Possible Role of Raf-1 Kinase in the Development of Cerebral Vasospasm and Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats.

This study aims to clarify the potential role of Raf-1 kinase in cerebral vasospasm (CVS) and early brain injury (EBI) after subarachnoid hemorrhage (SAH). Two experimental SAH models in rats, including cisterna magna double injection model for CVS study and prechiasmatic cistern single injection model for EBI study, were performed in this research. As a specific inhibitor of Raf-1, BAY 43-9006 was used in this study. In CVS study, time course study showed that the basilar artery exhibited vasospasm after SAH and became most severe at day 5, and the phosphorylation of Raf-1 had the same trends, while both vasospasm and the phosphorylation of Raf-1 induced by SAH were inhibited by BAY 43-9006 treatment. In addition, BAY 43-9006 treatment significantly reversed the phosphorylation of ERK1/2 and the activation of NF-κB induced by SAH and decreased the messenger RNA (mRNA) levels of IL-6 and IL-1ß. In EBI study, BAY 43-9006 treatment significantly suppressed the brain injury induced by SAH. Besides, BAY 43-9006 inhibited the phosphorylation of Raf-1 and ERK1/2; decreased the protein levels of COX-2, VEGF, and MMP-9; and reversed the activation of NF-κB induced by SAH. These results demonstrate that Raf-1 kinase contributes to CVS and EBI after SAH by enhancing the activation of the Raf-1/ERK1/2 and Raf-1/NF-κB signaling pathways, and that the inhibition of these pathways might offer new treatment strategies for CVS and EBI.

The therapeutic value of proanthocyanidin in experimental cerebral vasospasm following subarachnoid hemorrhage.

AIM: We aimed to examine the basilar artery in an experimental subarachnoid hemorrhage (SAH) model both from the ultrastructural and radiological aspects and tried to evaluate the benefit of proanthocyanidin (PC), an antioxidant matter, in reducing vasospasm. MATERIAL AND METHODS: A total of 21 male New Zealand rabbits were divided into three groups after performing angiography. Group I: Control group, Group II: Subarachnoid hemorrhage group, Group III: Subarachnoid hemorrhage and Proanthocyanidin group (100 mg/kg/ day). Rabbits were sacrificed on the 5th day after angiography. A segment of the basilar artery, 4 mm in length, was prepared for ultrastructural examination. RESULTS: There was no ultrastructural or radiological abnormality in the basilar artery in Group 1. In the basilar artery digital subtraction angiography (DSA) of Group 2, a statistically significant decrease in basilar artery calibration was found (p < 0.05). There was no damage in the endothelium, subendothelium, tunica media and adventitia of the basilar artery ultrastructurally but a statistically significant narrowing in lumen diameter was seen. There was also no difference in basilar artery calibration in the DSA of Group 3 (p > 0.05). No damage was seen in the basilar artery ultrastructurally. CONCLUSION: Proanthocyanidin with its strong antioxidant effect and possible vasomotor action can significantly attenuate SAH-induced vasospasm.

Morphological changes of cerebral vessels and expression patterns of MMP-2 and MMP-9 on cerebrovascular wall of alcoholic rats.

Alcohol abuse increases the incidence of cerebral accidents, which correlates with cerebrovascular structural changes. The present study was designed to observe the cerebrovascular remodeling of drinking rats with light microscopy and transmission electron microscopy (TEM). Short-term alcohol administration induced apparent amplification of perivascular spaces around small vessels in brain tissue, while long-term administration caused pathological changes of basilar arteries (BAs), including endothelial exfoliation, inner elastic lamina (IEL) fragmentation and thickening of tunica media and adventitia. In addition, the relationship between cerebrovascular remodeling and MMP-2 and MMP-9 synthesized by endothelial cells and vascular smooth muscle cells was explored by immunohistochemistry. The two protein expression in cerebral vessels changed dynamically, peaking at 1-2 weeks after treatment, and decreasing as treatment continued. These results suggest that MMP-2 and MMP-9 may play a significant role in blood-brain barrier disruption after alcohol abuse. But the chronic changes of cerebral arteries resulted from drinking are not coincident with time course of MMP-2 and MMP-9 expression in situ.

Treatment with ginsenoside rb1, a component of panax ginseng, provides neuroprotection in rats subjected to subarachnoid hemorrhage-induced brain injury.

