Blood-brain barrier dysfunctions following systemic injection of kainic acid in the rat.

Changes in blood-brain barrier (BBB) permeability and cerebral metabolic activity following intravenous injection of kainic acid (KA; 6, 12 mg/Kg) in rats were assessed by calculating respectively a blood-to-brain transfer constant (Ki) for [14C]alpha-aminoisobutyric acid and local cerebral glucose utilization (LCGU) values, at different times (1 h, or acute seizures phase, and 48 h, or chronic pathology phase) after the induction of seizures. A significant increase in the local permeability of the BBB was observed 1 h after the injection of KA 6 mg/Kg (eliciting no significant changes in cerebral metabolic activity, except within the frontal cortex and the hippocampus) and 12 mg/Kg (which induced a marked and widespread enhancement of LCGU). On the contrary, during the pathology phase, persistent regional increases in Ki values were evidenced in rats treated with the lowest dose of the convulsant, but not in rats injected with KA 12 mg/Kg (a dose able to cause extensive neuronal damage). Thus one can speculate that: 1) KA-induced regional changes in the permeability of the BBB are not correlated with changes in neuronal activity; 2) opening of the BBB is not reliably associated with neuronal injury.     

Morpho-functional effects of kainic acid injection into the cat's pulvinar-lateralis posterior nucleus complex

The histological, electroencephalographic, behavioral changes as well as the changes in the intensity threshold of stimulation necessary to induce contralateral turning were studied in 16 cats, in which kainic acid (KA) was injected locally into the pulvinar-lateralis posterior nucleus complex (P-LP). In 13 cats a stainless-steel tube with two attached electrodes was implanted in P-LP, and electrodes were also implanted in the ipsilateral dorsal hippocampus, the superior colliculus and the caudate nucleus. KA was injected through the tube using a 10 microliters Hamilton syringe. In other 3 cats, KA was injected stereotaxically through the needle of the Hamilton syringe and two electrodes were implanted in these areas after withdrawal of the syringe. The intensity thresholds of stimulation required to induce turning behavior were controlled before and after KA administration in the 13 cats with an implanted tube and only after KA injections in the three cats without a tube; in these instances the current threshold of the contralateral P-LP served as control. The histological results showed a moderate KA damage of the P-LP, with destruction of neuronal soma and gliosis and additional involvement, in all the experiments, of the dorsal hippocampus; however, passage fibers were spared by the lesions. A dose-dependent epileptic effect of KA was seen, which was slight with the 3 micrograms dose and intense with 6 micrograms. The EEG recording showed a prominent and almost simultaneous epileptic involvement of the hippocampus and the P-LP after KA, with less involvement of the other implanted structures. Turning behavior induced by electrical stimulation of the P-LP was suppressed when the electrode tip was located inside the lesioned area. When the electrode tip was placed inside a slight or moderate damaged tissue, a significative increase in current threshold was found, and finally when the tip of the electrode was outside the lesioned area no change in threshold was observed. These findings do not contradict our previous hypothesis of an intrinsic cholinergic mechanism involved in the turning response evoked by P-LP electrical stimulation, although it cannot be excluded that fibers coming presumably from the superior colliculus or pretectum may contribute to the response.     

Effects of kainic acid injection into the substantia nigra on the paroxysmal amygdaloid phenomena

Nigral injection of kainic acid determines an inhibition of the after-discharges evoked in the amygdala by stimulation of the ipsilateral piriform cortex and later an increase of the AD duration. The role of the substantia nigra in the control both of the paroxysmal phenomena localized in the amygdala and of its spontaneous activity is emphasized.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

IRFI 042 reduces oxidative stress against kainic acid toxicity in the rat brain

We investigated if IRFI 042, an analog of vitamin E, protects the brain against oxidative stress induced by intraperitoneal administration of Kainic acid (KA) (10 mg/kg); sham brain injury rats were used as controls. Animals received either IRFI 042 (20 mg/kg) or its vehicle 30 min before KA injection and after 6 h were sacrificed to measure malonildyaldheide (MDA) and glutathione levels (GSH) in the diencephalon. Behavioral changes were also monitored. Intraperitoneal administration of IRFI decreased MDA (micromol/g wet tissue: KA + vehicle = 22.5 ± 4.2; KA + IRFI = 17.1 ± 1; P < 0.005) and prevented GSH loss (nmol/g wet tissue: KA + vehicle = 0.41 ± 0.1; KA + IRFI = 1.86 ± 0.2; P < 0.005) in the diencephalon. The latency of occurrence of behavioral signs increased from 39 ± 1 to 62 ± 6 min in IRFI 042 group. The data suggest that IRFI 042 might protect against KA‐induced oxidative stress.     

