Long-lasting changes in the density of nitrergic neurons following kainic acid administration and chronic hypoxia

Using histochemical analysis (NADPH-diaphorase) we have investigated the influence of intraperitoneal administration of kainic acid (KA), hypoxia and combination of both these factors on neurons of the hippocampus and on the primary auditory cortex (PAC) in male rats of the Wistar strain. Kainic acid was administered to 18-day-old animals, which were exposed to long-lasting repeated hypoxia from the 2nd till the 17th day of age in a hypobaric chamber (for 8 hours a day). At the age of 1 year, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostate sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in CA1 and CA3 areas of the hippocampus, in the dentate gyrus and in the PAC. Both, hypoxia and KA lowered the number of NADPH-diaphorase positive neurons in the hilus, dorsal and ventral blades of the dentate gyrus, CA1 and CA3 areas of the hippocampus. On the contrary, KA given to the hypoxic animals increased the number of NADPH-diaphorase positive neurons in the dorsal blade of the dentate gyrus and PAC.     

The long-term memory retrieval following kainic acid administration

Effect of the neurotoxin kainic acid to the food-procuring task were studied in Wistar rats. A single injection of the acid in subconvulsive dose (8 mg/kg) impaired the task performance within some weeks but not immediately after the treatment. Higher doses of kainic acid (10 mg/kg) impaired the task performance within a few hours after treatment for up to 10 days. The treatment did not prevent rat's learning of a new task in the same experimental chamber. The revealed deficit in the long-term memory retrieval might be explained by specific effects of kainic acid upon the hippocampal system.     

Effects of in vivo administration of kainic acid on the extracellular amino acid pool in the rabbit hippocampus

The effect of local administration of kainic acid in the rabbit hippocampus was studied; the hippocampus was perfused continuously in the freely moving animal with an implanted 0.3-mm dialysis fiber. The pattern of endogenous amino acids in the perfusate, reflecting extracellular amino acids, was monitored with liquid chromatography separation and fluorimetric detection of amino acid derivatives. Kainic acid was included in the perfusion medium for up to 70 min at 0.1-1.0 mM and, with time, induced epileptiform activity. Endogenous glutamic acid, taurine, and phosphoethanolamine levels were increased selectively at the lower perfusion concentrations of kainic acid. Long perfusion periods with higher concentrations increased the levels of virtually all amino acids. Perfusion of the hippocampus with depolarizing concentrations of potassium gave an amino acid response partly similar to that seen with kainic acid treatment. However, one notable difference between the two responses was that the extracellular concentration of glutamine, although not influenced by kainic acid, was significantly decreased after high potassium concentrations. These results confirm previous notions that kainic acid has a primarily excitatory effect, one manifestation of this effect being the release of glutamic acid.     

Changes in the number of nitrergic neurons following kainic acid administration and repeated long-term hypoxia

Using histochemical analysis (NADPH-diaphorase) we have been investigating the influence of intraperitoneal administration of kainic acid (KA), hypoxia and combination of both these factors on neurons of the hippocampus and on the primary auditory cortex (PAC) in male rats of the Wistar strain. Kainic acid was administered to 18-day-old animals, which were exposed to long-lasting repeated hypoxia from the 2nd till the 17th day of age in a hypobaric chamber (for 8 h a day). At the age of 22 or 90 days, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostate sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the hippocampus, in the dentate gyrus and in the PAC. In 22-day-old animals both hypoxia and KA increased the number of NADPH-diaphorase positive neurons in the hilus, CA1, CA3 areas of the hippocampus and in the PAC. On the contrary, KA given to hypoxic animals lowered the number of NADPH-diaphorase positive neurons in the dentate gyrus. In 90-day-old animals, hypoxia and KA given to both normoxic and hypoxic animals lowered the number of NADPH-diaphorase positive neurons in some areas of the central nervous system.     

Morphological changes of the terminations of afferent pathways in a neurodegenerative lesion induced by kainic acid

30 days after kainic acid injection into the rat ventrobasal thalamus, lemniscal afferents were labeled using wheat-germ agglutinin conjugated to HRP. They appeared considerably swollen in the area where neuronal post-synaptic targets had been eliminated. Electron microscopic analysis of the lesioned tissue revealed the presence of large profiles containing numerous organelles, particularly smooth endoplasmic reticulum, and giving rise to thin excrescences filled with neurofilaments. Since these morphological features are typical of regenerating "growth cones", we conclude that afferent terminals deprived of their post-synaptic targets undergo morphological changes preparing them for new synapses.     

