Grafting of glial cell line-derived neurotrophic factor secreting cells for hypoxic-ischemic encephalopathy in neonatal rats

OBJECTIVE: It has been reported that an infarcted area is reduced by the injection of glial cell line-derived neurotrophic factor into brain parenchyma after hypoxic/ischemic insult in neonatal rats. For use of glial cell line-derived neurotrophic factor in humans, we have developed a system for the delivery of a constant supply of glial cell line-derived neurotrophic factor to the brain. The aim of this study was to examine the neuroprotective effect of glial cell line-derived neurotrophic factor with the use of this delivery system. STUDY DESIGN: Baby hamster kidney cells were transfected with human glial cell line-derived neurotrophic factor complementary DNA, encapsulated in semipermeable hollow fibers, and implanted into the left cerebrum of 12-day-old Wistar rats (glial cell line-derived neurotrophic factor group, 11 rats). Nontransfected baby hamster kidney cells served as controls (control group, 9 rats). Two days after implantation, the rats received a hypoxic/ischemic stress, with a modification of Levine's method. Seven days later the rats were killed, and coronal brain slices were cut 2, 4, 6, 8, and 10 mm from the anterior pole. The cortex, hippocampus, striatum, and thalamus were evaluated for damage severity. The serum concentrations of glial cell line-derived neurotrophic factor were also determined. RESULTS: The left brain hemispheric area was significantly larger; the neuronal damage to each brain region was significantly less, and the serum glial cell line-derived neurotrophic factor concentrations were significantly higher in the glial cell line-derived neurotrophic factor group, compared with the control group. CONCLUSION: Grafting of encapsulated glial cell line-derived neurotrophic factor-secreting cells is a promising way to protect the neonatal brain from hypoxic/ischemic insult.     

电刺激小脑顶核对缺氧缺血新生大鼠学习记忆能力的影响

目的探讨电刺激小脑顶核对缺氧缺血新生大鼠学习记忆功能的影响。方法 7日龄Wist-ar大鼠（36只）随机分为缺氧缺血性脑损伤组和假手术组,缺氧缺血性脑损伤组又分为模型组和电刺激组,采用结扎左侧颈总动脉并吸入氮氧混合气体2h制作新生大鼠缺氧缺血性脑损伤动物模型,分别于手术第2天相同时间点给予无电刺激装置（假手术组）,连接装置但不接通（模型组）,连接装置并且接通（电刺激组）。各组12只动物,随机分为7d干预组（6只）和14d干预组（6只）,两组分别于7、14d干预后,（1）用Y-型迷宫检测脑损伤鼠的学习记忆功能;（2）苏木精-伊红染色法光镜下观察脑神经细胞和神经纤维的病理变化;（3）免疫组织化学检测脑皮质和海马区STAT3蛋白水平的表达。结果 （1）Y-型迷宫测试结果模型组总时间较电刺激组增多,模型组主动回避反应率和正确反应率低于电刺激组（P〈0.05）;模型组总时间与假手术组相比,显著增加;模型组主动回避反应率和正确反应率也明显低于假手术组,差异均有统计学意义（P〈0.05）。（2）假手术组脑组织各部位的结构及细胞层次清楚、形态正常,未见明显损伤性改变。模型组脑组织可见明显的毛细血管出血,细胞核固缩、核碎裂明显,模型建立成功。电刺激组神经细胞变性坏死较少,多数细胞形态相对正常。（3）电刺激组子鼠脑皮质及海马周围信号转导和转录激活蛋白3（STAT3）蛋白较模型组增多（P〈0.05）。结论电刺激小脑顶核可促进脑损伤鼠学习记忆能力的恢复。     

Hypoxic-ischemic tolerance induced by hyperthermic pretreatment in newborn rats

OBJECTIVE: We investigated the effect of hyperthermic pretreatment 24 hours before hypoxic-ischemic exposure on neuronal cell damage in 7-day-old rats. METHODS: Newborn rats were separated on postnatal day 7 into two groups: 1) preheated (those exposed to 2 hours of hyperthermic pretreatment at 42-43C) (n = 29), and 2) nonheated (n = 20). At 24 hours after the hyperthermic stress, rats from both groups were subjected to left carotid artery ligation followed by 2 hours of hypoxia (8% oxygen/92% nitrogen) at 33C. All rats were killed 1 week after hypoxia-ischemia, and brains were extracted for histologic study. A different group of 7-day-old rats (n = 8) was placed in the same hyperthermic environment as mentioned above for 2 hours, and 24 hours after heat exposure brains were extracted for immunohistochemistry of heat-shock protein 70. RESULTS: The total incidence of hypoxic-ischemic brain damage significantly decreased in the preheated group (12 of 25 [48%]) compared with the nonheated group (17 of 20 [85%]; P < .03). The induction of immunoreactive heat-shock protein 70 was observed mainly in glial and vascular endothelial cells and, in a lesser amount, in neuronal cells of the cerebral cortex and hippocampus. CONCLUSION: Incidence of hypoxic-ischemic brain damage is consistently reduced by 2 hours of hyperthermic pretreatment in 7-day-old rats.     