OBJECTIVE: recent trials have shown Ginsenoside Rb1 (GRb1), an active component of a well known Chinese medicine Panax Ginseng, plays a significant role in improving the complications seen after an ischemic brain event. In the present study, we investigated the use of GRb1 as a treatment modality to reduce brain edema, reduce arterial vasospasm, and improve neurobehavioral function after subarachnoid hemorrhage-induced brain injury (SAH) in rats. METHOD: male Sprague-Dawley rats weighing between 250 and 300 g were randomly assigned to three groups: (1) Sham group (n = 10), (2) Vehicle group (SAH + no treatment; n = 12); (3) Treatment group (SAH + GRb1 treatment at 20 mg/kg; n = 11). Subarachnoid hemorrhage was induced using the modified double hemorrhage model followed by treatment administration intravenously. Post-operative assessment included neurobehavioral testing using the spontaneous activity scoring system, brain water content, and histological examination of the basilar artery. RESULTS: post-operative findings indicated treatment with GRb1 had significantly reduced brain edema and improved neurobehavioral functioning. In addition, histological examination revealed a significant reduction in basilar artery vasospasm and lumen thickness with treatment. CONCLUSION: the results of the study suggest that GRb1 treatment reduces brain edema, improves neurobehavioral function, and blocks vasculature thickening and spasm after SAH in rats. Given the novelty of the study, further research will be needed to confirm the benefits of treatment and mechanisms behind neuroprotection.

Preventive effects of intraperitoneal selenium on cerebral vasospasm in experimental subarachnoid hemorrhage.

Vasospasm is an important cause of morbidity and mortality with subarachnoid hemorrhage (SAH). The effect of intraperitoneal administration of selenium, which is an antioxidant on cerebral vasospasm was investigated in an experimental model. By means of intracisternal blood injection model, SAH was induced in 24 rabbits, which were randomly divided into 3 groups (group 1= control group, group 2=SAH alone group, and group 3=SAH plus selenium group). Basilar artery angiography was performed on day 0 and day 3 as described. Intraperitoneal selenium (0.05 mg/kg) treatment was started after the induction of SAH and administered once a day. Three days later, the animals were killed and the basilar artery was examined histologically for the luminal diameter and thickness of the arterial muscular wall. The mean values for the measurements of angiographic luminal diameter, pathologic luminal area, muscular wall thickness derived from the blind observer were analyzed statistically. There was no statistically significant difference in basal angiographic luminal diameter evaluation between groups 1-2-3 (P>0.005). But in third day angiography; comparison of group 2 and group 1-3 showed statistically significant differences (P<0.001). In pathologic investigation; there was statistically significant difference in luminal area and muscular wall thickness of the basilar artery between groups 1, 2, and 3 (P<0.005). Intraperitoneal selenium treatment was found effective by increasing the angiographic diameter; pathologic luminal area and reducing muscular wall thickness measurements. This is the first study to show that intraperitoneal administration of selenium is effective in preventing vasospasm after SAH in rabbits.

Effect of a chloride channel inhibitor, 5-nitro-2- (3-phenylpropylamino)-benzoate, on endothelin-1 induced vasoconstriction in rabbit basilar artery.

AIM: Endothelin-1 (ET-1) has been implicated in the pathophysiology of cerebral vasospasm. Chloride (Cl-) channels exist in vascular smooth muscle and activation of these channels leads to depolarization and contraction. The aim of the present study was to test the effect of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a Cl- channel antagonist, on the ET-1-induced cerebral vasospasm in rabbit basilar artery and thus investigate the contribution of Cl- channels. MATERIAL AND METHODS: Thirty rabbits were divided into five groups and received intra-arterial injection of isotonic saline (Group I, n=6), ET-1 (group II, n=6), ET-1 plus NPPB (Group III, n=6), dimethylsulfate (DMSO4) (Group IV, n = 6) and NPPB (Group V, n=6). Pre and post injection basilar artery diameters were measured in each group and transmission electron microscopic investigations on basilar arteries were performed. RESULTS: The mean pre-injection and post-injection vessel diameters were 0.8833 mm and 0.7000 mm in ET-1 group, 0.6833 mm and 0.8500 mm in ET-1 + NPPB group. NPPB administered prior to ET-1 injection, prevented the ET-1-induced vasoconstriction. Additionally, NPPB prevents the ET-1 induced changes in vessel wall and neurons in the brain stem. CONCLUSION: The results of this study add further insights to our armamentarium against cerebral vasospasm.

