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The role of pineal microRNA-325 in regulating circadian rhythms after neonatal hypoxic-ischemic brain damage.

ABSTRACT: Circadian rhythm disorder is a common, but often neglected, consequence of neonatal hypoxic-ischemic brain damage (HIBD). However, the underlying molecular mechanisms remain largely unknown. We previously showed that, in a rat model of HIBD, up-regulation of microRNA-325 (miR-325) in the pineal gland is responsible for the suppression of Aanat, a key enzyme involved in melatonin synthesis and circadian rhythm regulation. To better understand the mechanism by which miR-325 affects circadian rhythms in neonates with HIBD, we compared clinical samples from neonates with HIBD and samples from healthy neonates recruited from the First Affiliated Hospital of Soochow University (Dushuhu Branch) in 2019. We found that circulating miR-325 levels correlated positively with the severity of sleep and circadian rhythm disorders in neonates with HIBD. Furthermore, a luciferase reporter gene assay revealed that LIM homeobox 3 (LHX3) is a novel downstream target of miR-325. In addition, in miR-325 knock-down mice, the transcription factor LHX3 exhibited an miR-325-dependent circadian pattern of expression in the pineal gland. We established a neonatal mouse model of HIBD by performing double-layer ligation of the left common carotid artery and exposing the pups to a low-oxygen environment for 2 hours. Lhx3 mRNA expression was significantly down-regulated in these mice and partially rescued in miR-325 knockout mice subjected to the same conditions. Finally, we showed that improvement in circadian rhythm-related behaviors in animals with HIBD was dependent on both miR-325 and LHX3. Taken together, our findings suggest that the miR-325-LHX3 axis is responsible for regulating circadian rhythms and provide novel insights into the identification of potential therapeutic targets for circadian rhythm disorders in patients with neonatal HIBD. The clinical trial was approved by Institutional Review Board of Children's Hospital of Soochow University (approval No. 2015028) on July 20, 2015. Animal experiments were approved by Animal Care and Use Committee, School of Medicine, Soochow University, China (approval No. XD-2016-1) on January 15, 2016.

SUBMITTER: Sha N

PROVIDER: S-EPMC8343300 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundReducing neuroinflammatory damage is an effective strategy for treating white-matter damage (WMD) in premature infants. Caffeine can ameliorate hypoxia-ischemia-induced brain WMD; however, its neuroprotective effect and mechanism against hypoxic-ischemic WMD remain unclear.MethodsWe used 3-day-old Sprague-Dawley rats to establish a model of cerebral hypoxia-ischemia-induced brain WMD after unilateral common carotid artery ligation and hypoxia exposure (8% O2 + 92% N2) for 2.5 h. Mechanism experiments were conducted to detect M1/M2 polarization and activation of microglia and NLRP3 inflammasome.ResultsCaffeine inhibited NLRP3 inflammasome activation, reduced microglial Iba-1 activation, inhibited microglia M1 polarization, and promoted microglia M2 polarization by downregulating CD86 and iNOS protein expression, inhibiting the transcription of the proinflammatory TNF-α and IL-1β, upregulating CD206 and Arg-1 expression, and promoting the transcription of the anti-inflammatory factors IL-10 and TGF-β. Importantly, we found that these caffeine-mediated effects could be reversed after inhibiting A2aR activity.ConclusionsCaffeine improved long-term cognitive function in neonatal rats with hypoxic-ischemic WMD via A2aR-mediated inhibition of NLRP3 inflammasome activation, reduction of microglial activation, regulation of the phenotypic polarization of microglia and the release of inflammatory factors, and improvement of myelination development.ImpactThe direct protective effect of caffeine on hypoxic-ischemic white-matter damage (WMD) and its mechanism remains unclear. This study elucidated this mechanism using neonatal rats as an animal model of hypoxia-ischemia-induced cerebral WMD. The findings demonstrated caffeine as a promising therapeutic tool against immature WMD to protect neonatal cognitive function. We found that caffeine pretreatment reduced WMD in immature brains via regulation of microglial activation and polarization by adenosine A2a receptor, thereby, providing a scientific basis for future clinical application of caffeine.

