J Mol Neurosci DOI 10.1007/s12031-014-0271-1

Upregulation of EHD2 after Intracerebral Hemorrhage in Adult Rats Kaifu Ke & Ying Rui & Lei Li & Heyi Zheng & Wei Xu & Xiang Tan & Jianhua Cao & Xiaoyan Wu & Gang Cui & Maohong Cao

Received: 24 October 2013 / Accepted: 24 February 2014 # Springer Science+Business Media New York 2014

Abstract EHD2, a member of the Eps15 homology domain (EH domain) family, is important for protein interactions during vesicular trafficking. Previous studies have proved that EHD2 can regulate trafficking from the plasma membrane in the process of endocytosis. However, its function in central nervous system diseases is still with limited understanding. In this frame, we found that EHD2 expression was upregulated in the perihematomal caudate in adult rats after intracerebral hemorrhage (ICH). Double immunofluorescence staining revealed that EHD2 was colocalized with neurons and activated microglias after ICH. Besides, we detected that neuronal apoptosis markers (TUNEL and caspase-3), and microglial activation marker (CD68), also known as a marker of macrophage, were colocated with EHD2. The vitro study also indicated that EHD2 was linked with neuronal apoptosis and microglial phagocytosis. All our findings suggested that EHD2 might be involved in the pathophysiology of ICH. Keywords EHD2 . Intracerebral hemorrhage . Microglia . Phagocytosis . Rat

Introduction Intracerebral hemorrhage (ICH) is a subtype of stroke with high morbidity and mortality that causes devastating neurologic outcome. To meet this formidable challenge, considerable research has been carried out to study its pathophysiologic

Kaifu Ke and Ying Rui contributed equally to this work. K. Ke : Y. Rui : L. Li : H. Zheng : W. Xu : X. Tan : J. Cao : X. Wu : G. Cui : M. Cao (*) Department of Neurology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province 226001, People’s Republic of China e-mail: [email protected]

process in recent years (Wang 2010; Xi et al. 2006), but the understanding of the exact mechanism is limited. ICH causes brain injury through primary physical disruption of adjacent tissue and the mass effect. Moreover, secondary injury, such as brain edema and inflammation, also occurs (Wang 2010). After ICH, neurons undergo apoptosis in the region of peri-ICH and have associations with the activation of caspase-3 (Gong et al. 2001). Microglias, acting as inflammatory cell in brain, are significant in the pathology of ICH. The resistant microglias were activated on the onset of ICH, and they may secrete the inflammatory mediators, clear the death neurons and blood (Wang and Dore 2007). In this process, the vesicular trafficking played an essential role. The Eps15 homology domain (EH domain), is a conserved domain which is important for protein interactions during vesicular trafficking (Benjamin et al. 2011). The mammalian genome encodes four EH domain-containing proteins, EHD14 (18-EHDs [EH (Eps15 homology)-domain-containing proteins] which have been proved to play a crucial role in different stages of endocytosis. They differ in localization and function. EHD1 is localized in the endocytic-recycling compartment which regulates recycling of different ligands. EHD3 is localized in tubular structures of the endocytic recycling compartment. It participates in the transport from the early endosome to the endocytic recycling compartment, regulating endosome-to-Golgi transport and maintaining Golgi morphology. EHD4 is localized in the plasma membrane, and it disrupts nerve growth factor (NGF) receptor internalization in PC12 cells under dominant negative forms. EHD2 is also localized in plasma membrane which regulates trafficking from the plasma membrane and recycling back to it (Benjamin et al. 2011). It is highly expressed in heart, placenta, lung, and skeletal muscle. Fainter bands are seen in brain, liver, kidney, and pancreas (Pohl et al. 2000). But whether it is involved in the pathophysiological process of ICH and its function remains unknown.

J Mol Neurosci

In our study, we have constructed the ICH model and detected the expression and localization of EHD2 in the perihematomal rat brains. Furthermore, we have detected the relationship between EHD2 expression and neuronal apoptosis as well as microglia activation in both vivo and vitro. What we found might be a new insight in the pathophysiology of ICH.

