Establishment of a hypoxia ischemia reperfusion brain damage model in neonatal rats

Objective: Rice-Vannucci model has been widely used as HIE（Hypoxic ischemic encephalopathy ） animal model in the past forty years, but it does not mimic reperfusion injury that occurs during HIE. The aim of the present study was to establish a new neonatal rat model by simulating hypoxia ischemia reperfusion brain damage (HIRBD) through "common carotid artery (CCA) muscle bridge". Methods: Sixty 7-day-old male Sprague-Dawley rats were randomly assigned to group A (HIRBD groups, n=36), group B (Rice-Vannucci group, n=12), and group C (sham-operated group, n=12). Rats in group A were assigned to 3 subgroups (A1-A3, 12 animals/subgroup). Dynamic changes in cerebral blood flow (CBF) were evaluated by the laser speckle imaging system. The status of the CCA was observed under a stereomicroscope. Changes in body weight, gross morphology as well as pathological sections of brain tissue were examined to evaluate the feasibility of the model. Results: The results indicated that CCA muscle bridge successfully blocked the CBF. CBF was restored after removal of the CCA muscle bridge in HIRBD groups. The CCA was in good condition after removing the muscle bridge, and blood supply was not affected. Changes in body weight, gross morphology and pathological sections of brain tissue indicated that ischemia reperfusion induced by the CCA muscle bridge method caused varying degrees of brain damage. Conclusion: CCA muscle bridge method is effective for establishing a reliable, stable, and reproducible neonatal rat model for study of HIRBD.

Claudin-18 expression under hypoxia in neonatel lungs of brochopulmonary dysplasia model rats

Background: Bronchopulmonary dysplasia (BPD) is characterized by impaired alveolar and microvascular development. Claudin-18 is the only known lung-specific tight junction protein affecting alveolar epithelium development and transdifferentiation. Objective: To explore the changes in claudin-18 expression, alveolar epithelial cell (AEC) marker proteins, the canonical Wnt pathway, and their possible regulatory relationships in a hyperoxia-induced BPD rat model. Methods: The BPD neonatal rat model was established by exposure to hyperoxia (85%). Hematoxylin and eosin (HE) staining was used to confirm the establishment of the BPD model. The mRNA levels were assessed using quantitative real-time polymerase chain reaction, while protein expression levels were determined using western blotting, immunohistochemical staining, and immunofluorescence . Results: As confirmed by HE staining, the BPD neonatal rat model was successfully established. Compared with the air group, claudin-18 and claudin-4 expression decreased in the hyperoxia group. The expression of β-catenin of the Wnt signaling decreased, whereas that of p-GSK-3β increased. Expression of the AEC Ⅱ marker SFTPC decreased initially and then increased, whereas that of the AEC Ⅰ marker Podoplanin increased on day 14 (P < 0.05). Conclusions: Claudin-18 downregulation during hyperoxia may affect lung development and maturation, which may result in hyperoxia-induced BPD. Additionally, claudin-18 is associated with the canonical Wnt pathway and alveolar epithelial transdifferentiation.

Glucocorticoid receptor antagonist alters corticosterone and receptor sensitive mRNAs in the hypoxic neonatal rat

Hypoxia, a common stressor with preterm birth, increases morbidity and mortality associated with prematurity. Glucocorticoids (GC) are administered to the preterm infant to improve oxygenation; prolonged use of GCs remains controversial. We evaluated a selective glucocorticoid receptor (GR) antagonist (CORT113176) in our neonatal rat model of human prematurity to assess how fasting and hypoxia-induced increases in neonatal corticosterone affects endogenous hormones and endocrine pancreas function. Neonatal rat pups at postnatal day (PD) 2, PD8, and PD15 were pretreated with CORT113176 and, after 60 min of separation and fasting, exposed to hypoxia (8% O2) or control (normoxia) for 30 or 60 minutes while fasting was continued. Plasma corticosterone, ACTH, glucose, and insulin were measured and fasting HOMA-IR (index of insulin resistance) calculated. Glucocorticoid and insulin receptor sensitive gene mRNAs were analyzed in liver, muscle, and adipose to evaluate target tissue biomarkers. CORT113176 pretreatment augmented baseline and hypoxia-induced increases in corticosterone and attenuated hypoxia-induced increases in insulin resistance at PD2. Normoxic and hypoxic stress increased the hepatic GR sensitive gene mRNAs, Gilz and Per1; this was eliminated by pretreatment with CORT113176. CORT113176 pretreatment decreased baseline insulin receptor sensitive gene mRNAs Akt2, Irs1, Pik3r1, and Srebp1c at PD2. We show that CORT113176 variably augments the stress-induced increases in corticosterone concentrations (attenuation of negative feedback) and that GR is critical for hepatic responses to stress in the hypoxic neonate. We also propose that measurement of Gilz and Per1 mRNA expression may be useful to evaluate the effectiveness of GR antagonism.

