Ischemic preconditioning reduces ischemic brain injury by suppressing nuclear factor kappa B expression and neuronal apoptosis

Ischemic stroke induces a series of complex pathophysiological events including blood-brain barrier disruption, inflammatory response and neuronal apoptosis. Previous studies demonstrate that ischemic preconditioning attenuates ischemic brain damage via inhibiting blood-brain barrier disruption and the inflammatory response. Rats underwent transient (15 minutes) occlusion of the bilateral common carotid artery with 48 hours of reperfusion, and were subjected to permanent middle cerebral artery occlusion. This study explored whether ischemic preconditioning could reduce ischemic brain injury and relevant molecular mechanisms by inhibiting neuronal apoptosis. Results found that at 72 hours following cerebral ischemia, myeloperoxidase activity was enhanced, malondialdehyde levels increased, and neurological function was obviously damaged. Simultaneously, neuronal apoptosis increased, and nuclear factor-κB and cleaved caspase-3 expression was significantly increased in ischemic brain tissues. Ischemic preconditioning reduced the cerebral ischemia-induced inflammatory response, lipid peroxidation, and neurological function injury. In addition, ischemic preconditioning decreased nuclear factor-κB p65 and cleaved caspase-3 expression. These results suggested that ischemic preconditioning plays a protective effect against ischemic brain injury by suppressing the inflammatory response, reducing lipid peroxidation, and neuronal apoptosis via inhibition of nuclear factor-κB and cleaved caspase-3 expression.     

5-Hydroxymethylfurfural from wine-processed Fructus corni inhibits hippocampal neuron apoptosis

Previous studies have shown that 5-hydroxymethylfurfural, a compound extracted from wine- processed Fructus corni, has a protective effect on hippocampal neurons. The present study was designed to explore the related mechanisms. Our study revealed that high and medium doses （10, 1 μmol/L） of 5-hydroxymethylfurfural could improve the morphology of H2O2-treated rat hippocampal neurons as revealed by inverted phase-contrast microscopy and transmission electron microscopy. MTT results showed that incubation with high and medium doses of 5-hydroxymethylfurfural caused a significant increase in the viability of neuronal cells injured by H2O2. Flow cytometry assays con- firmed that H2O2 could induce cell apoptosis, while high and medium doses of 5-hydroxymethylfurfural had a visible protective effect on apoptotic rat hippocampal neurons. Real-time PCR and western blot analysis showed that high and medium doses of 5-hydroxymethylfurfural prevented H2O2-induced up-regulation of p53, Bax and caspase-3 and an- tagonized the down-regulation of Bcl-2 induced by H2O2 treatment. These results suggested that 5-hydroxymethylfurfural could inhibit apoptosis of cultured rat hippocampal neurons injured by H2O2 via increase in Bcl-2 levels and decrease in p53, Bax and caspase-3 protein expression levels.     

Toll-like receptor 4-mediated signaling participates in apoptosis of hippocampal neurons

The phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway is considered important for cellsurvival and has been shown to mediate various anti-apoptotic biological effects. This ...     

Brilliant blue G attenuates lipopolysaccharidemediated microglial activation and inflammation

Previous studies have confirmed that oxidized adenosine triphosphate, a P2X7 receptor antagonist, attenuates lipopolysaccharide-mediated microglial activation and inflammatory expression following neuronal damage in rat brain. NaCl and temperature may affect the potency of oxidized adenosine triphosphate. Brilliant blue G is a derivative of a widely used food additive and has little toxicity. This study explored the effects of brilliant blue G, a selective P2X7 receptor antagonist, on microglial activation and inflammation. Results demonstrated that brilliant blue G inhibited the release of cyclooxygenase-2 and interleukin-6 in BV2 cells. Immunofluorescence displayed that brilliant blue G could suppress lipopolysaccharide-induced microglial activation. This study used RNA interference to block P2X7 receptor expression and found that small interfering RNA also suppressed the release of cyclooxygenase-2 and interleukin-6 in BV2 cells. These results suggested that downregulation of the P2X7 receptor by brilliant blue G was involved in the inhibition of microglial activation and inflammation.     

