阻断子宫动脉建立FGR大鼠模型的研究

目的通过暂时阻断妊娠期大鼠子宫血供的方法建立子宫缺血引起胎儿生长受限的动物模型。方法根据大鼠子宫动脉是卵巢动脉的一个分支的解剖特点,于孕鼠妊娠第15天时施行手术暂时阻断卵巢动脉并于第21天行剖宫产术,术后称量新生胎仔体重及胎盘、脑、心、肝、肺、肾等重要脏器重量,对比各组间新生胎仔的预后的不同,并对照研究阻断血供10、20、30及40 min对胎仔的不同影响。结果妊娠晚期阻断孕鼠卵巢动脉20min可成功构建胎儿生长受限模型,这种方法与阻断动脉血流30或40 min相比,手术时间短,技术要求不高,胎仔死亡率与对照组差异无显著性(P>0.05)。各实验组较对照组新生胎仔体重及胎盘、各重要脏器重量均明显降低(P<0.05)。结论通过阻断卵巢动脉从而阻断子宫动脉血流,成功建立缺血缺氧性FGR孕鼠模型。该模型重复性好,操作简便,并可成功设立同体对照,为进行FGR相关的产科理论研究提供了一个有利的技术平台。     

小鼠左肺原位移植模型的建立

目的通过显微外科技术建立小鼠原位肺移植模型,为肺移植研究提供动物模型。方法采用C57BL/6小鼠作为供、受体,行同基因小鼠原位左肺移植,使用Cuff套管法进行气管及血管吻合。术后7、14、21、28 d取移植肺及原肺,行HE染色,评价肺移植后效果。结果学习曲线后,共30例小鼠移植,手术成功率89%,小鼠成活率100%。供体手术时间:(35.2±9.81)min,受体手术时间:(24.6±7.42)min,冷缺血时间是:(46.6±8.92)min,热缺血时间是:(17.2±3.08)min。同基因移植物大体及病理无明显改变,病理显示与原肺无差别。结论本技术能够方便快捷建立小鼠肺移植模型,成功率高,可重复性强,符合原位肺移植临床生理,是研究肺移植发病机制和治疗的良好动物模型。     

改良法建立妊娠期高血压疾病大鼠模型

目的寻找理想的妊娠期高血压疾病动物模型。方法60只雌性Wistar大鼠分为六组,分别为正常妊娠组（A组）、左旋亚硝酸精氨酸甲酯（L-NAME）腹腔注射组（B组）、生理盐水注射组（C组）、假手术组（D组）、双侧子宫动脉结扎术组（E组）以及L-NAME腹腔注射联合腹主动脉缩窄术组（F组）,对大鼠血压、尿蛋白值以及胎鼠、胎盘、胎鼠头的重量进行监测并观察肾脏、胎盘的病理改变。结果F组孕鼠于孕13d即出现了蛋白尿、血压升高,较B组及E组出现时间早（P〈0.05）,其胎鼠体重、胎头重量下降较各组更明显（P〈0.05）,肾小球小动脉管壁增厚、管腔狭窄;胎盘出现血管间膜增厚、纤维蛋白沉积等妊娠高血压疾病的典型病理改变。结论L-NAME腹腔注射联合腹主动脉缩窄术是更为理想的建立妊娠高血压疾病动物模型的方法。     

