siRNA对体外培养大鼠海马神经元细胞红蛋白基因表达的抑制

【目的】 研究siRNA(small interfering RNA)对细胞红蛋白(Cytoglobin, CYGB)基因表达的抑制作用。 【方法】 取新生大鼠海马做神经元原代培养,NeuN免疫荧光抗体鉴定神经元纯度后,通过阳离子脂质体转染试剂将体外化学合成的针对CYGB基因的siRNA转入细胞中,以未转入siRNA细胞及转入阴性siRNA细胞为对照,通过转染荧光对照siRNA检测转染效率,用RT-PCR方法检测siRNA对CYGB表达的抑制作用。 【结果】 成功体外培养海马神经元,转染荧光对照siRNA结果示转染效率在70%左右,RT-PCR结果提示与阴性对照及空白对照相比,转染siRNA-78 后24 h(P＜0.01),48 h(P＜0.001)及60 h(P＜0.001)CYGB mRNA的表达明显减弱,72 h后表达无明显减少。而转染siRNA-79,siRNA-80在各时间点与对照相比没有明显差别(P＞0.05)。 【结论】 特异性针对CYGB的siRNA-78转染体外培养的大鼠海马神经元24~60 h能显著下调CYGB基因的表达。     

Fetal and neonatal iron deficiency causes volume loss and alters the neurochemical profile of the adult rat hippocampus

Abstract

Objective

Perinatal iron deficiency results in persistent hippocampus-based cognitive deficits in adulthood despite iron supplementation. The objective of the present study was to determine the long-term effects of perinatal iron deficiency and its treatment on hippocampal anatomy and neurochemistry in formerly iron-deficient young adult rats.

Methods

Perinatal iron deficiency was induced using a low-iron diet during gestation and the first postnatal week in male rats. Hippocampal size was determined using volumetric magnetic resonance imaging at 8 weeks of age. Hippocampal neurochemical profile, consisting of 17 metabolites indexing neuronal and glial integrity, energy reserves, amino acids, and myelination, was quantified using high-field in vivo ¹H NMR spectroscopy at 9.4 T (N = 11) and compared with iron-sufficient control group (N = 10).

Results

The brain iron concentration was 56% lower than the control group at 7 days of age in the iron-deficient group, but had recovered completely at 8 weeks. The cross-sectional area of the hippocampus was decreased by 12% in the formerly iron-deficient group (P = 0.0002). The hippocampal neurochemical profile was altered: relative to the control group, creatine, lactate, N-acetylaspartylglutamate, and taurine concentrations were 6–29% lower, and glutamine concentration 18% higher in the formerly iron-deficient hippocampus (P < 0.05).

Discussion

Perinatal iron deficiency was associated with reduced hippocampal size and altered neurochemistry in adulthood, despite correction of brain iron deficiency. The neurochemical changes suggest suppressed energy metabolism, neuronal activity, and plasticity in the formerly iron-deficient hippocampus. These anatomic and neurochemical changes are consistent with previous structural and behavioral studies demonstrating long-term hippocampal dysfunction following perinatal iron deficiency.     

砷对大鼠脑海马c-fos、c-jun表达和细胞调亡影响

目的 观察砷对大鼠脑海马区c-fos、c-jun蛋白表达和细胞凋亡影响,探索砷对大脑神经毒性机制。方法 将56只SD大鼠随机分成4组,即低、中、高剂量染砷组(亚砷酸钠:5、10、20 mg/kg)和对照组,染砷组灌胃染毒,对照组给予蒸馏水,连续15 d,处死大鼠;免疫组化法和DNA 断裂原位末端标记法(TUNEL)法分别测定大鼠脑海马区c-fos、c-jun蛋白表达和细胞凋亡指数。结果 低、中、高剂量组大鼠脑海马区c-fos、c-jun蛋白表达阳性率分别为(54.86±10.35)%、(64.18±6.23)%、(73.69±5.52)%和(0.434±0.044)%、(0.620±0.067)%、(0.711 ±0.026)%,细胞凋亡指数分别为(37.80±5.28)%、(48.49±5.25)%、(72.30±4.81)%,与对照组比较差异有统计学意义(P<0.01),且呈剂量-反应关系;经相关性分析显示,大鼠海马区c-fos、c-jun蛋白表达阳性率与细胞凋亡指数均呈正相关,c-fos与c-jun蛋白表达阳性率也呈正相关。结论 砷可诱导大鼠脑海马区c-fos、c-jun蛋白表达,促使细胞凋亡增加;过度的c-fos、c-jun表达可能是砷致大脑神经毒性的原因之一。     

