神经酰胺对小鼠皮层神经元乳酸脱氢酶代谢的影响

目的研究神经酰胺对小鼠皮层神经元乳酸脱氢酶(LDH)代谢的影响。方法在培养的小鼠皮层神经元上清中分别加入50、100、200、500、1000、2000nmol/L的神经酰胺,分别作用0、1、4、8、12、16、24、36h,测定LDH浓度,计算其代谢率和漏出率。结果可显著改变乳酸脱氢酶(LDH)在细胞内外的分布,随神经酰胺作用时间的延长或剂量的增加,细胞外LDH含量显著升高,但神经酰胺对细胞总LDH代谢无影响。结论神经酰胺可增加LDH漏出率,但对细胞总LDH代谢无影响。     

Calbindin overexpression buffers hippocampal cultures from the energetic impairments caused by glutamate

A dramatic rise in free cytosolic calcium concentration is thought to be a central event in the pathogenesis of glutamate excitotoxicity in neurons. We have previously demonstrated that gene transfer of the calcium-binding protein calbindin D28k via a Herpes simplex amplicon vector decreases the rise in intracellular calcium and promotes cell survival following glutamatergic challenge. This study explores the effect of calbindin transgene expression on cellular metabolism following glutamate excitotoxicity. Because excitotoxic insults are often energetic in nature, and because calcium sequestering and extrusion place heavy energy demands on a cell, we hypothesized that calbindin overexpression may help preserve cellular energy levels during an insult. We overexpressed calbindin in primary hippocampal cultures, using a Herpes simplex amplicon vector system. We found that calbindin overexpression protected neurons from the decline in ATP levels, mitochondrial potential and metabolic rate following a glutamatergic insult. These results indicate that calbindin expression helps preserve cellular energy state following glutamate excitotoxicity. This illustrates the energetic load placed on neurons by increased free cytosolic calcium and may help explain the neuroprotective effects of calbindin.     

移植的神经干细胞在脑损伤区壳聚糖载体中的存活与分化

目的:探讨移植的NSCs在大鼠脑损伤区壳聚糖载体中的存活、分化情况及其对TBI大鼠认知功能的影响。方法:低温冷冻干燥法制备壳聚糖多孔支架。将从鼠胚前脑中分离的NSCs扩增、标记BrdU。Feeney法制备SD大鼠TBI模型,随机分为3组:损伤对照组清创后不做移植;NSCs 支架移植组行壳聚糖作载体的NSCs移植;NSCs 支架 NGF移植组行壳聚糖作载体的NSCs移植,并在其中加入NGF。术后1、2、3个月行避暗回避和跳台试验。脑切片行Nissl染色、BrdU与NF-200免疫荧光双标染色。结果:两移植组的认知功能在术后1、2、3个月较损伤对照组明显改善,含NGF的移植组改善更加显著。两移植组术后1、2、3个月在移植区均可见BrdU与NF-200免疫荧光双标细胞,含NGF移植组中的双标细胞数量多、胞体大、突起多且长。结论:大鼠TBI后移植的外源性NSCs可以在脑损伤区壳聚糖载体中长期存活并向神经元分化,可以改善大鼠的认知功能;NGF对其具有促进作用。     

马桑内酯致痫大鼠海马区GABA和Glu在神经元和神经胶质内时空变化研究

目的观察马桑内酯致癫痫鼠脑海马内不同时间段兴奋性神经递质谷氨酸与抑制性神经递质γ-氨基丁酸在神经元与神经胶质之间的时空变化情况。方法采用免疫细胞化学方法对癫痫发生不同时间段的鼠脑海马区进行GABA和Glu的标记。结果癫痫发作前期及间歇期，海马区GABA和Glu在神经胶质内聚集明显，而癫痫发作期，GABA和Glu在神经元内聚集明显。结论癫痫发作过程中，GABA和Glu存在一个从神经胶质转移到神经元后再转移到神经胶质的过程，这个时空变化提示可以从阻断这种转移方面开辟癫痫治疗的新途径。     

蛋白酶体抑制剂对中脑脑片的毒性作用及其机制研究

目的:观察蛋白酶体抑制剂对体外培养的大鼠中脑脑片黑质多巴胺(DA)能神经元的早期毒性作用,利用这种新型的组织模型探讨蛋白酶体功能异常在帕金森(PD)发病中的作用。方法:制备Wistar大鼠体外中脑黑质脑片的长期培养体系,加入蛋白酶体抑制剂lactacystin(0.1,0.5,1.0,5.0μmol/L)作用24 h后,测定培养基中乳酸脱氢酶(LDH)活力水平观察脑片活力。TH免疫组化观察黑质细胞变性缺失,α-synuclein免疫组化观察α-synuclein的表达,TUNEL法检测多巴胺能神经元凋亡。结果:经0.1,0.5,1.0和5.0μmol/L浓度的lactacystin作用24 h后,脑片黑质部位TH阳性神经元数量减少,α-synu-clein表达率增加,凋亡检测显示,5.0μmol/L的lactacystin组部分黑质细胞出现TUNEL染色阳性。结论:蛋白酶体抑制剂lactacystin对脑片中DA能神经元具有毒性作用,能诱导黑质细胞凋亡,其作用具有浓度依赖性。蛋白酶体功能缺陷在帕金森病发病机制中可能发挥重要作用。     