Important role of 72-kd heat shock protein expression in the endothelial cell in acquisition of hypoxic-ischemic tolerance in the immature rat

OBJECTIVES: Hypoxic-ischemic tolerance can be induced in neonatal rats through hyperthermic preconditioning. The purposes of this study were to determine the interval between hyperthermic preconditioning and a subsequent hypoxic-ischemic insult that would provide optimal neuroprotection against the insult and to examine the relationship between tolerance induction and heat shock protein expression. STUDY DESIGN: On postnatal day 7 Wistar rat pups were separated into the following 2 groups: a heated group (those exposed to 15 minutes of hyperthermic pretreatment at a brain temperature of 41.5 degrees C-42.0 degrees C) and an unheated control group. At 6, 12, 24, 48, and 72 hours after the hyperthermic stress, rats from both groups were exposed to left carotid artery ligation followed by 2 hours of hypoxia (8% oxygen and 92% nitrogen) at 33 degrees C. Twenty animals from each group were used at each time point. All rats were killed at 1 week after hypoxia-ischemia, at which time the brains were processed and neuronal damage in the cortex and hippocampus was assessed histologically. Another set of 7-day-old rats (n = 30) was studied immunohistochemically at 6, 12, 24, 48, and 72 hours after the same hyperthermic treatment. Expression of 72-kd heat shock protein was measured in neuronal, glial, and vascular endothelial cells. RESULTS: Hyperthermia-induced hypoxic-ischemic tolerance was observed at 6, 12, and 24 hours but not at 48 and 72 hours after hyperthermic preconditioning. Heat shock protein 72 expression in the vascular endothelial cells, rather than in the glial or neuronal cells, was most strongly associated with hypoxic-ischemic tolerance. CONCLUSION: These findings suggest that heat shock protein 72 in endothelial cells plays an important role in the acquisition of hypoxic-ischemic tolerance at postnatal day 7, a time when maximal angiogenesis occurs and the blood-brain barrier is still immature.     

Effects of hyperthermia on hypoxic-ischemic brain damage in the immature rat: its influence on caspase-3-like protease

OBJECTIVE: Recent clinical studies suggested that intrapartum maternal fever is a strong independent risk factor for neonatal encephalopathy. With use of a well-studied rat model of neonatal hypoxic-ischemic encepalopathy, this study investigated the hypothesis that intraischemic hyperthermia accelerates and worsens brain injury in immature animals and examined whether apoptotic cell death machinery is involved in the underlying mechanisms. STUDY DESIGN: Seven-day-old rats underwent a combination of left common carotid artery ligation and exposure to 8% oxygen for 15 minutes (n = 32 rats). During the 15-minute hypoxic insult, body temperature was elevated to 40 degrees C in 16 animals (hyperthermic hypoxic insult group), and was maintained at 37 degrees C in 16 animals (normothermic hypoxic insult group). Then both groups were placed in the same chamber in a water bath at 37 degrees C for 24 hours and finally returned to the mothers. Caspase-3-like activity was assessed 36 hours after the hypoxic-ischemic insult. One week later, microtubule-associated protein-2 immunostaining was used to examine neuronal damage. RESULTS: Intraischemic hyperthermia was shown to activate the caspase-3 activity 36 hours after hypoxia-ischemia while caspase-3 was activated insignificantly in the normothermic hypoxic insult group at that time. The hyperthermic hypoxic insult group also showed a reduced microtubule-associated protein-2-positive area 7 days after hypoxia-ischemia compared with that in the normothermia group. CONCLUSION: Hyperthermia during hypoxia-ischemia makes the immature brain inordinately susceptible to hypoxic-ischemic insult and causes brain injury, even if hypoxic-ischemic insult is so mild that it causes no or little injury by itself. This effect may be mediated by the escalation of the apoptotic cell death pathway in the immature animal.     

