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

2021; Elsevier BV; Volume: 191; Issue: 7 Linguagem: Inglês

10.1016/j.ajpath.2021.04.009

1525-2191

Tomohisa Akamatsu, Takehiro Sugiyama, Takuya Oshima, Yoshinori Aoki, Ayumi Mizukami, Keiji Goishi, Hiroyuki Shichino, Norihiro Kato, Naoto Takahashi, Yu‐ichi Goto, Akira Oka, Masayuki Itoh,

Immune Response and Inflammation

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) is associated with the pathophysiology of nHIE. In this study, morphologic change and microglial activation under the nHIE condition and LOX-1 treatment were investigated. The microglial activity and proliferation were assessed with a novel morphologic method, immunostaining, and quantitative PCR in the rat brains of both nHIE model and anti–LOX-1 treatment. Circumference ratio, the long diameter ratio, the cell area ratio, and the roundness of microglia were calculated. The correlation of the morphologic metrics and microglial activation in nHIE model and anti–LOX-1 treated brains was evaluated. LOX-1 was expressed in activated ameboid and round microglia in the nHIE model rat brain. In the evaluation of microglial activation, the novel morphologic metrics correlated with all scales of the nHIE-damaged and treated brains. While the circumference and long diameter ratios had a positive correlation, the cell area ratio and roundness had a negative correlation. Anti–LOX-1 treatment attenuated morphologic microglial activation and proliferation, and suppressed the subsequent production of inflammatory mediators by microglia. In human nHIE, round microglia and endothelial cells expressed LOX-1. The results indicate that LOX-1 regulates microglial activation in nHIE and anti–LOX-1 treatment attenuates brain injury by suppressing microglial activation. 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) is associated with the pathophysiology of nHIE. In this study, morphologic change and microglial activation under the nHIE condition and LOX-1 treatment were investigated. The microglial activity and proliferation were assessed with a novel morphologic method, immunostaining, and quantitative PCR in the rat brains of both nHIE model and anti–LOX-1 treatment. Circumference ratio, the long diameter ratio, the cell area ratio, and the roundness of microglia were calculated. The correlation of the morphologic metrics and microglial activation in nHIE model and anti–LOX-1 treated brains was evaluated. LOX-1 was expressed in activated ameboid and round microglia in the nHIE model rat brain. In the evaluation of microglial activation, the novel morphologic metrics correlated with all scales of the nHIE-damaged and treated brains. While the circumference and long diameter ratios had a positive correlation, the cell area ratio and roundness had a negative correlation. Anti–LOX-1 treatment attenuated morphologic microglial activation and proliferation, and suppressed the subsequent production of inflammatory mediators by microglia. In human nHIE, round microglia and endothelial cells expressed LOX-1. The results indicate that LOX-1 regulates microglial activation in nHIE and anti–LOX-1 treatment attenuates brain injury by suppressing microglial activation. Neonatal hypoxic-ischemic encephalopathy (nHIE) following asphyxia is a major brain injury in infants and occurs in approximately 1.5 per 1000 births.1Kurinczuk J.J. White-Koning M. Badawi N. Epidemiology of neonatal encephalopathy and hypoxic–ischaemic encephalopathy.Early Hum Dev. 2010; 86: 329-338Crossref PubMed Scopus (664) Google Scholar Despite therapeutic hypothermia, one-quarter of infants with moderate or severe nHIE die, and 30% of the survivors suffer from severe neurologic sequelae such as cerebral palsy, developmental disorders, epilepsy, and deafness.2Shankaran S. Laptook A.R. Ehrenkranz R.A. Tyson J.E. McDonald S.A. Donovan E.F. Fanaroff A.F. Poole W.K. Wright L.L. Higgins R.D. Finer N.N. Carlo W.A. Duara S. Oh W. Cotton C.M. Stevenson D.K. Stoll B.J. Lemons J.A. Guillet R. Jobe A.H. Human Development Neonatal Research NetworkWhole-body hypothermia for neonates with hypoxic-ischemic encephalopathy.N Engl J Med. 2005; 353: 1574-1584Crossref PubMed Scopus (2017) Google Scholar, 3Gluckman P.D. Wyatt J.S. Azzopardi D. Ballard R. Edwards A.D. Ferriero D.M. Polin R.A. Robertson C.M. Thoresen M. Whitelaw A. Gunn A.J. Selective head cooling with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial.Lancet. 2005; 365: 663-670Abstract Full Text Full Text PDF PubMed Scopus (1752) Google Scholar, 4Azzopardi D.V. Strohm B. Edwards A.D. Dyet L. Halliday H.L. Juszczak E. Kapellou O. Levene M. Marlow N. Porter E. Thoresen M. Whitelaw A. Brocklehurst P. 