Canine Double-hemorrhage Model Research Articles

Double cisterna magna blood injection model of experimental subarachnoid hemorrhage in dogs.

Several animal subarachnoid hemorrhage (SAH) models have been proposed to study the etiology and treatment for cerebral vasospasm. We describe the experimental procedures of a canine double-hemorrhage model of SAH and discuss the pathophysiological parameters and occurrence of angiographic delayed cerebral vasospasm using magnetic resonance (MR) imaging and digital subtraction angiography. Autologous blood was injected twice on days 1 and 3 into the cerebellomedullary cistern of 36 female beagles. All animals showed delayed angiographic vasospasm in the vertebrobasilar arteries on day 7. The degree of vasospasm was 29-42% of the arterial diameter. However, this model showed no symptomatic vasospasm or ischemic changes detected by MR imaging. This animal model can produce reproducible delayed vasospasm without detectable cerebral infarction on MR imaging. This model allows evaluation of the effect of treatment on delayed vasospasm in the same animals. The canine double-hemorrhage model of SAH is suitable for the quantitative and chronological study of delayed angiographic vasospasm, but not for investigating early brain injury and delayed cerebral ischemia.

MTOR Inhibitors Reduce Cerebral Vasospasm in a Subarachnoid Double‐Hemorrhage Canine Model

mTOR pathway promotes cell proliferation under environmental stress. In the present study, we investigated the role of mTOR pathway in subarachnoid hemorrhage brain injury. Thirty dogs were randomly divided into 5 groups: sham, SAH, SAH+DMSO, SAH + Rapamycin and SAH+AZD8055. An established canine double‐hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on days 0 and 2. Angiography was performed at days 0 and 7. Clinical behavior, histology, immunohistochemistry, and Western blot of mTOR, P70S6K1, 4E‐BP1 and PCNA in the basilar arteries were examined. Severe angiographic vasospasm was obtained in the SAH and SAH+DMSO dogs compared with in Sham respectively. In the SAH+ RAPA and SAH+ AZD8055 groups, a moderate vasospasm, mTOR, P70S6K1, 4E‐BP1 and PCNA increased in the sample of spastic basilar arteries (p<0.05). Rapamycin and AZD8055 attenuated angiographic vasospasm while improving appetite and activity scores (p<0.05) on days 5 to 7. Rapamycin and AZD8055 significantly reduced the level and expression of mTOR, P70S6K1, 4E‐BP1 and PCNA (p<0.05). In conclusion, mTOR molecular signaling pathway play a significant role in cerebral vasospasm (VSMCs proliferation) following SAH, and mTOR inhibitor has potential to become an attractive strategy to treat vasospasm following SAH.

Mammalian target of rapamycin (mTOR) inhibition reduces cerebral vasospasm following a subarachnoid hemorrhage injury in canines

Mammalian target of rapamycin (mTOR) pathway is a serine/threonine protein kinase that plays a vital role in regulating growth, proliferation, survival, and protein synthesis among cells. In the present study, we investigated the role of the mTOR pathway following subarachnoid hemorrhage brain injury — specifically investigating its ability to mediate the activation of cerebral vasospasm. Additionally, we investigated whether key signaling pathway molecules such as the mTOR, P70S6K1, and 4E-BP1 play a role in the process. Thirty dogs were randomly divided into 5 groups: sham, SAH (subarachnoid hemorrhage), SAH+DMSO (dimethyl sulfoxide), SAH+Rapamycin and SAH+AZD8055. An established canine double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on days 0 and 2. Angiography was performed at days 0 and 7. Clinical behavior, histology, immunohistochemistry, and Western blot of mTOR, P70S6K1, 4E-BP1 and PCNA (proliferating cell nuclear antigen) in the basilar arteries were examined. In the SAH and SAH+DMSO groups, severe angiographic vasospasm was obtained (34.3±19.8%, 38.4±10.3) compared with that in Sham (93.9±5.0%) respectively. mTOR, P70S6K1, 4E-BP1 and PCNA increased in the sample of spastic basilar arteries (p<0.05). In the SAH+RAPA and SAH+AZD8055 groups, Rapamycin and AZD8055 attenuated angiographic vasospasm (62.3±15.9% and 65.2±10.3%) while improving appetite and activity scores (p<0.05) on days 5 through 7. Rapamycin and AZD8055 significantly reduced the level and expression of mTOR, P70S6K1, 4E-BP1 and PCNA (p<0.05). In conclusion, our study suggests that the mTOR molecular signaling pathway plays a significant role in cerebral vasospasm following SAH, and that inhibition of the mTOR pathway has the potential to become an attractive strategy to treat vasospasm following SAH.

