Neurokinin-1 receptor antagonism in a rat model of subarachnoid hemorrhage: prevention of upregulation of contractile ETB and 5-HT1B receptors and cerebral blood flow reduction.

OBJECT: Cerebral vasospasm following subarachnoid hemorrhage (SAH) leads to reduced cerebral blood flow (CBF) and to cerebral ischemia, in some cases even producing infarction and long-term disability. The goal of the present study was to investigate the hypothesis that inhibition of neurokinin-1 receptors (NK1Rs) by administration of L-822429 blunts the decrease in CBF as well as cerebrovascular receptor upregulation in an animal model of SAH. METHODS: Subarachnoid hemorrhage was induced in rats by injection of 250 microl of blood into the prechiasmatic cistern. The NK1R inhibitor L-822429 was injected intracisternally 30 minutes and 24 hours after the induction of SAH. Two days after SAH induction, the basilar arteries were harvested, and contractile responses to endothelin-1 (ET-I, an ETA- and ETB-receptor agonist) and 5-carboxamidotryptamine (a 5-hydroxytryptamine- I1 [5-HT1]-receptor agonist) were investigated using sensitive myographs. To determine whether NKIR inhibition had an influence on local CBF after post-SAH, a quantitative autoradiographic technique was used. After SAH, the vascular receptor phenotype was changed in cerebral arteries through upregulation of contractile ET, and 5-HT1B receptors, while regional and total CBF were markedly reduced. Treatment with the selective NK1R inhibitor L-822429 prevented both the receptor upregulation and the reduction in regional and global CBF. CONCLUSIONS: The data reveal the coregulation of vascular receptor changes and blood flow effects, and also show that interaction with a small-molecule NK1R antagonist is a promising area of focus for the development of specific treatments for SAH.

Experimental subarachnoid hemorrhage induces changes in the levels of hippocampal NMDA receptor subunit mRNA.

NMDA receptors may play a crucial role in nerve cell death following subarachnoid hemorrhage (SAH). Changes in NMDA receptor-mediated transmission appear before neuronal death in rodent models of transient ischemia, and NMDA receptor function is known to be dependent on subunit composition. Here, we have investigated whether mRNA expression of the NMDA receptor subunits is altered in the hippocampal formation 3-5 h following experimental SAH, and correlated these early alterations to subsequent delayed cell death. SAH was induced by intraluminal perforation of the internal carotid artery intracranially, and cerebral blood flow (CBF) was bilaterally monitored by laser-Doppler flowmetry. Early changes in NMDA receptor subunit mRNA and early nerve cell death were analyzed at 3-5 h after SAH, and delayed nerve cell death was analyzed at 2-7 days after SAH. Duration of ipsilateral CBF reduction below 30% of baseline (CBF30) was predictive of ipsilateral delayed nerve cell death in the CA1 2-7 days after SAH. At CBF30 > 9 min, we found downregulation of mRNA for NR2A, NR2B, and NR3B at 3-5 h after SAH, whereas the levels of NR1 mRNA were unaffected. The downregulation of NR2A and NR2B mRNA may result in a reduced NMDA receptor function. Such reduction may be sufficient to provide neuroprotection in the dentate gyrus, where no cell death appears, but insufficient to rescue neurons in the hippocampus proper following SAH.

Delayed cell death related to acute cerebral blood flow changes following subarachnoid hemorrhage in the rat brain.

OBJECT: The authors tested the hypotheses that subarachnoid hemorrhage (SAH) leads to delayed cell death with the participation of apoptotic-like mechanisms and is influenced by the degree of acute decrease in the cerebral blood flow (CBF) following hemorrhage. METHODS: Subarachnoid hemorrhage was induced in rats by endovascular perforation of the internal carotid artery or injection of blood into the prechiasmatic cistern. Cerebral blood flow was measured using laser Doppler flowmetry for 60 minutes. Brain sections stained with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) showed DNA fragmentation at 2 and 7 days after both methods of inducing SAH in one third to two thirds of the surviving animals in the different experimental groups. More than 80% of the TUNEL-positive cells were neuron-specific nuclear protein-positive (neurons), but immunoreactivity to glial fibrillary acidic protein (astrocytes) and transferrin (oligodendrocytes) were markedly decreased in TUNEL-positive areas. Most of the TUNEL-positive cells displayed chromatin condensation and/or blebs and immunostained for increased Bax; approximately 50% of them were immunoreactive to cleaved caspase-3 and a few to Bcl-2. The duration of the acute CBF decrease below 30% of the baseline level was related to the degree of TUNEL staining. CONCLUSIONS: Subarachnoid hemorrhage resulted in delayed cell death in a large proportion, but not all, of the surviving animals. The acute CBF decrease was related to the degree of subsequent cell death. These findings indicated the relevance of apoptotic-like pathways. There appears to be a temporal therapeutic window during which adequate treatment might reduce the final damage following SAH.