Medium-intensity acute exhaustive exercise induces neural cell apoptosis in the rat hippocampus

The present study assessed the influence of medium-intensity (treadmill at a speed of 19.3 m/min until exhaustion) and high-intensity (treadmill at a speed of 26.8 m/min until exhaustion) acute exhaustive exercise on rat hippocampal neural cell apoptosis. TUNEL staining showed significantly increased neural cell apoptosis in the hippocampal CA1 region of rats after medium- and high-intensity acute exhaustive exercise, particularly the medium-intensity acute exhaustive exercise, when compared with the control. Immunohistochemistry showed significantly increased expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax in the hippocampal CA1 region of rats after medium- and high-intensity acute exhaustive exercise. Additionally, the ratio of Bax to Bcl-2 increased in both exercise groups. In particular, the medium-intensity acute exhaustive exercise group had significantly higher Bax and Bcl-2 protein expression and a higher Bax/Bcl-2 ratio. These findings indicate that acute exhaustive exercise of different intensities can induce neural cell apoptosis in the hippocampus, and that medium-intensity acute exhaustive exercise results in greater damage when compared with high-intensity exercise.     

Viability of primary cultured retinal neurons in a hyperglycemic condition

The retina of Wistar rats within 1-3 days of birth were dissociated into a retinal cell suspension using 0.05% trypsin digestion. The cell suspension was incubated in Dulbecco’s modified Eagle’s medium for 24 hours, followed by neurobasal medium for 5-7 days. Nissl staining showed that 79.86% of primary cultured retinal cells were positive and immunocytochemical staining showed that the purity of anti-neurofilament heavy chain antibody-positive cells was 71.53%, indicating that the primary culture system of rat retinal neurons was a reliable and stable cell system with neurons as the predominant cell type. The primary cultured retinal neurons were further treated with 0, 5.5, 15, 25, and 35 mM glucose for 24, 48, and 72 hours. The thiazolyl blue tetrazolium bromide test and flow cytometry showed that with increasing glucose concentration and treatment duration, the viability of retinal neurons was reduced, and apoptosis increased. In particular, 35 mM glucose exhibited the most significant effect at 72 hours. Thus, rat retinal neurons treated with 35 mM glucose for 72 hours can be used to simulate a neuronal model of diabetic retinopathy.     

Baicalin influences the dendritic morphology of newborn neurons in the hippocampus of chronically stressed rats

Chronic stress models, established in adult Sprague-Dawley rats through a 14-day subcutaneous injection of 40 mg/kg corticosterone, once per day, were given a daily oral feeding of 50 mg/kg baicalin. The study was an attempt to observe the effect of baicalin on neurogenesis in chronically stressed rats. Results showed that subcutaneous injection of corticosterone significantly decreased the total number of doublecortin-positive neurons in the hippocampus. The reduced cell number caused by corticosterone was mainly due to the decrease of class II doublecortin-positive neurons, but the class I doublecortin-positive neurons were unaffected. Baicalin treatment increased the number of both class I and class II doublecortin-positive neurons. In addition, doublecortin-positive neurons showed less complexity in dendritic morphology after corticosterone injection, and this change was totally reversed by baicalin treatment. These findings suggest that baicalin exhibits a beneficial effect on adult neurogenesis.     

ClC-3 chloride channel in hippocampal neuronal apoptosis

Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area fol- lowing ischemic brain injury. In this study, an apoptotic model in rat hippocampal neurons was es- tablished by 0.5 mmol/L 3-morpholinosyndnomine （SIN-l）, a nitric oxide donor. The models were then cultured with 0.1 mmol/L of 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS; the chloride channel blocker）for 18 hours. Neuronal survival was detected using the 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining. Western blot analysis and immunochemilumi- nescence staining were applied to determine the changes of activated caspase-3 and CIC-3 channel proteins. Real-time PCR was used to detect the mRNA expression of CIC-3. The results showed that SIN-1 reduced the neuronal survival rate, induced neuronal apoptosis, and promoted CIC-3 chloride channel protein and mRNA expression in the apoptotic neurons. DIDS reversed the effect of SIN-I. Our findings indicate that the increased activities of the CIC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide.     

Chloride channel blocker 4,4-diisothiocyanatostilbene-2,2’-disulfonic acid inhibits nitric oxide-induced apoptosis in cultured rat hippocampal neurons

Apoptosis in cultured rat hippocampal neurons was induced using the nitric oxide donor 3-morpholinosydnonimine, and cells were treated with the chloride channel blocker, 4,4-diisothiocyanatostilbene-2,2’-disulfonic acid. Results showed that the survival rate of neurons was significantly increased after treatment with 4,4-diisothiocyanatostilbene-2,2’-disulfonic acid, and the rate of apoptosis decreased. In addition, the expression of the apoptosis-related proteins poly(adenosine diphosphate-ribose)polymerase-1 and apoptosis-inducing factor were significantly reduced. Our experimental findings indicate that the chloride channel blocker 4,4- diisothiocyanatostilbene-2,2’-disulfonic acid can antagonize apoptotic cell death of hippocampal neurons by inhibiting the expression of the apoptosis-related proteins poly(adenosine diphosphate-ribose)polymerase-1 and apoptosis-inducing factor.     

