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Diaphragmatic Pacing for the Prevention of Sudden Unexpected Death in Epilepsy Purnell BS, Braun A, Fedele D, Murugan M, Boison D. Brain Commun. 2022;4(5):fcac232. doi:10.1093/braincomms/fcac232. PMID: 36196086; PMCID: PMC9525001 Sudden unexpected death in epilepsy is the leading cause of epilepsy related death. Currently, there are no reliable methods for preventing sudden unexpected death in epilepsy. The precise pathophysiology of sudden unexpected death in epilepsy is unclear; however, convergent lines of evidence suggest that seizure-induced respiratory arrest plays a central role. It is generally agreed that sudden unexpected death in epilepsy could be averted if the patient could be rapidly ventilated following the seizure. The diaphragm is a muscle in the chest which contracts to draw air into the lungs. Diaphragmatic pacing is a surgical intervention which facilitates normal ventilation in situations, such as spinal cord injury and sleep apnoea, in which endogenous respiration would be inadequate or non-existent. In diaphragmatic pacing, electrodes are implanted directly onto diaphragm or adjacent to the phrenic nerves which innervate the diaphragm. These electrodes are then rhythmically stimulated, thereby eliciting contractions of the diaphragm which emulate endogenous breathing. The goal of this study was to test the hypothesis that seizure-induced respiratory arrest and death can be prevented with diaphragmatic pacing. Our approach was to induce respiratory arrest using maximal electroshock seizures in adult, male, C57BL6 mice outfitted with EEG and diaphragmatic electrodes (n = 8 mice). In the experimental group, the diaphragm was stimulated to exogenously induce breathing. In the control group, no stimulation was applied. Breathing and cortical electrographic activity were monitored using whole body plethysmography and EEG, respectively. A majority of the animals that did not receive the diaphragmatic pacing intervention died of seizure-induced respiratory arrest. Conversely, none of the animals that received the diaphragmatic pacing intervention died. Diaphragmatic pacing improved postictal respiratory outcomes (two-way ANOVA, P < 0.001) and reduced the likelyhood of seizure-induced death (Fisher's exact test, P = 0.026). Unexpectedly, diaphragmatic pacing did not instantly restore breathing during the postictal period, potentially indicating peripheral airway occlusion by laryngospasm. All diaphragmatically paced animals breathed at some point during the pacing stimulation. Two animals took their first breath prior to the onset of pacing and some animals had significant apnoeas after the pacing stimulation. Sudden unexpected death in epilepsy results in more years of potential life lost than any other neurological condition with the exception of stroke. By demonstrating that seizure-induced respiratory arrest can be prevented by transient diaphragmatic pacing in animal models we hope to inform the development of closed-loop systems capable of detecting and preventing sudden unexpected death in epilepsy. [ABSTRACT FROM AUTHOR]

Abstract: The retrotrapezoid nucleus (RTN) regulates breathing in a CO2‐ and state‐dependent manner. RTN neurons are glutamatergic and innervate principally the respiratory pattern generator; they regulate multiple aspects of breathing, including active expiration, and maintain breathing automaticity during non‐REM sleep. RTN neurons encode arterial P C O 2/pH via cell‐autonomous and paracrine mechanisms, and via input from other CO2‐responsive neurons. In short, RTN neurons are a pivotal structure for breathing automaticity and arterial P C O 2 homeostasis. The carotid bodies stimulate the respiratory pattern generator directly and indirectly by activating RTN via a neuronal projection originating within the solitary tract nucleus. The indirect pathway operates under normo‐ or hypercapnic conditions; under respiratory alkalosis (e.g. hypoxia) RTN neurons are silent and the excitatory input from the carotid bodies is suppressed. Also, silencing RTN neurons optogenetically quickly triggers a compensatory increase in carotid body activity. Thus, in conscious mammals, breathing is subject to a dual and interdependent feedback regulation by chemoreceptors. Depending on the circumstance, the activity of the carotid bodies and that of RTN vary in the same or the opposite directions, producing additive or countervailing effects on breathing. These interactions are mediated either via changes in blood gases or by brainstem neuronal connections, but their ultimate effect is invariably to minimize arterial P C O 2 fluctuations. We discuss the potential relevance of this dual chemoreceptor feedback to cardiorespiratory abnormalities present in diseases in which the carotid bodies are hyperactive at rest, e.g. essential hypertension, obstructive sleep apnoea and heart failure. [ABSTRACT FROM AUTHOR]