Prolonged in utero meconium exposure impairs spatial learning in the adult rat. Central Prize Award

OBJECTIVE: The purpose of this study was to examine the effects of prolonged in utero meconium exposure on adult learning and memory, as measured by the Morris water maze. STUDY DESIGN: Timed pregnant Long-Evans rats were studied. On gestational day 20 (term, 21 days of gestation), laparotomy was performed, and each maternal animal received an injection of clear amniotic fluid or meconium-stained amniotic fluid into each gestational sac. The laparotomy incision was closed, and the animals received postoperative monitoring through delivery. On postnatal days 145 to 148, the offspring underwent Morris water maze testing. The mean (+/-SEM) for the latency time was reported for each day's trial and compared between groups. RESULTS: There were significant differences between meconium-stained amniotic fluid group and clear amniotic fluid group in the mean time to platform on day 1 (82.7 +/- 1.8 seconds vs 75.9 +/- 3.0 seconds; P=.04), day 2 (60.5 +/- 3.5 seconds vs 47. 8 +/- 4.6 seconds; P=.03), and day 3 (56.5 +/- 4.5 seconds vs 34.7 +/- 4.4 seconds; P=.001). However, there were no differences on days 4 and 5. There were also no differences between recall and response learning trials that were done after a 12-day retention period. CONCLUSION: In the absence of hypoxia or infection, prolonged in utero meconium exposure is associated with a delay of spatial learning in the adult rat.     

Creatine protects the immature brain from hypoxic-ischemic injury

OBJECTIVE: We tested the neuroprotective effects of creatine against hypoxic-ischemic injury in the immature brain. METHODS: Hippocampal slices were prepared from fetal guinea pigs at 0.9 gestation and incubated in artificial cerebrospinal fluid (aCSF) equilibrated with carbogen. Slices were subjected to oxygen-glucose deprivation (OGD) for 30 or 40 minutes. Two hours after OGD, adenosine triphosphate (ATP) and protein synthesis were analyzed. Creatine (3 mM) was applied to tissue slices of the study groups 2 hours before the insult. In a second set of experiments 7-day-old Wistar rats were anesthetized, and the left carotid artery was ligated. After 1 hour of recovery the pups were subjected to a hypoxic gas mixture (8% oxygen and 92% nitrogen) for 80 minutes. Seven days later the brains of the neonates were removed and analyzed for hypoxic-ischemic injury. The rat pups of the test group were treated with creatine (3 g/kg subcutaneously) before (-64 hours, -40 hours, and -16 hours) and after (+3 hours) the hypoxic-ischemic insult, with zero time corresponding to the start of hypoxia, whereas the animals of the control group received the solvent. RESULTS: Creatine significantly improved the recovery of protein synthesis 2 hours after OGD in hippocampal slices but had no effect on ATP levels. Whereas seven animals of the control group developed severe cystic cerebral infarction, only mild to moderate damage was observed in the rat pups of the study group. In contrast to creatine-treated pups, the volume of the ipsilateral hemisphere was considerably smaller than that of the contralateral one in control animals (104 +/- 22 versus 138 +/- 14 mL, P<.001). Except at the frontal level (A 6.0 mm), neuronal cell injury was significantly lower in the cortex of the animals that had received creatine. This was also true for the evaluated subfields in the hippocampus. CONCLUSION: We conclude that creatine protects the immature brain from hypoxic-ischemic injury.     

缺氧缺血性脑病的性别差异性研究进展

新生儿缺氧缺血性脑病(hypoxic-ischemic encephalopathy,HIE)是指围产期窒息缺氧导致的脑缺氧缺血性损害,可遗留不同程度的神经系统后遗症。动物研究表明,缺氧缺血性损伤后,炎性反应、氧化应激和细胞死亡途径等关键病理生理过程中存在明显的性别差异,雌性动物对亚低温、促红细胞生成素、脑源性营养因子和别嘌呤醇等治疗效果也明显优于雄性。临床研究发现男性HIE患儿病情更重、预后更差。基于性别的治疗干预很有可能在围产期脑损伤中提供更好的神经保护。本文总结了目前HIE性别差异性的相关证据,以期为临床治疗提供新思路。     