Magnetic Resonance Imaging Detects and Predicts Early Brain Injury after Subarachnoid Hemorrhage in a Canine Experimental Model

The canine double hemorrhage model is an established model to study cerebral vasospasm, the late sequelae of subarachnoid hemorrhage (SAH). The present study uses magnetic resonance imaging (MRI) to examine the recently reported early brain injury after SAH. Double hemorrhage SAH modeling was obtained by injecting 0.5 mL/kg of autologous arterial blood into the cisterna magna of five adult mongrel dogs on day 0 and day 2, followed by imaging at day 2 and day 7 using a 4.7-Tesla (T) scanner. White matter (WM) showed a remarkable increase in T2 values at day 2 which resolved by day 7, whereas gray matter (GM) T2 values did not resolve. The apparent diffusion coefficient (ADC) values progressively increased in both WM and GM after SAH, suggestive of a transition from vasogenic to cytotoxic edema. Ventricular volume also increased dramatically. Prominent neuronal injury with Nissl's staining was seen in the cortical GM and in the periventricular tissue. Multimodal MRI reveals acute changes in the brain after SAH and can be used to non-invasively study early brain injury and normal pressure hydrocephalus post-SAH. MR can also predict tissue histopathology and may be useful for assessing pharmacological treatments designed to ameliorate SAH.

Gene expression in a canine basilar artery vasospasm model: a genome-wide network-based analysis

To investigate the changes of gene expression on the cerebral vasospasm after subarachnoid hemorrhage, we used genome-wide microarray for a canine double-hemorrhage model and analyzed the data by using a network-based analysis. Six dogs were assigned to two groups of three animals: control and hemorrhage. The effects were assessed by the changes in gene expressions in the artery 7 days after the first blood injection. Among 23,914 genes, 447 and 66 genes were up-regulated more than two- and fivefold, respectively, and 332 and 25 genes were down-regulated more than two- and fivefold, respectively. According to gene ontology, genes related to cell communication (P = 5.28E-10), host–pathogen interaction (7.65E-8), and defense–immunity protein activity (0.000183) were significantly overrepresented. The top high-level function for the merged network derived from the network-based analysis was cell signaling, revealing that the subgroup that regulates the quantity of Ca2+ to have the strongest association significance (P = 4.75E-16). Canine microarray analysis followed by gene ontology profiling and connectivity analysis identified several functional groups and individual genes responding to cerebral vasospasm. Ca2+ regulation may play a key role in these gene expression changes and may be involved in the pathogenesis of cerebral vasospasm.

Gene Transfer of Extracellular Superoxide Dismutase Failed to Prevent Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage

We examined the therapeutic effect of human extracellular superoxide dismutase (ECSOD) gene transfer in the prevention of delayed cerebral vasospasm after experimental subarachnoid hemorrhage (SAH) because it was reported ECSOD relieved early-stage vasospasm. Twenty mongrel dogs were divided randomly into 4 groups to serve as control, SAH, SAH+adenovirus ECSOD (AdECSOD), and SAH+no transgene (AdBglII) groups, respectively. An established canine double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on day 0 and day 2. Angiography was performed at day 0 and day 7. Clinical behavior, cerebrospinal fluid (CSF) ECSOD activity, CSF leukocyte count, morphology, and human ECSOD expression (RT-PCR) in the basilar arteries were evaluated. Severe vasospasm was obtained in SAH, SAH+AdECSOD, and SAH+AdBglII gene-transferred dogs, and the residual diameters of the basilar artery were 41+/-1%, 39+/-4%, and 49+/-4%, respectively. Increased CSF activity of ECSOD was obtained in SAH+AdECSOD (162+/-23 U/mL) when compared with SAH (26+/-2) and SAH+AdBglII (25+/-3) dogs. RT-PCR confirmed successful gene transfer in the basilar arteries from SAH+AdECSOD dogs. Increased leukocyte counts were observed in the CSF and in the subarachnoid space, especially in SAH+AdECSOD and SAH+AdBglII dogs. Gene transfer of human ECSOD failed to prevent delayed cerebral vasospasm.

