Respiratory Motor Plasticity Research Articles

Aging Impairs Phrenic Long‐Term Facilitation in Rats by an Adenosine‐Dependent Mechanism

Phrenic long‐term facilitation (pLTF) is a form of respiratory plasticity elicited by acute intermittent hypoxia (AIH). AIH activates competing intracellular signaling cascades, initiated by phrenic motor neuron serotonin 2 and adenosine 2A (A2A) receptors, respectively. With moderate AIH (mAIH), the serotonin‐dependent Q pathway to phrenic motor facilitation dominates, with an adenosine constraint. Consequently, spinal A2Areceptor inhibition enhances mAIH‐induced pLTF. In middle‐aged male, and geriatric acyclic female rats, pLTF is attenuated through unknown mechanisms. We hypothesize that basal spinal adenosine levels are elevated in aged rats, thereby undermining mAIH‐induced pLTF. Thus, we predict that spinal A2A receptor inhibition in aged male rats will restore pLTF to levels equal to young male rats with A2A receptor inhibition. A selective A2A receptor antagonist (MSX‐3; 10 uM, 12 uL) or vehicle (0.9% sterile saline) were delivered intrathecally (C4) in anesthetized, vagotomized, paralyzed and ventilated young (3‐6 months) and aged (20‐22 months) male Sprague‐Dawley rats prior to mAIH (3, 5 min hypoxic episodes; arterial Po2= 45‐55 mmHg). In young males, mAIH elicits ~ 60% pLTF at 60 min post‐AIH. Spinal A2A receptor inhibition significantly enhanced mAIH‐induced pLTF in young males (119 + 5%; n = 6) vs vehicle controls (55 + 9%; n = 6; p < 0.001) and time controls (12 + 4%; n = 6; p < 0.001). In aged males, A2A receptor inhibition enhanced mAIH‐induced pLTF to a greater extent (186 + 19%; n = 7) vs vehicle controls (25 + 11%; n = 7; p < 0.001) and time controls (3 + 5%; n = 4; p < 0.001). Measured basal adenosine levels in ventral C3‐C5 homogenates were significantly elevated in aged vs young males (p = 0.008). Preliminary data suggest aged female rats (16‐18 months) also have enhanced mAIH‐induced pLTF with A2A receptor inhibition (173+ 24%; n = 3) vs vehicle controls (53 + 7%; n = 3) and time controls (5 + 17%; n = 2) in persistent estrus. These findings advance our understanding of mechanisms whereby age undermines mAIH‐induced phrenic motor plasticity. Understanding age and sex effects on AIH‐induced respiratory motor plasticity is crucial as we harness therapeutic AIH to treat severe neuromuscular disorders that compromise breathing.

Acute Intermittent Hypoxia Preconditioning Elicits Age and Sex‐Dependent Changes in Molecules Known to Regulate Phrenic Motor Plasticity

Acute intermittent hypoxia (AIH) elicits a form of respiratory motor plasticity known as phrenic long‐term facilitation (pLTF); preconditioning with daily AIH (dAIH) enhances pLTF, demonstrating metaplasticity. Two additional factors impacting pLTF expression are age and sex: whereas moderate AIH‐induced pLTF decreases with age in males, it increases in middle aged females. Unfortunately, little is known concerning mechanisms of dAIH preconditioning or age‐dependent sexual dimorphism in pLTF. Moderate AIH elicits 2 distinct signaling cascades in phrenic motor neurons initiated by 5HT2A/2B receptors (Q pathway) versus adenosine 2A or 5HT7 receptors (S pathway), respectively. The Q and S pathways interact via mutual cross‐talk inhibition, a powerful regulator of pLTF expression. To begin exploring the hypotheses that dAIH preconditioning and age/sex effects on pLTF can be accounted for by shifts in molecules regulating the Q and S pathways, we assessed mRNA of key molecules known to regulate pLTF in ventral cervical spinal homogenates from segments containing the phrenic motor nucleus from young (3 month) and middle‐aged (12 month) male and female Sprague‐Dawley rats. Rats (n=8/group) were exposed to sham or dAIH for 14 days (15, 1 min hypoxic episodes/day), and sacrificed 24 hours after the last AIH exposure. Tissues were harvested, preserved in RNA stabilizing solution (<10 min), and stored (‐80°C) until mRNA extraction. mRNA levels of molecules that regulate pLTF were assessed via RT PCR, including: serotonin 2A (Htr2a) & 2B (Htr2b); adenosine 2A (Adora2a); exchange protein activated by cAMP (Epac); PKA catalytic subunit (Prkaa1); PKA regulatory subunit (Prkar1a); fractalkine (Cx3cl1); phosphodiesterase type 4 (Pde4b); Gp91 and p47(NOX genes); and the Pkcδisoform. We also quantified protein expression in phrenic motor neurons (via immunofluorescence) for BDNF and adenosine‐1A & ‐2A receptors in rats fixed with 4% paraformaldehyde (n=4/group; analysis via MATLAB codes to quantify optical density in labeled motor neurons). We found significantly more Pde4b, Adora2a, and Pkcδ mRNA in young and older females versus age matched males; Epacwas also elevated, but only in young females (p<0.001). p47 was increased in older rats of both sexes (p<0.01). p47, Cx3cl1, Adora2a and Pkcδ mRNA were reduced by dAIH in older females (p<0.05), but not other groups. There were age‐dependent increases in adenosine 1A (p <0.0001) and 2A (p <0.0001) receptors, and BDNF (p<0.001) protein post‐AIH in both sexes. Thus, there are age, sex and dAIH preconditioning effects on molecules known to regulate the Q and S pathways to phrenic motor facilitation. These novel findings advance our understanding of pLTF, and inform translational research concerning the therapeutic potential of dAIH to treat breathing deficits in individuals of different ages or sex.

