Elsevier

Experimental Neurology

Volume 230, Issue 2, August 2011, Pages 240-247
Experimental Neurology

Blood–brain barrier disruption following subarchnoid hemorrhage may be faciliated through PUMA induction of endothelial cell apoptosis from the endoplasmic reticulum

https://doi.org/10.1016/j.expneurol.2011.04.022Get rights and content

Abstract

The blood–brain barrier (BBB) plays a vital role as both a physiologic and physical barrier in regulating the movement of water from the vasculature to the brain. During a subarachnoid hemorrhage (SAH), the BBB is disrupted by a variety of mediators, one of which can result in endothelial cell death. As a result, in the present study, we investigated the role of PUMA (p53 upregulated modulator of apoptosis) following SAH injury in rats. Specifically evaluating whether through the endoplasmic reticulum (ER), PUMA could orchestrate the induction of endothelial cell apoptosis and cause a disruption in the blood–brain barrier integrity. One hundred twelve male Sprague–Dawley rats were randomly divided into 4 groups: sham, SAH, SAH + control siRNA, SAH + PUMA siRNA. Outcomes measured include mortality rate, brain edema, BBB disruption, and neurobehavioral testing. We also used Western blotting techniques to measure the expression of key pro-apoptotic proteins such as BAX, BAK, and DRP1. PUMA siRNA treatment significantly reduced the mortality rate, cerebral edema, neurobehavioral deficits, and BBB disruption as measured by Evans blue assay following SAH injury. The T2WI images showed there was an increase in vasogenic edema in the brain following SAH, which could be alleviated by PUMA siRNA. Immunohistochemical staining and Western blot analysis demonstrated an increased expression of PUMA, BAX, BAK, GRP78 and DRP1 in the microvascular endothelial cells of the hippocampus, which was accompanied with endothelium apoptosis. This study showed that PUMA induced endothelial cell apoptosis may in fact play a significant role in BBB disruption following SAH and its mediation may be through the endoplasmic reticulum. By blocking the activity of PUMA using siRNA, we were able to prevent the accumulation of cerebral edema that occurs following BBB disruption. This translated into a preservation of functional integrity and an improvement in mortality.

Research highlights

► The activated PUMA protein was involved in BBB breakdown through, ER pathway partly. ► The expressions of PUMA, BAX, BAK, GRP78 and DRP1 were accompanied with apoptosis. ► PUMA siRNA reduced the mortality, brain water, and Evans blue content after SAH.

Introduction

Subarachnoid hemorrhage (SAH) is a devastatingly tragic stroke subtype with a significantly high morbidity and mortality rate. Despite promising therapeutic approaches such as triple-H therapy and calcium channel blockades, curative treatment that can reverse the damages following SAH injury is not available. This is partly attributed to the poor strategic approach when dealing with the impact of the initial bleeding in SAH; this includes a rise in intracranial pressure, decrease in cerebral blood flow, and a disruption of the blood–brain barrier (BBB) causing further brain swelling, edema formation and/or accumulation.

One of the main factors attributed to BBB disruption and subsequent brain edema accumulation is apoptotic cell death—especially among endothelial cells which play a vital role in maintaining an adequate barrier. Recently it has been shown that PUMA (p53 upregulated modulator of apoptosis), a direct transcriptional target of p53, is a an essential mediator of cell death and plays a key functional role in the process of p53-mediated apoptosis (Jeffers et al., 2003; Yan et al., 2008). At times of cell damage, the expression of PUMA is up-regulated and binds to and neutralizes almost all prosurvival members of the the Bcl-2 family (Chipuk et al., 2005). Hence it begs the questions whether PUMA is a key orchestrator of early brain injury following SAH.

As a result, the present study was designed to investigate the role of PUMA following SAH injury in rats. Specifically evaluating whether through the endoplasmic reticulum (ER), PUMA could orchestrate the induction of endothelial cell apoptosis and cause a disruption in the blood–brain barrier integrity. In order to test these aims, we used a PUMA siRNA and compared outcome results of mortality rate, brain edema, BBB disruption, neurobehavioral tests versus those animals not treated with the siRNA. We also used Western blotting techniques to measure the expression of key pro-apoptotic proteins such as BAX, BAK, and DRP1. To support our hypothesis of ER invovlvment, GRP78, a molecular chaperone located in the lumen of the ER, was measured.

Section snippets

Materials and methods

All protocols for this study were evaluated and approved by the Animal Care Committee at Peking University Health Sciences Center and the Guidelines for the Use of Animals in Neuroscience Research by the Society for Neuroscience.

PUMA siRNA reduced mortality and neurobehavioral deficits following SAH injury

Animal mortality was measured 24 hours following SAH injury and was presented in a percentage form (Fig. 1A). In the respective groups, the mortality rates were as follow: SAH group 40.42% (19/47 rats), control siRNA 39.13% (18/46 rats), PUMA siRNA 17.65% (6/34 rats), and 0% (0/28 rats) in the sham group. Statistical analysis revealed that there was a significant difference of mortality between the SAH, control siRNA group and PUMA siRNA group (p < 0.05). However, no statistical significance in

Discussion

The present study was designed to investigate the role of PUMA (p53 upregulated modulator of apoptosis) following SAH injury in rats; specifically evaluating whether through the endoplasmic reticulum, PUMA could orchestrate the induction of endothelial cell apoptosis and cause a disruption in the blood–brain barrier integrity. This study made the following novel observations: (1) SAH-induced brain injury resulted in a significant increase in mortality, brain edema, and neurobehavioral deficits

Conclusion

In conclusion, this study showed that PUMA induced endothelial cell apoptosis may in fact play a significant role in BBB disruption following SAH and its mediation may be through the endoplasmic reticulum. By blocking the activity of PUMA using siRNA, we were able to prevent the accumulation of cerebral edema that occurs following BBB disruption. This translated into a preservation of functional integrity and an improvement in mortality. Obviously, more studies evaluating the exact mechanistic

Acknowledgments

This work was partially supported by the National Natural Science Foundation of China (30901548 and 30971527). We would also like to recognize and thank Professor Han Hongbin and Dr. He Qingyuan for their support with the MRI.

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