Personal Statement

I am a neurologist with specialty board certification in vascular neurology. I have a laboratory effort funded by the National Institutes of Health (NIH), VA and American Heart Association focused on translational preclinical stroke modeling, pharmacology and vascular biology. Our lab has recently focused on the role of thrombin mediating cytotoxicity in the brain and the molecular mechanisms of protease activated receptors (PAR)-1-mediated neuroprotection. Also, we recently rekindled a long-standing interest in preclinical models of therapeutic hypothermia, motivated by my role as principal investigator (PI) of the largest clinical trial of therapeutic hypothermia for stroke, the ICTuS program. We have considerable experience with a variety of animal models, behavioral testing, histology and cell biology. I have also run large, multicenter clinical trials for industry and NIH.

My original contribution in science involved both preclinical and clinical aspects of thrombolysis for acute ischemic stroke. I joined the laboratory of Justin Zivin, MD, at the time he published his seminal demonstration in Science that rt-PA powerfully protected neurological function after cerebral embolism (Zivin et al, Science. 1985;230:1289). Prior to this demonstration, thrombolysis with streptokinase appeared dangerous, and few were willing to study another lytic drug without further safety data, especially with respect to hemorrhagic transformation. During my postdoctoral fellowship, I created a model of post-embolic hemorrhage and demonstrated three key findings: first, that heparin anticoagulation did not significantly worsen hemorrhage after transformation (later, others in the lab showed that perireperfusion hypertension does promote hemorrhage); second, that streptokinase but not rt-PA did promote transformation; and, third, that tenecteplase was a potent and likely preferable alternative to rt-PA. All of these data contributed to the development of rt-PA as a clinical therapeutic. I was honored to be selected as the San Diego PI of the National Institute of Neurological Disorders and Stroke for rt-PA Acute Stroke Trial, during which I helped draft the protocol and main results for the trial. I wrote, produced and directed the NIH Stroke Scale training and certification video that has now been viewed by more than half a million people around the world, and I oversaw the clinimetric validation of these video tools. I was the principal author of the key subgroup analysis of the main study results.

Perhaps the greatest problem in animal stroke models is the variation in stroke size across animals that receive an identical lesion. Efforts to control lesion reproducibility include cumbersome anesthetic and surgical setups. From my earliest model, I sought to harness the heterogeneity in stroke size because it reflects a clinical reality: human stroke is highly variable. To reduce the sample size of animals needed to show significant therapeutic effects, I sought a way to define lesion size before treatment began. By giving a bolus of two MDa dextran conjugated to fluorescein isothiocyanate (FITC) at the moment of reperfusion, we hoped to label the ischemic zone prior to interventions, and then compare the area of FITC-dextran leakage to final stroke size, measured with histology. This work was funded over several years but unfortunately the strategy did not work as hoped. During these studies, however, I noted that the area of FITC-dextran leakage reproducibly and accurately defined an area of irrecoverable necrosis, the core of the infarct. We also noted a startling effect of vascular endothelial growth factor, used originally in these studies to manipulate the leakage area for proof-of-concept, which caused angiogenesis. The fortuitous observation led to several studies of angiogenesis as we explored the role of putative neuroprotective growth factors. We eventually discovered that ischemic brain produces angiogenic growth factors to open capillaries and to promote entry of phagocytic macrophages, a phenomenon we labeled "the clean-up hypothesis." We showed convincingly that the angiogenesis documented around infarcts serves only in the cleanup of necrosis, and is not a protective or neo-restorative effect.

During the vascular leakage studies, we became aware of work showing that thrombin powerfully opens the blood-brain barrier by a direct effect on endothelial cells and then kills neurons and astroctyes. Given our interest in leakage, we began working with thrombin. We also were aware of the seemingly contradictory effect of very-low-dose thrombin to protect the brain. One of my doctoral students, Bo Chen, PhD, noted that neurons expressed prothrombin message, an effect we dismissed at first as lab error. After confirming his results, and finding traces of a similar finding in the literature, we began to ask what the purpose could be of neuronal expressed thrombin. We showed that thrombin powerfully kills cells in a dose-dependent manner via the PAR-1 receptor. Thrombin activity is associated with direct neuron killing during stroke. Thrombin antagonists, such as argatroban, are powerful neuroprotectants, and a clinical trial is underway to study PAR-1 agent 3K3A-APC, for which I serve as the national PI. Argatroban itself is under study as stroke therapy, and I served as the Los Angeles site PI. Although these translational results were gratifying, we were left with the question, why does the brain make the serum clotting factor prothrombin? In a series of elegant in vitro experiments, my colleague Padmesh Rajput, PhD, and I have demonstrated that stressed neurons secrete thrombin into the culture media; oxygen glucose deprivation of neuronal conditioned media causes astrocyte activation; such astrocyte activation can be reproduced with low doses of thrombin and is blocked by either thrombin inhibitor or by knocking down or knocking out PAR-1 from the astrocytes. We then created an inducible neuronal-targeted prothrombin knockout animal. After inducing the neuronal prothrombin knockout, the region of MCAo-induced infarction in these animals is significantly enlarged, and there is greatly reduced evidence of astrocyte activation in the ischemic bed. Taken together, these results suggest the novel hypothesis that neurons produce prothrombin as a distress signal to adjacent astroctyes, which respond with the astrocyte protective response. We are actively engaged in multiple studies to support/refute this hypothesis; extend it to other elements in the neurovascular unit; and identify the active agent(s) that constitute the astrocyte protective response.

Therapeutic hypothermia is the most powerful neuroprotectant ever documented in stroke models. We showed the protective effect of a single degree Celsius (C) in our quantal bioassay model. Therapeutic hypothermia is of proven benefit for victims of cardiac arrest or neonatal hypoxic-ischemic injury. For more than a decade, I have been working on the ICTuS program (P50NS044148) to deliver therapeutic hypothermia to patients. In the first trial, ICTuS, we showed that therapeutic hypothermia could be safely delivered with an endovascular cooling catheter. In the ICTuS-L program, we safely combined endovascular cooling with rt-PA. In the ICTuS 2/3 trial we attempted to document efficacy of therapeutic hypothermia and thrombolysis. The trial was stopped early, however, after several trials of intra-arterial neurothrombectomy were overwhelmingly positive. We have revised the ICTuS 3 protocol to accommodate neurothrombectomy, but during analysis of the small ICTuS 2 dataset we noted a troubling trend (not statistically significant) toward pneumonia risk during cooling. Also, other trials were published showing lack of efficacy for therapeutic hypothermia in head trauma, and no discernible difference between 33°C versus 36°C target temperatures. All these results led us to question whether we were using therapeutic hypothermia correctly in treating these illnesses. Based on our parallel observations of astrocyte protection of neurons, we asked whether cooling could disrupt the astrocyte-mediated protection of neurons. In fact, we showed that hypothermia interferes with the astrocytes, in a graded, temperature-dependent manner. We then showed that ultra-fast cooling to 33°C for a short time was more powerful in the MCAo model than were longer cooling periods. These exciting and novel data, if confirmed, suggest a reason for the failures of therapeutic hypothermia in some clinical trials. Moreover, our data suggests testable hypotheses about the effects of temperature in modulating neurovascular unit protection during ischemia.

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