Shape and size of the nasopharyngeal airway is controlled by muscles innervated facial, glossopharyngeal, vagal, and hypoglossal cranial nerves. Contrary to brainstem networks that drive facial, vagal and hypoglossal nerve activities (FNA, VNA, HNA) the discharge patterns and origins of glossopharyngeal nerve activity (GPNA) remain poorly investigated.Here, an in situ perfused brainstem preparation (n=19) was used for recordings of GPNA in relation to phrenic (PNA), FNA, VNA and HNA. Brainstem transections were performed (n=10/19) to explore the role of pontomedullary synaptic interactions in generating GPNA.GPNA generally mirrors FNA and HNA discharge patterns and displays pre-inspiratory activity relative to the PNA, followed by robust inspiratory discharge in coincidence with PNA. Postinspiratory (early expiratory) discharge was, contrary to VNA, generally absent in FNA, GPNA or HNA. As described previously FNA and HNA discharge was virtually eliminated after pontomedullary transection while an apneustic inspiratory motor discharge was maintained in PNA, VNA and GPNA. After brainstem transection GPNA displayed an increased tonic activity starting during mid-expiration and thus developed prolonged pre-inspiratory activity compared to control.In conclusion respiratory GPNA reflects FNA and HNA which implies similar function in controlling upper airway patency during breathing. That GPNA preserved its pre-inspiratory/inspiratory discharge pattern in relation PNA after pontomedullary transection suggest that GPNA premotor circuits may have a different anatomical distribution compared HNA and FNA and thus may therefore hold a unique role in in preserving airway patency.

Background Sepsis has a high mortality rate due to multiple organ failure. However, the influence of peripheral inflammation on brainstem autonomic and respiratory circuits in sepsis is poorly understood. Our working hypothesis is that peripheral inflammation affects central autonomic circuits and consequently contributes to multiorgan failure in sepsis. Methods In an Escherichia coli ( E. coli )–fibrin clot model of peritonitis, we first recorded ventilatory patterns using plethysmography before and 24 h after fibrin clot implantation. To assess whether peritonitis was associated with brainstem neuro-inflammation, we measured cytokine and chemokine levels in Luminex assays. To determine the effect of E. coli peritonitis on brainstem function, we assessed sympatho-respiratory nerve activities at baseline and during brief (20 s) hypoxemic ischemia challenges using in situ-perfused brainstem preparations (PBPs) from sham or infected rats. PBPs lack peripheral organs and blood, but generate vascular tone and in vivo rhythmic activities in thoracic sympathetic (tSNA), phrenic and vagal nerves. Results Respiratory frequency was greater ( p < 0.001) at 24 h post-infection with E. coli than in the sham control. However, breath-by-breath variability and total protein in the BALF did not differ. IL-1β ( p < 0.05), IL-6 ( p < 0.05) and IL-17 ( p < 0.04) concentrations were greater in the brainstem of infected rats. In the PBP, integrated tSNA ( p < 0.05) and perfusion pressure were greater ( p < 0.001), indicating a neural-mediated pathophysiological high sympathetic drive. Moreover, respiratory frequency was greater ( p < 0.001) in PBPs from infected rats than from sham rats. Normalized phase durations of inspiration and expiration were greater ( p < 0.009, p < 0.015, respectively), but the post-inspiratory phase ( p < 0.007) and the breath-by-breath variability ( p < 0.001) were less compared to sham PBPs. Hypoxemic ischemia triggered a biphasic response, respiratory augmentation followed by depression. PBPs from infected rats had weaker respiratory augmentation ( p < 0.001) and depression ( p < 0.001) than PBPs from sham rats. In contrast, tSNA in E. coli -treated PBPs was enhanced throughout the entire response to hypoxemic ischemia ( p < 0.01), consistent with sympathetic hyperactivity. Conclusion We show that peripheral sepsis caused brainstem inflammation and impaired sympatho-respiratory motor control in a single day after infection. We conclude that central sympathetic hyperactivity may impact vital organ systems in sepsis.

