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Metabolomic Profiling Reveals That Reprogramming of Cerebral Glucose Metabolism is Involved in Ischemic PreconditioningInduced Neuroprotection in a Rodent Model of Ischemic Stroke Jianliang Geng, Yue Zhang, Sijia Li, Shuning Li, Jiankun Wang, Hong Wang, jiye Aa, and Guangji Wang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00339 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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Journal of Proteome Research

Metabolomic Profiling Reveals That Reprogramming of Cerebral Glucose Metabolism is Involved in Ischemic Preconditioning-Induced Neuroprotection in a Rodent Model of Ischemic Stroke

Jianliang Geng, †

†, ‡

Wang, Jiye Aa,

†

†

†

†

†

Yue Zhang, Sijia Li, Shuning Li, Jiankun Wang, Hong

*, †

Guangji Wang

*, †

Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China

‡

College of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China

* Corresponding author. E-mail address: [email protected] Tel (Fax): +86 25 83302827 [email protected] Tel (Fax): +86 25 83271176

ABSTRACT Ischemic tolerance renders the brain resistant to ischemia-reperfusion (I/R) injury as a result of the activation of endogenous adaptive responses triggered by various types of preconditioning. The complex underlying metabolic mechanisms responsible for the neuroprotection of cerebral ischemic preconditioning (IPC) remain elusive. 1

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Herein, gas chromatography-mass spectrometry (GC-MS) technique was applied to delineate the dynamic changes of brain metabolome in a rodent model of ischemic stroke (transient occlusion of the middle cerebral artery, tMCAO), alone or after pretreatment with nonlethal ischemic tolerance induction (transient occlusion of the bilateral common carotid arteries, tBCCAO). Metabolomic analysis showed that accumulation of glucose (concentration increased more than 4 fold) and glycolytic intermediates is the prominent feature of brain I/R induced metabolic disturbance. IPC attenuated brain I/R damage by subduing post-ischemic hyperglycolysis, increasing PPP flux and promoting the utilization of β-hydroxybutyrate. The expression analysis of pivotal genes and proteins involved in relevant metabolic pathways revealed that the downregulation of AMP-activated protein kinase (AMPK)-mediated glucose transporter-1 (GLUT-1) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) and reduced mRNA levels of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits were associated with IPC-induced metabolic flexibility which allows the brain more capable to withstand severe I/R insults. The present study provided mechanistic insights into the metabolic signature of IPC and indicated that adaptively modulate brain glucose metabolism could be an effective approach for the therapeutic intervention of ischemic stroke.

KEYWORDS: Metabolomic profiling, Ischemic stroke, Ischemic preconditioning, Glucose metabolism reprogramming, Neuroprotection 2


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INTRODUCTION Ischemic stroke is a serious global public health problem that causing high mortality rates and long-term disability.1, 2 Limitations in the clinical application of thrombolytics and recurrent failures in neuroprotective drug development highlight the need for new therapeutic strategies in ischemic stroke treatment. Ischemic preconditioning (IPC), which is also termed ischemic tolerance, refers to a phenomenon in which the tissue or organ is rendered resistant to the deleterious effects of prolonged ischemia by previous exposure to brief periods of noninjurious stimulation. As an effective endogenous protective mechanism, ischemic tolerance has attracted extensive interest in the field of cardiac-cerebral vascular disease research, since it was first discovered by Murry et al. 3 During recent decades, the neuroprotective properties of IPC have been demonstrated in animal models of cerebral ischemia.4-6 In addition, large clinical trials showed that patients who suffer from transient ischemic attacks (TIA) before ischemic stroke have more favorable outcome than do patients without TIA.7 These findings provided robust evidence for the existence of cerebral preconditioning in humans. The molecular signaling cascades involved in endogenous neuroprotection are numerous and extremely complex. A multitude of biomolecules could act as stimuli, mediators, messengers or effectors during the adaptive response of the body to ischemic stress. Understanding the metabolic signatures of IPC is crucial not only for identifying potential therapeutic targets that can be modulated by pharmacological interventions, but also for facilitating the clinical translation of brain self-protective 3



