Simultaneous gradual lengthening of both proximal and distal nerve stumps for repair of peripheral nerve defect in rats

We investigated nerve regeneration following the repair of a segmental nerve defect induced by direct end-to-end neurorrhaphy after simultaneous gradual lengthening of both proximal and distal nerve stumps in rats. A 15-mm-long nerve segment was resected from the sciatic nerve of each rat. The proximal and distal nerve stumps, respectively, were directly lengthened at a rate of 1 mm/day using a custom-made external nerve-lengthening device. After being lengthened for 14 days, both nerve stumps were refreshed, and direct end-to-end neurorrhaphy was performed. For a control, 15-mm nerve grafting was performed immediately after nerve resection. Nerve regeneration was evaluated by motor nerve conduction velocity, muscle contraction force, and histological studies at 6, 8, and 14 weeks after initial nerve resection in both groups. As a result, at 8 and 14 weeks, the motor nerve conduction velocity was significantly higher in the nerve-lengthening group than in the autografting group. In addition, at 14 weeks, the tetanic force and wet weight of the gastrocnemius muscle were significantly higher in the nerve-lengthening group than in the autografting group. Histologically, the mean axonal diameter of myelinated nerve fibers and the total number of myelinated nerve fibers were also significantly higher in the nerve-lengthening group than in the autografting group for each evaluation period. It appears that the simultaneous gradual lengthening of both proximal and distal nerve stumps might have potential application in the repair of peripheral nerve defects.     

Corticotropin-releasing factor mRNA increases in the inferior olivary complex during harmaline-induced tremor

This study reports that corticotropin-releasing factor (CRF) expression within the inferior olivary complex (IOC) of the cat is increased 8 h after administration of the tremor-inducing β-carboline harmaline. Following harmaline treatment, hybridization of an oligodeoxynucleotide complementary to CRF mRNA increased significantly in the dorsal accessory olive, subnuclei A and C of the medial accessory olive and the dorsal cap of Kooy, a subnucleus thought previously to be unresponsive physiologically to harmaline. At this early time point, greater increases in CRF mRNA hybridization were present in the caudal than the rostral IOC. These results support published reports that harmaline-mediated effects are more profound within the caudal than the rostral IOC, but also suggest that harmaline mediates cellular responses in inferior olivary neurons which are not related to activation of rhythmic firing.     

Memantine attenuates the increase in striatal preproenkephalin mRNA expression and development of haloperidol-induced persistent oral dyskinesias in rats

Tardive dyskinesia (TD) is a serious motor side effect of long-term neuroleptic treatment that may persist after drug withdrawal. Alterations in striatal enkephalinergic neurons due to excessive glutamatergic activity is a possible pathogenetic mechanism. We studied the effect of the NMDA antagonist memantine in a rat model of TD, in which vacuous chewing movements (VCM) were induced by 20 weeks of haloperidol administration. The striatal density of preproenkephalin mRNA was measured and the number of neurons estimated. Haloperidol induced persistent VCM that was associated with increased striatal expression of preproenkephalin mRNA. Memantine inhibited the development of haloperidol-induced persistent VCM and attenuated the increase in preproenkephalin mRNA expression. This suggests that glutamate-mediated up-regulation of striatal enkephalin plays a role in the development of haloperidol-induced persistent oral dyskinesias.     

