The Impact of Inflammation on Thermal Hyperpnea: Relevance for Heat Stress and Febrile Seizures.

Extreme heat caused by climate change is increasing transmission of infectious diseases resulting in a sharp rise in heat-related illness and mortality. Understanding mechanistic link between heat, inflammation and disease is thus important for public health. Thermal hyperpnea, and consequent respiratory alkalosis is crucial in febrile seizures and convulsions induced by heat stress in humans. Here we address what causes thermal hyperpnea in neonates and how is it affected by inflammation. TRPV1, a heat-activated channel is sensitized by inflammation and modulates breathing, and thus may play a key role. To investigate whether inflammatory sensitization of TRPV1 modifies neonatal ventilatory responses to heat stress, leading to respiratory alkalosis and an increased susceptibility to hyperthermic seizures we treated neonatal rats with bacterial lipopolysaccharide, and breathing, arterial pH, in-vitro vagus nerve activity, and seizure susceptibility were assessed during heat stress in the presence or absence of a TRPV1 antagonist (AMG-9810) or shRNA-mediated TRPV1 suppression. Lipopolysaccharide-induced inflammatory preconditioning lowered the threshold temperature and latency of hyperthermic seizures. This was accompanied by increased tidal volume, minute ventilation, expired CO2, and arterial pH (alkalosis). Lipopolysaccharide exposure also elevated vagal spiking and intracellular calcium levels in response to hyperthermia. TRPV1 inhibition with AMG-9810 or shRNA reduced the lipopolysaccharide-induced susceptibility to hyperthermic seizures and altered the breathing pattern to fast shallow breaths (tachypnea), making each breath less efficient and restoring arterial pH. These results indicate that inflammation exacerbates thermal hyperpnea-induced respiratory alkalosis associated with increased susceptibility to hyperthermic seizures, primarily mediated by TRPV1 localized to vagus neurons.

Mechanisms of infantile epileptic spasms syndrome: What have we learned from animal models?

The devastating developmental and epileptic encephalopathy of infantile epileptic spasms syndrome (IESS) has numerous causes, including, but not limited to, brain injury, metabolic, and genetic conditions. Given the stereotyped electrophysiologic, age-dependent, and clinical findings, there likely exists one or more final common pathways in the development of IESS. The identity of this final common pathway is unknown, but it may represent a novel therapeutic target for infantile spasms. Previous research on IESS has focused largely on identifying the neuroanatomic substrate using specialized neuroimaging techniques and cerebrospinal fluid analysis in human patients. Over the past three decades, several animal models of IESS were created with an aim to interrogate the underlying pathogenesis of IESS, to identify novel therapeutic targets, and to test various treatments. Each of these models have been successful at recapitulating multiple aspects of the human IESS condition. These animal models have implicated several different molecular pathways in the development of infantile spasms. In this review we outline the progress that has been made thus far using these animal models and discuss future directions to help researchers identify novel treatments for drug-resistant IESS.

Febrile seizures lead to prolonged epileptiform activity and hyperoxia that when blocked prevents learning deficits.

OBJECTIVE: In adult brain tissue, oxygen levels typically remain in the normoxic zone, but status epilepticus results in hyperoxia, whereas brief self-terminating seizures lead to postictal hypoxia. The dynamic changes in oxygen levels and the underlying mechanisms are unknown in juveniles with febrile seizures. METHODS: Eight-day-old female and male rat pups were implanted with an electrode and oxygen-sensing optode in the hippocampus and then received once daily injections of lipopolysaccharide for 4 days to induce an immune response. Local partial pressure of oxygen (pO2 ) and local field potentials were recorded before, during, and after a heat-induced febrile seizure. Separate groups of pups received injections of vehicle or drugs targeting cyclooxygenase (COX)-1, COX-2, L-type calcium channels (LTCCs), and cannabinoid receptor type 1 (CB1) and transient receptor potential vanilloid-1 (TRPV1) receptors prior to febrile seizure induction to determine pO2 mechanisms. Following febrile seizures, a subset of pups were raised to young adulthood and then tested for learning impairments using the novel object recognition task. RESULTS: Febrile seizures resulted in predictable oxygen dynamics that were related to behavioral seizures and epileptiform activity. During a behavioral seizure, pO2 rapidly increased, rapidly decreased, and then returned to near baseline. When the behavioral seizure terminated, oxygen levels climbed into the hyperoxic zone during a time of prolonged epileptiform activity. When epileptiform activity terminated, oxygen levels slowly returned to baseline. A COX-1 antagonist prevented hyperoxia, whereas a COX-2 antagonist did not. An LTCC antagonist exacerbated hyperoxia. Boosting levels of an endocannabinoid also exacerbated hyperoxia, whereas blocking CB1 receptors and TRPV1 receptors reduced hyperoxia. Inhibiting TRPV1 receptors during a febrile seizure prevented learning deficits in young adult female rats. SIGNIFICANCE: Brain oxygenation during and following a febrile seizure has a distinct pattern and multiple mechanisms. Brain oxygen dynamics may be an important consideration in the development of treatments for febrile seizures.