Phrenic Motor Neuron Fractalkine Expression After Intermittent Hypoxia and Spinal Cord Injury

Acute intermittent hypoxia (AIH) elicits spinal respiratory motor plasticity, and has emerged as a promising therapeutic approach to restore lost respiratory (and non‐respiratory) motor function following cervical spinal cord injury (cSCI). Conversely, most individuals with cSCI experience sleep disordered breathing, generating chronic intermittent hypoxia (CIH) at intensities sufficient to trigger pathology. Since glia play a critical role in regulating neuroplasticity, they may play a key role in differentiating responses to therapeutic (AIH) versus pathogenic (CIH) intermittent hypoxia. Motor neuron‐microglial interactions regulate AIH‐induced phrenic motor plasticity. Specifically, phrenic motor neuron fractalkine, a chemokine unique to neurons released during hypoxia activates its high affinity receptor, CX3CR1—which is unique to microglia in the CNS. In response, microglia release ATP/adenosine, inhibiting AIH‐induced phrenic motor plasticity. Thus, if intermittent hypoxia or cSCI impact phrenic motor neuron fractalkine expression, they may directly impact plasticity and the potential of therapeutic AIH. In this study, we tested the hypothesis that phrenic motor neuron fractalkine expression is attenuated by daily AIH (but not CIH) in spinally intact rats, but not in rats with cSCI. Fractalkine was assessed in phrenic motor neurons of male rats with/without C2 hemisection (12 weeks post‐injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5‐min episodes per day, 10.5% O2; 5‐min intervals); and 3) moderate CIH (2‐min 10.5% O2 episodes; 2‐min normoxic intervals; 8 hr/day). Fourteen days before hemisection, intrapleural Cholera toxin B injections were made to retrogradely label phrenic motor neurons. Fractalkine expression was assessed in 40 mm sections using a custom MATLAB algorithm to assess immunofluorescence optical density within labeled phrenic motor neurons. Spinally intact and injured rats have differential responses to daily AIH vs CIH. Whereas injured rats had no change in phrenic motor neuron fractalkine expression after daily AIH or CIH, fractalkine expression was marginally reduced in intact rats following daily AIH (p = 0.07; cohen’s d= 132%), but not CIH (p = 0.88; cohen’s d= 23%). Thus, daily (therapeutic) AIH may decrease motor neuron fractalkine and microglial adenosine release, thereby enhancing moderate AIH‐induced phrenic motor plasticity.

Acute intermittent hypercapnic-hypoxia elicits central neural respiratory motor plasticity in humans.

Acute intermittent hypoxia (AIH) elicits long-term facilitation (LTF) of respiration. Although LTF is observed when CO2 is elevated during AIH in awake humans, the influence of CO2 on corticospinal respiratory motor plasticity is unknown. Thus, we tested the hypotheses that acute intermittent hypercapnic-hypoxia (AIHH): (1) enhances cortico-phrenic neurotransmission (reflecting volitional respiratory control); and (2) elicits ventilatory LTF (reflecting automatic respiratory control). Eighteen healthy adults completed four study visits. Day 1 consisted of anthropometry and pulmonary function testing. On Days 2, 3 and 4, in a balanced alternating sequence, participants received: AIHH, poikilocapnic AIH, and normocapnic-normoxia (Sham). Protocols consisted of 15, 60s exposures with 90s normoxic intervals. Transcranial (TMS) and cervical (CMS) magnetic stimulation were used to induce diaphragmatic motor-evoked potentials and compound muscle action potentials, respectively. Respiratory drive was assessed via mouth occlusion pressure (P0.1 ), and minute ventilation measured at rest. Dependent variables were assessed at baseline and 30-60min after exposures. Increases in TMS-evoked diaphragm potential amplitudes were observed following AIHH vs. Sham (+28±41%, P=0.003), but not after AIH. No changes were observed in CMS-evoked diaphragm potential amplitudes. Mouth occlusion pressure also increased after AIHH (+21±34%, P=0.033), but not after AIH. Ventilatory LTF was not observed after any treatment. We demonstrate that AIHH elicits central neural mechanisms of respiratory motor plasticity and increases resting respiratory drive in awake humans. These findings may have important implications for neurorehabilitation after spinal cord injury and other neuromuscular disorders compromising breathing. KEY POINTS: The occurrence of respiratory long-term facilitation following acute exposure to intermittent hypoxia is believed to be dependent upon CO2 regulation - mechanisms governing the critical role of CO2 have seldom been explored. We tested the hypothesis that acute intermittent hypercapnic-hypoxia (AIHH) enhances cortico-phrenic neurotransmission in awake healthy humans. The amplitude of diaphragmatic motor-evoked potentials induced by transcranial magnetic stimulation was increased after AIHH, but not the amplitude of compound muscle action potentials evoked by cervical magnetic stimulation. Mouth occlusion pressure (P0.1 , an indicator of neural respiratory drive) was also increased after AIHH, but not tidal volume or minute ventilation. Thus, moderate AIHH elicits central neural mechanisms of respiratory motor plasticity, without measurable ventilatory long-term facilitation in awake humans.

