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 lateral sclerosis, respectively). Phrenic motor neurons are the final link between the central nervous system and muscle, and their respective motor units (groups of muscle fibers innervated by a single motor neuron) represent the smallest functional unit of the neuromuscular ventilatory system. Compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE) are established electrophysiological approaches that enable the longitudinal assessment of motor unit integrity in animal models over time but have mostly been applied to limb muscles. Therefore, the objectives of this study are to describe an approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic MUNE, motor unit size (represented as SMUP), and CMAP, and then to demonstrate the utility of these approaches in a motor neuron loss model. Sensitive, objective, and translationally relevant biomarkers for neuronal injury, degeneration, and regeneration in motor neuron injury and diseases can significantly aid and accelerate experimental research discoveries to clinical testing.

Minimally Invasive Murine Laryngoscopy for Close-Up Imaging of Laryngeal Motion during Breathing and Swallowing.

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to impair laryngeal function, which results in difficulty breathing (dyspnea), swallowing impairment (dysphagia), and/or voice impairment (dysphonia). Dysphagia, in particular, can lead to aspiration pneumonia and associated morbidity, recurrent hospitalization, and early mortality. Despite these serious consequences, existing treatments for laryngeal dysfunction are largely aimed at surgical and behavioral interventions that unfortunately do not typically restore normal laryngeal function, thus highlighting the urgent need for innovative solutions. To bridge this gap, we have been developing an experimental endoscopic approach to investigate laryngeal dysfunction in murine (i.e., mouse and rat) models. However, endoscopy in rodents is quite challenging due to their small size relative to current endoscope technology, anatomical differences in the upper airway, and the necessity for anesthesia to optimally access the larynx. Here, we describe a novel transoral laryngoscopy approach that permits close-up, unobstructed video imaging of laryngeal motion in mice and rats. Critical steps in the protocol include precise anesthesia management (to prevent overdosing that abolishes swallowing and/or risks respiratory distress-related mortality) and micromanipulator control of the endoscope (for stable video recording of laryngeal motion by a single researcher for subsequent quantification). Importantly, the protocol can be performed over time in the same animals to study the impact of various pathological conditions specifically on laryngeal function. A novel advantage of this protocol is the ability to visualize airway protection during swallowing, which is not possible in humans due to epiglottic inversion over the laryngeal inlet that obstructs the glottis from view. Rodents therefore provide a unique opportunity to specifically investigate the mechanisms of normal versus pathological laryngeal airway protection for the ultimate purpose of discovering treatments to effectively restore normal laryngeal function.

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

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 (n = 13); (2) control + exercise (n = 10); (3) CTB-SAP + sham exercise (n = 13); and (4) CTB-SAP + exercise (n = 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 (via videofluoroscopy), strength (via force-lickometer), and structure [via 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.

Impact of Limb Phenotype on Tongue Denervation Atrophy, Dysphagia Penetrance, and Survival Time in a Mouse Model of ALS.

Current treatments for dysphagia in ALS do not target the underlying tongue weakness and denervation atrophy that is prevalent in spinal and bulbar ALS cases. To address this clinical gap, we studied the low copy number SOD1-G93A (LCN-SOD1) mouse model of ALS to quantify the impact of limb phenotype on tongue denervation atrophy, dysphagia penetrance, and survival time in preparation for future treatment-based studies. Two male LCN-SOD1 breeders and 125 offspring were followed for limb phenotype inheritance, of which 52 (30 LCN-SOD1 and 22 wild-type/WT, both sexes) underwent characterization of dysphagia penetrance (via videofluoroscopic swallow study; VFSS) and survival time at disease end-stage (15-20% body weight loss). From these, 16 mice (8/genotype) underwent postmortem histological analysis of the genioglossus for evidence of denervation atrophy. Results revealed that both breeders displayed a mixed (hindlimb and forelimb) ALS phenotype and sired equal proportions of hindlimb vs. mixed phenotype offspring. Dysphagia penetrance was complete for mixed (100%) versus incomplete for hindlimb (64%) phenotype mice; yet survival times were similar. Regardless of limb phenotype, LCN-SOD1 mice had significantly smaller genioglossus myofibers and more centralized myonuclei compared to WT mice (p < 0.05). These biomarkers of denervation atrophy were significantly correlated with VFSS metrics (lick and swallow rates, p < 0.05) but not survival time. In conclusion, both LCN-SOD1 phenotypes had significant tongue denervation atrophy, even hindlimb phenotype mice without dysphagia. This finding recapitulates human ALS, providing robust rationale for using this preclinical model to explore targeted treatments for tongue denervation atrophy and ensuing dysphagia.

