Change of L-type voltage-gated Ca2+ channel current during focal brain ischemia in diabetic rats

AIM: To observe the change of L-type voltage-gated Ca²⁺ channel current during focal brain ischemia in the normal rats and the rats with diabetes mellitus (DM). METHODS: Combination of high-fat diet with streptozotocin (STZ) was used to establish DM animal model. The operation of middle cerebral artery occlusion (MCAO) with monofilament on the rats was performed. The animals were divided into sham operation group, MCAO 1 h group, MCAO 3 h group, MCAO 6 h group, MCAO 24 h group, DM sham operation group, DM+MCAO 1 h group, DM+MCAO 3 h group, DM+MCAO 6 h group and DM+MCAO 24 h group. The score of neural function was determined to judge the degree of palsy in the rats in MCAO 24 h group and DM+MCAO 24 h group. The changes of L-type voltage-gated Ca²⁺ channel current of cortex neurons during ischemia were measured using the whole-cell patch clamp technique. RESULTS: The rats in DM+MCAO 24 h group awaked slowly, and the degree of semiplegia was more serious than that in the rats in MCAO 24 h group. The score of neural function in DM+MCAO group was higher than that in MCAO group (P<0.05). The longer the ischemic time was, the higher L-type voltage-gated Ca²⁺ channel current was observed in MCAO group and DM+MCAO group (P<0.05). L-type voltage-gated Ca²⁺ channel current in DM+MCAO group was higher than that in MCAO group at each time point(P<0.05). CONCLUSION: The aggratation of ischemic injury during DM+MCAO is probably associated with Ca²⁺ overload induced by calcium channel opening and current increasing.     

Foam cell formation and intracellular Ca2+ alteration

AIM:These studies aimed at exploring the alteration of intracellular Ca²⁺ level in the course of macrophage-derived foam cell formation as well as its mechanism.METHODS:Foam-like cell was generated by peritoneal macrophage of C57BL/6J mouse, which is susceptible to atherosclerosis, incubated in 10 mg·L^-1 oxidized low density lipoprotein for 96 hours. With the technique of Ca²⁺ fluorescent indicator and the assay of NADH-oxidizing coupling spectrum-alteration, the intracellular Ca²⁺ level and membranous Ca²⁺-ATPase activity of the above foam-like cell were determined.RESULTS:The foam-like macrophage Ca²⁺ level was 2.7 times higher than the control macrophage, and the former Ca²⁺-ATPase activity was 24% of the later.CONCLUSION:The results suggested that macrophage-derived foam cell formation was connected with slow Ca²⁺ entry or release, which possibly derived from long-lasting opening of membranous Ca²⁺ channels at the early stage and irreversible inactivating of membranous Ca²⁺ pump at the late stage.     

Nerve growth factor attenuates cochlear injury induced by brain ischemia and reperfusion in rats

AIM: To investigate the effect of nerve growth factor (NGF) on auditory and cochlear damage induced by brain ischemia and reperfusion. METHODS: The rats with brain ischemia and reperfusion were divided into two groups at random. In the experimental group, the rats were injected intramuscularly with NGF . In control group, the animals were injected with normal saline instead of NGF. Then the hearing loss and cochlear structural changes of rats in both groups were compared. RESULTS: It was found that the hearing loss of rats in NGF group were less significantly than that of control group ( P< 0.01) after 60 min and 24 h reperfusion following 30 min ischemia. Scanning and transmission microscopy showed that the damage of the outer hair cells and the spiral neurons of rats in NGF group was much slighter than that of control group. CONCLUSION: NGF prevents auditory and cochlear injury induced by brain ischemia and reperfusion.     

Changes in amino acid neurotransmitters during cerebral ischemia and reperfusion in awake rats

AIM: To study the dynamic changes of amino acid neurotransmitters during cerebral ischemia and reperfusion in awake rats. METHODS: Model of cerebral ischemia and reperfusion in awake rats was replicated. Glutamate (Glu), aspartate (Asp), γ-aminobutyric acid (GABA), glycine (Gly), taurine (Tau), alanine (Ala), serine (Ser), threonine (Thr) and glutamine (Gln) concentrations were measured with microdialysis, and excitotoxic index (EI) was calculated in dialysates of hippocampus, neo-cortex and striatum. RESULTS: The significant increases in extracellular not only excitatory amino acid neurotransmitters-Glu and Asp, and their neuromodulator-Gly, but also inhibitory amino acid neurotransmitter-GABA and its neuromodulator-Tau and Ala were observed. However, the EI, representing the balance of excitatory and inhibitory neurotransmitters, was significantly increased during ischemia. CONCLUSION: The results suggest that the elevated Glu and Asp levels during ischemia are insufficient to independently engender ischemic damage, and other neurotransmitters or other factors may play an important role in modulating the excitotoxic effects of Glu and Asp.     