Ginsenoside-Rd, a new voltage-independent Ca2+ entry blocker, reverses basilar hypertrophic remodeling in stroke-prone renovascular hypertensive rats.

The total saponins of Panax notoginseng have been clinically used for the treatment of cardiovascular diseases and stroke in China. Our recent study has identified ginsenoside-Rd, a purified component of total saponins of P. notoginseng, as an inhibitor to remarkably inhibit voltage-independent Ca(2+) entry. We deduced a hypothesis that the inhibition of voltage-independent Ca(2+) entry might contribute to its cerebrovascular benefits. Ginsenoside-Rd was administered to two-kidney, two-clip (2k2c) stroke-prone hypertensive rats to examine its effects on blood pressure, cerebrovascular remodeling and Ca(2+) entry in freshly isolated basilar arterial vascular smooth muscle cells (BAVSMCs). Its effects on endothelin-1 induced Ca(2+) entry and cellular proliferation were assessed in cultured BAVSMCs. The results showed that, in vivo, ginsenoside-Rd treatment attenuated basilar hypertrophic inward remodeling in 2k2c hypertensive rats without affecting systemic blood pressure.During the development of hypertension, there were time-dependent increases in receptor-operated Ca(2+) channel (ROCC)-, store-operated Ca(2+) channel (SOCC)- and voltage dependent Ca(2+) channel (VDCC)-mediated Ca(2+) entries in freshly isolated BAVSMCs. Ginsenoside-Rd reversed the increase in SOCC- or ROCC- but not VDCC-mediated Ca(2+) entry. In vitro, ginsenoside-Rd concentration-dependently inhibited endothelin-1 induced BAVSMC proliferation and Mn(2+) quenching rate within the same concentration range as required for inhibition of increased SOCC- or ROCC-mediated Ca(2+) entries during hypertension. These results provide in vivo evidence showing attenuation of hypertensive cerebrovascular remodeling after ginsenoside-Rd treatment. The underlying mechanism might be associated with inhibitory effects of ginsenoside-Rd on voltage-independent Ca(2+) entry and BAVSMC proliferation, but not with VDCC-mediated Ca(2+) entry.

Melatonin reduces experimental subarachnoid hemorrhage-induced oxidative brain damage and neurological symptoms.

Oxidative stress has detrimental effects in several models of neurodegenerative diseases, including subarachnoid hemorrhage (SAH). This study investigated the putative neuroprotective effect of melatonin, a powerful antioxidant, in a rat model of SAH. Male Wistar albino rats were divided as control, vehicle-treated SAH, and melatonin-treated (10 mg/kg, i.p.) SAH groups. To induce SAH, 0.3 mL blood was injected into cisterna magna of rats. Forty-eight hours after SAH induction, neurological examination scores were measured and the rats were decapitated. Brain tissue samples were taken for blood-brain barrier (BBB) permeability, brain water content, histological examination, or determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO), and Na+-K+-ATPase activities. Formation of reactive oxygen species in brain tissue samples was monitored by using a chemiluminescence (CL) technique. The neurological examination scores were increased in SAH groups on the second day of SAH induction and SAH caused a significant decrease in brain GSH content and Na+-K+-ATPase activity, which was accompanied with significant increases in CL, MDA levels, and MPO activity. On the other hand, melatonin treatment reversed all these biochemical indices as well as SAH-induced histopathological alterations, while increased brain water content and impaired BBB were also reversed by melatonin treatment. This study suggests that melatonin, which can easily cross BBB, alleviates SAH-induced oxidative stress and exerts neuroprotection by preserving BBB permeability and by reducing brain edema.

On the presence of neurotrophin p75 receptor on rat sympathetic cerebrovascular nerves.

