Increased brain levels of cholecystokinin octapeptide after kainic acid-induced seizures in the rat

Pronounced changes in the content of cholecystokinin octapeptide (CCK-8) have been found after limbic seizures induced by i.p. injection of kainic acid. Three hours after injection of the toxin a significant decrease in CCK-8 was observed in the frontal cortex and amygdala/pyriform cortex reflecting an increased release during acute seizures. A persistent decrease in the content of the peptide in the amygdala/pyriform cortex suggests destruction of the respective neurons. In the substantia nigra and in the striatum and, more moderately, in the hippocampus and frontal cortex increases in CCK-8 were observed 10 days after injection of kainic acid suggesting an increased synthesis or decreased release of the peptide in these brain areas subsequently to the acute seizures.     

Increased apoptosis and inflammation after focal brain ischemia in mice lacking connexin43 in astrocytes

Astrocytes secrete cytokines and neurotrophic factors to neurons, consistent with a neurosupportive role for astrocytes. However, in ischemic or metabolic insults, the function of astrocytic gap junctions composed mainly from connexin43 (Cx43) remains controversial. We have previously shown that heterozygous Cx43 null mice subjected to middle cerebral artery occlusion exhibited significantly enhanced stroke volume and apoptosis compared to wild-type mice. In this study, we used mice in which the human GFAP promoter-driven cre transgene deletes the floxed Cx43 gene in astrocytes, excluding the effects from reduced Cx43 expression in many other cell types as well as astrocytes. We induced focal brain ischemia in mice lacking Cx43 in astrocytes [Cre(+)] and control littermates [Cre(-)]. Cre(+) mice showed a significantly increased stroke volume and enhanced apoptosis, detected by terminal dUTP nick-end labeling and caspase-3 immunostaining, compared to Cre(-) mice. Inflammatory response assessed by the microglial marker CD11b was amplified in the penumbra of Cre(+) mice compared to that of Cre(-) mice. Our results suggest that astrocytic gap junctions could be important for the regulation of neuronal apoptosis and the inflammatory response after stroke. These findings support the view that astrocytes play a critical role in neuroprotection during ischemic insults.     

Temporal profile of connexin 43 expression after photothrombotic lesion in rat brain

Following focal ischemic injury, several mechanisms lead to secondary expansion of the affected area and therefore increase the initial damage. We thoroughly investigated the expression of astrocytic connexin 43 (Cx43) after photothrombosis in rat brain. The temporal profile of Cx43 mRNA as well as protein expression was studied in remote, structurally uninjured cortical and hippocampal areas. The hippocampal formation revealed an increased number of Cx43 mRNA positive astrocytes and an up-regulated protein expression exclusively in the ipsilateral stratum oriens. We assume a participation of this region in glia scar formation. While Cx43 mRNA positive cells were transiently increased, immunoreactivity was reduced in the somatosensory cortex of injured hemispheres. The observed decrease of Cx43 protein in the post-ischemic cerebral cortex implies an impairment of gap junctional intercellular communication which might be detrimental to the brain.     

Transplantation of cultured astrocytes attenuates degenerative changes in rats with kainic acid-induced brain damage

Viability of astrocyte grafts introduced into CA1 pyramidal layer of the left dorsal hippocampus after injection of kainic acid into this brain region and the effects of these grafts on the hippocampus and amygdala were studied on Wistar rats. In rats with astrocyte grafts the degree of destruction in fields CA1-CA2 of the dorsal and ventral hippocampus, fields CA3-CA4 of the ventral hippocampus, and central and basolateral amygdala was lower compared to animals with kainic acid-induced hippocampal damage and control rats; destructions in the dentate fascia were absent. Our results suggest that astrocyte grafts stimulate neurogenesis in the mature brain of recipient rats with kainic acid-induced brain damage. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 140, No. 12, pp. 627–632, December, 2005     

Differential changes in tachykinins after kainic acid-induced seizures in the rat.