Animal Models in Epileptic Spasms and the Development of Novel Treatment Options.

SUMMARY: The infantile spasms (IS) syndrome is a catastrophic developmental epileptic encephalopathy syndrome characterized by an age-specific expression of epileptic spasms that are associated with extremely abnormal, oftentimes described as chaotic, interictal EEG pattern known as hypsarrhythmia. Patients with IS generally have poor neurodevelopmental outcomes, in large part because of the frequent epileptic spasms and interictal EEG abnormalities. Current first-line treatments such as adrenocorticotropic hormone or vigabatrin are often ineffective and are associated with major toxic side effects. There is therefore a need for better and safer treatments for patients with IS, especially for the intractable population. Hope is on the horizon as, over the past 10 years, there has been robust progress in the development of etiology-specific animal models of IS. These models have been used to identify potential new treatments for IS and are beginning to provide some important insights into the pathophysiological substrates for this disease. In this review, we will highlight strengths and weaknesses of the currently available animal models of IS in addition to new insights into the pathophysiology and treatment options derived from these models.

Seizure modulation by the gut microbiota and tryptophan-kynurenine metabolism in an animal model of infantile spasms.

BACKGROUND: The infantile spasms syndrome is an early-onset epileptic encephalopathy presenting in the first 2 years of life, often with severe developmental consequences. The role of the gut microbiota and metabolism in infantile spasms remains unexplored. METHODS: Employing a brain injury neonatal rat model of infantile spasms intractable to anticonvulsant medication treatments, we determined how the ketogenic diet and antibiotics affected specific microbial communities and the resultant circulating factors that confer spasms protection in the infantile spasms model. To confirm a role of kynurenine metabolism pathway in spasms protection, indoleamine 2,3-dioxygenase 1 was pharmacologically inhibited and comprehensive metabolomics was applied. FINDINGS: We show that antibiotics reduced spasms and improved the effectiveness of the ketogenic diet when given in combination. Examination of the gut microbiota and metabolomics showed the downregulation of indoleamine 2,3-dioxygenase 1 and upregulation of hippocampal kynurenic acid, a metabolite with antiepileptic effects. To further test the involvement of indoleamine 2,3-dioxygenase 1, a specific antagonist 1-methyltryptophan and minocycline, an antibiotic and inhibitor of kynurenine formation from tryptophan, were administered, respectively. Both treatments were effective in reducing spasms and elevating hippocampal kynurenic acid. A fecal microbiota transplant experiment was then performed to examine the contribution of the gut microbiota on spasm mitigation. Transplant of feces of ketogenic diet animals into normal diet animals was effective in reducing spasms. INTERPRETATION: These results highlight the importance of tryptophan-kynurenine metabolism in infantile spasms and provide evidence for new-targeted therapies such as indoleamine 2,3-dioxygenase 1 inhibition or microbiota manipulation to promote kynurenic acid production as a strategy to reduce spasms in infantile spasms. FUNDING: This study was funded by the Alberta Children's Hospital Research Institute and the Owerko Centre.

The link between brain acidosis, breathing and seizures: a novel mechanism of action for the ketogenic diet in a model of infantile spasms.