Anatomical localization in hippocampus of tetrahydro-beta-carboline-induced alcohol drinking in the rat

Guide cannulae for unilateral or bilateral micro-injection were implanted stereotaxically into the dorsal hippocampus of the male adult Sprague-Dawley rat. Following post-operative recovery, the animal's individual preference for ethyl alcohol in concentrations from 3-30% (v/v) was tested over a 9-day period by a three-bottle, two-choice technique. Following this pre-screen, 3.0 microliter of 1,2,3,4-tetrahydro-beta-carboline (TH beta C) hydrochloride, a benzodiazepine receptor antagonist, was infused in a concentration of 25-200 ng into the hippocampus of each unrestrained rat twice a day for three to six days. After the first two days of infusion, the 9-day preference test for alcohol drinking was begun and continued identically as in the earlier test. A third alcohol preference test during which no injections were given was conducted at an interval of two weeks following the second. The micro-injection of TH beta C into certain sites in the hippocampus enhanced alcohol consumption from 0.5-2.0 g/kg during the 9-day test interval. The magnitude of this elevated intake was dependent on the site of infusion and was more pronounced when intermediate concentrations of 7-12% alcohol were offered to the rat. At sites in coronal planes encompassing AP 3.0 and AP 3.5, the micro-injection of TH beta C enhanced alcohol drinking significantly in 75% of the animals; however, when delivered at sites in coronal planes AP 1.0 through AP 2.5, TH beta C augmented alcohol drinking significantly in 15% of the rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

微波辐射后大鼠海马组织中水通道蛋白4的表达

目的 探讨微波辐射后大鼠海马组织中水通道蛋白4(AQP4)的表达.方法 采用0、10、30和100mW/cm2微波辐射70只雄性Wistar大鼠,于辐射后6 h及1、3、7、14 d活杀大鼠取海马,采用免疫组化和Western blot方法 检测大鼠海马组织中AQP4蛋白表达的变化,原位杂交、反转录-聚合酶链反应(RT-PCR)观察AQP4基因水平的改变.结果 10、30、100 mW/cm2微波辐射后大鼠海马组织中AQP4表达异常,其蛋白水平于10、30mW/cm2组呈先增加后恢复的过程,100mW/cm2组则呈进行性增加改变,与假辐射组比较,差异有统计学意义(P<0.01).10 mW/cm2组于辐射后6 h AQP4 mRNA即升高(0.51±0.02),与对照组比较,差异有统计学意义(P<0.01);30、100 mW/cm2组均在7 d时达最大值(分别为0.46±0.02、0.43±0.08),与对照组比较,差异有统计学意义(P<0.01,P<0.05).结论 微波辐射可导致大鼠海马组织中AQP4表达增加,此改变有可能参与了微波辐射致血脑屏障通透性升高的过程及脑水肿的发生.     

Ethanol administration results in a prolonged decrease in blood ionized calcium levels in the rat.

Previous studies have shown that ethanol decreases the level of ionized calcium (iCa) in the blood, and appears to prevent a compensatory increase in parathyroid hormone level. We have shown, however, that the presence of ethanol interferes with the measurement of blood iCa by the most commonly used iCa analyzer. It is impossible to interpret ethanol-induced alterations in Ca-regulating hormone levels without accurate measurement of blood iCa, thus the purpose of this study was to determine if ethanol decreases blood iCa levels independent of methodological artifacts. The time course of ethanol's effect and the relationship between iCa and blood ethanol concentration (BEC) were also examined. Rats (n=22) received ethanol (1.5 or 3 g/kg body weight) or saline by intraperitoneal injection. Blood samples were obtained by tail nick at 0, 2, 6, 24, 48 h and 8 days postinjection, and analyzed for iCa, pH, and BEC. Blood iCa and pH were measured using the I-Stat Clinical Analyzer, whose performance is not affected by the presence of ethanol. Ethanol administration resulted in a decrease in blood iCa levels. The magnitude and time course of the decrease varied with dose of ethanol, being greater and more prolonged with the higher dose, and blood iCa levels were not fully recovered at 48 h postinjection. No significant relationship was found between individual iCa and BEC values. This study confirms that ethanol decreases blood iCa levels, independent of methodological artifacts. Prolonged disruptions in Ca homeostasis resulting from ethanol consumption could have implications for long-term bone health.     