Changes in concentrations of the tachykinins substance P, neurokinin A and neurokinin B were investigated in rat brains after kainic acid-induced seizures. Two different antisera, one detecting substance P specifically and one recognizing neurokinins A and B but not substance P, were used. Subsequently to the acute seizures (3 h after kainic acid) significant decreases (by 25-40%) in total neurokinin (A + B) and substance P immunoreactivities were observed in the frontal cortex, dorsal hippocampus and striatum. Depending on the brain area neurokinin immunoreactivity recovered 1-3 days after injection of the toxin and was significantly increased in the frontal cortex (by 40-60%) and the hippocampus (by 100-300%) after 10-60 days. Further analysis by high pressure liquid chromatography revealed that increases in both neurokinin A and neurokinin B concentrations contributed to the increases in total neurokinin immunoreactivity 30 days after kainic acid. At the same time significantly increased levels were also observed for substance P in the frontal cortex (by 30%). Furthermore, increases were also observed in the concentrations of neuropeptide K and gamma-preprotachykinin-A(72-92) in the frontal cortex and the hippocampus 30 days after the kainic acid treatment.     

miR-301a-3p对大鼠星形胶质细胞Cx43转录后的靶向调控作用

目的:研究大鼠星形胶质细胞中微小RNA-301a-3p(miR-301a-3p)对缝隙连接蛋白43(Cx43)表达的靶向调控作用及其作用位点。方法:合成miR-301a-3p agomir和miR-301a-3p antagomir,转染至星形胶质细胞,Western blot检测各组细胞中Cx43蛋白的表达情况;构建重组载体wt-pEZX-MT05-Cx43和mut-pEZX-MT05-Cx43,采用双萤光素酶报告基因实验验证miR-301a-3p的靶基因;构建表达载体pcDNA3.1-Cx43,通过回复实验分析miR-301a-3p对细胞凋亡的影响。结果:将miR-301a-3p agomir转染到星形胶质细胞后,Western blot检测显示,与对照组相比,Cx43蛋白表达显著降低(P<0.05)。双萤光素酶报告基因实验结果表明,miR-301a-3p能够与Cx43的3′-UTR结合,对其表达产生负调控;将不含Cx43 3′-UTR的重组载体pcDNA3.1-Cx43转染星形胶质细胞后,能够回复miR-301a-3p对Cx43蛋白表达的负调控作用,引起细胞凋亡。结论:Cx...     

Immunocytochemical mapping of endomorphin-2-immunoreactivity in rat brain

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) is a novel endogenous opioid with high affinity and selectivity for the μ-opioid receptor. Immunocytochemical studies have located this peptide in spinal cord, brainstem and selected brain regions. However, there are disagreements regarding its distribution between published reports. Furthermore, the distributions reported for the endomorphins resemble that of neuropeptide FF, suggesting that some of the previous findings might be due to cross-reactivity with the latter substance. In the present study, the distribution of endomorphin-2-immunoreactivity (ir) was examined throughout the entire rat brain using an affinity-purified antiserum that appeared not to cross-react with neuropeptide FF. Endomorphin-2-ir cell somata were most prominent in the hypothalamus and the nucleus of the solitary tract (NTS). Endomorphin-2-ir varicose fibers were observed in such areas as the bed nucleus of the stria terminalis, the septal nuclei, the periaqueductal gray, the locus coeruleus, the lateral parabrachial nucleus, the NTS, and the substantia gelatinosa of the medulla. More modest immunoreactivity was seen in substantia nigra, nucleus raphe magnus, the ventral tegmental area, the pontine nuclei and the amygdala. Fibers were also observed in the ventral cerebellum. Of note was the negligible immunoreactivity in the striatum, a region known to express high levels of μ-opioid receptors. Thus, endomorphin-2-ir was widely, but not uniformly, distributed throughout the central nervous system and was associated largely, but not exclusively, with regions expressing μ-opioid receptors. Based on its distribution, it may have a role in the control of neuroendocrine, cardiovascular and respiratory functions, and mood, feeding, sexual behavior and pain.     

2-Chloroadenosine prevents kainic acid-induced toxicity in rat striatum

The stable adenosine analogue, 2-chloroadenosine was found to afford a dose-dependent protection (effective dose range: 12.5-50 nmol) against the characteristic neurotoxic actions of kainic acid (2.2 nmol) in rat striatum. The two agents were co-injected directly into the striatum and the extent of the excitotoxin-induced lesion assessed two weeks later in 40 microns coronal sections stained with thionin, or by histochemical staining for NADPH-dependent diaphorase. These findings provide further information concerning the possible mechanism(s) underlying kainic acid-induced neurotoxicity and also yield an insight into a possible neuroprotective role for endogenous adenosine-like substances.     