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Microglial physiology and pathophysiology: insights from genome-wide transcriptional profiling.Immunity. 2016; 44: 505-515Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar Microglia maintain central nervous system homeostasis through the clearance of dying or dead cells, apoptosis, the elimination of excess axons, the promotion of neuroaxonal growth, axonal guidance, and neuronal differentiation, and the regulation of embryonic cortical precursor cell development, astrocyte proliferation, and angiogenesis.8Walter L. Neumann H. Role of microglia in neuronal degeneration and regeneration.Semin Immunopathol. 2009; 31: 513-525Crossref PubMed Scopus (110) Google Scholar,9Czeh M. Gressens P. Kaindl A.M. The yin and yang of microglia.Dev Neurosci. 2011; 33: 199-209Crossref PubMed Scopus (239) Google Scholar Activated microglia alter their own morphology, proliferate, and secrete inflammatory mediators. Morphologically, although physiologically resting microglia show ramified shapes, small cell bodies, and thin, long processes, activated microglia show ameboid or round shapes, large cell bodies, and thick, short processes.10Lehrmann E. Christensen T. Zimmer J. Diemer N.H. Finsen B. Microglial and macrophage reactions mark progressive changes and define the penumbra in the rat neocortex and striatum after transient middle cerebral artery occlusion.J Comp Neurol. 1997; 386: 461-476Crossref PubMed Scopus (0) Google Scholar,11Thored P. Heldmann U. Gomes-Leal W. Gisler R. Darsalia V. Taneera J. Taneera J. Nygren J.M. Jacobsen S.-E.W. Ekdahl C.T. Kokaia Z. Lindvall O. Long-term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke.Glia. 2009; 57: 835-849Crossref PubMed Scopus (275) Google Scholar Like macrophages, functional M1 microglia secrete proinflammatory mediators, and M2 microglia secrete anti-inflammatory mediators. In nHIE, activated microglia are polarized to the M1 or M2 phenotype, and M1 activation enhances proinflammatory functions, resulting in secondary neuroinflammation and neuronal apoptosis.12Bhalala U.S. Koehler R.C. Kannan S. Neuroinflammation and neuroimmune dysregulation after acute hypoxic-ischemic injury of developing brain.Front Pediatr. 2015; 2: 144Crossref PubMed Scopus (74) Google Scholar,13Xiong X.-Y. Liu L. Yang Q.-W. Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke.Prog Neurobiol. 2016; 142: 23-44Crossref PubMed Scopus (296) Google Scholar Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor that was identified as a receptor of oxidized low-density lipoprotein (ox-LDL).14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Aiba Y. Tanaka T. Miwa S. Katsura Y. Kita T. Masaki T. An endothelial receptor for oxidized low-density lipoprotein.Nature. 1997; 386: 73-77Crossref PubMed Scopus (1144) Google Scholar Ischemia reperfusion injury and cytokines, as well as ox-LDL, induce LOX-1 expression in endothelial cells, thrombocytes, macrophages, and vascular smooth muscle cells.15Mehta J.L. Chen J. Hermonat P.L. Romeo F. Novelli G. Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1): a critical player in the development of atherosclerosis and related disorders.Cardiovasc Res. 2006; 69: 36-45Crossref PubMed Scopus (378) Google Scholar,16Navarra T. Del Turco S. Berti S. Basta G. The lectin-like oxidized low-density lipoprotein receptor-1 and its soluble form: cardiovascular implications.J Atheroscler Thromb. 2010; 17: 317-331Crossref PubMed Scopus (68) Google Scholar Activation of the LOX-1 intracellular pathway induces reactive oxygen species, NF-kappaB, monocyte chemoattractant protein-1 (MCP-1), caspase 9, and caspase 3, resulting in oxidative stress and apoptosis.17Li D. Mehta J.L. Intracellular signaling of LOX-1 in endothelial cell apoptosis.Circ Res. 2009; 104: 566-568Crossref PubMed Scopus (67) Google Scholar The authors reported that LOX-1 expression was increased in the brain in the nHIE model rat and that anti–LOX-1 neutralizing antibody administration attenuated the lesion area of infarction, edema, and neural apoptosis in the brain in nHIE model rat.18Akamatsu T. Dai H. Mizuguchi M. Goto Y.-I. Oka A. Itoh M. LOX-1 is a novel therapeutic target in neonatal hypoxic-ischemic encephalopathy.Am J Pathol. 2014; 184: 1843-1852Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar However, which neural cells express LOX-1 and the role of LOX-1 in nHIE remain unclear.18Akamatsu T. Dai H. Mizuguchi M. Goto Y.-I. Oka A. Itoh M. LOX-1 is a novel therapeutic target in neonatal hypoxic-ischemic encephalopathy.Am J Pathol. 