Infantile spasms (IS) syndrome is a catastrophic, epileptic encephalopathy of infancy that is often refractory to current antiepileptic therapies. The ketogenic diet (KD) has emerged as an alternative treatment for patients with medically intractable epilepsy, though the prospective validity and mechanism of action for IS remains largely unexplored. We investigated the KD's efficacy as well as its mechanism of action in a rodent model of intractable IS. The spasms were induced using the triple-hit paradigm and the animals were then artificially reared and put on either the KD (4:1 fats: carbohydrate + protein) or a control milk diet (CM; 1.7:1). 31Phosphorus magnetic resonance spectroscopy (31P MRS) and head-out plethysmography were examined in conjunction with continuous video-EEG behavioural recordings in lesioned animals and sham-operated controls. The KD resulted in a peripheral ketosis observed both in the blood and urine. The KD led to a robust reduction in the frequency of spasms observed, with approximately a 1.5-fold increase in the rate of survival. Intriguingly, the KD resulted in an intracerebral acidosis as measured with 31P MRS. In addition, the respiratory profile of the lesioned rats on the KD was significantly altered with slower, deeper and longer breathing, resulting in decreased levels of expired CO2. Sodium bicarbonate supplementation, acting as a pH buffer, partially reversed the KD's protective effects on spasm frequency. There were no differences in the mitochondrial respiratory profiles in the liver and brain frontal cortex measured between the groups, supporting the notion that the effects of the KD on breathing are not entirely due to changes in intermediary metabolism. Together, our results indicate that the KD produces its anticonvulsant effects through changes in respiration leading to intracerebral acidosis. These findings provide a novel understanding of the mechanisms underlying the anti-seizure effects of the KD in IS. Further research is required to determine whether the effects of the KD on breathing and intracerebral acid-base balance are seen in other paediatric models of epilepsy.

Impaired cardiorespiratory responses to hypercapnia in neonatal mice lacking PAC1 but not VPAC2 receptors.

The evidence is mounting for a role for abnormal signaling of the stress peptide pituitary adenylate cyclase activating polypeptide (PACAP) and its canonical receptor PAC1 in the pathogenesis of sudden infant death syndrome. In this study, we investigated whether the PACAP receptors PAC1 or VPAC2 are involved in the neonatal cardiorespiratory response to hypercapnic stress. We used head-out plethysmography and surface ECG electrodes to assess cardiorespiratory responses to an 8% hypercapnic challenge in unanesthetized and spontaneously breathing 4-day-old PAC1 or VPAC2 knockout (KO) and wild-type mouse pups. We demonstrate that compared with WTs, breathing frequency (RR) and minute ventilation ([Formula: see text]) in PAC1 KO pups were significantly blunted in response to hypercapnia. Although heart rate was unaltered in PAC1 KO pups during hypercapnia, heart rate recovery posthypercapnia was impaired. In contrast, cardiorespiratory impairments in VPAC2 KO pups were limited to only an overall higher tidal volume (VT), independent of treatment. These findings suggest that PACAP signaling through the PAC1 receptor plays a more important role than signaling through the VPAC2 receptor in neonatal respiratory responses to hypercapnia. Thus deficits in PACAP signaling primarily via PAC1 may contribute to the inability of infants to mount an appropriate protective response to homeostatic stressors in childhood disorders such as SIDS.

Impaired neonatal cardiorespiratory responses to hypoxia in mice lacking PAC1 or VPAC2 receptors.

The stress peptide pituitary adenylate cyclase activating polypeptide (PACAP) and its specific receptor PACAP type 1 receptor (PAC1) have been implicated in sudden infant death syndrome (SIDS). PACAP is also critical to the neonatal cardiorespiratory response to homeostatic stressors identified in SIDS, including hypoxia. However, which of PACAP's three receptors, PAC1, vasoactive intestinal peptide receptor type 1 (VPAC1), and/or vasoactive intestinal peptide receptor type 2 (VPAC2), are involved is unknown. In this study, we hypothesized that PAC1, but not VPAC2, is involved in mediating the cardiorespiratory response to hypoxia during neonatal development. To test this hypothesis, head-out plethysmography and surface ECG electrodes were used to assess the cardiorespiratory variables of unanesthetized postnatal day 4 PAC1 and VPAC2-knockout (KO) and wild-type (WT) mice in response to a 10% hypoxic challenge. Our results demonstrate that compared with WT pups, the early and late hypoxic rate of expired CO2 (V̇co2), V̇co2 and ventilatory responses were blunted in PAC1-KO neonates, and during the posthypoxic period, minute ventilation (V̇e), V̇co2 and heart rate were increased, while the increase in apneas normally associated with the posthypoxic period was reduced. Consistent with impaired cardiorespiratory control in these animals, the V̇e/V̇co2 slope was reduced in PAC1-KO pups, suggesting that breathing was inappropriately matched to metabolism. In contrast, VPAC2-KO pups exhibited elevated heart rate variability during hypoxia compared with WT littermates, but the effects of the VPAC2-KO genotype on breathing were minimal. These findings suggest that PAC1 plays the principal role in mediating the cardiorespiratory effects of PACAP in response to hypoxic stress during neonatal development and that defective PACAP signaling via PAC1 may contribute to the pathogenesis of SIDS.

