Striatal extracellular dopamine levels are not increased by hyperglycemic exacerbation of ischemic brain damage in rats

We tested the hypothesis that hyperglycemic exacerbation of incomplete forebrain ischemia is mediated by increased extracellular dopamine levels. Normoglycemic and hyperglycemic Sprgue—Dawley rats (eight each) with previously placed coaxial striatal microdialysis probes underwent 12 min of forebrain carotid artery occlusion and trimethaphan-induced hypotension. Microdialysis was performed before, during and for 6 h after ischemia, then perfusion-fixation was performed. Hyperglycemic rats had more severe postischemic damage in the caudate-putamen, neocortex, and hippocampus. Extracellular striatal dopamine levels were increased by ischemia, but were unaffected by hyperglycemia. These data show that hyperglycemic exacerbation of ischemic striatal damage does not depend on elevated extracellular dopamine levels.     

Calcium dysregulation induces apoptosis-inducing factor release: Cross-talk between PARP-1- and calpain- signaling pathways

Recent discoveries show that caspase-independent cell death pathways are a pervasive mechanism in neurodegenerative diseases, and apoptosis-inducing factor (AIF) is an important effector of this mode of neuronal death. There are currently two known mechanisms underlying AIF release following excitotoxic stress, PARP-1 and calpain. To test whether there is an interaction between PARP-1 and calpain in triggering AIF release, we used the NMDA toxicity model in rat primary cortical neurons. Exposure to NMDA resulted in AIF truncation and nuclear translocation, and shRNA-mediated knockdown of AIF resulted in neuroprotection. Both calpain and PARP-1 are involved with AIF processing as AIF truncation, nuclear translocation and neuronal death were attenuated by calpain inhibition using adeno-associated virus-mediated overexpression of the endogenous calpain inhibitor, calpastatin, or treatment with the PARP-1 inhibitor 3-ABA. Activation of PARP-1 is necessary for calpain activation as PARP-1 inhibition blocked mitochondrial calpain activation. Finally, NMDA toxicity induces mitochondrial Ca²⁺ dysregulation in a PARP-1 dependent manner. Thus, PARP-1 and mitochondrial calpain activation are linked via PARP-1-induced alterations in mitochondrial Ca²⁺ homeostasis. Collectively, these findings link the two seemingly independent mechanisms triggering AIF-induced neuronal death.     

Mechanisms of pH recovery from intracellular acid loads in the leech connective glial cell.

We used double-barrelled, neutral carrier, pH-sensitive microelectrodes to study the mechanisms by which the intracellular pH (pHi) is regulated in the connective glial cells of the medicinal leech. In HEPES-buffered, nominally CO2/HCO3(-)-free solutions the recovery of pHi from intracellular acidosis is Na(+)-dependent and reduced by at least half in the presence of amiloride, suggesting the action of Na+:H+ exchange. The rate of pHi recovery by this mechanism can be increased by raising the extracellular buffering power or by increasing extracellular pH. The presence of CO2/HCO3(-)-greatly increases the rate of pHi recovery from intracellular acidosis. This CO2/HCO3(-)-stimulated recovery is also dependent on external Na+, largely Cl(-)-independent, inhibited by DIDS, and accompanied by membrane hyperpolarization. This is consistent with it being mediated by the electrogenic cotransport of Na+ and HCO3- into the cells. A Cl(-)-dependent component to Na(+)- and HCO3(-)-dependent regulation is most easily explained by the added presence of a Na(+)-dependent exchange of HCO3- and Cl-.     

Alteration of mitochondrial calcium homeostasis by ammonia-induced activation of NMDA receptors in rat brain in vivo