缺血缺氧再灌注对胎鼠肾组织环氧化酶表达的影响

目的观察宫内缺血缺氧再灌注后,胎鼠肾组织COX蛋白表达及其AA代谢产物PGI2、PGE2和TXA2含量的变化,探讨COX在宫内窘迫胎鼠肾损伤发病机制中的作用.方法制备胎鼠子宫内缺血缺氧再灌注模型(缺血缺氧组：缺血缺氧30min;再灌注组：缺血缺氧30min后,分别再灌注30min,2h,6h,12h,24h,30h)缺血氧组：取胎鼠18只;再灌注组各时间点分别取胎鼠18只;对照组取胎鼠20只.将肾组织匀浆后采用Western免疫印迹和放免法检测COX蛋白表达及其PGI2、PGE2和TXA2含量的变化.同时HE染色观察肾组织病理学改变.结果子宫内缺血缺氧再灌注后胎肾组织COX-2蛋白表达上调,PGI2的稳定代谢产物6-Keta-PGF1α及PGE2均于再灌注2h开始增高(P<0.05).其中6-keta-PGF1α增加迅速,于再灌注12h达假手术组的6倍(P<0.01),PGE2于再灌注24h达假手术组的2倍(P<0.01),而TXB2增加幅度不大,无统计学意义.结论宫内缺血缺氧再灌注选择性地诱导胎肾COX-2蛋白表达增强,COX-2可能通过PGI2和PGE2对缺血性胎肾损伤具有保护作用,因此,在围产期肾损伤不宜应用COX-2抑制剂.     

昆明小鼠实验性自身免疫性脑脊髓炎模型的建立

用豚鼠脊髓匀浆液和不完全弗氏佐剂制备免疫原,联合腹腔注射百日咳毒素,建立昆明小鼠实验性自身免疫性脑脊髓炎动物模型.发病小鼠临床神经系统症状逐渐加重,脑组织经HE染色后,可见血管周围有大量炎性细胞浸润,呈袖套状改变;大脑白质可见胶质细胞增生形成胶质细胞结节以及神经细胞坏死等病理改变.正常小鼠中枢神经系统功能及病理检查均未见异常.此模型为进一步阐明多发性硬化的发病机制打下基础.     

延迟5分钟剖宫产造全脑缺氧缺血新生大鼠模型

目的建立围产期全脑缺氧缺血性损伤的新生大鼠模型。方法 SD雌性大鼠妊娠21 d时,颈椎脱臼法处死,用止血钳夹闭双侧子宫角血管5 min后,剖宫产取出新生大鼠,交由代乳鼠喂养。结果造模组雌性大鼠9只,共娩出新生大鼠91只,出生3 d内死亡7只,死亡率7.7%。新生大鼠出生第2天进行翻身实验,第14天进行悬吊实验和斜坡实验,造模组和其余各组均有显著性差异。新生大鼠出生后21 d,取脑组织切片行HE染色,显示大脑皮层典型的缺氧缺血性损伤,与正常组相比,可见神经细胞明显的病理形态学改变。结论采用延迟5min剖宫产和代乳鼠喂养的方法,操作简便,并结合行为学测试筛选行为异常者,可建立稳定可靠、可供长期实验使用的围产期全脑缺氧缺血性损伤的新生大鼠模型。     

博来霉素诱导G57BL/6与ICR小鼠肺间质纤维化模型的比较

目的探讨C57BL/6与ICR小鼠在博来霉素(BLM)致肺纤维化过程中的种属差异。方法 8周龄雌性C57BL/6小鼠19只,ICR小鼠16只,分别经尾静脉一次性注射BLM150mg/kg,观察每组小鼠体重、生存率及肺组织病理改变。结果①C57BL/6与ICR小鼠最低体重分别发生在静脉注射处置后的7d和5d,最低体重分别为注射前的65.46%和73.21%,两组间无显著的统计学差异。②C57BL/6与ICR小鼠的生存率分别为36.84%和56.25%,两组间存在显著的统计学差异。③C57BL/6小鼠BLM注射后28d,在胸膜下及血管周围形成广泛、稳定的间质纤维化病理改变,而ICR小鼠肺组织未见明显纤维化形成。C57BL/6小鼠肺纤维化病理评分明显高于ICR小鼠(P0.001)。结论 BLM诱导的肺纤维化作用在C57BL/6与ICR小鼠间存在着明显的种属差异。C57BL/6小鼠较ICR小鼠更适于复制博来霉素诱导的肺纤维化动物模型。     

