The effect of MK-801 on cortical spreading depression in the penumbral zone following focal ischaemia in the rat.

Cortical spreading depression (CSD) is a transient depression of neuronal activity that spreads across the cortical surface. In the present studies, we have investigated CSD activity in the penumbral zone following permanent middle cerebral artery (MCA) occlusion in the rat (n = 16/group), using double-barreled Ca(2+)-sensitive microelectrodes. Measurements of CSD activity were made for 3 h in each animal. During this time, a varying number of spontaneous CSDs were seen in the control group (total was 30, with a range of 0-7/rat). These CSDs were of varying duration: "small" (approximately 1 min) and "big" (5-45 min) CSDs. During a CSD, the extracellular [Ca2+] decreased to 0.11 +/- 0.07 mM (mean +/- SD). After 3 h, the extracellular [Ca2+] in the cortex (penumbral zone) was either normal (10/16 rats) or lowered to 0.5 mM (2/16 rats) or to 0.1 mM (4/16 rats). In the caudate nucleus (ischaemic core area), all rats had an extracellular [Ca2+] of approximately 0.1 mM when measured after the 3 h recording period. Neuropathological evaluation of the brains of the animals, which had been allowed to survive for 24 h after MCA occlusion, revealed ischaemic damage in the dorsolateral cortex and caudate nucleus. Administration of the noncompetitive NMDA antagonist, MK-801 (3 mg/kg i.p.), 30 min after MCA occlusion resulted in 24 and 29% reductions in the volume of hemispheric and cortical damage, respectively, which was highly significant (p less than 0.0001); no protection was seen against caudate damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical changes and gene expression following traumatic brain injury: Role of spreading depression

The effects of spreading depression-like DC depolarizations on biochemical changes and gene expression were examined following trau-matic neocortical lesions, as induced by transcranial cold injury. The surrounding of traumatic cold lesions was characterized by increased glu-cose and lactate contents, without major disturbances of protein synthesis or energy state. A transient pH decrease by 0.4 units was noticed 1 h post-injury, which shifted towards alkaline values by 3 h. These changes were similar in animals with spontaneous spreading depression-like DC shifts (n = 14) and those without spreading depressions (n = 7), but there was a marked difference in the gene response. In injured animals without SD, only a short-lasting response of c-fos, junB, c-jun and MKP-1 mRNAs as well as c-Fos protein was bilaterally found in the piri-form cortex, and - with ipsilateral dominance - the dentate gyrus and hippocampal CA3/4 fields at 1 h after lesioning. In injure d animals with spreading depressions, on the contrary, a strong elevation was seen in layers II-IV and VI of the injury-remote ipsilateral cerebral cortex, which persisted over as long as 6 h. The expression of c-fos, junB and MKP-1 mRNAs was closely related to the time interval between the last spreading depression and the end of the experiments. Levels were highest shortly after transient DC shifts, and decreased thereafter mono-exponentially with half-lives of 48, 75 and 58 min for c-fos, junB and MKP-1 mRNAs, respectively. Thus, spreading depression is a prominent factor influencing the trauma-related gene response, but - in contrast to focal ischemia - it does not aggravate the metabolic dysfunction.     

Biochemical changes and gene expression following traumatic brain injury: Role of spreading depression

The effects of spreading depression-like DC depolarizations on biochemical changes and gene expression were examined following trau-matic neocortical lesions, as induced by transcranial cold injury. The surrounding of traumatic cold lesions was characterized by increased glu-cose and lactate contents, without major disturbances of protein synthesis or energy state. A transient pH decrease by 0.4 units was noticed 1 h post-injury, which shifted towards alkaline values by 3 h. These changes were similar in animals with spontaneous spreading depression-like DC shifts (n = 14) and those without spreading depressions (n = 7), but there was a marked difference in the gene response. In injured animals without SD, only a short-lasting response of c-fos, junB, c-jun and MKP-1 mRNAs as well as c-Fos protein was bilaterally found in the piri-form cortex, and - with ipsilateral dominance - the dentate gyrus and hippocampal CA3/4 fields at 1 h after lesioning. In injure d animals with spreading depressions, on the contrary, a strong elevation was seen in layers II-IV and VI of the injury-remote ipsilateral cerebral cortex, which persisted over as long as 6 h. The expression of c-fos, junB and MKP-1 mRNAs was closely related to the time interval between the last spreading depression and the end of the experiments. Levels were highest shortly after transient DC shifts, and decreased thereafter mono-exponentially with half-lives of 48, 75 and 58 min for c-fos, junB and MKP-1 mRNAs, respectively. Thus, spreading depression is a prominent factor influencing the trauma-related gene response, but - in contrast to focal ischemia - it does not aggravate the metabolic dysfunction.     