细胞外信号调节激酶信号转导通路活化在新生大鼠缺氧缺血性脑损伤中的作用

目的 观察细胞外信号调节激酶(extracellular-signal regulated kinase,ERK)在新生大鼠缺氧缺血性脑损伤中的作用.方法 Wistar大鼠随机分为假手术组、缺氧缺血组和PD98059(ERK抑制剂)组,通过结扎右侧颈总动脉,恢复2 h后,吸入含8%氧气的氧氮混合气体2 h的方法 建立新生大鼠缺氧缺血性脑损伤模型,PD98059组结扎右侧颈总动脉前10 min股静脉注射PD980592 mg/kg.各组新生大鼠于模型制作后6 h剥离结扎侧海马及皮层脑组织,分别测定脑组织丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活性和细胞凋亡水平.Western印迹法检测ERK1和ERK2,Bcl-2和Bax蛋白的表达.组间差异比较采用方差分析及q检验.结果 缺氧缺血组细胞凋亡率明显高于假手术组[(18.80±1.37)%和(3.53±0.34)%](q=6.06,P＜0.01),PD98059组细胞凋亡率[(15.53±0.64)%]低于缺氧缺血组(q=3.87,P＜0.01).缺氧缺血组MDA含量为(342.9±10.8)μmol/L,明显高于假手术组[(181.5±17.0)μmol/L](q=6.35,P＜0.01)和PD98059组[(252.0±17.1)μmol/L](q=5.28,P＜0.01),而SOD活性[(34.8±4.3)U/ml]明显低于假手术组[(63.4±4.3)U/ml](q=4.99,P＜0.01)和PD98059组[(51.5±3.8)U/ml](q=4.17,P＜0.01).3组总ERK1和ERK2蛋白表达差异无统计学意义.缺氧缺血组磷酸化ERK1和磷酸化ERK2蛋白表达明显高于假手术组(q分别=3.82和4.08,P均＜0.01)和PD98059组(q分别=4.79和5.12,P均＜0.01).缺氧缺血后Bcl-2和Bax蛋白表达明显高于假手术组(q分别=3.55和3.42,P均＜0.01);PD98059组较缺氧缺血组抗凋亡蛋白Bcl-2表达增加,促凋亡蛋白Bax表达降低(q分别=3.71和5.86,P均＜0.01).结论 ERK激活通过调节凋亡蛋白表达参与了新生大鼠缺氧缺血性脑损伤的发生.

Abstract：

Objective To investigate the effects of extracellular-signal regulated kinase (ERK) on hypoxic-ischemic brain damage of neonatal rats.Methods The hypoxic-ischemic brain damage model of neonatal Wistar rats was established as following:first the right common carotid artery of the rats was ligated;2 h after operation,the rats began to inhale 8%-oxygen oxygen-nitrogen gas mixture lasting for 2 h.Rats were randomly divided into three groups:sham-group,hypoxic-ischemic group and ERK inhibitor PD98059 group (the rats were injected PD98059 2 mg/kg 10 min before the ligation).Six hours after the models were done,hippocampi and cortex of the ligation side of rats in the three groups were collected,and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured.Apoptosis of neuron was assessed by TUNEL staining.The expression of ERK1,ERK 2,Bcl-2 and Bax were examined by Western blot.The differences among the groups were analyzed with ANOVA and q test.Results Compared with the sham-group,the MDA level [(342.9± 10.8) μmol/L vs (181.5± 17.0) μmol/L,q= 6.35,P＜0.01) and the apoptosis rate of neuron [(18.80±1.37)% vs (3.53±0.34)%,q=6.06,P＜0.01) of hypoxic-ischemic group was higher,and SOD level was lower [(34.8±4.3) U/ml vs (63.4±4.3) U/ml,q=4.99,P＜0.01].While the apoptosis rate of neuron [(15.53±0.64) %] and MDA level [(252.0± 17.1) μmol/L] of PD98059 group were lower than those of hypoxic-ischemic group(q=3.87 and 5.28,P＜0.01respectively),the SOD level [(51.5 ± 3.8) U/ml] was higher than that of hypoxic-ischemic group (q=4.17,P＜0.01).There were no differences of ERK1 and ERK2 expressions among the three groups.The phosphorylated ERK1 and ERK2 levels of hypoxic-ischemic group were higher than those of sham-group (q=3.82 and 4.08,P＜0.01) and PD98059 group (q=4.79 and 5.12,P＜0.01).The expression of Bcl-2 and Bax of hypoxic-ischemic group were higher than those of sham-group (q=3.55 and 3.42,P＜0.01).Compared with hypoxic-ischemic group,Bcl-2 expression (q=3.71,P＜0.01) of PD98059 group was higher,and Bax expression (q=5.86,P ＜ 0.01) was lower.Conclusions ERK is involved in hypoxic-ischemic brain damage of neonatal rats through regulating the expression of apoptosis protein.     

Effects of 3-h hypothermia after neonatal hyperthermic hypoxic-ischemic encephalopathy in rat models on behavioral prognosis and anatomical and histological features after growth

Objective: To clarify the effects of 3-h hypothermia on learning ability and motor function after growth, employing neonatal rat models with hyperthermic hypoxic-ischemic encephalopathy (HIE).

Methods: We divided all rats into three groups: N (adult rats after neonatal hyperthermic HIE without subsequent 3-h hypothermia), H (adult rats after neonatal hyperthermic HIE with subsequent 3-h hypothermia) and Sham (S) groups. We evaluated their malfunctions with the rota-rod test and the step-down passive avoidance test. We also analyzed the cerebrum width and the hippocampal CA1 area of the insulted hemisphere.