therapy. Although several regulatory pathways that focus on cellular metabolism have been explored to explain the beneficial effects in the preconditioned brain, 8-10 the intricate metabolic mechanisms that underlie the IPC process remain elusive. The brain is a high energy consuming organ that depends on aerobic metabolism to fulfill its normal function. Glucose is the obligatory metabolic substrate for energy production, ribonucleotide biosynthesis and redox balance maintenance. Alterations in glucose metabolism have been identified as an important pathological mechanism of ischemic stroke. Experimental studies and clinical surveys have confirmed that preischemic hyperglycemia greatly aggravates postischemic neuronal damage, which is also known as “the glucose paradox of cerebral ischemia” .11, 12 Moreover, hyperglycolysis, which is defined as an abnormal increase in glucose uptake and metabolism in the ischemic penumbra, has been proven to be detrimental to neuronal survival.13 In addition to being metabolized to provide energy, glucose is also involved in the generation and elimination of reactive oxygen species (ROS), which result in oxidative damage to membrane lipids, proteins, and nucleic acids. 14, 15 Reduced nicotinamide adenine dinucleotide phosphate (NADPH), as a product of the oxidative pentose phosphate pathway (oxPPP), provides an essential redox equivalent for reduced glutathione (GSH) regeneration and enhances the antioxidant defense capacity.16 Meanwhile, over-activated NADPH oxidase (NOX) generates numerous ROS and further lead to neuronal death in the reperfusion stage.17 Metabolic depression is one of the main features of brain ischemic tolerance. 18, 19 Preconditioning stimuli elicit neuroprotective responses in both the early and delayed 4


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time windows by regulating the expression of a series of genes and proteins involved in cerebral metabolism and thereby slowing brain energy consumption rates and preserving normal mitochondrial function. 20, 21 These observations suggest a potential correlation between brain glucose metabolism and IPC-induced neuroprotection. To explore the metabolic regulatory mechanism of brain ischemic tolerance, an analytical platform based on gas chromatography-mass spectrometry (GC-MS) was employed to characterize the dynamic changes of brain metabolomic profile in the rodent models during I/R injury and IPC intervention. On the basis of comprehensive metabolomic analyses, we further demonstrated that IPC confers neuroprotection by reprogramming cerebral glucose metabolism. Our results provided insights into the neuroprotective effects of brain IPC.

MATERIALS AND METHODS Animals and Surgical Procedures Male Sprague-Dawley rats, aged 6-7 weeks, weighing 240-250 g, were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All the rats were provided water and standard chow ad libitum and acclimated to the facilities in a temperature-controlled environment (25°C) with a 12-h light-dark cycle for 3-4 days. The animal protocols used in this experiment were approved by the Animal Ethical Committee of China Pharmaceutical University. All experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (China Pharmaceutical University). The rats were randomly divided into six groups, including sham-operation group (Control), brain ischemia 1.5 h group (I-1.5 h), brain 5



ischemia 1.5 h and reperfusion 1 h group (I/R-1 h), brain ischemia 1.5 h and reperfusion 24 h group (I/R-24 h), brain IPC 24 h group (IPC-24 h) and brain IPC with ischemia 1.5 h and reperfusion 24 h group (IPC+I/R-24 h). Focal cerebral I/R injury was induced by transient occlusion of the middle cerebral artery (tMCAO), as previously described by Zea Longa et al.,22 with minor modifications. Briefly, the rats were anesthetized with an intraperitoneal injection of 10% aqueous choral hydrate (300 mg/kg, Sinopharm Chemical Reagent, China). A local anesthetic solution of 2 % lidocaine hydrochloride (10 mg/kg, Otsuka Pharmaceutical, China) was subcutaneously injected into the area of prospective incision site. Then, right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were surgically exposed through an incision along the midline of the neck. The right ECA was isolated and coagulated. A 30-mm length of monofilament with a silicon-coated tip (Jialing Biotech Co., Ltd., China) was introduced into the ECA lumen and gently advanced to the MCA from the CCA bifurcation via the ICA until slight resistance was felt. After 1.5 h of ischemia, focal cerebral blood flow was restored (reperfusion) by withdrawal of the inserted suture and the neck incision was carefully closed. IPC was induced by five cycles of 3-min transient occlusion of the bilateral common carotid arteries (BCCAO) with microsurgical clips, with each followed by 5 min of reperfusion. A heating pad was used to maintain the body temperature of the rats during surgery. The sham operation was identical except for the insertion of the monofilament into the ECA and the occlusion of the CCAs. 6


