Inhibition of prostaglandin E2 receptor EP3 mitigates

thrombin-induced brain injury Introduction

Ranking 5th among all causes of death in the US, stroke has long been a major public health
issue, killing approximately 133,000 Americans and 6.2 million people worldwide annually. In
the present day, stroke rates have been rising steadily since 1995, with hospitalizations of men
for strokes nearly doubled from 1995 through 2012. Such a drastic increase in hospitalization
rates has heightened the need for a novel therapeutic treatment for strokes.
Intracerebral Hemorrhage (ICH), otherwise known as Hemorrhagic stroke, accounts for
approximately 15% of all strokes. In this form of stroke, high blood pressure or external brain
trauma causes thin walled arteries in the brain to tear, resulting in a ruptured artery within the
brain. As blood spills into the brain, the area that the artery originally circulated is deprived of
oxygen-rich blood, leading to neuronal death. This damage is only furthered by the release of
toxic molecules by microglia and other brain cells.
Initial impacts of stroke, however, are nearly impossible to target as a result of the time frame
efficacy of the drugs that target those specific responses. Therefore, it is only logical to develop
treatment methods which focus on inhibition of the signaling pathways that induce the secondary
injuries from stroke.
Prostaglandin E2 (PGE2) is a compound has been proven to be involved in inflammation and
secondary injury after brain insult, including that of ICH. Additional, PGE2 receptor EP3,
expressed by neurons and activated glial cells, has been known to be associated with neuronal
death. Commonly, EP3 signaling is conducted through the G12/13 protein, resulting in activation
of the small G-protein Rho. Activation of these proteins constitutes the Rho-ROCK pathway, a
signaling pathway which is thought to contribute to cell migration and inflammation. As a result,
reduction of PGE2 production or reception would therefore be vital to minimizing brain damage
after ICH.
Thrombin is an enzyme that causes blood clots through conversion of fibrinogen to fibrin. It has
long been confirmed to be a major contributor towards ICH injury by promoting blood-brain
barrier disruption, neuroinflammation, and brain edema. Furthermore, thrombin has also been
shown to stimulate PGE2 production. Therefore, it was likely that PGE2 reception played a role
in thrombin-induced brain injury. However, the role of EP3R in the inflammatory response after
ICH had yet to be explored. Consequently, we investigated the function of EP3R in thrombin
toxicity and the possibility that the Rho-ROCK pathway is involved.
Our novel findings suggest that inhibition of PGE2 receptor EP3 has mitigating effects on
thrombin-induced brain injury. The results show that EP3 inhibition was able to mitigate
thrombin-induced brain injury in the forms of reducing brain lesion volume, brain edema, and
neurobehavioral deficits. Specifically, treatment with 3 mg/kg EP3R antagonist AE240 reduced
brain lesion volume by 36%, while treatment with EP3R agonist AE248 increased the injury
volume by 14%, indicating that inhibition of EP3R would diminish secondary brain injury and
vice versa. Moreover, inhibition of EP3 also resulted in less cell death after thrombin injection,
enabling greater preservation of cell viability. One major reason for this is due to the reduction of
the immune inflammatory response in the brain. Testing revealed that EP3 inhibition reduced M1
Microglia activation and MMP-9 activity, two major factors that contribute to the damaging
inflammatory response that occurs after stroke, while also promoting M2 Microglia polarization,
a phenotype of microglia which plays a beneficial role after stroke.
Further experimentation revealed that the protective effects of EP3 inhibition may be achieved
through the aforementioned Rho-Rock signal pathway. Knowing that this pathway has been
associated with neurotoxicity, we evaluated the total expression of the RhoA protein in the brain
after thrombin injection, observing that RhoA expression, and therefore expression of the Rho-
Rock signaling pathway, had been elevated. EP3 inhibition, however, decreased RhoA
expression, while EP3 re-activation had the opposite effect, reversing the impacts of the previous
EP3 inhibition and increasing RhoA expression. These results support the novel discovery that
EP3R-mediated toxicity involves the Rho–Rock pathway.
To summarize, our results indicate that EP3 inhibition plays a protective role, and that the Rho -
Rock pathway might contribute to thrombin induced neurotoxicity. Together, all these findings
demonstrate that promoting the neuroprotective M2 microglia phenotype and inhibiting the
RhoA–ROCK2–pMYPT1 signaling pathway through Ep3 inhibition could be a potential
therapeutic option for treating ICH.
These findings are described in an article titled Inhibition of Prostaglandin E2 Receptor EP3
Mitigates Thrombin-Induced Brain Injury, which was recently published in the journal Cerebral
Blood Flow & Metabolism in 2016. This work was conducted by Drs. Xiaoning Han, Xi Lan,
Qiang Li, Yufeng Gao, Jian Wang, and others and was supported by grants from the National
Institutes of Health, Johns Hopkins University, and the American Heart Association.