神经肽对小鼠急性脑缺血保护作用的研究

结扎小鼠一侧颈总动脉,建立急性脑缺血动物模型。术前腹腔给予人胚胎脑组织提取的生物活性多肽类物质神经肽,术后一小时测定脑组织中乳酸脱氢酶（ＬＤＨ）活性,同时进行组织学和超微结构观察。结果显示给药组小鼠脑组织ＬＤＨ活性明显高于未给药组,给药组小鼠脑组织脱水不明显,神经细胞中无明显空泡,细胞内超微结构改变较未给药组轻。本研究说明种经肽对脑组织急性缺血具有保护作用。     

小鼠胎脑组织活体转基因技术的建立

目的:建立实用的小鼠活体脑组织基因转染技术。方法:将EGFP质粒(CAGS启动子)注射到胎龄16 d(E16d)的胎鼠侧脑室,用镊形电极隔着子宫壁夹住胎鼠头部,在45 V电压下给予5次电脉冲刺激,每次刺激50ms,间隔1 s;转染后不同天数将胎鼠脑组织完整取出,以4%PFA固定后冰冻切片,进行激光共聚焦照相。结果:EGFP质粒被转入小鼠活体脑组织细胞中并获得表达,动物存活率为90%,GFP阳性率高于80%。结论:通过对麻醉剂、电脉冲刺激、质粒浓度、术中术后处理等多种实验条件的摸索,建立了实用的小鼠胎脑组织活体转基因技术。     

异柠檬酸脱氢酶（IDH）在小鼠胚胎发育中的变化

本文采用淀粉凝胶电泳对异柠檬酸脱氢酶(IDH)在小鼠发育中的改变进行了研究,结果证明肝脏中IDH的改变很有代表性。妊娠10天胚胎、出生前一天胎鼠和出生后一天胎鼠之间IDH的带有明显变化,可作为小鼠不同发育时期有关基因表达的指标。     

新生小鼠缺氧缺血性脑损伤模型的制作研究

目的：通过对动物模型的制作模拟新生儿围产期缺氧缺血性脑损伤,研究其脑组织病理变化,为新生儿缺氧缺血性脑损伤的病理生理的研究以及进一步有效的治疗提供实验基础。方法：将40只7d新生昆明小鼠分四组,分别为正常组（A组）、单侧颈总动脉结扎组（B组）、单侧颈总动脉结扎＋缺氧组（C组）和双侧颈总动脉结扎组（D组）。单侧颈总动脉结扎组（B组）行右侧颈总动脉结扎;单侧颈总动脉结扎＋缺氧组（C组）行右侧颈总动脉结扎后将其置于20℃的恒温50mL密闭容器中,分不同的时间将其取出;双侧颈总动脉结扎组（D组）行双侧颈总动脉结扎,各组术后均送回母鼠身边继续母乳喂养,三天后再作病理检测。结果：行单侧颈总动脉结扎加缺氧60min时,小鼠结扎侧皮质及海马区出现病理改变,随着缺氧时间延长（90rain、100min、120min）病变范围逐渐扩大,病理改变越明显。结论：本实验显示单侧颈总动脉结扎同时缺氧一定时间可以导致小鼠脑组织损伤,脑细胞发生病理改变,且皮层及海马区域的神经细胞对缺氧缺血最为敏感,从而为进一步研究新生儿缺氧缺血性脑损伤提供了较为可靠的模型。     

The Effect of Hypothermia Therapy on Cortical Laminar Disruption following Ischemic Injury in Neonatal Mice

