Conscious Pigs Research Articles

Muscle protein catabolism and splanchnic arginine consumption drive arginine dysregulation during Pseudomonas Aeruginosa induced early acute sepsis in swine.

Human sepsis is characterized by increased protein breakdown and changes in arginine and citrulline metabolism. However, it is unclear whether this is caused by changes in transorgan metabolism. We therefore studied in a Pseudomonas aeruginosa induced pig sepsis model the changes in protein and arginine related metabolism on whole body (Wb) and transorgan level. We studied 22 conscious pigs for 18 hours during sepsis, induced by infusing live bacteria (Pseudomonas aeruginosa) or after placebo infusion (control). We used stable isotope tracers to measure Wb and skeletal muscle protein synthesis and breakdown, as well as Wb, splanchnic, skeletal muscle, hepatic and portal drained viscera (PDV) arginine and citrulline disposal and production rates. During sepsis, arginine Wb production (p=0.0146), skeletal muscle release (p=0.0035) and liver arginine uptake were elevated (p=0.0031). Wb de novo arginine synthesis, citrulline production, and transorgan PDV release of citrulline, glutamine and arginine did not differ between sepsis and controls. However, Wb (p<0.0001) and muscle (p<0.001) protein breakdown were increased, suggesting that the enhanced arginine production is predominantly derived from muscle breakdown in sepsis. In conclusion, live-bacterium sepsis increases muscle arginine release and liver uptake, mirroring previous pig endotoxemia studies. In contrast to observations in humans, acute live-bacterium sepsis in pigs does not change citrulline production or arterial arginine concentration. We therefore conclude that the arginine dysregulation observed in human sepsis is possibly initiated by enhanced protein catabolism and splanchnic arginine catabolism, while decreased arterial arginine concentration and citrulline metabolism may require more time to fully manifest in patients.

Repeated Vagus nerve electrical stimulation alleviates constipation in a porcine model of opioid-induced constipation

Background: Chronic constipation (CC) is highly prevalent and affects approximately 15% of persons in the United States. Opioid-induced constipation (OIC) in particular, is a significant complication affecting up to 80% of non-cancer patients receiving opioids. Current treatments for CC suffer from adverse effects, poor compliance, and low effcacy. Progress in treatment of CC is hampered in part by the lack of studies in models of higher translational value and due to limited means to assess gut motility non-invasively. To palliate this lack of, we recently developed and validated a model of opioid-induced constipation in pigs using chronic administration of the mu-opioid agonist, loperamide[1]. Neuromodulation, of the autonomic nervous system, is now regarded as the future therapy for refractory diseases such as refractory OIC. Our previous work in naïve-anesthetized pigs indicate that acute electrical stimulation of the celiac branch of the Vagus nerve (CBVN) can increase colonic motility. It is, however, unknown whether repeated CBVN stimulation (R-CBVNS) in conscious pigs can relieve chronic constipation induced by repeated opioid exposure. Aims: To evaluate the effect of R-CBVNS in a porcine model of opioid-induced constipation induced by chronic administration of loperamide. Methods: Female minipig Yucatan (35-50 kg, 5-7 months) were surgically equipped with a cuff electrode around the CBVN and a cecal cannula. Fourteen days later, pigs received daily oral administration of loperamide at 0.2, 0.4 or 3 mg/kg/day in regular diet mixed with palatant (bananas, marshmallows or yogurt mixed with honey) for 14-30 days. After 2 days of loperamide, the effect of concurrent R-CBVNS (30 min/day, 2 Hz, 1 mA, 300 us, 30s on/90s off) was tested on chronic OIC. The intracolonic pressure signals were collected on day 2, 7 and 14, by manometric probes placed in the proximal (pc) and distal colon (dc) with a sampling frequency of 100Hz in awake pigs fitted with chronic cecal cannulas. The long-term effects of R-CBVNS was also tested on the highest dose of loperamide by monitoring motility on day 21 and day 28, 1 and 2 weeks respectively after discontinuing R-CBVNS. In the distal colon, four probes were inserted through the anus and aligned at 10, 13, 16, and 19 cm proximal to the anal verge, denoted as D10, D13, D16, D19. In the proximal colon, four probes were inserted about 10, 13, 16, and 19 cm below the ceco-colic junction, denoted as P10, P13, P16, and P19. Analysis of the intracolonic pressure signals was performed by custom scripts developed in the MATLAB version 2020b. The colon luminal pressure spectral analysis, signal power spectrum density as well as peak frequency and contraction bursts were characterized. Defecation response and stool water content changes (SWC, Bristol scale) were monitored. Results: Loperamide altered the power of dominant manometric contraction frequency band (2-7cpm) increasing it in the pc but reducing it in the dc suggesting colon-region specific responses (0.4 and 3mg/kg/day). R-CBVNS in pigs treated with 0.2, 0.4 or 3 mg/kg/day (for 14 days) increased primarily the proximal colon contraction frequency power during and post stimulation period. The increase in the proximal colon contraction power was mainly seen in the pig colon dominant frequency band (2-6 cpm). The effect of R-CBVNS was less apparent on distal colon motility. R-CBVNS improved stool shape and tended to increase stool water content, when compared to loperamide without concurrent stimulation. Moreover, after 2 weeks of R-CBVNS, the effect lasted one week after the stimulation protocol ended. Conclusions: OIC in pigs is characterized by delayed GI transit and decreased stool water content, alterations of colonic motility patterns including selective suppression of 5-6 cpm peak frequency while inducing 3 cpm peak akin to that reported in some CC patients. R-CBVNS demonstrates its effectiveness in alleviating chronic constipation in a swine model of OIC via restoring primary colonic functions, with a quasi-return of basal motility, but also probably through a pro-secretory effect as evidenced by an increase in stool water content. Reference: 1. Atmani, K., et al., A porcine model of opioid-induced constipation: colon motility characterization and evidence for intestinal opioid receptor modulation. Physiology, 2023. 38(S1): p. 5734528. Supported by NIH SPARC OT2-OD024899. 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.

