大鼠脑cDNA文库的构建

采用简单高效的ｃＤＮＡ合成技术制备Ｗｉｓｔａｒ大鼠脑ｃＤＮＡ基因文库，以纯化的ｐｏｌｙ（Ａ）＋－ＲＮＡ为模板，含ＮｏｔＩ切点的ｏｌｉｇｏ－（ｄＴ）１５为引物，在反转录酶的作用下，合成第一股单链ｃＤＮＡ；用Ｅ。ｃｏｌｉＲＮａｓｅＨ除去模板ＲＮＡ，并以Ｅ．ｃｏｌｉＤＮＡ聚合酶Ｉ，Ｅ．ｃｏｌｉＤＮＡ连接酶和Ｔ４ＤＮＡ聚合酶催化合成ｃＤＮＡ第二条链，即成为双链ｃＤＮＡ；此双股ｃＤＮＡ除０．５μｇ用于插入ｐＳＰＯＲＴＩ载体，转入Ｅ．ｃｏｌｉＤＨ５α，建成ｃＤＮＡ文库外，其余保存在－２０℃，以此ｃＤＮＡ为模板，应用ＰＣＲ方法，先后克隆了谷氨酸脱羧酶（ＧＡＤ，１８００ｂＰ）、神经元特异性烯醇化酶（ＮＳＥ，１３４０ｂＰ），甲状腺激素受体（Ｔ３－ｒｅｃｅｐｔｏｒ，１２３０ｂｐ）、胆囊收缩素（ＣＣＫ，３４５ｂｐ）的全编码基因．

Gastric mucosal damage in water immersion stress:Mechanism and prevention with GHRP-6

AIM:To investigate the mechanism of gastric mucosal demage induced by water immersion restraint stress(WRS) and its prevention by growth hormone releasing peptide-6(GHRP-6).METHODS:Male Wistar rats were subjected to conscious or unconscious(anesthetized) WRS,simple restraint(SR),free swimming(FS),non-water fluid immersion,immersion without water contact,or rats were placed in a cage surrounded by sand.To explore the sensitivity structures that influence the stress reaction besides skin stimuli,a group the rats had their eyes occluded.Cervical bilateral trunk vagotomy or atropine injection was performed in some rats to assess the parasympathetic role in mucosal damage.Gastric mucosal lesions,acid output and heart rate variability were measured.Plasma renin,endothelin-1 and thromboxane B2 and gastric heat shock protein 70 were also assayed.GHRP-6 was injected [intraperitoneal(IP) or intracerebroventricular(ICV)] 2 h before the onset of stress to observe its potential prevention of the mucosal lesion.RESULTS:WRS for 6 h induced serious gastric mucosal lesion [lesion area,WRS 81.8 ± 6.4 mm 2 vs normal control 0.0 ± 0.0 mm 2,P < 0.01],decreased the heart rate,and increased the heart rate variability and gastric acid secretion,suggesting an increase in vagal nervecarrying stimuli.The mucosal injury was inversely correlated with water temperature(lesion area,WRS at 35 ℃ 56.4 ± 5.2 mm 2 vs WRS at 23 ℃ 81.8 ± 6.4 mm 2,P < 0.01) and was consciousness-dependent.The injury could not be prevented by eye occlusion,but could be prevented by avoiding contact of the rat body with the water by dressing it in an impermeable plastic suit.When water was replaced by vegetable oil or liquid paraffin,there were gastric lesions in the same grade of water immersion.When rat were placed in a cage surrounded by sand,there were no gastric lesions.All these data point to a remarkable importance of cutenuous information transmitted to the high neural center that by vagal nerves reaching the gastric mucosa.FS alone also induced serious gastric injury,but SR could not induce gastric injury.Bilateral vagotomy or atropine prevented the WRS-induced mucosal lesion,indicating that increased outflow from the vagal center is a decisive factor in WRS-induced gastric injury.The mucosal lesions were prevented by prior injection of GHRP-6 via IP did,but not via ICV,suggesting that the protection is peripheral,although a sudden injection is not equivalent to a physiological release and uptake,which eventually may affect the vagal center.CONCLUSION:From the central nervous system,vagal nerves carry the cutaneous stimuli brought about by the immersion restraint,an experimental model for inducing acute gastric erosions.GHRP-6 prevents the occurrence of these lesions.

