Endogenous neuroprotection through preconditioning
Preconditioning is the phenomenon in which a low dose of an otherwise harmful stimulus given prior to injury induces tolerance to the injury, resulting in reduced damage. We have shown that preconditioning the brain against ischemic injury can be achieved through systemic administration of innate immune activators such as Toll-like receptor (TLR) agonists. We strive to decipher the endogenous neuroprotective pathways engaged during preconditioning using both in vitro and in vivo models of ischemic injury. These endogenous strategies provide a unique window into the powerful mechanisms of survival that have evolved to protect the brain from ischemic injury providing essential information for the development of clinical therapeutics.
Preconditioning reprograms the response to injury
Our genomic studies suggest that diverse preconditioning stimuli achieve neuroprotection through a process we refer to as ‘genomic reprogramming.’ Preconditioning with LPS (TLR4 ligand), CpG (TLR9 ligand) or brief ischemia (IP) induces a shared brain response to stroke not evident in non-preconditioned mice. Promoter region analysis of the shared genes revealed an over- representation of interferon regulatory sequences suggesting that the ischemic response in preconditioned mice is mediated via interferon regulatory factors (IRF). The induction of IRF-transcribed genes only in the preconditioned animals denotes a reprogrammed response to injury that may contribute to neuroprotection.
Computational genomic network integration
We have utilized advanced computational analysis of our transcriptomic data to identify gene expression patterns that correlate with protection against stroke. This analysis has flourished due to our collaboration with scientists at Pacific NW National Labs in Richland, WA. Using data obtained from over 100 microarrays we have constructed a genomic network that infers the relationships between genes based on their regulation over time and treatment. Topographical analysis of this network has identified important features of the network such as ‘bottleneck’ genes, which serve as critical links between gene clusters. These types of genes are believed to represent highly significant points in a biological system and may reveal critical mediators of the neuroprotective response.
Leukocytes AND brain cell activation required for protection
The systemic administration of TLR agonists as preconditioners suggest that the neuroprotective response is mediated by circulating immune cells (leukocytes) known to be highly activated by these inflammatory mediators. Utilizing transgenic mice in which TLR signaling was restricted to either circulating leukocytes or resident cells, we found that preconditioning required a TLR signal on BOTH leukocytes and another cell population in order to induce neuroprotection. Mice deficient in TLR signaling on either cell population were no longer protected following TLR agonist (CpG) preconditioning. This surprising finding suggests that communication between leukocytes and the brain, likely at the neurovascular endothelium (i.e. blood brain barrier), is essential for protection.
Systemic and brain parenchymal TLR9 is required for CpG- induced protection. Chimeric mice were injected with CpG (Black bars) or vehicle (White bars) 72h prior to MCAO. Values are group means ± SEM; **p<0.01.
Advanced imaging to study the neuromuscular endothelium
To understand the need for multiple cell types in coordinating the neuroprotective response initiated by TLR preconditioning we are using advanced imaging techniques to monitor leukocyte trafficking in the brain in live animals preconditioned with TLR agonists. Using a two-photon microscope and targeted labeling we can image the brain vasculature and monitor leukocyte endothelial interactions, blood brain barrier permeability and extravasation into the brain. These tools will provide functional information regarding the cellular communication necessary to translate the systemic TLR preconditioning signal from the periphery to the brain culminating in resistance to ischemic injury.
Above: In vivo imaging of the neurovascular endothelium. Left: Time lapse imaging of extravasation of fluorescently tagged dextran (40kD) through the brain endothelium in response to TLR preconditioning. Right: Real time imaging of leukocytes (red) interacting with the brain endothelium following TLR preconditioning.
Mechanisms of systemic preconditioning-induced protection
Our extensive mechanistic studies have provided new insights into preconditioning-induced protection. We were the first to propose the now widely held view that preconditioning occurs in three phases: a priming phase that initiates protection, a refractive phase in which the animal is resistant to injury, and a neuroprotective phase that consists of a reprogrammed response to injury. Deciphering this multifaceted process is the goal of our basic research program, laying the groundwork for translation of these important endogenous mechanisms for the protection of patients.
Translating basic research into potential clinical therapeutics
In collaboration with investigators at the Oregon National Research Primate Center we have developed a unique non-human primate stroke model in rhesus macaques that results in reproducible cortical injury. We have used this model to demonstrate that preconditioning with the TLR9 agonist CpG results in significant reduction in ischemic injury and stroke-related behavioral deficits. We are continuing studies to support the clinical development of CpG and other neuroprotective agents for prophylactic treatment of 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 and Drug Administration (FDA), a process that is required in order to gain permission to conduct Phase I clinical trials in humans.