Although the presence of neurotrophin p75 receptor on sympathetic nerves is a well-recognised feature, there is still a scarcity of details of the distribution of the receptor on cerebrovascular nerves. This study examined the distribution of p75 receptor on perivascular sympathetic nerves of the middle cerebral artery and the basilar artery of healthy young rats using immunohistochemical methods at the laser confocal microscope and transmission electron microscope levels. Immunofluorescence methods of detection of tyrosine hydroxylase (TH) in sympathetic nerves, p75 receptor associated with the nerves, and also S-100 protein in Schwann cells were applied in conjunction with confocal microscopy, while the pre-embedding single and double immunolabelling methods (ExtrAvidin and immuno-gold-silver) were applied for the electron microscopic examination. Immunofluorescence studies revealed "punctuate" distribution of the p75 receptor on sympathetic nerves including accompanying Schwann cells. Image analysis of the nerves showed that the level of co-localization of p75 receptor and TH was low. Immunolabelling applied at the electron microscope level also showed scarce co-localization of TH (which was intra-axonal) and p75. Immunoreactivity for p75 receptor was present on the cell membrane of perivascular axons and to a greater extent on the processes of accompanying Schwann cells. Some Schwann cell processes were adjacent to each other displaying strong immunoreactivity for p75 receptor; immunoreactivity was located on the extracellular sites of the adjacent cell membranes suggesting that the receptor was involved in cross talk between these. It is likely that variability of locations of p75 receptor detected in the study reflects diverse interactions of p75 receptor with axons and Schwann cells. It might also imply a diverse role for the receptor and/or the plasticity of sympathetic cerebrovascular nerves to neurotrophin signalling.

High fat and high fructose diet induced intracranial atherosclerosis and enhanced vasoconstrictor responses in non-human primate.

The present study examined the effect of high fat and high fructose (HFF) diet on the development of atherosclerosis and vascular contractile responses in the cerebral artery and thoracic aorta in non-human primates. Female cynomolgus monkeys (age: 3 to 4 years) were divided into normal control diet (N=5) and HFF diet groups (N=5). Twenty-eight weeks after feeding the HFF diet, total cholesterol and low-density lipoprotein-cholesterol in serum were significantly increased in the HFF diet group compared to the control group. The ultrastructural analyses of the basilar artery and aorta demonstrated the infiltration of lipid-laden foam cells and the appearance of lipid droplet-filled smooth muscle cells in the monkeys fed with the HFF diet. In terms of vascular reactivity, there was significantly greater vasoconstriction of the aorta and basilar artery in response to 5-hydroxytryptamine in the HFF diet group compared to the normal diet-fed group. In addition, KCl-induced vasoconstriction of the basilar arteries was also significantly enhanced in the HFF diet group compared to the normal diet-fed monkeys. In all, our present study has demonstrated that changes in the vascular responsiveness of the cerebral artery and its cellular architecture may manifest into cerebrovascular complications consistent with a pathological state normally observed with the onset and progression of atherosclerosis.

Prevention and reversal of vasospasm and ultrastructural changes in basilar artery by continuous infusion of CGS 35066 following subarachnoid hemorrhage.

Endothelin-1, a potent vasoconstrictive peptide, has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). The goal of this study was to evaluate the effect of continuous intravenous infusion of a highly selective endothelin-converting enzyme-1 inhibitor, CGS 35066, on the prevention and reversal of cerebral vasospasm following SAH. New Zealand white rabbits were subjected to SAH by injecting autologous arterial blood into the cisterna magna. Infusion of CGS 35066 at dosages of 1, 3, or 10 mg/kg/ day was initiated either 1 hr and 24 hrs later in the prevention and reversal protocols, respectively. Animals were sacrificed by perfusion-fixation 48 hrs after SAH induction. The cross-sectional areas of basilar arteries were measured using computer-assisted videomicroscopy. Ultrastructural changes in basilar arteries were determined using electron microscopy. CGS 35066 significantly prevented and reversed the arterial narrowing after SAH in all three groups. The mean cross-sectional areas of arteries from animals in both the prevention and reversal protocol groups that received 10 mg/kg/day of CGS 35066 did not differ significantly from those of the healthy controls. Histological studies of the basilar artery in the 10 mg/kg/day treatment group did not show pathomorphological changes, such as corrugation of the endothelium seen at 2 days after SAH induction or vacuole formation in the endothelial cells noted in the vehicle-treated SAH group. These findings suggest that CGS 35066 is a promising therapeutic agent for the prevention and reversal of cerebral vasospasm after SAH. It also prevents the pathological changes in vascular walls due to SAH.