Beta-endorphin administered into the lateral ventricle of the brain causes pulmonary platelet trapping in rabbits

Human beta-endorphin was injected into the cerebrospinal fluid in rabbits by means of a needle inserted into the lateral ventricle of the brain. Control rabbits received an equal amount of saline. beta-Endorphin induced a significant pulmonary platelet trapping compared to control. beta-Endorphin had no effect on arterial blood pressure, heart rate, platelet aggregability ex vivo or fibrinolytic activity (fibrinolytic plates). The plasma activity of antithrombin III, kallikrein-like activity and kallikrein inhibitor determined by means of chromogenic substrates was not influenced by beta-endorphin.     

Specific binding of kainic acid to purified subcellular fractions from rat brain

The subcellular distribution of kainic acid (KA) binding sites in rat brain has been studied using a microcentrifugation assay. KA did not bind to myelin or brain cytosol and had few or no binding sites in the nuclear fraction. However, it bound to microsomal components (K _d =128–136 nM; 2.5–4.8 pmol/mg protein), purified synaptic plasma membranes (SPM) (K _d =45–71 nM; 5.8–6.5 pmol/mg), and purified cell-body and intraterminal mitochondria (K _d =11–31 nM; 0.4–1.1 pmol/mg). Bound KA could be totally displaced byl-glutamate orl-aspartate, but several putative antagonists of these amino acids (nuciferin, compound HA-966, 2-amino-4-phosphonobutyrate, and 2-amino-3-phosphonoproprionate) failed to displace KA or did so at very high concentrations (4 mM). Glutamic acid diethyl ester (GDEE) andd,l--aminoadipate (-AA) were more effective (IC₅₀, 0.2–0.8 mM) and showed differential effects in their capacity to displace KA bound to the various subcellular fractions. Thus, GDEE only displaced 40–60% of the KA bound by SPM or mitochondria and did not prevent the binding of KA to microsomes. -AA, on the other hand, was more effective in preventing the binding of KA at high concentrations and displaced between 80 and 100% of the drug. Both compounds showed biphasic curves of KA displacement from synaptic plasma membranes and mitochondria. The overall results indicate the presence of multiple binding sites for KA in brain cells and suggest that KA does not act exclusively at synaptic glutamate receptors. The mechanism of KA action is most likely quite complex, and the drug probably acts at multiple binding sites affecting a number of processes.     

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。     

Outcome of bovine herpesvirus 4 infection following direct viral injection in the lateral ventricle of the mouse brain

A recombinant bovine herpesvirus 4 (BoHV-4EGFPDeltaTK), obtained by the insertion of an EGFP gene into the TK locus of DN 599 BoHV-4 strain, was injected into the lateral ventricle of the brain of mice and a clinical score was evaluated for 90 days. Although BoHV-4 was not neuro-pathogenic, BoHV-4EGFPDeltaTK transduction capability was analyzed. EGFP expression was localized in close proximity to the border of the ventricles and EGFP-positive cells were found to co-localize with ependymal cells. Although most of the cells had a polarized morphology, they were not neurons. EGFP-positive cells were seen to spread in tangentially oriented rows within the rostral migratory stream (RMS). Co-localization of EGFP signal with anti-GFAP antibody showed that they were glial cells. EGFP-positive cells were observed until 31 days post-injection and then disappeared completely. Virus isolation was possible at an early post-injection time (3 days), but then virus titer was below the detection limits at later times. Viral DNA, however, could be detected until 21 days post-injection. Thus, in this report we showed that (i) BoHV-4EGFPDeltaTK did not replicate in the mouse brain, (ii) is not pathogenic and (iii) gene transfer can be obtained in long-lived cells belonging to the RMS after BoHV-4EGFPDeltaTK injection within the lateral ventricle.     

