Sieck, Gary C.

Acute administration of estrogen induces vasodilation in several vascular beds including coronary artery smooth muscle (CSM). We tested the hypothesis that the response of CSM to estrogen is mediated via the binding of estrogen to estrogen receptors (ER) and that the response will be influenced by hormonal status. Coronary smooth muscle was prepared from gonadally intact (INT) and. ovariectomized (OVX) female pigs. Immunocytochemistry and receptor binding assays were performed to confirm presence vs. absence of ER. In INT CSM cells, but not OVX CSM, acute administration of 17β‐estradiol resulted in a concentration‐dependent decrease in [Ca 2+ ] i responses to endothelin‐1. [Ca 2+ ] i responses to 17β‐estradiol were comparable between groups. The effect of 17β‐estradiol was inhibited by the ER antagonist ICI 182,780. 17β‐estradiol decreased [Ca 2+ ] i in INT (but not OVX) CSM cells when sarcoplasmic reticulum (SR) Ca 2+ reuptake was inhibited, but to a lesser extent than when extracellular Ca 2+ was present. This effect was abolished by extracellular lanthanum. These data suggest that 17β‐estradiol modulates [Ca 2+ ] i in CSM via a likely non‐genomic and necessary ER signaling cascade that mediates both plasma membrane Ca 2+ influx and efflux, but does not involve SR Ca 2+ release. Supported by NIH grants UL1RR024150‐01(Mayo Clinic CTSA awards to YSP & CMP) and HL74309 (GCS)

Diving under cold conditions may add an additional risk for bubble production and decompression sickness. Additionally, there is sparse information regarding hemodynamic changes occurring during a dive and its effects on the outcome of a dive. Thus, detailed recordings of cardiovascular changes at different core temperatures during diving were studied. Anesthetized rats were monitored with a pressure‐volume catheter in the left ventricle and the femoral artery. Rats were randomly assigned to be normothermic controls, to perform a dry hyberbaric dive at constant core temperature (37ºC) or a dry hyperbaric dive with a core temperature of 35ºC during decompression and the early post dive period. The rats were compressed to 600 kPa, maintained for 45 min breathing air, and decompressed linearly to the surface at a rate of 50 kPa·min −1 . Immediately after surfacing, right ventricle and pulmonary artery were insonated for bubble detection using ultrasound. Compression increased mean arterial pressure, pulse pressure, stroke volume, cardiac output, LV end‐systolic volume, LV end‐systolic pressure, LV dp/dt max, and total peripheral resistance in the rats. Most of these changes gradually declined and returned to baseline after decompression. However, rewarming to 37ºC after a 2ºC drop in core temperature caused a significant reduction in SV and CO, but this did not have an effect on bubble production and survival.

Upper cervical spinal cord injury disrupts descending inspiratory‐related drive to phrenic motoneurons (PMNs) paralyzing the diaphragm muscle (DIAm). There is gradual recovery of rhythmic DIAm activity ipsilateral to injury suggesting strengthened spared synaptic inputs to PMNs. We previously showed that intrathecal treatment with brain‐derived neurotrophic factor (BDNF) enhances recovery of rhythmic DIAm activity after C2 spinal cord hemisection (C2SH). We hypothesized that TrkB signaling in PMN is necessary for functional recovery post‐C2SH. Using a recently validated intrapleural technique, daily injections of TrkB siRNA were administered to adult male Sprague‐Dawley rats. Bilateral DIAm EMG activity was chronically recorded before and after C2SH to assess recovery of rhythmic DIAm activity. In the vehicle‐treated group, functional recovery was evident in ~10% of C2SH rats at 7D and ~40% of rats at 14D post‐injury. In contrast, none of the TrkB siRNA treated rats displayed functional recovery up to 14D post‐C2SH. Transport of intrapleural Cy3‐labeled siRNA was verified in retrogradely‐labeled phrenic motoneurons. Our results indicate that BDNF/TrkB signaling at PMNs is necessary for functional recovery of rhythmic DIAm activity after C2SH. Supported by NIH grant R01 HL096750 and Mayo Clinic