The aim of the present work was to assess the effects of activation of NMDA receptors in rat brain in vivo on calcium homeostasis in isolated non-synaptic brain mitochondria. We have shown recently that acute intoxication with large doses of ammonia leads to activation of NMDA receptors in rat brain in vivo. In the present work we injected rats with ammonium acetate to activate NMDA receptors in vivo and isolated non-synaptic mitochondria to assess calcium homeostasis. We also tested whether blocking NMDA receptors with MK-801 prevents effects on calcium homeostasis induced by ammonium injection. It is shown that activation of NMDA receptors in rat brain in vivo leads to a rapid increase in intramitochondrial calcium content followed by a reduction in the calcium capacity and calcium uptake rate in rat brain mitochondria. Activation of NMDA receptors resulted in increased spontaneous calcium efflux from rat brain mitochondria and in a strong inhibition of Na-induced and tert-butylhydroperoxide-induced calcium efflux. All these effects were prevented by previous blocking of NMDA receptors by injection of MK-801. Cyclosporin A did not affect any of the above parameters, indicating that the mitochondrial permeability transition pore does not play a role in calcium efflux under any of the conditions studied. The results reported indicate that ammonia-induced activation of NMDA receptors in rat brain in vivo alters mitochondrial calcium homeostasis at several different steps.     

Estrogen attenuates nuclear factor-kappa B activation induced by transient cerebral ischemia

The protective effects of estrogens have been widely reported in a number of animal and cell culture models, but the molecular mechanisms of this potent neuroprotective activity are not well understood. Both in vitro and in vivo studies indicate that in the central nervous system and peripheral tissues, estrogen treatment reduces cytokine production and inflammatory responses. Nuclear factor-kappa B (NFkappaB) plays an essential role in the regulation of post-ischemic inflammation, which is detrimental to recovery from an ischemic stroke. We investigated the role of NFkappaB in neuronal survival in rats that received transient middle cerebral artery (MCA) occlusion, and observed that this transient cerebral ischemia induced substantial apoptosis and inflammatory responses, including IkappaB phosphorylation, NF-kappaB activation and iNOS over-expression. 17 beta-estradiol (E2) treatment produced strong protective effects by reducing infarct volume, neuronal apoptosis, and inflammatory responses. These findings provide evidence for a novel molecular and cellular interaction between the sex hormone and the immunoresponsive system. These studies also provide evidence that suppression of post-ischemic inflammation may play a critical role in estrogen-mediated neuroprotection.     

Acid-induced changes of brain protein buffering

Excessive cellular acidosis is thought to enhance destruction of brain from ischemia. Protein denaturation may contribute to such injury although the behavior of brain proteins to acidosis is poorly defined. As a first approach to detect acid-induced changes in brain proteins and to characterize buffer content, homogenates were acidified for 20 min (as low as pH 3.1), returned to baseline pH (6.9), and then titrated. Titration curves show a significant(P < 0.0001) and permanent increase in buffer content compared to controls when pH of acid exposure was 4.5−3.7 or less. Since acidity of pH 4.5 is rarely, if ever, achieved in vivo, protein denaturation from acidity alone is unlikely to account for necrosis of brain from ischemia.     

Origin of extracellular dopamine increase induced by lactic acid striatal perfusion monitored by microdialysis in the awake rat.

In previous studies we showed that a striatal lactic acid perfusion-induced lactacidosis produces a diphasic increase in extracellular dopamine (DA). In the present study, different pharmacological reagents were used to determine the origin of accumulated DA. Our data show that both DA accumulations were totally suppressed by tetrodotoxin and nicardipine, indicating a relationship with membrane depolarization and a Ca(2+)-dependent effect. The first DA peak was largely reduced by a specific inhibitor of DA uptake such as GBR-12935, and the second was totally suppressed by tyramine and reserpine and lowered and delayed by GBR-12935. These results compared to data in the literature suggest that the first increase in extracellular DA resulted mainly from a release of cytosolic DA by reversal of the DA transporter, while the second was mainly due to a release of vesicular DA by exocytosis. These data indicate that lactic acid perfusion helps clarify the mechanisms involved in this process and could be useful for the study of new treatments against the hyperactive dopaminergic reaction occuring during ischemia.     

Calcium deposits in the thalamus following repeated cerebral ischemia and long-term survival in the gerbil

We investigated the long-term changes in the gerbil brain following three episodes of 2-min forebrain ischemia at 1-h intervals in comparison with a 6-min period of ischemia. The animals were sacrificed after 1 month and 6 months. Following either ischemic insult, the hippocampal CA1 region showed a loss of pyramidal neurons together with a diffuse calcium accumulation as shown by alizarin red S staining. Three 2-min ischemic insults additionally produced neuronal damage in the striatum and thalamus. The thalamic damage was accompanied by an accumulation of small calcium granules after 1 month and large calcium concretions after 6 months. Calcium staining in the striatum was weak. Thus, the thalamic neuronal damage was accompanied by an active process of calcification, which has not been described in experimental cerebral ischemia models. The observations show that repeated ischemic insults produce different long-term effects in different brain regions.     