Abstract Introduction The glossopharyngeal nerve is important for gas exchange in fish, but in mammals concentration is focused on the hypoglossal nerve (Dewald et al, 2018). In the present study, we hypothesize the discharge pattern of glossopharyngeal nerve activity (GPNA) will relate to the 3-phase respiratory motor pattern apparent in the vagal, hypoglossal, and phrenic nerves in mammals. Methods We recorded GPNA from in situ arterially perfused brainstem preparations of juvenile rats (n=10) and compared these data to simultaneously recorded phrenic (PNA), vagal (VNA), and hypoglossal (HNA) nerve activities. We report the timing of GPNA relative to onset and duration of PNA, HNA, and VNA for at least 32 respiratory cycles in each preparation. Results Preparations had a eupnea-like 3-phase respiratory motor pattern (Dutschmann et al 2009). The average respiratory cycle length was 4.30s ± 0.61 (Mean±SD). The inspiratory discharge duration analyzed from PNA was 0.73s ± 0.15; the postinspiratory discharge duration analyzed from VNA, 2.85s ± 0.69; and a late expiratory phase duration, 0.87s ± 0.46. The GPNA started significantly before the onset of PNA with a pre-inspiratory discharge duration of 0.53s ± 0.23 (ANOVA p=0.0001). Overall the pre-inspiratory discharge of GPNA was similar compared to pre-inspiratory discharge of HNA (0.36s ± 0.21), postinspiratory discharge duration of GPNA (0.53s ± 0.12) was also comparable to HNA (0.59s ± 0.26), but significantly shorter compared to VNA (2.85s ± 0.69; p=0.0001). Conclusion Both GPNA and HNA displayed robust, characteristic pre-inspiratory/inspiratory discharge patterns in every preparation. Clinically, the activation pattern of the GPNA may be as crucial as that of the HNA, for maintaining a healthy, patent upper airway for breathing during sleep. Support (if any) The Japanese Respiratory Society Fellowship Grants, NIH/NHLBI

Introduction: Biometrics of common physiologic signals can reflect health status. We have developed analytics to measure the predictability of ventilatory pattern variability (VPV, Nonlinear Complexity Index (NLCI) that quantifies the predictability of a continuous waveform associated with inhalation and exhalation) and the cardioventilatory coupling (CVC, the tendency of the last heartbeat in expiration to occur at preferred latency before the next inspiration). We hypothesized that measures of VPV and CVC are sensitive to the development of endotoxemia, which evoke neuroinflammation. Methods: We implanted Sprague Dawley male rats with BP transducers to monitor arterial blood pressure (BP) and recorded ventilatory waveforms and BP simultaneously using whole-body plethysmography in conjunction with BP transducer receivers. After baseline (BSLN) recordings, we injected lipopolysaccharide (LPS, n = 8) or phosphate buffered saline (PBS, n =3) intraperitoneally on 3 consecutive days. We recorded for 4–6 h after the injection, chose 3 epochs from each hour and analyzed VPV and CVC as well as heart rate variability (HRV). Results: First, the responses to sepsis varied across rats, but within rats the repeated measures of NLCI, CVC, as well as respiratory frequency (fR), HR, BP and HRV had a low coefficient of variation, (<0.2) at each time point. Second, HR, fR, and NLCI increased from BSLN on Days 1–3; whereas CVC decreased on Days 2 and 3. In contrast, changes in BP and the relative low-(LF) and high-frequency (HF) of HRV were not significant. The coefficient of variation decreased from BSLN to Day 3, except for CVC. Interestingly, NLCI increased before fR in LPS-treated rats. Finally, we histologically confirmed lung injury, systemic inflammation via ELISA and the presence of the proinflammatory cytokine, IL-1β, with immunohistochemistry in the ponto-medullary respiratory nuclei. Discussion: Our findings support that NLCI reflects changes in the rat’s health induced by systemic injection of LPS and reflected in increases in HR and fR. CVC decreased over the course to the experiment. We conclude that NLCI reflected the increase in predictability of the ventilatory waveform and (together with our previous work) may reflect action of inflammatory cytokines on the network generating respiration.