Chapter 16 - Respiratory neuroplasticity: Mechanisms and translational implications of phrenic motor plasticity

Widespread appreciation that neuroplasticity is an essential feature of the neural system controlling breathing has emerged only in recent years. In this chapter, we focus on respiratory motor plasticity, with emphasis on the phrenic motor system. First, we define related but distinct concepts: neuromodulation and neuroplasticity. We then focus on mechanisms underlying two well-studied models of phrenic motor plasticity: (1) phrenic long-term facilitation following brief exposure to acute intermittent hypoxia; and (2) phrenic motor facilitation after prolonged or recurrent bouts of diminished respiratory neural activity. Advances in our understanding of these novel and important forms of plasticity have been rapid and have already inspired translation in multiple respects: (1) development of novel therapeutic strategies to preserve/restore breathing function in humans with severe neurological disorders, such as spinal cord injury and amyotrophic lateral sclerosis; and (2) the discovery that similar plasticity also occurs in nonrespiratory motor systems. Indeed, the realization that similar plasticity occurs in respiratory and nonrespiratory motor neurons inspired clinical trials to restore leg/walking and hand/arm function in people living with chronic, incomplete spinal cord injury. Similar application may be possible to other clinical disorders that compromise respiratory and non-respiratory movements.

Acute intermittent hypoxia and respiratory muscle recruitment in people with amyotrophic lateral sclerosis: A preliminary study

Respiratory failure is the main cause of death in amyotrophic lateral sclerosis (ALS). Since no effective treatments to preserve independent breathing are available, there is a critical need for new therapies to preserve or restore breathing ability. Since acute intermittent hypoxia (AIH) elicits spinal respiratory motor plasticity in rodent ALS models, and may restore breathing ability in people with ALS, we performed a proof-of-principle study to investigate this possibility in ALS patients. Quiet breathing, sniff nasal inspiratory pressure (SNIP) and maximal inspiratory pressure (MIP) were tested in 13 persons with ALS and 10 age-matched controls, before and 60 min post-AIH (15, 1 min episodes of 10% O2, 2 min normoxic intervals) or sham AIH (continuous normoxia). The root mean square (RMS) of the right and left diaphragm, 2nd parasternal, scalene and sternocleidomastoid muscles were monitored. A vector analysis was used to calculate summated vector magnitude (Mag) and similarity index (SI) of collective EMG activity during quiet breathing, SNIP and MIP maneuvers. AIH facilitated tidal volume and minute ventilation (treatment main effects: p < 0.05), and Mag (ie. collective respiratory muscle activity; p < 0.001) during quiet breathing in ALS and control subjects, but there was no effect on SI during quiet breathing. SNIP SI decreased in both groups post-AIH (p < 0.005), whereas Mag was unchanged (p = 0.09). No differences were observed in SNIP or MIP post AIH in either group. Discomfort was not reported during AIH by any subject, nor were adverse events observed. Thus, AIH may be a safe way to increase collective inspiratory muscle activity during quiet breathing in ALS patients, although a single AIH presentation was not sufficient to significantly increase peak inspiratory pressure generation. These preliminary results provide evidence that AIH may improve breathing function in people with ALS, and that future studies of prolonged, repetitive AIH protocols are warranted.

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. 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.

Adenosine A2a receptors modulate TrkB receptor-dependent respiratory plasticity in neonatal rats

Neuroplasticity is a fundamental property of the respiratory control system, enabling critical adaptations in breathing to meet the challenges, but little is known whether neonates express neuroplasticity similar to adults. We tested the hypothesis that, similar to adults, tyrosine receptor kinase B (TrkB) or adenosine A2a receptor activation in neonates are independently sufficient to elicit respiratory motor facilitation, and that co-induction of TrkB and A2a receptor-dependent plasticity undermines respiratory motor facilitation. TrkB receptor activation with 7,8-dihydroxyflavone (DHF) in neonatal brainstem-spinal cord preparations induced a long-lasting increase in respiratory motor output in 55 % of preparations, whereas adenosine A2a receptor activation with CGS21680 only sporadically induced respiratory motor plasticity. CGS21680 and DHF co-application prevented DHF-dependent respiratory motor facilitation, whereas co-application of MSX-3 (adenosine A2a receptor antagonist) and DHF more rapidly induced respiratory motor plasticity. Collectively, these data suggest that mechanisms underlying respiratory neuroplasticity may be only partially operational in early neonatal life, and that adenosine A2a receptor activation undermines TrkB-induced respiratory plasticity.

Acute morphine blocks spinal respiratory motor plasticity via long-latency mechanisms that require toll-like receptor 4 signalling.