Upregulative effect of CGRP on expression of CREB and phospho-CREB protein during focal cerebral ischemia/reperfusion in rat parietal cortex

AIM: To investigate the effect of calcitonin gene related peptide (CGRP) on the expression of cyclic AMP response element binding protein (CREB) and phosphorylated-CREB in rat parietal cortex during focal cerebral ischemia/reperfusion (I/R).METHODS: Focal cerebral ischemia/reperfusion model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The expressions of CREB and phospho-CREB in the parietal cortex in different groups (sham group, focal cerebral ischemia/reperfusion group and CGRP group) were detected with immunohistochemistry and Western blotting, and the positive products were analyzed by image analysis system.RESULTS: There was definite expression of CREB in right parietal cortex in sham group, while it was lesser in I/R group than that in sham group, but it became more in CGRP group than that in I/R group (P<0.05). Phospho-CREB was barely detected in right parietal cortex in sham group and it became more in I/R group than that in sham group. The expression of phospho-CREB increased in CGRP group than that in I/R group of the right parietal cortex (P<0.05).CONCLUSION: CGRP upregulates the expression of CREB and phospho-CREB in the ischemic neurons of the parietal cortex during focal cerebral ischemia/reperfusion and CREB probably involves in the mechanism of protective role of CGRP to ischemic neurons.     

Stability of cerebral focal ischemia/reperfusion model induced by monofilament in Kunming mice

AIM: To study the stability of mouse cerebral ischemia/reperfusion model induced by the method of monofilament. METHODS: Sixty male Kunming mice were divided into 3 groups according to the body weight: group A (18-21 g), group B (22-28 g) and group C (30-35 g). Ischemia/reperfusion (I/R) model was made by middle cerebral artery occlusion (MCAO) with nylon monofilament. To evaluate the mouse MCAO model, the method of PRM2 laser Doppler was used to detect the cerebral blood flow, the neurological deficit scores were determined by Longa standard and infarction volume was detected with TTC staining. The content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were also measured by ELISA. RESULTS: The successful rates of model establishment in both group A and group B were higher than that in group C (P＜0.05), especially the highest in group B . The mortality in group A was significantly higher than that in group B and group C (P＜0.05). The behavior scores and cerebral infarct volume in group A and group B were significantly higher than those in group C (P＜0.05). Obvious brain injury and neurological deficits were also observed in group A and group B with the higher content of MDA and the lower activity of SOD in the cerebral cortex of the injury side. CONCLUSION: There are three important factors to ensure the success and stability of MCAO mouse model induced by monofilament, i.e. the diameter of monofilament matching the body weight of the mice, the suitable length of monofilament within the blood vessel, as well as the maintaining of proper room temperature during experiment. The MDA content and SOD activity are also effective indexes for evaluating the cerebral I/R injury.     

Production of nitric oxide and change of nitric oxide synthase activity in brains mitochondria of the rats with focal cerebral ischemia/reperfusion

AIM and METHOD:To determine the production of nitric oxide(NO) and change of NO synthase(NOS) activity in mitochondria isolated from the rat brains of the ischemia/reperfusion rat model produced by transient occlusion of middle cerebral artery on the following time points:2 h after occlusion of artery and 30 min,2 h, 4h after reperfusion.RESULTS:After the occlusion of middle cerebral artery,the respiratory control rate(RCR) of mitochondria significantly decreased and slightly increased at 4h after reperfusion.Meantime,the production of NO in mitochondria increased significantly.But with the increase of perfusion, production of NO gradually decreased and reached normal level as in the control group.It also shows that cerebral ischemia increased NOS's activity significantly in the mitochondria and still kept a higher level than the control group although it decreased gradually after reperfusion.But the iNOS's activity did not show obvious change.The change of total NOS's activity depends on the change of cNOS's activity.CONCLUSION: The activation of NO/NOS system in the mitochondria might play an important role in the reperfusion injury during reperfusion of ischemic brain.     

Effect of cholecystokinin-octapeptide on focal cerebral ischemia/reperfusion injury in rats