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

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

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.

Axon and Myelin Sheath Segmentation in Electron Microscopy Images using Meta Learning.

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.

Phrenic motor neuron survival below cervical spinal cord hemisection.

Cervical spinal cord injury (cSCI) severs bulbospinal projections to respiratory motor neurons, paralyzing respiratory muscles below the injury. C2 spinal hemisection (C2Hx) is a model of cSCI often used to study spontaneous and induced plasticity and breathing recovery post-injury. One key assumption is that C2Hx dennervates motor neurons below the injury, but does not affect their survival. However, a recent study reported substantial bilateral motor neuron death caudal to C2Hx. Since phrenic motor neuron (PMN) death following C2Hx would have profound implications for therapeutic strategies designed to target spared neural circuits, we tested the hypothesis that C2Hx minimally impacts PMN survival. Using improved retrograde tracing methods, we observed no loss of PMNs at 2- or 8-weeks post-C2Hx. We also observed no injury-related differences in ChAT or NeuN immunolabeling within labelled PMNs. Although we found no evidence of PMN loss following C2Hx, we cannot rule out neuronal loss in other motor pools. These findings address an essential prerequisite for studies that utilize C2Hx as a model to explore strategies for inducing plasticity and/or regeneration within the phrenic motor system, as they provide important insights into the viability of phrenic motor neurons as therapeutic targets after high cervical injury.

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

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 1) A2A receptor-dependent at 7 days and 2) 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% O2) following bilateral, intrapleural injections at 7 days and 28 days of 1) CTB-SAP (25 µg) or 2) unconjugated CTB and SAP (control). Intrathecal C4 delivery included either the 1) A2A receptor antagonist (MSX-3; 10 µM; 12 µL) or 2) 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 (P < 0.05), whereas pLTF was abolished following 5-HT receptor inhibition in 28-day, not 7-day, CTB-SAP rats versus controls (P < 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 (P < 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.NEW & NOTEWORTHY 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.

Mechanisms of severe acute intermittent hypoxia-induced phrenic long-term facilitation.

Moderate acute intermittent hypoxia (mAIH; 35-55 mmHg PaO2) elicits phrenic long-term facilitation (pLTF) by a mechanism that requires activation of Gq protein-coupled serotonin type 2 receptors, MEK/ERK MAP kinase, and NADPH oxidase activity and is constrained by cAMP-PKA signaling. In contrast, severe AIH (sAIH; 25-35 mmHg PaO2) elicits Gs protein-coupled adenosine type 2 A receptor-dependent pLTF. Another Gs protein-coupled receptor, serotonin 7 receptors, elicits phrenic motor facilitation (pMF) by a mechanism that requires exchange protein activated by cyclic AMP (EPAC) and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and is constrained by NADPH oxidase activity. Here, we tested the hypothesis that the same downstream signaling mechanisms giving rise to serotonin 7 (vs. serotonin 2) receptor-induced pMF underlie sAIH-induced pLTF. In anesthetized rats, sAIH-induced pLTF was compared after pretreatment with intrathecal (C4) injections of inhibitors for: 1) EPAC (ESI-05); 2) MEK/ERK (UO126); 3) PKA (KT-5720); 4) PI3K/Akt (PI828); and 5) NADPH oxidase (apocynin). In partial agreement with our hypothesis, sAIH-induced pLTF was abolished by ESI-05 and PI828 and marginally enhanced by apocynin but, surprisingly, was abolished by UO126 and attenuated by KT-5720. Mechanisms of sAIH-induced pLTF reflect elements of both Gq and Gs pathways to pMF, likely as a consequence of the complex, cross-talk interactions between them.NEW & NOTEWORTHY Distinct mechanisms give rise to pLTF induced by moderate and severe AIH. We demonstrate that, unlike moderate AIH, severe AIH-induced pLTF requires EPAC and PI3K/Akt and is marginally constrained by NADPH oxidase activity. Surprisingly, sAIH-induced pLTF requires MEK/ERK activity similar to moderate AIH-induced pLTF and is reduced by PKA inhibition. We suggest sAIH-induced pLTF arises from complex interactions between dominant mechanisms characteristic of moderate versus severe AIH-induced pLTF.

Tongue and hypoglossal morphology after intralingual cholera toxin B-saporin injection.