While respiratory complications following opioid use are mainly mediated via activation of mu opioid receptors, long-latency off-target signalling via innate immune toll-like receptor 4 (TLR4) may impair other essential elements of breathing control such as respiratory motor plasticity. In adult rats, pre-treatment with a single dose of morphine blocked long-term facilitation (LTF) of phrenic motor output via a long-latency TLR4-dependent mechanism. In the phrenic motor nucleus, morphine triggered TLR4-dependent activation of microglial p38 MAPK - a key enzyme that orchestrates inflammatory signalling and is known to undermine phrenic LTF. Morphine-induced LTF loss may destabilize breathing, potentially contributing to respiratory side effects. Therefore, we suggest minimizing TLR-4 signalling may improve breathing stability during opioid therapy. Opioid-induced respiratory dysfunction is a significant public health burden. While respiratory effects are mediated via mu opioid receptors, long-latency off-target opioid signalling through innate immune toll-like receptor 4 (TLR4) may modulate essential elements of breathing control, particularly respiratory motor plasticity. Plasticity in respiratory motor circuits contributes to the preservation of breathing in the face of destabilizing influences. For example, respiratory long-term facilitation (LTF), a well-studied model of respiratory motor plasticity triggered by acute intermittent hypoxia, promotes breathing stability by increasing respiratory motor drive to breathing muscles. Some forms of respiratory LTF are exquisitely sensitive to inflammation and are abolished by even a mild inflammation triggered by TLR4 activation (e.g. via systemic lipopolysaccharides). Since opioids induce inflammation and TLR4 activation, we hypothesized that opioids would abolish LTF through a TLR4-dependent mechanism. In adult Sprague Dawley rats, pre-treatment with a single systemic injection of the prototypical opioid agonist morphine blocks LTF expression several hours later in the phrenic motor system - the motor pool driving diaphragm muscle contractions. Morphine blocked phrenic LTF via TLR4-dependent mechanisms because pre-treatment with (+)-naloxone - the opioid inactive stereoisomer and novel small molecule TLR4 inhibitor - prevented impairment of phrenic LTF in morphine-treated rats. Morphine triggered TLR4-dependent activation of microglial p38 MAPK within the phrenic motor system - a key enzyme that orchestrates inflammatory signalling and undermines phrenic LTF. Morphine-induced LTF loss may destabilize breathing, potentially contributing to respiratory side effects. We suggest minimizing TLR-4 signalling may improve breathing stability during opioid therapy by restoring endogenous mechanisms of plasticity within respiratory motor circuits.

Single-session effects of acute intermittent hypoxia on breathing function after human spinal cord injury

After spinal cord injury (SCI) respiratory complications are a leading cause of morbidity and mortality. Acute intermittent hypoxia (AIH) triggers spinal respiratory motor plasticity in rodent models, and repetitive AIH may have the potential to restore breathing capacity in those with SCI. As an initial approach to provide proof of principle for such effects, we tested single-session AIH effects on breathing function in adults with chronic SCI. 17 adults (13 males; 34.1 ± 14.5 years old; 13 motor complete SCI; >6 months post injury) completed two randomly ordered sessions, AIH versus sham. AIH consisted of 15, 1-min episodes (hypoxia: 10.3% O2; sham: 21% O2) interspersed with room air breathing (1.5 min, 21% oxygen); no attempt was made to regulate arterial CO2 levels. Blood oxygen saturation (SpO2), maximal inspiratory and expiratory pressures (MIP; MEP), forced vital capacity (FVC), and mouth occlusion pressure within 0.1 s (P0.1) were assessed. Outcomes were compared using nonparametric Wilcoxon's tests, or a 2 × 2 ANOVA. Baseline SpO2 was 97.2 ± 1.3% and was unchanged during sham experiments. During hypoxic episodes, SpO2 decreased to 84.7 ± 0.9%, and returned to baseline levels during normoxic intervals. Outcomes were unchanged from baseline post-sham. Greater increases in MIP were evident post AIH vs. sham (median values; +10.8 cmH2O vs. -2.6 cmH2O respectively, 95% confidence interval (−18.7) – (−4.3), p = .006) with a moderate Cohen's effect size (0.68). P0.1, MEP and FVC did not change post-AIH. A single AIH session increased maximal inspiratory pressure generation, but not other breathing functions in adults with SCI. Reasons may include greater spared innervation to inspiratory versus expiratory muscles or differences in the capacity for AIH-induced plasticity in inspiratory motor neuron pools. Based on our findings, the therapeutic potential of AIH on breathing capacity in people with SCI warrants further investigation.

CO 2 Effects on Intermittent Hypoxia‐Induced Respiratory Motor Plasticity in Humans: In Progress Study

