Age-Specific Adult Rat Brain MRI Templates and Tissue Probability Maps

Age-specific resources in human MRI mitigate processing biases that arise from structural changes across the lifespan. There are fewer age-specific resources for preclinical imaging, and they only represent developmental periods rather than adulthood. Since rats recapitulate many facets of human aging, it was hypothesized that brain volume and each tissue's relative contribution to total brain volume would change with age in the adult rat. Data from a longitudinal study of rats at 3, 5, 11, and 17 months old were used to test this hypothesis. Tissue volume was estimated from high resolution structural images using a priori information from tissue probability maps. However, existing tissue probability maps generated inaccurate gray matter probabilities in subcortical structures, particularly the thalamus. To address this issue, gray matter, white matter, and CSF tissue probability maps were generated by combining anatomical and signal intensity information. The effects of age on volumetric estimations were then assessed with mixed-effects models. Results showed that herein estimation of gray matter volumes better matched histological evidence, as compared to existing resources. All tissue volumes increased with age, and the tissue proportions relative to total brain volume varied across adulthood. Consequently, a set of rat brain templates and tissue probability maps from across the adult lifespan is released to expand the preclinical MRI community's fundamental resources.

Immunohistochemistry localises myosin-7a to cochlear efferent boutons

Background: Myosin 7a is an actin-binding motor protein involved in the formation of hair-cell stereocilia both in the cochlea and in the vestibular system. Mutations in myosin 7a are linked to congenital hearing loss and are present in 50% of Type-1 Usher syndrome patients who suffer from progressive hearing loss and vestibular system dysfunction. Methods: Myosin 7a is often used to visualise sensory hair cells due to its well characterised and localised expression profile. We thus conducted myosin-7a immunostaining across all three turns of the adult rat organ of Corti to visualise hair cells. Results: As expected, we observed myosin 7a staining in both inner and outer hair cells. Unexpectedly, we also observed strong myosin 7a staining in the medial olivocochlear efferent synaptic boutons contacting the outer hair cells. Efferent bouton myosin-7a staining was present across all three turns of the cochlea. We verified this localisation by co-staining with a known efferent bouton marker, the vesicular acetylcholine transporter. Conclusions: In addition to its role in stereocilia formation and maintenance, myosin 7a or certain myosin-7a expression variants might play a role in efferent synaptic transmission in the cochlea and thus ultimately influence cochlear gain regulation. Our immunohistochemistry results should be validated with other methods to confirm these serendipitous findings.

Cardiac-targeted PIASy Gene Silencing Mediates deSUMOylation of Caveolin-3 and Prevents Ischemia/reperfusion-induced Nav1.5 Down-regulation and Ventricular Arrhythmias

Background: Abnormal myocardial expression and function of Nav1.5 causes lethal ventricular arrhythmias during myocardial ischemia-reperfusion (I/R). PIASy mediated Caveolin-3 (Cav-3) SUMO modification affects Cav-3 binding to ligand Nav1.5. PIASy activity is increased after myocardial I/R, whether or not this may be attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias remains unclear. Methods: Using recombinant adeno-associated virus subtype 9 (AAV9), rat cardiac PIASy was silenced by intraventricular injection of PIASy shRNA. Two weeks later, the hearts were subjected to I/R, and electrocardiography was performed to assess malignant arrhythmias. Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurement. Results: We found that PIASy was upregulated by I/R, with increased SUMO2/3 modification of Cav-3, reduced membrane Nav1.5 density, and increased ventricular arrhythmia frequency. These effects were significantly reversed by PIASy silencing. In addition, PIASy silencing enhanced Cav-3 binding to Nav1.5 and prevented I/R-induced Nav1.5 re-localization. Using in vitro models of HEK293T cells and isolated adult rat cardiomyocytes exposed to hypoxia/reoxygenation (H/R), this reserch further confirmed that PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R. Mutation of the SUMO Consensus Sites Lysine in Cav-3 (K38R or K144R) alters the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells. Conclusions: I/R-induced cardiac PIASy activation contributes to Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5- related ventricular arrhythmias. Cardiac-targeted PIASy gene silencing mediates deSUMOylation of Cav-3 and prevents I/R-induced Nav1.5 down-regulation and ventricular arrhythmias in rats, identifying PIASy as a potential therapeutic target for relevant life-threatening arrhythmias in patients with ischemic heart diseases.