INTRODUCTION: We recently developed an inducible model of dysphagia using intralingual injection of cholera toxin B conjugated to saporin (CTB-SAP) to cause death of hypoglossal neurons. In this study we aimed to evaluate tongue morphology and ultrastructural changes in hypoglossal neurons and nerve fibers in this model. METHODS: Tissues were collected from 20 rats (10 control and 10 CTB-SAP animals) on day 9 post-injection. Tongues were weighed, measured, and analyzed for microscopic changes using laminin immunohistochemistry. Hypoglossal neurons and axons were examined using transmission electron microscopy. RESULTS: The cross-sectional area of myofibers in the posterior genioglossus was decreased in CTB-SAP-injected rats. Degenerative changes were observed in both the cell bodies and distal axons of hypoglossal neurons. DISCUSSION: Preliminary results indicate this model may have translational application to a variety of neurodegenerative diseases resulting in tongue dysfunction and associated dysphagia.

Differential mechanisms are required for phrenic long-term facilitation over the course of motor neuron loss following CTB-SAP intrapleural injections.

Selective elimination of respiratory motor neurons using intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB-SAP) mimics motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. This CTB-SAP model allows us to study the impact of motor neuron death on the output of surviving phrenic motor neurons. After 7(d) days of CTB-SAP, phrenic long-term facilitation (pLTF, a form of respiratory plasticity) is enhanced, but returns towards control levels at 28d. However, the mechanism responsible for this difference in magnitude of pLTF is unknown. In naïve rats, pLTF predominately requires 5-HT2 receptors, the new synthesis of BDNF, and MEK/ERK signaling; however, pLTF can alternatively be induced via A2A receptors, the new synthesis of TrkB, and PI3K/Akt signaling. Since A2A receptor-dependent pLTF is enhanced in naïve rats, we suggest that 7d CTB-SAP treated rats utilize the alternative mechanism for pLTF. Here, we tested the hypothesis that pLTF following CTB-SAP is: 1) TrkB and PI3K/Akt, not BDNF and MEK/ERK, dependent at 7d; and 2) BDNF and MEK/ERK, not TrkB and PI3K/Akt, dependent at 28d. Adult Sprague Dawley male rats were anesthetized, paralyzed, ventilated, and were exposed to acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) following bilateral, intrapleural injections at 7d and 28d of: 1) CTB-SAP (25 µg), or 2) un-conjugated CTB and SAP (control). Intrathecal C4 delivery included either: 1) small interfering RNA that targeted BDNF or TrkB mRNA; 2) UO126 (MEK/ERK inhibitor); or 3) PI828 (PI3K/Akt inhibitor). Our data suggest that pLTF in 7d CTB-SAP treated rats is elicited primarily through TrkB and PI3K/Akt-dependent mechanisms, whereas BDNF and MEK/ERK-dependent mechanisms induce pLTF in 28d CTB-SAP treated rats. This project increases our understanding of respiratory plasticity and its implications for breathing following motor neuron death.

5-HT2A/B receptor expression in the phrenic motor nucleus in a rat model of ALS (SOD1G93A).

Despite respiratory motor neuron death, ventilation is preserved in SOD1G93A rats. Compensatory respiratory plasticity may counterbalance the loss of these neurons. Phrenic long-term facilitation (pLTF; a form of respiratory plasticity) in naïve rats is 5-HT2 and NADPH oxidase-dependent. Furthermore, 5-HT2A, not 5-HT2B, receptor-induced phrenic motor facilitation is NADPH oxidase-independent in naïve rats. pLTF is NADPH oxidase-dependent in pre-symptomatic, but not end-stage, SOD1G93A rats. Here, we hypothesized that in the putative phrenic motor nucleus (PMN) of SOD1G93A rats vs. wild-type littermates: 1) pre-symptomatic rats would have greater 5-HT2B receptor expression that decreases at end-stage; and 2) 5-HT2A receptor expression would increase from pre-symptomatic to end-stage. Putative PMN 5-HT2A receptor expression was reduced when comparing across (but not within) pre-symptomatic vs. end-stage groups (p < 0.05). In contrast, putative PMN 5-HT2B receptor expression was increased when comparing across pre-symptomatic vs. end-stage groups, and within end-stage groups (p < 0.05). These data suggest a potential role for 5-HT2 receptors in pLTF and breathing in SOD1G93A rats.