实验性脑出血大鼠脑内Caspase-3mRNA的表达作用研究

目的动态观察实验性脑出血大鼠脑内血肿周围神经细胞凋亡情况和Caspas-3蛋白及mRNA表达水平的变化,探讨脑H{血后血肿周围神经细胞损伤机制。方法健康雄性Wistar大鼠随机分为生理盐水对照组、假手术组和脑出血模型组,分为术后3h,6h,12h,24h,48h,3d,5d,7d共8个时相点,采用尾状核注射自体非抗凝动脉血复制大鼠脑出血模型,术后制作冰冻切片,对切片进行TUNEL染色,以及Caspase-5免疫组化和原位杂交染色,之后利用图像分析仪,观察阳性细胞数。结果脑出血后3h血肿周围尚无凋亡细胞出现,6h有凋亡发生,以后逐渐增多,3d达高峰后逐渐下降,生理盐水对照组仅有少量TUNEL阳性细胞。假手术组及生理盐水对照组3h无Caspase-3蛋白和mRNA表达,生理盐水对照组6h以后有少量表达,脑出血模型组在6hCaspase-3蛋白和mRNA开始表达,3d时Caspase-3的蛋白达到高峰,5d以后逐渐下降,24hCaspase-3mRNA表达达高峰,5d以后逐渐下降。脑出血后血肿周围脑组织Caspase-3蛋白的表达与TUNEL阳性细胞数呈正相关（r=0．515,P〈0．05）;Caspase-3蛋白表达与mRNA表达呈正相关（r=0．625,P〈0．05）。结论脑出血后血肿周围神经细胞损伤有凋亡机制参与,在出血后6h发生凋亡,第3天达高峰。Caspase-3的表达在Caspase-5蛋白水平变化趋势与脑m血后细胞凋亡的趋势一致,Caspase-3的mRNA水平的表达高峰时间先于Caspase-3蛋白的表达及凋亡的发生,提示Caspase-3的表达决定脑出血后细胞凋亡的发生,在脑出血后细胞凋亡中起促进作用。     

Localization of tissue plasminogen activator mRNA in adult rat brain

The distribution of tissue plasminogen activator (tPA) messenger RNA in rat brain was studied using in situ hybridization with ³⁵S UTP-labeled RNA probes derived from a fulllength tPA cDNA. Sense strand controls produced low, even backgrounds, with small elevations in the hippocampus. Full-length antisense probes produced strong signals over cerebral ventricular ependyma (including ependyma of the subcommissural organ), meninges, blood vessels, and Purkinje cell layer of the cerebellum, as well as strong signals over scattered cells throughout the brain. Some of these scattered labeled cells were large with lightly stained nuclei, while others were small with darkly stained nuclei. The large labeled cells, which were probably neurons, constituted 8% and 8% of cells in the brain stem and neocortex, respectively, and 100% of Purkinje cells. The small cells, which were present in all areas of the brain, constituted 3–11 % of cells in individual brain areas.     

Constitutive expression of c-myb mRNA in the adult rat brain

In this study, we demonstrated the c-myb mRNA expression in the adult rat brain using an in situ hybridization technique. We found c-myb mRNA signals in the various regions of the forebrain and midbrain including the cerebral cortex, thalamus, hippocampus, hypothalamus, superior and inferior colliculi and central gray. In the cerebellum, a diffuse signal was found in the granular layer while some positive cells were detected in the molecular layer as well. In addition, a number of cells showed intense signals in many nuclei of the medulla oblongata. The constitutive expression of c-myb mRNA in the different kinds of neural cells suggests that this gene might be involved in the normal function of these neurons.     

KA致痫大鼠脑组织中N-cadherin mRNA的表达及意义

目的探讨癫痫鼠脑组织中N-cadherin mRNA的表达及意义。方法通过原位杂交方法对KA致痫鼠脑组织中N-cadherin mRNA的表达进行检测。结果对照组、手术对照组颞叶及海马均可见明显的N-cadherin mRNA的表达，但海马区明显高于颞叶。在充分点燃后12h，N-cadherin mRNA的表达明显下降，CA3区下降的最早且明显，在点燃后7d逐渐恢复，在14d时接近正常值，在28d高于对照组、手术对照组。结论N-cadherin是突触重建的重要的调控因子，是新的突触形成的介质，在癫痫的发生、发展中起着重要的调控作用。     