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At the end of the study, the animals were transcardially perfused with precooled saline under deep anesthesia. Then, the forebrain was immediately removed and the ischemic penumbra area was dissected on ice, according to the literature.23 The samples were then, snap-frozen in liquid nitrogen to quench metabolism and stored at -80 °C until extraction. The experimental scheme is illustrated in Figure1.

Figure 1. Schematic representation of the experimental protocols.

GC-MS Based Metabolomic Profiling Brain tissue (30 mg) was loaded into a 2-mL beating tube with three steel beads. An 800-µL aliquot of precooled methanol-water (4:1 v/v) containing 1,2-13C2-myristic acid (5 µg/mL, Sigma-Aldrich) was added to the sample. The tissue was homogenized using a bead beater (Retsch, MM400, Haan, Germany) with 30 s of 7



vibration at 6,500 Hz for 3 cycles. Then, the homogenates were centrifuged at 15,000 g for 10 min at 4 °C. A 200-µL aliquot of the supernatant was transferred into the autosampler vials and evaporated to dryness in a SpeedVac concentrator (Thermo Fisher Scientific, SavantTM SC250EXP, Holbrook, USA). An optimized two-step method was applied for sample derivatization.24 First, 30 µL of a 1% methoxyamine pyridine solution (Sigma-Aldrich) was added to the residue and incubated for 16 h at room temperature for oximation. Then, the analytes were trimethylsilylated using 30 µL of N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA, Sigma-Aldrich) containing 1% v/v trimethylchlorosilane (TMCS) as a catalyst. After trimethylsilylation for 1 h, 30 µL of n-heptane (Merck) containing methyl stearate (30 µg/mL, Sigma-Aldrich) was added to each GC vial to monitor the stability of the GC-MS instruments. The final mixture (90 µL in total) was vortexed for 1 min and was then ready for GC-MS analysis. The derivatized samples were analyzed using GC coupled to a mass spectrometer (Shimadzu GCMS-QP2010 Ultra, Kyoto, Japan) equipped with an automatic sampler (Shimadzu AOC-20i, Kyoto, Japan). A 0.5-µL sample aliquot was injected into the Rtx-5MS capillary column (0.25 mm × 30 m × 0.25 µm, Restek, PA, USA) in splitless mode. The injector temperature was set at 270 °C. The septum purge was turned on, with a flow rate of 2.0 mL/min. Helium was used as the carrier gas, with a flow rate of 1.0 mL/min. The column temperature was initially maintained at 70 °C for 2 min, was then raised to 320 °C at a rate of 20 °C/min, and was held for 2 min. Each run lasted a total of 23 min, and solvent cutting acquisition started at 4.5 8


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min. The mass spectrometer ion source temperature was 250 °C, and ionization was achieved with a 70-eV electron beam. Mass spectra were obtained from m/z 50 to 700 in full scan mode, and the acceleration voltage was turned on after a solvent delay of 170 s. The quality control (QC) samples pooled from each brain sample were prepared and analyzed with the same procedure described above. Three QCs were used to balance the instrument system before running. The samples were analyzed in a random sequence and the QC samples were injected at regular intervals (every 6 samples) to monitor the stability of the analysis in each batch. The GC-MS data were processed with Shimadzu GC postrun analysis software for baseline correction, denoising, peak smoothing and alignment, and characteristic ion extraction. The metabolites were identified by comparing their mass spectra with those in the National Institute of Standards and Technology standard compound database 14.0 (NIST) and the Wiley standard compound database 9.0 (Weily-VCH Verlag Gmbh & Co. KGaA) or with those of reference standards. The peak areas of identified compounds were quantified by the characteristic ions and were normalized by the internal standards before multivariate statistical analysis. Metabolomic Data Processing The resulting GC-MS data set, including compound names, grouping, sample names (observations) and peak areas (variations) was imported into the SIMCA-P software package (version 14.1, Umetrics, Sweden) for multivariate data analysis. Principal component analysis (PCA) with unit variance (UV) scaling was performed to 9



