Research ReportThrombin-induced autophagy: A potential role in intracerebral hemorrhage
Highlights
► Thrombin causes autophagy in brain and cultured astrocytes. ► Hirudin, an inhibitor of thrombin, reduces intracerebral hemorrhage-induced autophagy. ► 3-Methyladenine, an inhibitor of autophagy, attenuates autophagic vacuole accumulation after thrombin exposure. ► 3-Methyladenine aggravates thrombin-induced cell death.
Introduction
Thrombin, a serine protease generated by the cleavage of prothrombin, is an essential component of the coagulation cascade. As such, it is produced in the brain immediately after a cerebral hemorrhage to induce hemostasis. However, thrombin has multiple effects in brain injury. Thus, evidence indicates that thrombin contributes to early brain injury following intracerebral hemorrhage (ICH) and cerebral ischemia (Xi et al., 2003, Xi et al., 2006). In contrast to these early effects, thrombin is also associated with brain recovery after ICH (Yang et al., 2008).
Autophagy is a cellular degradation process in which cellular proteins and organelles are sequestered in double membrane vesicles known as autophagosomes, delivered to lysosomes, and digested by lysosomal hydrolases (Wang and Klionsky, 2003). Autophagy plays an important role in cellular homeostasis, and it is involved in a number of human diseases (Adhami et al., 2006, Komatsu et al., 2006, Rubinsztein et al., 2005). We have demonstrated that autophagy occurs after ICH and iron has a role (Gong et al., 2011, He et al., 2008) and most autophagic brain cells are astrocytes (He et al., 2008). It is known that thrombin and iron are two major factors causing brain injury after ICH (Xi et al., 2006). However, it is unclear whether thrombin also causes autophagic cell death after ICH and whether modifying thrombin-induced autophagy might affect brain injury or recovery after ICH. The purpose of the current study was, therefore, to investigate whether thrombin causes autophagy in brain (in vivo) and astrocytes (in vitro). This was examined using electron microscopy and three markers of autophagy. Light chain 3 is a marker for the detection of autophagosomes. Light chain 3 has two forms: type I (LC3-I) is cytosolic and type II (LC3-II) is membrane-bound. During autophagy, LC3-II is increased by conversion from LC3-I (Kabeya et al., 2000). Cathepsin D is a protein known to mediate autophagy (Araki et al., 2006, Klionsky et al., 2008) and monodanysylcadaverine (MDC) staining is a marker of autophagic vacuoles.
Section snippets
Thrombin increased the conversion of LC3-I to LC3-II and upregulated cathepsin D in brain
The time-course study showed that ratio of LC3-II to LC3-I (Western blotting) in the ipsilateral basal ganglia was increased by thrombin injection (Fig. 1). The conversion of LC3-I to LC3-II in the ipsilateral basal ganglia was significantly higher in the thrombin-treated group at day 1 (ratio: 0.8 ± 0.1 vs. 0.2 ± 0.1 in the saline group, p < 0.01, Fig. 1) or day 3 (ratio: 0.9 ± 0.1 vs. 0.4 ± 0.2 in the saline group, p < 0.05, Fig. 1). Thrombin also induced upregulation of cathepsin D (Fig. 2). The levels
Discussion
In the current study, we found: 1) thrombin causes autophagy in brain and cultured astrocytes; 2) hirudin, an inhibitor of thrombin, reduces ICH-induced autophagy; and 3) 3-MA, an inhibitor of autophagy, reduces MDC-labeled vacuoles accumulation after thrombin exposure and aggravates thrombin-induced cell death. The results suggest that thrombin has a role in autophagy after ICH.
Thrombin is a serine protease and an essential component in the coagulation cascade. It is produced immediately in
Animal preparation and intracerebral infusion
The University of Michigan Committee on the Use and Care of Animals approved the protocols for these studies. Male Sprague–Dawley rats (weighing 275 to 350 g, Charles River Laboratories, Portage, MI, USA) were anesthetized with pentobarbital (45 mg/kg, i.p.). A polyethylene catheter (PE-50) was then inserted into the right femoral artery to monitor arterial blood pressure and blood gasses, and to obtain blood for intracerebral blood infusion. Rectal temperature was maintained at 37.5 ± 0.5 °C using
Acknowledgments
This study was supported by grants , , from the National Institutes of Health (NIH) and 0840016N from American Heart Association (AHA). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH and AHA.
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