Conscious Animals Research Articles

In vivo stimulation of locus coeruleus: effects on amygdala subnuclei

The locus coeruleus (LC) is the major noradrenergic nucleus and sends projections to almost all brain areas. A marked increase in norepinephrine release has been demonstrated in several brain areas in response to exposure to acute stressful stimuli, especially those innervated by LC projections. One of the brain areas innervated by LC neurons is the amygdala, a structure highly involved in emotional processes and memory formation. The aim of this study was to increase knowledge of the functional connectivity between the LC and the amygdala subnuclei. To reach this objective, we evaluated c-fos immunoreactive cells in amygdala nuclei following direct electrical stimulation of the LC in conscious animals. This analysis of c-Fos immunoreactivity could inform whether there are differences in activity of the amygdala subnuclei related to LC electrical stimulation in conscious animals. Our results showed a marked increase in c-fos activity in these amygdala subnuclei both ipsilateral and contralateral to LC electrical stimulation in vivo. Therefore, our study provides evidence that in vivo electrical stimulation of LC is able to activate the amygdala subnuclei as measured by c-fos expression.

Neurophysiological assessment of animal welfare

Livestock industries such as the pork industry are striving to continuously improve the welfare of animals. Inherent to the success of this is the ability to rigorously assess the welfare of animals in the field. While much progress has been made towards the development of methodology to assess the welfare of animals, there have been major challenges to establishing practical and definitive procedures to assess the welfare of animals. These include, but are not limited to, establishing a universally accepted definition of animal welfare and the choice of measures that are taken from the animal to assess its welfare. Measures of biological functioning and affective (emotional) state of the animal have been common, but there have been many limitations in terms of practical application. Some of the reasons for this include the choice of physiological measures, which are often restrictive in providing information about welfare, affective measures being restricted to specific behavioural measures and the biological-functioning and affective-states approaches being undertaken in isolation. Biological and affective functioning are integrated and controlled by the brain. Many of the regions of the brain involved in the regulation of biological and emotional functioning have been identified. Furthermore, there is considerable knowledge about the roles and interactions among the neurophysiological systems in these brain regions. We propose a strategy to use this knowledge to develop procedures to assess animal welfare. The initial phase is to identify the neural pathways that regulate the physiological and emotional processes that allow animals to adapt and cope. The next phase is to determine the activity of these pathways in conscious animals in the field. This requires the identification of biomarkers of specific neuronal activity that can be measured in the conscious animal in the field. Emerging technologies are offering promise in the identification of such biomarkers and some of these are already applicable to the pig. There is now the opportunity to apply this strategy within the pork industry to assess the welfare of pigs throughout the value chain.

Threshold Effect of G9a/Glp on Peripheral Nerve Injury Induced Hypersensitivity.

BackgroundPrevious studies disclosed the pivotal role of methyltransferase complex G9a/Glp in the pathogenesis of neuropathic hypersensitivity induced by peripheral nerve injury. We observed that higher dose of G9a inhibitor improved nociceptive behavior, but the lower dose worsened pain. The aim of this study is to extensively observe the differential effect of various dosages of G9a/Glp inhibitors on nerve injury-induced allodynia.Materials and methodsAfter approval by the institutional ethical committee on pain research in conscious animals, C57BL/6 mice were used for measuring nociceptive behavior evoked with von Frey filaments after spared nerve injury. G9a/Glp inhibitor BIX01294 or UNC0638 was injected through the pre-buried intrathecal catheter. The dose–response curves of behavioral changes were depicted when inhibitors were administered once in bolus at the 14th day post spared nerve injury. Withdrawal behaviors were compared during the 49 days’ observation window after spared nerve injury with various dosages of inhibitors injected intrathecally for 14 days.ResultsDose–behavior curves of a single bolus of both BIX01294 and UNC0638 displayed a “V”-shaped responses of allodynia withdrawal from lower through higher dose when measured at the 14th day post spared nerve injury. A threshold dose of 10.0 µg for BIX01294 and 80.0 µg for UNC0638 significantly worsened allodynia. However, daily bolus intrathecal injection for 14 days of both inhibitors lower or higher than these threshold doses prominently improved nociceptive behavior, producing contrasting results. On the same animal, threshold dose followed by a lower or higher dose with a 14 days’ interval also showed contrast effect on nociceptive behavior, and a lower or higher dose to threshold dose sequence of inhibitor administration was vice versa.ConclusionsMethyltransferase complex G9a/Glp has a threshold role in mediating peripheral nerve injury-induced hypersensitivity at its low level versus high level through inhibiting and facilitating the nociceptive behavior, respectively.

