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

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

Regional distribution of CerPrP in the CNS of diseased Tg(CerPrP)1536^+/− mice infected with mouse RML prions, in vivo-adapted Cer/RML prions, and PMCA-adapted Cer/RML prions.

<p>PK-treated histoblotted coronal sections though, from top to bottom, the region of the septum, hippocampus, anterior midbrain, posterior midbrain, pons, and oblongata from diseased Tg(CerPrP)1536^+/− mice inoculated with mouse RML prions, Cer/RML-4827, or PMCA Cer/RML prions. Sections were prepared from two different Tg(CerPrP)1536^+/− mice inoculated with mouse RML prions (#4825 and #5302) and two different Tg(CerPrP)1536^+/− mice inoculated with PMCA Cer/RML prions (#5294 and #5295). Lateral areas of the posterior midbrain section of mouse #4825 were lost during tissue processing. Histoblots of sections through the anterior and posterior midbrain from an asymptomatic age-matched Tg(CerPrP)1536^+/− mouse inoculated with PBS, shown to the left, demonstrate the specificity of immunostaining with Hum-P anti-PrP recombinant Fab.</p

Assessment of the conformational stability of PrP^Sc in the brains of diseased mice.

<p>In A and C, densitometric analysis of immunoblots shows the percentage of protease-resistant CerPrP^Sc as a function of GdnHCl concentration. The sigmoidal dose-response was plotted using a four-parameter algorithm and non-linear least-square fit. Each point shown is the mean value derived from densitometric quantification of PK-resistant PrP in three diseased Tg(CerPrP)1536^+/− mouse brain extracts in each study group. Error bars indicate the standard error of the mean which, in some cases, was smaller than the size of the symbols used to indicate the mean. A, Tg(CerPrP)1536^+/− mice inoculated with 04-22412 CWD prions (green filled circles), or PMCA-derived CWD prions (green filled squares); C, Tg(CerPrP)1536^+/− mice inoculated with Cer/RML-4827 prions (blue filled circles), or PMCA Cer/RML prions (blue filled squares). For comparison, the conformational stability of MoPrP^Sc in the brains of wild type FVB mice infected with RML prions is shown (black diamonds). In B and D, representative immunoblots of protease-resistant PrP following PK treatment are shown. The mean GdnHCl_1/2 value, representing the concentration at which half the PrP^Sc in each series was denatured, is also shown.</p

Immunohistochemical detection of CerPrP^Sc and spongiform degeneration in the brains of diseased Tg(CerPrP)1536^+/− mice.

<p>A, B, and D are sections through the hippocampus of non-diseased or diseased Tg(CerPrP)1536^+/− mice; section C is from the cerebral cortex. A, absence of spongiform pathology and immunohistochemically-reactive PrP in the hippocampus of an asymptomatic PBS-inoculated Tg(CerPrP)1536^+/− mouse; B, accumulation of plaques in the hippocampus of diseased Tg(CerPrP)1536^+/− mouse inoculated with naturally occurring 04-22412 CWD prions; C, accumulation of plaques in the cerebral cortex of a diseased Tg(CerPrP)1536^+/− mouse inoculated with naturally occurring 04-22412 CWD prions; D, accumulation of plaques in the hippocampus of diseased Tg(CerPrP)1536^+/− mouse inoculated with PMCA CWD prions; E, high magnification of a large plaque aggregate rimmed by vacuoles; F, absence of spongiform pathology and immunohistochemically reactive PrP in the medulla of asymptomatic PBS-inoculated Tg(CerPrP) 1536^+/− mouse; G, diffuse PrP accumulation in the medulla of diseased Tg(CerPrP)1536^+/− mouse #5297 inoculated with mouse RML prions; H, high magnification of a section thought the hippocampus of diseased Tg(CerPrP)1536^+/− mouse #5300 showing PrP accumulation in small plaques; I, diffuse PrP accumulation in the medulla of a diseased Tg(CerPrP)1536^+/− mouse inoculated with PMCA Cer/RML prions. Hematoxylin was used as counterstain. Bar = 100 µm in A–D; Bar = 50 µm in E–I.</p