Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1–Related Microglial Activation in Neonatal Hypoxic-Ischemic Encephalopathy

Neonatal hypoxic-ischemic encephalopathy (nHIE) is a major neonatal brain injury. Despite therapeutic hypothermia, mortality and sequelae remain severe. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) associated with the pathophysiology of nHIE. In this study, morphologic change microglial activation under nHIE condition LOX-1 treatment were investigated. activity proliferation assessed novel method, immunostaining, quantitative PCR in rat brains both model anti–LOX-1 treatment. Circumference ratio, long diameter cell area roundness microglia calculated. correlation metrics treated was evaluated. expressed activated ameboid round brain. evaluation activation, correlated all scales nHIE-damaged brains. While circumference ratios had positive correlation, ratio negative correlation. 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Oka Itoh target encephalopathy.Am Pathol. 2014; 184: 1843-1852Abstract (12) However, which express role unclear.18Akamatsu Scholar,19Schwarz D.A. Barry Mackay K.B. Manu Naeve G.S. Vana Verge Conlon Foster A.C. Maki Identification differentially genes induced ischemic stroke.Brain Res Mol Brain 2002; 101: 12-22Crossref (49) LOX-1–expressing morphologically effect pathologic lesions assessed. All experiments study approved animal experiment ethical committees National Center Neurology Psychiatry Global Health Medicine. Rats housed 12-hour:12-hour light/dark cycle food water available ad libitum. Seven-day–old Sprague-Dawley pups (CLEA Japan, Tokyo, Japan) used divided into three groups: control (CTL), (HIE), (a-LOX-1). CTL group, underwent no surgical procedures kept at 36°C 2 hours. HIE a-LOX-1 groups, anesthetized, then exposed, ligated, cut left common carotid artery, previously described.18Akamatsu Scholar,20Rice 3rd, Vannucci Brierley J.B. influence immaturity damage rat.Ann 1981; 9: 131-141Crossref (1877) 21Patel S.D. Pierce Ciardiello S.J. pre-clinical studies neuroprotection.Biochem Soc Trans. 42: 564-568Crossref (38) 22Yao Zhang He X. Wang Jiang K. Zhao Establishment identification hypoxia-ischemia rats.Biomed Rep. 4: 437-443Crossref (8) After dam 1 hour, exposed 8% O2 hours maintained body temperature 36°C. 60 μg/kg intraperitoneally injected just (HI) insult every 12 HI until sacrifice (anti–LOX-1 treatment), Hippocampal tissues assessment study. anesthetized perfused intracardially phosphate-buffered saline (PBS) biochemical assessments 4% paraformaldehyde PBS immunohistochemical assessments, collected 24, 48, 72 7 days insult. For 4°C 24 embedded optimal cutting compound (Sakura Finetek Japan Co., frozen −80°C. hippocampus excised brains, immediately preserved coronal sections (16-μm thickness) cryostat (CM3050S; Leica Biosystems, Wetzlar, Germany). performed hypothalamus level. histopathologic analyses, MAP2 staining terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis performed. photographs taken fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). To assess stained rabbit polyclonal anti-MAP2 (Merck Millipore, Darmstadt, Germany) donkey anti-rabbit IgG conjugated DyLight 488 (Jackson ImmunoResearch Laboratories, West Grove, PA), regarded lost infarction. intact areas hemispheres calculated ImageJ software version 1.48 (NIH, Bethesda, MD; https://imagej.nih.gov/ij). insulted hemisphere compared (intact ratio) among groups hours, evaluate hippocampus, TUNEL assay an ApopTag fluorescein situ detection kit Millipore) antifade solution containing DAPI. TUNEL+ DAPI+ counted ×400 magnification. numbers number (apoptosis incubated primary antibodies goat (R&D Systems, Minneapolis, MN), anti-Iba1 (Wako Pure Chemical Industries, Japan), Millipore), mouse monoclonal anti-GFAP (Cell Signaling Technology, Beverly, MA), anti-MBP (Dako Corporation, Carpinteria, CA), anti–Ki-67 (Leica Nussloch, 16 anti-goat Alexa Fluor 598 Laboratories), anti-mouse 568 (Thermo Fisher Scientific, Rockford, IL), room hour. Finally, sealed DAPI observed (BZ-X700). addition, confirm distribution brain, immunostaining biotin-conjugated (Nichirei additional chromogen 