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO₂/H⁺ changes. Their activity is also sensitive to neuromodulatory inputs from multiple respiratory centers, and thus they serve as a key nexus of respiratory control. However, molecular mechanisms that control their activity and susceptibility to neuromodulation are unknown. Here, we show in vitro and in vivo that KCNQ channels are critical determinants of RTN neural activity. In particular, we find that pharmacological block of KCNQ channels (XE991, 10 μm) increased basal activity and CO₂ responsiveness of RTN neurons in rat brain slices, whereas KCNQ channel activation (retigabine, 2- 40 μm) silenced these neurons. Interestingly, we also find that KCNQ and apamin-sensitive SK channels act synergistically to regulate firing rate of RTN chemoreceptors; simultaneous blockade of both channels led to a increase in CO₂ responsiveness. Furthermore, we also show that KCNQ channels but not SK channels are downstream effectors of serotonin modulation of RTN activity in vitro. In contrast, inhibition of KCNQ channel did not prevent modulation of RTN activity by Substance ? or thyrotropin-releasing hormone, previously identified neuromodulators of RTN chemoreception. Importantly, we also show that KCNQ channels are critical for RTN activity in vivo. Inhibition of KCNQ channels lowered the CO₂ threshold for phrenic nerve discharge in anesthetized rats and decreased the ventilatory response to serotonin in awake and anesthetized animals. Given that serotonergic dysfunction may contribute to respiratory failure, our findings suggest KCNQ channels as a new therapeutic avenue for respiratory complications associated with multiple neurological disorders. [ABSTRACT FROM AUTHOR]

Key points The retrotrapezoid nucleus (RTN) is an important site of chemoreception, i.e. the mechanism by which the brain regulates breathing in response to changes in tissue CO2/H+., Mechanisms underlying RTN chemoreception appear to involve direct CO2/H+-mediated neuronal activation and indirect neuronal activation by CO2-evoked ATP release (i.e. purinergic signalling) from astrocytes., Here, we show in vitro and in vivo that purinergic signalling in the RTN contributes to ∼30% of RTN chemoreception., Purinergic drive in the RTN involves gap junction hemichannels but not P2Y1 receptors., These results clearly indicate that purinergic signalling contributes to integrated output of the RTN during hypercapnia and thus is an important determinant of respiratory drive. [ABSTRACT FROM AUTHOR]

Central chemoreception is the mechanism by which CO2/pH sensors regulate breathing in response to tissue pH changes. There is compelling evidence that pH-sensitive neurons in the retrotrapezoid nucleus (RTN) are important chemoreceptors. Evidence also indicates that CO2/H+-evoked adenosine 5′-triphosphate (ATP) release in the RTN, from pHsensitive astrocytes, contributes to chemoreception. However, mechanism( s) by which RTN astrocytes sense pH is unknown and their contribution to chemoreception remains controversial. Here, we use the brain slice preparation and a combination of patch-clamp electrophysiology and immunohistochemistry to confirm that RTN astrocytes are pH sensitive and to determine mechanisms by which they sense pH. We show that pH-sensitive RTN glia are immunoreactive for aldehyde dehydrogenase 1L1, a marker of astrocytes. In HEPES buffer the pH-sensitive current expressed by RTN astrocytes reversed near EK+ (the equilibrium potential for K+) and was inhibited by Ba2+ and desipramine (blocker of Kir4.1-containing channels), characteristics most consistent with heteromeric Kir4.1–Kir5.1 channels. In bicarbonate buffer, the sodium/bicarbonate cotransporter also contributed to the CO2/H+-sensitive current in RTN astrocytes. To test the hypothesis that RTN astrocytes contribute to chemoreception by a purinergic mechanism, we used fluorocitrate to selectively depolarize astrocytes while measuring neuronal activity. We found that fluorocitrate increased baseline activity and pH sensitivity of RTN neurons by a P2-receptor– dependent mechanism, suggesting that astrocytes may release ATP to activate RTN chemoreceptors. We also found in bicarbonate but not HEPES buffer that P2-receptor antagonists decreased CO2 sensitivity of RTN neurons. We conclude that RTN astrocytes sense CO2/H+ in part by inhibition of a Kir4.1–Kir5.1-like current and may provide an excitatory purinergic drive to pH-sensitive neurons. [ABSTRACT FROM AUTHOR]