Introduction: Our laboratory investigates changes in the respiratory pattern during systemic inflammation in various rodent models. The endogenous cannabinoid system (ECS) regulates cytokine production and mitigates inflammation. Inflammation not only affects cannabinoid (CB) 1 and CB2 receptor gene expression (Cnr1 and Cnr2), but also increases the predictability of the ventilatory pattern. Objectives: Our primary objective was to track ventilatory pattern variability and transcription of Cnr1 and Cnr2 mRNA, and of Il1b, Il6, and tumor necrosis factor-alpha (Tnfa) mRNAs at multiple time points in central and peripheral tissues during systemic inflammation induced by peritonitis. Methods: In male Sprague Dawley rats (n=24), we caused peritonitis by implanting a fibrin clot containing either 0 or 25×106 Escherichia coli intraperitoneally. We recorded breathing with whole-animal plethysmography at baseline and 1 h before euthanasia. We euthanized the rats at 3, 6, or 12 h after inoculation and harvested the pons, medulla, lung, and heart for gene expression analysis. Results: With peritonitis, Cnr1 mRNA more than Cnr2 mRNA was correlated to Il1b, Il6, and Tnfa mRNAs in medulla, pons, and lung and changed oppositely in the pons, medulla, and lung. These changes were associated with increased predictability of ventilatory pattern. Specifically, nonlinear complexity index correlated with increased Cnr1 mRNA in the pons and medulla, and coefficient of variation for cycle duration correlated with Cnr1 and Cnr2 mRNAs in the lung. Conclusion: The mRNAs for ECS receptors varied with time during the central and peripheral inflammatory response to peritonitis. These changes occurred in the brainstem, which contains the network that generates breathing pattern and thus, may participate in ventilatory pattern changes during systemic inflammation.

Sepsis is distinguished by an overwhelming uncontrolled inflammatory response to a pathogen (e.g. Escherichia coli, E. coli). The interval from the onset of sepsis to treatment is a critical factor for patient survival. We hypothesize that ventilatory pattern variability (VPV; autocorrelation coefficient (r), Mutual Information, (MI), Sample Entropy (SampEn) and nonlinear complexity index (NLCI)), respiratory frequency (fR), and cardio-ventilatory coupling (CVC) are biometrics that vary with neural inflammation and may signal the onset of a sepsis. Male, Sprague Dawley rats (n=35) received surgically implanted pressure transducers (DSI telemetry; arterial blood pressure, (BP)), allowed 2wks to recover, then placed unanesthetized in whole-body plethysmography (Buxco) set on a receiver (EMKA) for baseline (0h) recordings. Then, we implanted them with a thrombin-fibrinogen pellet, which was either sterile (D0) or contained 25 x 106 CFU E. coli (D25). After inoculation, we recorded ventilation at 3h (D0 n=5, D25 N=5), 6h (D0 n=3, D25 n=5), 15h (D0 n=4, D25 n=4), 24h (D0 n=4, D25 n=5) and analyzed fR, VPV & CRC. We tested for differences with a 2-way, repeated-measures ANOVA, both within groups between 0h and each hour and between D0 and D25 at each hour. fR did not differ from 0h or between D0 and D25 by 3, 6 and12h; but increased by 24h (D0 and D25 (p<001)). Similarly, VPV became more predictable by 15h D25 (r at 1 cycle length, D0 0.59 ± 0.08 vs 0.69 ± 0.201, p<0.001; MI, D0 0.37 ± 0.100 vs D25 0.49 ± 0.11 bits, p<.001; and NLCI, D0 0.08 ± 0.05 vs D25 0.15 ± 0.04 p<0.001). CVC changed, in the distribution (χ2) of the intervals between the peak of the R-wave the transition from expiration-to-inspiration phase switch (EIPS) by 15h for D25 (D0, 66.3 ± 53.1 vs D25, 31.2 ± 19.1, p< 0.001). Transformed relative Shannon entropy (tRSE) for the R-wave-to-EIPS intervals did not differ at any time point. Heart rate (HR) changed at 15hr for D25 (D0, 400 ± 47 vs D25, 445 ± 33, p<0.001). These results suggest that fR, VPV and CVC biometrics change as systemic infection progresses and may be useful measures of sepsis progression over time in critically ill patients.

Little is known regarding gender differences in response to systemic infection. Progesterone peaks during proestrus and is associated with increased respiratory frequency and reduced cytokine expression during systemic infection compared to nonproestrus. We hypothesized that hormone fluctuations during the estrus cycle may not only affect respiration, but also the systemic inflammation impact on respiratory pattern. We compared baseline and changes in Ventilatory Pattern Variability (VPV, Autocorrelation coefficient (r at one‐cycle length), Mutual Information (MI), Sample Entropy (SampEn) and Nonlinear Complexity Index (NLCI)) in proestrus and nonproestrus in Sprague Dawley rats (n=44) during peritonitis and compared male (n=24) to females.

