Cortical spreading depression aggravates early brain injury in a mouse model of subarachnoid hemorrhage

Cortical spreading depression (CSD) has been observed during the early phase of subarachnoid hemorrhage (SAH). However, the effect of CSD on the cerebral blood flow (CBF) and cerebral oxyhemoglobin (CHbO) during the early phase of SAH has not yet been assessed directly. We, therefore, used laser speckle imaging and optical intrinsic sinal imaging to record CBF and CHbO during CSD and cerebral cortex perfusion (CCP) at 24 hours after CSD in a mouse model of SAH. SAH was induced by blood injection into the prechiasmatic cistern. When CSD occurred, the change trend of CBF and CHbO in Sham group and SAH group was the same, but ischemia and hypoxia in SAH group was more significant. At 24 hours after SAH, the CCP of CSD group was lower than that of no CSD group, and the neurological function score of CSD group was lower. We conclude that induction of CSD further aggravates cerebral ischemia and worsens neurological dysfunction in the early stage of experimental SAH. Our study underscores the consequence of CSD in the development of early brain injury after SAH.     

大鼠局灶性脑缺血后KCl诱导皮层扩散性抑制的体光学成像

采用线栓法制备大鼠大脑中动脉栓塞(middlecerebralarteryocclusion,MCAO)模型,在额叶皮层用KCl诱导产生皮层扩散性抑制(corticalspreadingdepression,CSD)。MCAO4h后,利用550nm内源信号光学成像(opticalintrin-sicsignalimaging,OISI)监测局灶性脑缺血后大鼠顶-枕叶皮层内源光信号变化。成像1h内观测到一系列诱导CSD波(14±3次),CSD波局限于顶-枕叶皮层中央区域扩展,以光强的显著下降为特征;而旁侧区域光强无明显改变,不具备CSD波特征,表明CSD波未传播到该区域。随后TTC染色证明上述旁侧区域已经梗死。实验表明:MCAO后4h,皮层区域旁侧部分会梗死;CSD波的OIS变化可用来区分缺血梗死区和外周供血较为完整区域(未梗死区)。     

Physiological studies of cortical spreading depression

Cortical spreading depression (CSD) produces propagating waves of transient neuronal hyperexcitability followed by depression. CSD is initiated by K+ release following neuronal firing or electrical, mechanical or chemical stimuli. A triphasic (30-50 s) cortical potential transient accompanies localized transmembrane redistributions of K+, glutamate, Ca2+, Na+, Cl- and H+. Accumulated K+ in the restricted interstitial space can cause both further neuronal depolarisation and inward movement of K+ into astrocytes that buffers this increased extracellular K+ concentration ([K+])o. However, astrocyte interconnections may then propagate the CSD wave by K+ liberation through an opening of remote K+ channels by volume, Ca2+ or N-methyl-D-aspartate receptor activation. Changes in cerebral blood volume and in apparent water diffusion co-efficient (ADC) accompanying CSD were first studied using magnetic resonance imaging (MRI) in whole lissencephalic brains. Diffusion-weighted echoplanar imaging in gyrencephalic brains went on to demonstrate CSD features that paralleled classical migraine aura. The ADC activity persisted minutes/hours post KCl stimulus. Pixelwise analyses distinguished single primary events and multiple, spatially restricted, slower propagating, secondary events whose detailed features varied with the nature of the originating stimulus. These ADC changes varied reciprocally with T2*-weighted (i.e. referring to spin-spin relaxation times) waveforms reflecting local blood flow. There followed prolonged decreases in cerebral blood flow culminating in late cerebrovascular changes blocked by the antimigraine agent sumatriptan. CSD phenomena have possible translational significance for human migraine aura and other cerebral pathologies such as the peri-infarct depolarisation events that follow ischaemia and brain injury.     

一种新的具有多肽性质的β-肾上腺素能受体激动剂

从蚯蚓中分离出一种能增强离体豚鼠心耳收缩作用的物质。用分子筛层析,阴离子交换层析及高效液相色谱法进行了纯化,进一步分析表明,它是分子量约为30,000daltons的多肽(称为蚯这多肽制剂),可被肾上腺素能受体阻断剂——心得安对抗,能竞争性抑制~3H标记心得舒与大鼠心肌膜制剂内β-肾上腺素能受体的结合,为一种新的β-肾上腺素能受体激动剂。     

Spreading depression-induced preconditioning in the mouse cortex: differential changes in the protein expression of ionotropic nicotinic acetylcholine and glutamate receptors

Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in alpha-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12-15-fold increase in alpha7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the alpha4 nAChR subunit was not altered. These data point to the alpha7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating alpha7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).     