Characterizing candidate decompression rates for hypobaric hypoxic stunning of pigs. Part 1: Reflexive behavior and physiological responses.

Alternatives to carbon dioxide (CO2) stunning for the commercial slaughter of pigs are urgently needed because there is robust evidence that exposing pigs to hypercapnic environments is associated with pain, fear, and distress. Hypobaric hypoxia (via gradual decompression, also known as Low Atmospheric Pressure Stunning or LAPS) has been validated in poultry as a humane option, but its potential to improve the welfare of pigs at slaughter is unknown. We investigated the potential of hypobaric hypoxia to reliably elicit a non-recovery state in anesthetized weaner-grower pigs within a commercially viable timeframe. We determined the effect of candidate decompression rates (40, 60, 80, 100 ms-1, at two cycle durations 480 s and 720 s) on a range of physiological and reflexive behavioral indicators of hypoxia and death. We found that the decompression rates tested caused a 100% death rate. As expected, the decompression rate had overarching effects on behavioral and physiological markers of hypoxia and death, with faster decompression rates resulting in shorter latencies to cardiac arrest and cessation of breathing. We observed a higher proportion of pigs displaying repeated and prolonged whole-body movements (likely indicative of convulsive activity) at higher frequencies when we applied the slowest decompression rate (40 ms-1) compared to all other rates. Since these responses may impact the carcass and meat quality, the slower rate of decompression (40 ms-1) should be excluded as a candidate decompression rate. Furthermore, given the marginal effects of decompression rate on physiological indicators of death and reflexive behavioral parameters, we also recommend that the fastest rate tested (100 ms-1) is excluded in further study on conscious pigs (to prevent conscious animals from being exposed to unnecessary faster decompression rates which may compromise animal welfare). This work represents a necessary proof of principle step and confirms the potential of gradual decompression for stunning purposes in pigs. Importantly, however, the data presented provide no information on the welfare outcomes associated with decompression in conscious pigs. Subsequent work should focus on the comprehensive welfare assessment of intermediate decompression rates to determine the potential of hypobaric hypoxia to provide a humane stunning method for pigs.

Characterizing candidate decompression rates for hypobaric hypoxic stunning of pigs. Part 2: Pathological consequences.