Intermittent hypoxia (IH) elicits respiratory motor plasticity (known as ventilatory long-term facilitation [LTF]) in humans and other mammals. However, awake humans exhibit ventilatory LTF only when CO2 levels are raised slightly above eupneic levels in a sustained or intermittent fashion during the IH protocol. In this ongoing study, we seek to determine the effect of CO2 on IH-induced respiratory motor plasticity by examining multiple, distinct mechanisms contributing to the ventilatory response during and following acute intermittent hypercapnic hypoxia (IHH). We hypothesized that IHH elicits a prolonged increase in cortico-diaphragmatic conduction, resting neural drive to breathe, and respiratory motor output versus poikilocapnic IH and a normoxic, normocapnic control (CTRL). Five healthy young adults (age = 32 ± 5 years; 3 female) completed four study visits. Day 1 consisted of general subject characterization. On Days 2, 3 and 4 (separated by ≥72 h), subjects were randomly assigned to receive either: IHH (PETO2 = 55 ± 4 Torr, PETCO2 = 44 ± 3 Torr), IH (PETO2 = 55 ± 3 Torr, PETCO2 = 37 ± 2 Torr), or CTRL (PETO2 = 107 ± 3 Torr, PETCO2 = 36 ± 2 Torr). Protocols consisted of 15 episodes of 60 s exposure with 90 s breathing ambient room air. Cardiovascular responses (heart rate, blood pressure and oxygen saturation) were monitored throughout. Cortico-diaphragmatic conduction was quantified by recording motor evoked potentials (MEPs) using transcranial magnetic stimulation and surface EMG electrodes. Resting neural drive to breathe was assessed using the mouth occlusion pressure technique (i.e. P0.1). Respiratory motor output was measured as minute ventilation () during resting isocapnic conditions (PETCO2 maintained within 1 mmHg of baseline) and adjusted based on the rate of metabolic CO2 production (/). Dependent variables were assessed at baseline, and 30-60 min post. Following IHH, IH and CTRL, the mean change in MEP amplitude was +34 ± 48%, +9 ± 21%, and +2 ± 22%; the mean change in P0.1 was +26 ± 17%, +15 ± 16%, and +7 ± 16%; and the mean change in was +0.3 ± 1.0 L·min-1, +0.3 ± 1.1 L·min-1, and +0.3 ± 0.8 L·min-1. When adjusted for , the mean change in / was -0.5 ± 1.0, +0.4 ± 1.8, and -0.2 ± 1.1. Based on these preliminary data in 5 subjects, we suggest that IHH elicits greater increases in cortico-diaphragmatic conduction and resting neural drive to breathe vs. poikilocapnic IH and CTRL. Conversely, our IHH protocol does not elicit ventilatory LTF. We reason that measures such as MEP amplitude and P0.1 are less sensitive to behavioral influences vs. , and may provide unique insights into mechanisms of IH-induced respiratory motor plasticity in awake healthy humans.

Daily administration of ketoprofen restores AIH‐induced phrenic long term facilitation with prolonged chronic intermittent hypoxia

Acute intermittent hypoxia (AIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long-term facilitation (pLTF). Preconditioning with a single day of chronic intermittent hypoxia (8 hours, 2 min hypoxic episodes with 2 min intervals) abolishes AIH-induced pLTF through a mechanism that requires systemic inflammation and spinal p38 MAP kinase activation (Huxtable et al., 2015). pLTF is restored by high-dose, systemic administration of the non-steroidal anti-inflammatory drug, ketoprofen, or spinal p38 MAP kinase inhibition. Similarly, preconditioning with 7 days of CIH (8 hours per day, 2 min episodes, 2 min intervals) abolishes pLTF, but we observed that phospho-p38 within phrenic motor neurons is unchanged from control rats (Gonzalez-Rothi and colleagues, unpublished). Thus, mechanisms whereby 7 days of CIH undermines AIH-induced pLTF are unclear. Since CIH induces time-dependent spinal inflammatory gene expression profiles over time (Smith et al., 2013), we hypothesize that prolonged CIH (versus a single day) blunts LTF through an inflammation-dependent mechanism that is independent of p-38MAP kinase. Thus, in ongoing experiments, the impact of anti-inflammatory drugs on AIH-induced pLTF in rats preconditioned with prolonged CIH (7 days). pLTF was restored when ketoprofen was administered each day just prior to the IH7 exposure (70% increase, p=.0013 vs. vehicle), but not when a single dose of ketoprofen was delivered just prior to assessment of pLTF (19% increase, p=0.9975 vs. vehicle). Upcoming experiments will explore the role of p38 MAP kinase with prolonged CIH exposure. These experiments are the first to explore shifting mechanisms whereby prolonged CIH and associated inflammation undermine phrenic motor plasticity. Such experiments are essential since chronic intermittent hypoxia is a hallmark of sleep apnea, a condition highly prevalent in individuals living with neurological disorders (spinal cord injury, ALS, etc.). It is therefore important to identify and mitigate factors which may undermine the potential for therapeutic interventions (i.e., repetitive AIH) to harness endogenous plasticity and ultimately improve breathing.

Protocol Specific Effects of Daily Acute Intermittent Hypoxia on Pro‐Plasticity Gene Expression in the Ventral Cervical Spinal Cord