AIM:To examine the effect of cholecystokinin-octapeptide (CCK-8) on focal cerebral ischemia/reperfusion injury and its underlying mechanisms.METHODS:By using the suture model of focal cerebral ischemia and reperfusion, the effects of intracerebroventricular (icv) injection of CCK-8 and proglumide, nonselective CCK receptors antagonist, on the infarct size, regional cerebral blood flow (rCBF), and the levels of nitric oxide (NO), malondialdehyde (MDA) were observed in different brain regions of rats subjected to 1 h focal cerebral ischemia followed by 24 h reperfusion.RESULTS:(1) pretreatment with different doses of CCK-8 (0.3 μg,1.0 μg,2.0 μg or 4.0 μg) could attenuate the infarct size, but the statistically significant effects of CCK-8 were obtained only at the doses of 1.0 μg and 2.0 μg(P＜0.05). The neuroprotective effects of CCK-8 were blocked by pretreatment with proglumide. Administration of proglumide alone could worsen the ischemia/reperfusion injury. (2) CCK-8 (1.0 μg) inhibited the increase in NO, MDA levels in the ischemic core, and also inhibited the increase in NO level in the ischemic penumbra. The rCBF in the CCK-8 group was significantly higher than the normal value at 24 h after reperfusion (P＜0.05).CONCLUSIONS:These results suggest that both endogenous and exogenous CCK-8 alleviate focal cerebral ischemia/reperfusion injury. Such an action may be associated with inhibition of free radical-induced injuries and the improvement in rCBF.     

Effect of Ba2+ concentration on L-type Ca2+ channel in hypothalamic neurons

AIM:To investigate the effect of Ba²⁺ concentration on L-type of Ca²⁺ channel in hypothalamic neurons.METHODS:The cell acute isolation technique and cell-attached patch-clamp technique were used.RESULTS:The slope conductance of L-type Ca²⁺ channel were 28.6 pS (110 mmol/L) and 19.1 pS (10 mmol/L), and the open probability (NP₀) obviously different with different Ba²⁺ concentration as carrier. CONCLUSION:Ba²⁺ concentration had the obvious effect on the L-type Ca²⁺ channel.     

Protective effect of ischemic preconditioning on rat brain with ischemia/reperfusion injury

AIM:To study whether ischemic preconditioning(IPC) has a protective effect against ischemia/reperfusion(I/R) injury in brain, and the possible relationship between IPC and the regulating function of microcirculation.METHODS:The I/R models were established both in I/R and IPC groups of Sprague-Dawley rats. Additional procedure was performed of short term cerebral ischemic preconditioning in IPC group 24 hours before I/R. Skull windows were performed through which microcirculation features were measured before ischemia, during ischemia, and reperfusion. Finally, brains were cut into slices and stained with red tetrazoline(TTC).RESULTS:Most TTC stained brains in I/R group presented irregular palely red areas which were few in IPC group. Compared with I/R group, IPC group presented relatively increase in accumulated length of capillaries, mean cerebral microcirculatory perfusion, and microcirculatory velocity in ischemic and reperfusion phase. There was no-reflow phenomenon in I/R group in reperfusion phase, which was substituted by the course of increasing reperfusion in IPC group.CONCLUSIONS:IPC could relieve the reduction of tissue perfusion during ischemia and the no-reflow phenomenon during reperfusion by improving the regulating function of microcirculation, which relatively promote the opening of capillaries and accelerating of microvascular flow, therefore protect brain from I/R injury.     

Protective effect of spermine on rats with acute cerebral ischemia/reperfusion injury

AIM: To study the effects and the possible mechanisms of exogenous spermine on the rats with acute transient focal cerebral ischemia/reperfusion (I/R) injury.METHODS: The rat model of focal cerebral ischemia/reperfusion was established by middle cerebral artery occlusion (2 h) and reperfusion (2 h). Healthy adult SD rats were divided into 5 groups;sham group,I/R group and spermine(4,20 and 40 mmol/L)groups.The degree of cerebral injury was evaluated by neurological deficit score, infracted volume, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. The morphological changes of the brain were observed by HE staining and electron microscopy. RESULTS: Compared with I/R group, the neurological deficit score, infracted volume and the content of MDA were decreased, the SOD activity was increased and the ultrastructural changes were improved in spermine-treated groups. CONCLUSION: Exogenous spermine has a protective effect against acute focal cerebral ischemia/reperfusion injury. The mechanisms may be related to scavenging free radical by spermine.     

Relationship between morphologic changes in neuron or neuroglial cells and expression of TNF-α and c-Myc in cortex after focal cerebral ischemia/reperfusion in MCAO rats

AIM: To investigate the relationship between morphologic changes in neuron or neuroglial cells and expression of tumor necrosis factor α (TNF-α) and c-Myc in cortex after focal cerebral ischemia/reperfusion in MCAO rats. METHODS: The focal cerebral ischemia/reperfusion model was established by intraluminal thread occlusion of the middle cerebral artery (MCAO). The middle cerebral arteries of rats were occluded for 2 hours and reperfused for 1, 3 and 7 days. Using the techniques of immunohistochemical staining and optical microscopy, the morphologic changes in neuron or neuroglial cells were observed in the cortex of frontal or parietal lobe; the cell types which dynamicaly expressed TNF-α, c-Myc in the different period were also observed. RESULTS: The degeneration or necrosis of neuron or neuroglial cells were observed at the center of infarction, it was very serious at 3 d after reperfussion. Astrocyte and microglial cell proliferation were observed at the broder of infarction. TNF-α and c-Myc positive cells, most of which were astrocytes and microglial cells, increased significantly at 3 d after reperfusion. CONCLUSION: TNF-αand c-Myc may play an important role in the regulation of neuron or neuroglial cells after focal cerebral ischemia with reperfusion.     

