Sex hormone supplementation improves breathing and restores respiratory neuroplasticity following C2 hemisection in rats.

In addition to loss of sensory and motor function below the level of the lesion, traumatic spinal cord injury (SCI) may reduce circulating steroid hormones that are necessary for maintaining normal physiological function for extended time periods. For men, who comprise nearly 80% of new SCI cases each year, testosterone is the most abundant circulating sex steroid. SCI often results in significantly reduced testosterone production and may result in chronic low testosterone levels. Testosterone plays a role in respiratory function and the expression of respiratory neuroplasticity. When testosterone levels are low, young adult male rats are unable to express phrenic long-term facilitation (pLTF), an inducible form of respiratory neuroplasticity invoked by acute, intermittent hypoxia (AIH). However, testosterone replacement can restore this respiratory neuroplasticity. Complicating the interpretation of this finding is that testosterone may exert its influence in three possible ways: 1) directly through androgen receptor (AR) activation, 2) through conversion to dihydrotestosterone (DHT) by way of the enzyme 5α-reductase, or 3) through conversion to 17ß-estradiol (E2) by way of the enzyme aromatase. DHT signals via AR activation similar to testosterone, but with higher affinity, while E2 activates local estrogen receptors. Evidence to date supports the idea that exogenous testosterone supplementation exerts its influence through estrogen receptor signaling under conditions of low circulating testosterone. Here we explored both recovery of breathing function (measured with whole body barometric plethysmography) and the expression of AIH-induced pLTF in male rats following C2-hemisection SCI. One week post injury, rats were supplemented with either E2 or DHT for 7 days. We hypothesized that E2 would enhance ventilation and reveal pLTF following AIH in SCI rats. To our surprise, though E2 did beneficially impact overall breathing recovery following C2-hemisection, both E2 supplementation and DHT restored the expression of AIH-induced pLTF 2 weeks post-SCI.

Low level CO2 supplementation maintains isocapnia and reveals ventilatory long-term facilitation in rats.

Acute, intermittent hypoxia (AIH) induces ventilatory long-term facilitation (vLTF) in awake, freely behaving rats under poikilocapnic and isocapnic experimental conditions. Establishing pre-clinical methods for vLTF induction that more closely align with successful protocols in humans and anesthetized rats would minimize dissonance in experimental findings and improve translational aspects of vLTF. Here, we tested several levels of low-dose CO2 supplementation during and after AIH to determine 1) the lowest amount of inspired CO2 that would maintain isocapnia in rats during a vLTF protocol, and 2) the net impact of supplemental CO2 on vLTF expression. Rats received one of four levels of inspired CO2 (0%, 0.5%, 1% or 2%) administered during AIH and for the 60 min following AIH to quantify vLTF. Our findings indicated that 2% inspired CO2 was sufficient to maintain isocapnia across the AIH protocol and reveal significant vLTF. These findings provide evidence-based support for using 2% supplemental CO2 during and after AIH when assessing vLTF in rats.

A transcriptome dataset for gonadectomy-induced changes in rat spinal cord.

Circulating sex steroid hormones are critical for neural function and development of neuroplasticity in many regions of the central nervous system. In the spinal cord, our knowledge of steroid hormone influence mostly derives from mechanistic studies of pain processing in dorsal spinal cord circuits; less is known regarding hormonal influence of ventral spinal motor function. Gonadectomy (surgical removal of the testes in males and ovaries in females) rapidly and persistently reduces circulating sex steroids in both females and males, providing a means to interrogate the role of hormones on neural function. Here we provide a next-generation RNA sequencing (RNA-seq) data set to evaluate the impact of gonadectomy on the transcriptome of ventral spinal cord tissue of adult female and male rats.

The long-term impact of ovariectomy on ventilation and expression of phrenic long-term facilitation in female rats.