Methods and Materials Animals and the ICH Model Experiments were performed in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (National Research Council 1996, USA); all animal procedures were approved by the Department of Animal Center, Medical College of Nantong University. Male Sprague–Dawley rats (n=45, 6–8 weeks) with an average body weight of 250 g (220–275 g) were used in this study. Animals were housed under a 12 h light/dark cycle in a pathogen-free area with free access to water and food. Intracerebral hemorrhage (ICH) model was used as described previously with minor modifications (Jiang et al. 2002). The rats were anesthetized with chloral hydrate (Liew et al. 2012). Autologous blood or saline (sham group) 20 μl were infused into the right caudate nucleus stereotactically at a rate of 10 μl/min through a 26gauge needle (coordinates: 0.2 mm anterior, 5.5 mm ventral, and 3.5 mm lateral to the bregma) using a microinfusion pump. The needle was removed 10 min after injection, the skin incision closed, and the animals were allowed to recover. Finally, five rats were lost in the ICH group (Li et al. 2013). Behavioral Tests ICH-induced neurological deficits were assessed using forelimb-placing and corner-turn tests (Hua et al. 2002). Both forelimb-placing and corner-turn tests are widely used to evaluate the neurological deficits when injured in sensorimotor cortex and basal ganglia. In the vibrissae-elicited forelimb-placing test, test of each forelimb was conducted by brushing the respective vibrissae on the edge of a table top once per trial and repeated for ten times. A score of 1 was given if the rat placed its forelimb onto the edge of the table in response to vibrissae stimulation. The percentage of successful placing responses was recorded. A higher score indicates better function (Ohnishi et al. 2011; Okauchi et al. 2010). In the corner-turn test, the rat was allowed to proceed into a corner of which the angle was 30°. To exit the corner, the animal could turn to either the left or the right, and each movement was recorded. This task was repeated 10–15 times, and the percentage of right turns was reckoned. Impairment leads to a greater % of right turns than the scores of control animals (Okauchi et al. 2010).

Western Blot Analysis Rats were given chloral hydrate and sacrificed at different time points postoperatively (n=3 for each time point), the parenchyma tissue surrounding the hematoma (extending 3 mm to the hematoma) as well as an equal part of the contralateral, the left tissue were dissected out and immediately frozen at -80 °C until use. To prepare lysates, frozen perihematomal tissue samples were weighed and minced with eye scissors in ice. The samples were then homogenized in lysis buffer (1 % NP-40, 50 mmol/l Tris, pH 7.5, 5 mmol/l EDTA, 1 % SDS, 1 % sodium deoxycholate, 1 % Triton-X100, 1 mmol/l PMSF, 10 μg/ml aprotinin, and 1 μg/ml leupeptin) and centrifuged at 12,000 rpm and 4 °C for 20 min to collect the supernatant. After determined protein concentration with the Bradford assay (Bio-Rad), protein samples were undergone sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidine diflouride filter (PVDF) membrane by a transfer apparatus at 350 mA for 2.5 h. The membrane was blocked with 5 % nonfat milk in TBS-T for 2 h and incubated with primary antibody against EHD2 (anti-mouse, 1:300; Santa Cruz), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; anti-rabbit, 1:500, Santa Cruz) at 4 °C overnight (Shen et al. 2008a). After incubating with an anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody, protein was visualized using an enhanced chemiluminescence system (ECL, Pierce). Sections and Immunohistochemistry The rats were deeply anesthetized with chloral hydrate (0.4 g/kg) and perfused pericardially with 500 ml 0.9 % saline and then 4 % paraformaldehyde following at different survival times (n=5 per time point). After perfusion, the brains were removed and postfixed in the same fixative for 3 h and then replaced with 20 % sucrose for 2–3 days, following 30 % sucrose for 2–3 days. After treatment with sucrose solutions, the tissues were embedded in optimum cutting temperature (OCT) compound. Then, 7 μm frozen cross-sections were prepared and examined. All sections were stored at -20 °C until use. Sections were removed from the freezer, kept in an oven at 37 °C for 30 min, and rinsed in 0.01 M PBS for 5 min. Then we blocked the sections with confining liquid consisting of 10 % donkey serum, 1 % bovine serum albumin (BSA), 0.3 % Triton X-100, and 0.15 % Tween-20 for 2 h at room temperature, then incubated with anti-EHD2 antibody (mouse, 1:75, Santa Cruz) overnight at 4 °C. After incubated with the primary and the second reagents as the second antibody for 20 and 30 min, respectively, at 37 °C, the reaction sections were incubated with the liquid mixture [0.02 % daminobenzidine tetrahydrochloride (DAB), 3 % H2O2, and 0.1 % PBS]. Finally, the sections were dehydrated and covered with coverslips. Slides were examined at×10 or×20 magnifications on a Leica light microscope

J Mol Neurosci

(Germany). We examined the sections and counted the cells with strong or moderate brown staining, weak or no staining, as positive or negative EHD2 cells, respectively, from each group at higher magnified images.

the proportion of MAP2-positive cells expressing EHD2, a minimum of 200 MAP2-positive cells were counted adjacent to the hematoma in each section. Then double-labeled cells for EHD2 and MAP2 were recorded. Two or three adjacent sections per animal were sampled. So did CD11b.