Effects of intestinal mucosal blood flow and motility on intestinal mucosa

AIM： To investigate the role of intestinal mucosal blood flow （IMBF） and motility in the damage of intestinal mucosal barrier in rats with traumatic brain injury. METHODS： Sixty-four healthy male Wistar rats were divided randomly into two groups： traumatic brain injury （TBI） group （n = 32）, rats with traumatic brain injury; and control group （n = 32）, rats with sham-operation. Each group was divided into four subgroups （n = 8） as 6, 12, 24 and 48 h after operation. Intestinal motility was measured by the propulsion ratio of a semi-solid colored marker （carbon-ink）. IMBF was measured with the laser-Doppler technique. Endotoxin and D-xylose levels in plasma were measured to evaluate the change of intestinal mucosal barrier function following TBI. RESULTS： The level of endotoxin was significantly higher in TBI group than in the control group at each time point （0.382 ± 0.014 EU/mL vs 0.102 ± 0.007 EU/mL, 0.466 ± 0.018 EU/mL vs 0.114 ± 0.021 EU/mL, 0.478± 0.029 EU/mL vs 0.112 ±- 0.018 EU/mL and 0.412± 0.036 EU/mL vs 0.108 ±0.011 EU/mL, P 〈 0.05）. D-xylose concentrations in plasma in TBI group were significantly higher than in the control group （6.68 ± 2.37 mmol/L vs 3.66 ±1.07 retool/L, 8.51 ± 2.69 mmol/L vs 3.15 ＋ 0.95 mmol/L, 11.68 ±3.24 mmol/L vs 3.78 ± 1.12 mmol/L and 10.23 ± 2.83 mmol/L vs 3.34 ± 1.23 mmol/L, P 〈 0.05）. The IMBF in TBI group was significantly lower than that in the control group （38.5 ± 2.8 PU vs 45.6 ± 4.6 PU, 25.2 ± 3.1 PU vs 48.2 ± 5.3 PU, 21.5 ± 2.7 PU vs 44.9 ± 2.8 PU, 29. 4 ± 3.8 PU vs 46.7 ± 3.2 PU） （P 〈 0.05）. Significant decelerations of intestinal propulsion ratio in T8I groups were found compared with the control group （0.48% ± 0.06% vs 0.62%± 0.03%, 0.37% ±0.05% vs 0.64% ± 0.01%, 0.39% ± 0.07% vs 0.63% =1= 0.05% and 0.46% ± 0.03% vs 0.65% ± 0.02%） （P 〈 0.05）. CONCLUSION： The intestinal mucosal permeability is increased obviously in TBI rats. Decrease of intestinal motility and IMBF occur early in TBI, both are important pathogenic factors for stress-related damage of the intestinal mucosal barrier in TBI.

Pentapeptide Any-GS Blocks Antinociceptive Effect of Poneratoxin in Rats

Poneratoxin （PoTX） is an insect neuropeptide isolated from ant venom. It was previously demonstrated that administration of synthetic PoTX into the lateral brain ventricle （icv） induced in rats significant antinociceptive effect. Moreover it was demonstrated that this effect was not mediated by opioid receptors. The aim of present study was to determine other probable mechanisms mediating antinociceptive effect of PoTX, above all： （1） to check if insect-derived pentapeptide Any-GS may influence on PoTX-induced analgesia in rats, and （2） to estimate the role of voltage-gated sodium channels in rat＇s brain in antinociceptive effect of PoTX. The study was performed on adult, female wistar rats, which a week before experiments were implanted with polyethylene cannulas into the lateral brain ventricle （icv）. Antinociceptive effect of PoTX applied directly icv was determined in rats by the test of the tail immersion. PoTX applied icv at the dose of 1 or 5 nmol induced significant antinociceptive effect in rats. Pretreatment rats with equimolar dose of 1 or 5 nmol of veratridine, an agent, which opens voltage-gated sodium channels in neurons of rat brain, did not modify effect of PoTX. On the other hand, prior icv administration of pentapeptide Any-GS significantly inhibited antinociceptive effect of both icv doses of 1 and 5 nmols of PoTX. The results of the present study demonstrated antagonistic effect Any-GS against PoTX-induced analgesia. Thus blocking effect Any-GS on PoTX-induced analgesia indicates that this insect peptide is a probable antagonist of PoTX.

