Cisternal Blood Injection Research Articles

Power suppression in EEG after the onset of SAH is a significant marker of early brain injury in rat models

We analyzed the correlation between the duration of electroencephalogram (EEG) recovery and histological outcome in rats in the acute stage of subarachnoid hemorrhage (SAH) to find a new predictor of the subsequent outcome. SAH was induced in eight rats by cisternal blood injection, and the duration of cortical depolarization was measured. EEG power spectrums were given by time frequency analysis, and histology was evaluated. The appropriate frequency band and recovery percentage of EEG (defined as EEG recovery time) to predict the neuronal damage were determined from 25 patterns (5 bands × 5 recovery rates) of receiver operating characteristic (ROC) curves. Probit regression curves were depicted to evaluate the relationships between neuronal injury and duration of depolarization and EEG recovery. The optimal values of the EEG band and the EEG recovery time to predict neuronal damage were 10–15 Hz and 40%, respectively (area under the curve [AUC]: 0.97). There was a close relationship between the percentage of damaged neurons and the duration of depolarization or EEG recovery time. These results suggest that EEG recovery time, under the above frequency band and recovery rate, may be a novel marker to predict the outcome after SAH.

Knockdown of sortilin improves the neurological injury and regional cerebral blood flow in rats after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a severe subtype of stroke. Sortilin protein is elevated in cerebrospinal fluid (CSF) of SAH patients. This study explored the mechanism of sortilin in SAH. SAH model was established by occipital cisternal blood injection. Neurological evaluation was performed on SAH rats using the Gracia scoring system and beam-balance tests. Regional cerebral blood flow (rCBF) and intracranial pressure (ICP) changes were measured using a laser Doppler blood flow monitor and an intraparenchymal Camino ICP probe. The correlation between rCBF changes and neurological deficit was analyzed using the Spearman method. Sortilin protein level in rat cerebral cortex and CSF was detected by Western blot. The Garcia score and beam-balance score of rats at 1, 12, 24, 48, and 72 h after SAH were lowered. Blood clots were observed on the ventral surface of the brain in SAH rats, around Willis ring, and ventral surface of brain stem, but no blood clots were found in the control group. At 1, 12, 24, 48, and 72 h after SAH in rats, the severity of SAH was aggravated, rCBF was decreased, and ICP was increased. The changes of rCBF in rat cerebral cortex at 1 and 72 h after SAH were correlated with the Garcia score. Sortilin was highly expressed in the cerebral cortex and CSF of SAH rats. Knockdown of sortilin improved the neurological injury and rCBF in rats. Sortilin was highly expressed in the cerebral cortex and CSF of SAH rats. Sortilin silencing improved neurological injury and CBF in rats.

Denervation injury of scalp hair due to trigeminal ganglion ischemia: the first experimental study

Aim: Scalp hairs are mainly innervated by sensitive fibers of trigeminal nerves. Ischemic neurodegeneration of trigeminal ganglion can cause denervation injury of scalp hairs. We investigated if there is a relationship between the degenerated neuron densities of trigeminal ganglion neuron densities and the numbers of degenerated hair follicles numbers following subarachnoid hemorrhage (SAH). 
 Material and Method: Five normal (n=5), five SHAM (n=5), and ten (n=10) male rabbits were chosen from formerly experimental SAH created by cisternal homologous blood injection (0.75cc) group, which followed for three weeks. Degenerated neuron numbers of trigeminal ganglion and atrophic hair follicles numbers in the frontal areas of the scalp were examined by stereological methods. Degenerated neuron densities of trigeminal ganglions and atrophic hair follicles numbers were analyzed by the Mann-Whitney U test.
 Results: The mean degenerated neuron densities trigeminal ganglions (n/mm3) and atrophic hair follicles (n/mm2) were determined as 5±2/m3 and12±4/mm2 in control; 12±3/m3 and 41±8/mm2 in Sham and, 168±23/m3 and 79±14/mm2 in the study group (p>0.001). In the post-hoc analysis, all groups differed significantly from each other. A linear association was observed between the degenerated neuron densities of trigeminal ganglions and atrophic hair follicles (r: 0.343, p: 0.007). 
 Conclusion: Trigeminal ganglion neurodegeneration may be an essential factor in hair follicles atrophy after SAH, which has not been mentioned in the literature so far.