Cortical Spreading Depolarization (CSD) Recorded from Intact Skin,from Surface of Dura Mater or Cortex: Comparison with Intracortical Recordings in the Neocortex of Adult Rats

In cerebral cortex of anesthetized rats single waves of spreading depolarization (CSD) were elicited by needle prick. CSD-related changes of DC (direct current) potentials were either recorded from the intact skin or together with concomitant changes of potassium concentration with K⁺-selective microelectrodes simultaneously at the surface of the dura mater or of the cortex ([K⁺]_s) and in the extracellular space at a cortical depth of 1200 µm. At the intact skin CSD-related DC-shifts had amplitudes of less than 1 mV and had only in a minority of cases the typical CSD-like shape. In the majority these DC-shifts rose and recovered very slowly and were difficult to identify without further indicators. At dura surface CSD-related DC shifts were significantly smaller and rose and recovered slower than intracortically recorded CSD. Concomitant increases in [K⁺]_s were delayed and reached maximal values of about 5 mM from a baseline of 3 mM. They rose and recovered slower than simultaneously recorded intracortical changes in extracellular potassium concentration ([K⁺]_e) that were up to 65 mM. The results suggest that extracellular potassium during CSD is diffusing through the subarachnoid space and across the dura mater. In a few cases CSD was either absent at the dura or at a depth of 1200 µm. Even full blown CSDs in this cortical depth could remain without concomitant deflections at the dura. Our data confirmed in principle the possibility of non-invasive recordings of CSD-related DC-shifts. For a use in clinical routine sensitivity and specificity will have to be improved.

     

Characteristics of the catecholamine and histamine receptor sites mediating accumulation of cyclic adenosine 3',5'-monophosphate in guinea pig brain

—Five areas of guinea pig brain were examined to determine the properties of the receptor sites mediating increases in [³H]adenosine 3′,5′-monophosphate (cyclic AMP). Both epinephrine and histamine were effective in causing increases in cyclic AMP in slices derived from cerebral cortex, hippocampus or amygdala, but not in diencephalon or brainstem. Stimulation of slices of cerebral cortex by either epinephrine or histamine resulted in a small, but reproducible, decrease in specific radioactivity of the [³H]-cyclic AMP produced, as did stimulation of the hippocampus by epinephrine. The catecholamine receptor was an α-adrenergic receptor in all three areas where epinephrine was effective; α-adrenergic stimulation, but not β-adrenergic stimulation, increased levels of [³H]-cyclic AMP. Furthermore, α-, but not β-adrenergic blocking agents, prevented the epinephrine- induced increase of both [³H]- and total cyclic AMP in cerebral cortex and hippocampus. Only antihistaminic agents were capable of antagonizing the histamine-induced increase of both [³H]- and total cyclic AMP in these two brain areas. The catecholamine receptor in the amygdala also appeared to be an α-adrenergic receptor. The effects of histamine and epinephrine together were far greater than the sum of effects of either hormone alone in both cerebral cortex and hippocampus.     

Insulin Stimulates Phosphatidylinositol 3-Kinase Activity in Rat Neuronal Primary Cultures

Abstract: The effect of cortical spreading depression (CSD) on cerebral protein synthesis (CPS) was examined. CSD was evoked in normal rats with KCI, and CPS was measured autoradiographically with [1-¹⁴C]leucine. Average rates (mean ± SD) of CPS in layers I-IV of cortex decreased significantly from 10.7 ± 0.6 (sham-operated controls; n = 4) to 6.7 ± 0.7 nmol/g/min (n = 4; p < 0.01) and in layers V-VI from 10.9 ± 0.5 to 9.4 ± 0.4 nmol/g/min (p < 0.05) during 60 min of repetitive CSD. Spreading depression did not affect CPS rates in other subcortical brain regions. These results indicate that KCl-evoked CSD induces inhibition but not suppression of cortical protein synthesis.     

