Keith R. Pennypacker

Targeting antioxidant enzyme expression as a therapeutic strategy for ischemic stroke

ABSTRACT During ischemic stroke, neurons and glia are subjected to damage during the acute and neuroinflammatory phases of injury. Production of reactive oxygen species (ROS) from calcium dysregulation in neural cells and the invasion of activated immune cells are responsible for stroke‐induced neurodegeneration. Scientists have failed thus far to identify antioxidant‐based drugs that can enhance neural cell survival and improve recovery after stroke. However, several groups have demonstrated success in protecting against stroke by increasing expression of antioxidant enzymes in neural cells. These enzymes, which include but are not limited to enzymes in the glutathione peroxidase, catalase, and superoxide dismutase families, degrade ROS that otherwise damage cellular components such as DNA, proteins, and lipids. Several groups have identified cellular therapies including neural stem cells and human umbilical cord blood cells, which exert neuroprotective and oligoprotective effects through the release of pro‐survival factors that activate PI3K/Akt signaling to upregulation of antioxidant enzymes. Other studies demonstrate that treatment with soluble factors released by these cells yield similar changes in enzyme expression after stroke. Treatment with the cytokine leukemia inhibitory factor increases the expression of peroxiredoxin IV and metallothionein III in glia and boosts expression of superoxide dismutase 3 in neurons. Through cell‐specific upregulation of these enzymes, LIF and other Akt‐inducing factors have the potential to protect multiple cell types against damage from ROS during the early and late phases of ischemic damage. HIGHLIGHTSOverview of antioxidants as treatment for stroke.Explanation of failed attempts to develop exogenous antioxidant treatment.Overview of endogenous antioxidant systems.Use of agents that activate these endogenous antioxidant systems to develop stroke therapeutic.

Correcting the Trajectory of Stroke Therapeutic Research

One of the most vital issues in stroke research is the paucity of animal studies that have translated into treatments for human stroke patients. The August and October issues of this journal were devoted to this topic and raise a number of points of concern to rectify this problem. In fact, NINDS is keenly aware and recently hosted the workshop BTranslational Stroke research: Vision and Opportunities,^which raised similar concerns. The introductory article encompassed this theme of transition of stroke research to develop strategies for clinical relevance [4]. Unfortunately, there are a great number of problems and challenges to translating experimental stroke therapies, and there is likely no one or simple solution. One major problem is that the preclinical studies are using a homogeneous population with a similar age and being treated at a specific time point. Whereas, human patients are genetically diverse with different ages and many are consuming an assortment of pharmaceuticals. Other confounding variables include the timing of the stroke and type of stroke. With this diversity of the human patient, we may be overlooking potential treatments that were effective in a specific human population but not clear because clinical studies were not designed specifically to analyze that subgroup. Several papers in both issues address the shortcomings with animal models reflecting the human condition. There is a necessity to interject co-morbidities, age, and sex into existing animal models to better mirror the heterogeneity of the human population. Most studies are performed using young male rats. However, therapeutic testing in animal stroke models should include assessment in subjects with co-morbidities, such as diabetes and hypertension, which are common to most stroke patients [7]. Furthermore, there are clear gender differences in stroke severity and in response to treatment. These sex differences in stroke have not been well studied at both preclinical and clinical levels [1]. One study has shown that the administration of uric acid with tPA clearly benefits women but not men. However, the study had to investigate the independent effect of sex to dissect out this positive effect on women [15]. While the uric acid experience is reflective of excellent stroke animal model to human translation, it also shows the vulnerability of translation. By not designing the clinical trial specifically with preplanned gender-based outcome analysis, investigators nearly missed the potentially profound beneficial effect of uric acid on stroke outcomes in women. Another vital avenue of research is the stroke immune response, which plays a critical role in the pathophysiology of stroke, and different rodent stains have disparities in their immune responses. These disparities affect translation between rodent species and between animal and human and have a critical impact on therapeutic translation of immunomodulatory therapies [2]. With that in mind, reverse-translational methodologies, which start at analyzing stroke in humans, may play a growing role. Should the neurointerventional suite become one of the * Keith R. Pennypacker keith.pennypacker@uky.edu

