Problem
Chronic intermittent hypoxia (CIH), a widely accepted model for sleep apnea, poses a significant risk for various cardiovascular diseases (CVDs). Notably, autonomic imbalance, specifically sympathetic overactivity and parasympathetic withdrawal, has been identified as a key contributor to CIH-induced cardiovascular diseases, including hypertension, arrhythmia, baroreflex impairment and heart failure. Previously, we showed that CIH reduced baroreflex control of heart rate and induced vagal cardiac motor neuronal loss in the brainstem. CIH activates the central nervous system circuitry, which in turn enhances sympathetic outflow. However, whether CIH induces remodeling of the peripheral cardiac sympathetic innervation remains undetermined.
Despite comprehensive knowledge of the functional changes associated with CIH, the potential structural remodeling of cardiac sympathetic innervation remains unclear. This knowledge gap highlights a critical research need to uncover the intricate mechanisms governing CIH-induced cardiovascular diseases and, in particular, to elucidate the structural alterations in cardiac sympathetic innervation. Addressing this gap will not only enhance our understanding of the sympathetic neuroanatomical remodeling but also pave the way for the development of more efficacious interventions for CIH-induced cardiovascular diseases in the future.
Solution
In response to the identified gaps in understanding the structural remodeling of catecholaminergic innervation within the atria following CIH exposure, an international collaboration of researchers has undertaken a comprehensive study. Employing a combination of state-of-the-art techniques, the study utilized flat-mounts of whole atrial tissues, processed and labeled with tyrosine hydroxylase (TH, a sympathetic marker). Neurolucida 360 and Zeiss Arivis Vision 4D, along with the MAPCORE scaffolding tool, were employed for tracing, digitizing, and integrating mapped data onto a 3D mouse heart scaffold. The outcomes of this collaborative effort revealed substantial remodeling of TH-IR innervation within the atria induced by CIH (Figure 1). This manifested as an increased regional density of innervation and number of axon branching points. Additionally, abnormal patterns of TH-IR axons encircling intrinsic cardiac ganglia were identified following exposure to CIH.
Utilizing mapping tools from the NIH SPARC program developed by Dr. Hunter’s group, it was possible for Dr. Cheng’s team to integrate the topographical distribution of catecholaminergic innervation in the atria onto a novel 3D scaffold. This represents the first instance to enable precise anatomical distribution and holistic quantitative comparison between normal and CIH conditions on a 3D heart scaffold (Figure 2).
Impact
This study supports the hypothesis that CIH induces autonomic imbalance, partly mediated by structural alterations in sympathetic efferent cardiac innervation. Furthermore, the scaffold mapping tool provided by NIH SPARC program enabled the development of novel 3D heart scaffolds with precise anatomical distribution and holistic quantitative comparison of catecholaminergic innervation in normal and CIH conditions. This will not only advance our understanding of CIH-induced autonomic remodeling but will also facilitate large-scale population studies including comparison across sex, age, and various pathological conditions. In the long-term, this will lay the groundwork for the development of more effective interventions to address CIH-induced cardiovascular diseases.
Figure 1: Depth-encoded montages and heatmaps of TH-IR axon innervation in the flat-mount of the whole left atrium. Depth is displayed as green-yellow (outermost) and green (middle and inner) layers. The z-stack range in the RA was from 0 to 64 μm (A), whereas, in CIH, it was from 0 to 87 μm (C). Heatmap of TH-IR axons showing the overall intensity distribution and the hot spot of the highest regions reflected mainly in the middle region of the left atrium. Pipeline created in Arivis Vision 4D that included background subtraction, enhancement, seeds and tubularity filters resulted in complete tracing of the entire right atrium in RA (B) and CIH (D). Scale bar = 1000 μm.
Figure 2: Irregular patterns of TH-IR axons around ICG following CIH TH-IR axons encircled individual FG-labelled neurons in ICG and exhibited swollen terminal structures (A). Tangled structures and aberrant TH-IR axons formed finger-like projections around ICG neurons (B). Scale bar = 10 μm.
Figure 3: Extensive mapping of TH-IR innervation of the flat-mount of the whole left and right atrium on the 3D heart scaffold. TH-IR axon innervation in the entire atria was traced and digitized with Neurolucida 360 and Arivis Vision 4D. Through the smooth fitting of corresponding parts of the anatomical scaffold to annotated features of the atrium with MAPCORE scaffolding tool, an optimal fit for all data set in room air (RA) and chronic intermittent hypoxia (CIH) conditions is achieved.