Hypothermia has been proposed as a treatment for reducing neuronal damage in the brain induced by hypoxic ischemia. In the developing brain, hypoxic ischemia-induced injury may give rise to cerebral palsy (CP). However, it is unknown whether hypothermia might affect the development of CP. The purpose of this study was to investigate whether hypothermia would have a protective effect on the brains of immature, 3-day old (P3) mice after a challenge of cerebral ischemia. Cerebral ischemia was induced in P3 mice with a right common carotid artery ligation followed by hypoxia (6% O₂, 37°C) for 30 min. Immediately after hypoxic ischemia, mice were exposed to hypothermia (32°C) or normothermia (37°C) for 24 h. At 4 weeks of age, mouse motor development was tested in a behavioral test. Mice were sacrificed at P4, P7, and 5 weeks to examine brain morphology. The laminar structure of the cortex was examined with immunohistochemistry (Cux1/Ctip2); the number of neurons was counted; and the expression of myelin basic protein (MBP) was determined. The hypothermia treatment was associated with improved neurological outcomes in the behavioral test. In the normothermia group, histological analyses indicated reduced numbers of neurons, reduced cortical laminar thickness in the deep, ischemic cortical layers, and significant reduction in MBP expression in the ischemic cortex compared to the contralateral cortex. In the hypothermia group, no reductions were noted in deep cortical layer thickness and in MBP expression in the ischemic cortex compared to the contralateral cortex. At 24 h after the hypothermia treatment prevented the neuronal cell death that had predominantly occurred in the ischemic cortical deep layers with normothermia treatment. Our findings may provide a preclinical basis for testing hypothermal therapies in patients with CP induced by hypoxic ischemia in the preterm period.     

Impact of Perinatal Systemic Hypoxic–Ischemic Injury on the Brain of Male Offspring Rats: An Improved Model of Neonatal Hypoxic–Ischemic Encephalopathy in Early Preterm Newborns

In this study, we attempted to design a model using Sprague-Dawley rats to better reproduce perinatal systemic hypoxic-ischemic encephalopathy (HIE) in early preterm newborns. On day 21 of gestation, the uterus of pregnant rats were exposed and the blood supply to the fetuses of neonatal HIE groups were thoroughly abscised by hemostatic clamp for 5, 10 or 15 min. Thereafter, fetuses were moved from the uterus and manually stimulated to initiate breathing in an incubator at 37 °C for 1 hr in air. We showed that survival rates of offspring rats were decreased with longer hypoxic time. TUNEL staining showed that apoptotic cells were significant increased in the brains of offspring rats from the 10 min and 15 min HIE groups as compared to the offspring rats in the control group at postnatal day (PND) 1, but there was no statistical difference between the offspring rats in the 5 min HIE and control groups. The perinatal hypoxic treatment resulted in decreased neurons and increased cleaved caspase-3 protein levels in the offspring rats from all HIE groups at PND 1. Platform crossing times and the percentage of the time spent in the target quadrant of Morris Water Maze test were significantly reduced in the offspring rats of all HIE groups at PND 30, which were associated with decreased brain-derived neurotrophic factor levels and neuronal cells in the hippocampus of offspring rats at PND 35. These data demonstrated that perinatal ischemic injury led to the death of neuronal cells and long-lasting impairment of memory. This model reproduced hypoxic ischemic encephalopathy in early preterm newborns and may be appropriate for investigating therapeutic interventions.     

Conserved aquaporin 4 levels associated with reduction of brain edema are mediated by estrogen in the ischemic brain after experimental stroke

Aquaporin 4 (AQP4), the most abundant water channel protein in the brain, is involved in brain edema induced by ischemic insults. To evaluate whether the neuroprotective effects of estrogen are associated with AQP4 expression and edema formation, changes in AQP levels and ischemic edema were examined in the brains of male and female mice subjected to transient middle cerebral artery occlusion. Infarct volume and edema formation were markedly less in females than in males. AQP4 expression in the ischemic cortex of females was relatively well preserved, whereas it was significantly decreased in males. These effects disappeared in ovariectomized females but were reversed by estrogen replacement. Furthermore, AQP4 expression was decreased with increased brain edema in females treated with ICI182,780, an estrogen receptor antagonist. These findings suggest that the estrogen effect on the reduction of ischemic brain edema is associated with the preserved level of AQP4 that is partly mediated by estrogen receptors.     

