Fig. 5: Cardiac hypertrophy and ganglion-specific neuronal degeneration after induction of chemogenetic oxidative stress in DAAO-TGCdh5 mice.
a presents representative echocardiographic images showing short-axis M-mode views of the left ventricle of D-alanine-fed (0.5 M D-alanine for 6 weeks) DAAO-TGCdh5 mice and Cre+/TG− control mice. b shows quantitative analyses of echocardiographic parameters from these mice that were quantitated by observers blinded to genotype and treatment. Measurements include left ventricular ejection fraction (EF; p = 0.0039), fractional shortening (FS; p = 0.0039), left ventricular end-diastolic volume (LVEDV; p = 0.0089), left ventricular end-systolic volume (LVESV; p = 0.0021), posterior wall thickness (PWT; p = 0.0279), and anterior wall thickness (AWT; p = 0.1655). *denotes p < 0.05; **denotes p < 0.01; and ***denotes p < 0.001 (Mann–Whitney test, unpaired two-tailed). Data are presented as mean ± standard error; there were n = 10 animals per group (for posterior wall thickness n = 9 per group). c shows representative images from fixed mouse heart tissue isolated from untreated DAAO-TGCdh5 mice; the first panel shows H&E staining; the next panels show (as noted) immunofluorescent staining with GFP antibodies (to detect the transgene) and VE-Cadherin antibodies (endothelial marker) followed by an overlay of the GFP and VE-Cadherin signals. The scale bars note 100 μm. Expression of the transgene is clearly seen in the blood vessels but not in cardiac muscle itself. d shows a pictorial representation of nodose and stellate ganglia, which are the major ganglia modulating sympathetic outflow to the heart (stellate ganglion) and parasympathetic sensory neurons (nodose ganglion). e shows silver staining of nodose ganglia cell isolated from D-alanine-fed (0.5 M D-alanine for 7 weeks) DAAO-TGCdh5 or control mice. Strongly positive silver staining is seen in the nodose ganglion from D-alanine-fed transgenic but not control mice. Scale bars, 100 μm. f, g show a series of photomicrographs and immunofluorescence images from isolated nodose ganglia (f) or stellate ganglia (g). The GFP stain for the transgene is strikingly positive in neurons of the nodose ganglia, but neurons in the stellate ganglia are negative for transgene protein expression; transgene expression in the representative stellate ganglion shown in (g) is restricted to GFP staining in a small blood vessel that can be seen coursing through the ganglion. Staining with antibodies directed against the sympathetic neuron marker tyrosine hydroxylase (TH) identify sparse nodose neurons, while most stellate neurons are positive for TH expression, as previously reported69. These images are representative of n = 3 independently-treated mice for each genotype. Scale bars, 100 μm.