NEW FINDINGS: What is the central question of this study? Would ovariectomy cause prolonged changes in ventilation and sustained loss of acute, intermittent hypoxia-induced neuroplasticity or would these outcomes be restored with time? What is the main finding and its importance? Our main findings demonstrate that ovariectomy elicits minimal alteration in overall breathing function but impairs acute, intermittent hypoxia-induced plasticity for ≤ 12 weeks. ABSTRACT: Sex hormones are necessary to enable respiratory neuroplasticity, including phrenic long-term facilitation (pLTF), a form of respiratory motor plasticity elicited by acute, intermittent hypoxia (AIH). Female rats exhibit a progressive increase in phrenic nerve amplitude after AIH characteristic of pLTF only during pro-oestrus, the stage of the oestrous cycle notable for elevated circulating oestradiol levels. Removal of the ovaries [ovariectomy (OVX)], the primary source of circulating oestradiol, also eliminates AIH-induced pLTF after 1 week. Ovariectomy is used routinely as a model to examine the impact of sex hormones on CNS structure and function, but the long-term impact of OVX is rarely examined. Extra-ovarian sites of oestradiol synthesis, including multiple CNS sites, have been identified and might possess the capacity to restore oestradiol levels, in part, over time, impacting respiratory function and the expression of respiratory neuroplasticity. We examined both ventilation in awake, freely behaving female rats, using barometric plethysmography, and the expression of AIH-induced pLTF in anaesthetized, ventilated female rats 2 and 12 weeks after OVX and compared them with age-matched ovarian-intact female rats. Our findings indicate that chronic OVX had little impact on baseline breathing or in the response to respiratory challenge (10% O2 , 5% CO2 , balance N2 ) during plethysmography. However, OVX rats at both 2 and 12 weeks demonstrated a persistent loss of AIH-induced pLTF relative to control animals (P < 0.01), suggesting that other sources of oestradiol synthesis were insufficient to restore pLTF. These data are consistent with our previous work indicating that oestradiol plays a key role in expression of AIH-induced respiratory neuroplasticity.

Impact of estrogen deficiency on diaphragm and leg muscle contractile function in female mdx mice.

Female carriers of Duchenne muscular dystrophy (DMD) presenting with DMD symptomology similar to males with DMD, such as skeletal muscle weakness and cardiomyopathy, are termed manifesting carriers. There is phenotypic variability among manifesting carriers including the age of onset, which can range from the first to fourth decade of life. In females, estrogen levels typically begin to decline during the fourth decade of life and estrogen deficiency contributes to loss of muscle strength and recovery of strength following injury. Thus, we questioned whether the decline of estrogen impacts the development of DMD symptoms in females. To address this question, we studied 6-8 month-old homozygous mdx female mice randomly assigned to a sham or ovariectomy (OVX) surgical group. In vivo whole-body plethysmography assessed ventilatory function and diaphragm muscle strength was measured in vitro before and after fatigue. Anterior crural muscles were analyzed in vivo for contractile function, fatigue, and in response to eccentric contraction (ECC)-induced injury. For the latter, 50 maximal ECCs were performed by the anterior crural muscles to induce injury. Body mass, uterine mass, hypoxia-hypercapnia ventilatory response, and fatigue index were analyzed by a pooled unpaired t-test. A two-way ANOVA was used to analyze ventilatory measurements. Fatigue and ECC-injury recovery experiments were analyzed by a two-way repeated-measures ANOVA. Results show no differences between sham and OVX mdx mice in ventilatory function, strength, or recovery of strength after fatigue in the diaphragm muscle or anterior crural muscles (p ≥ 0.078). However, OVX mice had significantly greater eccentric torque loss and blunted recovery of strength after ECC-induced injury compared to sham mice (p ≤ 0.019). Although the results show that loss of estrogen has minimal impact on skeletal muscle contractile function in female mdx mice, a key finding suggests that estrogen is important in muscle recovery in female mdx mice after injury.

Development of ventilatory long-term facilitation is dependent on estrous cycle stage in adult female rats.

