assessing-functional-recovery-eupneic-diaphragm-activity-following

Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Hemisection in Rats

Following cSCI, activation of the DIAm can be impacted depending on the extent of the injury. The present manuscript describes a unilateral C2 hemisection ...

Mayo Clinic

Current strategies for optimizing deep brain stimulation (DBS) therapy involve multiple postoperative visits. During each visit, stimulation parameters are adjusted until desired therapeutic effects are achieved and adverse effects are minimized. However, the efficacy of these therapeutic parameters may decline with time due at least in part to disease progression, interactions between the host environment and the electrode, and lead migration. As such, development of closed-loop control systems that can respond to changing neurochemical environments, tailoring DBS therapy to individual patients, is paramount for improving the therapeutic efficacy of DBS. Evidence obtained using electrophysiology and imaging techniques in both animals and humans suggests that DBS works by modulating neural network activity. Recently, animal studies have shown that stimulation-evoked changes in neurotransmitter release that mirror normal physiology are associated with the therapeutic benefits of DBS. Therefore, to fully understand the neurophysiology of DBS and optimize its efficacy, it may be necessary to look beyond conventional electrophysiological analyses and characterize the neurochemical effects of therapeutic and non-therapeutic stimulation. By combining electrochemical monitoring and mathematical modeling techniques, we can potentially replace the trial-and-error process used in clinical programming with deterministic approaches that help attain optimal and stable neurochemical profiles. In this manuscript, we summarize the current understanding of electrophysiological and electrochemical processing for control of neuromodulation therapies. Additionally, we describe a proof-of-principle closed-loop controller that characterizes DBS-evoked dopamine changes to adjust stimulation parameters in a rodent model of DBS. The work described herein represents the initial steps toward achieving a "smart" neuroprosthetic system for treatment of neurologic and psychiatric disorders.

A neurochemical closed-loop controller for deep brain stimulation: toward individualized smart neuromodulation therapies.

Injury to nerves innervating respiratory muscles such as the diaphragm muscle results in significant respiratory compromise. Electromyography (EMG) and transdiaphragmatic pressure (Pdi) measurements reflect diaphragm activation and force generation. Immediately after unilateral diaphragm denervation (DNV), ventilatory behaviors can be accomplished without impairment, but Pdi generated during higher force non-ventilatory behaviors is significantly decreased. We hypothesized that 1) the initial reduction in Pdi during higher force behaviors after DNV is ameliorated after 14 days, and 2) changes in Pdi over time after DNV are associated with concordant changes in contralateral diaphragm EMG activity and ventilatory parameters. In adult male rats, the reduced Pdi during occlusion (∼40% immediately after DNV) was ameliorated to ∼20% reduction after 14 days. Contralateral diaphragm EMG activity did not significantly change immediately or 14days after DNV compared to the pre-injury baseline for any motor behavior. Taken together, these results suggest that over time after DNV compensatory changes in inspiratory related muscle activation may partially restore the ability to generate Pdi during higher force behaviors.

Compensatory effects following unilateral diaphragm paralysis.

Respiratory muscles such as the diaphragm are active across a range of behaviors including ventilation and higher-force behaviors necessary for maintenance of airway patency, and minimal information is available regarding anesthetic effects on the capacity of respiratory muscles to generate higher forces. The purpose of the present study was to determine whether diaphragm EMG activity during lower-force behaviors, such as eupnea and hypoxia-hypercapnia, is differentially affected compared with higher-force behaviors, such as a sigh, in lightly anesthetized animals. In adult male rats, chronically implanted diaphragm EMG electrodes were used to measure the effects of low-dose ketamine (30 mg/kg) and xylazine (3 mg/kg) on root mean square (RMS) EMG amplitude across a range of motor behaviors. A mixed linear model was used to evaluate the effects of ketamine-xylazine anesthesia on peak RMS EMG and ventilatory parameters, with condition (awake vs. anesthetized), behavior (eupnea, hypoxia-hypercapnia, sigh), side (left or right hemidiaphragm), and their interactions as fixed effects and animal as a random effect. Compared with the awake recordings, there was an overall reduction of peak diaphragm RMS EMG across behaviors during anesthesia, but this reduction was more pronounced during spontaneous sighs (which require ~60% of maximal diaphragm force). Respiratory rates and duty cycle during eupnea and hypoxia-hypercapnia were higher in awake compared with anesthetized conditions. These results highlight the importance of identifying anesthetic effects on a range of respiratory motor behaviors, including sighs necessary for maintaining airway patency. NEW & NOTEWORTHY Respiratory muscles accomplish a range of motor behaviors, with forces generated for ventilatory behaviors comprising only a small fraction of their maximal force generating capacity. Induction of anesthesia exerts more robust effects on the higher-force diaphragm motor behaviors such as sighs compared with eupnea. This novel information on effects of low, sedative doses of a commonly used anesthetic combination (ketamine-xylazine) highlights the importance of identifying anesthetic effects on a range of respiratory motor behaviors.

