutic targets

Glutamate and pro-inflammatory cytokine release from hippocampal astrocytes following OGD/REOX.

<p><b>A.</b> Glutamate release in hippocampal astrocyte cultures was determined at 2(1 µM) or TBOA (100 µM) was present during REOX only. Data are mean ± SEM. N of 4 cultures were for all groups except for normoxia (n = 5). *p<0.05 vs. normoxic control, # p<0.05 vs. corresponding untreated. ND: not detectable. <b>Inset:</b> The reversal potential for the glutamate transport was plotted as a function of [Na⁺]_i. The known values for [Na]_o, [H]_o, [H]_i and [K]_o at baselines were used along with an assumed value for [Glu]_o of 0.01 µM, [Glu]_i of 5 mM and [K]_i of 70 mM. <b>B–D</b>. Release of innate immune cytokines in the culture medium of hippocampal astrocytes. HOE 642 (1 µM) was present during normoxia or REOX treatment. IL-1β (<b>B</b>) IL-6 (<b>C</b>), or TNF-α (<b>D</b>) were normalized to cell lysate protein and expressed as pg/mg protein. Data are mean ± SEM (n = 3). ND: not detectable. *p<0.05 vs. normoxic control. # p<0.05 vs. corresponding untreated.</p

Minimal cell death of reactive astrocytes after OGD/REOX.

<p>Hippocampal astrocytes were grown on 24 well culture dishes and subjected to 2(1 µM) was present only during REOX. At the end of REOX, cells were loaded with calcein-AM (green) and PI (red). <b>A</b>. Representative images of live (calcein positive) and dead (PI positive) cells of normoxia (a, à), 5 h REOX (b, b`), 5 h REOX+HOE 642 (c, c`), 24 REOX (d, d`) or 24 REOX+HOE 642 (e, è). <b>B</b>. Summary of reactive astrocyte death from six wells from each experimental condition. Data are mean ± SEM. n = 6.</p

Increased NHE1 activity in hippocampal astrocytes following OGD/REOX.

<p><b>A.</b> pH_i changes were determined by BCECF-AM dye in astrocytes subjected to NH₄⁺/NH₃ prepulse-mediated acid-loading. Either normoxic astrocytes (left panel) or astrocytes at 2 h OGD (right panel) were exposed to 30 mM NH₄Cl₃ (<b>a–c</b>), then returned to standard HCO₃^–free HEPES-MEM solution (<b>c–e</b>). After an initial acidification, pH_i recovery followed (<b>d–e</b>). The pH recovery rates were determined at ∼ 6.5 to normalize for the allosteric regulation of H⁺ on NHE1 activity. A slope of the pH_i changes following the prepulse was calculated as pH_i recovery rate (solid or dashed line). For HOE 642 treatment, the drug was present throughout the experiment. <b>B.</b> pH_i (left panel) and pH_i recovery rates (right panel) were summarized under normoxic control, 2 h OGD, or 1, 5, or 24 h REOX conditions. <b>C.</b> Summary data of NHE1-mediated recovery rates under normoxic and OGD/REOX conditions. The values of ∼20 cells from each coverslip/culture were averaged. The n values were the number of cultures under each condition and indicated as normoxia (9), normoxia+HOE 642 (6), 0 REOX (5), 0 REOX+HOE 642 (4), 1 h REOX (3), 1 h REOX+HOE 642 (4), 5 h REOX (5), 5 h REOX+HOE 642 (4), 24 h REOX (4) and 24 h REOX+HOE 642 (3). Data are expressed as mean ± SEM. *p<0.05 vs. corresponding untreated. # p<0.05 vs. corresponding untreated.</p

Pro-inflammatory cytokine release from LME-treated hippocampal astrocyte cultures following OGD/REOX.

<p>Release of innate immune cytokines was detected in the culture medium of hippocampal astrocytes at 2(1 µM) was present during normoxia or REOX treatment (with 5 mM LME). IL-1β (<b>A</b>), IL-6 (<b>B</b>), TNF-α (<b>C</b>). Cytokine content was normalized to cell lysate protein and expressed as pg/mg protein. Data are mean ± SEM (n = 3). *p<0.05 vs. normoxic control. # p<0.05 vs. corresponding untreated, ND: not detectable.</p