Identification and Verification of the Effect of miR-143 on the Cardioprotective Role of Phosphocreatine in Doxorubicin- Induced Cardiotoxicity by Integrated Bioinformatics Analysis

Purpose MicroRNAs (miRNAs) have been reported to play pivotal role in drugs-induced cardiotoxicity act as biomarkes, diagnostic tools and endogenous repressors of gene expression by lowering mRNA stability and interfering with mRNA translation. However, the effect of miRNAs on doxorubicin-induced cardiotoxicity still not clear. In the present study, we identified several key candidate miRNAs involving doxorubicin (DOX)-induced cardiotoxicity in rat myocardial tissues and adult rat cardiomyocytes from the Gene Expression Omnibus (GEO) database via integrated bioinformatics analysis, and the possible effect of miR-143 in the protection of DOX-induced cardiotoxicity by phosphocreatine was subsequently investigated in vivo and in vitro. Methods GSE36239 miRNA expression profiles of DOX-induced cardiotoxicity in rat myocardial tissues and adult rat cardiomyocytes (ARC) were extracted fromGEO datasets. |log2FC| > 1 and P < 0.05 were set as screening criteria, miRNAs expressed in myocardial tissues or ARC were selected as different expression miRNA (DEMs), and subsequently the key miRNAs were obtained from candidate DEMs between myocardial tissues and ARC with Venny 2.1 software. Target genes of miR-143 were predicted with Targetscan and miRBase in the species of homo sapiens, and candidate genes were obtained with Venny 2.1. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were carried out. Final, the expression and potential role of miR-143 were verified in DOX-induced cardiotoxicity of rat and cardiomyocytes H9c2. Results A total 24 DEMs were captured , including 15 up-regulated and 9 down-regulated genes in rat myocardial tissues and 42 DEMs were discovered, including 13 up-regulated and 29 down-regulated in ARC. Ultimately, 6 DEMs were determined in rat myocardial tissues and ARC by venny 2.1 software. 46 target genes of miR-143, one of the 6 DEMs, were captured from the predict results of Targetscan and miRBase with venny 2.1. The target genes were notably enriched in biological processes (BP) such as cell proliferation and migration. KEGG pathway analysis showed the target genes were enriched in HIF-1 and PI3K-Akt signaling pathway, which closely related to the oxidative stress and cardiomyocytes apoptosis. Further, western blot and RT-PCR results showed DOX-induced oxidative stress down-regulated the expression of miR-143 and Nrf2, SOD and BCL2, and up-regulated Bax and Cleaved caspase 3, while they could been reversed by the intervention of phosphocreatine (PCr) or N-acetyl-L-cystine (NAC) in DOX-induced cardiotoxicity in vivo and in vitro.Conclusion Our data showed that DOX-induced oxidative stress could decrease the expression of miR-143, promote apoptosis of cardiomyocytes, while PCr or NAC mediated antioxidation could reverse the expression down-regulation of miR-143, alleviated apoptosis in DOX-induced cardiotoxicity. Our findings elucidated the regulatory network involving miR-143 in DOX-induced cardiotoxicity, and might unveiled a potential biomarker and molecular mechanisms, which could be helpful to the diagnosis and treatment of DOX-induced cardiotoxicity.

Hippo Pathway Effector Tead1 Induces Cardiac Fibroblast to Cardiomyocyte Reprogramming

Background The conversion of fibroblasts into induced cardiomyocytes may regenerate myocardial tissue from cardiac scar through in situ cell transdifferentiation. The efficiency transdifferentiation is low, especially for human cells. We explored the leveraging of Hippo pathway intermediates to enhance induced cardiomyocyte generation. Methods and Results We screened Hippo effectors Yap (yes‐associated protein), Taz (transcriptional activator binding domain), and Tead1 (TEA domain transcription factor 1; Td) for their reprogramming efficacy with cardio‐differentiating factors Gata4, Mef2C, and Tbx5 (GMT). Td induced nearly 3‐fold increased expression of cardiomyocyte marker cTnT (cardiac troponin T) by mouse embryonic and adult rat fibroblasts versus GMT administration alone ( P <0.0001), while Yap and Taz failed to enhance cTnT expression. Serial substitution demonstrated that Td replacement of TBX5 induced the greatest cTnT expression enhancement and sarcomere organization in rat fibroblasts treated with all GMT substitutions (GMTd versus GMT: 17±1.2% versus 5.4±0.3%, P <0.0001). Cell contractility (beating) was seen in 6% of GMTd‐treated cells by 4 weeks after treatment, whereas no beating GMT‐treated cells were observed. Human cardiac fibroblasts likewise demonstrated increased cTnT expression with GMTd versus GMT treatment (7.5±0.3% versus 3.0±0.3%, P <0.01). Mechanistically, GMTd administration increased expression of the trimethylated lysine 4 of histone 3 (H3K4me3) mark at the promoter regions of cardio‐differentiation genes and mitochondrial biogenesis regulator genes in rat and human fibroblast, compared with GMT. Conclusions These data suggest that the Hippo pathway intermediate Tead1 is an important regulator of cardiac reprogramming that increases the efficiency of maturate induced cardiomyocytes generation and may be a vital component of human cardiodifferentiation strategies.