Preclinical imaging anesthesia in rodents.

Despite the outstanding progress achieved by preclinical imaging science, laboratory animal anesthesia remains quite stationary. Ninety percent of preclinical imaging studies are carried on small rodents (mice and rats) anesthetized by outdated injectable and/or inhalation agents. A need for imaging awake (conscious) animals is questionably registered mainly for brain research, for phMRI and for accomplishing pain and analgesia studies. A need for improving current rodent anesthesia protocols and for enforcing the 3Rs paradigm is sought. Patient monitoring throughout the procedure and recovery phases, as well as vital parameter's data must be recorded in basic consciousness states and during imaging sessions. A multidrug approach is suggested to overcome the limits of monoanesthesia and well-timed physiological data are required to ground findings and to interpret imaging data.

Isoflurane and Sevoflurane Induce Severe Hepatic Insulin Resistance in a Canine Model.

IntroductionAnesthesia induces insulin resistance, which may contribute to elevated blood glucose and adverse post-operative outcomes in critically ill patients, and impair glycemic control in surgical patients with diabetes. However, little is known about the mechanisms by which anesthesia impairs insulin sensitivity. Here we investigate the effects of anesthesia on insulin sensitivity in metabolic tissues.MethodsHyperinsulinemic-euglycemic clamps were performed in 32 lean (control diet; n = 16 conscious versus n = 16 anesthetized) and 24 fat-fed (6 weeks fat-feeding; n = 16 conscious versus n = 8 anesthetized) adult male mongrel dogs in conjunction with tracer methodology to differentiate hepatic versus peripheral insulin sensitivity. Propofol was administered as an intravenous bolus (3mg/kg) to initiate anesthesia, which was then maintained with inhaled sevoflurane or isoflurane (2–3%) for the duration of the procedure.ResultsAnesthesia reduced peripheral insulin sensitivity by approximately 50% in both lean and fat-fed animals as compared to conscious animals, and insulin action at the liver was almost completely suppressed during anesthesia such that hepatic insulin sensitivity was decreased by 75.5% and; 116.2% in lean and fat-fed groups, respectively.ConclusionInhaled anesthesia induces severe hepatic insulin resistance in a canine model. Countermeasures that preserve hepatic insulin sensitivity may represent a therapeutic target that could improve surgical outcomes in both diabetic and healthy patients.

Case Studies in Physiology: The Case of the Giant Giraffe.

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Central Angiotensin-II Increases Blood Pressure and Sympathetic Outflow via Rho Kinase Activation in Conscious Rabbits.

Elevated sympathetic tone and activation of the renin-angiotensin system are pathophysiologic hallmarks of hypertension, and the interactions between these systems are particularly deleterious. The importance of Rho kinase as a mediator of the effects of angiotensin-II (AngII) in the periphery is clear, but the role of Rho kinase in sympathoexcitation caused by central AngII is not well established. We hypothesized that AngII mediates its effects in the brain by the activation of the RhoA/Rho kinase pathway. Chronically instrumented, conscious rabbits received the following intracerebroventricular infusion treatments for 2 weeks via osmotic minipump: AngII, Rho kinase inhibitor Fasudil, AngII plus Fasudil, or a vehicle control. AngII increased mean arterial pressure over the course of the infusion, and this effect was prevented by the coadministration of Fasudil. AngII increased cardiac and vascular sympathetic outflow as quantified by the heart rate response to metoprolol and the depressor effect of hexamethonium; coadministration of Fasudil abolished both of these effects. AngII increased baseline renal sympathetic nerve activity in conscious animals and impaired baroreflex control of sympathetic nerve activity; again Fasudil coinfusion prevented these effects. Each of these end points showed a statistically significant interaction between AngII and Fasudil. Quantitative immunofluorescence of brain slices confirmed that Rho kinase activity was increased by AngII and decreased by Fasudil. Taken together, these data indicate that hypertension, elevated sympathetic outflow, and baroreflex dysfunction caused by central AngII are mediated by Rho kinase activation and suggest that Rho kinase inhibition may be an important therapeutic target in sympathoexcitatory cardiovascular diseases.

Inhalation anesthesia induced by isoflurane alters penicillin disposition in swine tissues.