Predictive role of trigeminal ganglion ischemia on scalp survival affected by temporal artery diameters: The first experimental study

Scalp arteries are mainly innervated by trigeminal, facial, and vagal nerves. The ischemic neurodegeneration of the trigeminal ganglion can impede scalp circulation via vasospasm-creating effects. This study was designed to investigate whether there is any link between the vasospasm index of deep temporal arteries and ischemic neuron densities of the trigeminal ganglion after subarachnoid hemorrhage. The study subjects included five normal control rabbits, six sham rabbits, and nine rabbits chosen from a formerly established experimental subarachnoid hemorrhage group created by cisternal homologous blood injection (0.75mL). These rabbits, all male, were followed up for 3 weeks. The trigeminal ganglion and deep temporal artery vasospasm indexes were examined by stereological methods. Ischemic neuron densities of the trigeminal ganglion and vasospasm index values of deep temporal arteries were compared statistically. Postmortem examinations showed important vasospasms of deep temporal arteries, foramen magnum herniations, and neurodegeneration of the trigeminal ganglion. The mean vasospasm index values and degenerated neuron densities of the trigeminal ganglion were determined as 1.03±0.13 and 10±3/mm3 (p > 0.5) in the control group, 1.21±0.18 and 35±9/mm3 in the sham group (p < 0.005 for sham vs. control), and 2.54±0.84 and 698±134/mm3 in the experimental group (p < 0.0005 for sham vs. control and p < 0.00001 for study vs. control). There was an inverse relationship between the vasospasm index values and the degenerated neuronal density of the trigeminal ganglion. The high degenerated neuron density in the trigeminal ganglion had a facilitative effect on temporal artery vasospasm. Trigeminal ganglion neurodegeneration may promote temporal artery vasospasms after subarachnoid hemorrhage, which has not been previously mentioned in the literature.

Neurocognitive Outcomes in a Cisternal Blood Injection Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) results in neurocognitive dysfunction and anxiety in humans and in animal models. Neurobehavioral tests such as the Morris Water Maze (MWM) and Elevated Plus Maze (EPM) tests are validated in several models of SAH but have not been tested in the murine cisternal blood injection SAH model. Adult C57BL/6 mice (n=16) were randomized into two groups. Group 1 (n=8) received sham surgery. Group 2 (n=8) underwent SAH with 60 µL of autologous blood injected into the cisterna magna. Mice were then tested using the Modified Garcia Score on post-operative day 2 (POD2), EPM on POD5 & POD16, and MWM on POD6-16.Brain tissues harvested on POD16 were stained with Fluoro-Jade C to identify neurodegeneration in the hippocampus and cortex and Iba-1 immunofluorescence staining for microglial activation in the dentate gyrus and CA1 region of the hippocampus. SAH mice showed increased escape latency on POD10. Swim distance was significantly increased on POD9-10 and swim speed was significantly decreased on POD6&POD10 in SAH mice. SAH mice exhibited a trend for lowered proportion of covered arm entries in EPM on POD16. Modified Garcia Score was not significantly different between the groups on POD2. The area of microglial activation in the dentate gyrus and CA1 region of the hippocampus was mildly increased but not significantly different at day 16 after SAH. Similarly, no significant differences were noted in the number of Fluoro-Jade C (+) cells in cortex or hippocampus. Cisternal single blood injection in mice produces mild neurocognitive deficits most pronounced in spatial learning and most evident 10 days after SAH.

