Effect of ketamine on aquaporin-4 expression and neuronal apoptosis in brain tissues following brain injury in rats

BACKGROUND： Aquaporin-4 （AQP-4） is closely related to the formation of brain edema. Neuronal apoptosis plays an important part in the conversion of swelled neuron following traumatic brain injury. At present, the studies on the protective effect of ketamine on brain have involved in its effect on aquaporin-4 expression and neuronal apoptosis in the brain tissues following brain injury in rats. OBJECTIVE： To observe the effect of ketamine on AQP-4 expression and neuronal apoptosis in the brain tissue following rat brain injury, and analyze the time-dependence of ketamine in the treatment of brain injury.DESIGN： Randomized grouping design, controlled animal tria SETTING ： Department of Anesthesiology, the Medical School Hospital of Qingdao University MATERIALS： Totally 150 rats of clean grade, aged 3 months, were involved and randomized into control group and ketamine-treated group, with 75 rats in each. Each group was divided into 5 subgroups separately at 6,12, 24, 48 and 72 hours after injury, with 15 rats at each time point. Main instruments and reagents： homemade beat machine, ketamine hydrochloride （Hengrui Pharmaceutical Factory, Jiangsu）, rabbit anti-rat AQP-4 polyclonal antibody, SABC immunohistochemical reagent kit and TUNEL reagent kit （Boster Co.,Ltd., Wuhan）. METHODS： This trial was carried out in the Institute of Cerebrovascular Disease, Medical College of Qingdao University during March 2005 to February 2006. A weight-dropping rat model of brain injury was created with Feeney method. The rats in the ketamine-treated group were intraperitoneally administered with 50 g/L ketamine （120 mg/kg） one hour after injury, but ketamine was replaced by normal saline in the control group. In each subgroup, the water content of cerebral hemisphere was measured in 5 rats chosen randomly. The left 10 rats in each subgroup were transcardiacally perfused with ketamine, then the brain tissue was made into paraffin sections and stained by haematoxylin and eosin. Neuronal morphology was observed. AQP-4 expression and neuronal apoptosis were measured with immunohistochemical method and TUNEL method respectively. MAIN OUTCOME MEASURES： Water content in brain tissue, neuronal morphology, the number of AQP-4 positive neurons and TUNEL positive neurons in rats of two groups at each time point after injury. RESULTS： Totally 150 rats entered the stage of result analysis. （1） Water content of brain tissue： The water content of brain tissue at each time point after injury in the ketamine-treated group was lower than that in the control group. There were very significant differences in water content at 12 and 24 hours after injury respectively between ketamine-treated group and control group [（77.34±2.35）% vs. （82.31 ±1.48）%; （78.01 ±2.21 ）% vs. （83.86±2.37）%, t=-4.001 6,4.036 7, both P 〈 0.01]. （2） Neuronal morphology： Pathological changes in traumatic region and peripheral region of injury in the ketamine-treated group were significantly lessened, and necrotic and apoptotic cells in the ketamine-treated group were also significantly reduced as compared with control group. （3） AQP-4 expression： AQP-4 positive neurons at each time point in the ketamine-treated group were significantly less than those in the control group. There were very significant differences in AQP-4 expression at 12 and 24 hours after injury between ketamine-treated group and control group [（34.17±4.74） /visual field vs. （43.42±5.65） /visual field;（40.83±3.17） /visual field vs. （58.88±6.23） /visual field,t=3.966 3,8.165 7, both P〈 0.01]. （4） Neuronal apoptosis： TUNEL positive neurons at each time point in the ketamine-treated group were less than those in the control group. There were very significant differences in the neuronal apoptosis at 12 and 24 hours after injury between ketamine-treated group and control group [（26.25±3.04） /visual field vs. （32.75±4.39） /visual field; （29.33± 4.02） /visual field vs. （39.83±5.61） /visual field,t=-3.849 3,5.169 2,both P 〈 0.01]. CONCLUSION： Ketamine can reduce brain edema, AQP-4 expression and neuronal apoptosis following brain injury in rats, and has obvious therapeutic effect on brain injury, especially at 12 and 24 hours after injury.

Effect and mechanism of adrenomedullin on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury

Objective To investigate the effect and mechanism of adrenomedullin ( AM ) on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury. Methods Thirty-two Wistar rats were randomly divided into 4 groups: control group,IRI group, empty plasmid group and AM group. One week after re-

Cytotoxic Responses and Apoptosis in Rat Kidney Epithelial Cells Exposed to Lead

The toxic effects of lead on normal rat kidney epithelial cells（NRK cells）may occur via various pathways.However,the role of intrinsic mitochondrial pathway in Lead-induced apoptosis in NRK cells has not been investigated.The purpose of our study was to investigate cytotoxic responses and cell apoptosis mediated by lead in NRK cells.