Vagal TRPV1 activation exacerbates thermal hyperpnea and increases susceptibility to experimental febrile seizures in immature rats.

Thermal hyperpnea, a pattern of breathing during hyperthermia that is characterized by an increase in tidal volume as well as breathing frequency, is known to lead to respiratory alkalosis. Thermal hyperpnea-induced respiratory alkalosis is linked to febrile seizures (FS). The heat-sensitive transient receptor potential vanilloid-1 (TRPV1) receptors are localized in, and implicated in the heat sensitivity of peripheral and central structures involved in the respiratory response to hyperthermia. We, therefore, hypothesize that TRPV1 activation increases susceptibility to experimental FS (EFS) in immature rats due to an exacerbated thermal hyperpnea. We found that peripheral, but not central TRPV1 activation had pro-convulsant effects. These pro-convulsant effects were associated with an increased rate of expired CO2 due to an exaggerated ventilatory response to hyperthermia. The TRPV1 antagonist, AMG-9810, and TRPV1 deletion abolished the pro-convulsant effects, while exposure to 5% CO2, bilateral vagotomy and DREADD (designer receptor exclusively activated by designer drugs)-mediated inhibition of TRPV1-containing cells in the vagal nodose ganglia significantly attenuated these effects. These findings suggest that vagal TRPV1-driven thermal hyperpnea likely increases susceptibility to FS in immature rodents. This identifies a novel peripheral anatomical and molecular target that should be considered when developing therapeutics for FS.

Pituitary adenylate cyclase-activating polypeptide drives cardiorespiratory responses to heat stress in neonatal mice.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has emerged as a principal and rate-limiting regulator of physiological stress responses in adult rodents and has been implicated in sudden infant death syndrome (SIDS). Recent studies show that PACAP plays a role in neonatal cardiorespiratory responses to hypoxia, hypercapnia, and hypothermia, but not hyperthermia, which is often associated with SIDS. Here we tested the hypothesis that, consistent with a role in SIDS, PACAP is involved in regulating the neonatal cardiorespiratory responses to severe heat. To address this, we used head-out plethysmography and surface ECG electrodes to study the cardiorespiratory physiology of conscious neonatal PACAP-null and wild-type mice at ambient temperatures of 32°C (baseline) and 40°C (heat stress). We also assessed body surface temperature as an indicator of cutaneous heat loss. Our results show that wild-type neonatal mice respond to heat stress by increasing ventilation (P = 0.007) and associated expired CO2 (P = 0.041), heart rate (P < 0.001), and cutaneous heat loss (P < 0.001). In PACAP-null neonates, this heat response is impaired, as indicated by a decrease in ventilation (P = 0.04) and associated expired CO2 (P = 0.006) and a blunted increase in heart rate (P = 0.001) and cutaneous heat loss (P = 0.0002). In addition, heart rate variability at baseline was lower in PACAP-null neonates than wild-type controls (P < 0.01). These results suggest that, during heat stress, PACAP is important for neonatal cardiorespiratory responses that help regulate body temperature. Abnormal PACAP regulation could, therefore, contribute to neonatal disorders in which the autonomic response to stress is impaired, such as SIDS.

TRPV1 deletion exacerbates hyperthermic seizures in an age-dependent manner in mice.