Diaphragm muscle activity across respiratory motor behaviors in awake and lightly anesthetized rats.

The diaphragm muscle (DIAm) is the primary inspiratory muscle in mammals and is active during ventilatory behaviors, but it is also involved in higher-force behaviors such as those necessary for clearing the airway. Our laboratory has previously reported DIAm sarcopenia in rats and mice characterized by DIAm atrophy and a reduction in maximum specific force at 24 months of age. In Fischer 344 rats, these studies were limited to male animals, although in other studies, we noted a more rapid increase in body mass from 6 to 24 months of age in females (~140%) compared to males (~110%). This difference in body weight gain suggests a possible sex difference in the manifestation of sarcopenia. In mice, we previously measured transdiaphragmatic pressure (Pdi) to evaluate in vivo DIAm force generation across a range of motor behaviors, but found no evidence of sex-related differences. The purpose of this study in Fischer 344 rats was to evaluate if there are sex-related differences in DIAm sarcopenia, and if such differences translate to a functional impact on Pdi generation across motor behaviors and maximal Pdi (Pdi ) elicited by bilateral phrenic nerve stimulation. In both males and females, DIAm sarcopenia was apparent in 24-month-old rats with a ~30% reduction in both maximum specific force and the cross-sectional area of type IIx and/or IIb fibers. Importantly, in both males and females, Pdi generated during ventilatory behaviors was unimpaired by sarcopenia, even during more forceful ventilatory efforts induced via airway occlusion. Although ventilatory behaviors were preserved with aging, there was a ~20% reduction in Pdi , which likely impairs the ability of the DIAm to generate higher-force expulsive airway clearance behaviors necessary to maintain airway patency.

Impact of aging on diaphragm muscle function in male and female Fischer 344 rats.

Midcervical spinal cord contusion injury results in tissue damage, disruption of spinal pathways, and motor neuron loss. Unilateral C4 contusion results in loss of 40%-50% of phrenic motor neurons ipsilateral to the injury (~25% of the total phrenic motor neuron pool). Over time after unilateral C4 contusion injury, diaphragm muscle (DIAm) electromyogram activity increases both contralateral and ipsilateral to the side of injury in rats, suggesting compensation because of increased activation of the surviving motor neurons. However, the impact of contusion injury on DIAm force generation is less clear. Transdiaphragmatic pressure (Pdi) was measured across motor behaviors over time after unilateral C4 contusion injury in adult male Sprague-Dawley rats. Maximum Pdi (Pdi) was elicited by bilateral phrenic nerve stimulation at 7 days postinjury. We hypothesized that Pdi is reduced following unilateral C4 contusion injury, whereas ventilatory behaviors of the DIAm are unimpaired. In support of our hypothesis, Pdi was reduced by ~25% after unilateral C4 contusion, consistent with the extent of phrenic motor neuron loss following contusion injury. One day after contusion injury, the Pdi amplitude during airway occlusion was reduced from ~30 to ~20 cmHO, but this reduction was completely reversed by 7 days postinjury. Ventilatory behaviors (~10 cmHO), DIAm-specific force, and muscle fiber cross-sectional area did not differ between the laminectomy and contusion groups. These results indicate that the large reserve capacity for DIAm force generation allows for higher-force motor behaviors to be accomplished despite motor neuron loss, likely reflecting changes in motor unit recruitment. NEW & NOTEWORTHY Respiratory muscles such as the diaphragm generate the pressures necessary to accomplish a variety of motor behaviors ranging from ventilation to near-maximal expulsive behaviors. However, the impact of contusion injury on diaphragm pressure generation across behaviors is not clear. The present study shows that contusion injury impairs maximal pressure generation while preserving the ability of the diaphragm to accomplish lower-force motor behaviors, likely reflecting changes in diaphragm motor unit recruitment.