2014; 184: 1843-1852Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar,19Schwarz D.A. Barry G. Mackay K.B. Manu F. Naeve G.S. Vana A.M. Verge G. Conlon P.J. Foster A.C. Maki R.A. Identification of differentially expressed genes induced by transient ischemic stroke.Brain Res Mol Brain Res. 2002; 101: 12-22Crossref PubMed Scopus (49) Google Scholar In this study, the LOX-1–expressing cells were morphologically activated microglia in the nHIE brain and anti–LOX-1 treatment suppressed microglia activation. The effect of anti–LOX-1 treatment on functional microglial activation and proliferation in pathologic lesions of the brain in rat and human nHIE was morphologically assessed. All experiments in this study were approved by the animal experiment ethical committees of the National Center of Neurology and Psychiatry and National Center for Global Health and Medicine. Rats were housed under a 12-hour:12-hour light/dark cycle with food and water available ad libitum. Seven-day–old Sprague-Dawley rat pups (CLEA Japan, Tokyo, Japan) were used and divided into three groups: control (CTL), nHIE (HIE), and anti–LOX-1 (a-LOX-1). In the CTL group, the pups underwent no surgical procedures and were kept at 36°C for 2 hours. In the HIE and a-LOX-1 groups, the pups were anesthetized, and the authors then exposed, ligated, and cut the left common carotid artery, as previously described.18Akamatsu T. Dai H. Mizuguchi M. Goto Y.-I. Oka A. Itoh M. LOX-1 is a novel therapeutic target in neonatal hypoxic-ischemic encephalopathy.Am J Pathol. 2014; 184: 1843-1852Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar,20Rice 3rd, J.E. Vannucci R.C. Brierley J.B. The influence of immaturity on hypoxic-ischemic brain damage in the rat.Ann Neurol. 1981; 9: 131-141Crossref PubMed Scopus (1877) Google Scholar, 21Patel S.D. Pierce L. Ciardiello A.J. Vannucci S.J. Neonatal encephalopathy: pre-clinical studies in neuroprotection.Biochem Soc Trans. 2014; 42: 564-568Crossref PubMed Scopus (38) Google Scholar, 22Yao D. Zhang W. He X. Wang J. Jiang K. Zhao Z. Establishment and identification of a hypoxia-ischemia brain damage model in neonatal rats.Biomed Rep. 2016; 4: 437-443Crossref PubMed Scopus (8) Google Scholar After resting with their dam for 1 hour, the pups were exposed to 8% O2 for 2 hours and maintained at a body temperature of 36°C. In the a-LOX-1 group, 60 μg/kg anti–LOX-1 neutralizing antibody was intraperitoneally injected just after hypoxic-ischemic (HI) insult and every 12 hours after HI insult until sacrifice (anti–LOX-1 treatment), as previously described.18Akamatsu T. Dai H. Mizuguchi M. Goto Y.-I. Oka A. Itoh M. LOX-1 is a novel therapeutic target in neonatal hypoxic-ischemic encephalopathy.Am J Pathol. 2014; 184: 1843-1852Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar Hippocampal tissues were used for morphologic assessment in this study. In all groups, the pups were anesthetized and perfused intracardially with phosphate-buffered saline (PBS) for biochemical assessments or 4% paraformaldehyde in PBS for immunohistochemical assessments, and then the brains were collected at 24, 48, and 72 hours and 7 days after HI insult. For immunohistochemical assessments, the brains were perfused with 4% paraformaldehyde in PBS at 4°C for 24 hours and then embedded in optimal cutting temperature compound (Sakura Finetek Japan Co., Tokyo, Japan) and frozen at −80°C. For biochemical assessments, the hippocampus was excised from the brains, and the tissues were immediately frozen and preserved at −80°C. The frozen brains were cut into coronal sections (16-μm thickness) with a cryostat (CM3050S; Leica Biosystems, Wetzlar, Germany). All immunohistochemical assessments were performed with sections at the hippocampus and hypothalamus level. For histopathologic analyses, MAP2 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis of the frozen sections were performed. All photographs were taken with a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). To assess infarction, the sections were stained with rabbit polyclonal anti-MAP2 antibody (Merck Millipore, Darmstadt, Germany) and donkey anti-rabbit IgG antibody conjugated with DyLight 488 (Jackson ImmunoResearch Laboratories, West Grove, PA), and the authors regarded the MAP2 negative area and lost area as the infarction. The intact areas in both hemispheres were calculated by ImageJ software version 1.48 (NIH, Bethesda, MD; https://imagej.nih.gov/ij). The ratio of the intact area in the insulted hemisphere was then compared with that in the intact hemisphere (intact area ratio) among the three groups at 24, 48, and 72 hours, as previously described.18Akamatsu T. Dai H. Mizuguchi M. Goto Y.-I. Oka A. Itoh M. LOX-1 is a novel therapeutic target in neonatal hypoxic-ischemic encephalopathy.Am J Pathol. 