Febrile seizures (FS) are the most common seizure disorder to affect children. Although there is mounting evidence to support that FS occur when children have fever-induced hyperventilation leading to respiratory alkalosis, the underlying mechanisms of hyperthermia-induced hyperventilation and links to FS remain poorly understood. As transient receptor potential vanilloid-1 (TRPV1) receptors are heat-sensitive, play an important role in adult thermoregulation and modulate respiratory chemoreceptors, we hypothesize that TRPV1 activation is important for hyperthermia-induced hyperventilation leading to respiratory alkalosis and decreased FS thresholds, and consequently, TRPV1 KO mice will be relatively protected from hyperthermic seizures. To test our hypothesis we subjected postnatal (P) day 8-20 TRPV1 KO and C57BL/6 control mice to heated dry air. Seizure threshold temperature, latency and the rate of rise of body temperature during hyperthermia were assessed. At ages where differences in seizure thresholds were identified, head-out plethysmography was used to assess breathing and the rate of expired CO2 in response to hyperthermia, to determine if the changes in seizure thresholds were related to respiratory alkalosis. Paradoxically, we observed a pro-convulsant effect of TRPV1 deletion (∼4min decrease in seizure latency), and increased ventilation in response to hyperthermia in TRPV1 KO compared to control mice at P20. This pro-convulsant effect of TRPV1 absence was not associated with an increased rate of expired CO2, however, these mice had a more rapid rise in body temperature following exposure to hyperthermia than controls, and the expected linear relationship between body weight and seizure latency was absent. Based on these findings, we conclude that deletion of the TRPV1 receptor prevents reduction in hyperthermic seizure susceptibility in older mouse pups, via a mechanism that is independent of hyperthermia-induced respiratory alkalosis, but possibly involves impaired development of thermoregulatory mechanisms, although at present the mechanism remain unknown.

Partial Raphe Dysfunction in Neurotransmission Is Sufficient to Increase Mortality after Anoxic Exposures in Mice at a Critical Period in Postnatal Development.

Sudden infant death syndrome (SIDS) cases often have abnormalities of the brainstem raphe serotonergic (5-HT) system. We hypothesize that raphe dysfunction contributes to a failure to autoresuscitate from multiple hypoxic events, leading to SIDS. We studied autoresuscitation in two transgenic mouse models in which exocytic neurotransmitter release was impaired via conditional expression of the light chain from tetanus toxin (tox) in raphe neurons expressing serotonergic bacterial artificial chromosome drivers Pet1 or Slc6a4. These used recombinase drivers targeted different portions of medullary raphe serotonergic, tryptophan hydroxylase 2 (Tph2)(+) neurons by postnatal day (P) 5 through P12: approximately one-third in triple transgenic Pet1::Flpe, hßactin::cre, RC::PFtox mice; approximately three-fourths inSlc6a4::cre, RC::Ptox mice; with the first model capturing a near equal number of Pet1(+),Tph2(+) versus Pet1(+),Tph2(low or negative) raphe cells. At P5, P8, and P12, "silenced" mice and controls were exposed to five, ∼37 s bouts of anoxia. Mortality was 5-10 times greater in "silenced" pups compared with controls at P5 and P8 (p = 0.001) but not P12, with cumulative survival not differing between experimental transgenic models. "Silenced" pups that eventually died took longer to initiate gasping (p = 0.0001), recover heart rate (p = 0.0001), and recover eupneic breathing (p = 0.011) during the initial anoxic challenges. Variability indices for baseline breathing distinguished "silenced" from controls but did not predict mortality. We conclude that dysfunction of even a portion of the raphe, as observed in many SIDS cases, can impair ability to autoresuscitate at critical periods in postnatal development and that baseline indices of breathing variability can identify mice at risk. SIGNIFICANCE STATEMENT: Many sudden infant death syndrome (SIDS) cases exhibit a partial (∼26%) brainstem serotonin deficiency. Using recombinase drivers, we targeted different fractions of serotonergic and raphe neurons in mice for tetanus toxin light chain expression, which prevented vesicular neurotransmitter release. In one model, approximately one-third of medullary Tph2(+) neurons are silenced by postnatal (P) days 5 and 12, along with some Pet1(+),Tph2(low or negative) raphe cells; in the other, approximately three-fourths of medullary Tph2(+) neurons, also with some Tph2(low or negative) cells. Both models demonstrated excessive mortality to anoxia (a postulated SIDS stressor) at P5 and P8. We demonstrated fatal vulnerability to anoxic stress at a specific time in postnatal life induced by a partial defect in raphe function. This models features of SIDS.