Requisite ischemia for spreading depolarization occurrence after subarachnoid hemorrhage in rodents

Spontaneous spreading depolarizations are frequent after various forms of human brain injury such as ischemic or hemorrhagic stroke and trauma, and worsen the outcome. We have recently shown that supply-demand mismatch transients trigger spreading depolarizations in ischemic stroke. Here, we examined the mechanisms triggering recurrent spreading depolarization events for many days after subarachnoid hemorrhage. Despite large volumes of subarachnoid hemorrhage induced by cisternal injection of fresh arterial blood in rodents, electrophysiological recordings did not detect a single spreading depolarization for up to 72 h after subarachnoid hemorrhage. Cortical susceptibility to spreading depolarization, measured by direct electrical stimulation or topical KCl application, was suppressed after subarachnoid hemorrhage. Focal cerebral ischemia experimentally induced after subarachnoid hemorrhage revealed a biphasic change in the propensity to develop peri-infarct spreading depolarizations. Frequency of peri-infarct spreading depolarizations decreased at 12 h, increased at 72 h and normalized at 7 days after subarachnoid hemorrhage compared with sham controls. However, ischemic tissue and neurological outcomes were significantly worse after subarachnoid hemorrhage even when peri-infarct spreading depolarization frequency was reduced. Laser speckle flowmetry implicated cerebrovascular hemodynamic mechanisms worsening the outcome. Altogether, our data suggest that cerebral ischemia is required for spreading depolarizations to be triggered after subarachnoid hemorrhage, which then creates a vicious cycle leading to the delayed cerebral ischemia syndrome.

Changes in number of water-filled vesicles of choroid plexus in early and late phase of experimental rabbit subarachnoid hemorrhage model: the role of petrous ganglion of glossopharyngeal nerve.

Cerebrospinal fluid (CSF) secretion may be increased in the early phases of subarachnoid hemorrhage (SAH), possibly via ischemic glossopharyngeal nerve discharges, and decreased due to glossopharyngeal nerve degeneration in the late phase of SAH; but this reflex pathway has not been definitively investigated. We studied the relationship between petrous ganglion of the glossopharyngeal nerve (GPN) and water vesicles of the choroid plexus (CP) in the early and late phases of SAH. This study was conducted on 30 rabbits, divided into four groups, with five rabbits in the control group (group I), five rabbits in the sham group (Group II), and 20 rabbits in the SAH group. In the SAH group, five of the animals were decapitated after 4 days of cisternal blood injections (Group III), and the other 15 animals were decapitated after 20 days of injections (Group IV). The Petrous Ganglia and CPs of lateral ventricles were removed and stained for stereological analysis. The mean number of follicles per cubic millimeter was 5.3 ± 1.2 the in control group (Group I), 4.5 ± 0.9 in the sham group (Group II), 16.60 ± 3.77 the in early decapitated group (Group III), and 4.30 ± 0.84 in the late decapitated group (Group IV). The mean number of degenerated neuron density of petrous ganglions was 6 ± 2, 50 ± 6, 742 ± 96, and 2.420 ± 350 in the control (Group I), sham (Group II), early decapitated (Group III), and late decapitated group (Group IV), respectively. The mean number of water vesicles was statistically different after SAH between the early decapitated group (group III) and the late decapitated group (group IV) (P < 0.05). We studied the relationship between petrous ganglion cells of the GPN and water vesicles of CP in the early and late phases of SAH, and found that CP vesicles are increased in the early phase of SAH due to irritation of GPN, and decreased in the late phase due to ischemic insult of the petrous ganglion and parasympathetic innervation of the CP.

Tenascin-C is induced in cerebral vasospasm after subarachnoid hemorrhage in rats and humans: a pilot study

Objective: Cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) has been hypothesized to occur because of both inflammation-mediated sustained contraction of smooth muscle cells and vascular remodeling. As our recent study showed that tenascin-C (TN-C), an extracellular matrix glycoprotein which is up-regulated in inflammatory states and is associated with tissue remodeling, causes vasospasm-like changes in arterial walls, we examined whether TN-C might be induced in relation to the occurrence of cerebral vasospasm experimentally and clinically.Methods: First, rat models were produced by means of a single cisternal injection of either autologous arterial blood or saline. Immunostaining for TN-C was performed with basilar arteries obtained from non-operated rats (n = 3) and on days 1-4 in SAH (n = 18) or saline-injected (n = 12) rats. Second, levels of TN-C were prospectively measured in serum in 31 consecutive patients diagnosed with aneurysmal SAH on days 1-12 and compared between those with and without subsequent cerebral vasospasm.Results: In SAH rats, marked induction of TN-C immunoreactivity was shown throughout the vasospastic arterial wall, especially in the smooth muscle cell layers, in comparison with control rats. In a clinical study, serum TN-C levels increased transiently, the extent being significantly greater in patients with subsequent vasospasm; the peak occurred 2·4 days before an increase in the mean transcranial Doppler velocity to 120 cm/s and 3·6 days before the onset of symptomatic vasospasm (n = 14).Discussion: This is the first study suggesting TN-C increases with close linkage to the occurrence of vasospasm after SAH.