Influence of globus pallidus on arm movements in monkeys. I. Effects of kainic acid-induced lesions

The role of basal ganglia output via the globus pallidus (GP) was examined in monkeys trained to make rapid arm-reaching movements to a visual target in a reaction-time task. When neurons in the globus pallidus were destroyed by injection of kainic acid (KA) during task execution, contralateral arm movement times (MT) were increased significantly, with little or no change in reaction times (RT). The slowed movements were associated with a generalized depression in the amplitude and rate of rise of electromyographic (EMG) activity in all the contralateral muscles studied at the wrist, elbow, shoulder, and back, but there was no change in the sequential activation of these muscles. The most profound and persistent increases in movement time occurred when neurons were destroyed in the ventrolateral and caudal aspects of the internal as well as external pallidal segment. These results suggest a role for globus pallidus output in scaling the magnitude and/or buildup of EMG activity without affecting the initiation or the sequential organization of the programmed motor output.     

Ischaemia-induced temporal expression of connexin43 in rat heart

 To investigate the regulation of cell-to-cell coupling in myocardial ischaemia, the three-dimensional expression of connexin43 (Cx43) during experimental ischaemia was examined using a confocal laser scanning microscope. After induction of myocardial infarction in rats, serial optical sections were obtained from the left ventricular myocardium at various times (3 h to 60 days after ligation). The expression of Cx43 was detected immunohistochemically with FITC-labelled anti-rat Cx43 antibody. Fluorescent dots of Cx43 remained along the intercalated disc and decreased in number around the infarct up to 12 h after ligation. Cx43-expression disappeared completely within 48 h after ligation. After day 4, and especially on days 8 and 15 after ligation, the edges of the cardiomyocytes bordering the infarcted area manifested numerous sarcoplasmic tentacles that reacted positively to anti-desmin antibody. Distinct expression of Cx43 was observed extensively on the tentacles, although no cardiomyocytes remained viable around them. By day 60 after ligation, atypical expression of Cx43 had disappeared. These findings suggest that ischaemia induces temporally abnormal expression of Cx43, which might be responsible for abnormal conduction around the infarct. Received: 4 April 1997 / Accepted: 19 June 1997     

发育大鼠心肌细胞间隙连接蛋白43表达特性

目的：探讨大鼠心肌细胞间隙连接蛋白43(Cx43)表达的发育特性。方法：应用SP免疫组化和图像分析方法,观察胎大鼠、出生1 d、5 d、10 d和25 d大鼠心肌细胞Cx43的蛋白表达。结果：(1)胎鼠心肌细胞Cx43呈斑点状遍布于心肌细胞侧—侧连接、细胞质内和闰盘处。(2)出生后1 d大鼠心肌细胞的Cx43分布与胎鼠相似,5 d和10 d组逐渐向闰盘处聚集。25 d时部分已聚集于端闰盘处,其余仍呈斑点状散在分布。(3)出生前后心室肌细胞Cx43分布有差异。分布密度总规律是：胎大鼠＞出生1 d＞5 d＞10 d＞25 d。结论：出生前后大鼠心肌细胞Cx43分布随心脏发育逐渐向闰盘处重排,其分布密度呈逐渐下降趋势。     

Effect of mannitol treatment on brain neurotransmitter markers in kainic acid-induced epilepsy

The effect of mannitol treatment on the behavioural, morphological and neurochemical brain damage induced after subcutaneously applied kainic acid (10 mg/kg) was studied in the rat. Mannitol at a dose of 1.5 g/kg was injected intravenously 10 min, 1.5 h and 3 h respectively after kainic acid administration. A protective effect of mannitol was observed only when mannitol was given 1.5 h after kainic acid application, i.e. within the early phase of kainic acid-induced brain oedema development. At this time period, mannitol prevented the development of kainic acid-induced seizures as well as irreversible brain lesions and neurochemical changes, the latter being reduction of noradrenaline levels in amygdala/pyriform cortex measured 3 h, and reduction of glutamate decarboxylase and choline acetyltransferase activities measured 3 days after kainic acid treatment. Similarly loss of glutamate decarboxylase activity in dorsal hippocampus induced by kainic acid was prevented by mannitol treatment. It is concluded that by washing out brain oedema, mannitol treatment may prevent propagation of seizures and brain damage in the kainic acid model of epilepsy.     