Detoxification enzymes following intrastriatal kainic acid

A complete explanation of the neurotoxicity that follows kainic acid (KA) injection into the rat striatum is lacking. An assessment of the chronological course after intrastriatal KA injection of the activities of enzymes preferentially concentrated in glia or involved in the detoxification of oxygen metabolites is accomplished. An enhancement of the specific activities of glutathione peroxidase (GP) and catalase is found without an alteration in the specific activity of superoxide dismutase (SOD). There is no increase in the in vivo striatal levels of malondialdehyde, a putative indicator of lipid peroxidation, the expected result of cell membrane damage from oxygen metabolites. Understanding the mechanism and importance of the preferential induction of the activities of the detoxification enzymes will require further study.     

杏仁核注射红藻氨酸诱导大鼠边缘叶癫痫发作时海马中Smac和XIAP蛋白的表达

应用红藻氨酸(kainic acid,KA)诱导的大鼠边缘叶癫痫发作模型,检测第二个线粒体源的半胱天冬蛋白酶激活物,直接与凋亡抑制蛋白结合的低等电点蛋白(second mitochondrial activator of caspases／direct inhibitor of apoptosis protein-binding protein of low isoelectric point[PI],Smac／DIABLO)和X染色体连锁的凋亡抑制蛋白(X-chromosome-linked inhibitor of apoptosis protein,XIAP)在癫痫大鼠海马神经元表达。单侧杏仁核内注射KA诱导癫痫发作,1h后用安定终止发作,然后分别用TUNEL染色和cresyl violet染色观察海马神经元存活和凋亡的变化,用免疫荧光和Western blot检测海马Smac／DIABLO、XIAP和半胱天冬蛋白酶-9(caspase-9)的表达。结果表明,发作终止2h时KA注射同侧海马CA3区细胞浆内Smac／DIABLO蛋白表达增加,4h时caspase-9出现裂解片断,8h时出现TUNEL阳性细胞,24h时达高峰。脑室内注射caspase-9抑制剂z-LEHD-fluoromethyl ketone(z-LEHD-fmk)可减少TUNEL阳性细胞,增加存活神经元。发作后KA注射同侧海马CA3区神经元caspase-9免疫反应性增强,Smac／DIABLO和XIAP弥散于整个神经元内。对侧海马未检测到TUNEL阳性细胞及Smac／DIABLO和XIAP蛋白的上述变化。以上结果提示,癫痫发作可诱导Smac／DIABLO蛋白从线粒体向细胞浆的移位、XIAP亚细胞分布改变和caspase-9的激活,Smac／DIABLO、XIAP和caspase-9可能参与了癫痫神经元损伤的病理生理机制,caspase-9可能是潜在的治疗靶点。     

Neuroprotective effects of lactation against kainic acid treatment in the dorsal hippocampus of the rat

Marked hippocampal changes in response to excitatory amino acid agonists occur during pregnancy (e.g. decreased frequency in spontaneous recurrent seizures in rats with KA lesions of the hippocampus) and lactation (e.g. reduced c-Fos expression in response to N-methyl-d,l-aspartic acid but not to kainic acid). In this study, the possibility that lactation protects against the excitotoxic damage induced by KA in hippocampal areas was explored. We compared cell damage induced 24 h after a single systemic administration of KA (5 or 7.5 mg/kg bw) in regions CA1, CA3, and CA4 of the dorsal hippocampus of rats in the final week of lactation to that in diestrus phase. To determine cellular damage in a rostro-caudal segment of the dorsal hippocampus, we used NISSL and Fluorojade staining, immunohistochemistry for active caspase-3 and TUNEL, and we observed that the KA treatment provoked a significant loss of neurons in diestrus rats, principally in the pyramidal cells of CA1 region. In contrast, in lactating rats, pyramidal neurons from CA1, CA3, and CA4 in the dorsal hippocampus were significantly protected against KA-induced neuronal damage, indicating that lactation may be a natural model of neuroprotection.     

Morphological changes in the rat kidney following long-term diabetes

The morphological basis of diabetic nephropathy has been studied using light and electron microscopy. Kidneys of streptozotocin-induced diabetic rats were examined on the light microscope at 4 weeks and 8 months after induction of diabetes mellitus. In addition, the 8-month diabetic kidneys were examined with the electron microscope. Renal hypertrophy was evidenced by the increase in the weight of kidneys of diabetic rats. Whilst the diabetic kidneys were approximately twice as large after 4 weeks they were only 30% larger compared to age-matched controls after 8 months of induction of diabetes. After 4 weeks, light microscopy revealed dilated tubules within the cortex of the diabetic kidneys. Light microscopy showed a significant amount of destruction of the distal convoluted tubules while electron microscopy revealed a spectrum of damage that included basement membrane thickening, loss of podocytic foot processes, disruption of tubular basal infoldings and their related mitochondria and fibrosis of the tubules 8 months after induction of diabetes. It is concluded that renal hypertrophy persists after a prolonged occurrence of diabetes but the extensive damage and loss of renal tissue including the loss of the foot processes of podocytes might be partly responsible for the clinical presentation of diabetic nephropathy.     

Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration

Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier.     

杏仁核注射红藻氨酸诱导的边缘叶发作癫痫大鼠海马 Bad去磷酸化和Bcl-2表达上调

应用红藻氨酸(kainic acid,KA)诱导的人鼠边缘叶发作癫痫模型,检测Bad(Bcl-2-associated death protein)、14-3-3、磷酸化Bad、Bcl-XL和Bcl-2在癫痫人鼠海码神经元的表达。单侧杏仁核内注射KA诱导癫痫发作,持续记录脑电和局部脑血流(regional cerebral blood flow,r-CBF),发作1h后静脉注射30mg／kg安定终止发作,然后分别用cresyl violet染色和TUNEL染色观察海马神经元存活和凋亡的变化;用免疫荧光、Western blot和免疫沉淀俭测海马Bad、14-3-3、磷酸化Bad、Bcl-XL和Bcl-2的表达。结果表明,发作终止8h时出现TUNEL阳忡细胞,24h时达高峰;发作诱导Bad去磷酸化,去磷酸化的Bad与分了伴侣蛋F114-3-3解离,然后Bad与Bcl-XL结合：磷酸化Bad表达减少而Bcl-2表达增加;发作前后r-CBF无明显变化。以上结果提示,癫痫发作诱导Bad的去磷酸化和Bcl-2表达上调,Bad的上磷酸化可能具有损伤作用,而Bcl-2的表达上调则对癫痫神经元损伤具有保护作用,但与脑缺血无关。     

Differential subcellular redistribution of protein kinase C isozymes in the rat hippocampus induced by kainic acid

Protein kinase C (PKC) consists of a family of Ca2+/phospholipid-dependent isozymes that has been implicated in the delayed neurotoxic effects of glutamate in vitro. In the present study, we assessed the effect of the glutamate analogue kainic acid (KA) on the subcellular expression of PKC isozymes in the hippocampus (HPC) in the period preceding (0.5, 1.5, 12, and 24 h) and during (120 h) hippocampal necrosis using western blot analysis and PKC isozyme-specific antibodies. Before subcellular fractionation (cytosol + membrane), hippocampi were microdissected into "HPC" (fields CA1-CA3) and "dentate gyrus" (DG; granule cells + hilus) regions. Four general patterns of alterations in PKC isozyme expression/distribution were observed following KA treatment. The first pattern was a relative stability in expression following KA treatment and was most apparent for cytosol PKCalpha (HPC + DG) and membrane (HPC) and cytosol (DG) PKCbetaII. The second pattern, observed with PKCgamma and PKCepsilon, was characterized by an initial increase in expression in both membrane and cytosolic fractions before seizure activity (0.5 h) followed by a gradual decrease until significant reductions are observed by 120 h. The third pattern, exhibited by PKCdelta, involved an apparent translocation, increasing in the membrane and decreasing in the cytosol, followed by down-regulation in both fractions and subsequent recovery. The fourth pattern was observed with PKCzeta only and entailed a significant reduction in expression before and during limbic motor seizures followed by a dramatic fivefold increase in the membrane fraction during the period of hippocampal necrosis (120 h). Although these patterns did not segregate according to conventional PKC isozyme classifications, they do indicate dynamic isozyme-specific regulation by KA. The subcellular redistribution of PKC isozymes may contribute to the histopathological sequelae produced by KA in the hippocampus and may model the pathogenesis associated with diseases involving glutamate-induced neurotoxicity.     

Morphological changes in rat skeletal muscle following reinnervation

Morphological changes appearing in the course of muscle regeneration after reinnervation of denervated M. soleus (slow) and M. tibialis anterior (fast) rat skeletal muscle were investigated. It was found that pathological changes typical for denervation atrophy (seen on the 10th day after crushing the sciatic nerve) and symptoms of regeneration (beginning about the 15th day) were much more pronounced in the soleus than in the tibialis muscle. Some stages of regeneration in the soleus muscle could be distinguished. The contractile material destructions were the first pathological changes that disappeared after the beginning of regeneration. In the second stage other denervation changes disappeared and intensive regeneration of muscle fibres was observed. In the next stage regeneration slowed down, and the reduction of the excess of muscle nuclei was visible. Four months after crushing the nerve, regeneration proceeded to completion with only some traces of the passed processes: in the soleus muscle, chains of sarcolemmal nuclei, satellite cells and newly formed muscle fibres were more often seen than in contralateral muscle; in the tibialis, collagen depots were present around the vessels and between muscle fascicles.