Most studies in comparative immunology involve investigations into the detailed mechanisms of the immune system of a non-model organism. Although this approach has been insightful, it has promoted a deep understanding of only a handful of species, thus inhibiting the recognition of broad taxonomic patterns. Here, we call for investigating the immune defenses of numerous species within a pointillist framework, that is, the meticulous, targeted collection of data from dozens species and investigation of broad patterns of organismal, ecological, and evolutionary forces shaping those patterns. Without understanding basic immunological patterns across species, we are limited in our ability to extrapolate and/or translate our findings to other organisms, including humans. We illustrate this point by focusing predominantly on the biological scaling literature with some integrations of the pace of life literature, as these perspectives have been the most developed within this framework. We also highlight how the more traditional approach in comparative immunology works synergistically with a pointillist approach, with each approach feeding back into the other. We conclude that the pointillist approach promises to illuminate comprehensive theories about the immune system and enhance predictions in a wide variety of domains, including host-parasite dynamics and disease ecology.

Currently, neuraxial analgesia is the most effective method of analgesia during childbirth. However, the leading side effect, hypotension, makes it crucial to understand the existing treatments for spinal anesthesia-induced hypotension. This study attempts to elucidate anesthesia-induced hypotension and existing therapies. Researchers performed a scoping review using PubMed and Rayyan to select articles. Inclusion criteria were peer-reviewed articles written in English within the last five years, and nulligravida/primigravida women under 35 years old who reside in the USA. Researchers revealed the different means of treatment to reduce the incidence of spinal anesthesia-induced hypotension in mothers during C-sections. Norepinephrine and epinephrine showed to be more effcacious in maintaining blood pressure while avoiding unfavorable sequelae for the mother following delivery. The study outcome of participants receiving fixed-rate infusions of either norepinephrine or phenylephrine favored the use of norepinephrine. These patients exhibit lower rates of bradycardia ( p=0.004), contributing to a lesser need for bolus rescue of atropine ( p=0.01). Using colloid solutions during C-section compared to crystalloid solutions also revealed a lower incidence of hypotension with a p-value of < 0.00001. Modalities such as wrapping and subsequent elevation of the lower extremities also displayed a significantly higher systolic and diastolic blood pressure and ephedrine dose than the control group. Pharmacological treatment modalities are more effective than non-pharmacological treatments at preventing maternal hypotension. Epinephrine was the most effective treatment for maintaining maternal blood pressure, heart rate, and cardiac output. Further studies should be done to determine the optimal epinephrine dosage. There was no funding provided for this research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The central CO2-chemoreflex is crucial in regulating breathing, providing stimulation at rest and during challenges to homeostasis. The retrotrapezoid nucleus (RTN), located in the ventral brainstem, is essential to this process. Impaired RTN function is linked to hypoventilation syndromes, including congenital central hypoventilation syndrome (CCHS) characterized by apnea and blunted responsiveness to inhaled CO2. Intriguingly, progestins stimulate breathing and CO2-chemoreflexes, and the high-affnity ligand etonogestrel (ETO) has been proposed as a treatment option in CCHS. Our objectives were to address two important gaps-in-knowledge: 1) assess ETO’s ability to “rescue” breathing in a rat model of central CO2-chemoreflex impairment, and 2) determine ETO’s site of action. We generated a model of CO2-chemoreflex impairment in adult female Sprague-Dawley rats using targeted injections of substance-P conjugated saporin toxin. Resulting RTN lesions were classified by the number of surviving chemosensitive neurons vs. non-surgical controls. We first administered systemic ETO (acute/vehicle:1h; or chronic/sham: 28 days) and measured resting ventilation and chemoreflexes (hypercapnia, 7.2% CO2; hypoxia, 10% O2) using whole-body plethysmography. In separate ongoing experiments, ETO was infused directly into target brain regions expressing progesterone receptors. We observed a positive relationship between lesion size and CO2-chemoreflex