苯甲酸利扎曲普坦对皮质扩布性抑制及中脑导水管周围灰质c-Fos表达的影响

目的 观察苯甲酸利扎曲普坦对大鼠皮质扩布性抑制(cortical spreading depression,CSD)和CSD引起中脑导水管周围灰质(periaqueduetal grey,PAG)内c-Fos表达的影响.方法 实验选用的SD大鼠随机分为3组,A组:氯化钾刺激组;B组:氯化钾刺激+苯甲酸利扎曲普坦组;C组:氯化钠刺激组.对各组大鼠使用氯化钾(或氯化钠)刺激,以观察CSD出现的个数和波幅;第1次刺激2 h后使用免疫组织化学法检测各组PAG内不同层面c-Fos阳性神经元.结果 C组未记录到CSI).A组CSD个数(10.70±3.23)较B组(6.10±2.56)显著增多(t=-3.528,P<0.01);A组CSD波幅[17.33(95%CI 11.45～23.11)mV]较B组[11.82(95%CI 9.24～14.70)mV]显著增加(Z=-4.360,P<0.01).A组各层面c-Fos阳性细胞数较B、C组显著增多(P<0.05).结论 苯甲酸利扎曲普坦对CSD有抑制作用,可使CSD引起PAG内神经元兴奋作用减弱.     

Noradrenergic agonists and antagonists influence migration of cortical spreading depression in rat-a possible mechanism of migraine prophylaxis and prevention of postischemic neuronal damage.

Cortical spreading depression (CSD) is thought to be a neuronal mechanism that expands the penumbra zone after focal brain ischemia and that causes migraine aura. Both adrenergic agonists and antagonists significantly influence the size of the penumbra zone and decline the frequency of migraine. To study whether these compounds act by influencing CSD, we applied different drugs topically to an area of the exposed cortex of anesthetized adult rats and observed the migration of CSD-related DC potential deflections across the treated area. The adrenergic agonist norepinephrine (1 mmol/L) and the alpha(2)-agonist clonidine (0.56 mmol/L) blocked reversibly the migration of CSD. The beta-blocker propranolol (250 micromol/L to 1 mmol/L) dose-dependently diminished migration velocity or even blocked migration of CSD. The CSD blockade by the alpha(2)-antagonist yohimbine (1.75 mmol/L) was because of its action on inhibitory 5-HT(1A) receptors. None of the substances in the concentrations used had influence on regional cerebral blood flow or on systemic arterial blood pressure. The data suggest that the interference of these compounds with CSD may contribute to their beneficial therapeutic effect. The effect of beta-receptor antagonists in human migraine needs further exploration, since these drugs also work in migraine without aura.     

脑皮层扩散性抑制对大鼠蛛网膜下腔出血后皮层微循环的影响

目的 探讨脑皮层扩散性抑制（CSD）对蛛网膜下腔出血（SAH）模型小鼠脑皮层微循环的影响。方法 将30只雄性成年C57小鼠随机分为正常组（n=10,制作透明颅窗、诱导窗加KCl）、对照组（n=10,制作透明颅窗及SAH模型、诱导窗加NaCl）和观察组（n=10,制作透明颅窗及SAH模型、诱导窗滴KCl）。采用颈动脉内穿刺法构建SAH模型。使用激光散斑血流成像和内源信号成像技术记录脑皮层微循环和CSD。结果 观察组、正常组出现CSD,对照组未产生CSD。观察组CSD后脑皮层血流明显下降（P<0.05）、血管明显收缩（P<0.05）,其中CSD后5 d较显著。正常组CSD后血管明显舒张（P<0.05）、皮层血流明显增加（P<0.05）。对照组无明显变化（P>0.05）。结论 KCl能诱导产生CSD;CSD能使正常小鼠脑皮层血流增加和血管扩张;CSD使SAH后脑皮层血流减少和血管收缩。     