2014; 184: 1843-1852Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar To evaluate apoptosis in the hippocampus, a TUNEL assay was performed with an ApopTag fluorescein in situ apoptosis detection kit (Merck Millipore) and antifade solution containing DAPI. All TUNEL+ cells and DAPI+ cells in the hippocampus were counted at ×400 magnification. The ratio of the TUNEL+ and DAPI+ cell numbers were compared to the DAPI+ cell number (apoptosis ratio) in the hippocampus among the three groups at 24, 48, and 72 hours. The sections were incubated with primary antibodies of goat polyclonal anti–LOX-1 (R&D Systems, Minneapolis, MN), rabbit polyclonal anti-Iba1 (Wako Pure Chemical Industries, Osaka, Japan), rabbit polyclonal anti-MAP2 (Merck Millipore), mouse monoclonal anti-GFAP (Cell Signaling Technology, Beverly, MA), rabbit polyclonal anti-MBP (Dako Corporation, Carpinteria, CA), and mouse monoclonal anti–Ki-67 (Leica Biosystems, Nussloch, Germany) at 4°C for 16 hours. The sections were then incubated with secondary antibodies of donkey anti-goat IgG conjugated with Alexa Fluor 488 and 598 (Jackson ImmunoResearch Laboratories), donkey anti-rabbit IgG conjugated with DyLight 488 (Jackson ImmunoResearch Laboratories), and goat anti-mouse IgG conjugated with Alexa 568 (Thermo Fisher Scientific, Rockford, IL), at room temperature for 1 hour. Finally, the sections were sealed with antifade solution containing DAPI and observed with a fluorescence microscope (BZ-X700). In addition, to confirm the distribution of LOX-1–expressing cells in the brain, immunostaining was performed with an anti–LOX-1 antibody and a biotin-conjugated anti-goat IgG antibody (Nichirei Co., Tokyo, Japan), additional staining with the chromogen 3-amino-9-ethyl carbazole substrate (Nichirei Co.), and counterstaining with hematoxylin. For morphologic assessments, the sections were stained with anti-Iba1 antibody, four 1-mm2 fields in the CA1 region were observed at ×400 magnification, and the area, circumference, and long and short diameter of all Iba1+ cells (microglia) in each field were measured (Hybrid Cell Count function; Keyence). To evaluate microglial activation, each Iba1+ cell was observed for microglia changing their shape from ramified to ameboid and ultimately round according to the immunoreactive intensity with an anti-Iba1 antibody (Figure 1A). The ratio of the circumference to the cell area (circumference ratio), the ratio of the long diameter to the cell area (long diameter ratio), the ratio of the cell area to the rectangle, which was composed of the long and short diameters (cell area ratio), and the roundness (the ratio of the short diameter to the long diameter) of microglia (Figure 1B) in all groups were then calculated at 24, 48, and 72 hours after HI insult. For all assessments, microglia were selected showing the nucleolus in the stained sections. The average of four fields was regarded as one data point for each pup. For the assessments of microglial proliferation, all Iba1+ cells, Ki-67 and Iba1 double-positive (Ki-67+/Iba1+) cells, and all DAPI+ cells in the hippocampus were counted. The ratio of the number of Ki-67+/Iba1+ cells to the number of Iba1+ cells and the ratio of the number of Iba1+ cells to the number of DAPI+ cells in all groups were calculated at 24, 48, and 72 hours and 7 days after HI insult. RNA was extracted from the hippocampus with an RNeasy Mini Kit (Qiagen, Venlo, the Netherlands) and reverse-transcribed with a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). To elucidate the correlation between LOX-1 expression and the functional activation of microglia, the expression levels of Olr1 (oxidized low-density lipoprotein receptor and pro- and anti-inflammatory genes were measured by quantitative PCR in all groups at 24, 48, and 72 hours after HI insult using a LightCycler 480 with SYBR Green I Master, a LightCycler 480 System II, and LightCycler 480 Gene Scanning Software version 1.5.0 (Roche Diagnostics, Basel, Switzerland). The standard reference gene rat glyceraldehyde 3-phosphate dehydrogenase (gapdh) was used as a normalization reference. The primer sequences of the probes were as follows: Olr1 forward primer, 5′-AAACTATGCCTCCTGTCTGACC-3′; Olr1 reverse primer, 5′-TTCATGCAGCAACAGAAGGC-3′; Il1beta forward primer, 5′-ACCTATGTCTTGCCCGTGGA-3′; Il1beta reverse primer, 5′-GCAGGTCGTCATCATCCCAC-3′; Il6 forward primer, 5′-CCCAACTTCCAATGCTCTCCT-3′; Il6 reverse primer, 5′-GGATCGGTCTTGGTCCTTAGCC-3′; Ccl2 [chemokine (C-C motif) ligand] forward primer, 5′-GGCCAGCCCAGAAACCAGCC-3′; Ccl2 reverse primer, 5′-AGCAGCAGGTGAGTGGGGCA-3′; Tnf (tumor necrosis factor α) forward primer, 