Twelve healthy swine were dosed with penicillin G intramuscularly. Fluids and tissues samples were collected at the end of two periods of general anesthesia, performed 24h apart. Tissue samples were collected by minimally invasive laparoscopy under general anesthesia at 8 and 28h postdose. Four nonanesthetized, penicillin-treated pigs were euthanized at 8h postdose, and a second set of four similarly treated control pigs were sacrificed 28h postdose. Liver penicillin tissue concentrations from animals that underwent anesthesia and laparoscopic tissue collection had tissue concentrations that were higher than nonanesthetized pigs at both time points. Urine, plasma, kidney, skeletal, and cardiac muscle showed no differences between the two groups. Laparoscopic tissue collection under general anesthesia in swine induces physiological changes that cause alterations in tissue pharmacokinetics not seen in conscious animals.

PP.09.03] PROTEOMIC ANALYSIS OF KIDNEYS FROM SPONTANEOUSLY HYPERTENSIVE AND NORMAL RATS REVEALS CLIC-4, AS A PUTATIVE BIOMARKER IN HYPERTENSIVE NEPHROPATHY

Objective: Hypertensive nephrosclerosis is a major cause of declining kidney function. We aimed to identify novel markers associated with pathogenesis and development of hypertensive nephrosclerosis in experimental animals. Design and method: We compared kidney tissues from spontaneously hypertensive rats (SHR) and Wistar Kyoto normotensive rats (WKY) using comparative proteomic analysis at 6 (development of HTN), 13 (establish HTN), and 20 (beginning of albuminuria) weeks of age. BP was measured in conscious animals using a tail-cuff technique. 2-D Gel Electrophoresis was used to identify stained protein spots differing between hypertensive and normotensive rats. Each spot was excised manually, digested with trypsin and analyzed with a MALDI-TOF/TOF mass spectrometer. The acquired spectra were processed with the Flexanalysis v 2.2 software and protein identification through peptide mass fingerprinting was performed by the use of the MASCOT Server v 2.0. Western blot analysis was used to confirm the proteomic data on differentially expressed proteins in kidneys from SHR. To identify in which specific cell type of renal parenchyma the differences in protein expression occur, we performed immunohistochemical and immunofluorescence studies. Results: The proteomic analysis of SHRs and control kidney tissue yielded a significant number of differentially expressed proteins and altered biological pathways. Overexpression of ClC-4 in SHR kidney tissue is associated with the development of hypertension and establishment of hypertensive nephrosclerosis. Besides its pathogenetic significance, ClC-4 may be a novel diagnostic and therapeutic marker in essential hypertension. Conclusions: The proteomic analysis of SHRs and control kidney tissue yielded a significant number of differentially expressed proteins and altered biological pathways. Overexpression of ClC-4 in SHR kidney tissue is associated with the development of hypertension and establishment of hypertensive nephrosclerosis. Besides its pathogenetic significance, ClC-4 may be a novel diagnostic and therapeutic marker in essential hypertension.

ISH NIA OS-04 P2X3 RECEPTOR ACTIVITY IN THE CAROTID BODY (CB) OF SPONTANEOUSLY HYPERTENSIVE (SH) RATS CONTRIBUTES TO INCREASED CHEMOREFLEX HYPERSENSITIVITY AND HYPERTENSION

Objective: Arterial hypertension is associated with increases in both peripheral chemoreceptor reflex sensitivity and sympathetic nerve activity (SNA). We have shown that CB resection in SH rats reduces arterial pressure (AP; McBryde et al. 2013). Since P2X3 receptors exist in the rat CB and P2X3 receptor activity is associated with afferent hyperreflexia (Ford et al. 2015), we hypothesized that P2X3 receptor would be upregulated in the CB of SH vs normotensive (Wistar) control rats and that a selective P2X3 receptor antagonist (AF-219) would reduce chemoreflex hypersensitivity, AP and SNA in SH rats. Design and Method: SH and Wistar rats were implanted with radio-telemetry devices to record AP and renal SNA (RSNA) in conscious animals. Animals were infused with AF-219 (8 mg/kg/h) or vehicle i.v. for 1 h. The CB was stimulated with sodium cyanide (120 μg/kg i.v.). Western blotting and immunohistochemistry revealed the level of expression and localisation of P2X3 receptor protein in the CB, respectively. Results: In the CB, P2X3 receptor protein was upregulated four fold in SH vs Wistar rat (P < 0.05). Glomus cells of SH rat (identified as expressing tyrosine hydroxylase) and afferent nerve fibres were immunopositive for P2X3 receptors. In contrast, in Wistar rats, glomus cells expressing P2X3 receptors were fewer and P2X3 was not detected in nerve fibres. Infusion of AF-219 reduced: CB hyperreflexia (P < 0.05); AP (systolic −28 ± 3mmHg; P < 0.001) and RSNA (34%; P < 0.01). No changes in chemoreflex sensitivity, AP or RSNA were detected after vehicle or AF-219 infusion in Wistar rats. Conclusions: We surmise that upregulation of P2X3 receptors in the CB of SH rats contributes to chemoreflex hypersensitivity. Further, antagonism of P2X3 receptors lowers both AP and RSNA substantially in SH rats. Thus, P2X3 receptor antagonists may provide a novel way to attenuate CB over-activity and offer a novel approach for controlling hypertension. Supported by: Afferent Pharmaceuticals

