Activated autophagy pathway in experimental subarachnoid hemorrhage

Abstract

Recent results have suggested a role for autophagy in acute brain injury but an involvement in subarachnoid hemorrhage (SAH) has not been investigated. Although, autophagy is a regulated process essential for cellular homeostasis, it may represent an additional type of cell death mechanism. This study employed a modified endovascular perforation rat model under guidance by intracranial pressure monitoring to investigate whether autophagy pathway is involved in the early brain injury following SAH. Sham-operated control rats underwent an identical procedure without vessel perforation. Electron microscopy was performed to examine the ultrastructural changes in neural cells after SAH. Additionally, microtubule-associated protein light chain-3 (LC3), cathepsin-D and beclin-1 were investigated by Western blot analysis and immunohistochemistry. Electron microscopically, there was a marked increase in autophagosomes and autolysosomes in neurons at Day 1 following SAH. Although LC3 could be detected in sham-operated control rats, the conversion of LC3-I to LC3-II was significantly increased at Day 1 (P < 0.01) and Day 3 (P < 0.05). The time-course of beclin-1 expression paralleled the LC3 conversion. Cathepsin-D expression was also elevated at Day 1 (P < 0.01). Immunohistochemical study with antibodies against cathepsin-D and beclin-1 showed numerous positive stained cells after SAH, especially in deep layers of the fronto-basal cortex. Double immunolabeling revealed beclin-1 expression predominantly in neurons. This present study showed that the autophagy pathway is activated in neurons in the acute phase after SAH.

Introduction

Subarachnoid hemorrhage (SAH) is a devastating disease causing high morbidity and mortality especially within the first few days (Broderick et al., 1994). Many survivors of SAH experience persistent cognitive deficits, with an effect on functional status and quality of life (Hütter et al., 2001). Although considerable advances have been made in endovascular techniques, diagnostic methods, and surgical and perioperative management paradigms, outcome for patients with SAH still remains poor (Bederson et al., 2009). Recently, the term “early brain injury” has been generated referring to the immediate injury within 72 h and includes secondary events to SAH before the development of cerebral vasospasm (Kusaka et al., 2004). The identification of the mechanisms underlying brain injury during this acute period has received much less attention than delayed cerebral vasospasm and therefore, therapeutic options are generally limited (Cahill et al., 2006).

Apoptosis has been reported to be involved in acute brain injury after experimental SAH and might explain the serious impact of this disease on short- as well as long-term outcomes (Matz et al., 2000, Sabri et al., 2008). More recently, increased autophagic changes in neuro-glial cells have been demonstrated in several experimental settings of acute brain injury (Carloni et al., 2008, He et al., 2008, Liu et al., 2008). However, the occurrence of autophagy in SAH has not been examined. Although, autophagy is a physiological process with removal of long-lived proteins and organelles through an autophagosomal–lysosomal pathway that is essential for cell homeostasis and survival, it can promote cell death (Wang and Klionsky, 2003). Therefore, its function in acute pathologic diseases of brain is unclear and there is controversy over whether activated autophagic pathway represents a mechanism of cell death or may be a rescue mechanism activated after injury as part of an endogenous neuroprotective response (Carloni et al., 2008).

Due to similarities in pathophysiological changes to those found with aneurysmal rupture in human, the endovascular perforation SAH rat model is considered most suitable for studying early brain injury following SAH (Bederson et al., 1995, Lee et al., 2009). In this study, a modified endovascular perforation technique was employed for SAH induction in the rat to investigate whether autophagic pathway is activated in early brain injury following SAH.