Diaphragm muscle function following midcervical contusion injury in rats.

The diaphragm muscle (DIAm) is the primary inspiratory muscle in mammals and is highly active throughout life displaying rhythmic activity. The repetitive activation of the DIAm (and of other muscles driven by central pattern generator activity) presents an opportunity to analyze these physiological data on a per-event basis rather than pooled on a per-subject basis. The present study highlights the development and implementation of a graphical user interface-based algorithm using an analysis of critical points to detect the onsets and offsets of individual respiratory events across a range of motor behaviors, thus facilitating analyses of within-subject variability. The algorithm is designed to be robust regardless of the signal type (e.g., EMG or transdiaphragmatic pressure). Our findings suggest that this approach may be particularly beneficial in reducing animal numbers in certain types of studies, for assessments of perturbation studies where the effects are relatively small but potentially physiologically meaningful, and for analyses of respiratory variability.

Automated evaluation of respiratory signals to provide insight into respiratory drive.

The neuromuscular junction (NMJ) mediates neural control of skeletal muscle fibers. Neurotrophic signaling, specifically brain derived neurotrophic factor (BDNF) acting through its high-affinity tropomyosin related kinase B (TrkB) receptor is known to improve neuromuscular transmission. BDNF/TrkB signaling also maintains the integrity of antero- and retrograde communication between the motor neuron soma, its distal axons and pre-synaptic terminals and influences neuromuscular transmission. In this study, we employed a novel rat chemogenetic mutation ( ), in which a 1-naphthylmethyl phosphoprotein phosphatase 1 (1NMPP1) sensitive knock-in allele allowed specific, rapid and sustained inhibition of TrkB kinase activity. In adult female and male   rats, treatment with either 1NMPP1 (TrkB kinase inhibition) or DMSO (vehicle) was administered in drinking water for 14 days. To assess the extent of neuromuscular transmission failure (NMTF), diaphragm muscle isometric force evoked by nerve stimulation at 40 Hz (330 ms duration trains repeated each s) was compared to isometric forces evoked by superimposed direct muscle stimulation (every 15 s). Chronic TrkB kinase inhibition (1NMPP1 group) markedly worsened NMTF compared to vehicle controls. Acute BDNF treatment did not rescue NMTF in the 1NMPP1 group. Chronic TrkB kinase inhibition did not affect the apposition of pre-synaptic terminals (labeled with synaptophysin) and post-synaptic endplates (labeled with α-Bungarotoxin) at diaphragm NMJs. We conclude that inhibition of BDNF/TrkB signaling in   rats disrupts diaphragm neuromuscular transmission in a similar manner to   mice, likely  a pre-synaptic mechanism independent of axonal branch point failure.

Brain derived neurotrophic factor/tropomyosin related kinase B signaling impacts diaphragm neuromuscular transmission in a novel rat chemogenetic model.