大鼠脑缺血再灌流后Bcl-2、Caspase-3 mRNA水平表达与大脑皮层及纹状体区炎性细胞浸润的影响

目的探讨大鼠脑缺血再灌流后Bcl-2蛋白、Caspase-3mRNA的表达及炎性细胞浸润与神经细胞凋亡的关系。方法将54只Wistar大鼠随机分为二组：假手术组，缺血再灌流组。采用原位末端标记(TUNEL)、免疫组化和原位杂交技术分别观察脑缺血再灌流后不同时间点神经细胞凋亡及损伤的变化与Bcl-2、Caspase-3mRNA表达。结果Bcl-2表达于缺血再灌流12～24h迭高峰，再灌流2～4d呈下降趋势，至16d略高于假手术组；Caspase-3mRNA于缺血再灌流12～24h迭高峰，2～4d呈降低趋势，至16d略高于假手术组。结论脑缺血再灌流后细胞凋亡介导神经细胞损伤、坏死是一个渐进的动态演变过程。Bcl-2蛋白、Caspase-3mRNA表达在押制细胞凋亡和介导神经细胞损伤等方面起非常重要的作用。     

树鼩脊髓——延髓交界部腹外侧核团含胰岛素神经元的观察

本文用免疫过氧化物酶间接法对广西野生树鼩脊髓——延髓交界部含胰岛素神经元进行定位观察,发现脊髓——延髓交界部腹外侧核团的神经元呈强阳性反应.该处的神经元为大、中型的多突神经元.免疫反应物主要出现在核周部.同一脑片的其他神经元及神经纤维则为阴性反应.这个结果是前人未曾报道过的.     

Repetitive audiogenic seizures cause an increased acoustic response in inferior colliculus neurons and additional convulsive behaviors in the genetically-epilepsy prone rat

Previous studies indicate that daily repetition of audiogenic seizures (AGS) leads to audiogenic ‘kindling’ with increased seizure duration and additional seizural behaviors. The present study examined the neuronal correlates of this phenomenon. Extracellular single neuron firing and concomitant convulsive behaviors associated with 14 repetitive AGS were evaluated in the genetically epilepsy-prone rat severe seizure strain (GEPR-9). An increase in the number of acoustically-evoked action potentials in neurons of the central nucleus of inferior colliculus OW was observed by the second day of AGS repetition, and peaked at day four. The ICc responses remained at similar enhanced level through day 14. ICc neuronal responses were completely absent for approximately two min post-ictally after a single AGS in all animals, but 80% of the animals undergoing repetitive AGS consistently exhibited neuronal firing in this post-ictal period. Post-tonic clonus and an increased duration of post-ictal behavioral depression were also observed with repetitive AGS. The increased ICc neuronal firing was observed prior to the appearance of the post-tonic clonus component of repetitive AGS. This suggests that the ICc neuronal firing increase may subserve, at least, the initial increase in AGS severity. However, changes in neuronal firing in nuclei of the neuronal network for AGS efferent to the ICc may be responsible for the increased AGS severity that occurs after the fourth day of AGS repetition.     

Some perspectives on monoamine-opioid peptide interaction in rat central nervous system

Light microscopic immunocytochemistry was employed to investigate possible sites of interaction between the endogenous opioid peptides and monoamines in the rat central nervous system. The opioid and related peptides examined included beta-endorphin (beta-END), alpha-MSH (alpha-MSH) and leucine-enkephalin (Leu-ENK). The monoamines were examined using antisera generated against tyrosine hydroxylase, dopamine-beta-hydroxylase as well as serotonin. Due to the long-tract nature of the central monoamine projections as well as beta-END/alpha-MSH fiber systems, serial section analyses were performed utilizing parasagittal brain sections. Many areas rich in both the monoamines as well as opioid peptides were investigated. These included several thalamic and hypothalamic nuclei, several limbic structures, mesencephalic periaqueductal gray, brain stem noradrenergic cell groups and their rostral projections, the dopaminergic nigrostriatal system, and the serotonergic raphe nuclei and their projections. The results suggest a more intimate linkage between the monoamines and the opioid peptides than previously realized. Some of the intricacies of monoamine-opioid peptide interaction, in particular those pertaining to their possible role in pain and analgesia, catalepsy, and neuroendocrine effects are also discussed.     

Dopamine1 receptor agonists reverse opioid respiratory network depression, increase CO2 reactivity

In adult pentobarbital-anesthetized and unanesthetized decerebrate cats, the D(1)R agonists (6-chloro-APB, SKF-38393, dihydrexidine) given intravenously restored phrenic nerve and vagus nerve respiratory discharges and firing of bulbar post-inspiratory neurons after the discharges were abolished by the micro-opioid receptor agonist fentanyl given intravenously. Reversal of opioid-mediated discharge depression was prevented by the D(1)R antagonist SCH23390. Iontophoresis of the micro-opioid receptor agonist DAMGO depressed firing of medullary bulbospinal inspiratory neurons. Co-iontophoresis of SKF-38393 did not restore firing and had no effect on bulbospinal inspiratory neuron discharges when applied alone. The D(1)R agonists given intravenously prolonged and intensified phrenic nerve and bulbospinal inspiratory neuron discharges. They also increased reactivity to CO(2) by lowering the phrenic nerve apnea threshold and shifting the phrenic nerve-CO(2) response curve to lower et(CO(2)) levels. Intravenous fentanyl on the other hand decreased CO(2) reactivity by shifting the phrenic nerve apnea threshold and the response curve to higher et(CO(2)) levels. Fentanyl effects on reactivity were partially reversed by D(1)R agonists.