Differentiation renders susceptibility to excitotoxicity in HT22 neurons

HT22 is an immortalized mouse hippocampal neuronal cell line that does not express cholinergic and glutamate receptors like mature hippocampal neurons in vivo.This in part prevents its use as a model for mature hippocampal neurons in memory-related studies.We now report that HT22 cells were appropriately induced to differentiate and possess properties similar to those of mature hippocampal neurons in vivo,such as becoming more glutamate-receptive and excitatory.Results showed that sensitivity of HT22 cells to glutamate-induced toxicity changed dramatically when comparing undifferentiated with differentiated cells,with the half-effective concentration for differentiated cells reducing approximately two orders of magnitude.Moreover,glutamate-induced toxicity in differentiated cells,but not undifferentiated cells,was inhibited by the N-methyl-Daspartate receptor antagonists MK-801 and memantine.Evidently,differentiated HT22 cells expressed N-methyl-D-aspartate receptors,while undifferentiated cells did not.Our experimental findings indicated that differentiation is important for immortalized cell lines to render post-mitotic neuronal properties,and that differentiated HT22 neurons represent a better model of hippocampal neurons than undifferentiated cells.     

An optimal dose of tea polyphenols protects against global cerebral ischemia/reperfusion injury

Previous studies addressing the protection of tea polyphenols against cerebral ischemia/ reperfusion injury often use focal cerebral ischemia models, and the optimal dose is not unified. In this experiment, a cerebral ischemia/reperfusion injury rat model was established using a modified four-vessel occlusion method. Rats were treated with different doses of tea polyphenols (25, 50, 100, 150, 200 mg/kg) via intraperitoneal injection. Results showed that after 2, 6, 12, 24, 48 and 72 hours of reperfusion, peroxide dismutase activity and total antioxidant capacity in brain tissue gradually increased, while malondialdehyde content gradually decreased after tea polyphenol intervention. Tea polyphenols at 200 mg/kg resulted in the most apparent changes. Terminal deoxynucleotidyl transferase-mediated nick end labeling and flow cytometry showed that 200 mg/kg tea polyphenols significantly reduced the number and percentage of apoptotic cells in the hippocampal CA1 region of rats after cerebral ischemia/reperfusion injury. The open field test and elevated plus maze experiments showed that tea polyphenols at 200 mg/kg strengthened exploratory behavior and reduced anxiety of cerebral ischemia/reperfusion injured rats. Experimental findings indicate that tea polyphenols protected rats against cerebral ischemia/ reperfusion injury and 200 mg/kg is regarded as the optimal dose.     

Luteolin attenuates neuronal apoptosis in the hippocampi of diabetic encephalopathy rats

Luteolin (3’,4’,5,7-tetrahydroxyflavone) has powerful anti-apoptotic and antioxidant properties. This study aimed to investigate the effects of luteolin on hyperglycemia-mediated apoptosis in the hippocampi of rats with streptozotocin-induced diabetic encephalopathy after injection into the tail veins, and the molecular mechanisms involved. Biochemistry and terminal deoxynucleotidyl transferase mediated dUTP nick end labelling detection results showed that luteolin treatment (given twice daily for 15 days) significantly inhibited hyperglycemia-mediated apoptosis, decreased malondialdehyde levels and increased glutathione levels in the hippocampi of streptozotocin-induced diabetic rats. Western blot analysis revealed that luteolin also inhibited the expression of apoptosis-related factors and cytochrome c release from mitochondria. Luteolin also improved the learning and memory abilities of rats with diabetic encephalopathy in a water maze test. Further western blot analysis revealed that luteolin treatment facilitated neuronal cell survival through activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, an extracellular signal pathway involved in the suppression of cell apoptosis and promotion of cell survival. These experimental findings indicate that luteolin can inhibit apoptosis of hippocampal nerve cells in rats with diabetic encephalopathy, and that this effect is mediated by an indirect antioxidative effect, the inhibition of activation of apoptosis-related factors and the activation of phosphatidylinositol 3-kinase/Akt signal pathway.     