Utilization of Cyclocreatine Phosphate, an Analogue of Creatine Phosphate, by Mouse Brain During Ischemia and Its Sparing Action on Brain Energy Reserves

Abstract: Brains of mice fed the creatine analogue cyclocreatine accumulated 10 γmol/g fresh wt. of cyclocreatine, of which 93% occurred as the synthetic phosphagen, cyclocreatine-P (l-carboxymethyl-2-imino-3-phosphonoimidazolidine). In brains containing cyclocreatine-P^2-, creatine-P (phosphocreatine) levels were lowered 40%; levels of ATP, P₁, and glucose were not altered: glutamate levels were lowered 17%: and aspartate levels were lowered 56%, relative to controls. When cyclocreatine was removed from the diet, brain cyclocreatine levels decreased with a half-life of 17 to 28 days. Ischemia was initiated in brains by decapitation of mice previously injected with the centrally acting muscle relaxant mephenesin. The initial creatine-P pool of 2-3 γmol/g was completely depleted within 1 min in ischemic brains of both control and cyclocreatine-fed mice. In brains of cyclocreatine-fed mice, the much larger cyclocreatine-P pool of 9.3 γmol/g decreased to 6 γmol/g after 2 min and to 2.2 γrnol/g after 4 min of ischemia, with a correspondingly increased accumulation of P₁. Levels of total cellular ATP were sustained slightly longer during ischemia in brains containing cyclocreatine-P. Available energy reserves of control brains were almost completely depleted after 2 min of ischemia, whereas generation and utilization of high-energy phosphate continued for more than 3 min after initiation of ischemia in brains of cyclocreatine-fed mice. These data suggest that during ischemic episodes cyclocreatine-P can function as a supplemental reservoir of high-energy phosphate and prolong the time required to exhaust the available energy stores of ischemic brain.     

31P-NMR studies on the energy metabolism of the living rat brain using a surface coil method

We have examined the changes of the energetic metabolic state of rat and mouse brains under hypoxic hypoxia or ischemia using a 31P-NMR spectrometer with a surface coil. The NMR spectrometer has a super-conducting magnet providing a homogeneous magnetic field of 6.3 tesla. A probe was remodelled to accommodate an experimental animal in it. The animals were anesthetized with 1.0% or 1.5% halothane throughout the experiments. The optimal measurement conditions were a 90 degrees pulse width of 20 microsecond, and a 2 sec pulse repetition time. 200 acquisitions of FIDs was required for high spatially resolved signals. The Pcr/ATP ratio of the live, anesthetized rat brains was 1.76 +/- 0.46 (n = 8) for cerebra and 1.63 +/- 0.11 (n = 4) for cerebella. That of gerbil brains was 1.23 +/- 0.09 (n = 4). The Pcr/ATP ratio did not show any significant changes under both the conditions of hypoxic hypoxia or ischemia. The value of Pcr/Pi ratio decreased in the hypoxic conditions. The level of Pcr. of rat cerebrum decreased by 76.8 +/- 10.5% at 10% oxygen and by 57.3 +/- 15.7% at 5% oxygen compared with the value of 20% oxygen. The ATP level in the rat brain also decreased according to the degree of hypoxia. Cerebral ischemia was produced in the gerbil by ligation of the common carotid artery. The levels of Pcr. and ATP were severely depressed in the ischemic hemisphere but those of the intact side remained normal.     

Neonatal Semax and saline injections induce open-field behavior changes in mice of different genotypes