Preconditioning with daily acute intermittent hypoxia (dAIH) increases respiratory drive and enhances the capacity for AIH induced respiratory motor plasticity. Since dAIH is emerging as an important therapeutic modality to improve breathing (and limb movements) after cervical spinal cord injury and with other neurological disorders, it is critical to optimize dAIH protocols to maximize functional benefits, and to understand fundamental mechanisms of this plasticity. Here, we compared several dAIH protocols and their impact on ventral cervical mRNA for pro-plasticity molecules known to drive or regulate AIH-induced respiratory motor plasticity. Our general hypothesis is that distinct dAIH protocols differentially affect molecular changes near the phrenic motor nucleus, particularly molecules associated with the serotonin-induced Q pathway to phrenic motor facilitation. Thus, we compared mRNA expression following 14 day exposures to 5 unique dAIH protocols in the ventral C3-C6 spinal cord of adult male Sprague-Dawley rats (n = 6/group). Protocols tested included: 1) normoxia controls (30 or 50 min); 2) 5, 5 min episodes of 10.5% O2, 5 min normoxic intervals (5/5); 3) 5, 1 min episodes of 10.5% O2, 5 min normoxic intervals (1/5); 4) 5,1 min episodes of 10.5% O2 and 5% CO2, 5 min intervals (1/5 with CO2); and 5) 15, 1 min hypoxic episodes, 1 min intervals (1/1). Rats were exposed to dAIH between 8:00AM and 11:00AM using exposure cylinders (6 L/min per cylinder). Rats were sacrificed 24 hours after the last hypoxic exposure, and spinal tissues were harvested, dissected and preserved in RNA later solution (<10 min). Spinal tissues were stored at -80oC until mRNA extraction and cDNA synthesis. RT PCR was used to quantify mRNA levels of: brain derived neurotrophic factor (Bdnf), neurotrophic tyrosine receptor kinase-2 (Ntrk2), the catalytic subunit protein kinase A (Prkaa1) the regulatory subunit of PKA (Prkar1a), as well as serotonin 2A (Htr2a), 2B (Htr2b), and 7 (Htr7), and adenosine 2A (Adora2a) receptors. 1/1 dAIH elicited the most prominent effects on steady state mRNA levels. After 14-days of 1/1 dAIH, we detected significant increases in Htr2a (p<0.05), and the regulatory subunit of PKA (p<0.0001) mRNA expression. Thus, we hypothesize dAIH 1/1 will be most effective in eliciting meaningful functional changes in animals with injury or disease. These findings are highly relevant for translational studies in rodent models or humans with injury/disease by suggesting optimal repetitive AIH protocols that enable us to harness respiratory plasticity as a treatment for life-threatening respiratory deficits caused by spinal injury, ALS, MS, stroke or other neuromuscular disorders.

Fractalkine signaling in the cervical spinal cord orchestrates microglia‐neuron interactions regulating intermittent hypoxia‐induced phrenic motor plasticity

Microglia are innate immune cells with many non-immune functions in the healthy nervous system. For example, microglia engage in bidirectional communication with central nervous system neurons, regulating diverse processes including neuronal architecture, excitability, and synaptic plasticity. Although neuron-microglia interactions are known to regulate several forms of neuroplasticity, their role in regulating respiratory motor plasticity is unknown. Acute intermittent hypoxia (AIH) elicits a form of plasticity in the phrenic motor system known as phrenic long-term facilitation (pLTF) — a progressive, long-lasting increase in phrenic motor output following AIH. Whereas AIH consisting of moderate hypoxic episodes elicits pLTF by a serotonin-dependent, adenosine-constrained mechanism, severe AIH elicits pLTF by an adenosine-dominant mechanism. Here, we test the hypothesis that communication between cervical spinal neurons and microglia differentially regulates moderate versus severe AIH-induced pLTF. Specifically, we hypothesized that neuron-microglia signaling modulates pLTF expression by: 1) neuronal fractalkine (CX3CL1) release and activation of its receptor, CX3CR1, which is uniquely expressed in CNS microglia; and 2) fractalkine-induced ATP release, and breakdown to adenosine, which differentially regulates moderate and severe AIH-induced pLTF. In anesthetized, adult male Sprague Dawley rats, cervical spinal CX3CR1 inhibition enhanced serotonin-dependent moderate AIH-induced pLTF, but blocked adenosine-dependent, severe AIH-induced pLTF. Further, spinal CX3CR1 activation with recombinant fractalkine induced phrenic motor facilitation in a dose-dependent manner— an effect that required both cervical spinal ATP breakdown and adenosine 2A receptor activation. We thus propose a hypothetical model whereby hypoxia triggers dose-dependent phrenic motor neuron fractalkine release that activates CX3CR1 on nearby microglia. Subsequent microglial ATP release and eventual breakdown increases extracellular adenosine and undermines serotonin-dependent pLTF, but drives severe AIH-induced (adenosine-dependent) pLTF by activating phrenic motor neuron adenosine 2A receptors. These findings enhance our understanding of how spinal, intercellular interactions regulate AIH-induced plasticity in the phrenic motor circuit.