Selective calcification of rat brain lesions caused by systemic administration of kainic acid

Dystrophic calcification of previously damaged areas of nervous tissue occurs in a wide range of human diseases. The relationship between astroglial and microglial reactions and deposits of calcium salts was studied for up to five months in rats with a brain lesion produced by systemic administration of kainate. The morphology and atomic composition of the calcium salt deposits was also studied. Two types of lesions, sclerotic and liquefactive, were observed. In sclerotic lesions hyperplasia and hypertrophy of astrocytes partially substituted for the lost neurons, reaching a maximum in about twenty-five days after treatment. In liquefactive lesions, the astrocytic reaction occurred only around the liquefactive area. Microglial reaction was similar in both types of lesion and reached its highest expression in about twenty-five days. Calcium deposits were observed in the sclerotic but not in the liquefactive lesions. Clearly distinguishable granules of calcium salts were observed in sclerotic lesions under scanning electron microscopy after only five days post-injection. The size of calcified granules increased with time reaching 40 micro m or more in diameter at five months. The atomic composition of these deposits, studied by X-ray microanalysis, showed a time-dependent increase in calcium concentration. While there was no clear relationship between astroglial and microglial reactions and calcium salt deposits, the systemic injection of kainate produced progressively larger and more concentrated calcium deposits in sclerotic, but not in liquefactive lesions.     

Accumulation of labeled gamma-aminobutyric acid into rat brain and brain synaptosomes after i.p. injection

The accumulation of labeled GABA into brain and brain nerve endings was studied in the adult rat after i.p. injection of large doses of neurotransmitter (740 mg/Kg). In the first 5–30 minutes after the injection the exogenous neurotransmitter reaches a stable plasma level of around 5 mM. The accumulation of radioactive GABA into the brain presents a latency of a few minutes from the time of the injection. Thereafter, the accumulation of the neurotransmitter is almost linear with time. Once in the brain tissue labeled GABA is in part broken down. The exogenous neurotransmitter is taken up in GABA-ergic nerve endings with a steep increase between 20 and 30 minutes after the injection. From a quantitative point of view, the data show that the brain accumulation of labeled GABA at 30 minutes post injection is minimal in the respect of the steady state average concentration of the endogenous neurotransmitter (0.014%). However, the amount of radioactive GABA which accumulates in the nerve endings, at the same post injection time, is around 7% of the endogenous neurotransmitter in that comparment. The data thus show a selective enrichment of exogenous systemic GABA in a physiologically important compartment of the brain.     

Cardiovascular effects produced by choline injected into the lateral cerebral ventricle of the unanesthetized rat

Intracerebroventricular (icv) injection of choline (50–150 μg) causes a transient increase in blood pressure and a more prolonged decrease in heart rate (HR) in conscious rats. The bradycardia results from a centrally mediated increase in vagal tone. The cardiovascular effects do not appear to involve endogenous brain acetylcholine since there is no significant difference in the responses induced by choline before and after icv injection of hemicholinium-3. Intracerebroventricular ventricular injection of atropine or mecamylamine, alone, failed to influence the choline effect. However, atropine and mecamylamine, given together, abolished the reduction of HR, but still failed to modify the pressor response. The changes in blood pressure and HR appear to be due to effects of choline on post-synaptic receptors in different brain regions.     

Osteopontin in kainic acid-induced microglial reactions in the rat brain

The present study was performed to investigate the spatial and temporal expression of osteopontin (OPN) mRNA in the rat brain after kainic acid-induced seizures, and to determine whether this phenomenon is associated spatiotemporally with the microglial reaction. The expression of OPN mRNA was detected using an in situ hybridization technique and Northern blot analysis. Following intraperitoneal injection of kainic acid (10 mg/kg), OPN mRNA was expressed in selective vulnerable areas, including the hippocampus, thalamus, hypothalamus, amygdala, and entorhinal cortex. Comparison of the morphology and localization with the established microglial marker OX-42 in the adjacent sections positively identified the OPN-expressed cells as microglia. Furthermore, double labeling experiments revealed that OPN mRNA expression was confined to ameboid-like cells among microglia stained with GSI-B4, an another microglial marker. These findings from a rat model of seizure support the notion that OPN can be synthesized in a subpopulation of reactive microglial cells. It can therefore be assumed that in the response of the brain to excitotoxic injury, synthesis of OPN occurs generally in a subset of activated microglia.     

