Joan R. Coates, DVM, MS, DACVIM (Neurology)
Cerebrovascular accidents (CVAs) are a major cause of disability in humans and dogs. CVAs are classified based on 2 major causes: vascular obstruction and rupture of blood vessels that lead to infarction and hemorrhage, respectively. In humans, head injury is the most common cause of traumatic subarachnoid hemorrhage (SAH) and aneurysmal rupture accounts for more than 80% of nontraumatic SAH.1 Cerebral vasospasm is considered a major complication of SAH and continues to account for increased morbidity and mortality following aneurysmal subarachnoid hemorrhage (SAH).2 Vasospasm has been classified based on clinical and radiographic findings. Clinical vasospasm may resolve spontaneously or progress to cerebral infarction. Angiographic vasospasm is documented in about 60% of patients following SAH but becomes symptomatic in only half of those. Delayed ischemic deficits develop in about 16% of patients with SAH. In 1978, Weir and his colleagues established that cerebral vasospasm in humans begins at about day 3 after SAH, was maximal at 6-8 days, and was gone by day 12.3 The mainstay of therapy for cerebral vasospasm is the combination of hypervolemia, hemodilution, and hypertension ("triple-H therapy"). 2 If conventional therapy is unsuccessful then endovascular therapy is pursued.
Causes of cerebral infarction or hemorrhage have been classified in dogs.4 Increased use of magnetic resonance imaging has brought forth greater awareness of CVAs in veterinary medicine.5-7 Retrospective studies of dogs with CVAs have shown that dogs with other medical conditions had shorter survival times and more likely to have recurrence.8 CVAs can occur in the CNS of dogs from hemorrhage due to primary or secondary causes.9-10 The incidence of SAH and associated cerebral vasospasm in dogs is unknown but the process has been suspected.9,11
Pathophysiology of Cerebral Vasospasm
The pathophysiology of cerebral vasospasm is still not fully understood but endothelial dysfunction and inflammation play major roles in its development. Two salient points emerge from the existing literature concerning cerebral vasospasm. First, there is a decrease in nitric oxide (NO). The second is that there is an intense inflammatory response associated with persistent blood products in the subarachnoid space.
Impaired NO release combined with the inflammatory response forms a continuum that manifests itself as cerebral vasospasm. The initial etiology of vasospasm may result from an imbalance between vasodilation and vasoconstriction.12 Vascular endothelium regulates smooth muscle tone by generating NO and endothelial derived constriction factors.13 Disruption of endothelium or its relaxing factors may alter this balance, predisposing to vasospasm.14 NO depletion accompanies these changes, suggesting that SAH-induced endothelial dysfunction contributes to loss of NO. Furthermore, NO replacement reverses cerebral vasospasm in animal studies.15,16
Nitric oxide synthase, the primary source of NO in vascular tone regulation, metabolizes L-arginine to NO and citrulline. The endothelial isoform (eNOS) is constitutively expressed in cerebrovascular endothelium. NO is produced by a variety of sources such as vascular endothelium, neuron, glia, macrophages and white blood cells. Three isoforms of NOS have been identified as neuronal, endothelial and inducible. NO has a dual role in ischemic neuropathology: 1) beneficial--as a potent vasodilator; 2) cytotoxic--inhibition of enzyme systems such as complexes in the mitochondrial transport chain and formation of oxidant peroxynitrite. Immunoreactivity for eNOS mRNA and protein has been shown to decrease following SAH. Although the mechanism of nitric oxide synthase reduction is not known, it likely causes reduction of NO production and disruption of endothelial dependent vasodilation following SAH.