Ras Protein Contributes to Cerebral Vasospasm in a Canine Double-Hemorrhage Model

Mitogen-activated protein kinase (MAPK) has been shown to be involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). In the present study we examined the role of Ras protein, an upstream regulator of MAPK, and the effects of the inhibitors of Ras farnesyltransferase (FTase), FTI-277 and FTase inhibitor I, on angiographic vasospasm and clinical evaluations. Twenty-five dogs were randomly divided into 5 groups: control, SAH, SAH+dimethyl sulfoxide, SAH+FTI-277, and SAH+FTase inhibitor I. An established canine double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on days 0 and 2. Angiography was performed at days 0 and 7. Clinical behavior and the activation of Ras (GTP-Ras) and phosphorylated ERK1/2 of MAPK in the basilar arteries were examined. Severe vasospasm was obtained in the SAH and SAH+dimethyl sulfoxide dogs (42.5+/-2.5% and 38.9+/-2.4%, respectively). Enhanced GTP-Ras and phosphorylated ERK1/2 were observed in the spastic basilar arteries (P<0.05). Inhibitors of Ras FTase decreased GTP-Ras and phosphorylated ERK1/2, attenuated angiographic vasospasm, and improved appetite and activity scores. Ras contributes to cerebral vasospasm, and inhibitors of Ras FTase may have potential in the management of cerebral vasospasm.

Contribution of Src tyrosine kinase to cerebral vasospasm after subarachnoid hemorrhage.

Mitogen-activated protein kinase (MAPK) has been implicated in cerebral vasospasm after subarachnoid hemorrhage (SAH). This study was conducted to investigate whether Src tyrosine kinase, an upstream regulator of MAPK, is involved in cerebral vasospasm. An established canine double-hemorrhage model was used. Twenty-four dogs were divided into four groups: control, vehicle-treated, Src inhibitor PP2-treated, and Src inhibitor damnacanthal-treated groups. Vehicle (dimethyl sulfoxide), PP2, or damnacanthal was injected daily into the cisterna magna of 18 dogs at 3 to 6 days after induction of SAH. Angiography was performed on Day 0 (the day on which the first blood injection was administered to induce SAH) and on Day 7. Western blot analysis of Src and MAPK activation in basilar arteries (BAs) collected on Day 7 post-SAH was performed. Severe vasospasm was observed in the BAs of vehicle-treated dogs. Mild vasospasm was observed in all dogs treated with Src inhibitors. Phosphorylated Src and MAPK were increased after SAH and activation of these kinases in the BAs was abolished by PP2 and damnacanthal. The tyrosine kinase Src is an important upstream regulator of MAPK, and inhibition of Src might offer a new therapy in the management of cerebral vasospasm.

Effect of edaravone on cerebral vasospasm following experimental subarachnoid hemorrhage

The effect of the free radical scavenger edaravone on experimental cerebral vasospasm following subarachnoid hemorrhage (SAH) was investigated in a canine double hemorrhage model. Changes in the diameter of the basilar artery were assessed by serial angiography. The diameter ratio at day 7 was calculated as the percent of the basilar artery diameter of a given angiogram with respect to that of its control (day 0). The diameter ratios for the basilar artery following SAH in the control and vehicle-treated groups were 49.7% ± 3.9% (mean ± SEM) and 50.1% ± 1.7%, respectively. Edaravone was administered either by continuous intravenous injection for 7 days or by bolus injection for 7 days. Continuous administration of edaravone (1 mg/kg/hr or 10 mg/kg/hr) significantly attenuated the narrowing of the basilar artery following SAH. The diameter ratios in these groups were 71.3% ± 3.6% (1 mg/kg/hr) and 75.7% ± 1.7% (10 mg/kg/hr). Bolus administration of edaravone (3 mg/kg, every 12 hours) reduced the arterial narrowing following SAH. The diameter ratio on day 7 was 60.1% ± 3.3%, but the difference was not significant. These findings suggest that edaravone is effective in preventing cerebral vasospasm following SAH. Copyright © 2003 by National Stroke Association

Effect of a platelet-activating factor antagonist, E5880, on cerebrovasospasm following subarachnoid hemorrhage in a canine double-hemorrhage model

We investigated the effects of a platelet-activating factor (PAF) antagonist, E5880 (1-ethyl-2-[ N-(2-methoxy)benzoyl- N-[(2)-2-methoxy-3-(4-octadecycarbamoylox)piperidinocarbonyloxy-propyloxy]carbonyl]aminomethyl-pyridiniumchloride), on subarachnoid hemorrhage-induced prolongation of cerebral circulation time and decrease in the basilar artery diameter in a canine double-hemorrhage model. Animals were assigned to three groups, control (saline), E5880 1.2 mg/kg and E5880 2.4 mg/kg. For measurement of cerebral circulation time, regions of interest were chosen at the basilar artery and the straight sinus in order to obtain time–density curves. Cerebral circulation time was defined as the difference between the arterial and venous peaks. Cerebral circulation time and basilar artery diameter were assessed by intra-arterial digital subtraction angiography (IA-DSA) on Days 0, 2 and 7. The prolongation of cerebral circulation time following subarachnoid hemorrhage was significantly inhibited by intravenous administration of 2.4 mg/kg of E5880. Basilar artery constriction was also reduced by E5880. Thus, E5880 had preventive effects on the prolongation of cerebral circulation time and the vasoconstriction of basilar artery in this model. These results suggest that E5880 may have a preventive effect on neurological symptoms aggravated by cerebrovascular lesions following subarachnoid hemorrhage.