impairment (hypoxic response was intact). Twenty-one days of systemic ETO reversed CO2-impairment selectively in rats with moderate RTN lesions; large-lesion rats were refractory to ETO’s effects. These data suggest that ETO may enhance the CO2-chemoreflex by inducing neuroplasticity in brain structures with connectivity to surviving RTN chemosensitive neurons. Experiments are ongoing to parse out the site of ETO’s restorative action in the brain. T.A.J.:Canadian Lung Association BaO Fellowship, Canadian Institutes of Health Research (CIHR) Postdoctoral Fellowships. S.C.: Canadian Lung Association BaO Fellowship. S.P.: Women and Children’s Health Research Institute Innovation Grant, CIHR Project scheme grant. Nexplanon was kindly donated by Merck through the Investigator-Initiated Study Program (IISP # 55869; SP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GLP-1 (Glucagon-Like Peptide-1) is a type of gastrointestinal hormone that plays an integral role in regulating glucose homeostasis and appetite control. GLP-1 receptors are expressed throughout the body, and it has been suggested that GLP-1 can be detected by vagal afferent nerve activity, leading to changes in sympathetic nerve activity. However, the details of the effects of GLP-1 receptor stimulation on autonomic nerve activity have not been reported. Therefore, this study investigated the effects of different sites of GLP-1 receptor stimulation on autonomic nerve activity by administering GLP-1 receptor agonists via three routes: intravenous (IV), intraportal vein (iport), and intraperitoneal (iperi). Male Wistar rats were chronically implanted with electrodes, catheters for measuring cervical vagal activity, renal and lumbar sympathetic nerve activity, electroencephalogram, electromyogram, electrocardiogram, arterial pressure, sensors for tissue fluid glucose concentration, and catheters for drug administration in the vein, portal vein, and abdominal cavity. Exendin-4, a GLP-1 receptor agonist, was administered to conscious rats via one of the following routes: intravenous, intra-portal vein, or intra-abdominal. Cervical vagal activity was increased in all routes of Exendin-4 administration. Renal sympathetic nerve activity was rapidly decreased by Exendin-4 administration and gradually recovered to pre-administration levels. There were no significant differences in renal sympathetic nerve activity among the different routes of Exendin-4 administration. On the other hand, lumbar sympathetic nerve activity increased slowly after Exendin-4 administration through the iperi and iport routes, while it decreased initially, then gradually increased following administration through the IV route. These results demonstrate that stimulation of GLP-1 receptors by Exendin-4 increases vagal nerve activity, decreases renal sympathetic nerve activity, and produces regionally different responses in lumbar sympathetic nerve activity depending on the site of stimulation. JST (JPMJMS2023). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Environment-induced heat stress (EIHS) is caused by a sustained elevation in body temperature due to prolonged exposure to excess heat and humidity. Given the current and increasing frequency of EIHS and the large gap in knowledge regarding EIHS-mediated impacts to the myocardium, the purpose of this investigation was to determine the extent EIHS alters cardiac health. We hypothesized EIHS would cause myocardial injury and cellular dysfunction. To test this hypothesis, 13 w old male C57 mice were exposed to EIHS for 4 h or 8 h (n=7; 37.7 ± 0.10 °C, 38.3 ± 0.5 % humidity) or thermoneutral (TN) (n=7, 27.7 ± 0.15 °C, 42.5 ± 0.43% humidity) conditions, hearts were removed, weighed, and portions of the heart were fixed for histology or frozen for biochemical measures. Environment-induced HS increased subcutaneous temperature following 4 and 8 h by 0.9 °C (p< 0.01) compared to respective TN temperatures and decreased body weight by 6.8% (p=0.01) at 4 h and 13% (p<0.01) at 8 h compared to TN. Histological inspection revealed that EIHS increased anisokaryosis by 1.4-fold following 4 h (p<0.01) and by 75% following 8 h (p=0.01) compared to TN animals. Environment-induced HS increased relative abundance of malondialdehyde-modified proteins (35%, p=0.02), a marker of oxidative stress, and increased relative protein abundance of SOD2 (72%, p=0.05) following 4 h compared to TN. Eight hours of EIHS increased relative protein abundance of SOD1 (69%, p=0.01) compared to TN and increased catalase and GPX1 compared to TN (91%, p<0.01; 2-fold, p<0.01) and compared to 4 h of EIHS (72%, p=0.03; 34%, p=0.01), though MDA-modified proteins were similar to TN. Following 4 h of EIHS, markers of mitochondrial abundance, including electron