DBA/2, CBA mice, and their F1 hybrids (first series) and 101/HY and C3H mice (second series) were injected as neonates (2-7 days of life) with Semax (sc., 7 microg per animal). Semax is a peptide analogue of ACHT4-10 fragment which is resistant to degradation. The common feature of remote effects of both Semax and saline injections was the set of changes in the open-field behavior in adult (2.5- to 3-month-old) animals as compared to intact mice. Unexpectedly, the neonatal saline injections induced many changes in adult behavior, part of these effects being genotype-dependent. The most conspicuous shifts (genotype-dependent increase or decline) in freezing, grooming and rearing scores were displayed by DBA/2 and C3H mice, whereas the hole-poke frequencies were significantly changed in CBA and C3H mice. Squares crossed in the center of arena and rearing number were significantly increased in saline group of DBA/2 mice, whereas in Semax-injected DBA/2 group they were approximately equal to the level of intact mice. This means that the remote effects of noxious stimulation (injections of saline) were in some ways "compensated" as the result of concomitant peptide effect. At the same time, the numbers of freezing and grooming episodes were also increased in these groups. Because exploratory behavior and manifestations of anxiety increased or decreased simultaneously, it proves to be difficult to ascribe these changes to behavioral modulation along the "novelty seeking--anxiety" axis. In mice of other genotypes, changes in the same indices of the open-field behavior were revealed, but these changes were different in their direction. It was suggested that the complex patterns of postnatal behavior was the result of neonatal injections modulating subsequent brain development.     

Dynamic changes in the distribution and time course of blood–brain barrier-permeative nitroxides in the mouse head with EPR imaging: visualization of blood flow in a mouse model of ischemia

Electron paramagnetic resonance (EPR) imaging using nitroxides as redox-sensitive probes is a powerful, noninvasive method that can be used under various physiological conditions to visualize changes in redox status that result from oxidative damage. Two blood–brain barrier-permeative nitroxides, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP) and 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MCP), have been widely used as redox-sensitive probes in the brains of small animals, but their in vivo distribution and properties have not yet been analyzed in detail. In this study, a custom-made continuous-wave three-dimensional (3D) EPR imager was used to obtain 3D EPR images of mouse heads using MCP or HMP. This EPR imager made it possible to take 3D EPR images reconstructed from data from 181 projections acquired every 60 s. Using this improved EPR imager and magnetic resonance imaging, the distribution and reduction time courses of HMP and MCP were examined in mouse heads. EPR images of living mice revealed that HMP and MCP have different distributions and different time courses for entering the brain. Based on the pharmacokinetics of the reduction reactions of HMP and MCP in the mouse head, the half-lives of HMP and MCP were clearly and accurately mapped pixel by pixel. An ischemic mouse model was prepared, and the half-life of MCP was mapped in the mouse head. Compared to the half-life in control mice, the half-life of MCP in the ischemic model mouse brain was significantly increased, suggesting a shift in the redox balance. This in vivo EPR imaging method using BBB-permeative MCP is a useful noninvasive method for assessing changes in the redox status in mouse brains under oxidative stress.     

足月新生儿缺氧缺血性脑损伤大鼠模型的制作与鉴定

目的制作并鉴定研究足月新生儿缺氧缺血性脑损伤（HIBD）的大鼠模型,以期用于足月新生儿缺氧缺血性脑损伤发病机制及治疗的研究。方法40只新生10日龄SD大鼠分为对照组18只和实验组（HIBD组）22只,实验组行右侧颈总动脉结扎,缺氧（8%氧和92%氮气）2.5 h;对照组只分离右侧颈总动脉,不行结扎和缺氧处理。应用Longa评分法评价神经行为学改变;HE染色检测组织病理学改变;免疫组化检测凋亡标志蛋白cleavedcaspase-3（CC3）的表达;及TUNEL染色检测细胞凋亡情况。结果对照组大鼠全部存活,实验组死亡4只,死亡率18.1%。Longa评分分析实验组有不同程度神经功能缺损,与对照组比较差异有显著性（P〈0.05）;HE染色显示实验组均出现脑组织充血、水肿,缺血侧更重,细胞体肿胀,细胞排列紊乱,结构不清;免疫组化显示实验组CC3表达随损伤时间延长逐渐增加,与对照组比较差异有显著性（P〈0.01）;TUNEL染色显示实验组阳性细胞随时间延长逐渐增加,与对照组比较差异有显著性（P〈0.05）。结论该大鼠模型脑组织病变符合足月新生儿HIBD的病理学改变及神经行为学改变,可用于足月新生儿HIBD发病机制与防治措施的研究。