Co‐expression/co‐location of S100 proteins (S100B, S100A1 and S100A2) and protein kinase C (PKC‐β, ‐η and ‐ζ) in a rat model of cerebral basilar artery vasospasm

The cellular events leading to cerebral vasospasm after subarachnoid haemorrhages (SAH) involve a number of members of the protein kinase C (PKC) family. However, whereas calcium is thought to play a number of major roles in the pathophysiology of SAH, a number of PKCs function independently of calcium. We recently emphasized the potential role of the calcium-binding S100 proteins in a 'double haemorrhage' rat model of SAH-induced vasospasm. A number of S100 proteins are known to interfere directly with PKC, or indirectly with PKC substrates. We therefore investigated whether specific S100 proteins and PKCs are co-expressed/co-located in a rat model of SAH-induced vasospasm. SAH-induced vasospasm in rats (by means of a double cisternal injection of autologous blood from a rat femoral artery) distinctly modified the expression levels of calcium-dependent PKC-alpha and PKC-beta and calcium-independent PKC-eta and PKC-zeta in endothelial and smooth-muscle cells. The RNA levels of these four PKC isotypes were determined by quantitative RT-PCR. The present study reveals that, in endothelial cells, the S100B expression/location correlate well with those of PKC-eta, and those of S100A1 with PKC-beta. In smooth-muscle cells S100A2 expression/location correlate with those of PKC-eta, and those of S100B with PKC-zeta. The present data argue in favour of a joint action of the S100 protein network and the PKC signalling pathway during cerebral vasospasm.

Effects of the Ca++-permeable nonselective cation channel blocker LOE 908 on subarachnoid hemorrhage-induced vasospasm in the basilar artery in rabbits.

The Ca++ influx into vascular smooth-muscle cells (VSMCs) plays a fundamental role in the development and chronic effects of vasospasm after subarachnoid hemorrhage (SAH). The Ca++-permeable nonselective cation channels (NSCCs) are activated by several endothelium-derived constricting factors such as endothelin 1 (ET-1) and thromboxane A2. Moreover, the receptor-operated Ca++ channel blocker LOE 908 inhibits ET-1-induced extracellular Ca++ influx via NSCCs in the VSMCs of the basilar artery (BA) and the NSCC-dependent part of ET-1-induced vasoconstriction of BA rings. The purpose of the present study was to evaluate the in vivo role of LOE 908 on SAH-induced vasospasm. Forty-two Japanese white rabbits were assigned to seven groups. Treatment groups consisted of the following: 1) control rabbits without SAH that received a cisternal injection of saline; 2) rabbits with SAH that were subjected to the intravenous administration of saline; 3 through 6) rabbits with SAH that underwent the intravenous administration of 0.01. 0.1, 1, or 10 mg/kg LOE 908, respectively; and 7) rabbits without SAH that underwent the intravenous administration of 10 mg/kg LOE 908. Autologous blood was injected into the cisterna magna. The caliber of the BA was measured on angiographic studies before and after the cisternal injection of autologous blood. The intravenous injection of LOE 908 inhibited the magnitude of an SAH-induced vasosapsm. In addition, the concentration of LOE 908 required to relax vasospasm (1 mg/kg) correlated with that required to block Ca++ influx into VSMCs. The Ca++ channel blocker LOE 908 may inhibit the magnitude of an SAH-induced vasospasm by blocking the influx of Ca++ through NSCCs in rabbit BAs. Blocking the NSCCs may represent a new treatment for cerebral vasospasm after SAH.

Altered expression of P(2) receptor mRNAs in the basilar artery in a rat double hemorrhage model.