Brain lactic acidosis and ischemic cell damage: A topographic study with high-resolution light microscopy of early recovery in a rat model of severe incomplete ischemia

Summary Transient severe incomplete ischemia was induced in rats by a combination of bilateral carotid artery clamping and hypovolemic hypotension. Production of lactic acid in the ischemic brain was modified by preischemic administration of glucose or saline. After 30 min of ischemia and 5 or 90 min of recirculation, the animals were fixed by perfusion. High-resolution light microscopy based on whole hemisphere plastic sections revealed that the model produces a highly predictable ischemia in the telencephalon, with a more inconstant injury in the diencephalon, rostral brain stem, and cerebellum. The extent of injury correlates well with studies of local cerebral blood flow in the same model. The present study largely confirmed the opinion, based on the earlier study of the frontoparietal cortex, that the neuronal injury is predominantly of the pale type, although fair amounts of dark injury also appeared with predilection to the pyriform cortex, hippocampus, and occasionally the cerebellum. Excessive tissue lactic acidosis due to glucose pretreatment aggravated both types of neuronal injury. It was also accompanied by marked astrocytic edema as well as capillary obstruction in the group with long recirculation. A novel type of ischemic tissue change emerged, consisting of osmiophilic granules and whorls probably derived from damaged cell membranes.Supported by grants from the Finnish Medical Research Council, Research and Science Foundation of Lääke Oy, Swedish Medical Research Council, and US Public Health Service     

从缺血的病理生化改变探讨神经元损伤的机理

目的：探讨缺血性神经元损伤的机理。方法：用体培养海马神经元建立缺血模型,通过检测神经元致死率以及观察由缺血引起的细胞内生化代谢改变所致的神经元功能障碍,探讨缺血性神经元损伤的机理,结果：发现缺血组、缺葡萄糖组元致死率最高,且通过检测细胞膜表面磷脂酰丝氨酸的变化证实此二组神经元凋亡率也最高。缺血可引起元细胞骨架结构改变,使神经元功能遭到破坏,最终导致神经元不可逆损伤。结论：缺血的是生化改变通过不同的     

The role of transmembrane pH gradients in the lactic acid induced swelling of astrocytes

The swelling of astrocytes is an important component of the morbidity and mortality associated with ischemic brain trauma. In the ischemic brain, lactic acid levels rise dramatically with a concomitant acidification of the extracellular fluid. In this study we have measured the effects of elevated extracellular lactate and reduced extracellular pH (pHo) on astrocyte volume using the human astrocyte-derived cell line UC-11MG. Neither elevated lactate nor reduced pHo alone increased cell volume, but swelling of about 25% was measured when the cells were exposed simultaneously to 20 mM lactic acid and a reduced pHo of 6. The swelling was correlated with an approximately 4-fold increase in intracellular lactate as pHo was decreased from 8.0 to 6.0. As pHo was decreased intracellular pH also decreased, but much more slowly so that at acidic extracellular pH there was an inwardly directed proton gradient. The measured intracellular lactate concentrations closely followed the theoretical levels predicted by a model in which lactate transport is coupled to the inwardly directed proton gradient. Kinetic studies indicated that lactate transport is saturable with a Km of 3.8 mM, consistent with the model for facilitated cotransport of lactate with a proton or exchange of lactate for a hydroxyl ion. These data suggest that an important mechanism of postischemic astrocytic swelling is a proton driven, active accumulation of lactate to levels that result in a significant osmotic gradient of lactate at acidic pH.     