The role of the leukemia inhibitory factor receptor in neuroprotective signaling

ABSTRACT Several neurotropic cytokines relay their signaling through the leukemia inhibitory factor receptor. This 190 kDa subunit couples with the 130 kDa gp130 subunit to transduce intracellular signaling in neurons and oligodendrocytes that leads to expression of genes associated with neurosurvival. Moreover, activation of this receptor alters the phenotype of immune cells to an anti‐inflammatory one. Although cytokines that activate the leukemia inhibitory factor receptor have been studied in the context of neurodegenerative disease, therapeutic targeting of the specific receptor subunit has been understudied in by comparison. This review examines the role of this receptor in the CNS and immune system, and its application in the treatment in stroke and other brain pathologies.

Translational Evaluation of Acid/Base and Electrolyte Alterations in Rodent Model of Focal Ischemia

BACKGROUND AND PURPOSE Acid/base and electrolytes could provide clinically valuable information about cerebral infarct core and penumbra. We evaluated associations between acid/base and electrolyte changes and outcomes in 2 rat models of stroke, permanent, and transient middle cerebral artery occlusion. METHODS Three-month old Sprague-Dawley rats underwent permanent or transient middle cerebral artery occlusion. Pre- and post-middle cerebral artery occlusion venous samples for permanent and transient models provided pH, carbon dioxide, oxygen, glucose, and electrolyte values of ionized calcium, potassium, and sodium. Multiple regression determined predictors of infarct volume from these values, and Kaplan-Meier curve analyzed morality between permanent and transient middle cerebral artery occlusion models. RESULTS Analysis indicated significant differences in the blood gas and electrolytes between pre- to post-middle cerebral artery occlusion. A decrease in pH and sodium with increases in carbon dioxide, potassium, ionized calcium, and glucose changes were found in both middle cerebral artery occlusion models; while hematocrit and hemoglobin were significant in the transient model. pH and ionized calcium were predictors of infarct volume in the permanent model, as changes in pH and ionized calcium decreased, infarct volume increased. CONCLUSIONS There are acute changes in acid/base balance and electrolytes during stroke in transient and permanent rodent models. Additionally, we found pH and ionized calcium changes predicted stroke volume in the permanent middle cerebral artery occlusion model. These preliminary findings are novel, and warrant further exploration in human conditions.

The Blood And Clot Thrombectomy Registry And Collaboration (BACTRAC) protocol: novel method for evaluating human stroke

Background Ischemic stroke research faces difficulties in translating pathology between animal models and human patients to develop treatments. Mechanical thrombectomy, for the first time, offers a momentary window into the changes occurring in ischemia. We developed a tissue banking protocol to capture intracranial thrombi and the blood immediately proximal and distal to it. Objective To develop and share a reproducible protocol to bank these specimens for future analysis. Methods We established a protocol approved by the institutional review board for tissue processing during thrombectomy (www.clinicaltrials.gov NCT03153683). The protocol was a joint clinical/basic science effort among multiple laboratories and the NeuroInterventional Radiology service line. We constructed a workspace in the angiography suite, and developed a step-by-step process for specimen retrieval and processing. Results Our protocol successfully yielded samples for analysis in all but one case. In our preliminary dataset, the process produced adequate amounts of tissue from distal blood, proximal blood, and thrombi for gene expression and proteomics analyses. We describe the tissue banking protocol, and highlight training protocols and mechanics of on-call research staffing. In addition, preliminary integrity analyses demonstrated high-quality yields for RNA and protein. Conclusions We have developed a novel tissue banking protocol using mechanical thrombectomy to capture thrombus along with arterial blood proximal and distal to it. The protocol provides high-quality specimens, facilitating analysis of the initial molecular response to ischemic stroke in the human condition for the first time. This approach will permit reverse translation to animal models for treatment development.