3-amino-9-ethyl carbazole substrate Co.), counterstaining hematoxylin. antibody, four 1-mm2 fields CA1 region magnification, area, circumference, Iba1+ (microglia) each field measured (Hybrid Cell Count function; Keyence). changing shape ultimately according immunoreactive intensity (Figure 1A). (circumference ratio), (long rectangle, composed diameters (cell (the diameter) 1B) selected showing nucleolus sections. average one data point pup. Ki-67 Iba1 double-positive (Ki-67+/Iba1+) counted. Ki-67+/Iba1+ RNA extracted RNeasy Mini Kit (Qiagen, Venlo, Netherlands) reverse-transcribed high-capacity cDNA reverse transcription Scientific). elucidate between microglia, levels Olr1 (oxidized pro- using LightCycler 480 SYBR Green I Master, System II, Gene Scanning Software 1.5.0 (Roche Diagnostics, Basel, Switzerland). standard reference gene glyceraldehyde 3-phosphate dehydrogenase (gapdh) normalization reference. primer sequences probes follows: forward primer, 5′-AAACTATGCCTCCTGTCTGACC-3′; 5′-TTCATGCAGCAACAGAAGGC-3′; Il1beta 5′-ACCTATGTCTTGCCCGTGGA-3′; 5′-GCAGGTCGTCATCATCCCAC-3′; Il6 5′-CCCAACTTCCAATGCTCTCCT-3′; 5′-GGATCGGTCTTGGTCCTTAGCC-3′; Ccl2 [chemokine (C-C motif) ligand] 5′-GGCCAGCCCAGAAACCAGCC-3′; 5′-AGCAGCAGGTGAGTGGGGCA-3′; Tnf (tumor necrosis factor α) 5′-GGCCAATGGCATGGATCTCAAA-3′; 5′-AGCCTTGTCCCTTGAAGAGAAC-3′; Il10 5′-GCCAAGCCTTGTCAGAAATGA-3′; 5′-TTTCTGGGCCATGGTTCTCT; Tgfb1 (transforming growth factor-β1) 5′-TGGAGCCTGGACACACAGTA; 5′-GTAGTAGACGATGGGCAGTGG-3′. represented median expressions group. According manufacturer’s instruction DIG Probe Synthesis (Roche, Switzerland), hybridization distribution. First, DIG-labeled DNA made. probes' Olr1, 5′-TGACCCTGCCATGCCATGCT-3′; 5′-TGGGGATGGTGGAGGCCCTG-3′. 5-μm–thick 9-day–old refixed deparaffinized, washed Tris-buffered saline, denatured 200 mmol/L HCl, proteinase K (2 CaCl2, 20 μg/mL PBS). hybridized 55°C alkaline phosphatase-labeled anti-DIG (Roche) visualized 4-nitro blue tetrazolium chloride 5-bromo-4-chloro-3-indolyl phosphate. Each section bright-field illumination BX51 (Olympus, ×20, ×100, newborn victim without malformations any other abnormalities. fixed formalin, paraffin, 4-μm deparaffinization, anti–LOX-1, anti-Iba1, anti-MBP, anti-GFAP, anti-NeuN (EMD Billerica, MA) IgG, 598, 488, microscope. humans Ethical Committee permitted parents informed content. Statistical analyses SPSS Statistics 24.0 (IBM, Armonk, NY). Kolmogorov-Smirnov normality tests conducted outcome variables. Because not determined most variables, sample sizes (n = 4 9), nonparametric hereafter. expressions, Kruskal-Wallis test, followed U-tests Bonferroni corrections. rats, infarction group significantly lower than restored time points (Supplemental Figure S1, A C). higher decreased B D). Many hippocampal S2). type expressing LOX-1, double-staining 48 anti-MAP2, 2A). only (microglia), MAP2+ (neuron), GFAP+ (astrocyte), MBP+ (oligodendrocyte). Furthermore, 2B shows lesion, but area. parameters measured. shapes first ratios. decreased, those A–C). On hand, ratios, times Regarding roundness, degrees insult, there differences 3D). (Iba1+ cells) Ki-67+ investigate response Most 4A). total At 4B). 4C). Il1beta, Il-6, Tnf, makers (proinflammatory mediators), Tgf-beta1 markers (anti-inflammatory mediators) qualitatively analyzed 5). (Il1beta, Tnf-alpha, Ccl2) markedly increases (Il10 Tgfbeta1) genes, up-regulation Tgf-β1 mRNA diffusely neuron neocortex, amygdala complex, thalamus, basal ganglia, S3). postmortem 6A) 6B). 5 (round microglia), NeuN+ neurons, astrocytes, oligodendrocytes. exhibited phase feature distributed neurons cells. Interestingly, similar We propose method assessing during initial pathophysiological step, energy failure excitatory amino acids necrosis. It has been known earliest nHIE.23Ferrazzano Chanana Uluc Fidan Akture Kintner D.B. Cengiz Sun Age-dependent immature hypoxia-ischemia.CNS Neurol Disord Drug Targets. 2013; 12: 338-349Crossref (34) discrepancy suggests initially occurred various cytokines expression. reaction cascade initiated reperfusion, leading irreversible injury, death, long-lasting inflammation.24Drury P.P. 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