Obesity Exacerbates Rat Cerebral Ischemic Injury ...

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Ming-Hsiu Wu1,2 & Chung-Ching Chio3 & Kuen-Jer Tsai1 & Ching-Ping Chang4 & ..... Chen MP, Tsai JC, Chung FM, Yang SS, Hsing LL, Shin SJ, Lee YJ.
Mol Neurobiol DOI 10.1007/s12035-015-9305-0

Obesity Exacerbates Rat Cerebral Ischemic Injury through Enhancing Ischemic Adiponectin-Containing Neuronal Apoptosis Ming-Hsiu Wu 1,2 & Chung-Ching Chio 3 & Kuen-Jer Tsai 1 & Ching-Ping Chang 4 & Nan-Kai Lin 5 & Chao-Ching Huang 1,7 & Mao-Tsun Lin 6

Received: 26 February 2015 / Accepted: 11 June 2015 # Springer Science+Business Media New York 2015

Abstract A diet consisting of high levels of saturated fat has been linked to a dramatic rise in obesity. Long-term exposure to high fat, BWestern diet^ (WD), is detrimental to ischemic brain injury. Adiponectin receptor 1 (ADR-1) activation is also shown to exacerbate ischemic neuronal death. However, it is not known whether increasing percentages of adiponectin (APN)-containing neurons attenuates ischemic neuronal apoptosis by modulating ADRS. To explore the role of APN and its ADRs in the development of acute cerebral injury, we subjected WD and control diet (CD) rats to 1 h of middle cerebral artery occlusion followed by 23 h of reperfusion. Compared with CD rats, WD rats exhibited higher levels of brain infarct, neurologic deficits, brain edema, and apoptosis of APN-containing neurons; upregulation of both ADR-1 and P38 mitogen-activated protein kinase (P38MAPK); and downregulation of ADR-2 in ischemic brain tissues including * Chao-Ching Huang [email protected] * Mao-Tsun Lin [email protected] 1

The Institute of Clinical Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan

2

Division of Neurology, Department of Internal Medicine, Chi Mei Medical Center, Liouying, Tainan, Taiwan

3

Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan

4

Department of Biotechnology, Southern University of Science and Technology, Tainan, Taiwan

5

Li-Sheng Biotechnology Co., Ltd., Taipei, Taiwan

6

Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan

7

Department of Pediatrics, Wan-Fan Hospital, College of Medicine, Taipei Medical University, Taipei, Taiwan

frontal cortex, striatum, and hippocampus. Increasing percentages of APN-containing neurons by baculovirus-mediated administration of APN, in addition to reducing apoptosis of APN-containing neurons in ischemic brain tissues, significantly attenuated brain infarct and edema, neurologic deficits, and altered expression of ADR-1, P38MAPK, and ADR-2 in both WD and CD group rats. These data suggest a negative correlation between percentages of APN-containing neurons and cerebral ischemic injury. Obesity could exacerbate rat cerebral ischemic injury by enhancing apoptosis of APNcontaining neurons in ischemic brain tissues probably via modulating ADRs and P38MAPK. Keywords Adiponectin . Stroke . Obesity . Cerebral ischemia . P38 mitogen-activated protein kinase

Introduction Evidence has accumulated to indicate that elevating adiponectin (APN) in the plasma reduces the risk of ischemic stroke, whereas lowering plasma levels of APN enhances the risk of ischemic stroke [1, 2]. Increased 5-year mortality in stroke patients is associated with low plasma level of APN [3]. Plasma level of APN is negatively correlated with brain infarct and neurologic deficit [3]. Compared with extracranial atherosclerosis, intracranial atherosclerosis group has the lowest APN level [4]. In a mouse model, Nishimura et al. [5] showed that APN-deficient mice had significantly larger cerebral infarct and higher neurologic deficits after ischemia-reperfusion than did wild-type mice. They also demonstrated that infarct size in both APN-deficient and wild-type mice was reduced by supplementation of exogenous APN. However, it is not known whether there is a negative correlation between APN-containing neurons and ischemic injury.