Results: In the rota-rod test, the result of the N group was significantly worse than that of the S group. In the step-down passive avoidance test, the result of the N group was significantly worse than those of the S and H groups. The longest cerebrum width and the hippocampal CA1 area of the insulted hemisphere of the N group were significantly smaller than those of the S and H groups.

Conclusion: Neonatal hyperthermic hypoxic-ischemic insult restricts motor function and learning ability after growth, and such neuronal malfunctions can be relieved by hypothermia for 3 h soon after neonatal HIE.     

碱性成纤维细胞生长因子对新生大鼠脑缺血后神经新生的影响

目的 研究碱性成纤维细胞生长因子(bFGF)对新生大鼠脑缺血后神经新生的影响.方法 通过结扎3日龄新生SD大鼠双侧颈总动脉制备脑缺血模型,随机分为治疗组(36只):给予bFGF 10 ng/g侧脑室注射;对照组(36只)给予相同体积的生理盐水.另取36只新生大鼠,仅分离双侧颈总动脉,不结扎,不给予药物,作为假手术组.三组大鼠分别于术后第4、7、14天处死,获取脑组织标本,采用免疫荧光染色、Western印记和实时PCR方法,观察三组大鼠不同时点脑室下区巢蛋白(nestin)、Ⅲ型β-微管蛋白(Tuj1)、胶质纤维酸性蛋白(GFAP)和少突胶质细胞NG2蛋白及其mRNA的表达变化.结果 (1)免疫荧光染色:治疗组BrdU+/nestin+细胞数目术后7 d达高峰[(48.7±5.9)个/视野],高于同时点对照组[(32.2±3.1)个/视野]和假手术组[(17.3±1.6)个/视野],差异有统计学意义(P<0.01);治疗组BrdU+/Tuj1+细胞、BrdU+/GFAP+细胞、BrdU+/NG2+细胞数目术后14 d[分别为(92.6±9.7)、(58.2±6.1)、(57.3±5.4)个/视野]达高峰,高于同时点对照组[分别为(65.8±7.1)、(42.1±4.4)、(37.8±3.2)个/视野]和假手术组[分别为(35.3±3.1)、(33.6±3.4)、(22.4±2.1)个/视野],差异有统计学意义(P<0.01).(2)Western印迹和实时PCR:与免疫荧光染色结果一致,即治疗组nestin蛋白和mRNA表达术后7 d达高峰,高于同时点对照组和假手术组,差异有统计学意义(P<0.01);治疗组Tuj1、GFAP和NG2蛋白和mRNA术后14 d达高峰,高于同时点对照组和假手术组,差异有统计学意义(P<0.01).结论 bFGF可促进新生大鼠脑缺血后脑室下区神经干细胞的增殖以及向功能神经细胞(神经元、星形胶质细胞、少突胶质细胞)的分化,可能对新生大鼠脑缺血后神经细胞的再生具有促进作用.     

宫内脂多糖暴露后新生大鼠脑髓鞘基质蛋白、胶质纤维酸性蛋白、白细胞介素-1β、肿瘤坏死因子-α的表达变化

目的 探讨宫内脂多糖(lipopolysaeeharide,LPS)暴露后新生大鼠脑中炎症因子的表达与脑白质损伤的关系. 方法 向孕19 d SD大鼠宫内注射LPS(0.4μg/孕囊间)构建宫内感染大鼠模型作为LPS组(n=14),对照组给予同体积的无菌生理盐水(n=10).取1、3、7、11日龄新生大鼠脑标本(每组每时间点6只),通过免疫组织化学方法 检测髓鞘基质蛋白与胶质纤维酸性蛋白在新生大鼠脑组织中的表达;采用逆转录-聚合酶链反应技术检测脑组织白细胞介素-1β、肿瘤坏死因子-α mRNA的水平.组间差异比较采用t检验. 结果 (1)LPS组孕鼠胎盘组织HE染色可见显著炎性细胞浸润、间质明显增生、毛细血管腔变窄.(2)LPS组11日龄新生大鼠小脑、间脑髓鞘基质蛋白表达分别为1.29±0.76和1.71±0.49,均较对照组(分别为2.43±0.79和2.50±0.76)明显减少(P均<0.05);而LPS组11日龄新生大鼠海马、胼胝体、间脑和小脑中胶质纤维酸性蛋白的表达分别为2.71±0.49、2.40±0.55、1.50±0.55和2.80±0.45,均较对照组(分别为1.75±0.50、1.50±0.58、1.00±0.00和1.60±0.55)增强(P均<0.05).(3)LPS组与对照组1、3、7、11日龄新生大鼠脑组织中白细胞介素-1β与肿瘤坏死因子-α mRNA的表达差异无统计学意义(P均>0.05). 结论 宫内LPS暴露导致宫内炎性改变、影响胎盘血供,并致新生大鼠低出生体重,可能引起新生大鼠出现以低髓鞘化及反应性星形胶质化为特征的脑白质损伤.     