5′-GGCCAATGGCATGGATCTCAAA-3′; Tnf reverse primer, 5′-AGCCTTGTCCCTTGAAGAGAAC-3′; Il10 forward primer, 5′-GCCAAGCCTTGTCAGAAATGA-3′; Il10 reverse primer, 5′-TTTCTGGGCCATGGTTCTCT; Tgfb1 (transforming growth factor-β1) forward primer, 5′-TGGAGCCTGGACACACAGTA; Tgfb1 reverse primer, 5′-GTAGTAGACGATGGGCAGTGG-3′. The data are represented as the ratios to the median of the gene expressions at 24 hours after HI insult in the CTL group. According to the manufacturer’s instruction for the PCR DIG Probe Synthesis Kit (Roche, Basel, Switzerland), in situ hybridization of rat brain was performed to confirm LOX-1 distribution. First, the DIG-labeled DNA probes were made. The probes' primer sequences were as follows: the rat forward primer of Olr1, 5′-TGACCCTGCCATGCCATGCT-3′; the rat reverse primer of Olr1, 5′-TGGGGATGGTGGAGGCCCTG-3′. The 5-μm–thick coronal sections of the brain of 9-day–old Sprague-Dawley rat were refixed in 4% paraformaldehyde after deparaffinized, washed with Tris-buffered saline, denatured with 200 mmol/L HCl, and treated with proteinase K solution (2 mmol/L CaCl2, 20 μg/mL proteinase K in PBS). The sections were then hybridized with the probes at 55°C for 16 hours. After washed with Tris-buffered saline, the sections were incubated with an alkaline phosphatase-labeled anti-DIG antibody (Roche) and visualized with 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate. Each section was observed under bright-field illumination on a BX51 microscope (Olympus, Tokyo, Japan) at ×20, ×100, and ×400 magnification. To confirm LOX-1 expression in the human nHIE brain, immunohistochemical analysis was performed of a newborn victim of severe nHIE without malformations or any other abnormalities. The brain was fixed with formalin, embedded in paraffin, and then cut into 4-μm sections. After deparaffinization, the sections were incubated with primary antibodies of goat polyclonal anti–LOX-1, rabbit polyclonal anti-Iba1, rabbit polyclonal anti-MBP, mouse monoclonal anti-GFAP, and mouse monoclonal anti-NeuN (EMD Millipore, Billerica, MA) at 4°C for 16 hours. The sections were then incubated with secondary antibodies of anti-goat IgG, donkey anti-goat IgG conjugated with Alexa Fluor 488 and 598, donkey anti-rabbit IgG conjugated with DyLight 488, and goat anti-mouse IgG conjugated with Alexa 568 at room temperature for 1 hour. The sections were observed with a fluorescence microscope. The pathologic experiments in humans were approved by the Ethical Committee of the National Center of Neurology and Psychiatry and were permitted by the parents with informed content. Statistical analyses were performed using SPSS Statistics software version 24.0 (IBM, Armonk, NY). Kolmogorov-Smirnov normality tests were conducted for all outcome variables. Because normality was not determined for most variables, and sample sizes were not large (n = 4 to 9), nonparametric tests were conducted hereafter. For the assessment of morphologic activation, proliferation, and gene expressions, data among the three groups were compared using the Kruskal-Wallis test, followed by U-tests with Bonferroni corrections. To confirm the effect of HI insult and anti–LOX-1 treatment on the brains of neonatal rats, the area of infarction and the number of TUNEL+ cells were compared among the three groups, as previously described.18Akamatsu T. Dai H. Mizuguchi M. Goto Y.-I. Oka A. Itoh M. LOX-1 is a novel therapeutic target in neonatal hypoxic-ischemic encephalopathy.Am J Pathol. 2014; 184: 1843-1852Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar The intact area ratio of the HIE group was significantly lower than that of the CTL group, and the intact area ratio of the a-LOX-1 group was significantly restored compared with that of the HIE group at all time points (Supplemental Figure S1, A and C). The apoptosis ratio in the hippocampus in the HIE group was significantly higher than that of the CTL group, and the apoptosis ratio in the hippocampus in the a-LOX-1 group was significantly decreased compared with that of the HIE group at all time points (Supplemental Figure S1, B and D). Many LOX-1–expressing cells were observed in the hippocampal lesion (Supplemental Figure S2). To elucidate the type of neural cells expressing LOX-1, double-staining of the brain sections was performed at 48 hours after HI insult with anti–LOX-1 antibody and anti-MAP2, anti-GFAP, anti-MBP, and anti-Iba1 antibodies (Figure 2A). The expression of LOX-1 was observed only in Iba1+ cells (microglia), not in MAP2+ cells (neuron), GFAP+ cells (astrocyte), or MBP+ cells (oligodendrocyte). Furthermore, Figure 2B shows that microglia expressing LOX-1 were the ameboid or round microglia in