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The typical North American or BWestern^ diet (WD) has been linked to increased insulin resistance [6], obesity [7], dyslipidemia [8], arterial hypertension [9], and hyperglycemia [10]. These dysfunctions are associated with the incidence of stroke [11]. The hypothesis is further confirmed by an animal study showing that long-term exposure to WD has increased functional deficits and larger infarct following ischemia compared with those on a control diet (CD) [12]. Again, it is unknown whether obesity exacerbates ischemic injury through enhancing apoptosis of APN-containing neurons. Indeed, as shown in the present results, a negative correlation between percentage of APN-containing neurons and ischemic injury exists in a rat stroke model of middle cerebral artery occlusion (MCAO). In addition, obesity may exacerbate ischemic injury in stroke rats by enhancing apoptosis of APN-containing neurons.

Materials and Methods Subjects and Experimental Conditions Male Sprague–Dawley rats (Animal Resource Centers, Ministry of Science and Technology of the Republic of China) were used for this study. One group of rats were fed with balanced (CD; low-fat control for WD, Test Diet, 5TJS, Richmond, 1 N, USA), whereas WD groups of rats were fed with high fat diet (WD for rodents, Test Diet, 5TJN). The balanced CD consisted mainly of protein (16 % of energy, Kcal), fat (12 %), and carbohydrates (72 %). The WD consisted primarily of protein (16 %), fat (40 %), and carbohydrates (44 %). These animals were maintained the CD or WD for a period of 3 months. Rats were housed in groups of three on 12:12 h reverse light:dark cycle with lights off at 08:00 and fed food and water ad libitum. Ethics Statement This study strictly adhered to the recommendations in the Guide for the Care and Use of Laboratory Animals of the Department of Science and Technology of the Republic of China. The protocol was approved by the Chi Mei Medical Center Institutional Review Board for Animal Care and Use (Assurance Numbers: 101122405). All efforts were made to minimize the suffering of the experimental rats. Animals and Stroke Model Both CD group rats and WD group rats were anesthetized with ketamine (36.76 mg/ml), xylazine (5.659 mg/ml), and atropine (0.022 mg/ml) intramuscularly. Rectal temperature was maintained between 36.5 and 37.5 °C with a homeothermic blanket. Focal cerebral ischemia was induced by MCAO by the intraluminal filament technique, as described

previously [13, 14]. The filament was withdrawn 1 h after occlusion. In all animals, regional cerebral blood flow (CBF) was measured by laser Doppler (PF2B; Perimed, Stockholm, Sweden) with a flexible probe to confirm the achievement of consistent and similar levels of ischemic condition. Experimental Groups The WD or CD was initiated at ~6 weeks of age, and animals remained on their respective diets until experimental ending. Animals weighed ~354 g (~2.4 months of age) at the time of MCAO surgery. The rats were randomly assigned to one of eight groups: (1) CD group rats treated with sham operation (A); (2) WD group rats treated with operation (A’); (3) CD group rats treated with MCAO (B); (4) WD group rats treated with MCAO (B’); (5) MCAO-treated CD group rats pretreated with baculovirus-mediated APN (C); (6) MCAOtreated WD group rats pretreated with baculovirus-mediated APN (C’); (7) sham CD group rats pretreated with baculovirus-mediated APN (D); and (8) sham WD group rats pretreated with baculovirus-mediated APN (D’). In experiment 1, cortical, striatal, and hippocampal expressions of APN, APN R1, APN R2, and P38 MAPK in the ipsilateral brains were determined 72 h after operation in all eight groups of rats. In experiment 2, modified neurological severity scores (mNSS) were determined in all eight groups of rats 72 h postoperation. In experiment 3, cerebral infarction volumes and Evans Blue extravasations were determined in all eight groups of rats 72 h after operation. In experiment 4, numbers of colocalization of NeuN + APN + TUNEL positive cells in the cortex, striatum, and hippocampus were determined in all eight groups of rats 72 h after operation. In experimental 5, cortical, striatal, and hippocampal expressions of caspase-3 were determined in all eight groups of rats 72 h after operation. Intrastriatal Injection of Recombinant Baculovirus Expressing Adiponectin Bac-to-Bac system, kanamycin, gentamicin, tetracycline, Xgal, and IPTG were purchased from Invitrogen. Restriction enzyme BamH I, EcoR I, Sal I, and T4 polynucleotide kinase were purchased from New England Biolabs. Plasmids promoter CMV-IE was from the courtesy of S.T. Lee (STUST, Tainan, Taiwan). The full-length cDNA of the APN was constructed at Taiwan Academia Sinica under the aid of professor K.J. Tsai. Baculoviruses are insect pathogens and belong to DNA viruses with the capability of infecting more than 600 insects. Among them, Autographa californica multiple