The 3-hyroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, also referred to as statins, are potent inhibitors of cholesterol biosynthesis.17 Statins also directly up-regulate eNOS expression under cholesterol controlled conditions.18,19 An increase in eNOS mRNA, protein, and enzymatic activity has been demonstrated following statin treatment, resulting in increased cerebral blood flow.18 Selective up-regulation of eNOS activity by statin treatment may prevent eNOS depletion or even increase eNOS expression after SAH.18 Simvastatin upregulates NOS and has been shown to ameliorate cerebral vasospasm.16,20
Evidence suggests that an inflammatory response may be involved in the development of cerebral vasospasm.21,22 Intimal, medial, and subarachnoid infiltration by macrophages, neutrophils, and other inflammatory cells has been noted after SAH in human autopsy studies and experimentally in the dog.14 It has also been demonstrated that severe inflammation in the perivascular space of cerebral arteries can provoke moderate to severe and persistent vasospasm.23 Concentrations of circulating serum immunocomplexes are increased during cerebral vasospasm.24-26 Furthermore, a postmortem study has shown increased deposition of immunoglobulin and complement protein in the walls of human cerebral vessels exposed to subarachnoid blood clot.13 The time course for cerebral vasospasm parallels that of a delayed-type hypersensitivity or chronic allergic reaction when lymphocyte subsets in the CSF of rats after SAH are analyzed.27 Denaturation of the subarachnoid erythrocyte results in a massive infiltration of inflammatory and immunoreactive cells.28-30 The delay till the onset of cerebral vasospasm was 4 to 14 days which parallels a time-frame for immunological reaction against the aging human subarachnoid erythrocyte. Drugs that reduce inflammation such as corticosteroids improved outcome after phase 2 and phase 3 trials in humans and reduced cerebral vasospasm in animal models.31-33 Drugs that suppress cell mediated immunity such as FK506 do not reduce cerebral vasospasm.34 Cyclosporin A interferes with T-helper lymphocyte interleukin production. This limits the activation of the killer T lymphocytes ultimately resulting in immunosuppression. In both dog and nonhuman primate models, amelioration of cerebral vasospasm after administration of cyclosporine A has been reported.35,30 The results using cyclosporine in human trials have been conflicting.36,37
A Canine Model of Subarachnoid Hemorrhage Evaluating Treatments (Study Funded by the University of Missouri Research Board--Bulsara KR, Coates JR)
We proposed a two-hit theory for cerebral vasospasm. The first is that cerebrovascular nitric oxide synthase is decreased following SAH. This results in decreased nitric oxide (NO) production which compromises cerebral vasodilation.12,13,19,38 The persistent presence of deoxyhemoglobin in the subarachnoid space is the second and ultimate hit. Denaturation of the subarachnoid erythrocyte results in a massive inflammatory response with resultant vasoconstriction. The impaired vasodilation mechanism now allows unopposed vasoconstriction in a time-frame that parallels an immunological reaction against the aging subarachnoid erythrocyte.3 Research studies addressing cerebral vasospasm have frequently treated these issues independently.21,16,28,37 We hypothesize that upregulating nitric oxide synthase following SAH (simvastatin) and suppressing the inflammatory reaction (cyclosporine) will ameliorate cerebral vasospasm. If the combination of these drugs reduces cerebral vasospasm in a canine model to a greater extent than either one alone, it may have significant clinical implications. In this study, we tested the hypothesis that simultaneously upregulating nitric oxide synthase following SAH and suppressing the inflammatory reaction would ameliorate cerebral vasospasm to a greater extent than doing either alone.
We used a previously described double-hemorrhage model in dogs.30,34,39 Thirteen dogs were assigned to one of three groups: Control-untreated (n=5); Simvastatin (Zocor, Merck Inc., 20 mg/kg SID PO) only (n=4); simvastatin (20 mg/kg SID PO) and cyclosporine A (Sandimmune, Sandoz Inc., 6 mg/kg SID PO) (n=4). A double SAH model was induced in dogs by 2 injections (3 mls) of autologous blood into the cerebellomedullary cistern (CMC) 24 hours apart. Baseline basilar arterial angiogram and CMC CSF analysis were obtained. Drugs were administered 24 hours after the second injection for 10 days. CSF was collected from the CMC before each injection and on days 3, 7, and 10 and immediately analyzed and stored at -80°C. Angiograms were repeated on days 3, 7, and 10. Measurements of the basilar artery diameter were taken from just distal to the confluence of the vertebral arteries to just proximal to the bifurcation of the caudal cerebral arteries. The basilar artery was divided into 10 approximately equal segments in length of which individual vessel diameter measurements were obtained using units and scale of pixels in the PhotoshopTM program. A mean average of the narrowest regions of the basilar artery was taken for the 40%, 50% and 60% data points. Dogs were humanely sacrificed on day 10. CSF concentrations of neurotransmitters (GABA, glycine, glutamate and aspartate) also were investigated as biomarkers of neuronal injury.
We summarize results pertaining to angiography, CSF studies, and ophthalmic findings. Results may be confounded by small sample size and concurrent independent effects of hemorrhage and inflammation notably present in SAH models.