Project description:BackgroundAngiogenesis is an endogenous repair mechanism following hypoxic-ischemic brain damage (HIBD). Interestingly, recent studies have shown that angiogenesis can be regulated by telomerase reverse transcriptase (TERT), a critical component of telomerase. As telomerase reverse transcriptase can promote angiogenesis after stroke, we hypothesized that it could also promote angiogenesis after HIBD. To test this hypothesis, we developed in vivo and in vitro HIBD models in neonatal rats.MethodsTERT was overexpressed by lentivirus and adenovirus infection, and levels were measured using quantitative real-time polymerase chain reaction. We used a cell counting kit to quantify the proliferation rate of brain microvascular endothelial cells (BMECs), and immunofluorescence staining to measure CD34 expression levels. A microvessel formation assay was used to evaluate angiogenesis. Blood-brain barrier (BBB) integrity was assessed using immunohistochemical staining for ZO-1 and Evans Blue staining. Lastly, the expression level of Notch-1 was measured by western blotting.ResultsOverexpression of TERT promoted the proliferation of BMECs after hypoxic-ischemic damage in vitro. TERT overexpression increased the formation of microvessels in the neonatal brain after HIBD both in vivo and in vitro. Overexpression of TERT improved BBB integrity in the brains of neonatal rats after HIBD. In addition, the expression level of Notch-1 was increased in BMECs following oxygen glucose deprivation, and overexpression of TERT further increased Notch-1 expression levels in BMECs following oxygen glucose deprivation.DiscussionOur results reveal that telomerase reverse transcriptase promotes angiogenesis and maintains the integrity of the blood-brain barrier after neonatal hypoxic-ischemic brain damage. Furthermore, the Notch-1 signaling pathway appears to contribute to the angiogenic function of telomerase reverse transcriptase. This protective effect of telomerase reverse transcriptase opens new horizons for future investigations aimed at uncovering the full potential of telomerase reverse transcriptase as a promising new target for the treatment of hypoxic-ischemic encephalopathy.

Project description:ImportanceNeonatal hypoxic-ischemic encephalopathy (HIE) remains a significant cause of neurologic disability. Identifying infants suitable for therapeutic hypothermia (TH) within a narrow therapeutic time is difficult. No single robust biochemical marker is available to clinicians.ObjectiveTo assess the ability of a panel of candidate microRNA (miRNA) to evaluate the development and severity of encephalopathy following perinatal asphyxia (PA).Design, setting, and participantsThis validation study included 2 cohorts. For the discovery cohort, full-term infants with PA were enrolled at birth to the Biomarkers in Hypoxic-Ischemic Encephalopathy (BiHiVE1) study (2009-2011) in Cork, Ireland. Encephalopathy grade was defined using early electroencephalogram and Sarnat score (n = 68). The BiHiVE1 cohort also enrolled healthy control infants (n = 22). For the validation cohort, the BiHiVE2 multicenter study (2013-2015), based in Cork, Ireland (7500 live births per annum), and Karolinska Huddinge, Sweden (4400 live births per annum), recruited infants with PA along with healthy control infants to validate findings from BiHiVE1 using identical recruitment criteria (n = 80). The experimental design was formulated prior to recruitment, and analysis was conducted from June 2016 to March 2017.Main outcomes and measuresAlterations in umbilical cord whole-blood miRNA expression.ResultsFrom 170 neonates, 160 were included in the final analysis. The BiHiVE1 cohort included 87 infants (21 control infants, 39 infants with PA, and 27 infants with HIE), and BiHiVE2 included 73 infants (control [n = 22], PA [n = 26], and HIE [n = 25]). The BiHiVE1 and BiHiVE2 had a median age of 40 weeks (interquartile range [IQR], 39-41 weeks) and 40 weeks (IQR, 39-41 weeks), respectively, and included 56 boys and 31 girls and 45 boys and 28 girls, respectively. In BiHiVE1, 12 candidate miRNAs were identified, and 7 of these miRNAs were chosen for validation in BiHiVE2. The BiHiVE2 cohort showed consistent alteration of 3 miRNAs; miR-374a-5p was decreased in infants diagnosed as having HIE compared with healthy control infants (median relative quantification, 0.38; IQR, 0.17-0.77 vs 0.95; IQR, 0.68-1.19; P = .009), miR-376c-3p was decreased in infants with PA compared with healthy control infants (median, 0.42; IQR, 0.21-0.61 vs 0.90; IQR, 0.70-1.30; P = .004), and mir-181b-5p was decreased in infants eligible for TH (median, 0.27; IQR, 0.14-1.41) vs 1.18; IQR, 0.70-2.05; P = .02).Conclusions and relevanceAltered miRNA expression was detected in umbilical cord blood of neonates with PA and HIE. These miRNA could assist diagnostic markers for early detection of HIE and PA at birth.

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The role of pineal microRNA-325 in regulating circadian rhythms after neonatal hypoxic-ischemic brain damage.

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