Severe septic patients manifest respiratory failure and ventilator dependence, both of which we theorize relate to brainstem inflammation leading to dysfunction of the respiratory control network. Brainstem inflammation mirrors the systemic inflammation, associated with sepsis. Here, we hypothesize that dose dependent E. coli peritonitis induce neuroinflammation that alters the central respiratory network. For this AAALAC approved study, ventilatory pattern of awake pups (Sprague Dawley males, n=28, P24‐P27) were recorded using whole‐body plethysmography for 1h before (Baseline, Bsln) and 24h after implantation surgeries (T24). Fibrin clots containing either 5x106 (D5, n=10) or 2.5x106 (D2.5, n=9) or 0 (D0, n=9) E. coli cells were implanted in the peritoneal cavity. After 24h, ventilatory pattern was recorded and the animal immediately prepared for the in situ preparation where phrenic nerve (PNA), cervical vagus nerve (VNA), thoracic sympathetic chain (tSNA) activities, heart rate and perfusion pressure were recorded. Respiratory pattern was recorded for 10 min to assess differences in patterning between groups. Whole‐body plethysmography revealed that 24h after surgery implantation only the group of D5 (TE (time of expiration) 0.24±0.03, p=0.005; TTOT (Total time of respiratory cycle) 0.40±0.02, p=0.008; fR (respiratory frequency) 155± 11, p=0.015) differed compared to D2.5 (TE 0.31±0.02; TTOT 0.50±0.02; fR 122±6) and D0 (TE 0.32±0.02; TTOT 0.505±0.03; fR121± 6). Although no significant changes for the nonlinear complex index (NLCI) were identified between the groups, NLCI trended upwards for both D2.5 (0.100±0.02) and D5 (0.106±0.02) when compared to D0 (0.064±0.01, p=0.136). In the in situ brainstem‐spinal cord preparation both D2.5 (TPI (time of Post‐inspiration) 2.89±0.40, p=0.002; TTOT 4.25±0.34, p=0.002; fR 14.8± 0.90, p=0.002) and D5 (TPI 3.01±0.16, p<0.001; TTOT 4.37±0.18, p=0.001; fR13.90± 0.50, p=0.004) differed from D0 (TPI 3.01±0.41; TTOT 5.98±0.39; fR 10.60± 0.80), but did not differ from each other. From this data, we conclude that E. coli peritonitis induce changes in breathing pattern that persist in the in situ preparation. Further increase in inoculate does not lead to increased disordered breathing.

Cardio-ventilatory coupling refers to a heartbeat (HB) occurring at a preferred latency before the onset of the next breath. We hypothesized that the pressure pulse generated by a HB activates baroreceptors that modulates brainstem expiratory neuronal activity and delays the initiation of inspiration. In supine male subjects, we recorded ventilation, electrocardiogram, and blood pressure during 20-min epochs of baseline, slow-deep breathing, and recovery. In in situ rodent preparations, we recorded brainstem activity in response to pulses of perfusion pressure. We applied a well-established respiratory network model to interpret these data. In humans, the latency between HBs and onset of inspiration was consistent across different breathing patterns. In in situ preparations, a transient pressure pulse during expiration activated a subpopulation of expiratory neurons normally active during post-inspiration; thus, delaying the next inspiration. In the model, baroreceptor input to post-inspiratory neurons accounted for the effect. These studies are consistent with baroreflex activation modulating respiration through a pauci-synaptic circuit from baroreceptors to onset of inspiration.

Rationale: Acute Lung Injury (ALI) is associated with altered breathing patterns, which are in part mediated by brainstem inflammation.This manifests as decreased ventilatory pattern variability (VPV) and increased respiratory rate (fR).IL-1β expression is increased in the nucleus tractus solitarii (nTS) and area postrama (AP) in rats with ALI.IL-1β and IL-18 are both activated by caspase-1, but IL-1β can be activated independent of this process.We hypothesized that IL-18 will be co-expressed in the nTS and AP in rats 7d after induction of ALI.Increased expression of IL-18 would provide evidence that brainstem inflammation during ALI is mediated through the caspase-1 pathway.Methods: Ventilatory patterns were recorded in adult male Sprague Dawley Rats using continuous plethysmography (1h baseline) before undergoing intratracheal injection of 100 µL phosphate buffered saline (PBS) or bleomycin (3 u).Rats were monitored until day 7, when repeat plethysmography was performed.Plethysmography was analyzed for fR and VPV, using the non-linear complexity index (NLCI).Higher NLCI is associated with greater predictability in the waveform.After plethysmography, rats were euthanized, perfused and tissues were fixed in formalin.Brainstems were harvested and frozen.Brainstems will be mounted onto slides with a cryostat before immunohistochemistry is performed with IL-18.Staining will be compared between PBS and bleomycin groups.Results: At 7 days, the PBS group (n=6) was compared to the bleomycin group (n=3).Rats injected with bleomycin had more significant weight change (loss of 37.7g±17.5 vs gain of 17±8.9g),higher fR (196±69 vs 75±9), and a higher NLCI (0.28±0.15 vs 0.06±0.02).All three measures confirm lung injury was achieved in the bleomycin group.Conclusion: VPV is altered in rats 7d after induction of bleomycin-induced ALI.Brainstem cytokine expression will be assessed in these same animals.If staining shows that the bleomycin inoculated rats have higher expression of IL-18 in the AP and NTS, similar to previous staining of IL-1β, then the hypothesis that IL-1β is activated through the caspase-1 mechanism along with IL-18 is supported.If staining shows that the AP and NTS do not stain as highly for IL-18, then the hypothesis is not supported, and it suggests IL-1β may be activated by caspase independent mechanism.Results will further our understanding of IL-1β expression mediating brainstem inflammation in ALI.