Extracellular ATP might induce cerebral vasospasm after subarachnoid hemorrhage through P(2) receptor. To investigate the roles of P(2) receptor subtypes in vasospasm, we examined the changes in mRNA expression of P(2) receptor subtypes in basilar arteries from double cisternal blood injection rat models. One hundred male Sprague-Dawley rats, each weighing 350 to 400 g, were divided into 2 groups of 50. In the first group (n=50), the autologous arterial blood (0.2 to 0.3 mL) was injected into the cisterna magna on days 0 and 2. The rats were killed on day 3, 5, or 7 (n=10 in each group). In the sham group (n=10), the rats were injected with saline (0.3 mL) instead of blood. Ten rats were killed without blood or saline injection and served as control. The basilar arteries from rats in each group were used for reverse transcription and polymerase chain reaction. In another group of 50 rats, the same experiment was conducted, and the basilar arteries were collected for transmission electron microscopic study. In the subarachnoid hemorrhage groups, transmission electron microscopy showed the reduction in vessel perimeter on days 5 and 7 to be approximately 30% to 40%. The P(2X1) mRNA level was significantly decreased on day 3 and recovered on days 5 and 7. The P(2Y1) mRNA level was transiently increased on day 5, and the P(2Y2) mRNA level was elevated from day 5 to day 7 (P:<0.05). The differential expression of the P(2) receptors indicates that P(2X1) subtype might not play an important role in vasospasm. The upregulation of P(2Y1) and P(2Y2) receptors might enable ATP to produce contraction at low levels of concentration.

Quantitative analysis of gene expressions related to inflammation in canine spastic artery after subarachnoid hemorrhage.

The possible role of inflammatory reaction of the cerebral artery in the pathogenesis of cerebral vasospasm has been noted in recent studies. We quantitatively measured the levels of expression of genes related to inflammation in the spastic artery in a canine double-hemorrhage model. Twenty dogs were assigned to 4 groups: group D0, control; group D2, dogs killed 2 days after cisternal injection of blood; group D7, dogs given double cisternal injections of blood and killed 7 days after the first injection; and group D14. Angiography was performed twice: on the first day and before the animals were killed. Total RNA was extracted from the basilar artery. The expressions of interleukin (IL)-1alpha, IL-6, IL-8, IL-10, tumor necrosis factor-alpha, E-secretin, fibronectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1, transforming growth factor-ss, basic fibroblast growth factor, and collagen types I, III, and IV were examined with TaqMan real-time quantitative reverse transcription-polymerase chain reaction. Prolonged arterial narrowing peaking on 7 day was observed. There was a significant difference in vessel caliber between D0, D2, D7, and D14 groups (P:<0.0001). There were significant differences in mRNA expression in the basilar artery for IL-1alpha, IL-6, IL-8, ICAM-1, and collagen type I between D0, D2, D7, and D14 groups (P:=0.0079, 0. 0196, 0.0040, 0.0017, and <0.0001, respectively). The average level of mRNA was highest in D7 for IL-1alpha, IL-6, IL-8, and ICAM-1 (17-, 16-, 131-, and 1.7-fold compared with those of D0, respectively) and in D14 for collagen type I (10.9-fold). Increased expression of genes related to inflammation in the spastic artery suggests that inflammatory reaction of the cerebral artery is associated with sustained contraction.

Upregulation of rho A and rho kinase messenger RNAs in the basilar artery of a rat model of subarachnoid hemorrhage.

Rho A, a small guanosine triphosphate-binding protein, and rho kinases have been suggested to play an important role in the agonist-induced myofilament Ca++ sensitization and cytoskeletal organization of smooth-muscle cells. To discover their possible roles in the prolonged contraction seen in cerebral vasospasm, the authors investigated the messenger (m)RNA expressions of rho A and rho-associated kinases alpha and beta in the basilar artery (BA) of a rat double cisternal blood-injection model. An experimental subarachnoid hemorrhage (SAH) was achieved in rats by twice injecting autologous arterial blood into the cisterna magna of each animal. The mRNAs for rho A and rho-associated kinases alpha and beta of the rat BA were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). The cisternal blood injection induced a marked corrugation of elastic lamina and contraction of smooth-muscle cells observed with the aid of light and transmission electron microscopy in the rat BA on Days 3, 5, and 7. Results of the RT-PCR revealed that mRNAs for rho A and rho kinases alpha and beta were expressed in the rat BA and that they were significantly upregulated and reached their peaks on Day 5. The mRNA upregulation of these proteins indicates that activation of rho A/rho kinase-related signal transduction pathways is involved in the development of long-lasting contraction of cerebral arteries after SAH.