The impact of anesthetics and hyperoxia on cortical spreading depression

Cortical spreading depression (CSD), a transient neuronal and glial depolarization that propagates slowly across the cerebral cortex, is the putative electrophysiological event underlying migraine aura. It negatively impacts tissue injury during stroke, cerebral contusion and intracranial hemorrhage. Susceptibility to CSD has been assessed in several experimental animal models in vivo, such as after topical KCl application or cathodal stimulation. Various combinations of anesthetics and ambient conditions have been used by different laboratories making comparisons problematic and differences in data difficult to reconcile. We systematically studied CSD susceptibility comparing commonly used experimental anesthetics (isoflurane, α-chloralose, and urethane) with or without N₂O or normobaric hyperoxia (100% O₂ inhalation). The frequency of evoked CSDs, and their propagation speed, duration, and amplitude were recorded during 2 h topical KCl (1 M) application. We found that N₂O reduced CSD frequency when combined with isoflurane or urethane, but not α-chloralose; N₂O also decreased CSD propagation speed and duration. Urethane anesthesia was associated with the highest CSD frequency that was comparable to pentobarbital. Inhalation of 100% O₂ did not alter CSD frequency, propagation speed or duration in combination with any of the anesthetics tested. Our data show anesthetic modulation of CSD susceptibility in an experimental model of human disease, underscoring the importance of proper study design for hypothesis testing as well as for comparing results between studies.     

Differential effects of intrafrontocortical microinjections of dopamine agonists and antagonists on circling behavior of rats

Asymmetric posturing and circling behavior resulting from acute unilateral manipulation of central dopamine have been used to assess this neurotransmitter's contribution to motor control. Although providing extensive evidence for the involvement of mesolimbic and nigrostriatal dopamine in motor activity, this approach has not been used to study the mesocortical system. We now report circling behaviour following acute manipulation of frontal cortical dopamine. Unilateral microinjections of the agonists, (+)-amphetamine (12 and 25 micrograms in 1.0 microliter) and LY 141865 (12 micrograms in 1.0 microliter) resulted in contraversive circling. Conversely, unilateral intrafrontocortical microinjections of the antagonist, metoclopramide (25 and 100 micrograms in 1.0 microliter) resulted in ipsiversive circling in amphetamine (1.5 mg/kg, i.p.) pretreated rats. Lower central doses of each drug and vehicle injections had no significant effect. These results provide evidence for an excitatory influence of mesocortical dopamine on motor control. This finding may implicate frontal cortical dopamine in the extrapyramidal motoric side effects of chronic neuroleptic treatment which previously have been attributed to dopamine function in subcortical areas.     

Differential modulatory effects of norepinephrine on synaptically driven responses of layer V barrel field cortical neurons

The effects of norepinephrine (NE) and the alpha-1 agonist phenylephrine (PE) on synaptically evoked responses of electrophysiologically identified pyramidal neurons in layer V of rat somatosensory cortex were studied in brain slices using intracellular recording techniques. When added to the bathing medium NE (10 microM) tended to increase the synaptic responsiveness of regular spiking neurons and decrease the responsiveness of intrinsic burst neurons. NE had mixed effects on layer V cells which were characterized as intermediate types between regular spiking and intrinsic burst neurons. PE exerted a similar spectrum of actions on layer V cortical neurons. For both adrenergic agents the greatest facilitating effect was observed on responses to low intensity synaptic stimulation. These results suggest that NE exerts different modulatory actions on different electrophysiologically-defined classes of layer V sensory cortical neurons.     

Differential effects of CB1 neutral antagonists and inverse agonists on gastrointestinal motility in mice

