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|Year : 2017 | Volume
| Issue : 5 | Page : 1043-1045
Animal models for cerebral vasospasm: Where do we stand?
Dhananjaya I Bhat
Department of Neurosurgery, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India
|Date of Web Publication||6-Sep-2017|
Dhananjaya I Bhat
Department of Neurosurgery, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Bhat DI. Animal models for cerebral vasospasm: Where do we stand?. Neurol India 2017;65:1043-5
| » Introduction|| |
Cerebral vasospasm (CV) as a cause for delayed neurologic deterioration following aneurysmal subarachnoid haemorrhage (aSAH) was identified during the second half of the 20th century. Since then, animal models have been used extensively to try and understand the complex nature of this phenomenon and to evolve treatment strategies and develop new drugs to improve the outcome. Unfortunately despite the use of numerous animal models along with major advances in the field of medicine, science and technology, we are far from achieving even modest improvements in the clinical outcomes in patients with aSAH and delayed neurologic deterioration.
Ethics in animal experimentation
Non-human animals for experiments and research have been used from time immemorial. One of the first documented use of animals in the field of research in medicine was in the 6th century B.C. by Alcmaeon of Croton who determined that intelligence and sensory processing takes place in the brain, based on studies using dogs. Progressively over time, more and more animals have been subjected to experimentation for the “betterment “of homo sapiens. Use of non-human animals in the field of biomedical research will undoubtedly rake up heated arguments due to the ethical and moral issues associated with it. The animal rights activists argue that just because an animal cannot express itself with speech, it does not mean that it cannot feel pain, distress or suffering, and nature has not created man as “morally” superior, giving him the license to cause pain and harm to other animals for his welfare. Hence, at present, there are no methods to simulate and study the entire human body in health and disease using the in vitro techniques. Grammer: Hence, animal models are an indispensable part of the research armamentarium.
“The greatness of a nation and its moral progress can be judged by the way its animals are treated.”
Many countries have government approved legal regulatory bodies which overlook the care and use of animals in experimental works and ensure that the studies are done in an ethical and humane way so as to keep the pain and distress level to the minimum. In India, it is the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), which is a very strict regulatory body. It is a statutory body and comes under the Prevention of Cruelty to Animals Act 1960 with the mission “to take all such measures as may be necessary to ensure that animals are not subject to unnecessary pain or suffering before, during or after the performance of experiments on them.” All animal laboratories have to be registered under the CPCSEA and follow their guidelines. Each laboratory has its own Institutional Animal Ethics Committees (IAECs) which overlooks and approves research project proposals. As followed throughout the world, the CPCSEA strictly follows the principles of Russell and Burch (1959) which are the 3R's. Refinement: Improving all aspects of experiment and care so that the animal suffers the least; Reduction: Use as minimum number of animals as possible without compromising the scientific validity; and, Replacement: Avoid using animals if non-animal models like cell and tissue cultures and computer models are available. In addition, the CPCSEA for the first time in the world has included the 4th R which is Rehabilitation. They have stressed that it is the moral responsibility of the experimenter to take care of the animal even after the study is completed “with the sole intention of alleviating the pain/distress or suffering due to the physical, physiological and psychological trauma that the animals have been exposed to and to provide the animal a life distinctly different from laboratory housing and care, until the point of natural death.” This has been recognized by the Government of India and a national policy status has been given to it.