Expression of heparanase in nestin-positive reactive astrocytes in ischemic lesions of rat brain after transient middle cerebral artery occlusion

Heparanase is an enzyme that cleaves heparan sulfate proteoglycans, an important component of the extracellular matrix to generate heparan sulfate fragments, leading to the remodeling of the extracellular matrix and the basement membrane particularly during cancer metastasis. A growing body of evidence suggests that heparanase serves multiple functions in normal tissues including the central nervous system. In this study, we showed that heparanase is expressed in reactive astrocytes in the peri-infarct lesion of a rat brain whose middle cerebral artery was transiently occluded for 90 min. RT-PCR and Western blot analyses revealed that heparanase expression was markedly upregulated during the subacute phase of ischemia (from 3 to 7 days post-reperfusion (dpr)). As revealed by immunohistochemical study, heparanase was localized in astrocytes located in the peri-infarct region. Heparanase+ astrocytes expressed nestin that is known as a marker of reactive astrocytes. Infiltrated neutrophils were weakly heparanase+. After 7 dpr, the expression level of heparanase+ astrocytes considerably decreased. Therefore, the maximum expression of heparanase by astrocytes may correlate with the time of migration of reactive astrocytes toward the ischemic core, which may result in astrogliosis. These findings suggest a novel role of heparanase in the pathophysiology of brain ischemia.     

Altered expression of connexin43 and phosphorylation connexin43 in glioma tumors

In this study, we aim to evaluate the connexin (Cx43) and phosphorylation Cx43 (p-Cx43) expression of human glioma tumors and correlate their expression with degrees of malignancy and proliferation, apoptosis, and migration activity of tumors. Cx43 and p-Cx43 expression were examined by Western blot analysis and immunohistochemical staining. The U251 cell viability was measured by MTT analysis. The apoptosis and migration were also evaluated by flow cytometric analysis and fluoroblok transwell chambers, respectively. We found that the Cx43 expression were significantly downregulated in in malignant glioma (WHO grade III and IV), compared to the malignant glioma (WHO grade I and II) and the p-Cx43 expression levels of malignant glioma (WHO grade III and IV) were significantly increased (P<0.05), compared to the malignant glioma (WHO grade I and II) at immunohistochemical analysis. After treatment of cells with a specific inhibitor of PKC, MAPK, and PTK inhibitors, the cell viability and migration were significantly decreased, while the apoptosis was slightly induced. In conclusion, the Cx43 expression level is inversely correlated with the tumor grade and proliferation and migration activity of tumor. Higher p-Cx43 expression level in high tumor grade suggests that a complex mechanism is involved in the suppression of tumor growth by connexins.     

Protective effects of mossy fiber lesions against kainic acid-induced seizures and neuronal degeneration

The effects of a hippocampal mossy fiber lesion have been determined on neuronal degeneration and limbic seizures provoked by the subsequent intracerebroventricular administration of kainic acid to unanesthetized rats. Mossy fiber lesions were made either by transecting this pathway unilaterally or by destroying the dentate granule cells unilaterally or bilaterally with colchicine. All control rats eventually developed status epilepticus and each temporally discrete seizure that preceded status epilepticus was recorded from the hippocampus ipsilateral to the kainic acid infusion before the contralateral hippocampus. A mossy fiber lesion of the ipsilateral hippocampus prevented the development of status epilepticus in 26% of subjects and in 52% of subjects seizures were recorded from the contralateral hippocampus before the ipsilateral hippocampus. Unlike electrographic records from other treatment groups, those from rats which had received a bilateral colchicine lesion exhibited no consistent pattern indicative of seizure propagation from one limbic region to another. A bilateral, but not a unilateral, mossy fiber lesion also dramatically attenuated the behavioral expression of the seizures. Regardless of its effects on kainic acid-induced electrographic and behavioral seizures, a mossy fiber lesion always substantially reduced or completely prevented the degeneration of ipsilateral hippocampal CA3-CA4 neurons. This protective effect was specific for those hippocampal neurons deprived of mossy fiber innervation. Neurons in other regions of the brain were protected from degeneration only when the mossy fiber lesion also prevented the development of electrographic status epilepticus. These results suggest that the hippocampal mossy fibers constitute an important, though probably not an obligatory, link in the circuit responsible for the spread of kainic acid seizures. Degeneration of CA3-CA4 neurons appears to depend upon (1) the duration of hippocampal seizure activity and (2) an as yet undefined influence of or interaction with the mossy fiber projection which enhances the neurodegenerative effect of the seizures.     