Hesperidin Reduces Memory Impairment Associated with Adult Rat Hippocampal Neurogenesis Triggered by Valproic Acid

Treatment with valproic acid (VPA) deteriorates hippocampal neurogenesis, which leads to memory impairment. Hesperidin (Hsd) is a plant-based bioflavonoid that can augment learning and memory. This study aimed to understand the effect of Hsd on the impairment of hippocampal neurogenesis and memory caused by VPA. The VPA (300 mg/kg) was administered by intraperitoneal injection twice daily for 14 days, and Hsd (100 mg/kg/day) was administered by oral gavage once a day for 21 days. All rats underwent memory evaluation using the novel object location (NOL) and novel object recognition (NOR) tests. Immunofluorescent staining of Ki-67, BrdU/NeuN, and doublecortin (DCX) was applied to determine hippocampal neurogenesis in cell proliferation, neuronal survival, and population of the immature neurons, respectively. VPA-treated rats showed memory impairments in both memory tests. These impairments resulted from VPA-induced decreases in the number of Ki-67-, BrdU/NeuN-, and DCX-positive cells in the hippocampus, leading to memory loss. Nevertheless, the behavioral expression in the co-administration group was improved. After receiving co-administration with VPA and Hsd, the numbers of Ki-67-, BrdU/NeuN-, and DCX-positive cells were improved to the normal levels. These findings suggest that Hsd can reduce the VPA-induced hippocampal neurogenesis down-regulation that results in memory impairments.

Silencing long ascending propriospinal neurons after spinal cord injury improves hindlimb stepping in the adult rat

Long ascending propriospinal neurons (LAPNs) are a subpopulation of spinal cord interneurons that directly connect the lumbar and cervical enlargements. Previously we showed, in uninjured animals, that conditionally silencing LAPNs disrupted left-right coordination of the hindlimbs and forelimbs in a context-dependent manner, demonstrating that LAPNs secure alternation of the fore- and hindlimb pairs during overground stepping. Given the ventrolateral location of LAPN axons in the spinal cord white matter, many likely remain intact following incomplete, contusive, thoracic spinal cord injury (SCI), suggesting a potential role in the recovery of stepping. Thus, we hypothesized that silencing LAPNs after SCI would disrupt recovered locomotion. Instead, we found that silencing spared LAPNs post-SCI improved locomotor function, including paw placement order and timing, and a decrease in the number of dorsal steps. Silencing also restored left-right hindlimb coordination and normalized spatiotemporal features of gait such as stance and swing time. However, hindlimb-forelimb coordination was not restored. These data indicate that the temporal information carried between the spinal enlargements by the spared LAPNs post-SCI is detrimental to recovered hindlimb locomotor function. These findings are an illustration of a post-SCI neuroanatomical-functional paradox and have implications for the development of neuronal- and axonal-protective therapeutic strategies and the clinical study/implementation of neuromodulation strategies.

Enduring Effects of Conditional Brain Serotonin Knockdown, Followed by Recovery, on Adult Rat Neurogenesis and Behavior

Serotonin (5-hydroxytryptamine, 5-HT) is a crucial signal in the neurogenic niche of the hippocampus, where it is involved in antidepressant action. Here, we utilized a new transgenic rat model (TetO-shTPH2), where brain 5-HT levels can be acutely altered based on doxycycline (Dox)-inducible shRNA-expression. On/off stimulations of 5-HT concentrations might uniquely mirror the clinical course of major depression (e.g., relapse after discontinuation of antidepressants) in humans. Specifically, we measured 5-HT levels, and 5-HT metabolite 5-HIAA, in various brain areas following acute tryptophan hydroxylase 2 (Tph2) knockdown, and replenishment, and examined behavior and proliferation and survival of newly generated cells in the dentate gyrus. We found that decreased 5-HT levels in the prefrontal cortex and raphe nuclei, but not in the hippocampus of TetO-shTPH2 rats, lead to an enduring anxious phenotype. Surprisingly, the reduction in 5-HT synthesis is associated with increased numbers of BrdU-labeled cells in the dentate gyrus. At 3 weeks of Tph2 replenishment, 5-HT levels return to baseline and survival of newly generated cells is unaffected. We speculate that the acutely induced decrease in 5-HT concentrations and increased neurogenesis might represent a compensatory mechanism.