Fibulin-3 is necessary to prevent cardiac rupture following myocardial infarction.

Despite the high prevalence of heart failure in the western world, there are few effective treatments. Fibulin-3 is a protein involved in extracellular matrix (ECM) structural integrity, however its role in the heart is unknown. We have demonstrated, using single cell RNA-seq, that fibulin-3 was highly expressed in quiescent murine cardiac fibroblasts, with expression highest prior to injury and late post-infarct (from ~ day-28 to week-8). In humans, fibulin-3 was upregulated in left ventricular tissue and plasma of heart failure patients. Fibulin-3 knockout (Efemp1-/-) and wildtype mice were subjected to experimental myocardial infarction. Fibulin-3 deletion resulted in significantly higher rate of cardiac rupture days 3-6 post-infarct, indicating a weak and poorly formed scar, with severe ventricular remodelling in surviving mice at day-28 post-infarct. Fibulin-3 knockout mice demonstrated less collagen deposition at day-3 post-infarct, with abnormal collagen fibre-alignment. RNA-seq on day-3 infarct tissue revealed upregulation of ECM degradation and inflammatory genes, but downregulation of ECM assembly/structure/organisation genes in fibulin-3 knockout mice. GSEA pathway analysis showed enrichment of inflammatory pathways and a depletion of ECM organisation pathways. Fibulin-3 originates from cardiac fibroblasts, is upregulated in human heart failure, and is necessary for correct ECM organisation/structural integrity of fibrotic tissue to prevent cardiac rupture post-infarct.

Molecular and Functional Changes to Postsynaptic Cholinergic Signaling in the Vestibular Sensory Organs of Aging C57BL/6 Mice.

Cholinergic circuits in the central nervous system are vulnerable to age-related functional decline, but it is not known if aging impacts cholinergic signaling in the vestibular sensory organs, which are critically important to balance maintenance and visual gaze stability. We have previously shown cholinergic neurotransmission between vestibular efferent terminals and type II mechanosensory hair cells requires the alpha9 (Chrna9) nicotinic receptor subunit. Homozygous knockout of the alpha9 subunit causes vestibulo-ocular reflex adaptation deficits that mirror those observed in aged mice. This prompted examination of cholinergic signaling in the vestibular sensory organs of aged mice. We confirmed older (>24 months) mice had impaired performance in a balance beam task compared to young (3-4 months) adult mice. While there was no qualitative loss of cholinergic axon varicosities in the crista ampullaris of old mice, qPCR analysis revealed reduced expression of nicotinic receptor subunit genes Chrna1, Chrna9, and Chrna10 in the cristae of old relative to young mice. Functionally, single-cell patch clamp recordings taken from type II vestibular hair cells exposed to acetylcholine show reduced conductance through alpha9/10 subunit-containing nicotinic receptors in older mice, despite preserved passive membrane properties and voltage-activated conductances. These findings suggest that cholinergic signaling in the peripheral vestibular sensory organs is vulnerable to aging processes, manifesting in dynamic molecular and functional age-related changes. Given the importance of these organs to our everyday activities, and the dramatic increase in fall incidence in the older, further investigation into the mechanisms of altered peripheral vestibular function in older humans is warranted.

Extracellular Matrix Protein-1 as a Mediator of Inflammation-Induced Fibrosis After Myocardial Infarction.

Irreversible fibrosis is a hallmark of myocardial infarction (MI) and heart failure. Extracellular matrix protein-1 (ECM-1) is up-regulated in these hearts, localized to fibrotic, inflammatory, and perivascular areas. ECM-1 originates predominantly from fibroblasts, macrophages, and pericytes/vascular cells in uninjured human and mouse hearts, and from M1 and M2 macrophages and myofibroblasts after MI. ECM-1 stimulates fibroblast-to-myofibroblast transition, up-regulates key fibrotic and inflammatory pathways, and inhibits cardiac fibroblast migration. ECM-1 binds HuCFb cell surface receptor LRP1, and LRP1 inhibition blocks ECM-1 from stimulating fibroblast-to-myofibroblast transition, confirming a novel ECM-1-LRP1 fibrotic signaling axis. ECM-1 may represent a novel mechanism facilitating inflammation-fibrosis crosstalk.