We thank Dr Swen Hülsmann for his thoughtful and supportive letter to the editor regarding our recent publication (Dhingra et al. 2020). Dr Hülsmann (2021) noted the absence of a discussion of the role of the recently described post-inspiratory complex (PiCo) in the generation of the post-inspiratory phase of the breathing pattern. Indeed, post-inspiratory control of glottal adduction is an essential element controlling expiratory airflow during the eupneic three-phase breathing pattern and during both expulsive (coughing, sneezing, etc.) and non-respiratory behaviours (swallowing, vocalisation, etc.); and loss of airway-protective post-inspiratory glottal constriction, for instance during swallowing, may result in aspiration pneumonia in neurodegenerative diseases (Dutschmann et al. 2014; Anderson et al. 2016). In our study, it was striking that respiratory local field potentials (LFPs) occurring at the transition from inspiration (I) to post-inspiration (PI) were the most widely distributed of any respiratory phase transition. In general, LFPs are thought to largely reflect the summation of synchronous synaptic conductances because of their long-time constants. Therefore, our observation suggests that synaptic conductances within many respiratory network areas were maximal precisely at the I–PI transition. This observation certainly negates the conceptual model put forward in the ‘triple-oscillator hypothesis’ that interactions between only the pre-Bötzinger complex, PiCo and parafacial respiratory group/retro-trapezoid nucleus account for the three-phase respiratory motor pattern (Anderson & Ramirez, 2017). This conclusion is further underscored by our previous publication, which demonstrated at the motor output level that perfused brainstem preparations containing all elements of the ‘triple-oscillator hypothesis’, but lacking pontine components of the network, are insufficient to generate eupnea, and instead produce a pathologic apneustic inspiratory motor pattern expressed synchronously on vagal, hypoglossal and phrenic nerves (Jones & Dutschmann, 2016). As noted in Dr Hülsmann’s letter, Anderson et al. (2016) has 100+ citations. Yet, to our knowledge, only one of those studies sought to confirm its findings (Toor et al. 2019). Together, these two papers fail to demonstrate that the PiCo is necessary for the generation of the eupneic post-inspiratory motor pattern under intact network conditions, especially when compared with similar perturbations of the activity of the Kölliker–Fuse nuclei (KFn). Inhibition of the KFn with isoguvacine completely ablates post-inspiratory laryngeal, cervical vagal nerve activity (cVNA) (Dutschmann & Herbert, 2006; Dutschmann et al. 2021), whereas inhibition of the PiCo with isoguvacine only reduces the peak amplitude of cVNA (Toor et al. 2019). Modulation of KFn activity with DAMGO, a μ-opioid receptor agonist, also results in a complete ablation of post-inspiratory cVNA (Levitt et al. 2015), whereas modulation of PiCo activity with DAMGO only reduces the amplitude of cVNA (Anderson et al. 2016). Clearly, the pons is necessary for the expression of post-inspiratory cVNA, and hence the three-phase respiratory motor pattern, and its absence disrupts PI more than any medullary counterpart including the PiCo. On the other hand, our recent work suggests to us that focusing on the pons, medulla or any particular respiratory area in isolation might also be short-sighted since local modulation of excitability within any key respiratory network area is sufficient to severely disrupt the three-phase respiratory motor pattern (Dhingra et al. 2019a,b), and since LFPs associated with the inspiratory off-switch (e.g. I–PI transition) can be detected in areas even we did not previously consider (Dhingra et al. 2020). Alternatively, Toor et al. (2019) convincingly demonstrated that the PiCo is necessary for sequential swallowing in vivo and may relate more to the ventral swallowing group (for review, see Jean, 2001). This interpretation is consistent with the fact that breathing and swallowing are coupled via the post-inspiratory phase (Dick et al. 1993; Oku, 2020). Indeed, resuscitation of the eupneic three-phase respiratory motor pattern in the perfused brainstem preparation begins first with swallowing and subsequently with swallowterminated inspiratory bursts (Bautista et al. 2014). Further, inhibition of the KFn unmasks swallow-terminated inspiratory motor patterns (Bautista & Dutschmann, 2014; Bautista et al. 2014). Perhaps the primary physiological role of the PiCo is in swallowing? In our paper, we did not include a horizontal view of the dataset since this would not allow the reader to differentiate LFPs arising from dorsal versus ventral aspects of the volume. Instead, for completeness, we chose to provide the cycle-triggered average respiratory LFPs of all electrode sites in a representative recording in Fig. 2 (Dhingra et al. 2020). Examining the medial averaged LFP traces at the level of the facial nerve (Fig. 2F and G in Dhingra et al. 2020), there were some small amplitude LFPs at the I–PI transition. Perhaps these reflected the engagement of the PiCo in the transition from I to PI? However, as discussed above and in our paper, the striking feature of the dataset as a whole was the widespread engagement of respiratory network areas in the collective transition to PI, rather than the dominance of any one area. Taken together, this feature of our dataset and the absence of any other substantial evidence supporting either the PiCo as the sole respiratory area necessary for PI or the triple-oscillator hypothesis led to our decision to forgo a discussion of these latter topics. Instead, our discussion focused on what respiratory LFPs might reflect, how respiratory LFPs might arise and the broad features of their spatio-temporal patterning. In conclusion, understanding the network-level mechanisms underlying respiratory motor pattern formation in the intact brainstem clearly remains an open challenge for the field. We hope that others will see our efforts to develop an approach to map respiratory LFPs at the spatial scale of the intact network in single perfused brainstem preparations as a first step toward the goal of providing our field with the appropriate experimental tools necessary to tackle this outstanding challenge.