Changes of myocardial nuclear membrane Ca2+-ATPase function in ischemia/reperfusion injury

AIM: The changes of myocardial nuclear membrane Ca²⁺ -ATPase function was investigated in ischemia/reperfusion injury. METHODS: The model of myocardial ischemia/reperfusion injury was established in rats. Myocardial nuclei were purified with sucrose density centrifugation, the activity of Ca²⁺ -ATPase was measured and calcium uptake was assayed with [⁴⁵ Ca²⁺ ] . RESULTS: Plasma levels of malondialdehyde (MDA) and free fatty acid (FFA) in myocardial ischemia/reperfusion injury increased significantly( P<0.01 vs control). Ca²⁺ -ATPase activity and [⁴⁵ Ca²⁺ ] uptake was lower than normal at below 10 μmol/L, while higher at 50 μmol/L. CONCLUSION:These data indicate dysfunction of nuclear menbrane calcium pump and [⁴⁵ Ca²⁺ ] uptake function in myocardial ischemia/reperfusion injury.     

Salidroside increases release of intracellular free Ca2+ from sarcoplasmic reticulum in cultured rat cardiomyocytes

AIM: To observe the effects of salidroside on intracellular free Ca²⁺ concentration in cultured rat cardiomyocytes. METHODS: Primarily cultured cardiomyocytes of neonatal rats were divided into control group, different concentrations of salidroside groups and verapamil pretreatment+different concentrations of salidroside groups. The fluorescent intensity of intercellular free calcium concentration ([Ca²⁺]_i) in cultured cardiomyocytes of newborn rats loaded with fluo-3/AM(5 μmol/L) was measured by laser scanning confocal microscopy. RESULTS: Salidroside at concentrations of 15 mg/L, 30 mg/L and 60 mg/L elevated [Ca²⁺]_i in cultured rat cardiomyocytes with the peak values of 574.08±4.65, 591.86±3.64 and 618.66±4.27, respectively (all P<0.01), indicating that the effect of salidroside on the level of [Ca²⁺]_i was dose-dependent. In the presence of verapamil in D-Hanks solution, salidroside also elevated the fluorescent intensity of [Ca²⁺]_i in cardiomyocytes from 357.74±3.13, 387.17±2.37 and 391.43±1.34 to 480.86±3.98, 496.70±3.08 and 522.18±3.19, respectively (all P<0.01). CONCLUSION: Salidroside increases the release of [Ca²⁺]_i from sarcoplasmic reticulum in cultured rat cardiomyocytes.     

Cortical gene expression pattern in rat focal cerebral ischemia

AIM: To investigate the genes differential expression in cortex during rat focal cerebral ischemia.METHODS: cDNA microarray chips containing numerous cDNAs were used to investigate the gene expression pattern between samples of focal cerebral ischemia and sham-control operation rats. RESULTS: Two hundred and eleven genes differentially expressed were screened out, among these genes, up-and down-regulated genes were 199 and 12, respectively. CONCLUSIONS: The analysis of gene expression pattern of focal cerebral ischemia based on cDNA microarray can realize high-throughput screening of the genes associated with the focal cerebral ischemia. The differential expression of genes may be related to the pathogenesis of focal cerebral ischemic diseases.     

The mechanism of neuronal injury and repair after focal cerebral ischemia

AIM:To explore the mechanism of neuronal injury and repair by investigating the expression of caspase-3 and apurinic/apyrimidinic endonuclease (APE/Ref-1) after focal cerebral ischemia. METHODS:A model of middle cerebral artery occlusion in rats was performed. The expression of caspase-3P₂₀ and APE/Ref-1 was examined by immunohistochemistry staining, TUNEL was applied to detected DNA damage, and double labeling with TUNEL and APE/Ref-1 was used to determine the relationship between APE/Ref-1 and DNA damage. RESULTS:The active subunit P₂₀ of caspase-3 was predominantly expressed within ischemic penumbra. The peak time of caspase-3P₂₀ positive cells preceded the appearance of TUNEL. With aggravation of cerebral ischemia, APE/Ref-1 immunoreactive cells in penumbra were significantly decreased. CONCLUSION:The activation of caspase enzymatic cascade following cerebral ischemia leads to degradation in DNA, meanwhile, decrease in DNA repair molecules or the failure of DNA repair may deteriorate the course.