Effective improvement of the cerebral vasospasm after subarachnoid hemorrhage with low-dose nitroglycerin.

In the rabbit subarachnoid hemorrhage (SAH) model, the sensitivity of spastic basilar arteries to nitric oxide (NO) was enhanced whereas the endothelial function to release/produce NO became impaired, as described previously. We assumed from these results that low-dose NO might selectively dilate spastic arteries without influencing normal or systemic blood vessels; therefore, we investigated whether exogenous low-dose NO effectively improves cerebral vasospasm. Low-dose NO was derived from a small size of the tape containing nitroglycerin, which is not invasive and is clinically available. The experimental SAH was induced by injecting autologous blood into the cisterna magna of the rabbit. Experiments were performed on the following three groups: (a) SAH group with nitroglycerin tape (nitroglycerin group), (b) SAH group with placebo tape (placebo group), and (c) saline group injected with saline instead of blood. The tape containing 0.675 mg nitroglycerin was applied once daily for 2 days onto the skin area of the rabbit's ear. Angiograms were performed once before cisternal injection of blood and/or saline and again on day 2. On day 2 the basilar artery was isolated and sliced into 2-mm ring preparations. Relaxations of the basilar artery to acetylcholine, sodium nitroprusside, and calcium ionophore A23187, as well as the contractile responses to serotonin and endothelin- 1, were measured. The diameter of the basilar artery on day 2 was reduced to 69.6+/-2.2% (n = 7) before the injection of autologous blood. The angiographic vasospasm of the basilar artery was partially but significantly (p<0.0001) improved to the percentage diameter of 89.4+/-1.4% (n = 7) by the application of low-dose nitroglycerin, which did not affect the systemic blood pressure and heart rate. In the basilar artery preparations harvested from SAH rabbits on day 2, the impaired acetylcholine-induced endothelium-dependent relaxation was partially but significantly (p<0.001) improved in the nitroglycerin group. However, this group remained unaffected in the increased sensitivity to nitroglycerin and the contractile responses to serotonin and endothelin-1. Low-dose nitroglycerin tape effectively improved the cerebral vasospasm after SAH without any significant changes in the systemic circulation and would be one of the useful and noninvasive treatments for cerebral vasospasm. The results seem to be partially affected by the effective dilation of the spastic artery and the improvement of the impaired endothelium-dependent relaxation with low-dose NO.

Anti-oxidants prevent focal rat brain injury as assessed by induction of heat shock proteins (HSP70, HO-1/HSP32, HSP47) following subarachnoid injections of lysed blood

The initial aim of this study was to determine if the HSP70 (the main inducible heat shock protein), HO-1 (heme oxygenase-1, HSP32) and HSP47 (a collagen chaperone) stress proteins were induced in the same focal regions of rat brain following experimental subarachnoid hemorrhage (SAH). The next objective was to determine whether anti-oxidants prevented the stress gene expression in the focal regions. Lysed blood (150 μl) was injected into the subarachnoid space of adult, female Sprague–Dawley rats via the cisterna magna. Animals were sacrificed 24 h later. Immunocytochemistry showed focal regions of stress gene induction in most animals (13/21), HSP70 and HO-1 proteins being expressed in neurons, microglia and astrocytes and HSP47 being expressed in microglia. Co-induction of the same three stress proteins was observed in focal areas in the striatum and cerebellum as well. In the 13 animals with focal regions of stress gene induction there were 8.1±1.8 foci in cortex, 5.5±0.9 foci in striatum, and 11.7±7.3 foci in cerebellum in the brain of each animal. The focal regions of stress gene induction varied in size from 200 μm to 7 mm in diameter. Systemic administration of the tirilazad-like anti-oxidants U101033E ( n=8) and U74389G ( n=7) completely blocked stress protein induction in focal brain regions normally produced by cisternal injections of lysed blood. There were fewer drug treated animals (0/15) with focal areas of stress gene induction compared to non-drug (13/21) treated animals following the cisternal lysed blood injections ( p<0.01 using Fisher's probability test). This study shows that anti-oxidants prevent focal regions of injury as assessed by heat shock protein expression in a rat model of SAH.