Intralingual Administration of AAVrh10-miRSOD1 Improves Respiratory But Not Swallowing Function in a Superoxide Dismutase-1 Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by degeneration of motor neurons and muscles, and death is usually a result of impaired respiratory function due to loss of motor neurons that control upper airway muscles and/or the diaphragm. Currently, no cure for ALS exists and treatments to date do not significantly improve respiratory or swallowing function. One cause of ALS is a mutation in the superoxide dismutase-1 (SOD1) gene; thus, reducing expression of the mutated gene may slow the progression of the disease. Our group has been studying the SOD1G93A transgenic mouse model of ALS that develops progressive respiratory deficits and dysphagia. We hypothesize that solely treating the tongue in SOD1 mice will preserve respiratory and swallowing function, and it will prolong survival. At 6 weeks of age, 11 SOD1G93A mice (both sexes) received a single intralingual injection of gene therapy (AAVrh10-miRSOD1). Another 29 mice (both sexes) were divided into two control groups: (1) 12 SOD1G93A mice that received a single intralingual vehicle injection (saline); and (2) 17 non-transgenic littermates. Starting at 13 weeks of age, plethysmography (respiratory parameters) at baseline and in response to hypoxia (11% O2) + hypercapnia (7% CO2) were recorded and videofluoroscopic swallow study testing were performed twice monthly until end-stage disease. Minute ventilation during hypoxia + hypercapnia and mean inspiratory flow at baseline were significantly reduced (p < 0.05) in vehicle-injected, but not AAVrh10-miRSOD1-injected SOD1G93A mice as compared with wild-type mice. In contrast, swallowing function was unchanged by AAVrh10-miRSOD1 treatment (p > 0.05). AAVrh10-miRSOD1 injections also significantly extended survival in females by ∼1 week. In conclusion, this study indicates that intralingual AAVrh10-miRSOD1 treatment preserved respiratory (but not swallowing) function potentially via increasing upper airway patency, and it is worthy of further exploration as a possible therapy to preserve respiratory capacity in ALS patients.

Peripheral-to-central immune communication at the area postrema glial-barrier following bleomycin-induced sterile lung injury in adult rats.

The pathways for peripheral-to-central immune communication (P â†’ C I-comm) following sterile lung injury (SLI) are unknown. SLI evokes systemic and central inflammation, which alters central respiratory control and viscerosensory transmission in the nucleus tractus solitarii (nTS). These functional changes coincide with increased interleukin-1 beta (IL-1ß) in the area postrema, a sensory circumventricular organ that connects P â†’ C I-comm to brainstem circuits that control homeostasis. We hypothesize that IL-1ß and its downstream transcriptional target, cyclooxygenase-2 (COX-2), mediate P â†’ C I-comm in the nTS. In a rodent model of SLI induced by intratracheal bleomycin (Bleo), the sigh frequency and duration of post-sigh apnea increased in Bleo- compared to saline- treated rats one week after injury. This SLI-dependent change in respiratory control occurred concurrently with augmented IL-1ß and COX-2 immunoreactivity (IR) in the funiculus separans (FS), a barrier between the AP and the brainstem. At this barrier, increases in IL-1ß and COX-2 IR were confined to processes that stained for glial fibrillary acidic protein (GFAP) and that projected basolaterally to the nTS. Further, FS radial-glia did not express TNF-α or IL-6 following SLI. To test our hypothesis, we blocked central COX-1/2 activity by intracerebroventricular (ICV) infusion of Indomethacin (Ind). Continuous ICV Ind treatment prevented Bleo-dependent increases in GFAP + and IL-1ß + IR, and restored characteristics of sighs that reset the rhythm. These data indicate that changes in sighs following SLI depend partially on activation of a central COX-dependent P â†’ C I-comm via radial-glia of the FS.

Optimizing the Translational Value of Mouse Models of ALS for Dysphagia Therapeutic Discovery.