Ventilatory long-term facilitation (vLTF) is a form of respiratory plasticity characterized by a progressive and sustained increase in minute ventilation over time following acute, intermittent hypoxia (AIH). Though vLTF has been repeatedly demonstrated in adult males (rats and humans), few studies have assessed vLTF in adult females and no studies have explored differential expression of vLTF across the normal female estrous cycle. We recently reported that AIH-induced plasticity of phrenic motor output (phrenic long-term facilitation, pLTF), a phenotypically similar form of respiratory plasticity presenting as a sustained increase in phrenic nerve amplitude, develops in adult female rats only during the proestrus stage of the estrous cycle, notable for high levels of serum estrogen. Here, we tested the hypothesis that AIH-induced vLTF would also be estrous-stage dependent; developing in female rats during proestrus, but not estrus. Barometric plethysmography in adult (4-5 months), normally cycling female rats revealed a progressive increase in minute ventilation for 60 min following AIH (5 × 5 min episodes; 10% O2) during proestrus indicative of vLTF, while estrus rats showed no changes in minute ventilation over the same time period. The development of vLTF in proestrus rats was driven by changes in tidal volume production versus respiratory frequency consistent with prior studies. These data are the first to investigate differential vLTF expression across the estrous cycle in adult female rats and highlight the importance of female estrous cycle stage as a critical physiological variable to consider in studies of AIH-induced plasticity.

Pediatric Referrals to an Emergency Department From Urgent Care Centers.

OBJECTIVE: The aims of this study were to describe pediatric emergency department (ED) referrals from urgent care centers and to determine the percentage of referrals considered essential and serious. METHODS: A prospective study was conducted between April 2013 and April 2015 on patients younger than 21 years referred directly to an ED in central Pennsylvania from surrounding urgent care centers. Referrals were considered essential or serious based on investigations/procedures performed or medications/consultations received in the ED. RESULTS: Analysis was performed on 455 patient encounters (mean age, 8.7 y), with 347 (76%) considered essential and 40 (9%) considered serious. The most common chief complaints were abdominal pain (83 encounters), extremity injury (76), fever (39), cough/cold (29), and head/neck injury (29). Thirty-three percent of the patients received laboratory diagnostic investigations (74% serum, 56% urine), and 52% received radiologic investigations (67% x-ray, 17% computed tomography scan, 13% ultrasound, 11% magnetic resonance imaging). Forty-four percent of the patients received a procedure, with the most common being intravenous (IV) placement (66%); reduction, casting, or splinting of extremity fracture/dislocation (18%); and laceration repair (14%). The most common medications administered were IV fluids (33%), oral analgesics (30%), and IV analgesics (26%). Eighty-three percent of the patients were discharged home, 12% were hospitalized, and 4% had emergent surgical intervention. The most common primary diagnoses were closed extremity fracture (60 encounters), gastroenteritis (42), brain concussion (28), upper respiratory infection (24), and nonsurgical, unspecified abdominal pain (24). CONCLUSIONS: Many ED referrals directed from urgent care centers in our sample were considered essential, and few were considered serious. Urgent care centers should develop educational and preparedness strategies based on the epidemiology of emergencies that may occur.

Cyclooxygenase enzyme activity does not impair respiratory motor plasticity after one night of intermittent hypoxia.

Although inflammation is prevalent in many clinical disorders challenging breathing, we are only beginning to understand the impact of inflammation on neural mechanisms of respiratory control. We recently demonstrated one form of respiratory motor plasticity is extremely sensitive to even mild inflammation induced by a single night (8 h) of intermittent hypoxia (IH-1), mimicking aspects of obstructive sleep apnea. Specifically, phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (AIH) is abolished by IH-1, but restored by high doses of the non-steroidal anti-inflammatory drug, ketoprofen. Since a major target of ketoprofen is cyclooxygenase (COX) enzymes, we tested the involvement of COX in IH-1 suppression of pLTF using the selective COX inhibitor NS-398. Systemic COX inhibition (3 mg/kg, i.p., 3 h before AIH) had no effect on pLTF in normoxia treated rats (76 ±â€¯40% change from baseline, n = 6), and did not restore pLTF in IH-1 treated rats (-9 ±â€¯7% baseline, n = 6). Similarly, spinal COX inhibition (27 mM, 12 µl, i.t.) had no effect on pLTF in normoxic rats (76 ±â€¯34% baseline, n = 7), and did not significantly restore pLTF after IH-1 (37 ±â€¯18% baseline, n = 7). COX-2 protein is expressed in identified phrenic motor neurons of both normoxia and IH-1 exposed rats, but immunolabeling was minimal in surrounding microglia; IH-1 had no discernable effect on COX-2 immunoreactivity. We conclude that the inflammatory impairment of pLTF by IH-1 is independent of COX enzyme activity or upregulated COX-2 expression.

