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Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic ...

University of Missouri

Intrapleural injection of cholera toxin B conjugated to saporin (CTB-SAP) mimics respiratory motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Seven-day CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) primarily through TrkB and PI3K/Akt-dependent mechanisms [i.e., Gs-pathway, which can be initiated by adenosine 2A (A2A) receptors in naïve rats], whereas 28-day CTB-SAP rats elicit moderate pLTF though BDNF- and MEK-/ERK-dependent mechanisms [i.e., Gq-pathway, which is typically initiated by serotonin (5-HT) receptors in naïve rats]. Here, we tested the hypothesis that pLTF following CTB-SAP is ) A2A receptor-dependent at 7 days and ) 5-HT receptor-dependent at 28 days. Adult Sprague-Dawley male rats were anesthetized, paralyzed, ventilated, and exposed to acute intermittent hypoxia (AIH; 3-, 5-min bouts of 10.5% O) following bilateral, intrapleural injections at 7 days and 28 days of ) CTB-SAP (25 µg) or ) unconjugated CTB and SAP (control). Intrathecal C delivery included either the ) A2A receptor antagonist (MSX-3; 10 µM; 12 µL) or ) 5-HT receptor antagonist (methysergide; 20 mM; 15 µL). pLTF was abolished with A2A receptor inhibition in 7-day, not 28-day, CTB-SAP rats versus controls ( < 0.05), whereas pLTF was abolished following 5-HT receptor inhibition in 28-day, not 7-day, CTB-SAP rats versus controls ( < 0.05). In addition, 5-HT2A receptor expression was unchanged in CTB-SAP rats versus controls, whereas 5-HT2B receptor expression was decreased in CTB-SAP rats versus controls ( < 0.05). This study furthers our understanding of the contribution of differential receptor activation to pLTF and its implications for breathing following respiratory motor neuron death. The current study investigates underlying receptor-dependent mechanisms contributing to phrenic long-term facilitation (pLTF) following CTB-SAP-induced respiratory motor neuron death at 7 days and 28 days. We found that A2A receptors are required for enhanced pLTF in 7-day CTB-SAP rats, whereas 5-HT receptors are required for moderate pLTF in 28-day CTB-SAP rats. Targeting these time-dependent mechanisms have implications for breathing maintenance over the course of many neuromuscular diseases.

Divergent receptor utilization is necessary for phrenic long-term facilitation over the course of motor neuron loss following CTB-SAP intrapleural injections.

Intrapleural injections of cholera toxin B conjugated to saporin (CTB-SAP) selectively eliminates respiratory (e.g., phrenic) motor neurons, and mimics motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Additionally, microglial density increases in the phrenic motor nucleus following CTB-SAP. This CTB-SAP rodent model allows us to study the impact of motor neuron death on the output of surviving phrenic motor neurons, and the underlying mechanisms that contribute to enhancing or constraining their output at 7 days (d) or 28d post-CTB-SAP injection. 7d CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) through the Gs-pathway (inflammation-resistant in naïve rats), while pLTF is elicited though the Gq-pathway (inflammation-sensitive in naïve rats) in control and 28d CTB-SAP rats. In 7d and 28d male CTB-SAP rats and controls, we evaluated the effect of cyclooxygenase-1/2 enzymes on pLTF by delivery of the nonsteroidal anti-inflammatory drug, ketoprofen (IP), and we hypothesized that pLTF would be unaffected by ketoprofen in 7d CTB-SAP rats, but pLTF would be enhanced in 28d CTB-SAP rats. In anesthetized, paralyzed and ventilated rats, pLTF was surprisingly attenuated in 7d CTB-SAP rats and enhanced in 28d CTB-SAP rats (both p < 0.05) following ketoprofen delivery. Additionally in CTB-SAP rats: 1) microglia were more amoeboid in the phrenic motor nucleus; and 2) cervical spinal inflammatory-associated factor expression (TNF-α, BDNF, and IL-10) was increased vs. controls in the absence of ketoprofen (p < 0.05). Following ketoprofen delivery, TNF-α and IL-10 expression was decreased back to control levels, while BDNF expression was differentially affected over the course of motor neuron death in CTB-SAP rats. This study furthers our understanding of factors (e.g., cyclooxygenase-1/2-induced inflammation) that contribute to enhancing or constraining pLTF and its implications for breathing following respiratory motor neuron death.

Nonsteroidal anti-inflammatory drug (ketoprofen) delivery differentially impacts phrenic long-term facilitation in rats with motor neuron death induced by intrapleural CTB-SAP injections.