Background Cannabinoid type 1 (CB₁) receptors are involved in the regulation of gastrointestinal (GI) motility and secretion. Our aim was to characterize the roles of the CB₁ receptor on GI motility and secretion in vitro and in vivo by using different classes of CB₁ receptor antagonists. Methods Immunohistochemistry was used to examine the localization of CB₁ receptor in the mouse ileum and colon. Organ bath experiments on mouse ileum and in vivo motility testing comprising upper GI transit, colonic expulsion, and whole gut transit were performed to characterize the effects of the inverse agonist/antagonist AM251 and the neutral antagonist AM4113. As a marker of secretory function we measured short circuit current in vitro using Ussing chambers and stool fluid content in vivo in mouse colon. We also assessed colonic epithelial permeability in vitro using FITC‐labeled inulin. Key Results In vivo, the inverse agonist AM251 increased upper GI transit and whole gut transit, but it had no effect on colonic expulsion. By contrast, the neutral antagonist AM4113 increased upper GI transit, but unexpectedly reduced both colonic expulsion and whole gut transit at high, but not lower doses. Conclusions & Inferences Cannabinoid type 1 receptors regulate small intestinal and colonic motility, but not GI secretion under physiological conditions. Cannabinoid type 1 inverse agonists and CB₁ neutral antagonists have different effects on intestinal motility. The ability of the neutral antagonist not to affect whole gut transit may be important for the future development of CB₁ receptor antagonists as therapeutic agents.     

黄芩苷、栀子苷及其配伍治疗局灶性脑缺血的海马基因表达：一项基因网络功能分析

The compound traditional Chinese medicine Qingkailing,which is an ingredient used to treat cerebral ischemia,has been limited to studies concerning single genes or single pathways.Interactions and pharmacological mechanisms of the compound ingredients(baicalin and jasminoidin) remain poorly understood.In the present study,baicalin and jasminoidin,as well as the combination,were used to separately treat mouse models of cerebral ischemia.cDNA microarray analyses of 374 cerebral ischemia-related genes were utilized to determine changes in gene-expression profiles.Arraytrack 3.40 and Ingenuity Pathway Analysis(IPA) databases were utilized to analyze changes in gene molecular functions and network path functions.Baicalin or jasminoidin alone effectively reduced infarct area,and the combination resulted in significantly better outcomes.IPA showed inhibited cell apoptosis in the baicalin group and Ca2+ channel regulation in the jasminoidin group.The combination of baicalin and jasminoidin activated HTR3A and F5 expression,regulated Ca2+ channels,activated kappa light polypeptide gene enhancer inhibitor IKBKG in B cells to control IkB kinase/nuclear factor-kB cascade,suppressed activation of inflammatory cytokine interleukin-6 receptors and activated transduction of guanine-nucleotide-binding protein(G protein) signal.Results suggested that the combination of baicalin and jasminoidin resulted in similar molecular mechanisms to baicalin and jasminoidin alone.However,novel pharmacological actions of compatibility were detected,demonstrating significant protection against cerebral ischemia.     

Induction of tolerance to focal ischemia in rat brain: dissociation between cortical lesioning and spreading depression.

Cortical application of KCl has previously been shown to induce tolerance to a subsequent episode of cerebral ischemia. KCl triggers recurrent episodes of cortical spreading depression and produces a small lesion at the cortical application site. To determine whether a cortical lesion alone is sufficient to induce tolerance to ischemia, the authors used 5-mol/L NaCl to precondition rat brain 3 days before permanent occlusion of the middle cerebral artery. NaCl produced a small lesion at the application site without evoking cortical spreading depression. Preconditioning with 5-mol/L NaCl significantly attenuated the decrease in CBF after middle cerebral artery occlusion and reduced the volume of cortical infarction by 35%. The results show that a small cortical lesion, by itself, is sufficient to induce tolerance to ischemia.     

Inhibition of NR2A reduces calcitonin gene-related peptide gene expression induced by cortical spreading depression in rat amygdala

Despite robust evidence on the role of calcitonin gene-related peptide (CGRP) in migraine via both central and peripheral actions, relatively less is known about how CGRP in the limbic system is involved in migraine progression. This study investigated whether CGRP production machinery exists in the two key limbic regions including hippocampus and amygdala using cortical spreading depression (CSD) as a model of migraine and whether such alteration by CSD is sensitive to N-methyl-d-aspartate (NMDA) receptor regulation in rats. A single or repetitive CSD was induced by topical application of KCl and monitored using electrophysiological methods. The NR2A-containing NMDA receptor antagonist, NVP-AAM077, or its vehicle, was perfused into the contralateral cerebroventricular ventricle of rat. Quantitative PCR was used to measure CGRP mRNA levels in the ipsilateral and contralateral hippocampus and amygdala after CSD events and compared to respective sham treatments. The results showed that neither a single CSD nor repetitive CSD affected CGRP mRNA levels in both the contralateral and ipsilateral hippocampus at 24 h post CSD induction. Differently, significant elevation of CGRP gene expression was observed in the ipsilateral amygdala at 24 h post multiple CSD, but not contralateral side, and not post-single CSD. Further results showed that the CSD-induced CGRP gene expression in the amygdala was markedly reduced by NVP-AAM077 and this reduction corresponded to a reduced cortical susceptibility to CSD in rats. We conclude that repetitive CSD events induce CGRP gene expression in amygdala, which is sensitive to NR2A regulation.     