Active hexose correlated compound modulates LPS-induced hypotension and gut injury in rats

We hypothesized that AHCC; (Amino UP Chemical Co., Ltd., Sapporo, Japan), a mushroom mycelium extract obtained from liquid culture of Lentinula edodes, restores immune function in LPS-induced inflammation in the gut, especially when the nitric oxide signaling pathway is impaired. This is the first inter-disciplinary proposal to identify molecular mechanisms involved in LPS-induced immune dysfunction in the gut in conscious animals treated or non-treated with AHCC, a promoter of immune support. Specifically, we have tested the effects of AHCC on LPS-induced deleterious effects on blood pressure and gut injury in conscious rats. The time course of biological markers of innate/acquired immune responses, and inflammation/oxidative stress is fully described in the present manuscript. Rats were randomly assigned into 3 groups (N=6 per group). Group 1 received 10% of AHCC in drinking water for 5days; Group 2 received lipopolysaccharide (LPS; Escherichia coli 0111:B4 purchased from Sigma) only at 20mg/kg IV; Group 3 received combined treatments (AHCC + LPS). LPS was administered at 20mg/kg IV, 5days following AHCC treatment. We have demonstrated that AHCC decreased the LPS-deleterious effects of blood pressure and also decreased inflammatory markers e.g., cytokines, nitric oxide and edema formation. Finally, AHCC diminished lymphocyte infiltration, restoring gut architecture. Because AHCC was administered prior to LPS, our results indicate the potential impact of AHCC's prophylactic effects on LPS inflammation. Consequently, additional experiments are warrant to assess its therapeutic effects in sepsis-induced inflammation.

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

Vestibular nucleus neurons respond to hindlimb movement in the conscious cat.

The limbs constitute the sole interface with the ground during most waking activities in mammalian species; it is therefore expected that somatosensory inputs from the limbs provide important information to the central nervous system for balance control. In the decerebrate cat model, the activity of a subset of neurons in the vestibular nuclei (VN) has been previously shown to be modulated by hindlimb movement. However, decerebration can profoundly alter the effects of sensory inputs on the activity of brain stem neurons, resulting in epiphenomenal responses. Thus, before this study, it was unclear whether and how somatosensory inputs from the limb affected the activity of VN neurons in conscious animals. We recorded brain stem neuronal activity in the conscious cat and characterized the responses of VN neurons to flexion and extension hindlimb movements and to whole body vertical tilts (vestibular stimulation). Among 96 VN neurons whose activity was modulated by vestibular stimulation, the firing rate of 65 neurons (67.7%) was also affected by passive hindlimb movement. VN neurons in conscious cats most commonly encoded hindlimb movement irrespective of the direction of movement (n = 33, 50.8%), in that they responded to all flexion and extension movements of the limb. Other VN neurons overtly encoded information about the direction of hindlimb movement (n = 27, 41.5%), and the remainder had more complex responses. These data confirm that hindlimb somatosensory and vestibular inputs converge onto VN neurons of the conscious cat, suggesting that VN neurons integrate somatosensory inputs from the limbs in computations that affect motor outflow to maintain balance.

The integrative role of orexin/hypocretin neurons in nociceptive perception and analgesic regulation.

