Effects of Aging on Autophagy After Experimental Intracerebral Hemorrhage

Ye Gong, Yangdong He, Yuxiang Gu, Richard F. Keep, Guohua Xi, and Ya Hua

Abstract Intracerebral hemorrhage (ICH) causes severe brain injury in aged rats. Autophagy occurs in the brain after ICH, and the present study examined the effects of aging on autophagy after ICH. Aged (1822-month) and young (46month) male Fischer rats received an intracerebral injection of 100-mL autologous whole blood. Rats were killed at day 7 for Western blot analysis to measure microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosome, and cathepsin D, a lysosomal biomarker. Rats were killed at 11 weeks after ICH for brain histology. Age-related changes in neurological deficits were also examined. Western blotting showed that the LC3-I/LC3-II conversion ratio in the ipsilateral basal ganglia was higher in aged compared to young rats (p < 0.05). Perihematomal cathepsin D levels were also higher in aged rats (p < 0.05). Neurological deficits after ICH were more severe in aged rats, and they had a slower recovery of function (p < 0.05). In addition, there were more ferritin and OX-42 positive cells in the ipsilateral basal ganglia in aged than in young rats 11 weeks after ICH (p < 0.05). Brain atrophy was found in both young and aged rats. In conclusion, ICH causes more severe autophagy and neurological deficits in aged rats. Keywords Aging Autophagy Cerebral hemorrhage Ferritin

Introduction
Age is an important factor affecting brain injury after ischemic and hemorrhagic stroke. Intracerebral hemorrhage (ICH) causes more severe brain swelling and neurological deficits in aged than in young rats [1], but the mechanisms underlying the enhanced injury have not been well studied. Autophagy plays an important role in cellular homeostasis, and is involved in ICH and cerebral ischemia. Iron has an important role in autophagic cell death after ICH [2]. Light chain 3 (LC3) is a marker of autophagosomes. LC3 has two forms: type I is cytosolic and type II is membrane-bound. During autophagy, LC3-II is increased by conversion from LC3-I [3]. Cathepsin D is an enzyme in lysosomes that is associated with autophagy [4]. Ferritin, a naturally occurring iron chelator, is involved in maintaining brain iron homeostasis. Ferritin levels are upregulated in the brain after ICH [5, 6], and they may limit iron-induced brain injury. Microglia are cells within the brain that respond to injury such as cerebral ischemia and ICH [7, 8]. The present study examined the effect of aging on autophagic cell death after ICH. Neurological deficit and brain ferritin levels were also examined.

Materials and Methods

Y. Gong and Y. Gu Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA and Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China Y. He, R.F. Keep, and G. Xi Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA Y. Hua (*) Department of Neurosurgery, University of Michigan, Room 5018 BSRB, Ann Arbor, MI 48109-0532, USA e-mail: yahua@umich.edu

Animal Preparation and Intracerebral Infusion

Animal use protocols were approved by the University of Michigan Committee on the Use and Care of Animals. Male Fischer rats at different ages (46 and 1822 months old, Geriatrics Center, University of Michigan or NIH) were used in this study. The animals were anesthetized with pentobarbital (45 mg/kg i.p.). The right femoral artery was catheterized for continuous blood pressure monitoring and blood
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J.H. Zhang and A. Colohan (eds.), Intracerebral Hemorrhage Research, Acta Neurochirurgica Supplementum, Vol. 111, DOI: 10.1007/978-3-7091-0693-8_18, Springer-Verlag/Wien 2011

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sampling. Blood was obtained from the catheter for analysis of blood pH, PaO2, PaCO2, hematocrit, and blood glucose. Core temperature was maintained at 37C with use of a feedback-controlled heating pad. The rats were positioned in a stereotaxic frame (Kopf Instrument), and a cranial burr hole (1 mm) was drilled on the right coronal suture 3.5 mm lateral to the midline. Autologous whole blood (100 mL) was infused into the right caudate nucleus at a rate of 10 mL/min through a 26-gauge needle (coordinates: 0.2 mm anterior, 5.5 mm ventral and 3.5 mm lateral to the bregma) with the use of a microinfusion pump. The needle was removed, the burr hole was filled with bone wax, and the skin incision was closed with sutures after infusion. Animals were killed at 1 or 11 weeks after ICH and the brains used for immunohistochemistry or Western blot analysis.

by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a hybond-C pure nitrocellulose membrane (Amersham, Piscataway, NJ). Membranes were blocked in Carnation nonfat milk and probed with primary and secondary antibodies. The primary antibodies were mouse anticathepsin D antibody (Sigma, St Louis, MO; 1:1,000 dilution) and rabbit anti-MAP-LC3 antibody (Abgent Inc., San Diego, CA; 1:400 dilution). The secondary antibodies were goat anti-mouse and goat anti-rabbit IgG (BioRad; 1:2,500 dilution). Relative densities of bands were analyzed with NIH Image program (version 1.61).