Key points The functional neuroanatomy of the mammalian respiratory network is far from being understood since experimental tools that measure neural activity across this brainstem‐wide circuit are lacking. Here, we use silicon multi‐electrode arrays to record respiratory local field potentials (rLFPs) from 196–364 electrode sites within 8–10 mm3 of brainstem tissue in single arterially perfused brainstem preparations with respect to the ongoing respiratory motor pattern of inspiration (I), post‐inspiration (PI) and late‐expiration (E2). rLFPs peaked specifically at the three respiratory phase transitions, E2–I, I–PI and PI–E2. We show, for the first time, that only the I–PI transition engages a brainstem‐wide network, and that rLFPs during the PI–E2 transition identify a hitherto unknown role for the dorsal respiratory group. Volumetric mapping of pontomedullary rLFPs in single preparations could become a reliable tool for assessing the functional neuroanatomy of the respiratory network in health and disease. AbstractWhile it is widely accepted that inspiratory rhythm generation depends on the pre‐Bötzinger complex, the functional neuroanatomy of the neural circuits that generate expiration is debated. We hypothesized that the compartmental organization of the brainstem respiratory network is sufficient to generate macroscopic local field potentials (LFPs), and if so, respiratory (r) LFPs could be used to map the functional neuroanatomy of the respiratory network. We developed an approach using silicon multi‐electrode arrays to record spontaneous LFPs from hundreds of electrode sites in a volume of brainstem tissue while monitoring the respiratory motor pattern on phrenic and vagal nerves in the perfused brainstem preparation. Our results revealed the expression of rLFPs across the pontomedullary brainstem. rLFPs occurred specifically at the three transitions between respiratory phases: (1) from late expiration (E2) to inspiration (I), (2) from I to post‐inspiration (PI), and (3) from PI to E2. Thus, respiratory network activity was maximal at respiratory phase transitions. Spatially, the E2–I, and PI–E2 transitions were anatomically localized to the ventral and dorsal respiratory groups, respectively. In contrast, our data show, for the first time, that the generation of controlled expiration during the post‐inspiratory phase engages a distributed neuronal population within ventral, dorsal and pontine network compartments. A group‐wise independent component analysis demonstrated that all preparations exhibited rLFPs with a similar temporal structure and thus share a similar functional neuroanatomy. Thus, volumetric mapping of rLFPs could allow for the physiological assessment of global respiratory network organization in health and disease.