The goal of this study was to compare dysphagia phenotypes in low and high copy number (LCN and HCN) transgenic superoxide dismutase 1 (SOD1) mouse models of ALS to accelerate the discovery of novel and effective treatments for dysphagia and early amyotrophic lateral sclerosis (ALS) diagnosis. Clinicopathological features of dysphagia were characterized in individual transgenic mice and age-matched controls utilizing videofluoroscopy in conjunction with postmortem assays of the tongue and hypoglossal nucleus. Quantitative PCR accurately differentiated HCN-SOD1 and LCN-SOD1 mice and nontransgenic controls. All HCN-SOD1 mice developed stereotypical paralysis in both hindlimbs. In contrast, LCN-SOD1 mice displayed wide variability in fore- and hindlimb involvement. Lick rate, swallow rate, inter-swallow interval, and pharyngeal transit time were significantly altered in both HCN-SOD1 and LCN-SOD1 mice compared to controls. Tongue weight, tongue dorsum surface area, total tongue length, and caudal tongue length were significantly reduced only in the LCN-SOD1 mice compared to age-matched controls. LCN-SOD1 mice with lower body weights had smaller/lighter weight tongues, and those with forelimb paralysis and slower lick rates died at a younger age. LCN-SOD1 mice had a 32% loss of hypoglossal neurons, which differed significantly when compared to age-matched control mice. These novel findings for LCN-SOD1 mice are congruent with reported dysphagia and associated tongue atrophy and hypoglossal nucleus pathology in human ALS patients, thus highlighting the translational potential of this mouse model in ALS research.

Hypoglossal Motor Neuron Death Via Intralingual CTB-saporin (CTB-SAP) Injections Mimic Aspects of Amyotrophic Lateral Sclerosis (ALS) Related to Dysphagia.

Amyotrophic lateral sclerosis (ALS) is a devastating disease leading to degeneration of motor neurons and skeletal muscles, including those required for swallowing. Tongue weakness is one of the earliest signs of bulbar dysfunction in ALS, which is attributed to degeneration of motor neurons in the hypoglossal nucleus in the brainstem, the axons of which directly innervate the tongue. Despite its fundamental importance, dysphagia (difficulty swallowing) and strategies to preserve swallowing function have seldom been studied in ALS models. It is difficult to study dysphagia in ALS models since the amount and rate at which hypoglossal motor neuron death occurs cannot be controlled, and degeneration is not limited to the hypoglossal nucleus. Here, we report a novel experimental model using intralingual injections of cholera toxin B conjugated to saporin (CTB-SAP) to study the impact of only hypoglossal motor neuron death without the many complications that are present in ALS models. Hypoglossal motor neuron survival, swallowing function, and hypoglossal motor output were assessed in Sprague-Dawley rats after intralingual injection of either CTB-SAP (25 g) or unconjugated CTB and SAP (controls) into the genioglossus muscle. CTB-SAP treated rats exhibited significant (p ≤ 0.05) deficits vs. controls in: (1) lick rate (6.0 ±â€¯0.1 vs. 6.6 ±â€¯0.1 Hz; (2) hypoglossal motor output (0.3 ±â€¯0.05 vs. 0.6 ±â€¯0.10 mV); and (3) hypoglossal motor neuron survival (398 ±â€¯34 vs. 1018 ±â€¯41 neurons). Thus, this novel, inducible model of hypoglossal motor neuron death mimics the dysphagia phenotype that is observed in ALS rodent models, and will allow us to study strategies to preserve swallowing function.

Systemic inflammation inhibits serotonin receptor 2-induced phrenic motor facilitation upstream from BDNF/TrkB signaling.

Although systemic inflammation induced by even a low dose of lipopolysaccharide (LPS, 100 µg/kg) impairs respiratory motor plasticity, little is known concerning cellular mechanisms giving rise to this inhibition. Phrenic motor facilitation (pMF) is a form of respiratory motor plasticity elicited by pharmacological agents applied to the cervical spinal cord, or by acute intermittent hypoxia (AIH; 3, 5-min hypoxic episodes); when elicited by AIH, pMF is known as phrenic long-term facilitation (pLTF). AIH consisting of moderate hypoxic episodes (mAIH, arterial Po2 = 35-55 mmHg) elicits pLTF via the Q pathway to pMF, a mechanism that requires spinal serotonin (5HT2) receptor activation and new brain-derived neurotrophic factor (BDNF) protein synthesis. Although mild systemic inflammation attenuates mAIH-induced pLTF via spinal p38 MAP kinase activation, little is known concerning how p38 MAP kinase activity inhibits the Q pathway. Here, we confirmed that 24 h after a low LPS dose (100 µg/kg ip), mAIH-induced pLTF is greatly attenuated. Similarly, pMF elicited by intrathecal cervical injections of 5HT2A (DOI; 100 µM; 3 × 6 µl) or 5HT2B receptor agonists (BW723C86; 100 µM; 3 × 6 µl) is blocked 24 h post-LPS. When pMF was elicited by intrathecal BDNF (100 ng, 12 µl), pMF was actually enhanced 24 h post-LPS. Thus 5HT2A/2B receptor-induced pMF is impaired downstream from 5HT2 receptor activation, but upstream from BDNF/TrkB signaling. Mechanisms whereby LPS augments BDNF-induced pMF are not yet known. NEW & NOTEWORTHY These experiments give novel insights concerning mechanisms whereby systemic inflammation undermines serotonin-dependent, spinal respiratory motor plasticity, yet enhances brain-derived neurotrophic factor (BDNF)/TrkB signaling in phrenic motor neurons. These insights may guide development of new strategies to elicit functional recovery of breathing capacity in patients with respiratory impairment by reducing (or bypassing) the impact of systemic inflammation characteristic of clinical disorders that compromise breathing.