Dietary conjugated linoleic acid reduces rat adipose tissue cell size rather than cell number

We investigated the basis for the reduction in fat pad size in rats fed conjugated linoleic acid (CLA). In the first study, growing female Sprague-Dawley rats (initial weight150 g) were fed diets containing 0, 0.25 and 0.5 g/100 g diet of a purified (97% CLA) and 0.5% of a feed-grade (55% CLA) source of CLA for 5 wk to determine the effects on growth performance and fat mass. There was no effect of CLA on growth rate or food intake. Dietary CLA reduced retroperitoneal fat pad weight 13, 25 and 32% in rats fed 0.25 and 0. 5% of the pure CLA and 0.5% of the feed-grade CLA, respectively (P < 0.05). Similar effects were observed in the parametrial fat pad. The reduced pad size was due to smaller adipocyte size rather than a reduced cell number. Relative to the control group, mean cell volume was 15, 28 and 29% lower in tissue from rats fed 0.25 and 0.5% of the pure CLA and 0.5% of the feed-grade CLA, respectively (P < 0.01). In the second study, rats were fed CLA (0 vs. 0.5%) for 7 or 49 d. Reductions in fat pad weight were observed within 7 d. In addition, the effects of CLA on energy metabolism were studied in the chronically fed rats. There were no significant effects of CLA on oxygen consumption, CO(2) or heat production. During wk 4 of feeding, but not at other times, there was a 5% lower respiratory quotient in CLA-fed rats (P < 0.05). There was a time-dependent accumulation of CLA in adipose tissue and a decrease in monounsaturated fatty acids. These results suggest that the reduction in fat mass in rats fed CLA can be accounted for by a reduction in cell size rather than a change in cell number.     

Unexpected decrease in plasma high density lipoprotein cholesterol with weight loss.

High density lipoprotein (HDL) cholesterol is inversely related to coronary heart disease prevalence. Despite the fact that obese patients have lower plasma HDL-cholesterol concentrations, there are few prospective studies on the effect of weight loss on HDL-cholesterol. Consequently, plasma lipoprotein levels were measured in 15 obese females before and after a 10 week weight loss program. Mean weight loss was 8.6 +/- 3.9 kg (P less than 0.001). Total plasma cholesterol and low density lipoprotein-cholesterol did not change significantly. Plasma triglyceride levels decreased (P less than 0.05) as did HDL-cholesterol (P less than 0.02). A subgroup of 11 of the subjects had repeat lipid measurements 8 months after the start of treatment. Mean weight loss at this time was 12.8 +/- 0.8 kg (P less than 0.01). No subject had returned to her pretreatment weight but mean weight loss was not significantly different from the 10 week value. At 8 months all lipid values, including HDL-cholesterol, had returned to their pretreatment value. By multiple regression analysis HDL-cholesterol decreased with increasing relative weight but also decreased with increasing rate of weight loss. These results suggest that negative caloric balance produces a decrease in HDL-cholesterol that in prospective studies may obscure the inverse relationship between HDL-cholesterol and indices of obesity.     

A decrease in irreversibly sickled erythrocytes in sicle cell anemia patients given vitamin E

Patients with sickle cell anemia were given 450 IU of vitamin E (as alpha-tocopherol) per day for 6 to 35 weeks. Plasma tocopherol levels increased from 0.7 +/- 0.2 mg/g lipid pretreatment, to 2.3 +/- 0.3 mg/g lipid. The percentage of circulating irreversibly sickled red cells decreased from 25 +/- 3% pretreatment to 11 +/- 1% after vitamin E administration (P less than 0.001). The percentage of irreversibly sickled red cells remained below pretreatment levels as long as the vitamin was administered (up to 35 weeks). The biochemical and clinical implications of these observations are discussed.     