Chronic, closed‐loop, cervical epidural stimulation elicits diaphragm motor plasticity after spinal cord injury

Cervical spinal cord injury (cSCI) accounts for over half of all traumatic SCI (NSCISC, 2020). Unfortunately, respiratory insufficiency due to cSCI is the leading cause of mortality and morbidity after SCI. Electrical epidural stimulation (EES) can partially restore volitional locomotor function after SCI in humans while high-frequency EES activates respiratory muscles (DiMarco and Kowalski, 2009) and may lead to at least short-term plasticity (Gonzalez-Rothi et al., 2017). We've shown that our model of cervical closed-loop epidural stimulation (CL-ES) in awake, freely-behaving rats with C2 hemisection (C2HS) elicits short term plasticity in diaphragm motor output (Malone et al., FASEB J 2020). Here we show that this expression of plasticity may be bimodal in that there are “responders” and “non-responders” just as that seen in people with SCI (Tan et al., 2020; Lamy and Boakye, 2013). Rats were implanted with CL-ES electrodes at C4 and bilateral diaphragm EMG electrodes to record respiratory muscle activity in awake rats. Four groups were studied: C2HS + CL-ES (n=8); C2HS + no stim (n=6); Sham + CL-ES (n=6); Sham + no stim (n=6). As expected, increasing current (range: 50–750 μA; inspiratory-triggered) increased the magnitude of evoked potentials. Further, four days of chronic (12-20 hours/day) CL-ES elicited facilitation of ipsilesional evoked responses in 6 of 8 animals with C2HS. Data were calculated as maximum percent change from baseline (-/+) of the stimulus triggered averages at the current with the largest dispersion during sweeps. CL-ES in C2HS rats elicited facilitation in max percent change in stimulus triggered average: 1) peak amplitude (pA; 879% ± 455%) vs. Sham + CL-ES and Sham + no stim (109% ± 76%; p=0.04 and 92% ± 68% p=0.05, respectively) and 2) area under the curve (AUC; 555% ± 142%) vs. groups without stimulation (C2HS + no stim: 99% ± 52%, p=0.05; Sham + no stim: 73% ± 60%, p=0.038). The lack of robust facilitation in Sham + CL-ES rats suggests the possibility of an intact inhibitory modulatory network in healthy rats. These results are preliminary and highly variable, thus we can only speculate about mechanisms giving rise to this form of respiratory neuroplasticity. Ongoing RTqPCR experiments utilizing correlative measures of spinal neurotrophic factor expression (e.g. brain derived neurotrophic factor) and siRNA knock-down of relevant receptors on phrenic motor neurons (Dale et al., 2017) may give more insight. We hypothesize that facilitation of these evoked potentials may depend on activation of neurotrophic factor receptors within the phrenic motor network. This work demonstrates for the first time that CL-ES can elicit respiratory motor plasticity after cSCI, demonstrating potential for a neuroceutical therapy to address breathing impairment in individuals with spinal cord injury. Data presented as mean ± SEM.

Acute Neonatal Inflammation Differentially Activates Adult Microglia In Respiratory Neural Control Regions

Adult respiratory motor plasticity is abolished by neonatal inflammation, but is restored with an adult anti-inflammatory treatment, suggesting lasting inflammatory signaling underlies deficiencies in adult respiratory motor plasticity. While neonatal inflammation impairs adult hippocampal plasticity by increasing adult microglial number (resident immune cells of the brain) and inflammatory gene expression, it is unclear if adult microglia in respiratory control regions are similarly affected by neonatal inflammation. Thus, we hypothesized that acute neonatal inflammation would increase the number of and inflammatory transcriptomic profiles in adult microglia in respiratory control regions (medulla and cervical spinal cord). Using flow cytometry, a single neonatal inflammatory challenge (1 mg/kg LPS, i.p., postnatal day 4) significantly increased adult medullary microglial frequency in males (neonatal LPS: 25 ± 3% microglia, n=11; neonatal saline: 12 ± 2% microglia, n=11, p<0.0001), although the effect was not statistically significant in females (neonatal LPS: 21 ± 4% microglia, n=10; neonatal saline: 12 ± 3% microglia, n=11, p=0.0626). Interestingly, these differences were not observed in spinal microglia in either sex (male neonatal LPS: 13 ± 5% microglia, n=12; male neonatal saline: 11 ± 3% microglia, n=10, p=0.9; female neonatal LPS: 16 ± 6% microglia, n=12; female neonatal saline: 13 ± 5% microglia, n=12, p=0.9), suggesting a neonatal LPS challenge region-specifically increases adult microglia. Similar regional differences were also observed in preliminary transcriptomic data from adult females challenged acutely as neonates; more microglial genes were differentially increased in isolated medullary microglia (271 genes, n=3) than in spinal microglia (23 genes, n=6). In addition, preliminary data suggest potential sex differences in adult microglial inflammatory gene responses in respiratory control regions following an acute neonatal LPS challenge. More inflammatory genes were increased in adult female microglia (18 genes, n=9) than in male microglia (7 genes, n=7). In conclusion, neonatal inflammation region-dependently increased medullary microglial frequency in adult males, while medullary microglia have increased inflammatory transcriptomic profiles in females. These microglial changes may contribute to the mechanisms underlying abolition of adult respiratory motor plasticity following an early life neonatal inflammatory challenge.