Vasospasm animal models: Strengths and Limitations
One of the first reported animal model for aSAH and vasospasm was in dogs in 1961 in which Lougheed and Tom injected autologous blood in the chiasmatic cistern of dogs via a transoral route. Since then, a multitude of models have emerged; however, we are far from reaching any satisfactory conclusion regarding vasospasm, delayed neurologic deficits and its management. It is noteworthy that in the field of ischemic stroke, the scenario is contrasting. There has been a significant translation of basic research to clinical practice like the use of tissue plasminogen activator to treat ischemic stroke within the golden hour. One of the reasons for this status is that there is a standardized time-tested ischemic animal model which almost mimics the human disease. Unfortunately in the context of vasospasm, this kind of a model is lacking. Marbacher et al., in a review of vasospasm animal models, identified 66 models in 7 animals. Over the last several decades, due to ethical issues and logistics, more and more rat and mice models have emerged and there has been a decrease in the use of large animals like canines, pigs and non-human primates. All these models are capable of producing varying degrees of biphasic pattern of vasospasm (acute and delayed, akin to that which takes place in humans) and neurologic deficits; however, none of these models have been standardized nor have been universally accepted. In one set of animal model experiments, vasospasm was induced by injecting autologous blood through a cisternal puncture either in the cisterna magna or the prechiasmatic cistern or by doing a small craniotomy (e.g frontotemporal or transclival) and placing a blood clot over the vessels. In some models, a single injection is used, and in the others, the blood is injected twice with a 48-hour interval between the two injections. The experimental design, amount of blood injected, method of injection, methods to assess vasospasm, timing of assessment are all varied across experimental models. Zibly et al., have very well brought out this issue and have tried to develop a standardized swine model, which has reliably shown a predictable amount of vasospasm when the animal is injected with 12 and 15 ml blood in the cisternal space with a 48-hour gap between the 2 injections. However, it should be noted that all these models study only part of the entire disease process. They do not take into account the trauma to the brain during the initial bleed. Another animal model is the endovascular puncture model, initially started in larger animals like dogs, pigs and non-human primates. In this procedure, a suture filament (prolene 5'0) in guided to the internal carotid artery bifurcation endovascularly via the external carotid artery. On reaching the bifurcation, the vessel wall is jabbed and punctured. A sudden rise in intracranial pressure indicates the success of the puncture. This model may be argued as the closest mimic to human aSAH. However, it is difficult to standardize the procedure, control the bleed volume, and it has a high mortality rate of 35-50% (this also mimics the condition in humans). In addition, there are immense ethical issues and prohibitive costs when one is using large animals. Swine models may be a good alternative to dogs and primates as there are lesser ethical issues involved, and since they are large, they may serve as better models rather than rats and mice. However, over the last couple of decades, endovascular puncture methods have been adapted for smaller animals, and more and more, rat, and recently, mice models have emerged. In another method of inducing closed compartment vessel rupture, a small craniotomy is done, a ligature is placed around a vessel (for example, the anterior cerebral artery) and the end of the suture is brought out through the craniotomy defect. After a few days the suture is tugged out and the vessel ruptures, thus mimicking an aSAH.,
Animal models in which blood is placed around the extracranial vessels offer some insight into the pathological processes of vasoconstriction. However, these are far from ideal as, the microscopic structure of an extracranial vessel is different from that of intracranial arteries, along with other differences such as lack of cerebrospinal fluid around the vessel and inability to study neurologic effects in extracranial models. Computer models have been constructed to study the formation, flow dynamics of aneurysms and may help to predict rupture. However, at present, they are not able to predict and model vasospasm and the consequent neurologic injury.
| » Changing Trends|| |
It is seen in human subjects and animal models that many times the neurological status does not match with the degree of vasospasm. In the present paper also, the authors did not find any association between the degree of vasospasm and delayed neurologic deficits. In the recent times, researchers have been noticing this and are becoming more aware of the concept of early brain injury (EBI) which starts immediately after a bleed. There has been a shift of focus of research from delayed cerebral vasospasm to early brain injury. Whenever there is an aneurysmal rupture, there is injury to the vessel wall and a sudden increase in intracranial pressure, which may transiently reach the systolic blood pressure. This helps in hemostasis; however, the down side is that there is a significant transient decrease in cerebral blood flow, and sometimes even an arrest of cerebral circulation. This, along with a sustained and elevated intracranial pressure, causes ischemic brain damage. All these result in a failure of the energy-dependent cellular homeostatic mechanisms leading to cytotoxic edema, endothelial wall damage causing nitric oxide/nitric oxide synthase pathway disruption, oxidative stress, endothelin activation, and the release of many proinflammatory cytokines. This leads to neuronal and glial cell dysfunction and death. Delayed vasospasm may be precipitated by this EBI and further reduction of blood flow to the already compromised brain may result in neurologic deterioration. Inability to study this aspect of aSAH may be a limitation in the cisternal puncture models. The failure of clazosentan to overcome neurological ischemia and infarction occurring after subarachnoid hemorrhage (CONSCIOUS-1 trial); and, the failure of patients to show any improvement in mortality and morbidity following clazosentan administration despite reversal of large artery vasospasm in aSAH has been attributed to the biochemical and inflammatory changes which were not reversed by the drug. Therapies targeting these molecular mechanisms may be game changers in the management of delayed neurological deficits. In this regard, the endovascular puncture model is now being used in rats and mice and may be an ideal model to study vasospasm and delayed neurologic deterioration.,
| » Conclusions|| |
At present, what we need is an animal model which is reliable, stable, reproducible, and financially viable, keeping into account the strict experimental and ethical guidelines so that no animal is unnecessarily subjected to agony. It should mimic as close as possible the pathophysiologic changes seen in human aSAH. Once this ideal model has evolved it has to be standardized throughout the world and then only can we make some meaningful progress. In this regard, the endovascular puncture model seems to be gaining favor. Unfortunately, at present, there are no non-animal models which can be used to study early brain injury, delayed vasospasm and the consequent neurologic deficits., Until we discover them, we will have to keep searching for the ideal animal model.
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