Differential expressions of TGF-β1, HIF-1, VEGF, α-SMA and E-cadherin in renal tissues of a neonatal rat model of hydronephrosis

Purpose: To investigate the differential expressions of transforming growth factor-β1 (TGF-β1), hypoxia inductive factor-1 (HIF-1), vascular endothelial growth factor (VEGF), α-smooth muscle protein (α-SMA) and E-cadherin in renal tissues of neonatal rat model of hydronephrosis.Methods: The neonatal rats (90) were randomly divided into sham group and model group. The rats in the model group were further divided into two subgroups: week 1 and week 12 after relief of obstruction, with 30 rats in each group. Six rats were taken from each group for the determination of renal histopathological changes. Levels of TGF-β1, HIF-1, VEGF, α-SMA and E-cadherin in renal tissues were compared for different pathological grades and at different time points of obstruction relief.Results: With increase in Elder grade, the concentrations of TGF-β1, HIF-1, VEGF and α-SMA in renal tissues of hydronephrosis neonatal rats were gradually increased, while the expression level of Ecadherin gradually decreased (p < 0.05). However, the concentrations of TGF-β1, HIF-1, VEGF and α-SMA in renal tissues were significantly reduced, while the expression level of E-adherin was upregulated with time after relief of obstruction (p < 0.05).Conclusion: These findings are of great significance in determining the degree of kidney injury and recovery, and for the development of drugs for the treatment of renal injury.

Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model

The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC’s main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels—as seen during stress—promote NEC in formula-fed (FF) newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure—NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p = 0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.

The Effect of a Novel Glucocorticoid Receptor Antagonist (CORT113176) on Glucocorticoid and Insulin Receptor Sensitive Hepatic Gene (mRNA) Expression in a Neonatal Rat Model of Human Prematurity

Preterm birth is a global health problem the sequelae of which are not well understood. Hypoxia, a common stressor with prematurity, can affect blood glucose via stress-induced increases in glucocorticoids (GC). GCs are also administered to preterm infants to improve oxygenation; however, this is controversial. CORT113176 (Corcept Therapeutics) is a novel, selective glucocorticoid receptor (GR) antagonist that does not bind to the progesterone receptor. We have demonstrated that CORT113176 (in our rat model of preterm birth) increases baseline corticosterone (due to loss of GC negative feedback) and attenuates hypoxia-induced increases in insulin resistance implicating endogenous corticosterone in post-natal metabolic adaptations to stress. We now propose that CORT113176 is useful to evaluate the hepatic effects of endogenous GCs in our rat model of preterm birth by measuring critical GC and insulin receptor sensitive gene mRNAs. Postnatal day (PD) 2 rat pups of both sexes (N=5 per treatment/sex) were pretreated with CORT113176 (600 mg/kg IP) or vehicle. After 60 minutes, a group of pups were euthanized with livers collected and preserved in RNAlater (baseline). The remaining pups were separated from their dams, exposed to normoxia (control) or hypoxia (8% O2) for 60 minutes, and livers obtained. Total hepatic RNA was extracted, and mRNA expression was analyzed (RT-qPCR) for GC and insulin receptor sensitive genes: GC: Fkbp5, Gilz, Nr3c1 (Gr), Nr3c2 (Mr), Per1, Ttpa. INSULIN: Akt2, G6Pase, Igf1r, Insr, Irs1, Irs2, Pik3cb, Pik3r1, Srebp1c. CORT113176 decreased the expression of all baseline hepatic insulin receptor mRNAs in both sexes, except for G6Pase. Pik3r1 mRNA expression significantly decreased with 60 minutes of normoxic separation (fasting) in males and females compared to baseline and hypoxic separation; this was blocked by CORT113176. In the GC receptor sensitive panel, CORT113176 decreased basal Nr3c1 (Gr) mRNA. Normoxic and hypoxic separation increased Per1 and Gilz mRNA expression; this effect was blocked by CORT113176. Interestingly, Fkbp5 expression, a proposed clinical marker for GR antagonism, was not altered by CORT113176. The hepatic GC and insulin receptor sensitive gene mRNA panels we developed are sensitive to GR antagonism suggesting they may be a useful addition to Fkbp5. The increase in endogenous corticosterone, acting via GR, is critical in the hepatic response to stress in our neonatal rat model of hypoxia and prematurity.

Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model

The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC’s main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels—as seen during stress—promote NEC in formula-fed newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure—NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p=0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.