CYCLIC AMP IN THE RAT CEREBRAL CORTEX AFTER ACTIVATION OF NORADRENALINE NEURONS OF THE LOCUS COERULEUS

The levels of cyclic AMP in the rat brain were studied in vivo following destruction or stimulation of the noradrenergic pathway originating in the locus coeruleus. After chronic lesion of the locus coeruleus no alterations in cyclic AMP content were found. Electrical stimulation of the locus coeruleus produced an elevation of cyclic AMP in the cerebral cortex of chloral hydrate anaesthetized rats of 30%. Maximal increases were found after 15–60 s stimulation at a frequency of 30–100 Hz. This maximal response was slightly inhibited by phenoxybenzamine, an α-adrenergic blocking agent, and by the β-blocker propranolol. When the α and β blockers were administered together a highly significant decrease in cyclic AMP response was observed. Pretreatment of the rats with reserpinc +α methyl-p-tyrosine prevented the cyclic AMP response. In addition to the effect in the cerebral cortex, cyclic AMP-levels were also enhanced in the hippocampus, in the striatum and in the hypothalamus. These results suggest that the locus coeruleus regulates a small fraction of cerebral cyclic AMP levels, by both α- and β-adrenergic receptors.     

Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebal cortex plasma membranes

We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 +/- 1,469.2 microM-min in sham-treated (n = 8) to 569.0 +/- 707.8 microM-min in CSD-treated (n = 13) rats (p <0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down-regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post-CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down-regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down-regulation of these transporters may contribute to ischemic tolerance induced by CSD.     

Targeted tissue oxidation in the cerebral cortex induces local prolonged depolarization and cortical spreading depression in the rat brain

Spreading depression (SD) has been linked to several neurological disorders as epilepsy, migraine aura, trauma, and cerebral ischemia, which were also influenced by disorderliness of the brain redox homeostasis. To investigate whether local tissue oxidation directly induces SD, we oxidized a restricted local area of the rat cerebral cortex using photo-dynamic tissue oxidation (PDTO) technique and examined the cerebral blood flow (CBF) and direct current (DC) potential in and around the oxidized area. Intensive PDTO induced prolonged depolarization only in the photo-oxidized area, which led to global changes of CBF and DC potential: synchronous negative shifts of DC potential (with an amplitude of approximately 20 mV) and hyperperfusion of CBF occurred. The changes in DC potential and CBF spread at a rate of around 3mm/min beyond the oxidized area to the whole hemisphere of the cerebral cortex, indicating that intensive local oxidation induces SD in the rat brain.     

Variation of repetitive cortical spreading depression waves is related with relative refractory period: a computational study

Cortical spreading depression (CSD) is an important experimental model for diseases such as stroke, epilepsy and migraine. Previous observations indicated that the amplitude and velocity of the typical direct current potential shift during repetitive CSD waves were varying. The recovery state of the tissue was found related with the variation of successive CSD waves. A computational model in this paper aimed to investigate the role of relative refractory period of CSD. This model simulated that continuous injection of KCl solution induced repetitive CSD waves. The first CSD wave often had a larger amplitude and faster velocity than those of the succeeding secondary waves. The relative refractory period lasted much longer than the recovery of ions turbulence. If the induction interval was long enough for recovery, a series of CSD waves would have the same profile as the first one. In the relative refractory period, an early stimulation might lead to a late initiation of CSD, i.e., “haste makes waste”. The amplitude and velocity of CSD waves were found increasing with the initiation interval and asymptotic to those of the first CSD wave. This study verified that the propagation dynamics of CSD waves is modulated by the relative refractory period. It suggested that the refractory period is critical for preventing undesirable CSD waves.     

Cerebral Blood Flow Modulation by Basal Forebrain or Whisker Stimulation Can Occur Independently of Large Cytosolic Ca2+ Signaling in Astrocytes

We report that a brief electrical stimulation of the nucleus basalis of Meynert (NBM), the primary source of cholinergic projection to the cerebral cortex, induces a biphasic cerebral cortical blood flow (CBF) response in the somatosensory cortex of C57BL/6J mice. This CBF response, measured by laser Doppler flowmetry, was attenuated by the muscarinic type acetylcholine receptor antagonist atropine, suggesting a possible involvement of astrocytes in this type of CBF modulation. However, we find that IP3R2 knockout mice, which lack cytosolic Ca2+ surges in astrocytes, show similar CBF changes. Moreover, whisker stimulation resulted in similar degrees of CBF increase in IP3R2 knockout mice and the background strain C57BL/6J. Our results show that neural activity-driven CBF modulation could occur without large cytosolic increases of Ca2+ in astrocytes.     