Diffusion- and perfusion-weighted magnetic resonance imaging of focal cerebral ischemia and cortical spreading depression under conditions of mild hypothermia

In a model of experimental stroke, we characterize the effects of mild hypothermia, an effective neuroprotectant, on fluid shifts, cerebral perfusion and spreading depression (SD) using diffusion- (DWI) and perfusion-weighted MRI (PWI). Twenty-two rats underwent 2 h of middle cerebral artery (MCA) occlusion and were either kept normothermic or rendered mildly hypothermic shortly after MCA occlusion for 2 h. DWI images were obtained 0.5, 2 and 24 h after MCA occlusion, and maps of the apparent diffusion coefficient (ADC) were generated. SD-like transient ADC decreases were also detected using DWI in animals subjected to topical KCl application (n=4) and ischemia (n=6). Mild hypothermia significantly inhibited DWI lesion growth early after the onset of ischemia as well as 24 h later, and improved recovery of striatal ADC by 24 h. Mild hypothermia prolonged SD-like ADC transients and further decreased the ADC following KCl application and immediately after MCA occlusion. Cerebral perfusion, however, was not affected by temperature changes. We conclude that mild hypothermia is neuroprotective and suppresses infarct growth early after the onset of ischemia, with better ADC recovery. The ADC decrease during SD was greater during mild hypothermia, and suggests that the source of the ADC is more complex than previously believed.     

Prolonged induction of neuronal NOS expression and activity following cortical spreading depression (SD): implications for SD- and NO-mediated neuroprotection

Cortical spreading depression (CSD) is associated with various short- and long-term physiological and neurochemical changes and has been shown to confer an increased susceptibility to accompanying ischemic injury or provide protection against a subsequent experimental ischemia. Nitric oxide is involved in the processes of ischemic injury and under certain conditions mediates cellular protection. To investigate the possibility that CSD-induced alterations in nitric oxide synthase (NOS) expression and activity occur and might be associated with the time-dependent enhancement or prevention by CSD of ischemic damage, this study examined the spatiotemporal changes in nNOS expression and activity in cerebral cortex following CSD. Anesthetized rats had unilateral CSD induced by a 10-min topical application of KCl and were killed at various times thereafter. CSD increased both nNOS mRNA and protein levels throughout layers II-III of cortex. nNOS mRNA in the affected neocortex was significantly increased by 30-90% at 2, 7, and 14 days (P < or = 0.05) compared with contralateral levels, but was not significantly above control values at 1-6 h, 1 day, and 28 days after CSD induction. Levels of [3H]-L-N(G)-nitroarginine binding to NOS were increased by 40-170% 7, 14, and 28 days (P < or = 0.01) after CSD in a similar, but delayed, profile to nNOS mRNA. Levels of nNOS-immunoreactivity were also increased in both neurons and astrocytes of ipsilateral cortex 7 and 14 days after CSD--confirmed by double-immunofluorescence localization. Ex vivo NOS activity in layers I-III of ipsilateral cortex was also increased by 30-50% (P < or = 0.01) at 7 and 14 days after CSD, times coincident with reported maximal ischemic protection. These results demonstrate that nNOS is up-regulated by cellular depolarization/depression occurring during CSD, or by resultant stimuli and suggest that "CSD-conditioned" cortex may be capable of producing appropriate levels of NO to mediate or contribute to protective/adaptive responses to subsequent physical ischemic injury.     