Vesicular demyelination induced by raised intracellular calcium

Incubation of nerve with high concentrations of the divalent cation ionophore A23187 produces myelin vesiculation (Schlaepfer 1977). This observation has now been extended using segments of rat ventral or dorsal root incubated with high (19 μM, 10 μg/ml) or low (1–1.5 μM) concentrations of A23187, or another divalent ionophore, ionomycin. Low concentrations of A23187 induced no vesiculation within a 2-h period. However, subsequent incubation of these roots in fresh, ionophore-free medium for 20 h, resulted in a prominent vesicular demyelination at the Schmidt-Lanterman incisures and paranodes of many fibres. At this time (22 h) the Schwann cells associated with some demyelinating internodes appeared vital upon ultrastructural examination: the cells also excluded the nuclear dye nigrosin. High concentrations of A23187 induced a similar vesicular demyelination in affected fibres within only 15–20 min. While the Schwann cells continued to exclude nigrosin for a further 4 h, their ultrastructural appearance indicated that they were probably in the early stages of necrosis. Incubation of moribund root with the ionophore produced no myelin vesiculation. At all ionophore concentrations, the myelin vesiculation was dependent upon the presence of extracellular Ca²⁺, and could be modulated in severity by varying this concentration. Other divalent cations (Ba²⁺, Co²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Sr²⁺) could not substitute for Ca²⁺. The vesiculation induced by A23187 could be entirely prevented by the addition of Zn²⁺ (? 1 μM), Ni²⁺ (? 1–10 μM), Co²⁺ (? 100 μM) or Mn²⁺ (? 100 μM) to the bathing medium. A23187 applied to only part of an isolated internode resulted in a localization of the myelin disruption to that region. Ionomycin (? 1 μM), an ionophore with a greater selectivity for Ca²⁺ than A23187, also induced a prompt Ca²⁺-dependent myelin vesiculation. We conclude that vesicular demyelination can be initiated in vital Schwann cells by a raised intracellular Ca²⁺ concentration. Such demyelination does not necessarily lead to Schwann cell death. The possible relevance of the findings to vesicular demyelinating neuropathies is discussed, and a hypothesis regarding the mechanism of demyelination is advanced.     

The effects of extracellular pH and calcium manipulation on protein synthesis and response to anoxia/aglycemia in the rat hippocampal slice

Extracellular pH modulates the function of the N-methyl-

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-aspartate (NMDA) receptor, which may influence pathophysiological responses to glutamate. While damage due to oxygen and glucose deprivation or glutamate exposure is attenuated by acidification of the incubating medium of cultured neurons, neuron damage is enhanced in vivo following ischemia in hyperglycemic animals. A persistent inhibition of protein synthesis (to less than 5% of normoxic levels) is a reliable index of damage to neurons both in vivo and in the rat hippocampal slice. We explored the influence of extracellular pH and calcium manipulation on protein synthesis inhibition and energy failure due to anoxia/aglycemia or exposure to N-methyl-

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-aspartate in the rat hippocampal slice. Moderate acidification of the medium during anoxia/aglycemia did not reduce the damage to protein synthesis in hippocampal neurons (9% of normoxic levels) and did not alter basal ATP levels or the rate of ATP depletion during anoxia/aglycemia. However, when calcium levels were lowered during the acidification and following the anoxia/aglycemia, protein synthesis was almost completely protected (84% of normoxic levels). Calcium reduction itself also attenuated the protein synthesis inhibition due to anoxia/aglycemia (to 55.6% of normoxic controls), but the protection was not as complete. In contrast, moderate acidification of the medium significantly reduced the damage to protein synthesis due to a brief exposure to NMDA (37% of control with NMDA, 78.9% of control with acidification during NMDA), even in the presence of extracellular calcium. Alkalinization of the medium exacerbated the protein synthesis inhibition following anoxia/aglycemia, and significantly reduced basal ATP levels (to 52% of normoxic control levels). Thus, pH_o changes influence neuronal metabolism and response to anoxia/aglycemia. In addition, while acidification can reduce the excitotoxic damage caused by direct exposure to NMDA, it cannot reduce damage due to anoxia/aglycemia unless calcium is lowered concomitantly. Thus, both NMDA receptor activation and calcium are involved in the damage due to oxygen and glucose deprivation in the slice.     