Inducible cyclooxygenase expression in canine basilar artery after experimental subarachnoid hemorrhage.

Inducible cyclooxygenase (COX-2) has been found to play a pathological role in cerebral insult. We investigated the expression of COX-2 in the basilar artery after experimental subarachnoid hemorrhage (SAH). In a canine "two-hemorrhage" model of SAH, the basilar arteries were obtained on day 2 after a cisternal injection of autologous blood or on days 4, 6, 7, or 9 after the second injection. Basilar arteries also were obtained 12 hours after intracisternal injection a cytokine: interleukin (IL)-1 beta (0.03 microgram), IL-6 (3 micrograms), or IL-8 (10 micrograms). Western blotting with a polyclonal anti-COX-2 antibody was performed in these arteries. COX-2 protein was not demonstrated in the basilar artery in control animals without SAH. However, it was expressed in the basilar artery on days 2, 4, 6, and 7 after blood injection but not on day 9. Intracisternal injection of IL-1 beta, IL-6, or IL-8 also induced COX-2 in the basilar artery. COX-2 expression was detected in basilar arterial tissue in both acute and chronic stages after SAH. Elevation of inflammatory cytokines after SAH may be involved in the induction of COX-2, which may produce sufficient quantities of eicosanoids to affect hemodynamics after SAH.

Prevention of vasospasm after subarachnoid hemorrhage in dogs by continuous intravenous infusion of PD156707.

This randomized, blinded study tested the prophylactic effect of PD156707, a nonpeptide competitive antagonist of endothelin A receptors, against vasospasm after subarachnoid hemorrhage in dogs. Twenty-two dogs were allocated on day 0 to undergo cerebral angiography followed by injection of arterial blood (0.5 ml/kg) into the cisterna magna. Dogs had central venous catheters implanted for continuous infusion of drug vehicle (n = 10) or PD156707 (n = 12). Cisternal blood injection was repeated on day 2. Drug levels were measured in plasma on days 2, 4, 6, and 7 and in cerebrospinal fluid (CSF) on days 2 and 7. Angiography was repeated on day 7 to assess vasospasm. After angiography on day 7, acute effects of infusion of PD156707, 100 mg, or drug vehicle on established vasospasm were assessed. Analysis of physiological variables within (analysis of variance) groups across time and between (unpaired t-test) groups at each time showed that drug-treated animals had significantly increased heart rate on day 7 compared to day 0 (p < 0.005). Comparison of basilar artery diameters at day 7 showed that PD156707 significantly decreased the degree of basilar artery vasospasm (placebo: -47 +/- 5% reduction [mean +/- SE] versus PD156707: -28 +/- 7%, p < 0.05, unpaired t-test). There was, however, significant vasospasm when comparing within groups (paired t-test, placebo: p < 0.0001, PD156707: p < 0.005). Mean plasma PD156707 levels (322 +/- 123 ng/ml) were adequate to block responses of endothelin-1 on endothelin A receptors in vitro although CSF levels (11 +/- 7 ng/ml) were substantially lower. Infusion of PD156707 into the basilar artery on day 7 caused a small but significant 10 +/- 3% (paired t-test, p < 0.01) increase in diameter compared to placebo (3 +/- 3% increase, p = 0.32). This infusion also was associated with a substantial increase in CSF drug levels to 19 +/- 9 mg/ml. These results suggest that endothelin A receptors mediate some of the vasospasm that occurs after SAH in dogs and that blockade of these receptors may be a beneficial treatment for vasospasm.

Trigeminal nerve and brainstem catecholamine systems in cerebral vasospasm.