transport chain complexes II (69%, p=0.02) and V (36%, p=0.03), VDAC2 (79%, p=0.01) and PHB1 (65%, p=0.03) were increased compared to TN, and PDH (35%, p=0.04) and Cox IV (55%, p=0.01) were increased compared to 8 hrs of EIHS. Environment-induced HS appeared to alter mitochondrial fusion and fission as DRP1 (51%, p=0.05), phosphorylated (p)-DRP1 (47%, p=0.04), FIS1 (57%, p=0.01) and Mfn1 (26%, p=0.03) were increased compared to TN following 4 h and Mfn2 was increased (37%, p=0.02) following 8 h of EIHS compared to TN. Mitophagy markers BNIP3L/NIX and Pink1 were increased by EIHS following 4 h compared to TN (78%, p=0.02; 41%, p=0.03). Environment-induced HS increased autophagy markers ULK1 (36%, p<0.01), PI3K (80%, p<0.01), and ATG7 (49%, p<0.01) following 4 h compared to TN and ULK1 (33%, p<0.01) and ATG7 (41%, p=0.02) following 4 h compared to 8 h of EIHS. However, p-ULK1, Beclin, p-Beclin, ATG12, and ATG16 were similar between groups. Relative protein abundance of p62 and LC3 II were similar between groups; however, using a colchicine-inhibition approach, 8 h of EIHS caused a significant accumulation of p62, suggestive of increased flux. These data suggest that in a murine model of EIHS, 4 h was suffcient to cause cellular injury and dysfunction in the myocardium; however, as EIHS continued, it was associated with changes reflective of a cellular stress response that may contribute to acclimation. This work was supported by USDA 2020-02716. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Experimental and clinical studies indicate that a proatherogenic endothelial phenotype develops in the prediabetic, hyperglycemic state providing the platform for accelerated vascular disease with prediabetes and, in turn, type 2 diabetes. Prediabetes (fasting plasma glucose concentrations: 100-125 mg/dL) is associated with an increased risk of stroke. Circulating endothelial cell-derived microvesicles (EMVs) are now recognized as both a biomarker and mediator of vascular health and disease. In fact, EMVs have been shown to be involved in the pathogenesis of cerebrovascular dysfunction leading to increased stroke risk. Circulating EMVs are elevated in adults with prediabetes; whether EMVs associated with the prediabetic state promote endothelial abnormalities known to increase stroke risk is unknown. The experimental aim of this study was to determine the effect of EMVs isolated from adults with prediabetes on brain endothelial cell nitric oxide (NO) and endothelin (ET)-1 production. Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 20 mid-life adults (age:44-69 yr): 10 normoglycemic (NG; 8M/2F; age: 55±2 yr; BMI: 25.7±0.8 kg/m2; fasting plasma glucose: 87±2 mg/dL) and 10 prediabetic (PreD: 8M/2F; 55±2 yr; 27.1±1.5 kg/m2; 107±2 mg/dL). Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Expression of intracellular proteins of interest was determined by capillary electrophoresis immunoassay. Circulating EMV concentrations were significantly higher (~65%) in PreD vs NG (180±25 vs 110±13 EMV/μL) and significantly associated with plasma glucose concentrations (r=0.48; P=0.03). Phosphorylation is the primary posttranslational modification regulating eNOS enzyme activity. Phosphorylation of Ser1177 confers the greatest activation of eNOS; whereas, phosphorylation at Thr495 reduces eNOS activation. Expression of p-eNOS (Ser1177) was lower (47.6±2.6 vs 58.6±3.5 AU; P=0.02) and p-eNOS (Thr495) higher (82.6±4.4 vs 31.1±2.5 AU; p<0.001) in cells treated with EMVs from PreD vs NG. Concordantly, NO production was ~20% lower (6.0±0.2 vs 7.2±0.4 μmol/L; p<0.05) in cells treated with EMVs from PreD adults. PreD-associated EMVs also significantly increased (~25-70%) the expression of Big ET-1 (804.4±47.7 vs 656.7±30.7 AU) and endothelin converting enzyme (290.0±24.6 vs 169.6±19.0 AU) as well as ET-1 production (30.2±4.3 vs 24.2±2.0 pg/mL) in hCMECs. In summary, EMVs harvested from adults with PreD reduced endothelial nitric oxide synthase (eNOS) activation and NO production and increased ET-1 synthesis and release in brain endothelial cells in vitro. Increased risk and incidence of stroke associated with prediabetes may be mediated by circulating EMVs. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Recent studies point out the involvement of circulating extracellular vesicles (crEVs), in regulating neurotransmission via transferring proteins, lipids, and nucleic acids. Recently, we reported that plasma crEV proteins of either simian human immunodeficiency virus (SHIV) infected rhesus macaque or HIV-infected patient indicate a link to neuropathogenesis. Here, we tested the hypothesis that crEV proteins are indicative of dysfunction of synaptic vesicle (SV)-associated signaling pathways of SHIV-infected rhesus macaques. Methods: Plasma crEVs were isolated from SHIV-infected (SHIV-crEVs) and uninfected (CTL-crEVs) rhesus macaque and characterized by the ZetaView analyzer. Proteomic analysis of the isolated crEVs was performed using liquid chromatography/mass spectrometry (LC-MS/MS). Results: Our ZetaView analysis indicated that isolated crEVs were predominantly exosomes (particle size < 150 nm). In the LC-MS/MS study, 5,654 proteins were quantified, with 236 proteins (~ 4%) significantly differentially expressed between SHIV-crEVs and CTL-crEVs. Several SV and SV-signaling pathway associated proteins were quantified by tandem mass tags-based proteomic analysis both in SHIV-crEVs and CTL-crEVs. We observed that synaptogyrin, synaptotagmins, synaptobrevin, synaptosomal-associated proteins, syntaxin, and other SV-associated proteins were abundantly under expressed in SHIV-crEVs than in CTL-crEVs. Ingenuity Pathway Analysis demonstrated that proteins in SHIV-crEVs were involved in the deactivation of synaptogenesis, synaptic long-term protentiation/depression, and glutamate receptor signaling pathways. On the other hand, semaphorin neuronal repulsive signaling pathway and amyloid processing were activated in SHIV-crEVs. Bioinformatic analysis revealed that these proteins in SHIV-crEVs were involved in several CNS-related disorders/diseases such as abnormal morphology of synapses, motor dysfunction and movement disorder, seizure disorder, early/progressive neurological disorder, cognitive impairment, tauopathy, dementia, and Alzheimer’s disease. Conclusions: Our novel findings suggest that plasma crEVs could be an attractive noninvasive technique, which may elucidate the development of neuronal dysfunction and progression of neurological diseases. Funding: AG075988, HL148836, AG063345, AI110158, HL141143, HL168568, and the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of hypoglossal motoneurons (XII MNs) contributes to stiffening of the upper airway during inspiration. The magnitude of this excitation is decreased during sleep, likely due to a combination of withdrawal of noradrenaline and acetylcholine release at XII MNs. Acetylcholine acts at muscarinic acetylcholine receptors (mAChRs) and surprisingly mAChR activation has an excitatory effect on inspiratory bursting in neonatal preparations, which becomes inhibitory in adult preparations. mAChRs modulate several ion channels and characterizing muscarinic effect changes at the XII nucleus will help to explain the mechanism of the inhibitory shift over postnatal maturation. The hyperpolarization activated cyclic nucleotide gated (HCN) channel is positively modulated by mAChRs. HCN channels give rise to Ih, a mixed cation current activated at hyperpolarized membrane potentials that contribute to membrane depolarization. Ihincreases dramatically with postnatal maturation in XII MNs. Thus, we hypothesize that 1) the effects of muscarinic modulation of Ih increases with postnatal maturation, and 2) that muscarinic activation of Ih contributes to the net inhibitory component of muscarinic modulation of inspiratory bursting at XII motoneurons. To test our hypotheses, we used the rhythmic medullary slice preparation to test the functional effects of muscarinic modulation of Ih on inspiratory bursting in neonatal CD1 mice (postnatal day, P0-P4) in combination with pharmacological block of Ih with ZD7288 (25 μM, 150s). Local application of muscarine (100μM, 30s) in the XII motor nucleus increased inspiratory burst amplitude (189 ± 58% of baseline, n = 9). Contrary to our hypothesis, there was no statistically significant difference between inspiratory burst amplitude elicited by muscarine in the presence of ZD7288 (192 ± 47% of baseline, n = 9) versus with the aCSF vehicle control (174 ± 43% of baseline, p = 0.098). We next tested a higher ZD7288 concentration (100 μM) and longer application times (150s, 330s) in neonatal preparations. Preliminary data (n=2) indicate that ZD7288 may increase muscarinic potentiation of inspiratory burst amplitude at XII MNs (muscarinic potentiation (100μM, 30s): control — 112 ±65 % of baseline, aCSF vehicle control - 131 ± 38% of baseline; ZD7288 100μM, 150s - 148 ± 44% of baseline; ZD7288 100μM, 330s - 107 ± 72% of baseline). Future research will elucidate whether the Ih contribution to muscarinic modulation of inspiratory bursting increases with postnatal maturation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The sudden death induced by rapid intravenous (IVr) injection of overdose FNT in clinical settings is commonly observed in conscious humans and more often in the males than females. Our previous study has shown that IVr injection of FNT induces a long-lasting apnea associated with bradycardia and hypotension in