the lesion, but not the ramified microglia in the intact area. To morphologically evaluate the effect of anti–LOX-1 treatment on microglial activation, the sections were stained at 24, 48, and 72 hours after HI insult with an anti-Iba1 antibody, and the parameters of each Iba1+ cell were measured. Microglial shapes were first observed as their activation and measured the ratios. The circumference ratio and long diameter ratio decreased, and the cell area ratio and roundness increased according to the intensity of activation. Microglia in the HIE group had significantly lower circumference ratios and long diameter ratios and significantly higher cell area ratios at all time points than those of the CTL group (Figure 3, A–C). On the other hand, microglia in the a-LOX-1 group had significantly higher circumference ratios and long diameter ratios, and significantly lower cell area ratios at all times than those of the HIE group. Regarding roundness, although microglia in the HIE group had significantly increased degrees of roundness at 48 and 72 hours after HI insult, there were no differences among the three groups at 24 hours (Figure 3D). The number of microglia (Iba1+ cells) and Ki-67+ microglia in the hippocampus were counted to investigate microglial proliferation in response to HI insult and the effect of anti–LOX-1 treatment. Most microglia in the HIE group expressed Ki-67 (Figure 4A). The ratios of the number of Ki-67+ microglia to the number of total microglia were significantly higher in the HIE group than in the CTL group at 24 to 72 hours after HI insult. At 48 and 72 hours, the ratios in the a-LOX-1 group significantly decreased compared with those in the HIE group. At 7 days after HI insult, no differences were observed among the three groups (Figure 4B). The ratio of the number of total microglia to the number of total DAPI+ cells was suppressed in the a-LOX-1 group compared with the HIE group at 7 days after HI insult (Figure 4C). The expression of Olr1, and Il1beta, Il-6, Tnf, and Ccl2 as M1 microglial makers (proinflammatory mediators), and Tgf-beta1 and Il10 as M2 microglial markers (anti-inflammatory mediators) was qualitatively analyzed in the hippocampus to evaluate the effect of anti–LOX-1 treatment on the functional activation of microglia (Figure 5). The expression of Olr1 in the HIE group significantly increased at all times compared with that in the CTL group. Anti–LOX-1 treatment significantly suppressed the expression of Olr1 at 72 hours compared with that in the HIE group. The expression of proinflammatory genes (Il1beta, Il-6, Tnf-alpha, and Ccl2) in the HIE group was markedly increased compared with that in the CTL group. On the other hand, anti–LOX-1 treatment significantly suppressed the increases in proinflammatory genes at 72 hours. The expression of anti-inflammatory genes (Il10 and Tgfbeta1) was significantly increased in the HIE group compared with the CTL group. Regarding anti-inflammatory genes, anti–LOX-1 treatment suppressed only the up-regulation of Tgf-β1 at 72 hours. Olr1 mRNA was observed diffusely neuron in the neocortex, amygdala complex, thalamus, basal ganglia, and hippocampus, as well as endothelial cells, in the rat brain (Supplemental Figure S3). To confirm that microglia in human nHIE expressed LOX-1, the cortical sections of postmortem human brain were stained with severe nHIE. LOX-1 was expressed in neural cells (Figure 6A) and endothelial cells (Figure 6B). Figure 5 shows that LOX-1 was expressed in Iba1+ cells (round microglia), but not in NeuN+ neurons, GFAP+ astrocytes, or MBP+ oligodendrocytes. Anti–LOX-1 treatment exhibited effective neuroprotection in the acute phase of nHIE, as previously described.18Akamatsu T. Dai H. Mizuguchi M. Goto Y.-I. Oka A. Itoh M. LOX-1 is a novel therapeutic target in neonatal hypoxic-ischemic encephalopathy.Am J Pathol. 2014; 184: 1843-1852Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar In addition, LOX-1–expressing cells had a morphologic feature of activated microglia and anti–LOX-1 treatment suppressed microglial activation, proliferation, and subsequent production of inflammatory mediators. Olr1 mRNA diffusely distributed in neurons and endothelial cells. Interestingly, microglia in the human nHIE brain expressed LOX-1, similar to those in the nHIE rat brain. We propose a novel morphologic method for assessing microglial activation. In nHIE brain, during the initial pathophysiological step, energy and oxygen failure induces the accumulation of excitatory amino acids and necrosis. It has been known that microglial activation occurs earliest in the hippocampus in nHIE.23Ferrazzano P. Chanana V. Uluc K. Fidan E. Akture E. Kintner D.B. Cengiz P. Sun D. Age-dependent microglial activation in immature brains after hypoxia-ischemia.CNS Neurol Disord Drug Targets. 