Inosine inhibits apoptosis and cytochrome C mRNA expression in rat neurons after cerebral ischemia/reperfusion

BACKGROUND: It has been demonstrated that adenosine can induce glial cell to release cytochrome C, enhance expression of apoptotic gene bax, inhibit anti-apoptotic gene bcl-2, and activate caspase-3 to apoptosis; Whereas inosine can inhibit neuronal apoptosis which is similar to bcl-2. OBJECTIVE: To observe the effects of inosine on neuronal apoptosis and expression of cytochrome C mRNA in rats after focal cerebral ischemia/reperfusion, and analyze the pathway of its neuroprotective effect. DESIGN: A randomised controlled animal trial. SETTINGS: Department of Neurology, Rongcheng Second People's Hospital; Department of Neurology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Sixty-eight rats, weighing 230-280 g and clean grade, were used. TdT-mediated dUTP-biotin nick end labeling (TUNEL) and cytochrome C mRNA in situ hybridization kits and DAB staining kit were purchased from Wuhan Boster Biological Co., Ltd.; Inosine injection [200 mg (2 mL) each] from Qingdao First Pharmaceutical Factory. METHODS: The experiment was accomplished in the animal experimental center in Tongji Medical College of Huazhong University of Science and Technology from December 2003 to June 2005. ① Sixty-four rats were made into focal ischemia by middle cerebral artery occlusion (MCAO) with a nylon monofilament suture. The successfully induced rats were assigned to inosine group (n =32) and model group (n =32) at random. Rats in the inosine group were intraperitoneally administrated with inosine in dose of 100 mg/kg preoperatively, twice a day, 7 days in all. The rats in the control group were injected with the same dose of saline solution by the similar way preoperatively. Each group was randomized into ischemia /reperfusion 2, 6, 12, 24 hours, 2, 3, 7 and 14 days subgroups consisted of 4 rats. The other 4 rats were taken as the sham-operated group, the rats were given the same treatment except for not introduced the filament into the external carotid artery stump, and brain tissue was removed at 2 hours of reperfusion. ② In situ hybridization was performed to examine the expression of cytochrome C mRNA while TUNEL staining was made to characterize apoptosis. ③ The t test was used to compare the difference of measurement data. MAIN OUTCOME MEASURES: ① Neuronal apoptosis in the different regions of the ischemic brain tissue; ② Expression of cytochrome C mRNA in the different regions at different time points after MCAO. RESULTS: All the 68 rats were involved in the analysis of results. ① Neuronal apoptosis: A small number of TUNEL-positive cells were detected in the sham-operated brain and non-ischemic brain. The number of apoptotic cells in the ischemic cortex peaked at 24 hours of reperfusion [(72.00±1.98) cells] and that in the striatum peaked at 2 days [(94.75±3.57) cells], then decreased to the level of sham-operated group at 14 days. Inosine could reduce apoptotic cells from 12 hours to 7 days of reperfusion as compared with the model group (t =6.19-26.67, P < 0.01). ② Cytochrome C mRNA expression: There was weak expression of cytochrome C mRNA in both sham-operated brain and contralateral brain. Cytochrome C was detected at 2 hours of reperfusion in ischemic brain [(25.75±3.50), (39.75±2.49) cells], and strongly increased to a peak at 12 hours and 24 hours of reperfusion in cortex and striatum [(122.50±6.69), (119.25±5.12) cells], respectively. Furthermore, inosine could significantly decrease cytochrome C expression in cortex at 12 hours to 14 days of reperfusion after ischemic reperfusion and that in striatum at 12 hours to 3 days (t =8.67-43.26, P < 0.01). CONCLUSION: Inosine can exert a neuroprotective effect by inhibiting apoptosis and cytochrome C mRNA expression.

Apoptosis Induced by Zinc Deficiency in Rat Osteoblast:Possible Involvement of Protein Kinase C

Ra ostcobasts were isolated from the 21-day fetal rat calvchas. The cells were grown in DMEM Plus 10% FBS, and were treated for 24 h. with 10 μmol/L TPEN or 10 μmol/L TPEN supplemented with 10 μmol/L Zn2+. Apoptosis of osteoblasts were measured by fiow cytometry, electron microscopy and DNA fragmentation analyzed by gel elecmphoresis. In addition, IP3 production and PKC activity were measmed in ordr to show whether they are involved in apoptosis in osteoblast induced by alnc deficiency. The results showed that 10 μmol/L TPEN could induce apoptosis in osteoblast in 24 h. But cells ed with 10μmol/L TPEN supplemented with 10 μmol/L Zn2+ showed no apoptotic changs in 24 h. TPEN significantly reduced the formation of IP3 and PKC activity after 24 h incubation. No differences were observed between the cells treated with TPEN supPlemented with Zn2+ simulaneosly and the untreated cells. It can be inferred that apoptosis induced by ainc deficiency may be due to the decreased activity of PKC which is impaired by reduced formation of IP3.

