Endogenous neuroprotection through preconditioning

Figure one, titled Systematic administration of TLR ligands induces neuroprotection in mice, presents data for TLR4, TLR7 and TLR 9 with three pairs of bar graphs, measuring infarct percentages.

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. 

Figure two presents Venn diagram on left showing overlap of CpG, LPS and Ischemic preconditioning, pointing to web diagram on right.
A shared IRF driven reprogrammed response to stroke in preconditioned mice. Left: Venn diagram comparing the reprogrammed genes in each of the preconditioning paradigms at 24h post stroke. Right: Hypothesis Gene-TRE network of the 13 genes (in blue) common to all conditions showing the relationship of identified transcriptional regulatory elements (TREs; in red) to the regulated genes.

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.

Figure three displays two computational illustrations, side by side. Illustration on left: Identify points of constriction (bottlenecks) that serve as linker points between clusters of genes and may represent genes of biological significance. Illustration on right is titled, IFIT1: Computationally Identified Bottleneck Gene. It presents four diagrams comparing data on LPS plus stroke, CpG plus stroke, IP plus stroke and stroke alone, respectively.
Computational analysis of transcriptomic data. Left: Inferred network of gene interactions predicted by expression profile (time, treatment) in the brain of preconditioned and untreated animals exposed to stroke. Yellow circles denote bottleneck genes. These genes are considered to have high biological relevance in a system. Right: First-order network of IFIT1, an identified bottleneck in the inferred network. Computational analysis predicts IFIT1 is critical for the expression of a cluster of genes induced following stroke in preconditioned animals. Red circles: genes upregulated 24h following MCAO; blue circles: no change.

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.  

Figure four: Three pairs of bar graphs presenting Infarct Volume (percentage of Vehicle) for WT->WT, TLR9KO->WT and WT->TLR9KO.

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. 

Image on left: Time-lapse image tiled into four sections, showing 15 minutes, 30 minutes, 120 minutes and 180 minutes.
Image on right: Leukocytes interacting with the brain endothelium.

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.

Diagram presenting blood and brain information, prior to stroke and post stroke, over the three phases of preconditioning: Priming (systematic preconditioning), refractive phase (lasts multiple days), neuroprotection (reduced cerebral injury). In priming stage blood info says: induced cytokines and leukocyte activation. Brain info says: New gene transcription. In refractive phase, blood and brain have shared information: resistant to ischemic injury. In neuroprotection phase, blood info says: reprogrammed c

Translating basic research into potential clinical therapeutics

Two images, side by side, titled “Developed nonhuman primate stroke model.". On left is a photo of two doctors performing a procedure. On right is a figure with two images of a brain, one labeled saline, the other labeled CpG. Embedded text below two images of brain says, TLR9 agonist protects non-human primates from ischemic injury.

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.