Suramin-induced reversal of chronic cerebral vasospasm in experimental subarachnoid hemorrhage.

The naphthylsulfonate derivative suramin is an inhibitor of growth factor receptors (receptor tyrosine kinases) and G protein-coupled P2Y receptors. Both types of these receptors are suspected of being involved in cerebral vasospasm after subarachnoid hemorrhage (SAH). In the current study, the authors examined the therapeutic effects of suramin and a selective P2X-receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in the reversal of vasospasm in an established canine double-hemorrhage model. Twenty-four dogs underwent double blood injection into the cisterna magna, with injections given on Days 0 and 2. The dogs were divided randomly into three groups (six animals in each group) to be treated from Days 2 through 6 with the vehicle dimethyl sulfoxide, suramin, or PPADS. An additional group of six dogs received double blood injection without any treatment and served as an SAH control group. The animals were killed on Day 7. Angiography was performed on Day 0 before blood injection and again on Day 7 before the animals were killed. After the death of the animals, the basilar arteries (BAs) were collected for morphological studies and determination of tyrosine kinase expression, and the bloody cerebrospinal fluid (CSF) produced by the hemorrhages was collected for measurement of oxyhemoglobin and adenosine triphosphate (ATP). In the SAH control group, the mean diameter of the BAs on Day 7 was 46.23 +/- 6.32% of the value on Day 0 (which served as a reference of 100%). In the DMSO-treated group, the mean residual diameter of the BA was 47.77 +/- 0.8% on Day 7 compared with the value on Day 0. Suramin, but not PPADS, increased the residual diameter to 74.02 +/- 4.24% on Day 7. On Day 7 the level of ATP in the CSF was decreased and the level of oxyhemoglobin was increased, compared with values measured on Day 0. Suramin, but not PPADS, reduced tyrosine phosphorylation in the spastic BAs. By reducing tyrosine kinase activity, suramin may be useful in the treatment of cerebral vasospasm.

Morphological changes of cerebral arteries in a canine double hemorrhage model

Cerebral vasospasm is a major cause of morbidity and mortality in patients suffering from subarachnoid hemorrhage (SAH). Despite numerous studies, the pathogenesis of this deadly disorder is not clearly understood. Alterations in endothelial cells are a distinct morphological feature of cerebral vasospasm and some recent studies suggest that apoptosis might play a role in the cells’ death. The goal of the present study is to examine the time course of apoptosis in endothelial cells of spastic cerebral arteries following experimental subarachnoid hemorrhage. Fifteen dogs were used in the present study. Twelve of them were divided into three groups (four per group) and subjected to a double-hemorrhage method of SAH. Following SAH, groups were sacrificed respectively on days 3, 5, and 7. Three dogs served as controls without blood injection. The basilar arteries were studied with the transmission electron microscopy and with angiography. Angiographic vasospasm began on day 3 and peaked on day 7. In morphologic studies, control dogs did not demonstrate apoptotic-like changes in endothelial cells of the basilar arteries. Beginning with day 3, apoptotic-like changes were noted in endothelial cells and consisted of condensation of peripheral nuclear chromatin, blebbing of the cell membrane, and condensation of the cytoplasm. Such changes progressed with time and were maximally developed by day 7. This is the first study that demonstrates the time course of apoptotic-like changes in the endothelial cells in the vasospastic basilar artery. Apoptosis might play an important role in the pathogenesis of vasospasm.

Phosphatidylinositol 3-kinase inhibitor failed to reduce cerebral vasospasm in dog model of experimental subarachnoid hemorrhage.