Spinal muscular atrophy with respiratory distress type I (SMARD1) is a neurodegenerative disease defined by respiratory distress, muscle atrophy and sensory and autonomic nervous system defects. SMARD1 is a result of mutations within the IGHMBP2 gene. We have generated six Ighmbp2 mouse models based on patient-derived mutations that result in SMARD1 and/or Charcot-Marie Tooth Type 2 (CMT2S). Here we describe the characterization of one of these models, Ighmbp2D564N (human D565N). The Ighmbp2D564N/D564N mouse model mimics important aspects of the SMARD1 disease phenotype, including motor neuron degeneration and muscle atrophy. Ighmbp2D564N/D564N is the first SMARD1 mouse model to demonstrate respiratory defects based on quantified plethysmography analyses. SMARD1 disease phenotypes, including the respiratory defects, are significantly diminished by intracerebroventricular (ICV) injection of ssAAV9-IGHMBP2 and the extent of phenotypic restoration is dose-dependent. Collectively, this model provides important biological insight into SMARD1 disease development.

The Ighmbp2D564N mouse model is the first SMARD1 model to demonstrate respiratory defects.

The tongue plays a crucial role in the swallowing process, and impairment can lead to dysphagia, particularly in motor neuron diseases (MNDs) resulting in hypoglossal-tongue axis degeneration (e.g., amyotrophic lateral sclerosis and progressive bulbar palsy). This study utilized our previously established inducible rodent model of dysphagia due to targeted degeneration of the hypoglossal-tongue axis. This model was created by injecting cholera toxin B conjugated to saporin (CTB-SAP) into the genioglossus muscle of the tongue base for retrograde transport to the hypoglossal (XII) nucleus via the hypoglossal nerve, which provides the sole motor control of the tongue. Our goal was to investigate the effect of high-repetition/low-resistance tongue exercise on tongue function, strength, and structure in four groups of male rats: (1) control + sham exercise ( = 13); (2) control + exercise ( = 10); (3) CTB-SAP + sham exercise ( = 13); and (4) CTB-SAP + exercise ( = 12). For each group, a custom spout with adjustable lick force requirement for fluid access was placed in the home cage overnight on days 4 and 6 post-tongue injection. For the two sham exercise groups, the lick force requirement was negligible. For the two exercise groups, the lick force requirement was set to ∼40% greater than the maximum voluntary lick force for individual rats. Following exercise exposure, we evaluated the effect on hypoglossal-tongue axis function ( videofluoroscopy), strength ( force-lickometer), and structure [ Magnetic Resonance Imaging (MRI) of the brainstem and tongue in a subset of rats]. Results showed that sham-exercised CTB-SAP rats had significant deficits in lick rate, swallow timing, and lick force. In exercised CTB-SAP rats, lick rate and lick force were preserved; however, swallow timing deficits persisted. MRI revealed corresponding degenerative changes in the hypoglossal-tongue axis that were mitigated by tongue exercise. These collective findings suggest that high-repetition/low-resistance tongue exercise in our model is a safe and effective treatment to prevent/diminish signs of hypoglossal-tongue axis degeneration. The next step is to leverage our rat model to optimize exercise dosing parameters and investigate corresponding treatment mechanisms of action for future translation to MND clinical trials.

A Strength Endurance Exercise Paradigm Mitigates Deficits in Hypoglossal-Tongue Axis Function, Strength, and Structure in a Rodent Model of Hypoglossal Motor Neuron Degeneration.

Various neurological diseases affect the morphology of myelinated axons. Quantitative analysis of these structures and changes occurring due to neurodegeneration or neuroregeneration is of great importance for characterization of disease state and treatment response. This paper proposes a robust, meta-learning based pipeline for segmentation of axons and surrounding myelin sheaths in electron microscopy images. This is the first step towards computation of electron microscopy related bio-markers of hypoglossal nerve degeneration/regeneration. This segmentation task is challenging due to large variations in morphology and texture of myelinated axons at different levels of degeneration and very limited availability of annotated data. To overcome these difficulties, the proposed pipeline uses a meta learning-based training strategy and a U-net like encoder decoder deep neural network. Experiments on unseen test data collected at different magnification levels (i.e, trained on 500X and 1200X images, and tested on 250X and 2500X images) showed improved segmentation performance by 5% to 7% compared to a regularly trained, comparable deep learning network.

Article	Year
Divergent receptor utilization is necessary for phrenic long-term facilitation over the course of motor neuron loss following CTB-SAP intrapleural injections. Journal of neurophysiology\| PubMed ID: 34288779	2021
Nonsteroidal anti-inflammatory drug (ketoprofen) delivery differentially impacts phrenic long-term facilitation in rats with motor neuron death induced by intrapleural CTB-SAP injections. Experimental neurology\| PubMed ID: 34634309	2022
The Ighmbp2D564N mouse model is the first SMARD1 model to demonstrate respiratory defects. Human molecular genetics\| PubMed ID: 34726235	2022
A Strength Endurance Exercise Paradigm Mitigates Deficits in Hypoglossal-Tongue Axis Function, Strength, and Structure in a Rodent Model of Hypoglossal Motor Neuron Degeneration. Frontiers in neuroscience\| PubMed ID: 35844238	2022
Axon and Myelin Sheath Segmentation in Electron Microscopy Images using Meta Learning. IEEE Applied Imagery Pattern Recognition Workshop : [proceedings]. IEEE Applied Imagery Pattern Recognition Workshop\| PubMed ID: 37214276	2022

Catherine L. Smith

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