Enhanced connexin 43 expression following neural stem cell transplantation in a rat model of traumatic brain injury

Introduction

Reestablishment of functional networks after traumatic brain injury (TBI) has been proffered as one of the goals of neural stem cell (NSC) transplantation therapeutics. Gap junctions provide essential means for direct cellular communication by transferring small molecules and ions, which may provide insights into the interplay between grafted NSCs and host cells.

Material and methods

Thirty-six adult male Wister rats were used in this study. The controlled cortical impact (CCI) model of brain injury has been performed. Seventy-two hours after CCI injury, animals were randomly assigned to two groups: PBS- and NSC- transplanted group. NSCs-transplanted group received delivery of the NSCs suspension to the cortex below the injury cavity in the ipsilateral hemisphere. At 1, 2, and 4 weeks post-transplantation, we investigated the expression patterns of gap junction-associated connexin 43 (Cx43) in the transplant site and the border of CCI by immunohistochemistry, Western blot and RT-PCR.

Results

Our findings showed that Cx43 staining was significantly greater in the transplant site and the border of CCI in the NSCs-transplanted rats compared to the control rats at different time points (p < 0.01 at 1 week, p < 0.05 at 2 and 4 weeks). Significantly higher gene and protein expression of Cx43 was found in NSCs-transplanted rats compared to the control rats in the period of 4 weeks post-transplantation (p < 0.01), and remained at a higher level until 2 weeks with or without NSC transplantation.

Conclusions

It is proposed that gap junction-associated Cx43 might participate in NSCs’ beneficial effects via gap-junctional coupling by which grafted NSCs integrate into host neural tissue following transplantation after TBI.     

Brain-derived neurotrophic factor, nerve growth and neurotrophin-3 selected regions of the rat brain following kainic acid-induced seizure activity.

Changes in levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) in various regions of the rat brain following kainic acid-induced seizure activity were investigated. BDNF protein, as measured by a two-site enzyme immunoassay, increased transiently 12-24 h after the intraperitoneal administration of kainic acid to 61.6 ng/g wet weight in the hippocampus (approximately 10-fold increase), 19.5 ng/g in the piriform plus entorhinal cortex (approximately 10-fold) and 8.2 ng/g in the olfactory bulb (approximately 16-fold), and then rapidly decreased. Increases of 2- to 4-fold in levels of BDNF were also detected in the septum, cerebral cortex, striatum and hypothalamus, but not in the cerebellum. In contrast, levels of NGF and NT-3 decreased 24 h after the administration of kainic acid. Western and Northern blotting analyses of hippocampal tissues, respectively, revealed increase in levels of a 14-kDa protein corresponding to BDNF and its mRNA at both 4.2 and 1.4 kb. Hippocampal mRNAs for NGF and NT-3 increased and decreased, respectively, in kainic acid-treated rats. Immunohistological investigations showed that, in the hippocampus, the administration of kainic acid enhanced a homogeneous immunoreactivity of BDNF in the polymorph inner layer (the stratum radiatum of the CA3/CA4 regions and the hilar region) and in granule cells of the dentate gyrus. BDNF protein was found in neurons, but not at all in glial cells or in blood vessels, and was localized in the cytoplasm, the nucleoplasm and the primary dendrites of neurons as well as in perisynaptic extracellular spaces, but hardly in their axons. Our results show that kainic acid treatment increases levels of BDNF, but not NGF or NT-3, in various regions of the rat brain, other than the cerebellum. Also, the majority of BDNF newly synthesized by hippocampal granule neurons is secreted into the perisynaptic extracellular space in the polymorph inner layer of the dentate gyrus, supporting an autocrine-like role for the factor in synaptic functions.     

Electrophysiological properties of lemniscal afferents in rat after kainic acid lesions in the ventrobasal thalamus

Kainic acid (KA) has been largely used as a neurotoxin, and its axon-sparing effect being repeatedly emphasized, on the basis of anatomical and biochemical data. The present study examines this 'axon-sparing' effect from an electrophysiological point of view and demonstrates that lemniscal fibers retain the capacity to convey somesthetic information 5-60 days after an injection of KA in the ventrobasal complex of the thalamus depriving these afferent fibers of their target cells.