The level of wakefulness is one of the major factors affecting nociception and pain. Stress-induced analgesia supports an animal’s survival via prompt defensive responses against predators or competitors. Previous studies have shown the pharmacological effects of orexin peptides on analgesia. However, orexin neurons contain not only orexin but also other co-transmitters such as dynorphin, neurotensin and glutamate. Thus, the physiological importance of orexin neuronal activity in nociception is unknown. Here we show that adult-stage selective ablation of orexin neurons enhances pain-related behaviors, while pharmacogenetic activation of orexin neurons induces analgesia. Additionally, we found correlative activation of orexin neurons during nociception using fiber photometry recordings of orexin neurons in conscious animals. These findings suggest an integrative role for orexin neurons in nociceptive perception and pain regulation.

What's the Point of Self-consciousness? A Critique of Singer's Arguments against Killing (Human or Non-human) Self-conscious Animals

Singer has argued against the permissibility of killing people (and certain animals) on the grounds of the distinction between conscious and self-conscious animals. Unlike conscious animals, which can be replaced without a loss of overall welfare, there can be no substitution for self-conscious animals. In this article, I show that Singer's argument is untenable, in the cases both of the preference-based account of utilitarianism and of objective hedonism, to which he has recently turned. In the first case, Singer cannot theoretically exclude that a self-conscious being's stronger preferences may only be satisfied by killing another self-conscious being. In the second case, he fails to demonstrate that the rules of ordinary morality, demanding that killing be strictly forbidden, could not frequently be overruled by the principles of esoteric morality. In both cases, his theory cannot solve the classical utilitarian problem of prohibiting the killing of people in secret.

Optimum anesthesia for reliable urethral pressure profilometry in female dogs and goats.

To investigate the effects of propofol and isoflurane on urethral pressure profilometry of female dogs and goats, and to identify the method of anesthesia that least influences urethral pressure profilometry and to assess its reproducibility. The effects of premedication with midazolam, propofol sedation and isoflurane anesthesia were assessed in five female dogs. The effects of propofol and isoflurane were compared in seven goats, whereas in another group of 19 goats, the state of deep propofol sedation was compared with the state of recovery from propofol sedation. The coefficient of reproducibility and within-subject coefficient of variation were calculated to evaluate test-retest reproducibility. In conscious female dogs, maximal urethral closure pressure and functional area were significantly higher than under propofol or isoflurane (P = 0.04), but not different from the recovery state. In six of seven goats, maximal urethral closure pressure and functional area were higher when measured under propofol sedation than under isoflurane (median maximal urethral closure pressure, 69 vs 47 cmH2 O; P = 0.03). Maximal urethral closure pressure was lower under propofol than during recovery from propofol in 17 of 19 goats (median maximal urethral closure pressure, 54 vs 66 cmH2 O; P < 0.001). The test-retest coefficient of reproducibility for goats was 28 cmH2 O, and the within-subject coefficient of variation was 16%. In dogs, urethral pressure profilometry should be measured in conscious animals whenever possible. In goats, urethral pressure profilometry is least affected during recovery from propofol sedation, and it shows acceptable reproducibility under this condition.

Long-lasting bradypnea induced by repeated social defeat.

Repeated social defeat in the rat induces long-lasting cardiovascular changes associated with anxiety. In this study, we investigated the effects of repeated social defeat on breathing. Respiratory rate was extracted from the respiratory sinus arrhythmia (RSA) peak frequency of the ECG in rats subjected to social defeat for 4 consecutive days. Respiratory rate was recorded under anesthesia 6 days (D+10) or 26 days (D+30) after social defeat. At D+10, defeated (D) rats spent less time in the open arms of the elevated plus maze test, had heavier adrenal glands, and displayed bradypnea, unlike nondefeated animals. At D+30, all signs of anxiety had disappeared. However, one-half of the rats still displayed bradypnea (DL rats, for low respiratory rate indicated by a lower RSA frequency), whereas those with higher respiratory rate (DH rats) had recovered. Acute blockade of the dorsomedial hypothalamus (DMH) or nucleus tractus solitarii (NTS) 5-HT3 receptors reversed bradypnea in all D rats at D+10 and in DL rats at D+30. Respiratory rate was also recorded in conscious animals implanted with radiotelemetric ECG probes. DH rats recovered between D+10 and D+18, whereas DL rats remained bradypneic until D+30. In conclusion, social stress induces sustained chronic bradypnea mediated by DMH neurons and NTS 5-HT3 receptors. These changes are associated with an anxiety-like state that persists until D+10, followed by recovery. However, bradypnea may persist in one-half of the population up until D+30, despite apparent recovery of the anxiety-like state.

Multiscale entropy analysis of heart rate variability in heart failure, hypertensive, and sinoaortic-denervated rats: classical and refined approaches.