Phosphatidylinositol 3-kinase (PI3-kinase) is involved in smooth muscle contraction induced by growth factors and/or G protein-coupled receptor agonists. To evaluate the role of PI3-kinase in the pathogenesis of delayed vasospasm, we applied 2 PI3-kinase inhibitors to an established canine double-hemorrhage model of experimental subarachnoid hemorrhage. Twenty-four dogs underwent double blood injections via the cisterna magna on days 0 and 2. The dogs were killed on day 7. Dogs were treated with either vehicle (dimethyl sulfoxide), wortmannin, or LY294002 once per day on day 2 through day 6. Angiography was performed before blood injection and before the dogs were killed. The basilar arteries were collected for morphology, Western blot analysis, and PI3-kinase activity. The residual diameter of the basilar arteries in the dimethyl sulfoxide treatment group, which was compared with day 0 angiogram, decreased markedly on day 7 (the percentage of the residual diameter was 47.8+/-0.8%). Wortmannin and LY294002 did not significantly change residual diameter on day 7. Both PI3-kinase inhibitors abolished PI3-kinase activity compared with the vehicle treatment group. However, both PI3-kinase inhibitors failed to significantly attenuate PI3-kinase protein expression (Western blot) (P>0.05, ANOVA). Delayed treatment, which was to mimic the clinical situation, with PI3-kinase inhibitors failed to reverse vasospasm. PI3-kinase may not play an important role in the delayed vasospasm. The possible effect of PI3-kinase inhibitors in the early stage of vasospasm was not investigated in the present study.

Apoptosis in basilar endothelial cells in a canine double hemorrhage model.

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a combination of prolonged contraction and vessel remodeling that may lead to cerebral ischemia or infarction [2]. Morphological studies of cerebral arteries affected by vasospasm often show extensive necrosis of smooth-muscle cells [1] as well as desquamation and dystrophy of endothelial cells [7, 11]. The actual mechanisms of cellular death following cerebral vasospasm are not known. Recent studies of post-SAH vasospasm in this laboratory strongly suggest that endothelial cells may be dying through apoptotic pathways [8, 10].

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.

Morphological changes of cerebral penetrating arteries in a canine double hemorrhage model

BACKGROUND Morphological presentations of cerebral vasospasm, such as dystrophy and desquamation of endothelial cells, corrugation of the internal elastic layer, and necrotic changes in smooth muscle cells, are well defined in large cerebral arteries. This study was undertaken to examine pathological changes in cerebral penetrating arteries in a canine double hemorrhage model. METHODS Eighteen mongrel dogs were subjected to an autologous arterial blood (0.4 mL/kg) injection into the cisterna magna on day 0 and day 2 after withdrawal of an equivalent amount of cerebrospinal fluid. Angiogram was performed on day 0 before the blood injection and on the day the dogs were sacrificed. The dogs were divided into four groups: control (day 0) ( n = 4), hemorrhage and sacrificed on day 3 ( n = 4), day 5 ( n = 5), and day 7 ( n = 5). The penetrating arteries were removed and found to be spastic on days 3, 5, and 7, but not in the control group. RESULTS Endothelial dystrophy and partial desquamation were recorded in all dogs sacrificed on days 5 and 7. Condensation of chromatin, blebbing of the membrane, and condensation of cytoplasm were identified in many endothelial cells, features that are consistent with apoptosis. The morphological changes in the penetrating arteries were more pronounced on days 5 and 7. CONCLUSIONS Vasospasm occurred in cerebral penetrating arteries in a canine double hemorrhage model. The morphological change in penetrating arteries, especially apoptosis in endothelial cells, is consistent with an early phase of vasospasm. Vasospasm in a penetrating artery may contribute to the cerebral ischemia that occurs during vasospasm.

Prevention of cerebral vasospasm by a novel endothelin receptor antagonist, TA-0201.

This study was designed to examine the preventive effect of a novel endothelin (ET)-receptor antagonist TA-0201 on the cerebral vasospasm in a canine double-hemorrhage model. TA-0201 (10(-9)-10(-7) M) inhibited ET-1-induced vasoconstriction in the isolated canine basilar artery without endothelium in a concentration-dependent manner. Its pA2 value was 9.2 (ET(A) antagonism). In a canine double-hemorrhage model, intravenous treatment with TA-0201 (3 mg/kg, twice a day for 7 days) ameliorated the basilar artery narrowing significantly on day 7 compared with that in nontreated dogs. The reductions of the basilar artery diameter were 26.1+/-3.9% and 40.5+/-4.1% with and without TA-0201 treatment, respectively (p<0.05). Histologic study on day 7 indicated that treatment with TA-0201 inhibited vessel-wall damage such as disintegration of endothelium architecture and degeneration of medial smooth-muscle cells. We conclude that intravenous treatment with TA-0201 prevents the development of cerebral vasospasm and accompanying pathologic changes of the vessel wall, probably through blockade of ET(A) receptors.