A new experimental model in rats for study of the pathophysiology of subarachnoid hemorrhage.

A new experimental model of subarachnoid hemorrhage (SAH) in rats is described. A needle was stereotaxically placed in the prechiasmatic cistern and 300, 250 or 200 microl of blood was injected manually, keeping the intracranial pressure (ICP) at the mean arterial blood pressure (MABP) level. An acceptable mortality was observed only after injection of 200 microl of blood. In this group, MABP and ICP increased immediately after SAH, but soon approached baseline levels. The subarachnoid blood was mainly distributed in the basal cisternal system and its estimated volume was about 95% of the amount injected. This new model resembles clinical SAH, is very reproducible, easy to use and seems to be a suitable model for studies of the pathophysiology of SAH.

Experimental subarachnoid hemorrhage: cerebral blood flow and brain metabolism during the acute phase in three different models in the rat.

OBJECTIVE: To study the cerebral metabolism and its relationship to cerebral blood flow (CBF) acutely after subarachnoid hemorrhage (SAH). METHODS: SAH was induced in rats by endovascular perforation of the internal carotid artery, blood injection into the prechiasmatic cistern or the cisterna magna. CBF (measured by laser Doppler flowmetry), cerebral perfusion pressure, O(2) tension, and extracellular levels of glucose, lactate, and pyruvate were monitored during 90 minutes after SAH. CBF (assessed by (125)I-antipyrine autoradiography), arteriovenous O(2) difference, and cerebral metabolic rate of O(2) were calculated at 15 or 90 minutes after SAH. RESULTS: After a transient reduction, cerebral perfusion pressure normalized within 5 minutes after SAH in all groups. There was a transient global decrease in CBF after SAH: its duration depended on the severity of the hemorrhage. CBF of less than 20% of baseline was observed for at least 15 minutes in 25% and 14% of the animals after perforation and prechiasmatic SAH, respectively. In all SAH groups, O(2) tension was suddenly reduced to approximately 40% of baseline and gradually increased, reaching 70 to 90% of baseline 90 minutes after SAH. The cerebral metabolic rate of O(2) was reduced only at 15 minutes after perforation and prechiasmatic SAH, but arteriovenous O(2) difference was normal in all groups. During 30 minutes after perforation SAH, a 50% decrease in glucose and a threefold increase in lactate and pyruvate levels were observed. CONCLUSION: The data suggest that SAH induced an acute global decrease in CBF together with a depression in the cerebral metabolism. The degree of the changes was related to the severity of the hemorrhage. The metabolic derangements were not always explained by ischemic episodes.

Experimental subarachnoid hemorrhage: subarachnoid blood volume, mortality rate, neuronal death, cerebral blood flow, and perfusion pressure in three different rat models.

OBJECTIVE: To investigate which of three subarachnoid hemorrhage (SAH) models is the most suitable for studies of pathological and pathophysiological processes after SAH. METHODS: SAH was induced in rats via intracranial endovascular perforation (perforation model), blood injection into the cisterna magna (300 microl), or blood injection into the prechiasmatic cistern (200 microl). The subarachnoid blood volume was quantitatively measured. Cerebral blood flow (CBF) (as assessed with laser Doppler flowmetry), intracranial pressure, and mean arterial blood pressure were recorded for 90 minutes after SAH. Mortality was recorded, and neuronal death was assessed in animals that survived 7 days after SAH. RESULTS: The subarachnoid blood volume was close to the injected amount after prechiasmatic SAH. In the other models, the volume varied between 40 and 480 microl. The mortality rates were 44% in the perforation SAH group, 25% in the prechiasmatic SAH group, and 0% in the cisterna magna SAH group; the corresponding values for neuronal death were 11, 44, and 28%. Cerebral perfusion pressure approached baseline values within 5 minutes after SAH in all three models. CBF decreased to approximately 35% of baseline values immediately after SAH in all groups; it gradually increased to normal values 15 minutes after SAH in the cisterna magna SAH group and to 60 and 89% of baseline values 90 minutes post-SAH in the perforation and prechiasmatic SAH groups. CBF was significantly correlated with the subarachnoid blood volume. CONCLUSION: The prechiasmatic SAH model seems to be the most suitable for study of the sequelae after SAH; it produces a significant decrease in CBF, an acceptable mortality rate, and substantial pathological lesions, with high reproducibility. The CBF reduction is predominantly dependent on the amount of subarachnoid blood.

Subarachnoid hemorrhage enhances endothelin receptor expression and function in rat cerebral arteries.