Following spinal cord hemisection at C2 (C2SH), removal of descending excitatory drive to phrenic motoneurons results in ipsilateral diaphragm muscle (DIAm) paralysis. Over time, there is partial recovery of rhythmic phrenic activity post‐C2SH. Intrathecal delivery of brain‐derived neurotrophic factor (BDNF) enhances this functional recovery, with 100% of rats displaying rhythmic phrenic activity at 14 days (14D) post‐C2SH; but in humans intrathecal BDNF is associated with adverse effects that limit application. Stem cell transplantation may facilitate a targeted BDNF delivery and minimize side effects. In adult rats, bilateral DIAm EMG activity was chronically monitored before and after C2SH to assess recovery. Wild type, GFP‐expressing bone‐marrow derived mesenchymal stem cells (MSC‐wt) or BDNF‐GFP expressing MSC (MSC‐BDNF) were injected at C2. Seventy % of MSC‐BDNF treated rats displayed functional recovery at 7D and 100% at 14D post‐C2SH. Functional recovery in BDNF‐wt treated C2SH rats was not different from untreated C2SH controls (~10% at 7D and ~40% at 14D). Cellular incorporation of MSC into the spinal cord was evident by GFP immunoreactivity in both MSC groups. We conclude that targeted MSC‐based delivery of BDNF enhances functional recovery of DIAm respiratory activity following an upper cervical spinal cord injury. Supported by NIH grant HL096750 and Mayo Clinic.

In previous studies, we showed that the root mean square of DIAm EMG correlates with transdiaphragmatic pressure (Pdi) reflecting force generation. We also showed that there is a shift in the power spectral density towards higher frequencies with recruitment of more fatigable fast‐twitch motor units. Recruitment of motor units leads to summation of motor unit action potentials (MUAP) causing non‐stationarity of the mean square value. Accordingly, non‐stationarity of EMG reflects motor unit recruitment. In the present study, we assessed stationarity of DIAm EMG across different ventilator and non‐ventilatory behaviors using a reverse arrangement test for 10‐ms binned mean square values. Stationarity was confirmed if 80% of the mean square calculations were within 2 standard deviations. In adult male rats, EMG recordings were obtained during eupnea, hypoxia‐hypercapnia (5% CO 2 /10% O 2 ), airway occlusion, and sneezing. At the onset of DIAm activity for each behavior, the EMG was stationary for ~75 ms. The EMG was also stationary for ~150 ms around the peak RMS for all motor behaviors. However, the period of non‐stationarity of DIAm EMG varied across motor behaviors: eupnea and hypoxia‐hypercapnia: 50 ms; airway occlusion: 240 ms; sneezing: 500 ms). Therefore, the duration of non‐stationarity of DIAm EMG increased with increasing force output; thus, reflecting the extent of motor unit recruitment.

Unilateral C2 spinal cord hemisection (C2SH) removes premotor drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle (DIAm). In previous studies, we found that intrathecal brain‐derived neurotrophic factor (BDNF) increases the proportion of rats displaying recovery of ipsilateral DIAm activity over time. We hypothesized that enhancing expression of the tropomyosin‐related kinase receptor subtype B (TrkB) in phrenic motoneurons will promote functional recovery ipsilateral to C2SH. Adult rats were injected intrapleurally with AAV7 expressing TrkB.FL‐FLAG, and 3 weeks later C2SH was performed. A separate group of C2SH animals served as controls. Bilateral DIAm EMG activity was chronically monitored to assess recovery up to 2 weeks after C2SH. In AAV‐treated rats, delivery of TrkB.FL‐FLAG to phrenic motoneurons was evident by the presence of FLAG protein in the phrenic motor nucleus. At 7‐days post‐C2SH, the proportion of rats displaying ipsilateral DIAm EMG activity was 13% and 40% in the untreated control and AAV‐treated groups, respectively. At 14‐days post‐C2SH, all AAV‐treated rats displayed functional recovery compared to 40% of C2SH rats. We conclude that targeted delivery of TrkB.FL to phrenic motoneurons enhances functional recovery of DIAm respiratory activity following upper cervical spinal cord injury. Supported by NIH grant HL96750 and Paralyzed Veterans of America.

Aging is associated with respiratory complications and muscle dysfunction that may reduce the ability to perform expulsive, non‐ventilatory behaviors. We hypothesized that ventilatory function is impaired with aging and that the diaphragm muscle (DIAm) is susceptible to sarcopenia. Unrestrained‐awake ventilatory function, DIAm specific force, and fiber cross‐sectional area (CSA) were examined in a validated mouse model of advanced aging (expression of a hypomorphic allele ( H ) of BubR1 ), naturally aged (28 mo) and adult (5 mo) wild type ( wt ) mice (same genetic background). Minute ventilation and breathing frequency were decreased 17% in BubR1 H/H and 33% in aged wt mice vs. adult wt mice. Tidal volume was not different across groups. DIAm specific force was lower in both aged mice groups compared to the adult wt mice, being 62% lower in BubR1 H/H mice. DIAm fiber CSA from BubR1 H/H and aged wt mice was smaller than adult wt mice (23 and 10%, respectively). These results indicate that aging is associated with reduced ventilatory function and evidence of DIAm sarcopenia. In addition, aged mice recruit a greater percentage of DIAm fibers for eupneic breathing (greater fraction of maximum force). Together, these age‐related changes may limit the ability to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance, and thus contribute to respiratory complications. Supported by Mayo Clinic.