Neurobehavior effects in four strains of mice offspring exposed prenatally to alprazolam

OBJECTIVE: This study was performed to determine whether prenatal exposure to alprazolam affects offspring behavior in different strains of mice. STUDY DESIGN: Eight to 11 gravid mice of the C3H/He, C57BL/6, A/J, and DBA/2 strains were given either an anxiolytic dose of alprazolam (0.32 mg/kg) or a placebo by gavage on day 18 of an anticipated 19- to 21-day gestation. Neurobehavior tasks were conducted to assess anxiety, learning and memory, and social interaction. Data were analyzed by analysis of variance or a Fisher exact probability test. RESULTS: Anxiety in alprazolam-exposed offspring was reduced in C3H/He (P <.05) and A/J (P <.05) newborn infants by separation vocalization but may be increased in the C3H/He adult strain on the plus maze task. Learning was slower among C57BL/6 mice exposed to alprazolam (P <.01), whereas memory was reduced in exposed A/J and DBA/2 offspring (P <.05). Alprazolam exposure was associated with more aggression among C3H/He and C57BL/6 male offspring (P <.01) and with less group activity by C57BL/6 offspring (P <.05). CONCLUSION: Altered behaviors in several mouse strains after prenatal exposure to alprazolam suggests a vulnerability of GABA-benozdiazepine receptor formation in fetal brain development.     

三日龄大鼠缺氧缺血性脑损伤对大脑MRI影像和学习记忆能力的远期影响

目的 观察缺氧缺血(HI)对3日龄大鼠脑细胞凋亡及成年后头部MRI影像和学习记忆能力的影响.方法 应用凋亡基因芯片研究3日龄大鼠缺氧缺血性脑损伤后12 h与损伤7 d凋亡基因表达的差异.42日龄时进行大脑MRI检查,并在44日龄时采用水迷宫测试其学习和记忆能力.组间比较采用t检验和秩和检验.结果 与HI脑损伤后12 h相比,损伤后7 d表达上调的基因包括TNF及其受体家族中的Tnfsf10(TRAIL)、Tnfsf13(CD30)、Tnfrsf21、Tnfrsf11b;Caspase家族中的Caspase1、2、3和6;Bcl2家族中的促凋亡基因Bak1、Becn1、Bcl10和Bid3;死亡域TRADD和Myd88.而Caspase 8和抗凋亡基因Mapk8ip表达下调.42日龄时头部MRI检查显示HI组右侧大脑皮质面积较左侧和假手术组显著减小[侧脑室层(23.5±3.6)mm2、(33.0±4.3)mm2和(34.5±3.9)mm2(F=17.09,P<0.01);海马层(18.9±4.4)mm2、(29.1±5.0)mm2和(30.8±4.5)mm2(F=14.44,P<0.01)].HI组水迷宫试验上台时间在第4天为(52.7±35.9)s,明显长于假手术组的(17.8±8.9)s(P<0.01).记忆测试中,HI组大鼠经过站台的次数显著少于假手术组(T=292.5,P<0.05).结论 3日龄SD大鼠HI脑损伤后,神经细胞凋亡基因激活持续到损伤后7 d,涉及凋亡的外源性和内源性通路.神经细胞的凋亡会导致大脑皮层萎缩,可能影响动物成年后的空间学习和记忆能力.     