Dexamethasone preventing contractile and cytoskeletal protein changes in the rabbit basilar artery after subarachnoid hemorrhage.

OBJECT: The aim of this project was to study the perturbations of four smooth-muscle proteins and an extracellular protein, type I collagen, after subarachnoid hemorrhage (SAH) and to examine the possible preventive effects of dexamethasone. METHODS: Using a one-hemorrhage rabbit model, the authors first examined the effects of SAH on the expression of alpha-actin, h-caldesmon, vimentin, smoothelin-B, and type I collagen; second, they studied whether post-SAH systemic administration of dexamethasone (three daily injections) corrected the induced alterations. Measurements were obtained at Day 7 post-SAH. The proteins were studied by performing immunohistochemical staining and using a laser-scanning confocal microscope. Compared with control (sham-injured) arteries, the density of the media of arteries subjected to SAH was reduced for alpha-actin (-11%, p = 0.01) and h-caldesmon (-15%, p = 0.06) but increased for vimentin (+15%, p = 0.04) and smoothelin-B (+53%, p = 0.04). Among animals in which SAH was induced, arteries in those treated with dexamethasone demonstrated higher values of density for alpha-actin (+13%, p = 0.05) and h-caldesmon (+20%, p = 0.01), lower values for vimentin (-55%, p = 0.05), and nonsignificantly different values for smoothelin-B. The density of type I collagen in the adventitia decreased significantly after SAH (-45%, p = 0.01), but dexamethasone treatment had no effect on this decrease. CONCLUSIONS: The SAH-induced alterations in the density of three of four smooth-muscle proteins were prevented by dexamethasone treatment; two of these proteins--alpha-actin and h-caldesmon--are directly related to contraction. This drug may potentially be useful to prevent certain morphological and functional changes in cerebral arteries after SAH.

Evidence that estrogen suppresses rho-kinase function in the cerebral circulation in vivo.

BACKGROUND AND PURPOSE: Premenopausal women are less susceptible to cardiovascular diseases than men or postmenopausal women. Such disease states are often associated with increased vascular RhoA/Rho-kinase activity and decreased activity of nitric oxide (NO). This study tested whether female gender is associated with lower Rho-kinase activity or higher NO activity in cerebral arteries in vivo and whether estrogen contributes to any such gender differences. METHODS: Changes in basilar artery diameter were measured with the use of a cranial window preparation in anesthetized Sprague-Dawley rats. Some female rats were ovariectomized (OVX) and treated subcutaneously daily for 14 days with vehicle (dimethyl sulfoxide) or 17beta-estradiol. Vascular expression of RhoA or Rho-kinase was assessed by Western blotting. RESULTS: The Rho-kinase inhibitor Y-27632 was selectively approximately 3-fold more potent as a cerebral vasodilator in males versus females. Expression of total RhoA or Rho-kinase did not differ between males and females. In OVX rats, vasodilator responses to Y-27632 resembled responses in males. Treatment of OVX rats with 17beta-estradiol normalized the vasodilator effects of Y-27632 to be equivalent to responses in intact female controls. The NO synthase inhibitor N-nitro-l-arginine methyl ester caused approximately 50% greater constriction of the basilar artery in females versus males, but responses in OVX rats treated with either vehicle or 17beta-estradiol did not differ from those recorded in intact females. CONCLUSIONS: These data indicate that vascular Rho-kinase function is suppressed in females because of the effects of estrogen, whereas the higher NO activity in females is estrogen independent.

Basilar artery of the capybara (Hydrochaeris hydrochaeris): an ultrastructural study.

The present study investigated the ultrastructural features of the basilar artery of the largest rodent species, the capybara. The study suggests that the general ultrastructural morphological organization of the basilar artery of the capybara is similar to that of small rodents. However, there are some exceptions. The basilar artery of the capybara contains a subpopulation of 'granular' vascular smooth muscle cells resembling monocytes and/or macrophages. The possibility cannot be excluded that the presence of these cells reflects the remodelling processes of the artery due to animal maturation and the regression of the internal carotid artery. To clarify this issue, more systemic studies are required involving capybaras of various ages.

The attenuation of vasospasm by using a sod mimetic after experimental subarachnoidal haemorrhage in rats.