Brain‐derived neurotrophic factor (BDNF) acting through full‐length tropomyosin related kinase receptor subtype B (TrkB.FL) plays an important role in neuroplasticity following spinal cord injury. BDNF/TrkB.FL signaling in phrenic motoneurons is both necessary and sufficient to enhance spontaneous recovery of ipsilateral rhythmic phrenic activity after C2 spinal cord hemisection (SH), where premotor drive to phrenic motoneurons is removed. Phrenic motoneuron expression of the excitatory glutamatergic NMDA receptor correlates with the time course of spontaneous functional recovery post‐SH. We hypothesized that increased BDNF/TrkB.FL signaling in phrenic motoneurons enhances recovery of rhythmic phrenic activity after SH via regulation of glutamatergic receptor expression. Rats were injected intrapleurally with AAV7 expressing human TrkB.FL‐FLAG at 3 weeks prior to SH. FLAG was present in the cervical ventral horn region, and AAV7‐GFP expression was specifically in phrenic motoneurons. At 14 days post‐SH, quantitative real‐time RT‐PCR verified human TrkB.FL expression in microdissected phrenic motoneurons. Phrenic motoneuron expression of NMDA increased ~10 fold compared to control, suggesting that increased BDNF/TrkB.FL signaling magnified SH‐induced changes in phrenic motoneuron NMDA expression. Supported by HL96750 and T32‐HL105355, and Paralyzed Veterans of America.

Respiratory excitatory premotor drive from the medulla is widely distributed to phrenic motor neurons, and it is generally assumed that motor neuron recruitment order is determined by intrinsic, size‐related electrophysiological properties (Size Principle). During ventilatory behaviors, fatigue‐resistant diaphragm muscle (DIAm) motor units (smaller motor neurons) are recruited, but during more forceful non‐ventilatory behaviors such as airway occlusion, deep sighs and sneezing, additional more fatigable motor units are recruited. We hypothesized that a fixed recruitment order of DIAm motor units is maintained across different ventilatory and non‐ventilatory behaviors. Unitary motor unit discharge was recorded across eupnea, hypoxia‐hypercapnia (10% O 2 –5% CO 2 ), airway occlusion and deep sighs. The recruitment order displayed by motor units during eupnea and hypoxia‐hypercapnia was conserved during more forceful behaviors (82% of pairs maintained their recruitment order). Reversals of motor unit recruitment order occurred predominantly when the difference in onset times were within 20 ms. Onset discharge rates of motor units recruited during eupnea and hypoxia‐hypercapnia were lower than those of units recruited only during airway occlusion or deep sighs. These results support the Size Principle as an organizing principle of DIAm motor unit recruitment across a range of motor behaviors.

The diaphragm muscle (DIAm) is responsible for breathing and must provide sufficient force to accomplish both ventilatory and non‐ventilatory behaviors. Age‐related changes to the DIAm, especially sarcopenia, may limit the ability to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance, and thus contribute to respiratory complications. Age‐related changes in load (as a result of chest wall stiffening or lung compliance) may impact DIAm function. Adult male mice (6 and 24 months old) underwent measurements of respiratory system compliance and transdiaphragmatic pressure (Pdi) during: eupnea, hypoxia (10% O 2 ) –hypercapnia (5% CO 2 ), spontaneously occurring deep breaths, airway occlusion, and bilateral phrenic nerve stimulation. Overall respiratory system compliance decreased with age (increased load), while Pdi required for ventilatory behaviors did not change, consistent with the reserve capacity of the DIAm. The Pdi generated during deep breaths and airway occlusion were reduced 30–45% with age. These results indicate that DIAm dysfunction during aging is associated with reduced force to perform expulsive, non‐ventilatory behaviors. Reduced reserve capacity in old age may underlie increased susceptibility to lung infections and augment respiratory compromise associated with other age‐related diseases. Supported by T32‐HL‐105355 and the Mayo Clinic.