OverviewMy work involves both basic and translational research accomplished using a team-oriented approach involving a diverse array of collaborators working in unison to discover, characterize, and develop novel therapies. The goal of my research is to develop novel therapeutic treatments for ischemic brain injury by targeting various aspects of the injury process, including prophylactic, acute, and regenerative therapies. In the acute phase of stroke, very few therapeutics demonstrating efficacy in rodent models have subsequently translated to efficacy in humans. Likewise, few therapies exist that promote repair and regeneration in patients recovering from stroke. The paucity of treatments targeting stroke likely is a reflection of the severity of stroke-associated brain injury, as well as the limitations of rodent models to predict clinical outcomes in humans. To improve translation of therapies from animals to humans, a model using the nonhuman primate (NHP) was developed in the laboratory of Mary Stenzel-Poore, PhD at OHSU. Using this model in the rhesus macaque, we are currently collaborating on studies designed to advance novel therapeutic treatments more effectively to humans. In order to identify novel therapeutic strategies we also explore the pathophysiological mechanisms associated with stroke using mice and nonhuman primates. Using information from stroke in rodents, nonhuman primates, and humans as our guide, we continually work to dissect the critical path to injury and to design therapies to target these “bottlenecks” that appear to have a profound effect in patients with heart disease and stroke. We are also exploring novel imaging approaches to understand stroke in the NHP brain and identify protective mechanisms of our therapies. Using agents that allow us to track inflammatory cells by magnetic resonance imaging (MRI) and methods of infarct quantitation, we are advancing the field of preclinical stroke research while providing additional tools to learn more about experimental stroke pathology. Lastly, it is widely known that discrepancies between stroke incidence and severity in males and females exist. Data in rodent models as well as human clinical outcomes provide substantial evidence that a major component of gender differences involve sex hormones although other non-hormone factors also have been defined. Despite the fact that women are more affected by stroke with higher mortality and morbidity than men, no gender specific treatments have been developed. One reason for this could be the absence of good female-specific stroke models, particularly in the nonhuman primate. To address this important women’s health issue, we have begun to develop a stroke model in the female rhesus macaque and intend to expand our studies to include aging animals. The aged female macaque mimics a very important clinical stroke demographic, post-menopausal women, a group that is at high risk of stroke. In addition, our understanding of the effects of hormone therapy in aging women is inadequate. Women are often are prescribed hormones to treat menopausal symptoms or immune dysfunction and the effects of this treatment may be to increase stroke-associated mortality. However, no systematic study has evaluated the best treatment regimen in this high-risk stroke group. Using this model we will to address these important women’s health issues.
In DetailPreconditioning as a Neurotherapeutic Strategy. Many effective preventative therapies can be given before stroke, including treatments involving activators of the innate toll-like receptor (TLR) pathway. Yet none of these therapies are currently approved for clinical use as preventatives for stroke or ischemic brain injury. TLRs are a highly conserved receptor family that recognizes danger signals or constituents of pathogens associated with bacteria or viruses. These receptors, when stimulated, alert the immune system to possible infection or systemic insults. When stimulated at suboptimal levels, however, TLRs can also promote a state of “tolerance” that changes a cell’s response to a subsequent identical stimulus, a phenomenon called “homotolerance”. Similarly, TLR agonists when given as prophylactic agents in vitro or in vivo in models of ischemic stroke can “precondition” or prepare the brain to tolerate a subsequent ischemic injury days later, a phenomenon referred to as “heterotolerance”. TLR stimulation initiated 3 days prior to an ischemic event reduces injury by ~2-fold in animals subjected to severe stroke. We believe that this effect could have a profound importance for clinical medicine. Many patient populations are known to be at high risk of ischemic injury in the brain appearing as frank stroke or ischemic brain lesions. Patients undergoing cerebrovascular or cardiovascular procedures (e.g. stenting, endovascular coiling, cardiac bypass, heart valve replacement) are at risk of stroke (2-10%) and many have detectable ischemic lesions (>50%) in the brain when examined by magnetic resonance imaging (MRI) following the procedures. These patients could benefit from antecedent neuroprotective therapy using TLR agonists.