BACKGROUND: Delayed cerebral vasoconstriction and brain ischemia, are critical problems in the management of a patient affected by rupture of an intracranial aneurysm. Overexpression of Cu-Zn superoxide dismutase (Cu-Zn SOD) can reduce the extent of cerebral vasospasm. We, therefore investigated if vasospasm, can be prevented by a novel, stable, and cell permeable SOD mimetic, MnTBAP [Mn(III) tetrakis (4-benzoic acid) porphyrin] which permeates the biological membranes and scavenges superoxide anions and peroxynitrite. METHODS: 28 rats (225-250 g) were divided equally into four groups: group 1: control; group 2: SAH only; group 3: SAH plus placebo; and group 4: SAH plus MnTBAP. We used a double haemorrhage method to produce SAH. Starting six hours after SAH, 5 mg/kg MnTBAP (Calbiochem, Darmstadt-Germany; Cat. No 475870)) or an equal volume of 0.9% saline (37 degrees C) was administered by intraperitoneal injection twice daily for 5 days to groups 4 and 3 respectively. MnTBAP or 0.9% saline injections were continued up to fifth day after SAH and rats were sacrificed on the fifth day. Brain sections at the level of the pons were examined by light microscopy. Planimetric measurements were made for the cross-sectional areas of the lumen and the vessel wall (intima plus media) of the basilar artery by a micrometer. FINDING: Administration of MnTBAP significantly attenuated the vasoconstriction of the basilar artery in group 4 compared with the groups 2 and 3 (p<0.001). INTERPRETATION: These results suggest that this SOD mimetic (MnTBAP) attenuates delayed cerebral vasoconstriction following experimental SAH and that superoxide anions have a role in the pathogenesis of vasospasm after SAH.

Intra-arterial simultaneous administration of anandamide attenuates endothelin-1 induced vasospasm in rabbit basilar arteries.

BACKGROUND: Anandamide induces not only endothelium-dependent vasodilatation through cannabinoid receptors but also some endothelium- independent vasodilator effect by calcitonin gene-related peptide release through vanilloid receptors. Endothelin-1, a powerful vasoconstrictive peptide derived from endothelial cells, has been shown to be converted to its active form after cleaving by a vascular matrix metalloproteinase which is also involved in inactivation of calcitonin gene-related peptide. The purpose of this study was to investigate whether anandamide inhibits the acute vascular and morphological effects of Endothelin-1 applied intra-arterially on rabbit basilar arteries. METHOD: Fifteen albino rabbits were anaesthetised and underwent placement of a vertebral artery catheter for angiography of the basilar artery. Animals were divided, arbitrarily, into animals in which there was either intra-arterial injection of saline (Group I, n=5), Endothelin-1 (Group II, n=5) and Endothelin-1 and anandamide (Group III, n=5). The diameter of the basilar artery between the pre and post injection angiograms was measured in each of the three groups and transmission electron microscopic investigations on basilar arteries were performed. FINDINGS: Angiographic studies showed that simultaneous administration of anandamide significantly attenuated Endothelin-1 induced vasoconstriction. Furthermore, it was demonstrated that anandamide reversed the morphological changes induced by Endothelin-1 on the vessel wall. INTERPRETATION: These results indicated that anandamide overcomes the angiographic and morphological effects of intrarterially administered ET-1 induced vasospasm in rabbit basilar arteries probably by induction of CGRP related vasodilatation through vanilloid receptors and prevents the acute ET-1 induced ultrastructural vessel wall damage.

Epitope-dependent localization of estrogen receptor-alpha, but not -beta, in en face arterial endothelium.

Rapid, nongenomic effects of 17 beta-estradiol (E(2)) in endothelial cells are postulated to arise from membrane-associated estrogen receptors (ERs), which have not been visualized in vascular tissue. To identify membrane ERs, we used multiple site-directed ER alpha or ER beta antibodies to label en face rat cerebral and coronary arterial endothelia. Western blots revealed a novel 55-kDa ER alpha isoform. Three-dimensional images of cells labeled with these antibodies and markers for the nucleus and caveolin-1 were acquired with a wide-field microscope, deconvolved, and numerically analyzed. We found ER alpha in the nucleus and cell periphery, where one-third colocalized with caveolin-1. The receptor location was dependent on the epitope of the antibody. Human ovarian surface epithelium produced similar results; but in rat myometrium, the distribution was epitope independent and nuclear. ER beta distribution was predominantly intranuclear and epitope independent. A small amount of ER alpha colocalized with ER beta within the nucleus. The results were identical in both arterial preparations and insensitive to E(2). We postulate that the different ER alpha conformations at the membrane, in the nucleus, and between different cell types allow E(2) to trigger cell- and location-specific signaling cascades.