Comparison of central effects of noradrenaline and dopamine injected into the lateral brain ventricle in rats.

Noradrenaline and dopamine injected into the lateral brain ventricle exerted a significant effect on the behavior of rats. Both amines caused a slight rise in the basic locomotor activity which was significantly increased in the animals with inhibited monoamine oxidase activity. Besides that, they suppressed the behavior of rats in the open-field test, inhibited the conditioned avoidance response, decreased body temperature and increased amphetamine-induced motor hyperactivity. Noradrenaline, in contrast to dopamine, changed the intensity of amphetamine-induced stereotypy and prolonged the action of hypnotics. The central action of both catecholamines (in higher doses especially) seemed to have a biphasic course: in the first phase after administration depression was observed which was more pronounced after noradrenaline administration, in the second phase a stimulating effect b     

Expression and subcellular localization of thymosin beta15 following kainic acid treatment in rat brain

Thymosin β15 (Tβ15) is a pleiotropic factor which exerts multiple roles in the development of nervous system and brain diseases. In this study, we found that the expressions of Tβ15 mRNA and protein were substantially increased in several brain regions including hippocampal formation and cerebral cortex, following kainic acid (KA)-evoked seizures in rat. Interestingly, a subset of cortex neurons exhibited nuclear Tβ15 immunoreactivity upon KA treatment. Furthermore, translocation of Tβ15 from cytosol to nuclei was observed in cultured neurons or HeLa cells during staurosporine (STS)-induced apoptosis, which was also verified by time-lapse imaging of YFP-tagged Tβ15. It appeared that localization of Tβ15 is restricted to the cytosol in normal condition by its G-actin-interacting domain, because site-directed mutagenesis of this region resulted in the nuclear localization of Tβ15 in the absence of STS treatment. To explore the role of nuclear Tβ15, we enforced Tβ15 to localize in the nuclei by fusion of Tβ15 with nuclear localization signal (NLS-Tβ15). However, overexpression of NLS-Tβ15 did not alter the viability of cells in response to STS treatment. Collectively, these results suggest that nuclear localization of Tβ15 is a controlled process during KA or STS stimulation, although its functional significance is yet to be clarified.     

Distribution and persistence of kainic acid in brain.

The distribution of ³H-kainic acid in rat brain was studied as a function of time after injections of 5 nmoles into the neostriatum, substantia nigra or cerebellum. More than half of the injected material had disappeared from the injection site and the brain by 1/2 hour post injection. Under the conditions used very small amounts of radioactivity (corresponding to less than 7 pmol/ mg of tissue) were found in areas other than the injection site, suggesting that the histological damage reported in the hippocampus and pyriform cortex after striatal injections may be due to a secondary process not dependent on the presence of toxic concentrations of kainic acid in those areas. No radioactivity was found in the TCA-insoluble material nor did it appear that there was rapid metabolism of the bulk of the kainic acid.     

Activities of glutamine synthetase,glutaminase and arginase in kainic acid lesioned rat brain corpus striatum

Intrastriatal kainic acid (2 μg/μl) administration gave rise to significant increase in activities of glutamine synthetase and arginase along with a significant decrease in the activity of glutaminase in the lesioned striatal tissue 7 days after the administration of kainic acid. The increase in the activity of glutamine synthetase was attributed to the gliosis occurring in such lesions. The decrease in the activity of glutaminase was thought to be due to the loss of GABAergic neurons. The increase in arginase activity might be occurring in glial cells or in nerve endings. Although the earlier results indicated a low specific activity of arginase in glial cells, the observed increase in its activity might be partly due to its increase in proliferating glial cells, liberating ornithine for the formation of polyamines. However, it was also thought that a substantial increase may be occurring in the arginase present in the intact glutamatergic (corticostriate pathway) nerve endings, since it was earlier found that the synaptosomes of the rat brain had appreciably high activity of arginase. These results were discussed in relation to the probable roles of arginine and glutamine as the precursors for neurotransmitter pools of glutamate in striatum.