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

Cytokine production is regulated by the endogenous cannabinoid system (ECS) in many pro‐inflammatory conditions. Sepsis is a highly morbid condition that results from a dysregulated host response to infection. Our previous work showed that ventilatory pattern variability (VPV) decreased concomitantly with increased pro‐inflammatory cytokine production. However, the ECS has not been integrated with transcriptional regulation of pro‐inflammatory cytokines nor with VPV during the progression to sepsis. We hypothesized that decreases in VPV correlate with increased cannabinoid receptor (Cnr) 1 & 2 and pro‐inflammatory cytokine mRNAs. We implanted a fibrin clot that contained either 0 or 25×106E. coli in the peritoneal cavity of adult, male rats (n=24). Respiratory measures were obtained prior to clot implantation and again 1 hour (h) prior to euthanasia. At 3, 6, or 12 h following the clot implantation the rostral and caudal medulla, lung, heart and liver were harvested. VPV was computed with a group of linear and nonlinear dynamic measurements. Total RNA was extracted using Trizol reagent, and cDNA was synthesized using reverse transcription PCR. Quantitative real‐time PCR was performed in duplicate for each sample using TaqMan Gene® Expression Assays specific for 18S ribosomal RNA (18S), Glyceraldehyde 3‐phosphate dehydrogenase (Gapdh), beta actin (Actb), Cnr1 & 2, interleukin (IL) 1‐beta (Il1b) & Il6 and tumor necrosis factor‐alpha (Tnfa). The most stable reference gene combination was determined using NormFinder software and each target gene was normalized to the geometric mean of these reference genes. Relative quantitation of gene expression was performed using the comparative CT method (2−ΔΔCT method) with the 0 E. coli fibrin clot groups (n=4/group) at each time point as the comparative control. At 12 h after inoculation, expiratory duration decreased [F(2,18) = 4.01, p = 0.036] and the ventilatory waveform became more predictable (increased mutual information [F(2,18) = 5.56, p = 0.013] and decreased sample entropy [F(2,18) = 4.25, p = 0.031]). The fold change in cytokine and ECS gene expression varied with tissue. At 12 h after inoculation gene expression increased in heart and rostral medulla, while gene expression decreased in lung, caudal medulla and liver. These preliminary data and analyses support our hypothesis that decreased VPV observed at 12 h is associated with increased expression of ECS and pro‐inflammatory cytokines in heart and rostral medulla. In lung and caudal medulla, however, expression of pro‐inflammatory cytokines decreased, while Cnr1 increased in caudal medulla and Cnr2 increased in lung tissue. Further research is needed to determine how modulation of the ECS can effectively regulate proinflammatory cytokine production and changes in VPV.Support or Funding InformationNIH 5T32HL007913‐18, NIH U01 EB021960, VA Research Service I01BX004197

Sepsis is a life‐threatening disease characterized by dysregulated systemic inflammation and organ failure. We have associated an increase in predictability of the ventilatory pattern variability (VPV) with progressive development of systemic inflammation leading to sepsis. Vagal nerve stimulation (VNS) is a proposed electro‐therapeuty that reduces circulating pro‐inflammatory cytokines, like interleukin‐1 beta (IL‐1β), that are associated with systemic inflammation. We hypothesize that the anti‐inflammatory effects of VNS will be reflected in reduced predictability of VPV. Urethane‐anaesthetized adult Sprague Dawley male rats (350–400g n=17) were instrumented for blood pressure (BP), heart rate (HR), and airflow (pneumotach). The left cervical vagus was dissected, isolated and prepared for electrical stimulation via nerve cuff. Each rat was subjected to a staircase protocol of monophasic electrical stimulus pulses (0.6 mA, 0.1 ms pulse width, 10 Hz) to identify the maximal current that was subthreshold for cardiac and respiratory responses. Four experimental cohorts were then established: 1) (+stim/+E. coli) (n=4), 2) (−stim/+E. coli) (n=5), 3) (+stim/−E. coli) (n=2), 4) (−stim/−E. coli) (n=6). In +stim groups, a stimulus train was initiated 10 min before and continued for 20 min after an E. coli solution (60 μg/kg rat; 50,000 cells suspended in normal saline) was injected intravenously (IV). The effects of stimulation on the systemic inflammatory response were determined by comparing serum cytokine levels prior to stimulation to those 90 min after inoculation and the effects on VPV was quantified from non‐linear complexity index (NLCI) prior to the blood draws. Cervical VNS reduced expression of IL‐1β from 102.33 ± 49 pg/ml to 69.50 ± 28 pg/ml and reduced NLCI (0.57 ± 0.04 bits to 0.48 ± 0.03 bits; p < 0.001). VPV became more predictable with the infusion of E. Coli and VNS in the presence of E. coli infusion reduced IL‐1β expression and decreased NLCI. These data suggest that VNS impacts both peripheral and central inflammation during E. Coli infusion.Support or Funding InformationSupported by U01 EB021960, and VA I01BX004197