Nongenomic Actions of 17-ß Estradiol Restore Respiratory Neuroplasticity in Young Ovariectomized Female Rats.

Gonadal steroids modulate CNS plasticity, including phrenic long-term facilitation (pLTF), a form of spinal respiratory neuroplasticity resulting in increased phrenic nerve motor output following exposure to acute intermittent hypoxia (aIH; three 5 min episodes, 10.5% O2). Despite the importance of respiratory system neuroplasticity, and its dependence on estrogen in males, little is known about pLTF expression or mechanisms of estrogen signaling in females. Here, we tested the hypotheses that (1) pLTF expression in young, gonadally intact female rats would be expressed during estrous cycle stages in which 17ß-estradiol (E2) is naturally high (e.g., proestrus vs estrus), (2) pLTF would be absent in ovariectomized (OVX) rats and in physiological conditions in which serum progesterone, but not E2, is elevated (e.g., lactating rats, 3-10 d postpartum), and (3) acute E2 administration would be sufficient to restore pLTF in OVX rats. Recordings of phrenic nerve activity in female Sprague Dawley rats (3-4 months) revealed a direct correlation between serum E2 levels and pLTF expression in cycling female rats. pLTF was abolished with OVX, but was re-established by acute E2 replacement (3 h, intraperitoneal). To identify underlying E2 signaling mechanisms, we intrathecally applied BSA-conjugated E2 over the spinal phrenic motor nucleus and found that pLTF expression was restored within 15 min, suggesting nongenomic E2 effects at membrane estrogen receptors. These data are the first to investigate the role of ovarian E2 in young cycling females, and to identify a role for nongenomic estrogen signaling in any form of respiratory system neuroplasticity.SIGNIFICANCE STATEMENT Exposure to acute intermittent hypoxia induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity that improves breathing in models of spinal cord injury. Although pathways leading to pLTF are well studied in males and estradiol (E2) is known to be required, it has seldom been investigated in females, and underlying mechanisms of E2 signaling are unknown in either sex. We found that while ovariectomy abolished pLTF, it could be restored by acute systemic E2, or by intraspinal application of the membrane-impermeable E2 (BSA-conjugated E2; 15 min). The ability of nongenomic estrogen signaling within the cervical spinal cord to recover respiratory neuroplasticity in disorders of respiratory insufficiency suggests that membrane estrogen receptors may represent novel therapeutic targets to restore breathing in both sexes.

Mammalian target of rapamycin is required for phrenic long-term facilitation following severe but not moderate acute intermittent hypoxia.

Phrenic long-term facilitation (pLTF) is a persistent increase in phrenic nerve activity after acute intermittent hypoxia (AIH). Distinct cell-signaling cascades give rise to pLTF depending on the severity of hypoxemia within hypoxic episodes. Moderate AIH (mAIH; three 5-min episodes, PaO2 ∼35-55 mmHG) elicits pLTF by a serotonin (5-HT)-dependent mechanism that requires new synthesis of brain-derived neurotrophic factor (BDNF), activation of its high-affinity receptor (TrkB), and ERK MAPK signaling. In contrast, severe AIH (sAIH; three 5-min episodes, PaO2 ∼25-30 mmHG) elicits pLTF by an adenosine-dependent mechanism that requires new TrkB synthesis and Akt signaling. Although both mechanisms require spinal protein synthesis, the newly synthesized proteins are distinct, as are the neurochemicals inducing plasticity (serotonin vs. adenosine). In many forms of neuroplasticity, new protein synthesis requires translational regulation via mammalian target of rapamycin (mTOR) signaling. Since Akt regulates mTOR activity, we hypothesized that mTOR activity is necessary for sAIH- but not mAIH-induced pLTF. Phrenic nerve activity in anesthetized, paralyzed, and ventilated rats was recorded before, during, and 60 min after mAIH or sAIH. Rats were pretreated with intrathecal injections of 20% DMSO (vehicle controls) or rapamycin (0.1 mM, 12 µl), a selective mTOR complex 1 inhibitor. Consistent with our hypothesis, rapamycin blocked sAIH- but not mAIH-induced pLTF. Thus spinal mTOR activity is required for adenosine-dependent (sAIH) but not serotonin-dependent (mAIH) pLTF, suggesting that distinct mechanisms regulate new protein synthesis in these forms of spinal neuroplasticity.