Bilateral sensory disturbance after cortical spreading depression revealed by fluorescence imaging of voltage‐sensitive dye

Cortical spreading depression (CSD), a propagation wave of transient neuronal and glial depolarization followed by suppression of spontaneous brain activity, has been hypothesized to be the underlying mechanism of migraine aura and triggers the headache attack. Evidence from various animal models accumulates since its first discovery in 1944 and provides support for this hypothesis. In this paper, alterations of bilateral cortical responses are investigated in a mice migrainous model of CSD using voltage‐sensitive dye imaging under hindlimb and cortical stimulation. After CSD induction in the right hemisphere, bilateral sensory responses evoked by left hindlimb stimulation dramatically decreases, whereas right hindlimb stimulation can still activate bilateral responses with an increased response of the left hemisphere and a well‐preserved response of the right hemisphere. In addition, cortical neural excitability remains after CSD assessed by direct activation of the right hemisphere in spite of the sensory deficit under contralateral hindlimb stimulation. These results depict the sensory disturbance of bilateral hemispheres after CSD, which may be helpful in understanding how sensory disturbance occur during migraine aura.      

Cannabinoid modulation of electrically evoked pH and oxygen transients in the nucleus accumbens of awake rats

Cannabinoid receptors have been implicated in the regulation of blood flow in the cerebral vasculature. Because the nucleus accumbens (NAc) shows high levels of central cannabinoid receptor 1 (CB1) expression we examined the effects of cannabinoids on the local transient alkaline shifts and increases in extracellular oxygen induced by electrical stimulation of the medial forebrain bundle (MFB) in conscious animals. These changes result from increases in cerebral blood flow (CBF) and metabolism in the NAc that are evoked by the stimulation. Oxygen and pH changes were monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in the NAc of freely moving rats. Administration of the cannabinoid receptor agonist WIN55,212-2 potently inhibited extracellular oxygen and pH changes, an effect that was reversed and prevented by pre-treatment with the CB1 receptor antagonists SR141716A and AM251. The effects on pH following WIN55,212-2 were similar to those following nimodipine, a recognized vasodilator. When AM251 was injected alone, the amplitude of electrically evoked pH shifts was unaffected. Administration of AM404 and VDM11, endocannabinoid transport inhibitors, partially inhibited pH transients in a CB1 receptor-dependent manner. The present findings suggest that CB1 receptor activation modulates changes in two well-established indices of local blood flow and metabolism resulting from electrically evoked activation of ascending fibers. Although endogenous cannabinoid tone alone is not sufficient to modify these responses, uptake blockade demonstrates that the system has the potential to exert CB1-specific effects similar to those of full agonists.     

beta-adrenergic supersensitivity in rat brainstem during clonidine withdrawal

The clonidine withdrawal syndrome was studied in the rat by measuring β-adrenergic responses as isoproterenol stimulated cyclic 3′, 5′-Adenosine monophosphate accumulation in brain slices and β-adrenergic membrane receptors as [³H] dihydroalprenolol binding. Supersensitivity of cyclic AMP accumulation was evident in brain-stems of clonidine-treated animals 18 and 24 hours after the last dose, but not in cerebral cortex. In addition there was no indication of changes in either number or affinity of beta-receptors in brainstem. The similarity of these findings to changes in adenylate cyclase activity seen during opiate withdrawal is intriguing.     

Orexin A-induced anxiety-like behavior is mediated through GABA-ergic, α- and β-adrenergic neurotransmissions in mice