新生大鼠海马神经元细胞体外培养方法改良研究

目的探讨改良的新生大鼠海马神经元细胞体外培养方法,为新生儿缺血性脑损伤模型等海马神经元相关疾病研究奠定基础。 方法采用本研究自行设计的改良新生大鼠海马神经元细胞的体外培养方法,进行新生大鼠海马神经元细胞的体外培养。判断新生大鼠原代海马神经元细胞体外培养成功的标准为：荧光显微镜下,可见微管相关蛋白(MAP)2在海马神经元细胞体和树突中表达。该改良方法具体为：①以L-多聚赖氨酸和鼠尾胶原作为神经元细胞体外培养的生长基质,分离出生24 h内新生SD大鼠海马结构。将其运用单一胰蛋白酶水浴振荡消化后,吹打成细胞悬液,将海马神经元细胞以适当的密度种植于含10%胎牛血清DMEM培养基中,贴壁培养4 h后,更换为Neurobasal维持培养基继续培养,以后每3 d更换1/2培养液。②采用抗MAP2-5-异硫氰酸荧光素(anti-MAP2-FITC)免疫荧光法,对所获得的海马神经元细胞进行鉴定。 结果①新生大鼠海马神经元细胞的体外培养结果显示,新生大鼠海马神经元细胞于培养30 min时,大部分细胞贴壁生长,培养2 h后,细胞贴壁明显,少数细胞开始长出突起。于培养5 d后,神经元突起进一步增多,并形成神经细胞网络,培养7 d后,神经元细胞分化更加成熟,神经元细胞之间的突起联系更加紧密,可见密集的神经细胞网络。海马神经元细胞体外培养至第21天后,神经元细胞开始退化、变性,细胞体皱缩,残留细胞体痕迹,周围光晕消失,折光性减弱,突起融合而粗大杂乱,神经细胞网络粗大老化。②anti-MAP2-FITC免疫荧光法对所获得的海马神经元细胞进行鉴定的结果显示,在所有细胞中,免疫荧光染色MAP2阳性新生大鼠海马神经元细胞纯度达96.3%。 结论通过上述改良方法培养获得的新生大鼠海马神经元细胞生长状态良好,并具有较高的纯度,可为进一步进行新生儿缺血性脑损伤模型等海马神经元相关疾病的研究提供实验条件。     

Regional differences in cell loss associated with binge-like alcohol exposure during the first two trimesters equivalent in the rat.

Women who abuse alcohol during pregnancy may deliver offspring who could be diagnosed with fetal alcohol syndrome (FAS) or a less severe deficit involving cognitive and behavioral disorders. The severity of the deficits may involve the interaction of several known risk factors, such as alcohol consumption pattern or duration, the timing of alcohol consumption relative to critical windows of vulnerability, or the inherent differential vulnerability among the various brain regions to alcohol-induced brain injury. In this study, we explore the vulnerability of the different brain regions by making cell counts from multiple brain regions. Specifically, we used stereological cell-counting techniques to estimate the total cell numbers in the cerebellum (Purkinje and granule cells), olfactory bulb (mitral and granule cells), hippocampus (CA1 and CA3 cells), and dentate gyrus (granule cells). Groups of timed-pregnant Sprague-Dawley rats were assigned to one of five treatments: alcohol by intragastric intubation (2.25, 4.5, or 6.5 g/kg/day), nutritional control [pairfed and intubated=Pairfed) and intubated], and normal control (Chow). Treatments began on embryonic day 1 (E1) and continued through E20. On E33 (usually postnatal day 10), all offspring were perfused intracardially with saline followed by fixatives. Representative forebrains, cerebella, and olfactory bulb from each group were processed for cell counting. The optical dissector was used to obtain cell densities, while Cavalieri's principle was used to calculate the reference volume. The product of density and volume gave unbiased estimates of the total neuronal number within each brain region. Overall peak BACs (regardless of sampling day) for the three alcohol groups averaged 136, 290, and 422 mg/dl for the 2.25-, 4.5-, and 6.5-g/kg groups, respectively. The total number of cerebellar Purkinje cells was reduced in the 6.5-g/kg group relative to controls, while the total number of olfactory bulb mitral cells and hippocampal CA1 and CA3 pyramidal cells from all alcohol-treated groups was not different from controls. Total numbers of granule neurons were reduced in the cerebellum and olfactory bulb of offspring exposed to 4.5 or 6.5 g/kg/day, but granule cell numbers in the dentate gyrus were not affected by the prenatal alcohol treatment. Taken together with previous findings, these data demonstrate that prenatal alcohol exposure results in regional vulnerability of various brain structures and underscores the variability of deleterious effects of alcohol on brain development.     

Diet-induced effects on neuronal and glial elements in the middle-aged rat hippocampus

Consumption of a high-fat and/or high-cholesterol diet can have detrimental effects on the brain. In the present study, dietary treatment with saturated fats, trans fats, or cholesterol to middle-aged Fischer 344 rats resulted in alterations to serum triglyceride and cholesterol levels, organ weights, and hippocampal morphology. Previously, we demonstrated that a 10% hydrogenated coconut oil and 2% cholesterol diet resulted in worse performance on the 12-day water radial arm maze, increased cholesterol and triglyceride levels, and decreased dendritic microtubule associated protein 2 (MAP2) staining in the hippocampus. The diets administered herein were used to examine components from the previous diet and further examine their effects on hippocampal morphology. Specifically, neuronal morphology, dendritic integrity, fatty acid metabolism, microgliosis, and blood vessel structure in the hippocampus and/or adjacent structures were explored. Our results indicate alterations to peripheral and neural systems following each of the diets.