Basilar Arterial Angiogram
Neurologic examination was normal in all dogs following procedures. Decreased basilar artery diameter was seen on day 3 in the control and simvastatin/cyclosporine group. A return to baseline diameters was seen by day 7. An increase from baseline diameter was seen in the simvastatin group at day 10. The vasodilation effect seen in simvastatin is consistent with previous findings that simvastatin augments NO-mediated vasodilation and its effects may persist.40 Cyclosporine may interfere with the vasodilatory effects of simvastatin. Though there is no doubt that cyclosporine can effectively suppress the immune system, there is concern that it can also cause vasoconstriction. It has been shown to cause an acute release of prostanoid thromboxane in the myocardium resulting in coronary vasospasm.41 It also has been associated with endothelial injury and reversible vasospasm resulting in encephalopathy.42-45 The combination of simvastatin and cyclosporine does not ameliorate cerebral vasospasm in a canine model to a greater extent than simvastatin alone.46
We also determined effects of simvastatin and cyclosporine on cerebrospinal fluid (CSF) analysis in a canine model of subarachnoid hemorrhage (SAH) induced vasospasm. CSF analysis revealed significantly elevated total protein (TP) concentration and red blood cell count (RBCC) in all groups on day 1. Total nucleated cell count (TNCC) was significantly elevated in control-untreated and cyclosporine/simvastatin groups on day 3. Total protein, RBCC, and TNCC returned to baseline values by day 10. Decreases in vasospasm with treatment correlated with decreased CSF inflammation. Subarachnoid hemorrhage resulted in elevations in TP, RBCC and TNCC that returned to baseline values by day 10. This study demonstrates importance of careful interpretation of CSF analyses in dogs with SAH or blood contaminated samples.
Subarachnoid hemorrhage (SAH) is characterized by decreased cerebral blood flow, subsequent cerebral vasospasm and ischemia, and high mortality in people. Impaired endothelial and neuronal nitric oxide (NO) release further lead to inflammation and excitoxicity triggered by excitatory amino acids, glutamate and aspartate.47,48 We hypothesized that dogs with SAH have alterations in CSF concentrations of glutamate, aspartate, GABA and glycine which are indirectly but positively affected by use of cyclosporine and simvastatin. CSF concentrations of neurotransmitters were investigated as biomarkers of neuronal injury and indicators of beneficial treatments for CNS ischemia.
In the control group, glutamate significantly increased to highest levels by day 3 and then returned to baseline, whereas glycine, GABA and aspartate were not significantly altered from baseline at any time point. There was significant decremental effect of simvastatin alone and in combination with cyclosporine on day 3 glutamate concentrations when compared to the control group. A significant incremental effect of combination treatment on day 3 glycine levels was noted compared to control group. No significant differences in GABA and aspartate levels were noted between treatment groups on any of the sample days.
Although precise roles of these neurotransmitters have not been elucidated in pathophysiology of canine CNS ischemia, their alterations from baseline suggest further investigation. A combination of immunosuppression and NO synthase upregulation may be useful in ameliorating elevated glutamate levels in CNS ischemia.
We were able to document presence of scleral hemorrhage in dogs of the above described study. Complete ophthalmic examination was performed on all dogs within 1-3 days of treatment initiation. Bilateral scleral hemorrhage predominantly localized to the temporal and nasal aspects of the globe (at the 3 and 9 o'clock position) was detected in 11 of 13 dogs. In all cases, anterior and posterior segment examinations were unremarkable without evidence of vitreal or chorioretinal bleeding. Scleral hemorrhage was first observed within 1 to 3 days of SAH induction and resolved within 7-10 days. The clinical findings in dogs are compared to those in people with SAH (Terson's syndrome). Terson described intracranial bleeding as cause of vitreous hemorrhage and concluded this was a sign of SAH.49 Some have expanded the definition to include retinal hemorrhages. Mechanisms of vitreous and retinal hemorrhages in SAH remain controversial.50,51 In humans, it has been proposed that sudden increase in intracranial pressure passed via the optic nerve sheath obstructs the central retinal vein and retinochoroidal anastomosis. This leads to a subsequent rapid increase of the intraretinal and also intravitreal stasis, resulting in intraretinal and intravitreal hemorrhages.51,53 Anatomic variations in vascular supply to the eyes of people versus dogs likely account for differences in ophthalmic findings of SAH, specifically presence or absence of a central retinal artery and vein. To the authors' knowledge, this is the first documented report of the ophthalmic manifestations of SAH in dogs.54 Subarachnoid hemorrhage should be suspected in dogs that have sustained head trauma and present with scleral hemorrhage, particularly if the hemorrhage is bilateral and in the absence of other ophthalmic abnormalities.
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