The sex hormones, estrogen and progesterone, affect central respiratory control. Progesterone, increases respiratory frequency (fR). Sepsis, a disease process caused by a dysregulated systemic inflammatory response, also affects respiratory control that can be quantified by Ventilatory Pattern Variability (VPV). We hypothesized that natural hormone fluctuations during the female estrus cycle affect VPV and its differential control during sepsis. The estrus cycle of female Sprague Dawley rats (n = 19) was assessed by collecting, hematoxylin/eosin staining, and identifying vaginal epithelial cells. Proestrus, in which progesterone peaks, was determined by the absence of both leukocytes and cornified multinucleated epithelial cells. Rats were inoculated with fibrin pellets containing 0 (D0) or 100x 10^ 6 (D100) E. coli cells during proestrus. Before and 12 h after inoculation, we recorded the ventilatory pattern in conscious unrestrained rats using whole‐body plethysmography. VPV [coefficient of variation (CV) for the duration of the respiratory cycle (CV‐Ttot), autocorrelation function (AC), mutual information (MI), sample entropy (SampEn), and nonlinear complexity index (NLCI)] was compared across four experimental groups: 1) D0 proestrus rats (n = 3), 2) D100 proestrus rats (n = 7), 3) D0 non‐proestrus rats (n = 4), and 4) D100 non‐proestrus rats (n = 5) using one‐way analysis of variance (ANOVA). In the non‐proestrus groups, SampEn was greater in D0 than D100 (1.31±0.011 vs 1.14±0.002 Nats, p = 0.006), but in proestrus rats, SampEn did not vary with inoculation (D0, 1.26±0.003 vs D100, 1.26±0.004 Nats, NS). However, CV‐Ttot, AC and MI trended toward but did not reach significance given the low n. These preliminary data suggest that a decrease in VPV due to septicemia occurs in nonproestrus stages, whereas did not occur in proestrus.Support or Funding InformationFunded By: VA Research Service (I01BX004197); NIH (U01 EB021960)

Sepsis is a dynamic complex condition with high patient mortality and health‐care system cost for which the understanding of underlying pathophysiology is still evolving. We hypothesize that pattern variability of centrally controlled physiological rhythms, like cardiac and respiratory rhythms, decreases with an increase in systemic inflammation, such as sepsis. In our lab, we have previously demonstrated in an animal sepsis model that respiratory pattern predictability will increase with disease onset and progression. We describe a complementary IRB approved clinical study, in which physiological waveform data were recorded continuously from consented adult patients (n=43 to date) admitted to the medical intensive care unit (MICU). Serum was collected at intervals determined clinically and subsequently quantified using Luminex assays. The electrocardiogram data was analyzed for heart rate variability (HRV) indices, including Coefficient of Variation, Poincare Plots and Power Spectral Density (high frequency power (HFP)) for the hour preceding and succeeding each serum sampling. Of the 15 patients analyzed, we identified 18 intervals between successive blood draws with a significant change in IL‐6 serum concentration, defined as a 20% relative change accompanied by >20 pg/mL absolute change. In each of the 6 time intervals where IL‐6 was significantly increased there was an associated decrease in HRV and/or HFP. In 12 time intervals IL‐6 decreased; 7 of these were associated with an increase in HRV and/or HFP. Previous studies and our own studies on humans and rats conclude that decreases in the variability, or increases in the predictability, of physiological rhythms correlate to diseased state. Our working hypothesis is that neural inflammation in the brainstem alters central regulation of cardio‐respiratory rhythms and their coupling. As sepsis has had consistent and widespread negative impact on patients, the heath‐care system, and healthcare economy, it is imperative to identify and describe biometrics that serve as early warnings to clinicians.Support or Funding InformationNIH U01 EB021960‐01A1 and VA Research Service Merit Award I01BX004197‐01A1