visualize the dynamic change trends of the brain metabolome in different phases. An orthogonal partial least squares-discriminant analysis (OPLS-DA) model was constructed after Pareto scaling to identify the differential metabolites by S-plot and variable importance on projection (VIP) values. The differential metabolites were further evaluated by Student’s t-test to prove their statistical significance. Neurobehavioral Assessment Neurological deficits were assessed using a five-point scale as described in a previous study.25 Briefly, the scale was as follows: score of 0: normal (no neurobehavioral dysfunction); score of 1: slight (failure to fully extend the opposite forepaw when suspended vertically); score of 2: moderate (contralateral circling); score of 3: severe (leaning to the affected side); and score of 4: very serious (no autonomous activity and unconsciousness). The degree of neurological damage was evaluated 1.5 h after ischemia and at 24 h after reperfusion by an observer who was blinded to the experimental conditions. The rats with a neurological deficit score of 1 or above at 1.5 h after the MCAO operation was diagnosed with acute ischemic stroke and was selected for subsequent experiments. Measurement of the Infarct Area After neurological examination, the rats were rapidly perfused with normal saline. The brains were frozen at -20 °C for 20 min and sliced into 2-mm coronal sections for 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich) staining. The brain slices were incubated in a 1% TTC phosphate buffer solution at 37 °C for 30 min followed by 4% formalin (Phygene) overnight. After TTC staining, the viable tissues were 10


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stained red, while the infarcted tissues were remained white. The stained sections were scanned, and the infarct areas were statistically analyzed as percentages of the total slice areas using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Biochemical Assay and Histopathological Analysis Blood glucose concentrations were measured by a portable blood glucose meter (ACCU-CHEK® Active, Roche) using blood samples obtained by tail bleeding. The concentrations of brain NADPH and adenosine triphosphate (ATP) were determined by commercial fluorometric assay kits (Sigma-Aldrich). Brain glucose-6-phosphate dehydrogenase (G6PD) activity and phosphofructokinase (PFK-1) activity were measured by commercially available assay kits (Sigma-Aldrich) using a coupled enzyme reaction, which results in a colorimetric product that is proportional to the enzymatic activity. The GSH/ glutathione disulfide (GSSG) ratio of brain tissue was measured with a microplate assay kit (Jiancheng Bioengineering Institute, China). The brain NOX activity was determined by a colorimetric assay kit (Jiancheng Bioengineering Institute, China) using the enzymatic inhibition method. Detailed experimental manipulations were performed according to the manufacturer’s instructions. Histopathological changes were assessed by hematoxylin-eosin (H&E) staining. Briefly, the brains were rapidly removed after the rats were sacrificed. The ischemic hemispheres were dissected out, immersed in 10% phosphate-buffered formalin for 12 h of fixation, and then embedded in paraffin wax. A series of 5-µm thick sections 11



were sliced and stained with H&E to examine the microstructure of the cells. Determination of the Metabolic Flux of the PPP Flux through the oxidative and non-oxidative PPP generates m+1 lactate from 1, 2-13C glucose, while glycolysis generates only m+2 lactate as shown in Figure S1. Therefore, m+1 lactate / m+2 lactate reflects ratio of PPP overflow to glycolysis. The metabolic flux of the PPP (pentose cycle, PC %) was measured by a GC-MS platform using stable isotope-labeled 1,2-13C2 glucose (Cambridge Isotope Laboratories, USA) as a tracer. Briefly, 1,2-13C2 glucose was infused via the jugular vein at a constant rate of 1.5 mmol/kg/h using a syringe pump (BYZ-810, TSYD, China) for 1 h. Then, the rats were transcardially perfused with precooled saline.26 The ischemic penumbra were collected and immediately frozen in liquid nitrogen until extraction and analysis. The mass isotopomer distribution in lactate was calculated from the spectral intensities after subtracting the derivatized groups and natural 13C enrichment using the Isocor software.27 The equation (m1/m2)/(3+m1/m2) was used to calculate the PC %.28, 29 RNA Isolation and RT-PCR Analysis Total RNA was isolated from frozen rat brains using the RNAiso Plus reagent (Takara Bio). RNA concentrations and quality were verified by spectrophotometric method. cDNA was synthesized using a PrimeScript TM RT reagent kit with gDNA eraser (Takara Bio) according to the manufacturer's protocol. All cDNA samples were stored at -20 °C. Quantitative real-time PCR was conducted with the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), using the SYBR Green 12