All motor commands flow through motoneurons, which entrain control of their innervated muscle fibers, forming a motor unit (MU). Owing to the high fidelity of action potentials within MUs, their discharge profiles detail the organization of ionotropic excitatory/inhibitory as well as metabotropic neuromodulatory commands to motoneurons. Neuromodulatory inputs (e.g. norepinephrine, serotonin) enhance motoneuron excitability and facilitate persistent inward currents (PICs). PICs introduce quantifiable properties in MU discharge profiles by augmenting depolarizing currents upon activation (i.e. PIC amplification) and facilitating discharge at lower levels of excitatory input than required for recruitment (i.e. PIC prolongation).. Here, we introduce a novel geometric approach to estimate neuromodulatory and inhibitory contributions to MU discharge by exploiting discharge non-linearities introduced by PIC amplification during time-varying linear tasks. In specific, we quantify the deviation from linear discharge ('brace height') and the rate of change in discharge (i.e. acceleration slope, attenuation slope, angle). We further characterize these metrics on a simulated motoneuron pool with known excitatory, inhibitory, and neuromodulatory inputs and on human MUs (number of MUs; Tibialis Anterior: 1448, Medial Gastrocnemius: 2100, Soleus: 1062, First Dorsal Interosseus: 2296).. In the simulated motor pool, we found brace height and attenuation slope to consistently indicate changes in neuromodulation and the pattern of inhibition (excitation-inhibition coupling), respectively, whereas the paired MU analysis (Δ) was dependent on both neuromodulation and inhibition pattern. Furthermore, we provide estimates of these metrics in human MUs and show comparable variability in Δand brace height measures for MUs matched across multiple trials.. Spanning both datasets, we found brace height quantification to provide an intuitive method for achieving graded estimates of neuromodulatory and inhibitory drive to individual MUs. This complements common techniques and provides an avenue for decoupling changes in the level of neuromodulatory and pattern of inhibitory motor commands.

A geometric approach to quantifying the neuromodulatory effects of persistent inward currents on individual motor unit discharge patterns.

Brain derived neurotrophic factor (BDNF) signalling through its high-affinity tropomyosin receptor kinase B (TrkB) is known to have potent effects on motor neuron survival and morphology during development and in neurodegenerative diseases. Here, we employed a novel 1NMPP1 sensitive TrkB rat model to evaluate the effect of 14 days inhibition of TrkB signalling on phrenic motor neurons (PhMNs). Adult female and male TrkB rats were divided into 1NMPP1 or vehicle treated groups. Three days prior to treatment, PhMNs in both groups were initially labeled via intrapleural injection of Alexa-Fluor-647 cholera toxin B (CTB). After 11 days of treatment, retrograde axonal uptake/transport was assessed by secondary labeling of PhMNs by intrapleural injection of Alexa-Fluor-488 CTB. After 14 days of treatment, the spinal cord was excised 100 μm thick spinal sections containing PhMNs were imaged using two-channel confocal microscopy. TrkB inhibition reduced the total number of PhMNs by ∼16 %, reduced the mean PhMN somal surface areas by ∼25 %, impaired CTB uptake 2.5-fold and reduced the estimated PhMN dendritic surface area by ∼38 %. We conclude that inhibition of TrkB signalling alone in adult TrkB rats is sufficient to lead to PhMN loss, morphological degeneration and deficits in retrograde axonal uptake/transport.

Chemogenetic inhibition of TrkB signalling reduces phrenic motor neuron survival and size.

Aging results in increased neuromuscular transmission failure and denervation of the diaphragm muscle, as well as decreased force generation across a range of motor behaviors. Increased risk for respiratory complications in old age is a major health problem. Aging impairs autophagy, a tightly regulated multistep process responsible for clearing misfolded or aggregated proteins and damaged organelles. In motor neurons, aging-related autophagy impairment may contribute to deficits in neurotransmission, subsequent muscle atrophy, and loss of muscle force. Chloroquine is commonly used to inhibit autophagy. We hypothesized that chloroquine decreases transdiaphragmatic pressure (Pdi) in mice. Old mice (16-28 mo old;  = 26) were randomly allocated to receive intraperitoneal chloroquine (50 mg/kg) or vehicle 4 h before measuring Pdi during eupnea, hypoxia (10% O)-hypercapnia (5% CO) exposure, spontaneous deep breaths ("sighs"), and maximal activation elicited by bilateral phrenic nerve stimulation (Pdi). Pdi amplitude and ventilatory parameters across experimental groups and behaviors were evaluated using a mixed linear model. There were no differences in Pdi amplitude across treatments during eupnea (∼8 cm HO), hypoxia-hypercapnia (∼10 cm HO), or sigh (∼36 cm HO), consistent with prior studies documenting a lack of aging effects on ventilatory behaviors. In vehicle and chloroquine-treated mice, average Pdi was 61 and 46 cm HO, respectively. Chloroquine decreased Pdi by 24% compared to vehicle ( < 0.05). There were no sex or age effects on Pdi in older mice. The observed decrease in Pdi suggests aging-related susceptibility to impairments in autophagy, consistent with the effects of chloroquine on this important homeostatic process. Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

Chloroquine impairs maximal transdiaphragmatic pressure generation in old mice.

