Subcutaneous Neurotophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if any, therapies exist that target age-related laryngeal muscle dysfunction. Neurotrophins are involved in muscle innervation and differentiation of neuromuscular junctions (NMJs). It is thought that neurotrophins enhance neuromuscular transmission by increasing neurotransmitter release. The neuromuscular junctions (NMJs) become smaller and less abundant in aging rat laryngeal muscles, with evidence of functional denervation. We explored the effects of NTF4 for future clinical use as a therapeutic to improve function in aging human laryngeal muscles. Here, we provide the detailed protocol for systemic application and direct injection of NTF4 to investigate the ability of aging rat laryngeal muscle to remodel in response to NTF4 application. In this method, rats either received NTF4 either systemically via osmotic pump or by direct injection through the vocal folds. Laryngeal muscles were then dissected and used for histological examination of morphology and age-related denervation

Enhancement of Aging Rat Laryngeal Muscles with Endogenous Growth Factor Treatment

Clinical evidence suggests that laryngeal muscle dysfunction is associated with human aging. Studies in animal models have reported morphological changes consistent with denervation in laryngeal muscles with age. Life‐long laryngeal muscle activity relies on cytoskeletal integrity and nerve–muscle communication at the neuromuscular junction (NMJ). It is thought that neurotrophins enhance neuromuscular transmission by increasing neurotransmitter release. We hypothesized that treatment with neurotrophin 4 (NTF4) would modify the morphology and functional innervation of aging rat laryngeal muscles. Fifty‐six Fischer 344xBrown Norway rats (6‐ and 30‐mo age groups) were used to evaluate to determine if NTF4, given systemically (n = 32) or directly (n = 24), would improve the morphology and functional innervation of aging rat thyroarytenoid muscles. Results demonstrate the ability of rat laryngeal muscles to remodel in response to neurotrophin application. Changes were demonstrated in fiber size, glycolytic capacity, mitochondrial, tyrosine kinase receptors (Trk), NMJ content, and denervation in aging rat thyroarytenoid muscles. This study suggests that growth factors may have therapeutic potential to ameliorate aging‐related laryngeal muscle dysfunction

Cardiomyocyte Deletion of \u3ci\u3eBmal1\u3c/i\u3e Exacerbates QT- and RR-Interval Prolongation in \u3ci\u3eScn5a\u3c/i\u3e\u3csup\u3e+/ΔKPQ\u3c/sup\u3e Mice

Circadian rhythms are generated by cell autonomous circadian clocks that perform a ubiquitous cellular time-keeping function and cell type-specific functions important for normal physiology. Studies show inducing the deletion of the core circadian clock transcription factor Bmal1 in adult mouse cardiomyocytes disrupts cardiac circadian clock function, cardiac ion channel expression, slows heart rate, and prolongs the QT-interval at slow heart rates. This study determined how inducing the deletion of Bmal1 in adult cardiomyocytes impacted the in vivo electrophysiological phenotype of a knock-in mouse model for the arrhythmogenic long QT syndrome (Scn5a+/ΔKPQ). Electrocardiographic telemetry showed inducing the deletion of Bmal1 in the cardiomyocytes of mice with or without the ΔKPQ-Scn5a mutation increased the QT-interval at RR-intervals that were ≥130 ms. Inducing the deletion of Bmal1 in the cardiomyocytes of mice with or without the ΔKPQ-Scn5a mutation also increased the day/night rhythm-adjusted mean in the RR-interval, but it did not change the period, phase or amplitude. Compared to mice without the ΔKPQ-Scn5a mutation, mice with the ΔKPQ-Scn5a mutation had reduced heart rate variability (HRV) during the peak of the day/night rhythm in the RR-interval. Inducing the deletion of Bmal1 in cardiomyocytes did not affect HRV in mice without the ΔKPQ-Scn5a mutation, but it did increase HRV in mice with the ΔKPQ-Scn5a mutation. The data demonstrate that deleting Bmal1 in cardiomyocytes exacerbates QT- and RR-interval prolongation in mice with the ΔKPQ-Scn5a mutation

Chronic Wasting Disease Prions in Elk Antler Velvet

Residue 226 of cervid prion proteins may be a determinant of CWD pathogenesis

Disrupting the Circadian Clock Mechanism in Cardiomyocytes Exacerbates the LQT3-related phenotype in Scn5a(ΔKPQ/+) Mice