Double Immunofluorescent Staining Cell Culture and Transfection After air-dried for 1 h at room temperature, sections were rinsed in 0.01 M PBS for 5 min and then blocked with 10 % normal serum-blocking solution species the same as the secondary antibody, containing 3 % (w/v) bovine serum albumin (BSA) and 0.1 % Triton X-100 and 0.05 % Tween-20 2 h at RT in order to avoid unspecific staining. Then the sections were incubated with mouse primary antibodies for anti-EHD2 (mouse, 1:75; Santa Cruz), rabbit monoclonal primary antibodies anti-GFAP (a marker of astrocytes, 1:200; Sigma), antiMAP2 (a marker of neuron, 1:100; Abcam), anti-CD11b (microglial marker, 1:75; Santa Cruz), and rabbit primary antibodies for CD68 (1:100; Santa Cruz). Briefly, sections were incubated with all primary antibodies overnight at 4 °C, followed by a mixture of FITC- and TRITC-conjugated secondary antibodies for 2 h at 4 °C (Shen et al. 2008b). The stained sections were examined with a Leica fluorescence microscope (Leica, DM 5000B; Leica CTR 5000; Germany). Terminal Deoxynucleotidyl Transferase-Mediated Biotinylated-dUTp Nick-End Labeling Terminal deoxynucleotidyl transferase-mediated biotinylateddUTP nick-end labeling (TUNEL) staining was performed by use of the In Situ Cell Death Detection Kit, Fluorescence (Roche). Frozen tissue sections were rinsed with PBS and treated with 1 % Triton-100 for 2 min on ice. Slides were rinsed in PBS and incubated for 60 min at 37 °C with 50 μl of TUNEL reaction mixture. The negative control sections were incubated for 60 min at 37 °C with 50 μl label solution. After washing with PBS, the slides were analyzed with fluorescence microscopy (Leica DM 5000B, Germany). Quantitative Analysis Cell quantification in the border of the hematoma was performed in an unbiased manner according to the principles described. To avoid counting the same cell in more than one section, we counted every fifth section (50 μm apart). The number of EHD2-positive cells around the hematoma (3 mm from the injection site) was counted at 20× magnification. For each section, three separate perihematoma regions were examined. The cell counts in the three or four sections were then used to determine the total number of EHD2-positive cells per square millimeter. The number of cells double-labeled for EHD2 and the other phenotypic markers, such as MAP2, GFAP, and CD11b used in the experiment, were quantified. To identify

The HAP I cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) with 10 % FCS at 37 °C in a humidified incubator in the condition of 5 % CO2 and 95 % air. Confluent cultures were passaged by trypsinization. When cells were allowed to reach 80 % confluence, culture medium was switched to serum-free DMEM medium, and experiments were initiated 24 h later. LPS (100 ng/ml) was administered directly into culture medium with 1 % FCS for the indicated incubation times. Nontreated cells as controls were performed in all experiments. PC12 cells were cultured in Dulbecco’s modified Eagle’s medium with 10 % (v/v) fetal bovine serum, 5 % donor horse serum, and antibiotics at 37 °C under 5 % CO2 in humidified air. The cells were passed every 3–4 days. To study apoptosis, cells were seeded onto a poly-L-lysine-coated 60 mm dishes and incubated in a low concentration of serum (1 % horse serum) for 24 h prior to treatment with hemin in 100 μmol/l at different time points (Ke et al. 2013). Primerpairs for the EHD2 (NM_001024897.1) small interfering RNA (siRNA) expression vector targeted the sequence: 5′ GCUGGAGAUUUCUGAUGAATT 3′. For transient transfection, the EHD2 siRNA vector and the nonspecific vector were carried out using lipofectamine 2,000 (Invitrogen) and plus reagent in OptiMEM (Invitrogen). Transfected cells were used for the subsequent experiments 48 h after transfection. Statistical Analysis All data were analyzed with Stata 7.0 statistical software. All values are expressed as means±SEM. The statistical significance of differences between groups was determined by oneway analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison tests. p