OBJECTIVE: Inspired by organ culture-induced changes in the vascular endothelin (ET) receptor population, we investigated whether such changes occur in cerebral arteries in a rat subarachnoid hemorrhage (SAH) model. METHODS: SAH was induced with injection of 250 microl of blood into the prechiasmatic cistern. After 2 days, the middle cerebral artery, basilar artery, and posterior communicating artery were harvested. Pharmacological studies were performed in vitro, and levels of messenger ribonucleic acid (mRNA) were quantified in real-time reverse transcriptase-polymerase chain reaction assays. RESULTS: In the middle cerebral artery and basilar artery from rats with induced SAH, enhanced biphasic responses to ET-1 were observed. The -log(50% effective concentration) value for the high-affinity phase was approximately 12, compared with approximately 8.5 for sham-operated animals. At a concentration of ET-1 of 10(-9) mmol/L (approximately equal to the physiological concentration of ET-1 in the plasma), submaximal contractions of 50 to 75% of the contraction obtained through stimulation with 60 mmol/L K(+) were now observed. Quantitative mRNA studies with the same arteries demonstrated significant increases in the number of copies of ET(B) receptor mRNA but not ET(A) receptor mRNA. Evidence of functional ET(B) receptors was provided in antagonist studies. The posterior communicating artery did not exhibit significant changes. CONCLUSION: The altered receptor profile observed may represent the final stage in the series of events leading from SAH to actual spasm of the artery. The pharmacological data for the ET(B) receptor suggest complex interactions between normally present ET(A) receptors and up-regulated ET(B) receptors.

Subarachnoid hemorrhage-induced upregulation of the 5-HT1B receptor in cerebral arteries in rats.

OBJECT: Cerebral vasospasm following subarachnoid hemorrhage (SAH) leads to reduced blood flow in the brain. Inspired by organ culture-induced changes in the receptor phenotype of cerebral arteries, the authors investigated possible changes in the 5-hydroxytryptamine (HT) receptor phenotype after experimental SAH. METHODS: Experimental SAH was induced in rats by using an autologous prechiasmatic injection of arterial blood. Two days later, the middle cerebral artery (MCA), posterior communicating artery (PCoA), and basilar artery (BA) were harvested and examined functionally with the aid of a sensitive in vitro pharmacological method molecularly by performing quantitative real-time reverse transcription-polymerase chain reaction (PCR). In the MCA and BA the 5-HT1B receptor was upregulated, as determined through both functional and molecular analysis. In response to selective 5-HT1 receptor agonists both the negative logarithm of the 50% effective concentration was increased (one log unit in the MCA and one half unit in the BA), as was the agonist's potency (increased by 50% in the MCA and doubled in the BA). In addition, the authors found an approximately fourfold increase in the number of copies of messenger RNA coding for the 5-HT1B receptor as determined by quantitative real-time PCR. In the PCoA no upregulation of the 5-HT1B receptor was observed. CONCLUSIONS: Changes in the receptor phenotype in favor of contractile receptors may well represent the end stage in a sequence of events leading from SAH to the actual development of cerebral vasospasm. Insight into the mechanism of upregulation may provide new targets for developing specific treatment against cerebral vasospasm.

Inflammation in the brain after experimental subarachnoid hemorrhage.

OBJECTIVE: To study the occurrence of an inflammatory response in the brain after subarachnoid hemorrhage and its relation to the decrease in acute cerebral blood flow, subarachnoid blood proximity, and cell damage. METHODS: Subarachnoid hemorrhage was induced in rats via endovascular perforation of the internal carotid artery or injection of blood into the prechiasmatic cistern. Cerebral blood flow was measured by laser Doppler flowmetry for 60 minutes. After 2 and 7 days, the brains were analyzed by immunohistochemistry using the following antibodies: OX6, ED1, intercellular adhesion molecule 1, tumor necrosis factor alpha, interleukin-1beta, interleukin-6, inducible nitric oxide synthase, and nestin. Deoxyribonucleic acid fragmentation was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling. RESULTS: In approximately half of the surviving animals (0-92%, depending on the marker and subgroup), signs of inflammation were detected. The most common findings were increased immunoreactivity to nestin, ED1, OX6, intercellular adhesion molecule 1, and tumor necrosis factor alpha. There was great variability in the intensity and the location of the inflammatory reaction among the animals, but tissues in proximity to the extravasated blood seemed to be especially affected. A significant correlation between the duration of cerebral blood flow under 30% of the baseline and the degree of the inflammation was observed. There was a strong correspondence between areas showing deoxyribonucleic acid fragmentation and inflammation. CONCLUSION: Subarachnoid hemorrhage triggered an inflammatory reaction in the brain in a large fraction of the surviving animals, which may have contributed to cell death. Acute ischemic episodes and direct effect of blood seemed to be significant factors in its genesis.