The analysis of heart rate variability (HRV) by nonlinear methods has been gaining increasing interest due to their ability to quantify the complexity of cardiovascular regulation. In this study, multiscale entropy (MSE) and refined MSE (RMSE) were applied to track the complexity of HRV as a function of time scale in three pathological conscious animal models: rats with heart failure (HF), spontaneously hypertensive rats (SHR), and rats with sinoaortic denervation (SAD). Results showed that HF did not change HRV complexity, although there was a tendency to decrease the entropy in HF animals. On the other hand, SHR group was characterized by reduced complexity at long time scales, whereas SAD animals exhibited a smaller short- and long-term irregularity. We propose that short time scales (1 to 4), accounting for fast oscillations, are more related to vagal and respiratory control, whereas long time scales (5 to 20), accounting for slow oscillations, are more related to sympathetic control. The increased sympathetic modulation is probably the main reason for the lower entropy observed at high scales for both SHR and SAD groups, acting as a negative factor for the cardiovascular complexity. This study highlights the contribution of the multiscale complexity analysis of HRV for understanding the physiological mechanisms involved in cardiovascular regulation.

Cardiac Morphology and Function, and Blood Gas Transport in Aquaporin-1 Knockout Mice.

We have studied cardiac and respiratory functions of aquaporin-1-deficient mice by the Pressure-Volume-loop technique and by blood gas analysis. In addition, the morphological properties of the animals' hearts were analyzed. In anesthesia under maximal dobutamine stimulation, the mice exhibit a moderately elevated heart rate of < 600 min−1 and an O2 consumption of ~0.6 ml/min/g, which is about twice the basal rate. In this state, which is similar to the resting state of the conscious animal, all cardiac functions including stroke volume and cardiac output exhibited resting values and were identical between deficient and wildtype animals. Likewise, pulmonary and peripheral exchange of O2 and CO2 were normal. In contrast, several morphological parameters of the heart tissue of deficient mice were altered: (1) left ventricular wall thickness was reduced by 12%, (2) left ventricular mass, normalized to tibia length, was reduced by 10–20%, (3) cardiac muscle fiber cross sectional area was decreased by 17%, and (4) capillary density was diminished by 10%. As the P-V-loop technique yielded normal end-diastolic and end-systolic left ventricular volumes, the deficient hearts are characterized by thin ventricular walls in combination with normal intraventricular volumes. The aquaporin-1-deficient heart thus seems to be at a disadvantage compared to the wild-type heart by a reduced left-ventricular wall thickness and an increased diffusion distance between blood capillaries and muscle mitochondria. While under the present quasi-resting conditions these morphological alterations have no consequences for cardiac function, we expect that the deficient hearts will show a reduced maximal cardiac output.

Parainfluenza 3-Induced Cough Hypersensitivity in the Guinea Pig Airways.

The effect of respiratory tract viral infection on evoked cough in guinea pigs was evaluated. Guinea pigs were inoculated intranasally with either parainfluenza type 3 (PIV3) and cough was quantified in conscious animals. The guinea pigs infected with PIV3 (day 4) coughed nearly three times more than those treated with the viral growth medium in response to capsaicin, citric acid, and bradykinin. Since capsaicin, citric acid, and bradykinin evoked coughing in guinea pigs can be inhibited by drugs that antagonize the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), it was reasoned that the virally-induced hypertussive state may involve alterations in TPRV1 activity. PIV3 infection caused a phenotypic switch in tracheal nodose Aδ “cough receptors” such that nearly 50% of neurons began to express, de novo, TRPV1 mRNA. There was also an increase TRPV1 expression in jugular C-fiber neurons as determined by qPCR. It has previously been reported that tracheal-specific nodose neurons express the BDNF receptor TrkB and jugular neurons express the NGF receptor TrkA. Jugular neurons also express the artemin receptor GFRα3. All these neurotrophic factors have been associated with increases in TRPV1 expression. In an ex vivo perfused guinea pig tracheal preparation, we demonstrated that within 8 h of PIV3 infusion there was no change in NGF mRNA expression, but there was nearly a 10-fold increase in BDNF mRNA in the tissue, and a small but significant elevation in the expression of artemin mRNA. In summary, PIV3 infection leads to elevations in TRPV1 expression in the two key cough evoking nerve subtypes in the guinea pig trachea, and this is associated with a hypertussive state with respect to various TRPV1 activating stimuli.