2013; 12: 338-349Crossref PubMed Scopus (34) Google Scholar The discrepancy of gene expression with time suggests that LOX-1 activation initially occurred and induced various cytokines expression. The inflammatory reaction and apoptosis cascade are then initiated after reperfusion, leading to irreversible central nervous system injury, neuronal cell death, and long-lasting neuronal inflammation.24Drury P.P. Bennet L. Gunn A.J. Mechanisms of hypothermic neuroprotection.Semin Fetal Neonatal Med. 2010; 15: 287-292Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar In these irreversible neuroinflammatory and apoptosis processes, activated microglia play major roles in self-proliferation and the production of cytokines and chemokines. In this study, activated microglia expressed LOX-1 in nHIE and were morphologically shifted to an anti-inflammatory phenotype with suppression of self-proliferation by anti–LOX-1 treatment. This finding indicates that LOX-1 is a key molecule in the activation and proliferation of microglia in nHIE. IL-1beta, IL-6, MCP-1, and TNF-alpha are major proinflammatory (M1) mediators, and IL-10 and TGF-beta1 are major anti-inflammatory (M2) mediators, which activated microglial expression in response to neuroinflammation.9Czeh M. Gressens P. Kaindl A.M. The yin and yang of microglia.Dev Neurosci. 2011; 33: 199-209Crossref PubMed Scopus (239) Google Scholar,13Xiong X.-Y. Liu L. Yang Q.-W. Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke.Prog Neurobiol. 2016; 142: 23-44Crossref PubMed Scopus (296) Google Scholar,25Ma Y. Wang J. Wang Y. Yang G.-Y. The biphasic function of microglia in ischemic stroke.Prog Neurobiol. 2017; 157: 247-272Crossref PubMed Scopus (297) Google Scholar,26Bose S. Kim S. Oh Y. Moniruzzaman M. Lee G. Cho J. Effect of CCL2 on BV2 microglial cell migration: involvement of probable signaling pathways.Cytokine. 2016; 81: 39-49Crossref PubMed Scopus (16) Google Scholar During neuroinflammation, microglia produced IL-1beta, TNF-alpha, and TGF-beta1.15Mehta J.L. Chen J. Hermonat P.L. Romeo F. Novelli G. Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1): a critical player in the development of atherosclerosis and related disorders.Cardiovasc Res. 2006; 69: 36-45Crossref PubMed Scopus (378) Google Scholar Cytokines then autonomously up-regulate LOX-1 expression. It is known that LOX-1 activation of microglia promotes neuroinflammation and the vicious cycle of LOX-1 activation.27Zhang D. Sun L. Zhu H. Wang L. Wu W. Xie J. Gu J. Microglial LOX-1 reacts with extracellular HSP60 to bridge neuroinflammation and neurotoxicity.Neurochem Int. 2012; 61: 1021-1035Crossref PubMed Scopus (38) Google Scholar,28Ge X. Zhang D.-M. Li M.-M. Zhang Y. Zhu X.-Y. Zhou Y. Peng X. Shen A.-G. Microglial LOX-1/MAPKs/NF-κB positive loop promotes the vicious cycle of neuroinflammation and neural injury.Int Immunopharmacol. 2019; 70: 187-200Crossref PubMed Scopus (12) Google Scholar Therefore, we can hypothesize that anti–LOX-1 treatment induced neuroprotection by not only suppressing microglial activation-mediated neuroinflammation, but also inhibiting the vicious cycle of microglial LOX-1 activation. Interestingly, anti–LOX-1 treatment suppressed the production of both M1 and M2 mediators. Anti–LOX-1 treatment decreased the expression of the M2 mediator TGF-beta1, but not IL-10. Some studies have shown that TGF-beta1 acts as a proinflammatory mediator to exacerbate HI insult.29Lou Z. Wang A.-P. Duan X.-M. Hu G.-H. Song G.-L. Zuo M.-L. Yang Z.-B. Upregulation of NOX2 and NOX4 mediated by TGF-β signaling pathway exacerbates cerebral ischemia/reperfusion oxidative stress injury.Cell Physiol Biochem. 2018; 46: 2103-2113Crossref PubMed Scopus (59) Google Scholar It is well-known that microglial functions depend on the M1/M2 balance.9Czeh M. Gressens P. Kaindl A.M. The yin and yang of microglia.Dev Neurosci. 2011; 33: 199-209Crossref PubMed Scopus (239) Google Scholar,30Amici S.A. Dong J. Guerau-de-Arellano M. Molecular mechanisms modulating the phenotype of macrophages and microglia.Front Immunol. 2017; 8: 1520Crossref PubMed Scopus (79) Google Scholar,31Hu X. Li P. Guo Y. Wang H. Leak R.K. Chen S. Gao Y. Chen J. Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia.Stroke. 2012; 43: 3063-3070Crossref PubMed Scopus (877) Google Scholar These studies, together with the current results, suggest that anti–LOX-1 treatment strongly decreases the M1 aspect of microglial function. Activated microglia change their shape and show functional activations such as the proliferation, phagocytosis, and the production of inflammatory mediators.32Morrison H.W. Filosa J.A. A quantitative spatiotemporal analysis of microglia morphology during ischemic stroke and reperfusion.J Neuroinflammation. 