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nucleopolyhedrovirus (AcMNPV) has been studied extensively and employed in gene transfer widely [15]. Baculovirus can infect not only insect cells but also a wide range of mammalian cells. Under appropriate promoter, the encoded target gene can be properly translated [16]. The recombinant baculovirus expressing APN was made by Bac-to-Bac system. Using BamH I and EcoR I double digestion, CMV-IE was introduced into the pFastBac donor plasmid to form the baculovirus transfer vector pFastBac™ 1 [17]. The plasmids were named pFastBac™ 1-CMV-IE, and then it was constructed to express APN (NM_144744) sequence under the control of cytomegalovirus immediateearly (CMV-IE) promoter [18]. The plasmid design is in the order of CMV-IE-APN. The primers were designed as (EcoR I) 5′ ggAATTCCATgCTACTgTTgCAAgCgCTCCTgTT 3′ a n d ( S a l I ) 5 ′ C g g T C g A C C g T C A g T T g g TAT C ATggTAgAgAAggA 3′ (Tri-I Biotech). By gel electrophoresis for comparison of DNA content, insert and vector were mixed with the proportion of one to three. The DNA ligation was proceeded with T4 DNA ligase, at 16 °C overnight. The pFastBac™ 1-CMV-IE APN donor plasmids were transformed into MAX Efficiency Escherichia coli DH10Bac™ to form colonies [19]. The donor plasmids used the mini-attTn7 as the target site and a helper plasmid to form recombinant bacmids. The E. coli DG10Bac™ cells were cultured in 1.5 ml culture tube with 37 °C for 3.5 h and then shaken with 225 rpm to form extracts 100 μl on coated plates. The plate contained kanamycin, gentamicin, and tetracycline with concentration 0.2 %. A lacZ deletion was present on the chromosome while colonies were blue (Lac+) in the presence of a chromogenic substrate such as X-gal (Invitrogen™) and the inducer, IPTG (Invitrogen™). White colonies were selected to enhance extraction with recombinant basmid DNA. The recombinant bacmids were transfected into insert Spodoptera frugiperda Sfa cells by cellfectin II reagent for amplification of virus. The IPLB-Sf9 (Sf9) cell line was cultured at 26 °C in Grace’s insect medium (GibcoBRL), supplemented with 10 % heat-inactivated FBS. It was used for the generation and propagation of wild type and recombinant AcMNPV. All viral stocks were prepared according to the standard protocols described by O’Reilly [20]. At 4 days post-infection (dpi), the TNM-FH medium containing virus particles was harvested by centrifugation at 1000g for 5 min and stored at 4 °C without further ultracentrifugation. The virus titers were determined by quantitative PCR [21]. These recombinant baculovirus expressing APN was then used for intracerebral gene transfection. Modified Neurological Severity Scores Acute neurological injury was assessed in all groups of rats 72 h after surgery using a mNSS, a composite of the motor,

sensory, and reflex test scores [22]. One point was given for failure to perform a cash. The higher the score, the more severe the injury.

Western Blot Rats were measured for protein expression 72 h after MCAO. Proteins were harvested in ice-cold RIPA buffer containing protease inhibitors and phosphatase inhibitors. The tissue lysates were incubated on ice for 30 min, spun down, and the supernatant transferred to a new tube. Aliquots containing 100 μg of proteins were separated by 12 % SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5 % nonfat mild in PBS buffer containing 1 % Tween-20 for 1 h at room temperature, then incubated with one of the following primary antibodies: Actin (1:4000, Santa Cruz), APN (1:1000, cell signaling), APN R 1 (1:500, Bioss), APN R 2 (1:1000, abcam), Caspase-3 (1:1000, cell signaling), or P38 MAPK (1:1000, cell signaling) overnight at 4 °C. The membranes were then washed three times in PBS containing 1 % Tween-20 and incubated with the anti-mouse or antirabbit or anti-goat immunoglobulin G (1:10000, Santa Cruz) for 1 h at room temperature. Quantification of western blotting was performed using Image J software with normalization to total protein levels.