Neuroprotective effects of the CTK 01512-2 toxin against neurotoxicity induced by 3-nitropropionic acid in rats

The mitochondrial inhibitor 3-nitropropionic acid (3-NP) induces excitotoxicity. The authors hypothesized that CTK 01512-2, a recombinant peptide calcium channel N-type blocker, and the TRPA1 antagonist, could show neuroprotective effects. The male Wistar rats received 3-NP [25 mg/kg (i.p.) for 7 days], and a treatment of CTK 01512-2 was delivered intrathecally (i.t.), thrice a week. The neuroprotective effects were evaluated by [¹⁸F]FDG MicroPET analysis. The CTK 01512-2 toxin was able to reestablish similar glucose uptakes on the control animals. To detect the neurobehavioral effects from 3-NP, three protocols (6.25, 12.5, 18.75 mg/kg of 3-NP (i.p.), for 3, 4, and 6 days, respectively) were evaluated by performance tests (open field test, walk footprint, elevated plus-maze, Y-maze, and the object recognition test). Important disabilities in the gait of the rats were seen, as well as memory deficits, and anxious behavior in the animals that were treated with all 3-NP protocols. The dose of 18.75 mg/kg (for 3 days) showed the most pronounced behavioral effects and lethality, while the rats treated with 12.5 mg/kg (for 4 days) showed behavioral effects similar to the 6.25 mg/kg dose (for 6 days). The third protocol was then repeated and the rats were treated with the CTK 01512-2 toxin to be evaluated behaviorally again. The recombinant peptide prevented all of the gait-evaluated parameters that were induced by 3-NP at a 6.25 mg/kg dose, which displayed an improvement in the exploratory activities. Overall, these results have reinforced the positive effects of CTK 01512-2 against the behavioral changes that were induced by the mitochondrial inhibitor 3-NP.     

Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats

The effects of oligemia (moderate ischemia) on the brain need to be explored because of the potential role of subtle microvascular changes in vascular cognitive impairment and dementia. Chronic bilateral common carotid artery occlusion (BCCAO) in adult rats has been used to study effects of oligemia (hypoperfusion) using neuropathological and neurochemical analysis as well as behavioral tests. In this study, BCCAO was induced for 1 week, or 2, 4, and 6 months. Sensitive immunohistochemistry with marker proteins was used to study reactions of astrocytes (GFAP, nestin), and lectin binding to study microglial cells during BCCAO. Overt neuronal loss was visualized with NeuN antibodies. Astrocytes reacted to changes in the optic tract at all time points, and strong glial reactions also occurred in the target areas of retinal fibers, indicating damage to the retina and optic nerve. Astrocytes indicated a change in the corpus callosum from early to late time points. Diffuse increases in GFAP labeling occurred in parts of the neocortex after 1 week of BCCAO, in the absence of focal changes of neuronal marker proteins. No significant differences emerged in the cortex at longer time points. Nestin labeling was elevated in the optic tract. Reactions of microglia cells were seen in the cortex after 1 week. Measurements of the basilar artery indicated a considerable hypertrophy, indicative of macrovascular compensation in the chronic occlusion model. These results indicate that chronic BCCAO and, by inference, oligemia have a transient effect on the neocortex and a long-lasting effect on white matter structures.     

APETX2对氯化锂-匹鲁卡品诱导痫性发作大鼠的影响及机制探究

目的 探索APETX2对氯化锂-匹鲁卡品诱导痫性发作大鼠的行为学影响及可能的机制。方法 成年雄性SPF级SD大鼠18只,侧脑室置管后随机分为:癫痫组(9只)、APETx2组(9只),癫痫造模后观察2组癫痫大发作潜伏期及发作强度; APETx2处理原代培养海马神经元,动态观察其对钙成像的影响。结果 APETx2组的SD大鼠癫痫潜伏期延长,大发作程度减轻; APETx2处理原代培养海马神经元钙内流下降。结论 APETx2可抑制氯化锂-匹鲁卡品诱导SD大鼠痫性发作,减少酸诱导海马神经元钙离子浓度增加可能为机制之一。     

In vitro ischemia-induced intracellular Ca2+ elevation in cerebellar slices: a comparative study with the values found in hippocampal slices