Introduction: The pro-arrhythmic LQTS type 3 (LQT3) is caused by gain-of-function mutations in the cardiac Na+ channel Scn5a. LQT3 patients typically have an abnormally long heart rate-corrected QT-interval (QTc), but even patients with the same disease-causing mutation show a wide range of clinical phenotypes. This suggests additional factors influence the LQT3-related phenotype. Hypothesis: Many LQT3 patients show an increased incidence in life-threatening arrhythmias at night. We tested the hypothesis that disruption of the cardiomyocyte molecular clock that underlies circadian rhythms modifies the LQT3-related phenotype. Methods: We used in vivo ECG telemetry of control mice and mice that harbor an LQT3-causing mutation (Scn5aΔKPQ/+). All animals were genetically engineered to enable us to induce the deletion of Bmal1, a key component of the molecular clock, in adult cardiomyocytes. We calculated the RR-interval, QT-interval, and the QTc-interval using the correction formula from Mitchell et al. AJP 1998. Results: Before Bmal1 deletion, Scn5aΔKPQ/+ mice showed a prolongation in the RR, QT and QTc-intervals compared to control animals. Bmal1 deletion slowed the RR and QT-intervals in both groups, but the QTc-interval remained unchanged. Linear regression analysis revealed that the slope of the QT-RR relation in Scn5aΔKPQ/+ mice was double that of control animals and Bmal1 deletion increased the slope in both groups. Additionally, Bmal1 deletion lengthened the QT-interval at a lower RR-interval in Scn5aΔKPQ/+ animals compared to control. Conclusion: Inducing Bmal1 deletion in control and Scn5aΔKPQ/+ mice did not change the QTc interval, but increased the slope of the QT-RR relation so at slower RR-intervals there is a greater change in the QT-interval. Scn5aΔKPQ/+ mice showed the greatest QT prolongation at slow RR-intervals. We conclude that disruption in the molecular clock mechanism exacerbates the LQT3-related phenotype, especially at slow heart rates

Accelerated High Fidelity Prion Amplification Within and Across Prion Species Barriers

Experimental obstacles have impeded our ability to study prion transmission within and, more particularly, between species. Here, we used cervid prion protein expressed in brain extracts of transgenic mice, referred to as Tg(CerPrP), as a substrate for in vitro generation of chronic wasting disease (CWD) prions by protein misfolding cyclic amplification (PMCA). Characterization of this infectivity in Tg(CerPrP) mice demonstrated that serial PMCA resulted in the high fidelity amplification of CWD prions with apparently unaltered properties. Using similar methods to amplify mouse RML prions and characterize the resulting novel cervid prions, we show that serial PMCA abrogated a transmission barrier that required several hundred days of adaptation and subsequent stabilization in Tg(CerPrP) mice. While both approaches produced cervid prions with characteristics distinct from CWD, the subtly different properties of the resulting individual prion isolates indicated that adaptation of mouse RML prions generated multiple strains following inter-species transmission. Our studies demonstrate that combined transgenic mouse and PMCA approaches not only expedite intra- and inter-species prio

Western blot analysis showing amplification of protease-resistant CerPrP by serial PMCA.

<p>A: Serial PMCA of 04-22412 CWD using Tg(CerPrP)1536^+/− brain homogenate. CWD prions in a 10% brain homogenate of diseased mule deer 04-22412 were diluted 10-fold into 10% brain homogenate from perfused Tg(CerPrP)1536^+/− mice. Following a round of PMCA, the sample, containing amplified protease-resistant CerPrP, was diluted 10-fold into 10% brain homogenate from perfused Tg(CerPrP)1536^+/− mice for a further round of PMCA. This process of serial PMCA was repeated for 22 rounds. PK-treated samples from each of the first 10 rounds were analyzed by Western blotting. In the final lane, a sample from Tg brain homogenate without PK treatment was loaded. B: Serial PMCA of RML using Tg(CerPrP)1536^+/− brain homogenate. Mouse RML prions in a 10% brain homogenate from a diseased wild type FVB mouse were diluted 10-fold into 10% brain homogenate from perfused Tg(CerPrP)1536^+/− mice. Serial PMCA was repeated for 22 rounds. PK-treated samples from each of the first 7 rounds were analyzed by Western blotting. The unamplified RML seed that produced protease-resistant PrP following PMCA in round 1 was loaded in the first lane, while a sample from Tg brain homogenate without PK treatment was loaded in the final lane. C: Western blot quantification of protease-resistant PrP in inocula used to challenge Tg(CerPrP)1536^+/− mice. Samples were PK-treated as indicated. Ratios indicate the fold dilution of the original preparation. In the final lane, a sample from CWD brain homogenate without PK treatment was loaded.</p