Orexins are hypothalamic neuropeptides, which are involved in several physiological functions of the central nervous system, including anxiety and stress. Several studies provide biochemical and behavioral evidence about the anxiogenic action of orexin A. However, we have little evidence about the underlying neuromodulation. Therefore, the aim of the present study was to investigate the involvement of neurotransmitters in the orexin A-induced anxiety-like behavior in elevated plus maze (EPM) test in mice. Accordingly, mice were pretreated with a non-selective muscarinic cholinergic antagonist, atropine; a γ-aminobutyric acid subunit A (GABA-A) receptor antagonist, bicuculline; a D2, D3, D4 dopamine receptor antagonist, haloperidol; a non-specific nitric oxide synthase (NOS) inhibitor, nitro-l-arginine; a nonselective α-adrenergic receptor antagonist, phenoxybenzamine and a β-adrenergic receptor antagonist, propranolol 30 min prior to the intracerebroventricular administration of orexin A. The EPM test started 30 min after the i.c.v. injection of the neuropeptide. Our results show that orexin A decreases significantly the time spent in the arms (open/open + closed) and this action is reversed by bicuculline, phenoxybenzamine and propranolol, but not by atropine, haloperidol or nitro-l-arginine. Our results provide evidence for the first time that the orexin A-induced anxiety-like behavior is mediated through GABA-A-ergic, α- and β-adrenergic neurotransmissions, whereas muscarinic cholinergic, dopaminergic and nitrergic neurotransmissions may not be implicated.     

Effect of aging on neurotransmitter receptor binding in rat and human brain.

β-Adrenergic and GABA receptor binding were measured in brain areas of rats 3 to 24 months of age. While GABA receptor binding was not significantly different across age in any area, β-adrenergic receptor binding was significantly reduced in the cerebellum and brain stem, but not cerebral cortex, of 24-month-old animals. The loss in β-adrenergic receptor binding does not correlate in a temporal fashion with the reported decrease in norepinephrine-stimulated cyclic AMP accumulation in the cerebellum which occurs as early as 12 months of age. An age-related reduction in β-adrenergic binding was also noted in human cerebellar tissue obtained at autopsy, suggesting that the cerebellar dysfunction seen with aging may be related to a loss of cerebellar neurons which receive noradrenergic input.     

Antagonism of alpha- and beta-adrenergic-mediated accumulations of cyclic AMP in rat cerebral cortical slices by the beta-antagonist (-)alprenolol.

(?)Alprenolol, a compound reported to bind with a high degree of specificity and stereoselectivity to β-adrenergic receptors from rat cerebral cortex completely inhibited the accumulations of cyclic AMP elicited by maximally effective concentrations of norepinephrine and epinephrine at antagonist concentrations as low as 10^?5M. Other β-adrenergic antagonists such as (?)propranolol, (±)sotalol, and (+)alprenolol only partially antagonized accumulations of cyclic AMP elicited by these catecholamines even at 10-fold higher concentrations. α-Adrenergic antagonists such as phentolamine, phenoxybenzamine and clonidine only partially antagonized inhibited the accumulation of cyclic AMP elicited by methoxamine, a compound shown to stimulate the accumulation of cyclic AMP by interaction with α-adrenergic receptors. The results indicate that in brain tissue containing a mixed population of α- and β- adrenergic linked cyclic AMP generating systems, (?)alprenolol does not exhibit absolute specificity for β-receptors.     

Involvement of neurotransmitters in the action of apelin-13 on passive avoidance learning in mice

The widespread distribution of apelin-13 and apelin receptors in the brain suggests an important function of this neuropeptide in the brain that has not been explored extensively so far. In the present work, apelin-13 was found to facilitate the consolidation of passive avoidance learning in mice. In order to assess the possible involvement of transmitters in this action, the animals were pretreated with the following receptor blockers in doses which themselves did not influence the behavioral paradigm: phenoxybenzamine (a nonselective α-adrenergic receptor antagonist), propranolol (a β-adrenergic receptor antagonist), cyproheptadine (a nonselective 5-HT2 serotonergic receptor antagonist), atropine (a nonselective muscarinic acetylcholine receptor antagonist), haloperidol (a D2, D3 and D4 dopamine receptor antagonist), bicuculline (a γ-aminobutyric acid subunit A (GABA-A) receptor antagonist), naloxone (a nonselective opioid receptor antagonist), and nitro-l-arginine (a nitric oxide synthase inhibitor). Phenoxybenzamine, cyproheptadine, atropine, haloperidol, bicuculline and nitro-l-arginine prevented the action of apelin-13. Propranolol and naloxone were ineffective. The data suggest that apelin-13 elicits its action on the consolidation of passive avoidance learning via α-adrenergic, 5-HT2 serotonergic, cholinergic, dopaminergic, GABA-A-ergic and nitric oxide mediations.