Changes in levels of intracellular calcium ion ([Ca²⁺]_i) induced by in vitro ischemic conditions in gerbil cerebellar and hippocampal slices were investigated using a calcium imaging system and electron microscopy. When the cerebellar slice was perfused with a glucose-free physiological medium equilibrated with a 95% N₂/5% CO₂ gas mixture (in vitro ischemic medium), a large [Ca²⁺]_i elevation was region-specifically induced in the molecular laver of the cerebellar cortex (a dendritic field of Purkinje cells). When the hippocampal slice was perfused with in vitro ischemic medium, a large [Ca²⁺]_i elevation was region-specifically induced in CA1 field of the hippocampal slices. Electron microscopic examinations showed that the large [Ca²⁺]_i elevations occurred in Purkinje cells and CA1 pyramidal neurons. To isolate Ca²⁺ release from intracellular Ca²⁺ store sites, the slices were perfused with Ca²⁺-free in vitro ischemic medium. the increases in [Ca²⁺]_i in both cerebellar and hippocampal slices were significantly lower than those observed in the slices perfused with the Ca²⁺-containing in vitro ischemic medium. However, the suppression of the [Ca²⁺]_i-elevation in the molecular layer of the cerebellar slices was smaller than that in the CA1 field of the hippocampal slices. These results reinforce the hypothesis that calcium plays a pivotal role in the development of ischemia-induced neuronal death, and suggest that Ca²⁺ release from intracellular Ca²⁺ store sites may play an important role in the ischemia-induced [Ca²⁺]_i elevation in Purkinje cells.     

The p53-independent nuclear translocation of cyclin G1 in degenerating neurons by ischemic and traumatic insults

Cyclin G1 (CG1) was identified as a p53-transactivated target gene, and yet its physiological and pathological roles have been unclear. Here, we demonstrate that CG1 is translocated from cytoplasm to the nuclei of neurons in response to variety of injuries. In the normal matured rodent brain, CG1 immunoreactivity was hardly observed; however, some brain injuries exhibited intense CG1 immunoreactivity in the nuclei of the damaged neurons. Transient common carotid artery occlusion (CCAO) in the gerbil showed strong CG1-like immunoreactivity in the hippocampal CA1 neurons, and permanent middle cerebral artery occlusion (MCAO) in the mouse showed strong CG1-like immunoreactivity in the nuclei of neurons located in the ischemic brain regions. TUNEL staining did not exactly overlap with the CG1-positive cells, but overlapped highly with Fluoro-Jade B staining, a degeneration marker. Brain trauma caused by knife cut, cold injury, and kinate injection also showed CG1 accumulation in the neuronal nuclei located near the injury site. These observations were obtained in p53-deficient mice as well, suggesting that the accumulation of CG1 in the injured neurons is p53-independent. A similar nuclear translocation of endogenous CG1 was confirmed in a primary culture of cortical neurons when a toxic level of N-methyl-D-aspartate (NMDA) was applied. These results demonstrate that nuclear translocation of CG1 from cytoplasmic region occurs in damaged and degenerating neurons in a p53-independent manner, and the CG1 nuclear staining could be a good marker for the neurons received fatal damages.     

Effect of ethanol on lactic acid production by exercised normal muscle

Several studies have suggested that muscle lactic acid production is defective in alcoholic myopathy. However, normal controls have not been evaluated. To study the effects of ethanol on the production of lactic acid in normal exercised muscle, oral ethanol (1 g per kg body weight, 20% v/v) was given to eight nonalcoholic subjects. Forearm ischemic exercise with a total work load of 7.2 kg-m/min was performed just before and 90 min after ingestion of ethanol. At the time of exercise, the serum ethanol level was 95.7 ± 15.1 mg% (mean ± SD). Resting serum lactic acid was significantly higher after ingestion of ethanol (15.7 ± 4.5 mg%) than before (8.5 ± 4.7 mg%). Lactic acid generation with ischemic exercise was significantly lower after ethanol ingestion. The mean peak serum lactic acid level (expressed as percentage elevation above resting) was 330.5 ± 118.2% before and 127.9 ± 75.1% after (p <.05). These results indicate that a single oral dose of ethanol decreases lactic acid production by exercised muscle in normal subjects.