Cervical spinal injury compromises caudal spinal tissue oxygenation and undermines acute intermittent hypoxia-induced phrenic long-term facilitation

An important model of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic burst amplitude following acute intermittent hypoxia (AIH). Moderate AIH elicits pLTF by a serotonin-dependent mechanism known as the Q pathway to phrenic motor facilitation. In contrast, severe AIH (greater hypoxemia) increases spinal adenosine accumulation and activates phrenic motor neuron adenosine 2A receptors, thereby initiating a distinct mechanism of plasticity known as the S pathway. Since the Q and S pathways interact via mutual cross-talk inhibition, the balance between spinal serotonin release and adenosine accumulation is an important pLTF regulator. Spinal injury decreases spinal tissue oxygen pressure (PtO2) caudal to injury. Since AIH is being explored as a neurotherapeutic to restore breathing ability after cervical spinal injury, we tested the hypothesis that decreased PtO2 in the phrenic motor nucleus after C2 spinal hemisection (C2Hx) undermines moderate AIH-induced pLTF, likely due to shifts in the adenosine/serotonin balance. We recorded C3/4 ventral cervical PtO2 with an optode, and bilateral phrenic nerve activity in anesthetized, paralyzed and ventilated rats, with and without C2Hx. In intact rats, PtO2 was lower during severe versus moderate AIH as expected. In chronic C2Hx rats (> 8 weeks post-injury), PtO2 was lower during baseline and moderate hypoxic episodes, approaching severe AIH levels in intact rats. After C2Hx, pLTF was blunted ipsilateral, but observed contralateral to injury. We conclude that C2Hx compromises PtO2 near the phrenic motor nucleus and undermines pLTF, presumably due to a shift in the serotonin versus adenosine balance during hypoxic episodes. These findings have important implications for optimizing AIH protocols in our efforts to restore breathing ability with therapeutic AIH in people with chronic cervical spinal injury.

Baseline Arterial CO2 Pressure Regulates Acute Intermittent Hypoxia-Induced Phrenic Long-Term Facilitation in Rats.

Moderate acute intermittent hypoxia (mAIH) elicits a progressive increase in phrenic motor output lasting hours post-mAIH, a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF). mAIH-induced pLTF is initiated by activation of spinally-projecting raphe serotonergic neurons during hypoxia and subsequent serotonin release near phrenic motor neurons. Since raphe serotonergic neurons are also sensitive to pH and CO2, the prevailing arterial CO2 pressure (PaCO2) may modulate their activity (and serotonin release) during hypoxic episodes. Thus, we hypothesized that changes in background PaCO2 directly influence the magnitude of mAIH-induced pLTF. mAIH-induced pLTF was evaluated in anesthetized, vagotomized, paralyzed and ventilated rats, with end-tidal CO2 (i.e., a PaCO2 surrogate) maintained at: (1) ≤39 mmHg (hypocapnia); (2) ∼41 mmHg (normocapnia); or (3) ≥48 mmHg (hypercapnia) throughout experimental protocols. Although baseline phrenic nerve activity tended to be lower in hypocapnia, short-term hypoxic phrenic response, i.e., burst amplitude (Δ = 5.1 ± 1.1 μV) and frequency responses (Δ = 21 ± 4 bpm), was greater than in normocapnic (Δ = 3.6 ± 0.6 μV and 8 ± 4, respectively) or hypercapnic rats (Δ = 2.0 ± 0.6 μV and −2 ± 2, respectively), followed by a progressive increase in phrenic burst amplitude (i.e., pLTF) for at least 60 min post mAIH. pLTF in the hypocapnic group (Δ = 4.9 ± 0.6 μV) was significantly greater than in normocapnic (Δ = 2.8 ± 0.7 μV) or hypercapnic rats (Δ = 1.7 ± 0.4 μV). In contrast, although hypercapnic rats also exhibited significant pLTF, it was attenuated versus hypocapnic rats. When pLTF was expressed as percent change from maximal chemoreflex stimulation, all pairwise comparisons were found to be statistically significant (p < 0.05). We conclude that elevated PaCO2 undermines mAIH-induced pLTF in anesthetized rats. These findings contrast with well-documented effects of PaCO2 on ventilatory LTF in awake humans.

Novel Influences of Sex and APOE Genotype on Spinal Plasticity and Recovery of Function after Spinal Cord Injury.

Spinal cord injuries can abolish both motor and sensory function throughout the body. Spontaneous recovery after injury is limited and can vary substantially between individuals. Despite an abundance of therapeutic approaches that have shown promise in preclinical models, there is currently a lack of effective treatment strategies that have been translated to restore function after spinal cord injury (SCI) in the human population. We hypothesized that sex and genetic background of injured individuals could impact how they respond to treatment strategies, presenting a barrier to translating therapies that are not tailored to the individual. One gene of particular interest is APOE, which has been extensively studied in the brain because of its allele-specific influences on synaptic plasticity, metabolism, inflammation, and neurodegeneration. Despite its prominence as a therapeutic target in brain injury and disease, little is known about how it influences neural plasticity and repair processes in the spinal cord. Using humanized mice, we examined how the ε3 and ε4 alleles of APOE influence the efficacy of therapeutic intermittent hypoxia (IH) in inducing spinally-mediated plasticity after cervical SCI (cSCI). IH is sufficient to enhance plasticity and restore motor function after experimental SCI in genetically similar rodent populations, but its effect in human subjects is more variable (Golder and Mitchell, 2005; Hayes et al., 2014). Our results demonstrate that both sex and APOE genotype determine the extent of respiratory motor plasticity that is elicited by IH, highlighting the importance of considering these clinically relevant variables when translating therapeutic approaches for the SCI community.

Intermittent hypercapnic hypoxia: a model to study human respiratory motor plasticity?

Intermittent hypercapnic hypoxia: a model to study human respiratory motor plasticity?