The calmodulin-dependent protein kinase II inhibitor KN-93 protects rat cerebral cortical neurons from N-methyl-D-aspartic acid-induced injury

In this study, primary cultured cerebral cortical neurons of Sprague-Dawley neonatal rats were treated with 0.25, 0.5, and 1.0 μM calmodulin-dependent protein kinase II inhibitor KN-93 after 50 μM N-methyl-D-aspartic acid-induced injury. Results showed that, compared with N-methyl-D-aspartic acid-induced injury neurons, the activity of cells markedly increased, apoptosis was significantly reduced, leakage of lactate dehydrogenase decreased, and intracellular Ca(2+) concentrations in neurons reduced after KN-93 treatment. The expression of caspase-3, phosphorylated calmodulin-dependent protein kinase II and total calmodulin-dependent protein kinase II protein decreased after KN-93 treatment. And the effect was apparent at a dose of 1.0 μM KN-93. Experimental findings suggest that KN-93 can induce a dose-dependent neuroprotective effect, and that the underlying mechanism may be related to the down-regulation of caspase-3 and calmodulin-dependent protein kinase II expression.     

Inhibition of inflammatory mediator release from microglia can treat ischemic/hypoxic brain injury

Interleukin-1α and interleukin-1β aggravate neuronal injury by mediating the inflammatory reaction following ischemic/hypoxic brain injury. It remains unclear whether interleukin-1α and interleukin-1β are released by microglia or astrocytes. This study prepared hippocampal slices that were subsequently subjected to oxygen and glucose deprivation. Hematoxylin-eosin staining verified that neurons exhibited hypoxic changes. Results of enzyme-linked immunosorbent assay found that interleukin-1α and interleukin-1β participated in this hypoxic process. Moreover, when hypoxic injury occurred in the hippocampus, the release of interleukin-1α and interleukin-1β was mediated by the P2X4 receptor and P2X7 receptor. Immunofluorescence staining revealed that during ischemia/hypoxia, the P2X4 receptor, P2X7 receptor, interleukin-1α and interleukin-1β expression was detectable in rat hippocampal microglia, but only P2X4 receptor and P2X7 receptor expression was detected in astrocytes. Results suggested that the P2X4 receptor and P2X7 receptor, respectively, mediated interleukin-1α and interleukin-1β released by microglia, resulting in hippocampal ischemic/hypoxic injury. Astrocytes were activated, but did not synthesize or release interleukin-1α and interleukin-1β.     

Pre-moxibustion and moxibustion prevent Alzheimer＇s disease

The Alzheimer’s disease model in Wistar rats was established by injection of amyloid β-peptide (Aβ1-42 ) into the hippocampal CA1 region. Rats were treated with suspended moxibustion on Baihui (GV20) a...     

Expression of synaptosomal-associated protein-25 in the rat brain after subarachnoid hemorrhage

Synaptosomal-associated protein-25 is an important factor for synaptic functions and cognition. In this study, subarachnoid hemorrhage models with spatial learning disorder were established through a b...     

Optimal time for subarachnoid transplantation of neural progenitor cells in the treatment of contusive spinal cord injury

This study aimed to identify the optimal neural progenitor cell transplantation time for spinal cord injury in rats via the subarachnoid space. Cultured neural progenitor cells from 14-day embryonic rats, constitutively expressing enhanced green fluorescence protein, or media alone, were injected into the subarachnoid space of adult rats at 1 hour (acute stage), 7 days (subacute stage) and 28 days (chronic stage) after contusive spinal cord injury. Results showed that grafted neural progenitor cells migrated and aggregated around the blood vessels of the injured region, and infiltrated the spinal cord parenchyma along the tissue spaces in the acute stage transplantation group. However, this was not observed in subacute and chronic stage transplantation groups. O4- and glial fibrillary acidic protein-positive cells, representing oligodendrocytes and astrocytes respectively, were detected in the core of the grafted cluster attached to the cauda equina pia surface in the chronic stage transplantation group 8 weeks after transplantation. Both acute and subacute stage transplantation groups were negative for O4 and glial fibrillary acidic protein cells. Basso, Beattie and Bresnahan scale score comparisons indicated that rat hind limb locomotor activity showed better recovery after acute stage transplantation than after subacute and chronic transplantation. Our experimental findings suggest that the subarachnoid route could be useful for transplantation of neural progenitor cells at the acute stage of spinal cord injury. Although grafted cells survived only for a short time and did not differentiate into astrocytes or neurons, they were able to reach the parenchyma of the injured spinal cord and improve neurological function in rats. Transplantation efficacy was enhanced at the acute stage in comparison with subacute and chronic stages.