Translating TLR-induced Preconditioning to Human Clinical Use. In collaboration with investigators at the Oregon National Research Primate Center and Dr. Mary Stenzel-Poore, we continue the preclinical development of several preconditioning agents. We have used mouse and primate models to determine whether preconditioning with CpG or Poly ICLC induces protection against stroke injury. To our knowledge our studies are the first to show that a preconditioning neuroprotectant can result in efficacy in a primate species. Several additional studies using our human optimized CpG agents have independently confirmed these findings, further supporting clinical development of CpG for prophylactic neuroprotection in patients at high risk of stroke. As such, we have initiated Investigational New Drug (IND)-enabling studies in preparation for IND filing with the US Food & Drug Administration (FDA), a process that is required in order to gain permission to conduct Phase I clinical trials in humans. This effort entails safety studies in mice and monkeys and extensive characterization of the drug product prior to human use. We anticipate filing an IND with the FDA by 2015.
Figure 2.Stroke reduces physical activity level in the rhesus macaque. Animals show considerable reduction in physical activity vollowing stroke, as compared to baseline activity level. This parameter is a sensitive measure of stroke outcome and can be used in preclinical studies and as a measure of post-stroke motor recovery following longer durations of study. Shown here is an example actogram showing daily values of activity for a single animal undergoing stroke surgery. A detailed analysis of stroke-associated physical activity was published by our lab in Urbanski et al. (2012) Transl. Stroke Res. 3:442-451.
Figure 3: Strategy for integrated drug discovery and development program for ischemic injury. Using NHP model of stroke, we can examine the brain after stroke and determine key mediators of damage using “omics” approaches. These mediators would be targets for therapeutic intervention with inhibitors of pathological pathways. On the other hand (right), we can examine the brain after inducing TLR-mediated neuroprotected, or other preconditioning paradigms. These studies would identify necessary mediators of neuroprotection that could be enhanced using therapeutic approaches.
Stroke Gender Studies. Stroke kills more women today than breast cancer and AIDS combined and unfortunately none of the available clinical treatments are very effective. While the incidence of stroke is generally higher in men, women are more severely affected than men with reduced recovery, greater recurrence and increased mortality, accounting for over 60% of stroke-related death. Experimental and clinical data suggest that sex differences in stroke risk and outcome may be due to sex hormones (e.g. estrogen, progesterone. Furthermore, post-menopausal women have increased stroke severity, which is further exacerbated by advanced age. These findings suggest that age and gender are important and female sex hormones may be protecting younger women from stroke. Although gender differences are known to influence stroke outcomes, male animal models are often exclusively used to test new stroke therapies, rather than models with both genders. As such, our current understanding of stroke in women is exceedingly limited and would benefit from experimental models relevant to women. We have transferred our experience with male monkeys and stroke to female monkeys and have begun to assess these differences. We intend to eventually evaluate stroke in an aged female population to address susceptibility to ischemia in the brain in the post-menopausal female population.
Estrogen is thought to particularly influence diseases that increase as a function of age, such as stroke. Estradiol (E2) has many direct effects on the brain, starting during development and influencing plasticity in adults. With stroke, estrogen administration is protective in rodent experimental models. It is even capable of reducing damage in the more sensitive aged rodent. Paradoxically, clinical studies in post-menopausal women given estrogen replacement therapy (ERT) showed exacerbated stroke-associated mortality and no reduced risk of stroke compared to women not given ERT. These data suggest we have much to learn with regard to hormones and stroke and timing of estrogen treatment may be critical. We have preliminary data in surgically-menopausal adult, female rhesus monkeys that show less ischemic damage in the brain than intact males. We postulate the reason for this result may involve the neurosteroid synthesis pathway, an endogenous biosynthetic pathway that allows estrogens to be produced locally in the brain. Of interest, a precursor of this brain pathway is the adrenal steroid DHEA (Fig 4), a product that declines precipitously with age in men and women. Thus, older female animals gradually lose both potential sources of E2 upon aging, one produced by the ovaries and another produced by the brain itself via pathways involving DHEA (Fig 4B). Therefore, our hypothesis is that while young OVX animals lose ovarian estrogens, they still have high DHEA levels and can synthesize E2 in the brain, sparing estrogen-dependent function. This system may explain our preliminary stroke results in female macaques. We show that young adult females are still protected from stroke after OVX when compared to age-matched males, further suggesting a role for another source of estrogen (eg. brain neurosteroid synthesis) or nonhormone-related effects (eg. genetics) specific to young adults.
Figure 4: Impact of age and estrogen on cognitive function in female rhesus macaques. Estrogen replacement has a positive effect on cognitive functioning in aged monkeys after removal of ovaries (ovariectomy;OVX) (unpublished). Estrogen is derived from the ovary and can also be made in the brain via neurosteroid synthesis pathway using DHEA. (A) The depiction of a young brain with 2 sources of estrogen, so that if ovary is removed estrogen is not completely absent in the brain. (B) However, upon aging estrogens wane as both systems are eventually affected via decline in ovarian output and DHEA.