Immunofluorescence Staining Rats were measured with immunofluorescence staining 72 h after MCAO. After rats were perfusion with 0.9 % saline and 4 % formalin, the brain was taken out sequentially and soaked into 4 % formalin (Sigma, Chemical Co), 15 % sucrose in phosphate buffer (PB), and 30 % sucrose in PB. Then, the brain was sliced transversely in 4 mm thicknesses and frozen embedded. The brain sides were detected with neurons containing protein APN. That were covered with the mounting medium containing DAPI for the detection of total nuclei and covered with the APN, primary antibodies 1:200 (Bioss), and NeuN 1:100 (Millipore), second antibody anti-goat 488 mm, anti-mouse 588 mm. The other procedures for detecting apoptotic were performed by using a TUNEL kit (Clontech). The slides were cover with the neuronal antibody for detection of total neuron and covered with the APN antibody for detection of protein expression, secondary antibody anti-mouse 368 mm. Tissue pieces were checked with fluorescence microscopy after staining. At brain regions, including cortex, striatum, and hippocampus, we checked neurons expressing APN and the percentage of TUNEL-positive nuclei was calculated [23]. Fluorescence staining was viewed with a fluorescent microscope (Zeiss).

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Cerebral Infarction Assessment

Evans Blue Extravasation

Rats were with deep anesthesia and were transcardially perfused with heparinized 0.05 mol/l phosphate-buffered saline (PBS) followed by ice-cold 15 % sucrose in PBS. The brains were rapidly removed and frozen in liquid nitrogen and then sectioned for immunohistochemistry [24, 25]. Brain slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC) as detailed previously [23]. The infarct volume (mm3), as revealed by negative TTC stains indicating dehydrogenasedeficiency tissue, was measured in each slice and summed using computerized planimetry [26]. We also made correction of the distortion of infarct volume caused by brain edema in reference to the report of Lin et al. [14].

Evans Blue dye (Sigma-Aldrich, St. Louis, MO, USA; 10 %, 5 ml/kg) in 0.9 % saline was injected into the right femoral vein at 3 days after the onset of MCAO. After the dye had circulated for 2 h, rats were anesthetized generally and transcardially perfused with physiological saline. Brains were removed and each hemisphere was weighted, homogenized in PBS, and centrifuged at 2000×g for 15 min at 4 °C. After overnight incubation and centrifugation at 3500×g for 30 min at 4 °C, the supernatant was taken for spectrophotometric quantification (Thermo Fisher Sciencific Inc., Watham, MA, USA) of extravasated Evans Blue dye at 620 nm and quantified

Fig. 1 Elevating percentages of DAPI + Neu N + APN-specific stained cells by intrastriatal injection of baculovirus-mediated APN. Top panels; Immunofluence staining of % of DAPI + Neu N + APN-specific stained cells of ipsilateral frontal cortex (a), striatum (b), and hippocampus (c) from CD rats received sham operation (A’; CD + Sham), WD rats received sham operation (B’; WD + Sham), CD rats received MCAO (A; CD + MCAO), WD rats received MCAO (B; WD + MCAO), CD rats received MCAO and APN injection (C; CD + MCAO + APN), WD rats received MCAO and APN injection (D; WD + MCAO + APN), CD rats

received sham operation and APN (A^; CD + Sham + APN), and WD rats received sham operation and APN (B^; WD + Sham + APN). CD and WD group rats were subjected to 1 h of MCAO followed by 72 h of reperfusion. Intracerebral injection of recombinant baculovirusmediated APN was performed 3 days prior to MCAO or sham operation. Data are presented as mean±SD (n=8 for each group). *P