Effect of Synthetic Leucopyrokinin Analog [D-AlaS]-[2-8]-Leucopyrokinin （[D-AlaS]-[2-8]-LPK） on Opioid-lnduced Analgesia in Rats

The study was undertaken in order to evaluate effect of synthetic insect neuropeptide leucopyrokinin analog, [D-Ala5]-[2-8]-LPK, on analgesia induced by selective agonists of/a-, 6- and l〈-opioid receptors. The study was performed on male Wistar rats, which a week before the experiments were implanted with polyethylene cannulas into the lateral brain ventricle （icv）. Effect of prior administration of [D-Ala5]-[2-8]-LPK on analgesia induced in rats by next icv administration of equimolar dose of μ-, δ- and -opioid agonists： DAMGO, DPDPE and GR fumarate respectively, was evaluated. Antinociceptive effect was determined in rats by the test of the tail immersion. It was found that two doses of 5 and 10 nmols icv of [D-AlaS]-[2-8]-LPK inhibited analgesia in rats by equimolar doses of DAMGO. This analog also transiently （only in two time intervals） and in one dose of 10 nmols inhibited analgesia induced in rats by icv administration of equimolar DPDPE dose of 10 nmols icv. Obtained results indicate that [D-AlaS]-[2-8]-LPK inhibits antinociceptive effect of DAMGO and in part of DPDPE, i.e. mainly antagonized ~t-opioid receptors. These results correspond with results of our previous study that selective antagonists of μ- and δ-opioid receptors blocked antinociceptive effect of synthetic insect neuropeptide leucopyrokinin and of it active analog [2-8]-leucopyrokinin. We regard that [D-AIaS]-[2-8]-LPK, the first discovered antagonist of leucopyrokinin may be a useful as a probable tool substance in the study of biological effects of insect-derived peptides either in invertebrates or in mammals.

Effects of different frequencies of electro-acupuncture at Shuigou(GV 26) on recovery of motor function in rats with focal cerebra ischemic injury

OBJECTIVE:To investigate the effects of different frequencies of electro-acupuncture at Shuigou(GV 26) on the latent period and wave amplitude of motor evoked potentials(MEPs) in rats with focal cerebral infarction.METHODS:Fifty healthy male Wistar rats were randomly divided into five groups:controls,model,2 Hz Shuigou,50 Hz Shuigou and 100 Hz Shuigou.There were 10 rats in each group.Using a modification of a technique for middle cerebral artery occlusion,focal cerebral ischemic injury was induced in all rats except those in the control group.The rats in the control group received no treatment.After behavioral deficit had been evaluated using the Zausinger 6-point neurological function score,therats in the Shuigou groups underwent acupuncture and continuous wave stimulation at a frequency of 2 Hz,50 Hz or 100 Hz(intensity 1 mA) for 10 min twice daily for 3 days.The control and model groups underwent no intervention.Zausinger 6-point neurological function score and MEPs were measured 72 h after the start of treatment.RESULTS:The neurological function scores of the three Shuigou groups were significantly higher than those of the model group(P<0.05).There was no significant difference between sides in the latency and amplitude of MEPs in the model group(P> 0.05).The latency on the affected side in the model group was significantly longer than that in the control group(P<0.05) and the amplitude on affected side was significantly reduced(P<0.01).After 3 days of electro-acupuncture,the latency on the affected side in the 2 Hz Shuigou group was significantly shortened(P<0.05) and the amplitude was significantly increased(P<0.05) compared with the model group.CONCLUSION:Low frequency electro-acupuncture at Shuigou(GV 26) can promote recovery of motor function after focal cerebral ischemic injury in rats.