anesthetized rats (Zhuang et al. 2023 EB). This study aimed to determine whether a lethal dose of FNT could cause sudden death with the mortality being higher in male rats than female ones and whether the FNT-induced respiratory disorder, especially ventilatory arrest, but not cardiac arrest, was the trigger of the death. The rat previously instrumented with ECG electrodes and jugular vein cannulation was placed in a restraint tube that was connected to a plethysmograph chamber to record cardiorespiratory activities. We found that FNT at doses <2 mg/kg induced an immediate persistent apnea, lasting less than 1 min, associated with bradycardia. Following restoration of respiratory rhythm, the rats showed depressed VE and bradycardia for several hours and the cardiorespiratory variables recovered next day. A lethal dose of FNT at 3 mg/kg, compared to FNT at <2 mg/kg, induced an even longer apnea (lasting >1.5 min) accompanied with more severe bradycardia. This sustained apnea was usually interrupted by brief restoration of breathing, which was followed by ventilatory and cardiac arrest occurring at approximately 5 min and 10 min post injection respectively. Although the initial cardiorespiratory response to FNT appeared to be similar in both genders, the death rate was relatively higher in male rats than female ones. In conclusion, our study establishes an animal model of IVr injection of overdose FNT-induced sudden death occurring within minutes. The sudden death is the result of ventilatory, but not cardiac, arrest following a brief restoration of respiratory activity from a sustained apnea. Supported by NIH R01 grants HL163512 and DA059063. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: The intestinal tract is one of the most densely innervated organs in our body, containing extrinsic sensory and sympathetic neurons. With their cell bodies located outside of the gut, these neurons directly detect enteric pathogens and release immunomodulatory neurotransmitters or neuropeptides to communicate with immune cells in the gut. Specifically, sympathetic neurons secrete neurotransmitter norepinephrine, which activate β2 adrenergic receptors (β2ΑR) on immune cells to regulate immune functions. Recent studies from our lab, as well as others, have begun to highlight the neural regulation of immune responses during enteric infections. However, the precise pathways responsible for orchestrating these effects remain elusive. Hypothesis: We sought to determine whether extrinsic sensory and sympathetic neurons are implicated in host protective responses against enteric pathogen Citrobacter rodentium (C. rodentium). Methods: Mice were inoculated by oral gavage with C. rodentium (108 colony-forming units) or sterile luria broth. Bacterial colonization was quantified by homogenization of distal colon tissues and plating serial dilutions onto MacConkey agar. Relevant tissues were collected for qPCR, flow cytometry or histology. Genetically engineered Arc Targeted Recombination in Active Population (TRAP) mice were used to identify neurons that were activated during infection. These ArcTRAP mice received single intraperitoneal (i.p.) injection of 4-hydroxytamoxifen post-infection to “trap” active neurons, marking them tdTomato+. 6-hydroxydopamine (6OHDA) was used to chemically ablate sympathetic innervations to the colon via 3 consecutive daily i.p. injections. Results: At day 4 post-infection, there were significantly more active tdTomato+ neurons in the rostral ventrolateral medulla (RVLM) of the infected mice compared to the non-infected controls. This increase was more pronounced as the infection peaked at day 10, indicating that more neurons were being activated in the RVLM in response to C. rodentium in the gut. 6OHDA treatment significantly increased bacterial burden in distal colon at day 10 post-infection. This was associated with decreased tyrosine hydroxylase expression compared to the vehicle controls. Subsequently, qPCR revealed that there was increased pro-inflammatory cytokines expression in the colon of sympathectomized infected mice compared to the infected controls. Similarly, β2AR knockout significantly increased bacterial burden in distal colon at day 3 and 10 post-infection. Increased pro-inflammatory cytokines expression suggests an immunomodulatory role for β2AR during C. rodentium infection. Conclusions: These studies provided novel insights into the neuroimmune crosstalk that is crucial for acute enteric infections with C. rodentium. Specifically, these experiments may reveal how extrinsic neurons shape local immune responses in the gut. NIH NIAID R01AI150647; Graduate Student Support Program (UC Davis: School of Veterinary Medicine). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.