Behavioral Tests
For the behavioral tests, all animals were tested before and after surgery, and scored by experimenters who were blind to both neurological and treatment conditions. Three behavioral tests were used: forelimb placing, forelimb use asymmetry (cylinder) and corner turn tests [10]. (A) Forelimb Placing Test. Forelimb placing was scored using a vibrissae-elicited forelimb-placing test. Independent testing of each forelimb was induced by brushing the vibrissae ipsilateral to that forelimb on the edge of a tabletop once per trial for ten trials. Intact animals placed the forelimb quickly onto the countertop. Percent successful placing responses were determined. There is a reduction in successful responses in the forelimb contralateral to the site of the injection after ICH [10]. (B) Forelimb Limb-Use Asymmetry Test. Forelimb use during explorative activity was analyzed by videotaping rats in a transparent cylinder for 310 min depending on the degree of activity during the trial. Behavior was quantified by determining the occasions when the non-impaired (ipsilateral) forelimb was used as a percentage of total number of limb use observations on the wall (I). The occasions when the impaired forelimb (contralateral to the blood-injection site) was used as a percentage of total number of limb use observations on the wall (C), and the occasions when both forelimbs were used simultaneously as a percentage of total number of limb use observations on the wall (B). A single overall limb use asymmetry score was calculated as: Limb use asymmetry score = (I/(I + C+B))(C/(I+C + B)). (C) Corner Turn Test. The rat was allowed to proceed into a corner, the angle of which was 30. To the exit the corner, the rat could turn either to the left or the right, and this was recorded. This was repeated 1015 times, with at least 30 s between trials, and the percentage of right turns calculated. Only turns involving full rearing along either wall were included. The rats were not picked up immediately following each turn so that they did not develop an aversion for their prepotent turning response.

Immunohistochemistry
Rats were anesthetized with pentobarbital (60 mg/kg, i.p.) and perfused with 4% paraformaldehyde in 0.1 M pH 7.4 phosphate-buffered saline. Brains were removed, kept in 4% paraformaldehyde for 46 h, then immersed in 25% sucrose for 34 days at 4C. The brains were embedded in OCT compound (Sakura Finetek USA Inc.) and sectioned on a cryostat (18 mm thick). Immunohistochemistry was performed using the avidin-biotin complex technique as previously described [9]. The primary antibodies were rabbit anti-human ferritin IgG (1:400 dilution; DAKO) and mouse anti-rat CD11b (MRC OX42; 1:200 dilution; Serotec, Oxford, UK). Normal rabbit or mouse IgG was used for negative controls.

Cell Counts
Coronal sections from 1 mm anterior and 1 mm posterior to the blood injection site were used for cell counts. Three highpower images (40 magnification) were taken in the basal ganglia using a digital camera. Ferritin and OX-42 positive cells were counted.

Western Blot Analysis

Western blot analysis was performed as previously described [9]. Briefly, brain samples were sonicated with Western blot lysis buffer. The protein concentration was determined using a Bio-Rad Laboratories (Hercules, CA) protein assay kit. Fifty micrograms of protein from each sample was separated

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Statistical Analysis
Kruskal-Wallis test and ANOVA test were used. Values are mean SD. Statistical significance was set at p < 0.05.