Reduced cortical somatostatin gene expression in a rat model of maternal immune activation.

Alterations in GABAergic interneurons and glutamic acid decarboxylase (GAD) are observed in the brains of people with schizophrenia. Studies also show increased density of interstitial white matter neurons (IWMN), including those containing GAD and somatostatin (SST) in the brain in schizophrenia. Maternal immune activation can be modelled in rodents to investigate the relationship between prenatal exposure to infections and increased risk of developing schizophrenia. We reported that maternal immune activation induced an increase in density of somatostatin-positive IWMN in the adult rat offspring. Here we hypothesised that maternal immune activation induced in pregnant rats by polyinosinic:polycytidylic acid would alter SST and GAD gene expression as well as increase the density of GAD-positive IWMNs in the adult offspring. SST gene expression was significantly reduced in the cingulate cortex of adult offspring exposed to late gestation maternal immune activation. There was no change in cortical GAD gene expression nor GAD-positive IWMN density in adults rats exposed to maternal immune activation at either early or late gestation. This suggests that our model of maternal immune activation induced by prenatal exposure of rats to polyinosinic:polycytidylic acid during late gestation is able to recapitulate changes in SST but not other GABAergic neuropathologies observed in schizophrenia.

Late gestation immune activation increases IBA1-positive immunoreactivity levels in the corpus callosum of adult rat offspring.

Animal models of maternal immune activation study the effects of infection, an environmental risk factor for schizophrenia, on brain development. Microglia activation and cytokine upregulation may have key roles in schizophrenia neuropathology. We hypothesised that maternal immune activation induces changes in microglia and cytokines in the brains of the adult offspring. Maternal immune activation was induced by injecting polyriboinosinic:polyribocytidylic acid into pregnant rats on gestational day (GD) 10 or GD19, with brain tissue collected from the offspring at adulthood. We observed no change in Iba1, Gfap, IL1-ß and TNF-α mRNA levels in the cingulate cortex (CC) in adult offspring exposed to maternal immune activation. Prenatal exposure to immune activation had a significant main effect on microglial IBA1-positive immunoreactive material (IBA1+IRM) in the corpus callosum; post-hoc analyses identified a significant increase in GD19 offspring, but not GD10. No change in was observed in the CC. In contrast, maternal immune activation had a significant main effect on GFAP+IRM in the CC at GD19 (not GD10); post-hoc analyses only identified a strong trend towards increased GFAP+IRM in the GD19 offspring, with no white matter changes. This suggests late gestation maternal immune activation causes subtle alterations to microglia and astrocytes in the adult offspring.

Effects of Ageing on the Mitochondrial Genome in Rat Vestibular Organs.

BACKGROUND: Deterioration in vestibular function occurs with ageing and is linked to age-related falls. Sensory hair cells located in the inner ear vestibular labyrinth are critical to vestibular function. Vestibular hair cells rely predominantly on oxidative phosphorylation (OXPHOS) for energy production and contain numerous mitochondria. Mitochondrial DNA (mtDNA) mutations and perturbed energy production are associated with the ageing process. OBJECTIVE: We investigated the effects of ageing on mtDNA in vestibular hair and support cells, and vestibular organ gene expression, to better understand mechanisms of age-related vestibular deficits. METHODS: Vestibular hair and supporting cell layers were microdissected from young and old rats, and mtDNA was quantified by qPCR. Additionally, vestibular organ gene expression was analysed by microarray and gene set enrichment analyses. RESULTS: In contrast to most other studies, we found no evidence of age-related mtDNA deletion mutations. However, we found an increase in abundance of major arc genes near the mtDNA control region. There was also a marked age-related reduction in mtDNA copy number in both cell types. Vestibular organ gene expression, gene set enrichment analysis showed the OXPHOS pathway was down regulated in old animals. CONCLUSION: Given the importance of mtDNA to mitochondrial OXPHOS and hair cell function, our findings suggest the vestibular organs are potentially on the brink of an energy crisis in old animals.

Cell necrosis-independent sustained mitochondrial and nuclear DNA release following trauma surgery.