The neuromotor control of the diaphragm muscle (DIAm) is dynamic. The activity of the DIAm can be recorded via electromyography (EMG), which represents the temporal summation of motor unit action potentials. Our goal in the present study was to investigate DIAm neuromotor control during quiet spontaneous breathing (eupnea) in awake rats by evaluating DIAm EMG at specific temporal locations defined by motor unit recruitment and derecruitment. We evaluated the nonstationarity of DIAm EMG activity to identify DIAm motor unit recruitment and derecruitment durations. Combined with assessments of root mean square (RMS) and sum of squares (SS) EMG, the durations of these phases provide physiological information about the temporal aspects of motor control. During eupnea in awake rats ( = 10), the duration of motor unit recruitment comprised 61 ± 19 ms of the onset-to-peak duration (214 ± 62 ms) of the DIAm RMS EMG. The peak-to-offset duration of DIAm EMG activity was 453 ± 96 ms, with a terminating period of derecruitment of 161 ± 44 ms. The burst duration was 673 ± 128 ms. Both the RMS EMG amplitude and the SS EMG were higher at the completion of motor unit recruitment than at the start of motor unit derecruitment, suggesting that offset discharge rates were lower than onset discharge rates. Our analyses provide novel insights into the time domain aspects of DIAm neuromotor control and allow indirect estimates of the contribution of recruitment and frequency to RMS EMG amplitude during eupnea in awake rats. We characterized three phases of neuromotor control-motor unit recruitment, sustained activity, and derecruitment-based on statistical assessments of stationarity of the diaphragm muscle (DIAm) EMG activity in awake rats. Our findings may allow indirect estimates of the contribution of motor unit recruitment and frequency coding toward generating force and provide novel insights about the temporal aspects of DIAm neuromotor control and descending respiratory drive in unanesthetized animals.

Article	Year
A neurochemical closed-loop controller for deep brain stimulation: toward individualized smart neuromodulation therapies. Frontiers in neuroscience\| PubMed ID: 25009455	2014
Compensatory effects following unilateral diaphragm paralysis. Respiratory physiology & neurobiology\| PubMed ID: 28790008	2017
Diaphragm muscle activity across respiratory motor behaviors in awake and lightly anesthetized rats. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 29357493	2018
Impact of aging on diaphragm muscle function in male and female Fischer 344 rats. Physiological reports\| PubMed ID: 29981218	2018
Diaphragm muscle function following midcervical contusion injury in rats. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 30236045	2019
Automated evaluation of respiratory signals to provide insight into respiratory drive. Respiratory physiology & neurobiology\| PubMed ID: 35218924	2022
Brain derived neurotrophic factor/tropomyosin related kinase B signaling impacts diaphragm neuromuscular transmission in a novel rat chemogenetic model. Frontiers in cellular neuroscience\| PubMed ID: 36385943	2022
A geometric approach to quantifying the neuromodulatory effects of persistent inward currents on individual motor unit discharge patterns. Journal of neural engineering\| PubMed ID: 36626825	2023
Chemogenetic inhibition of TrkB signalling reduces phrenic motor neuron survival and size. Molecular and cellular neurosciences\| PubMed ID: 36958643	2023
Chloroquine impairs maximal transdiaphragmatic pressure generation in old mice. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 37823202	2023
Neuromotor control of spontaneous quiet breathing in awake rats evaluated by assessments of diaphragm EMG stationarity. Journal of neurophysiology\| PubMed ID: 37877195	2023

Obaid U. Khurram

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