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method. The primers were synthesized by Invitrogen (Life Technologies), and the sequences are shown in Table S1. The PCR thermocycling parameters were set as 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Each sample was run in duplicate and was normalized to the housekeeping gene β-actin. The replicates were averaged, and the fold induction was determined on by using ∆∆Ct-based fold-change calculations. Western Blot Analysis The total proteins were extracted from 30 mg of brain cortical tissues (penumbra) with 300 µL of RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail (Servicebio). The protein concentrations were determined using a BCA protein assay kit (Beyotime). A total of 20 µg of proteins was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene difluoride membranes. After treatment for 1 h with a blocking solution containing 5% nonfat dry milk, the membrane was incubated with the following specific primary antibodies against rabbit anti-6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3, 1:1000; Cell Signaling), rabbit anti-glucose transporter-1 (GLUT-1, 1:1000; Abcam), rabbit anti- adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPKα, 1:1000; Cell Signaling), rabbit anti-phospho-AMPKα (Thr172) (p-AMPKα, 1:1000; Cell Signaling) and rabbit anti-β-actin (1:1000; Servicebio) at 4 °C overnight. Afterwards, the membranes were washed three times with TBST at 10 min intervals and incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h 13



at room temperature. The densities of the bands were determined by using image analysis software (Gel-pro Analyzer 4.5). Superoxide Detection To detect ROS in vivo, dihydroethidium (DHE, Sigma-Aldrich) was prepared as a 1 mg/mL solution in 2% DMSO and administered at 2 mg/kg via the caudal vein.30 After 30 min, the rats were euthanized and perfused with cold saline. The chilled brain was sliced into 10-µm sections with a microtome cryostat (LEICA, CM1950). The fluorescence intensity was observed with a confocal fluorescence microscope (ZEISS, LSM 700) with excitation at 535 nm and emission at 580 nm. Sections of the cerebral cortex from the penumbra region were analyzed from each sample. Statistical Analysis All the data were presented as the means±SEM (standard error of the mean). The significance of differences between two groups was determined using an unpaired

t-test. One-way ANOVA was used with Bonferroni's multiple comparison test for comparisons among multiple groups (GraphPad Prism 7.0). A difference was considered statistically significant when p < 0.05.

RESULTS Neuroprotective Effects of IPC in a Rodent Ischemic Stroke Model To evaluate the neuroprotective effects of IPC, the cerebral infarct area, neurological deficit scores, and histopathological changes were assessed. The ischemic stroke model rats showed obvious behavioral symptoms of brain damage and infarct focus. IPC decreased the neurological deficit scores and the cerebral infarction of model rats, 14


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as shown in Figure 2A, 2B and 2C. The H&E staining revealed a series of morphologic abnormalities in neurons after brain I/R injury, such as cell swelling, nuclear hyperchromatism and pyknosis and encephalomalacia. IPC significantly improved these pathological changes but did not affect the normal tissue, as shown in Figure 2D.

Figure 2. The neuroprotective effects of IPC on brain I/R damage. The brains were harvested at 24h after reperfusion for evolution. (A) Representative brain sections stained with TTC. (B) Brain infracts area. (C) Neurological deficit scores. (D) Representative H&E staining of cortexes (×400). Data are expressed as mean ± SEM. (Asterisks, vs sham operation; number sign, vs I/R-24h, ** p

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