Analysis of PAC1 receptor gene variants in Caucasian and African American infants dying of sudden infant death syndrome.

AIM: Stress peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), has been implicated in sudden infant death syndrome (SIDS). The aim of this exploratory study was to determine whether variants in the gene encoding the PACAP-specific receptor, PAC1, are associated with SIDS in Caucasian and African American infants. METHODS: Polymerase chain reaction and Sanger DNA sequencing was used to compare variants in the 5'-untranslated region, exons and intron-exon boundaries of the PAC1 gene in 96 SIDS cases and 96 race- and gender-matched controls. RESULTS: The intron 3 variant, A/G: rs758995 (variant 'h'), and the intron 6 variant, C/T: rs10081254 (variant 'n'), were significantly associated with SIDS in Caucasians and African Americans, respectively (p < 0.05). Also associated with SIDS were interactions between the variants rs2302475 (variant 'i') in PAC1 and rs8192597 and rs2856966 in PACAP among Caucasians (p < 0.02) and rs2267734 (variant 'q') in PAC1 and rs1893154 in PACAP among African Americans (p < 0.01). However, none of these differences survived post hoc analysis. CONCLUSION: Overall, this study does not support a strong association between variants in the PAC1 gene and SIDS; however, a number of potential associations between race-specific variants and SIDS were identified that warrant targeted investigations in future studies.

Subtle alterations in breathing and heart rate control in the 5-HT1A receptor knockout mouse in early postnatal development.

We hypothesized that absence of the 5-HT(1A) receptor would negatively affect the development of cardiorespiratory control. In conscious wild type (WT) and 5-HT(1A) receptor knockout (KO) mice, we measured resting ventilation (Ve), oxygen consumption (Vo(2)), heart rate (HR), breathing and HR variability, and the hypercapnic ventilatory response (HCVR) at postnatal day 5 (P5), day 15 (P15), and day 25 (P25). In KO mice compared with WT, we found a 17% decrease in body weight at only P5 (P < 0.01) and no effect on Vo(2). Ve was significantly (P < 0.001) lower at P5 and P25, but there was no effect on the HCVR. Breathing variability (interbreath interval), measured by standard deviation, the root mean square of the standard deviation (RMSSD), and the product of the major (L) and minor axes (T) of the Poincaré first return plot, was 57% to 187% higher only at P5 (P < 0.001). HR was 6-10% slower at P5 (P < 0.001) but 7-9% faster at P25 (P < 0.001). This correlated with changes in the spectral analysis of HR variability; the low frequency to high frequency ratio was 47% lower at P5 but 68% greater at P25. The RMSSD and (L × T) of HR variability were ~2-fold greater at P5 only (P < 0.001; P < 0.05). We conclude that 5-HT(1A) KO mice have a critical period of potential vulnerability at P5 when pups hypoventilate and have a slower respiratory frequency and HR with enhanced variability of both, suggesting abnormal maturation of cardiorespiratory control.

Functional expression of pattern recognition receptors in tissues of the human female reproductive tract.

The human female reproductive tract (FRT) must balance the requirements of procreation with the demands of protection from pathogen invasion. We hypothesize that the FRT expresses functional pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) proteins that may mediate these tasks. Expression of PRRs was evaluated in FRT tissues by RT-PCR. PRR function within FRT tissue cells was determined by CXCL8 (IL-8) production in response to treatment with PRR agonists. We now report that TLRs7-9 are expressed in Fallopian tube, uterine endometrium, cervix and ectocervix, while TLR10 expression is restricted to Fallopian tube. NOD1 and NOD2 and the signal transducer RICK were detected in all FRT tissues. Stimulation of FRT tissue cells with PRR ligands resulted in secretion of CXCL8. Results of these studies indicate that PRRs are functionally expressed in FRT tissues, and suggest that these receptors mediate microbial recognition and immune defense in the reproductive tract.