2013; 10: 4Crossref PubMed Scopus (302) Google Scholar,33Zanier E.R. Fumagalli S. Perego C. Pischiutta F. De Simoni M.-G. Shape descriptors of the "never resting" microglia in three different acute brain injury models in mice.Intensive Care Med Exp. 2015; 3: 39Crossref PubMed Scopus (60) Google Scholar Morrison et al32Morrison H.W. Filosa J.A. A quantitative spatiotemporal analysis of microglia morphology during ischemic stroke and reperfusion.J Neuroinflammation. 2013; 10: 4Crossref PubMed Scopus (302) Google Scholar emphasized the importance of morphologic assessments in evaluating microglial activation and Zanier et al33Zanier E.R. Fumagalli S. Perego C. Pischiutta F. De Simoni M.-G. Shape descriptors of the "never resting" microglia in three different acute brain injury models in mice.Intensive Care Med Exp. 2015; 3: 39Crossref PubMed Scopus (60) Google Scholar reported the utility of the parameters of microglial forms in quantitatively assessing the morphologic activation of microglia. The current study improved their methods for accurately assessing microglial morphology, regardless of cellular size, and confirmed a significant correlation between LOX-1 and microglial morphology and functions. The circumference ratio, long diameter ratio, and cell area ratio well reflected the changes in microglial activity, as expected. However, roundness did not adequately reflect the change in microglial activity. It was thought that the ramified shape with radiant processes was regarded as round. Based on these morphologic data, we propose the use of the ratio of the circumference and long diameter to the cellular area, and the ratio of the cellular area to the rectangle as new parameters for the morphologic assessment of microglial activity. Moreover, round microglia and endothelial cells expressed LOX-1 in human nHIE brain, similar to that in the nHIE model brain. The expression level of the soluble form of LOX-1 (sLOX-1) in plasma increases with the severity of human nHIE,34Akamatsu T. Sugiyama T. Aoki Y. Kawabata K. Shimizu M. Okazaki K. Kondo M. Takahashi K. Yokoyama Y. Takahashi N. Goto Y.-I. Oka A. Itoh M. A pilot study of soluble form of LOX-1 as a novel biomarker for neonatal hypoxic-ischemic encephalopathy.J Pediatr. 2019; 206: 49-55.e3Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar and HI insult in the premature brain induces blood–brain barrier premiability.18Akamatsu T. Dai H. Mizuguchi M. Goto Y.-I. Oka A. Itoh M. LOX-1 is a novel therapeutic target in neonatal hypoxic-ischemic encephalopathy.Am J Pathol. 2014; 184: 1843-1852Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar Taken together, we hypothesize that plasma sLOX-1 is mainly derived from microglia in the nHIE and that LOX-1 initiates inflammatory signals in the nHIE brain. It is known that the microglial LOX-1/MAPKs/NF-kappaB pathway is involved in lipopolysaccharide-induced brain injury.28Ge X. Zhang D.-M. Li M.-M. Zhang Y. Zhu X.-Y. Zhou Y. Peng X. Shen A.-G. Microglial LOX-1/MAPKs/NF-κB positive loop promotes the vicious cycle of neuroinflammation and neural injury.Int Immunopharmacol. 2019; 70: 187-200Crossref PubMed Scopus (12) Google Scholar LOX-1 may be a novel therapeutic target in human nHIE or other neuroinflammatory diseases. However, a detailed intracellular pathway analysis of microglia is needed. In summary, the current study shows that activated microglia express LOX-1 and that anti–LOX-1 treatment exerts neuroprotection by suppressing microglial activation in nHIE rats, and a new morphologic method for assessing microglial activation is proposed. Furthermore, activated microglia in a human nHIE expressing LOX-1 indicates the potential of LOX-1 target molecules or chemicals for curative treatment. Microglial activation was evaluated with morphology and novel metrical scales and production levels of cytokines and chemokines, using nHIE model rat brain and anti–LOX-1–treated brain. This study revealed that LOX-1 regulated microglial activation in nHIE, and anti–LOX-1 treatment attenuated brain injury by suppressing microglial activation. The alternation of microglial activity between nHIE model rat brain and LOX-1–treated brain correlated with the results of novel morphologic methods; the circumference ratio, long diameter ratio, and the cell area ratio might reflect the microglial activation. Therefore, we propose novel morphologic metrical scales for the evaluation of microglial activity.