Early life and adult stress promote sex dependent changes in hypothalamic miRNAs and environmental enrichment prevents stress-induced miRNA and gene expression changes in rats.

BACKGROUND: The hypothalamus plays a key role in the stress response. While early life stress (ELS) increases susceptibility to psychiatric disorders including major depressive disorder (MDD), acute stress during adulthood can also precipitate MDD after ELS. AIM: Here, we tested the expression of miRNAs following ELS and susceptibility to depression-like behavior and whether sex or acute stress exacerbates this response. We also tested whether environmental enrichment (Enr) promotes early life and adult behavioral stress resilience and its effect on hypothalamic miRNA and gene expression. Following rat maternal separation (MS) as an ELS model, Enr from weaning through adulthood, and restraint (RS) as acute adult stress, we tested both animal behavior and miRNA expression in the hypothalamus. Target genes and their enrichment and ontology were analyzed using bioinformatic tools. Target gene expression changes were tested using qPCR, and miRNA promoter methylation was studied using methylated-DNA immunoprecipitation qPCR. RESULTS: MS, Enr, RS, and sex altered hypothalamic miRNAs, including several previously reported in MS literature: miRs-29, - 124, - 132, - 144, - 504. Sex had a significant effect on the greatest number of miRNAs. Also, Enr reversed downregulation of miR-29b-1-5p and -301b-3p in MS. qPCR showed that MAPK6 and MMP19, targets of miR-301b-3p, were upregulated in MS and reversed by Enr. Additionally, miR-219a was hypermethylated in MS coinciding with decreased miR-219a expression. CONCLUSIONS: This study found that sex plays a critical role in the hypothalamic miRNA response to both ELS and acute stress, with males expressing greater changes following postnatal stress. Moreover, enrichment significantly altered behavior as well as hypothalamic miRNA expression and their gene targets. Because of its role as the initiator of the autonomic stress response and connection to hedonic and motivational behavior, the hypothalamic miRNA landscape may significantly alter both the short and long-term behavioral response to stress.

MicroRNA Regulates Early-Life Stress-Induced Depressive Behavior via Serotonin Signaling in a Sex-Dependent Manner in the Prefrontal Cortex of Rats.

Background: The underlying neurobiology of early-life stress (ELS)-induced major depressive disorder is not clearly understood. Methods: In this study, we used maternal separation (MS) as a rodent model of ELS and tested whether microRNAs (miRNAs) target serotonin genes to regulate ELS-induced depression-like behavior and whether this effect is sex dependent. We also examined whether environmental enrichment prevents susceptibility to depression- and anxiety-like behavior following MS and whether enrichment effects are mediated through serotonin genes and their corresponding miRNAs. Results: MS decreased sucrose preference, which was reversed by enrichment. Males also exhibited greater changes in forced swim climbing and escape latency tests only following enrichment. S lc6a4 and H tr1a were upregulated in the frontal cortex following MS. In male MS rats, enrichment slightly reversed H tr1a expression to levels similar to control rats. miR-200a-3p and miR-322-5p, which target SLC6A4, were decreased by MS, but not significantly. An HTR1A-targeting miRNA, miR-320-5p, was also downregulated by MS and showed slight reversal by enrichment in male animals. miR-320-5p targeting of H tr1a was validated in vitro using SHSY neuroblastoma cell lines. Conclusions: Altogether, this study implicates miRNA interaction with the serotonin pathway in ELS-induced susceptibility to depression-related reward deficits. Furthermore, because of its recovery by enrichment in males, miR-320 may represent a viable sex-specific target for reward-related deficits in major depressive disorder.