Phrenic long-term facilitation following intrapleural CTB-SAP-induced respiratory motor neuron death.

Amyotrophic lateral sclerosis (ALS) is a devastating disease leading to progressive motor neuron degeneration and death by ventilatory failure. In a rat model of ALS (SOD1G93A), phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is enhanced greater than expected at disease end-stage but the mechanism is unknown. We suggest that one trigger for this enhancement is motor neuron death itself. Intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB-SAP) selectively kill respiratory motor neurons and mimic motor neuron death observed in SOD1G93A rats. This CTB-SAP model allows us to study the impact of respiratory motor neuron death on breathing without many complications attendant to ALS. Here, we tested the hypothesis that phrenic motor neuron death is sufficient to enhance pLTF. pLTF was assessed in anesthetized, paralyzed and ventilated Sprague Dawley rats 7 and 28 days following bilateral intrapleural injections of: 1) CTB-SAP (25 µg), or 2) un-conjugated CTB and SAP (control). CTB-SAP enhanced pLTF at 7 (CTB-SAP: 162 ±â€¯18%, n = 8 vs. Control: 63 ±â€¯3%; n = 8; p < 0.05), but not 28 days post-injection (CTB-SAP: 64 ±â€¯10%, n = 10 vs. Control: 60 ±â€¯13; n = 8; p > 0.05). Thus, pLTF at 7 (not 28) days post-CTB-SAP closely resembles pLTF in end-stage ALS rats, suggesting that processes unique to the early period of motor neuron death enhance pLTF. This project increases our understanding of respiratory plasticity and its implications for breathing in motor neuron disease.

Compensatory plasticity in diaphragm and intercostal muscle utilization in a rat model of ALS.

In SOD1G93A transgenic rat model of ALS, breathing capacity is preserved until late in disease progression despite profound respiratory motor neuron (MN) cell death. To explore mechanisms preserving breathing capacity, we assessed inspiratory EMG activity in diaphragm and external intercostal T2 (EIC2) and T5 (EIC5) muscles in anesthetized SOD1G93A rats at disease end-stage (20% decrease in body mass). We hypothesized that despite significant phrenic motor neuron loss and decreased phrenic nerve activity, diaphragm electrical activity and trans-diaphragmatic pressure (Pdi) are maintained to sustain ventilation. We alternatively hypothesized that EIC activity is enhanced, compensating for impaired diaphragm function. Diaphragm, EIC2 and EIC5 muscle EMGs and Pdi were measured in urethane-anesthetized, spontaneously breathing female SOD1G93A rats versus wild-type littermates during normoxia (arterial PO2 ~90mmHg, PCO2 ~45mmHg), maximal chemoreceptor stimulation (MCS: 10.5% O2/7% CO2), spontaneous augmented breaths and sustained tracheal occlusion. Phrenic MNs were counted in C3-5; T2 and T5 ventrolateral MNs were counted. In end-stage SOD1G93A rats, 29% of phrenic MNs survived (vs. wild-type), yet integrated diaphragm EMG amplitude was normal. Nevertheless, maximal Pdi decreased ~30% vs. wild type (p<0.01) and increased esophageal to gastric pressure ratio (p<0.05), consistent with persistent diaphragm weakness. Despite major T2 and T5 MN death, integrated EIC2 (100% greater than wild type) and EIC5 (300%) EMG amplitudes were increased in mutant rats during normoxia (p<0.01), possibly compensating for decreased Pdi. Thus, despite significant phrenic MN loss, diaphragm EMG activity is maintained; in contrast, Pdi was not, suggesting diaphragm dysfunction. Presumably, increased EIC EMG activity compensated for persistent diaphragm weakness. These adjustments contribute to remarkable preservation of breathing ability despite major respiratory motor neuron death and diaphragm dysfunction.