T2 relaxation time measurements in the brains of scalded rats

This study aimed to evaluate the T2 relaxation time of the brain in severely scalded rats using a magnetic resonance （MR） T2 mapping sequence, and to investigate the correlation between T2 relaxation time and plasma glucose level. Twenty-eight Wistar rats were randomly divided into the scalded group （n = 21）and control group （n = 7）. Magnetic resonance scans were performed with T1WI, T2WI, and T2-mapping sequences in the scalded group; the scans were performed 1 day prior to scalding and 1, 3, 5, and 7 days post-scalding; in addition, identical MR scans were performed in the control group at the same time points. T2-maps were generated and T2 relaxation times were acquired from the following brain regions： the hippocampus, thalamus, caudate-putamen, and cerebrum. Pathological changes of the hippocampus were observed. The plasma glucose level of each rat was measured before each MR scan, and a correlation analysis was performed between T2 relaxation time and plasma glucose level. We found that conventional T 1WI and T2WI did not reveal any abnormal signals or morphological changes in the hippocampus, thalamus, caudate-putamen,： or cerebrum post-scalding. Both the T2 relaxation times of the selected brain regions and plasma glucose levels increased 1, 3, and 5 days post-scalding, and returned to normal levels 7 days post-scalding. The most marked increase of T2 relaxation time was found in the hippocampus; similar changes were also revealed in the thalamus, caudate-putamen, and cerebrum. No correlation was found between T2 relaxation time and plasma glucose level in scalded rats. Pathological observation of the hippocampus showed edema 1, 3, and 5 days post-scalding, with recovery to normal findings at 7 days post-scalding. Thus, we concluded that T2 mapping is a sensitive method for detecting and monitoring scald injury in the rat brain. As the hippocampus is the main region for modulating a stress reaction, it showed significantly increased water content along with an increased plasma glucose level post-scalding.

Neuroprotective effect of Naomaitong extract following focal cerebral ischemia induced by middle cerebral artery occlusion in rats

OBJECTIVE: To examine the neuroprotective effect of extract from Naomaitong following focal cerebral ischemia reperfusion induced by occlusion of middle cerebral artery(MCA), and to determine the biochemical alterations in urine using proton nuclear magnetic resonance spectroscopy and principal component analysis.METHODS: Wistar rats were randomly assigned tothree groups: sham-operated group, MCA focal cerebral ischemia reperfusion model group, and active extract of Naomaitong treatment group. The model was established by an improved MCA occlusion(MCAO) method. Sham-operated rats received the same surgical procedure, but without occlusion. The Naomaitong treatment group were treated with active extract from Naomaitong at a dose of3.0 g·kg-·1d-1. Brain tissues and urine samples were collected from all groups for histopathological assessment and proton nuclear magnetic resonance spectroscopy-based metabonomics, respectively.RESULTS: Hematoxylin-eosin and triphenyl tetrazolium chloride staining of brain tissues showed a significant decrease in cerebral infarction area in the Naomaitong group. In model rats, metabonomic analyses showed increased urinary levels of glutamate, taurine, trimetlylamine oxide, betaine, and glycine, and reduced levels of creatinine and creatine.Naomaitong regulated the metabolic changes by acting on multiple metabolic pathways, including glycine metabolism, glutaminolysis, transmethylation metabolism and creatinine metabolism.CONCLUSION: These data demonstrate that extract from Naomaitong is neuroprotective against focal cerebral ischemia induced by MCAO, and can alleviate biochemical changes in urinary metabolism. Metabonomics may be a useful approach for assessing the biochemical mechanisms underlying the neuroprotective actions of extract from Naomaitong.