Differential inhibitory effects of platelet glycoprotein IIb/IIIa antagonists on aggregation induced by procoagulant agonists

INTRODUCTION: In addition to mediating the final common pathway of aggregation, the glycoprotein (GP) IIb/IIIa receptor participates in the activation of coagulation on the platelet surface. High-affinity conformation of GP IIb/IIIa in response to collagen-induced inside-out signalling seems to be mediated by GP VI(-FcRgamma) and reinforced by release of soluble mediators. METHODS: We assessed the effects of the three currently available GP IIb/IIIa antagonists--abciximab, tirofiban and eptifibatide--on platelet aggregation induced by various procoagulant and GP VI-related agonists, i.e. collagen-related peptide (CRP), convulxin and collagen fibrils, in PPACK-anticoagulated platelet-rich plasma. RESULTS: At concentrations that equally inhibited 80% of ADP-induced maximal aggregation abciximab-inhibited GP VI-mediated platelet responses to CRP or convulxin significantly more than the low-molecular-weight antagonists (CRP: abciximab 75+/-18%, tirofiban 41+/-7% and eptifibatide 41+/-6%; convulxin: abciximab 90+/-6%, tirofiban 64+/-20%, eptifibatide 61+/-14%, p<0.01 for all). In contrast, aggregation induced by collagen was equally abolished with all antagonists under the similar conditions. During CRP- or convulxin-triggered platelet activation, inhibition of fibrin polymerisation with GPRP potentiated the antiaggregatory effects of tirofiban and eptifibatide to reach that of abciximab. GPRP as such did not affect platelet aggregation. CONCLUSIONS: GP IIb/IIIa antagonists exhibit distinct inhibition profiles in platelet aggregation, depending on fibrin polymerization and calcium. Specifically, the ability of procoagulant platelet agonists to expose pre-activated and ligand-bound GP IIb/IIIa from the internal pool seems important.     

Region-specific immediate-early gene expression following the administration of corticotropin-releasing hormone in virgin and lactating rats

Central administration of corticotropin-releasing hormone (CRH) induces immediate-early gene (IEG) expression (c-fos and NGFI-B) in forebrain structures in a pattern similar to that observed following restraint stress. Lactating rats display modified neuroendocrine and behavioural responses to stress which have been hypothesized to be at least partially mediated through changes within the circuitry converging on the PVN, including CRH activated pathways. Quantitative measures of regional expression of c-fos and NGFI-B mRNA representative of two classical intracellular pathways, were used to define modification of the circuitry involved in the altered response to central CRH in the lactating female. Compared to saline controls, virgin female rats injected with 5 μg CRH i.c.v. displayed significantly increased immediate-early gene expression in the hypothalamic paraventricular nucleus (PVN), arcuate nucleus, lateral septum, bed nucleus of the stria terminalis, central, medial and cortical nuclei of the amygdala, and all subfields of the hippocampal formation. In lactating rats treated with CRH there was a significant increase in c-fos gene expression in the CeA and in the hippocampal subfields CA1, CA4 and dentate gyrus but not in the other areas examined. The i.c.v. administration of CRH significantly increased NGFI-B expression in the PVN, arcuate nucleus, medial amygdala and all hippocampal subfields of virgin rats. Lactating rats treated with CRH failed to show a significant increase in NGFI-B expression in the PVN, median eminence, arcuate nucleus, medial amygdala, CA2 and CA3 subfields of the hippocampus. These results further suggest that changes in specific neural circuits might at least partially underlie the modified responses to CRH and perhaps to stress in the lactating female.     

Differential effects of cortical speading depression on epileptic foci induced by various convulsants.

Interaction between epileptic foci and spreading depression (SD) was studied in the cerebral cortex of rats anesthetized with Nembutal. At comparable discharge rates, picrotoxin and penicillin caused complete and partial SD blockade respectively, strychnine and Metrazol were ineffective and Aldactone facilitated SD. Conversely, the duration of SD-induced blockade of epileptic activity was maximal for Aldactone and minimal for picrotoxin. Treatment of the picrotoxin focus with tetrodotoxin (10(-4)M) reduced the discharge rate and reinstated SD propagation into the focus. [K+]e measured with ion-selective K+ electrodes 1 mm below the cortical surface increased to 8 mM in penicillin foci blocking SD and remained below 5 mM in Aldactone foci. It is concluded that the differential effect of various convulsants on SD propagation depends on the potassium concentration in the depth of the focus rather than on the discharge rate or on the mechanism of the the epileptogenic effect.