Results
Physiological data were measured prior to intracerebral blood infusion. The physiological variables, including mean arterial blood pressure, blood pH, blood gases and blood glucose, were not different between the two groups. In young rats the ratio of LC3-II to LC3-I in the ipsilateral basal ganglia was higher than that in the contralateral basal ganglia 7 days after ICH (p < 0.05, Fig. 1a). The ratio of LC3-II to LC3-I in the ipsilateral basal ganglia of aging rats was much higher compared with that in young rats (1.76 0.16

vs. 1.14 0.03, p < 0.05, Fig. 1a). Cathepsin D protein levels in the ipsilateral basal ganglia 7 days after ICH were also significantly higher in aged compared to young rats (2,480 146 vs. 1,476 533 pixels, p < 0.05, Fig. 1b). Immunohistochemistry showed age-related differences in ferritin immunoreactivity and microglia activation in the ipsilateral basal ganglia 11 weeks after ICH. There were more ferritin-positive cells in the ipsilateral basal ganglia in aged rats (p < 0.05, Fig. 2a). Most ferritin positive cells were microglia-like. OX-42 staining confirmed more microglia in the ipsilateral basal ganglia in aged than in young rats (Fig. 2b). Aged rats had slow recovery after ICH. Marked neurological deficits were found 1 day after ICH in young and aged rats, and there were no significant differences between the groups for the initial behavioral deficits at day 1. Young rats showed better forelimb using asymmetry scores from day 3 (Fig. 3a), better forelimb placing scores from day 7 (Fig. 3b) and better corner turn scores from week 7 (Fig. 3c).

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Fig. 1 Western blot analysis showing LC3-I (18 kDa), LC3-II (16 kDa) (a) and cathepsin D levels (b) in the ipsilateral (ipsi) basal ganglia of aging (lanes 13) or young (lanes 46) rats and in the contralateral (contra) basal ganglia of young (lanes 79) rats 7 days after ICH. Values are mean SD, *p < 0.05 vs. the other groups

Young

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Fig. 2 Ferritin (a) and OX-42 (b) positive cells in the ipsilateral basal ganglia 11 weeks after ICH. Values are mean SD, n = 78, #p < 0.01 vs. young rats

116 Fig. 3 Forelimb use asymmetry (a), forelimb placing (b) and corner turn (c) tests were performed prior to ICH (P) and then at days 1, 3, 5 and weeks 111 after ICH. Values are mean SD, n = 78. *p < 0.05, #p <0.01 vs. young rats

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120 100 Forelimb use asymmetry (%) 80 60 40 20 0 20 Young Aged # #

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Discussion
ICH results in stronger autophagic responses in aged compared to young rats. 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. We examined ICH-induced autophagy in young and aged rats at day 7 after autophagy in the perihematomal area peaks at that time [2]. The function of autophagy after ICH still remains unclear, and autophagy could be beneficial or harmful. During autophagic processes, injured cellular components are degraded in the lysosome. Enhanced autophagy in aged ICH rats may result from severe brain injury. Our previous studies have demonstrated that brain swelling is more severe in aged compared to young rats and that aged animals have stronger microglial activation [1]. However, over-activated autophagy may actually cause neuronal death. Thus, there is evidence showing that autophagy in certain pathological situations can trigger and mediate programmed cell death [11, 12]. Iron accumulates in the brain after ICH, reaching very high levels. Iron causes brain injury after ICH and is not cleared from the brain for at least several weeks [6, 13, 14]. Our previous studies found that iron can induce autophagy in the brain and deferoxamine, an iron chelator, reduces ICHinduced autophagy. This suggests an important role of iron in autophagy following ICH [2]. In this study, there were more ferritin positive cells after ICH in aged rats. Ferritin is the iron storage protein and can be upregulated in the brain after ICH [6]. Iron may participate in the induction of autophagy in aged rats, and it would be interesting to know whether iron overload after ICH is more severe in aged rats. Microglia is activated in response to injury. In normal brain, microglia are quiescent, but after ICH they become highly phagocytic and are involved in clearing debris from areas of damage [15]. Microglia become progressively activated with age in humans [8, 16]. Inhibition of microglia activation by minocycline reduces ICH-induced brain injury [17]. Activated microglia secretes many toxic materials. In the present study we found that there were greater neurological deficits after ICH in aged rats. Future studies should determine if greater autophagy and microglia activation cause worse behavioral deficits in aged rats. Clarification of the mechanisms of brain injury after ICH in the aging brain should help develop new therapeutic strategies for hemorrhagic brain injury.
Acknowledgment This study was supported by grants NS-017760, NS-039866 and NS-057539 from the National Institutes of Health (NIH) and 0755717Z, 0840016N from the 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. Drs. Gong and Gu were supported by NSFC30872675 and NSFC30700864 from the China National Natural Science Foundation. Conflict of interest statement of interest. We declare that we have no conflict

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