Hypoxia triggers short term potentiation of phrenic motoneuron discharge after chronic cervical spinal cord injury.

Repeated exposure to hypoxia can induce spinal neuroplasticity as well as respiratory and somatic motor recovery after spinal cord injury (SCI). The purpose of the present study was twofold: to define the capacity for a single bout of hypoxia to trigger short-term plasticity in phrenic output after cervical SCI and to determine the phrenic motoneuron (PhrMN) bursting and recruitment patterns underlying the response. Hypoxia-induced short term potentiation (STP) of phrenic motor output was quantified in anesthetized rats 11 weeks following lateral spinal cord hemisection at C2 (C2Hx). A 3-min hypoxic episode (12-14% O2) always triggered STP of inspiratory burst amplitude, the magnitude of which was greater in phrenic bursting ipsilateral vs. contralateral to C2Hx. We next determined if STP could be evoked in recruited (silent) PhrMNs ipsilateral to C2Hx. Individual PhrMN action potentials were recorded during and following hypoxia using a "single fiber" approach. STP of bursting activity did not occur in cells initiating bursting at inspiratory onset, but was robust in recruited PhrMNs as well as previously active cells initiating bursting later in the inspiratory effort. We conclude that following chronic C2Hx, a single bout of hypoxia triggers recruitment of PhrMNs in the ipsilateral spinal cord with bursting that persists beyond the hypoxic exposure. The results provide further support for the use of short bouts of hypoxia as a neurorehabilitative training modality following SCI.

Intraspinal transplantation and modulation of donor neuron electrophysiological activity.

Rat fetal spinal cord (FSC) tissue, naturally enriched with interneuronal progenitors, was introduced into high cervical, hemi-resection (Hx) lesions. Electrophysiological analyses were conducted to determine if such grafts exhibit physiologically-patterned neuronal activity and if stimuli which increase respiratory motor output also alter donor neuron bursting. Three months following transplantation, the bursting activity of FSC neurons and the contralateral phrenic nerve were recorded in anesthetized rats during a normoxic baseline period and brief respiratory challenges. Spontaneous neuronal activity was detected in 80% of the FSC transplants, and autocorrelation of action potential spikes revealed distinct correlogram peaks in 87% of neurons. At baseline, the average discharge frequency of graft neurons was 13.0 ± 1.7 Hz, and discharge frequency increased during a hypoxic respiratory challenge (p<0.001). Parallel studies in unanesthetized rats showed that FSC tissue recipients had larger inspiratory tidal volumes during brief hypoxic exposures (p<0.05 vs. C2Hx rats). Anatomical connectivity was explored in additional graft recipients by injecting a transsynaptic retrograde viral tracer (pseudorabies virus, PRV) directly into matured transplants. Neuronal labeling occurred throughout graft tissues and also in the host spinal cord and brainstem nuclei, including those associated with respiratory control. These results underscore the neuroplastic potential of host-graft interactions and training approaches to enhance functional integration within targeted spinal circuitry.

Phrenic motoneuron discharge patterns following chronic cervical spinal cord injury.

Cervical spinal cord injury (SCI) dramatically disrupts synaptic inputs and triggers biochemical, as well as morphological, plasticity in relation to the phrenic motor neuron (PhMN) pool. Accordingly, our primary purpose was to determine if chronic SCI induces fundamental changes in the recruitment profile and discharge patterns of PhMNs. Individual PhMN action potentials were recorded from the phrenic nerve ipsilateral to lateral cervical (C2) hemisection injury (C2Hx) in anesthetized adult male rats at 2, 4 or 8 wks post-injury and in uninjured controls. PhMNs were phenotypically classified as early (Early-I) or late inspiratory (Late-I), or silent according to discharge patterns. Following C2Hx, the distribution of PhMNs was dominated by Late-I and silent cells. Late-I burst parameters (e.g., spikes per breath, burst frequency and duration) were initially reduced but returned towards control values by 8wks post-injury. In addition, a unique PhMN burst pattern emerged after C2Hx in which Early-I cells burst tonically during hypocapnic inspiratory apnea. We also quantified the impact of gradual reductions in end-tidal CO2 partial pressure (PETCO2) on bilateral phrenic nerve activity. Compared to control rats, as PETCO2 declined, the C2Hx animals had greater inspiratory frequencies (breaths∗min(-1)) and more substantial decreases in ipsilateral phrenic burst amplitude. We conclude that the primary physiological impact of C2Hx on ipsilateral PhMN burst patterns is a persistent delay in burst onset, transient reductions in burst frequency, and the emergence of tonic burst patterns. The inspiratory frequency data suggest that plasticity in brainstem networks is likely to play an important role in phrenic motor output after cervical SCI.