Cisternal blood injection in the rat and squirrel monkey produces a biphasic cerebral vasospasm, a decrease in cerebral blood flow (CBF) and an increase in glucose uptake (CMRglu) due to an anaerobic glucolysis actually representing a decrease in metabolism. Lesioning of the A2-nucleus, its ascending cathecolamine pathways or their projection site, the median eminence in the hypothalamus, prevents the occurrence of spasm. A unilateral postganglionic trigeminal lesion causes an ipsilateral constriction of the cerebral arteries while a preganglionic lesion does not affect the baseline arterial diameter. Both kinds of trigeminal lesions induce a global increase in glucose uptake of about 50% without influencing CBF. Following subarachnoid hemorrhage (SAH) the decrease in CBF in both groups of lesioned animals is similar to that seen in controls. After SAH there is no further change in CMRglu in the animals with a preganglionic lesion, while in the postganglionically lesioned animals there is an additional increase in CMRglu of about 50% as compared to controls or animals with a preganglionic lesion. Treatment with the peptidergic substance P (SP) antagonist, spantide, or gammaglobulin against SP prevents or significantly reduces the degree of spasm and the changes in flow and metabolism normally seen post-SAH. The non-peptidergic neurokinins NK1 and NK3 antagonists do not influence flow and metabolism in SAH animals. The NK2 seems to change both flow and metabolism post-SAH in rats.

Reduced expression of calponin in canine basilar artery after subarachnoid haemorrhage.

Calponin, an actin- and tropomyosin-binding protein, has been characterized as an inhibitory factor in the smooth-muscle actomyosin activity. The level of calponin was determined in canine basilar arteries in a double-haemorrhage model. Thirty dogs were assigned to three groups: day 0 group, control; day 2 group, dogs sacrificed 2 days after cisternal injection of blood; and day 7 group, dogs given double cisternal injections of blood and sacrificed 7 days after the first injection. Constriction of the basilar artery was confirmed by arterial angiography. Portions of the affected arteries or the corresponding region in control animals were solubilized for sodium dodecylsulphate-polyacrylamide gel electrophoresis and Western blotting. A major band corresponding to calponin was seen at 34 kD in the basilar artery extracts using chicken gizzard polyclonal antibodies. The densitometer values of the band on Coomassie blue-stained gels were expressed as percentages of day 0 control values. The signals of day 2 and day 7 samples were 47% +/- 20% and 23% +/- 12%, respectively (mean +/- standard deviation). The proportions of calponin to actin/tropomyosin in the day 0, day 2, and day 7 groups were 13% +/- 6%, 6% +/- 2%, and 4% +/- 2%, respectively. The reduced expression of calponin may be related to sustained contraction during cerebral vasospasm.

Trigeminal afferents and brainstem centers involved in the occurrence of cerebral vasospasm.

Cisternal injections of blood in the rat and squirrel monkey produce an angiographically demonstrable biphasic vasospasm with a maximal late spasm at two days in the rat and six days post-subarachnoid hemorrhage (SAH) in the monkey. The SAH induces a decrease in cerebral blood flow of about 25% and a corresponding increase in glucose uptake of between 30% and 50%. In about half of the animals low-flow areas were noted in the cortex and the basal ganglia with a corresponding marked increase in glucose uptake. Lesioning of the A2-nucleus, its ascending pathway or the median eminence prevents the occurrence of spasm. Similarly, treatment with a substance P antagonist or gammaglobulin against substance P prevents or significantly reduces the degree of spasm. A unilateral post-ganglionic trigeminal lesion causes an ipsilateral constriction of the cerebral arteries of 27%, while a preganglionic lesion does not affect the baseline diameter. A pre- or post-ganglionic trigeminal lesion induces an increase in glucose uptake globally of about 50% without influencing cerebral blood flow. Following SAH the decrease in blood flow in both groups of lesioned animals is similar to that seen in controls. After SAH there is no further change in glucose uptake in the animals with a preganglionic lesion, while in the post-ganglionically lesioned animals there is an additional increase in glucose uptake of about 50% as compared to controls or the animals with a preganglionic lesion.