Immunological and pathological outcomes of SARS-CoV-2 challenge after formalin-inactivated vaccine immunisation of ferrets and rhesus macaques

There is an urgent requirement for safe and effective vaccines to prevent novel coronavirus disease (COVID-19) caused by SARS-CoV-2. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferret and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine in ferrets or unvaccinated macaques. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen at seven days post infection in ferrets. This increased lung pathology was observed early in the infection (day 7) but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

Comparison of Rhesus and Cynomolgus macaques as an authentic model for COVID-19.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques, resembling the mild clinical cases of COVID-19 in humans. Immune responses against SARS-CoV-2 were also similar in both species and equivalent to those reported in milder infections and convalescent human patients. Importantly, we have devised a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the optimal study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of novel and repurposed interventions against SARS-CoV-2. Accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

Dose-dependent response to infection with SARS-CoV-2 in the ferret model: evidence of protection to re-challenge

In December 2019 an outbreak of coronavirus disease (COVID-19) emerged in Wuhan, China. The causative agent was subsequently identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread worldwide causing a pandemic. Currently there are no licensed vaccines or therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in development and repurposed drugs are being tested in the clinic. There is a vital need for authentic COVID-19 animal models to further our understanding of pathogenesis and viral spread in addition to pre-clinical evaluation of candidate interventions. Here we report a dose titration study of SARS-CoV-2 to determine the most suitable infectious dose to use in the ferret model. We show that a high (5x106 pfu) and medium (5x104 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral RNA shedding in both groups of six challenged animals, whilst a low dose (5x102 pfu) resulted in only one of six displaying signs of URT viral RNA replication. The URT shedding lasted up to 21 days in the high dose animals with intermittent positive signal from day 14. Sequential culls revealed distinct pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were observed although animals did present with a consistent post-viral fatigue lasting from day 9-14 in the medium and high dose groups. After virus shedding ceased, re-challenged ferrets were shown to be fully protected from acute lung pathology. The endpoints of URT viral RNA replication in addition to distinct lung pathology and post viral fatigue were observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 14-21 parallel observations reported in a minority of clinical cases.

Early initiation of extracorporeal membrane oxygenation improves survival in adult trauma patients with severe adult respiratory distress syndrome.

BACKGROUND: The use of extracorporeal membrane oxygenation (ECMO) in the trauma population has been reported to have a mortality benefit in patients with severe refractory hypoxic respiratory failure. This study compares the early initiation of ECMO for the management of severe adult respiratory distress syndrome (ARDS) versus a historical control immediately preceding the use of ECMO for trauma patients. METHODS: A retrospective study was conducted at a single verified Level I trauma center. The study population was limited to trauma patients diagnosed with severe ARDS using the Berlin definition (PaO2/FIO2 ratio < 100). Patients managed with ECMO versus conventional ventilation (CONV) were compared. The primary outcome of interest was mortality; secondary outcomes included hospital length of stay, intensive care unit-free days, and ventilator-free days. RESULTS: Fifteen ECMO patients managed from March 2013 to November 2014 were identified, as were 14 CONV patients managed from March 2012 to February 2013 who met the Berlin definition of severe ARDS. Data related to age, Injury Severity Scores (ISSs), admission lactic acid levels, base deficit, the number of transfused red blood cell units within the first 24 hours, and presence of severe traumatic brain injury were collected and were not statistically different between the groups. Likewise, Murray Lung Injury (MLI), Sequential Organ Failure Assessment (SOFA), and Acute Physiology and Chronic Health Evaluation II (APACHE II) scores determined at the onset of severe ARDS were not statistically different between the groups. Median hospital stay (CONV, 28.0 days [14.0-47.0]; ECMO, 43.5 days [30.0-93.0]; p = 0.15), intensive care unit-free days (CONV, 0.0 days [0.0-5.0]; ECMO, 5.0 days [0.0-7.0]; p = 0.26), and ventilator-free days (CONV, 0.0 days [0.0-10.0]; ECMO, 8.0 days [0.0-19.0]; p = 0.13) were not statistically different between the groups. Mortality in the ECMO group was significantly reduced compared with the CONV group (ECMO, 13.3%; CONV, 64%; p = 0.01). Timing from the onset of severe ARDS to ECMO intervention occurred at a mean 1.9 ± 1.4 days. CONCLUSION: Patients who were treated with ECMO for severe ARDS had an improved mortality compared with historical controls. ECMO should be considered at the early onset of severe ARDS to improve survival. LEVEL OF EVIDENCE: Therapeutic study, level IV.