Contribution of the spontaneous crossed-phrenic phenomenon to inspiratory tidal volume in spontaneously breathing rats.

Spinal cord hemisection at C2 (C2HS) severs bulbospinal inputs to ipsilateral phrenic motoneurons causing transient hemidiaphragm paralysis. The spontaneous crossed-phrenic phenomenon (sCPP) describes the spontaneous recovery of ipsilateral phrenic bursting following C2HS. We reasoned that the immediate (next breath) changes in tidal volume (V(T)) induced by ipsilateral phrenicotomy during spontaneous breathing would provide a quantitative measure of the contribution of the sCPP to postinjury V(T). Using this approach, we tested the hypothesis that the sCPP makes more substantial contributions to V(T) when respiratory drive is increased. Pneumotachography was used to measure V(T) in anesthetized, spontaneously breathing adult male rats at intervals following C2HS. A progressive increase in V(T) (ml/breath) occurred over an 8 wk period following C2HS during both poikilocapnic baseline breathing and hypercapnic respiratory challenge (7% inspired CO(2)). The sCPP did not impact baseline breathing at 1-3 days postinjury since V(T) was unchanged after ipsilateral phrenicotomy. However, by 2 wk post-C2HS, baseline phrenicotomy caused a 16 ± 2% decline in V(T); a comparable 16 ± 4% decline occurred at 8 wk. Contrary to our hypothesis, the phrenicotomy-induced declines in V(T) (%) during hypercapnic respiratory stimulation did not differ from the baseline response at any postinjury time point (all P > 0.11). We conclude that by 2 wk post-C2HS the sCPP makes a meaningful contribution to V(T) that is similar across different levels of respiratory drive.

Influence of vagal afferents on supraspinal and spinal respiratory activity following cervical spinal cord injury in rats.

C(2) spinal hemisection (C2HS) interrupts ipsilateral bulbospinal pathways and induces compensatory increases in contralateral spinal and possibly supraspinal respiratory output. Our first purpose was to test the hypothesis that after C2HS contralateral respiratory motor outputs become resistant to vagal inhibitory inputs associated with lung inflation. Bilateral phrenic and contralateral hypoglossal (XII) neurograms were recorded in anesthetized and ventilated rats. In uninjured (control) rats, lung inflation induced by positive end-expired pressure (PEEP; 3-9 cmH(2)O) robustly inhibited both phrenic and XII bursting. At 2 wk post-C2HS, PEEP evoked a complex response associated with phrenic bursts of both reduced and augmented amplitude, but with no overall change in the mean burst amplitude. PEEP-induced inhibition of XII bursting was still present but was attenuated relative to controls. However, by 8 wk post-C2HS PEEP-induced inhibition of both phrenic and XII output were similar to that in controls. Our second purpose was to test the hypothesis that vagal afferents inhibit ipsilateral phrenic bursting, thereby limiting the incidence of the spontaneous crossed phrenic phenomenon in vagal-intact rats. Bilateral vagotomy greatly enhanced ipsilateral phrenic bursting, which was either weak or absent in vagal-intact rats at both 2 and 8 wk post-C2HS. We conclude that 1) compensatory increases in contralateral phrenic and XII output after C2HS blunt the inhibitory influence of vagal afferents during lung inflation and 2) vagal afferents robustly inhibit ipsilateral phrenic bursting. These vagotomy data appear to explain the variability in the literature regarding the onset of the spontaneous crossed phrenic phenomenon in spontaneously breathing (vagal intact) vs. ventilated (vagotomized) preparations.