丰富环境暴露对大鼠惊厥性脑损伤保护作用的探讨

目的探讨丰富环境对大鼠惊厥性脑损伤是否具有保护作用。 方法2004 08西安交通大学医学院对30只21日龄雄性SD大鼠经水浴法诱导反复惊厥10次后随机均分为长程丰富环境暴露组(LG)、短程丰富环境暴露组(SG)和对照组(CG)。LG、SG组分别暴露于丰富环境中10d和5d，CG组不给予干预措施。观察各组大鼠在避暗试验(passive avoidance test,PAT)、Morris水迷宫(Morris water maze,MWM)及旷场试验(open field test,OFT)中学习记忆和情感行为的变化。 结果丰富环境暴露可使反复惊厥大鼠在PAT中的记忆潜伏期延长\[LG组为(32342±9838)s，SG组为(27652±9264)s，CG组为(19713±9511)s，P<001\]，错误反应次数减少(LG组为175±101，SG组为214±113，CG组为537±126，P<001)；在OFT中，丰富环境暴露可增强反复惊厥大鼠的兴奋性(LG组得分为6235±781，SG组为5367±766，CG组为4548±734，P<001)，提高大鼠对陌生环境的适应能力(LG组后肢性站立的次数为3337±474，SG组为2618±423，CG组为1719±431，P<001)，改善大鼠的紧张情绪(LG组粪便粒数为385±201，SG组为614±198，CG组为1162±212，P<001)；在MWM中，丰富环境暴露可使反复惊厥大鼠逃逸潜伏期缩短，搜寻策略改善(LG、SG及CG组直线式搜寻策略所占百分比依次为378%、336%及272%,χ2=4102,P<0005)。 结论丰富环境暴露可以改善反复惊厥大鼠的学习记忆和情感行为，对惊厥性脑功能损伤具有保护作用。     

缺氧缺血性脑损伤新生大鼠脑组织细胞红蛋白的表达

目的 探讨细胞红蛋白(eytoglobin,CYGB)在7日龄新生大鼠缺氧缺血性脑损伤(hypoxic-isehemie brain damage,HIBD)脑内的表达变化.方法 健康7日龄SD大鼠70只,按简单随机抽样法随机取50只制备HIBD模型作为HIBD组,再随机分为HIBD后0、4、12、24和48 h组,每组10只;假手术组10只,只分离左颈总动脉,不结扎,也不放入缺氧箱;另10只不做任何处理作为对照组.假手术组术后即断头取脑,对照组同时取脑,HIBD组在相应时间点取脑.采用免疫组织化学法和Western印迹法从蛋白水平分析CYGB在脑组织中的分布及表达特点. 结果 (1)免疫组织化学染色显示CYGB主要表达于大脑皮质、丘脑和海马神经元的胞浆,HIBD组CYGB表达数量及染色强度均高于对照组,其中缺氧24、48 h组表达最强.(2)Western印迹结果显示,HIBD后0、4、12、24和48 h CYGB平均吸光度分别为261.5土5.0,263.0土5.4,464.3±6.8,522.8±11.2,512.9±7.9,明显高于假手术组(117.6±7.0)及对照组(116.6±9.0),P<0.01.其中缺氧0和4 h组明显高于对照组(P<0.01);缺氧12 h组的表达明显高于缺氧0、4 h组(P<0.01);缺氧24、48 h组明显高于缺氧12 h组(P<0.01);缺氧0、4 h组间,缺氧24、48 h组间差异无统计学意义(P>0.05). 结论 7日龄新生大鼠HIBD后脑内CYGB表达明显增强,并且随着缺氧缺血发展呈现时相性变化,提示其可能在脑缺氧缺血的适应性调节过程中起重要作用.     

Multiple courses of antenatal betamethasone and cognitive development of mice offspring

OBJECTIVE: To measure the effect of multiple courses of antenatal betamethasone, used for lung maturation, on long-term cognition of mice offspring. METHODS: Forty gravid CD-1 mice were randomly assigned to receive one of four treatments (n = 10 per group): 0.1 mg betamethasone or saline placebo, given subcutaneously either once daily on gestational days 13-16 or twice daily on days 14 and 15. This dose of betamethasone given on gestational day 14 causes fetal lung maturation in mice. Three offspring per gender in each litter underwent standard cognitive tasks as juveniles and as adults. Analysis of variance or Kruskal-Wallis testing was used to compare data. RESULTS: Learning acquisition and memory were indistinguishable between the betamethasone-exposed and the corresponding placebo-exposed offspring when performing the following tasks: juvenile runway with adult memory, adult water runway and Morris spatial maze. This lack of difference in task performance between treatment groups persisted after controlling for gender and for each multiple-course regimen. CONCLUSION: Multiple courses of antenatal corticosteroids did not impact the mouse offsprings' long-term learning and memory.     

Mechanisms of hypoxic-ischemic injury in the term infant

The pathogenesis of hypoxic-ischemic brain injury in the term infant is multifactorial and complex. Over the past decade the investigative emphasis has turned to cellular and molecular mechanisms of injury, and it has been increasingly recognized that the neonatal brain differs vastly from the adult brain in terms of response to hypoxia-ischemia. This review will discuss the initiation and evolution of brain injury in the term neonate, and the inherent biochemical and physiologic qualities of the neonatal brain that make its response to hypoxia-ischemia unique. Attention will be given to specific areas of investigation including excitotoxicity, oxidative stress, and inflammation. The coalescence of these entities to a final common pathway of hypoxic-ischemic brain injury will be emphasized.     