BACKGROUND: Mitochondrial DNA (mtDNA), a potent proinflammatory damage-associated molecular pattern, is released in large titers following trauma. The effect of trauma surgery on mtDNA concentration is unknown. We hypothesized that mtDNA and nuclear DNA (nDNA) levels would increase proportionately with the magnitude of surgery and both would then decrease rapidly. METHODS: In this prospective pilot, plasma was sampled from 35 trauma patients requiring orthopedic surgical intervention at six perioperative time points. Healthy control subjects (n = 20) were sampled. DNA was extracted, and the mtDNA and nDNA were assessed using quantitative polymerase chain reaction. Markers of cell necrosis were also assayed (creatine kinase, lactate dehydrogenase, and aspartate aminotransferase). RESULTS: The free plasma mtDNA and nDNA levels (ng/mL) were increased in trauma patients compared with healthy controls at all time points (mtDNA: preoperative period, 108 [46-284]; postoperative period, 96 [29-200]; 7 hours postoperatively, 88 [43-178]; 24 hours, 79 [36-172]; 3 days, 136 [65-263]; 5 days, 166 [101-434] [healthy controls, 11 (5-19)]) (nDNA: preoperative period, 52 [25-130]; postoperative period, 100 [35-208]; 7 hours postoperatively, 75 [36-139]; 24 hours postoperatively, 85 [47-133]; 3 days, 79 [48-117]; 5 days, 99 [41-154] [healthy controls, 29 (16-54)]). Elevated DNA levels did not correlate with markers of cellular necrosis. mtDNA was significantly elevated compared with nDNA at preoperative period (p = 0.003), 3 days (p = 0.003), and 5 days (p = 0.0014). Preoperative mtDNA levels were greater with shorter time from injury to surgery (p = 0.0085). Postoperative mtDNA level negatively correlated with intraoperative crystalloid infusion (p = 0.0017). Major pelvic surgery (vs. minor) was associated with greater mtDNA release 5 days postoperatively (p < 0.05). CONCLUSION: This pilot of heterogeneous orthopedic trauma patients showed that the release of mtDNA and nDNA is sustained for 5 days following orthopedic trauma surgery. Postoperative, circulating DNA is not associated with markers of tissue necrosis but is associated with surgical invasiveness and is inversely related to intraoperative fluid administration. Sustained elevation of mtDNA levels could be of inflammatory origin and may contribute to postinjury dysfunctional inflammation. LEVEL OF EVIDENCE: Prospective study, level III.

The influence of cervical spinal cord compression and vertebral displacement on somatosympathetic reflexes in the rat.

BACKGROUND CONTEXT: One theory within chiropractic proposes that vertebral subluxation in the upper cervical region induces spinal cord compression sufficient to alter spinal cord efferent output. We report on the feasibility of three different experimental approaches to test this theory. METHODS: A high threshold electrical-evoked somatosympathetic reflex was recorded in adrenal or renal nerves of 10 anaesthetized adult male rats before and after (1) graded pressure was applied directly to the C1/C2 spinal cord segment in eight rats by the use of either direct compression or inflation of an extradural balloon and (2) displacement, less than a dislocation applied posterior to anterior, to the C2 vertebra in two rats. The latency and amplitude of the pre- and postintervention reflex responses were compared. RESULTS: The reflex amplitude was not significantly changed by pressure (26 mmHg) from an extra-dural balloon or direct compression of the dura mater onto the dorsal spinal cord. Additional pressure, at least sufficient to occlude the dorsal vessels, induced a significant reduction in the amplitude of the reflex, and this reduction persisted for 20 minutes after removal of the pressure (Dunn's method for all pairwise multiple comparison Q stat=3.437; critical value for k=6 with α=0.05 is 2.936). Maximal vertebral (C2) displacement (4 mm), without dislocation did not induce significant changes compared with the control period. CONCLUSIONS: Although this feasibility study suggests it is unlikely that upper cervical vertebral subluxation, displacement less than a dislocation, compromises the sympathetic outflow in the adrenal or renal nerves, further vertebral displacement studies are necessary to formally test this.