Iatrogenic Chlamydia infection-associated damage in the basilar arterial wall of the rat.

A random finding of intracranial vascular Chlamydia sp. infection in Wistar rats and its associated damage of the component tissues of the basilar arterial wall are described as seen with transmission electron microscopy. The pathological changes observed showed no apparent specificity except for "striding" of the endothelial cells on the elastic lamina, the phenomenon observed in all five rats with basilar arterial Chlamydia infection, and in only two out of 13 rats with no proof of the infection. No atherosclerotic changes were found in the infected artery in rats sacrificed 8 or 26 weeks after the presumed infection.

Posttreatment with adenovirus-mediated gene transfer of calcitonin gene-related peptide to reverse cerebral vasospasm in dogs.

OBJECT: Gene transfer to cerebral vessels is a promising new therapeutic approach for cerebral vasospasm after subarachnoid hemorrhage (SAH). This study was undertaken to explore whether a delayed treatment with adenovirus encoding the prepro-calcitonin gene-related peptide (CGRP), 2 days after initial blood injection, reduces cerebral vasospasm in a double-hemorrhage model of severe vasospasm in dogs. METHODS: In 20 dogs, arterial blood was injected into the cisterna magna on Days 0 and 2. Thirty minutes after the second blood injection, the animals received either adenovirus encoding the prepro-CGRP gene (AdCMVCGRP-treated group, eight dogs) or adenovirus encoding the beta-galactosidase gene (AdCMVbeta gal-treated group, six dogs) under the cytomegalovirus (CMV) promoter. One group of dogs did not receive treatment and served as controls (control SAH group, six dogs). Angiography was performed on Days 0 and 7 to assess cerebral vasospasm. On Day 7 following angiography, the animals were killed and their brains were stained with X-gal to detect the distribution of gene expression. Cerebrospinal fluid (CSF) was also tested for CGRP immunoreactivity. Severe vasospasm was observed in control SAH dogs on Day 7, and the mean basilar artery (BA) diameter was 53.4 +/- 5.5% of the value measured on Day 0. Treatment with AdCMVbeta gal did not alter vasospasm (the BA diameter was 55 +/- 3.9% of that measured on Day 0). The leptomeninges and adventitia of the BAs of dogs treated using AdCMVbeta gal demonstrated positive staining with X-gal. High levels of CGRP were measured in CSF from dogs that received AdCMVCGRP. In the group treated with AdCMVCGRP, vasospasm was significantly reduced (the BA diameter was 78.2 +/- 5.3% of that measured on Day 0, p < 0.05 compared with the control SAH group and the AdCMVbeta gal group). CONCLUSIONS: In a model of severe vasospasm in dogs, gene transfer of CGRP after injection of blood attenuated cerebral vasospasm after SAH.

Improved rat model for cerebral vasospasm studies.

While the rat has been used extensively in subarachnoid hemorrhage (SAH)-cerebral vasospasm studies, concerns exist whether this animal represents a usable model because its time course and pattern of cerebral vasospasm following SAH is not comparable to that observed in man. At present, our knowledge of the rat model is based almost exclusively on studies using a 'single hemorrhage' method. Since there is a positive correlation between severity of cerebral vasospasm, and volume of subarachnoid blood, an obvious question is whether the rat will show modifications in vascular responses when insulted by a second SAH. Here, an SAH was produced in rats using a 'double hemorrhage' method. Following SAH, cerebral arteries showed pathological alterations, significant decreases in luminal perimeter, and increases in arterial wall thickness, over a 7-day post-SAH period. The above vascular features are considered to be indicative of cerebral vasospasm and their presence over a 7-day post-SAH period represents a significant time extension when compared to a single hemorrhage. These modified vascular responses made the double hemorrhaged rat a much-improved animal model.