KCC3 and KCC4 expression in rat adult forebrain

Potassium chloride ion cotransporters (KCCs) are part of a family of transporters classically described as being involved in cell volume regulation. Recently, KCC2 has been shown to have a role in the development of the inhibitory actions of amine transmitters, whereas KCC3 also plays a fundamental role in the development and function of the central and peripheral nervous system. We have re-assessed the expression of each of the known KCCs in the rat forebrain using RT-PCR and in situ hybridisation histochemistry. As well as confirming the widespread expression of KCC1 and KCC2 throughout the brain, we now show a more restricted expression of KCC3a in the hippocampus, choroid plexus and piriform cortex, as well as KCC4 in the choroid plexus and the suprachiasmatic nucleus of the hypothalamus. The expression of KCC4 in the latter and KCC2 in the lateral hypothalamic and ventromedial hypothalamic nuclei suggests that these cotransporters may have selective roles in neuroendocrine or homeostatic functions. Finally, we demonstrate the existence of a truncated splice variation of KCC3a in the rat that appears to be expressed exclusively in neurons (as is KCC2), whereas the native form of KCC3a and KCC4 appears to be expressed in glial cells.     

Tissue-specific mRNA expression of a calcitonin receptor-like receptor during fetal and postnatal development

The distribution of calcitonin receptor-like receptor (CRLR) mRNA in developing rats was investigated by in situ hybridization. Signals were found in the piriform cortex, the central and basolateral amygdala and the amygdalostriatal transition area. Among peripheral organs, the CRLR was predominantly expressed in the lung. mRNA expression in blood vessels, liver, midgut, rectum and urethra was restricted to gestational days 16 and/or 20. The CRLR was thought to be a calcitonin gene-related peptide (CGRP) type 1 receptor (Aiyar et al., J. Biol. Chem., 271 (1996) 11325–11329). This contrasts with previously reported evidence that the CRLR is an orphan receptor with no identifiable interactions with CGRP and other related ligands (Flühmann et al., Biochem. Biophys. Res. Commun., 206 (1995) 341–347). In situ hybridization signals have not been detected in the cerebellum and the spleen known to present a high density of CGRP binding sites. The different regional distribution of CGRP receptor binding sites and CRLR mRNA implies the latter encoding a different CGRP receptor subtype.     

匹罗卡品致痫诱发大鼠齿状回颗粒细胞GAP-43 mRNA表达

目的 研究边缘系统癫痫发作后海马颗粒细胞生长相关蛋白（GAP-43）基因表达变化。方法 建立匹罗卡品急、慢性癫痫模型,用原位杂交方法定量检测不同时间点海马颗粒细胞GAP-43mRNA表达。结果 对照组颗粒细胞几乎不表达GAP-43mRNA,匹罗卡品致病后6～12h颗粒细胞表达GAP-43mRNA增高,15～30d呈现第2次高峰。结论 成年大脑海马颗粒细胞在致痫后发生可塑性变化,GAP-43mRNA表达是癫痫大鼠大脑结构性重组（颗粒细胞苔藓纤维出芽）的重要分子机制。     

Improved sciatic nerve regeneration by local thyroid hormone treatment in adult rat is accompanied by increased expression of SCG10

Thyroid hormone plays an important role in regulating the development and regeneration of the nervous system. Our previous work showed that local administration of triiodothyronine (T3) at the level of transected rat sciatic nerve increased the number and diameter of regenerated axons, but the mechanism underlying the improved regeneration is still unclear. Here, we have investigated the effect of T3 on the expression of SCG10, a regulator of microtubule dynamics in growth cones. After transection of adult rat sciatic nerves, silicone tubes were implanted and filled with T3 or phosphate-buffered solution. At various time points following surgery, the expression of SCG10 protein and mRNA was analyzed. Semi-quantitative Western blot analysis revealed that sciatic nerve transection induced a more than 20-fold upregulation of SCG10 protein in proximal nerve segments at 1 day post-lesion, while at this time point, SCG10 mRNA in dorsal root ganglion neurons was not increased yet. The increase in SCG10 protein and mRNA could be observed over 30 days. Local T3 treatment significantly enhanced the increase in SCG10 protein levels about two-fold in the different segments of transected nerve during the regeneration period. Also SCG10 mRNA levels in lumbar ganglia were enhanced. Immunohistochemical analysis showed that T3 treatment not only increased the number of SCG10 positive axons but also the intensity of their staining. These results suggest that SCG10 is involved in the regulation of regeneration. The stimulating effect of T3 on SCG10 expression could provide a mechanism by which T3 enhances peripheral nerve regeneration.     