Baicalin protects neonatal rat brains against hypoxicischemic injury by upregulating glutamate transporter 1 via the phosphoinositide 3-kinase/protein kinase B signaling pathway

Baicalin is a flavonoid compound extracted from Scutellaria baicalensis root.Recent evidence indicates that baicalin is neuroprotective in models of ischemic stroke.Here,we investigate the neuroprotective effect of baicalin in a neonatal rat model of hypoxic-ischemic encephalopathy.Seven-day-old pups underwent left common carotid artery ligation followed by hypoxia（8% oxygen at 37°C） for 2 hours,before being injected with baicalin（120 mg/kg intraperitoneally） and examined 24 hours later.Baicalin effectively reduced cerebral infarct volume and neuronal loss,inhibited apoptosis,and upregulated the expression of p-Akt and glutamate transporter 1.Intracerebroventricular injection of the phosphoinositide 3-kinase/protein kinase B（PI3 K/Akt） inhibitor LY294002 30 minutes before injury blocked the effect of baicalin on p-Akt and glutamate transporter 1,and weakened the associated neuroprotective effect.Our findings provide the first evidence,to our knowledge that baicalin can protect neonatal rat brains against hypoxic-ischemic injury by upregulating glutamate transporter 1 via the PI3 K/Akt signaling pathway.

Inhibitory effect of tea polyphenols on renal cell apoptosis in rat test subjects suffering from cyclosporine-induced chronic nephrotoxicity

Objective To investigate the inhibitory effect of tea polyphenols on renal cell apoptosis in rat test subjects suffering from cyclosporine A (CsA)-induced chronic nephrotoxicity.Methods Four groups of rats with CsA-induced chronic nephrotoxicity were respectively treated with vehicle olive oil, tea polyphenols, CsA and tea polyphenols plus CsA. At the end of the 28th day of treatment, 24 hours urine and blood samples were obtained, and the animals were then sacrificed. The serum and urine samples were analysed for creatinine clearance, and kidney tissue was used for pathologic analysis of renal tubular injury and interstitial fibrosis. The TUNEL assay, apoptosis-related enzyme caspase-3 mRNA detected by RT-PCR, and its enzymatic activity were analysed for the possible detections of cell apoptosis.Results CsA-treated rats displayed increased apoptosis of the tubular and interstitial cells, in comparison with vehicle-treated controls (18. 3±4. 6 vs 4. 8±1.3 cells/mm2, P < 0. 05 ) . In comparision with animals treated by CsA, animals treated with CsA plus tea polyphenols demonstrated significantly improved levels of creatinine clearance (0. 12 ±0. 03 vs 0. 22±0. 02 ml ·min-1·100g-1 body weight, P < 0. 05), tubular injury (2. 29 ±0. 43 vs 1. 42±0. 26, P < 0. 05), and interstitial fibrosis (2. 83±0. 20 vs 1. 46±0. 19, P <0. 05), and showed a statistically significant decrease in tubular and interstitial cell apoptosis (18. 3±4. 6 vs 7. 7±2.1 cells/mm2, P<0. 05). The expression of caspase-3 mRNA and caspase-3 activity was significantly higher in the CsA-treated group than that of the CsA plus tea polyphenols (TP)-treated group (P<0. 05).Conclusion These results suggested that tea polyphenols significantly inhibits apoptosis of the tubular and interstitial cells in rats with cyclosporine-induced chronic nephrotoxicity, and that tea polyphenols may be useful to prevent CsA-associated kidney toxicity.

Alterations of bcl-2, bcl x and bax protein expressions in area CA_3 of rat hippocampus following fluid percussion brain injury

Objective: To investigate the alterations of bcl 2 gene family in the area of CA 3 in rats and the molecular mechanism of neuronal apoptosis following traumatic brain injury. Methods: Male Sprague Dawley rats were subjected to lateral fluid percussion brain injury of moderate severity. bcl 2, bcl x, and bax protein expressions were detected by immunohistochemistry. Results: The immunoreactivity of bcl 2 and bcl x proteins decreased in the hippocampus ipsilateral impact site at 6 hours after injury, and this was the main cause of down regulation of the value of (bcl 2 +bcl x)/ bax. During the period of 1～3 days after injury, bax protein expression increased significantly, while bcl 2 and bcl x protein expressions decreased relatively slowly. The decreased value of (bcl 2+bcl x)/ bax was mainly due to the bax up regulation. Conclusions: The bcl 2 gene family is involved in neuronal apoptosis after traumatic brain injury, and the protein expression alterations of the bcl 2 gene family members lead to apoptosis of the neuronal cells.