血小板生长因子对缺氧缺血性脑损伤新生鼠脑细胞凋亡率和血清神经元特异性烯醇化酶的影响

目的 研究血小板源性生长因子(platelet derived growth factor,PDGF)对缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)的新生鼠神经细胞凋亡率及血清神经元特异性烯醇化酶(neuron-specific enolase,NSE)浓度的影响,进而探讨其对HIBD的神经保护作用. 方法 7日龄新生Wistar大鼠48只制备HIBD模型,并分为PDGF治疗组和生理盐水对照组,每组各24只.另取24只为假手术组.治疗组在缺氧缺血后即刻给PDGF-BB 50 ng/kg腹腔注射.对照组和假手术组腹腔注射等体积的生理盐水.每组于处置后12、24和72 h随机取8只处死,留血清标本,酶联免疫吸附法检测大鼠血清标本NSE浓度;取右侧大脑组织制备脑细胞悬液,双染法流式细胞仪检测脑细胞凋亡率.采用单因素方差分析及q检验进行统计学分析. 结果 (1)脑细胞凋亡率:治疗组[(6.09±0.70)％、(9.67±1.52)％和(14.15±1.52)％]和对照组[(8.00±1.10)％、(11.45±2.42)％和(22.90±2.03)％]3个时点的脑细胞凋亡率均较假手术组(2.11±0.54)％、(2.34±0.46)％和(2.21±0.49)％]显著增加(P均＜0.01或＜0.05),治疗组较对照组各时点脑细胞凋亡率均明显降低(P均＜0.01或＜0.05),3组大鼠在12、24、72 h时的组间比较差异均有统计学意义(F=39.01、66.60、194.20,P均＜0.01).(2)血清NSE浓度:各时点对照组[(10.04±0.19) μg/L、(9.33±0.15)μg/L和(8.36±0.16)μg/L]和治疗组[(8.43±0.17)μg/L、(6.73±0.16) μg/L和(6.12±0.13)μg/L]较假手术组[(4.22±0.53)μg/L、(3.96±0.60) μg/L和(3.59±0.55) μg/L]NSE浓度增加(P均＜0.01),治疗组较对照组各时点NSE浓度降低(P均＜0.01),3组大鼠在12、24、72h组间比较差异均有统计学意义(F=371.25、245.61、236.22,P均＜0.01). 结论 PDGF能抑制新生大鼠HIBD后神经细胞凋亡及降低血清NSE浓度,对HIBD新生大鼠有神经保护作用.     

The effects of fibroblast growth factor-2 and pluripotent astrocytic stem cells on cognitive function in a rat model of neonatal hypoxic-ischemic brain injury

Objective: This study aimed to determine the effect of pluripotent astrocytic stem cells (PASCs) and fibroblast growth factor-2 (FGF-2) on cognitive function in neonatal rats with hypoxic-ischemic brain injury (HIBI).

Methods: The study was performed on 7-d-old rats that were randomly divided into four groups. All rats, except those in the sham group, were kept in a hypoxic chamber containing 8% oxygen for 2?h after the ligation of the right carotid artery. Next, 5?d after HIBI was induced, PASCs were administered to the motor cortex, and FGF-2 was administered intraperitoneally to group AF; PASCs were administered to the motor cortex, and salt solution buffered with phosphate was administered intraperitoneally to group A; and fresh cell culture solution (medium) was administered to group M. Immunofluorescence was used to localize the administered PASCs in the brains of rats from groups A and AF. The Morris water maze tank (MWM) test was performed to assess the rats’ cognitive functions at week 12. The rats that were administered PASCs were observed for the development of neoplasms and autopsies were performed after 30 months.

Results: PASCs migrated to damaged brain regions surrounding the hippocampus in groups A and AF. The mean platform finding time (PFT) significantly decreased over time in each group on day 1–4 of MWM testing (p?<?0.001). On day 2–4, the mean PFT was shortest in group S followed by group AF. In group A, the PFT was significantly longer than in group S on day 3–4 (p?=?0.01 and 0.007, respectively). On day 5 of the MWM test, the time spent in the eastern quadrant (which previously contained the platform) was longest in group S followed by groups AF, A, and M; however, the differences between groups were not significant (p?=?0.51). After 30 months, none of the rats in groups A or AF had benign or malignant neoplasms.

Conclusions: Following the administration of PASCs in rats with experimentally induced HIBI, PASCs migrated to the injured brain regions; however, treatment with PASCs did not have a positive effect on cognitive function. The administration of FGF-2 together with PASCs resulted in positive cognitive results, although not at the level of significance.