大鼠局灶性脑缺血再灌注后早期生长反应基因-1 mRNA的表达

目的探讨早期生长反应基因-1( early growth response gene-1,Egr-1)mRNA在大鼠局灶性脑缺血再灌注后的表达情况。方法取10只健康雄性SD大鼠,体重200 ～250 g,应用原位杂交和RT-PCR方法检测大鼠局灶性脑缺血再灌注后Egr-1 mRNA的表达情况。结果 (1)原位杂交结果:假手术组神经元及胶质细胞为轻度阳性表达。缺血2 h再灌注2 h后,缺血侧Egr-1 mRNA细胞阳性表达明显增强。再灌注4 h时Egr-1 mRNA细胞阳性表达最高,再灌注22 h Egr-1 mRNA细胞阳性表达下降,至166 h时下降更加明显,但仍显著高于假手术组。(2)RT-PCR结果:大鼠脑缺血再灌注后缺血侧Egr-1 mRNA的表达明显高于假手术组,P<0.01。动态观察发现,脑缺血2h再灌注2 h后,Egr-1 mRNA即高表达,缺血2 h再灌注4 h达高峰,再灌注46 h已明显下降,但仍高于假手术组,P<0.01,随缺血再灌注时间延长,Egr-1 mRNA表达又逐渐增多,至再灌注166 h其表达亦明显高于假手术组,P<0.01。结论缺血再灌注后Egr-1 mRNA有规律性表达。     

血管性痴呆小鼠海马胆碱乙酰转移酶mRNA表达特征研究

目的探讨血管性痴呆小鼠海马神经元胆碱乙酰转移酶原位杂交变化特征及其在血管性痴呆发病中的作用。方法双侧颈总动脉线结反复缺血-再灌注法制备模型,利用跳台试验和水迷宫试验观测其行为学改变,采用原位杂交技术观测小鼠海马神经元胆碱乙酰转移酶mRNA的表达变化。结果模型组小鼠学习、记忆成绩较假手术明显降低(P<0.01),其海马胆碱乙酰转移酶mRNA表达也明显下降(P<0.01)。结论血管性痴呆小鼠学习、记忆成绩下降可能与其海马胆碱乙酰转移酶mRNA低水平表达有关。     

Chronic arthritis increases tyrosine hydroxylase mRNA levels in the pontine noradrenergic cell groups

In situ hybridization was used to examine the change of tyrosine hydroxylase(TH) mRNA levels in the pontine noradrenergic cell groups of chronic monoarthritic rats induced by adjuvant inoculation. The number of TH mRNA-expressing neurons and grains per labeled neuron in the A5, A6 and A7 cell groups on the ipsilateral and contralateral sides significantly increased 2 weeks after adjuvant inoculation into the left tibio-tarsal joint, compared to controls. These results suggest that noradrenalin in the pontine region may play a role in modulating chronic nociceptive stimuli.     

Brain-derived neurotrophic factor (BDNF) mRNA in rats with neonatal ibotenic acid lesions of the ventral hippocampus

Increasing evidence suggests that schizophrenia is a neurodevelopmental disorder with a progressive course characterized by worsening of symptoms and morphological alterations within the brain. This suggests that a neurodegenerative component may exist in schizophrenia. The role of brain-derived neurotrophic factor (BDNF) in neurodevelopment, cell viability and synaptic plasticity led to the investigation of BDNF as a potential candidate molecule in the pathophysiology of schizophrenia. BDNF mRNA was examined by in situ hybridization in the prefrontal cortex and hippocampus of animals with neonatal ibotenic acid lesions of the ventral hippocampus, a putative neurodevelopmental animal model of schizophrenia. Results demonstrate that animals with neonatal ibotenic acid lesions of the ventral hippocampus have reduced basal levels of BDNF mRNA. It is possible that alterations in this trophic factor render animals more susceptible to neurodegenerative insults.