Pigs are commonly stunned pre-slaughter by exposure to carbon dioxide (CO2), but this approach is associated with significant welfare concerns. Hypobaric hypoxia, achieved with gradual decompression (also known as Low Atmospheric Pressure Stunning or LAPS) may be an alternative, allowing the retention of welfare friendly handling approaches and group stunning. Although validated in poultry, the feasibility and welfare consequences of gradual decompression for pigs are unknown. Here, we characterize pathological changes in 60 pigs resulting from exposure to a range of candidate decompression curves (ranging from 40 to 100 ms-1 ascent equivalent, with two cycle durations 480 and 720 s). To protect welfare, we worked on unconscious, terminally anesthetized pigs which were subject to detailed post-mortem examinations by a specialized porcine veterinary pathologist. All pigs were killed as a result of exposure to decompression, irrespective of cycle rate or length. Pigs showed no external injuries during ante-mortem inspections. Exposing pigs to decompression and the unavoidable subsequent recompression resulted in generalized congestion of the carcass, organs and body cavities including the ears, oral cavity, conjunctivae and sclera, mucosa of other external orifices (anus and vulva), nasal planum, nasal cavities including nasal conchae, frontal sinuses, cranium, meninges, brain, larynx, trachea, lungs, heart, parietal pleura of the thoracic cavity, peritoneum of the abdominal cavity, stomach, small intestine, caecum, colon, liver, spleen and kidneys and representative joint cavities in the limbs (stifles and elbows). Various severities of hemorrhage were observed in the conjunctivae and sclera, mucosa of other external orifices (anus and vulva), nasal cavities including nasal conchae, frontal sinuses, cranium, meninges, brain, larynx, tracheal lumen, lungs, parietal pleura of the thoracic cavity, liver, spleen and kidneys and representative joint cavities in the limbs (stifles and elbows). In general, faster decompression rates produced higher scores, but in the conjunctivae, sclera and kidneys, faster decompression rates were associated with marginally lower congestion scores. There was considerable individual variation in pathological scores across all body regions. The congestion and hemorrhage observed could translate into welfare harms in conscious pigs undergoing this type of stunning, depending when in the cycle the damage is occurring, but no welfare related conclusions can be drawn from the responses of unconscious pigs. Since recompression is always required, its effects cannot be separated from decompression, however cessation of cardiac activity several minutes before recompression should have eliminated any haemodynamic effects relating to cardiac function and blood pressure. This study represents the first systematic attempt to identify candidate rate profiles to underpin future explorations of decompression as a stunning method for pigs. These pathological findings also inform discussions about the likely carcass quality implications of this novel stunning method.

A New Electrode Design for Direct Bladder Wall Stimulation: A Pilot Minipig Study with Chronic Testing

(1) Aims: To test a newly designed helical-wire hook electrode implanted in the bladder wall to induce contraction and promote voiding. (2) Methods: In three minipigs with a created lesion of the sacral spinal cord, four electrodes were implanted in the bladder wall, ventral to the trigone. Stimulation tests were conducted initially in conscious pigs, and later after general anesthesia. (3) Results: Electrical stimulation in the conscious animals on postoperative days 4 and 7 at 40 Hz was limited to 10 mA, because of abdominal, leg, and anal contractions with animal discomfort; bladder contractions were not induced. Electrical stimulation on postoperative days 9 and 28 at 60 mA under anesthesia induced sustained vesical wall contractions with bladder pressure variations, but without voiding. Simultaneous abdominal contractions occurred, with strong leg and anal contractions. Subsequent stimulation with a single set of electrodes or at 20 Hz induced less vesical pressure response. At autopsy, the electrodes had not migrated, and extraction forces were high, at 7.9 ± 0.9 Newtons (n = 12). (4) Conclusions: Our 28-day study has confirmed the utility of the new electrode design, preventing migration from the bladder wall and making it suitable for long-term electrode implants.

Characterization of Lower Urinary Tract Dysfunction after Thoracic Spinal Cord Injury in Yucatan Minipigs.

There is an increasing need to develop approaches that will not only improve the clinical management of neurogenic lower urinary tract dysfunction (NLUTD) after spinal cord injury (SCI), but also advance therapeutic interventions aimed at recovering bladder function. Although pre-clinical research frequently employs rodent SCI models, large animals such as the pig may play an important translational role in facilitating the development of devices or treatments. Therefore, the objective of this study was to develop a urodynamics protocol to characterize NLUTD in a porcine model of SCI. An iterative process to develop the protocol to perform urodynamics in female Yucatan minipigs began with a group of spinally intact, anesthetized pigs. Subsequently, urodynamic studies were performed in a group of awake, lightly restrained pigs, before and after a contusion-compression SCI at the T2 or T9-T11 spinal cord level. Bladder tissue was obtained for histological analysis at the end of the study. All anesthetized pigs had bladders that were acontractile, which resulted in overflow incontinence once capacity was reached. Uninjured, conscious pigs demonstrated appropriate relaxation and contraction of the external urethral sphincter during the voiding phase. SCI pigs demonstrated neurogenic detrusor overactivity and a significantly elevated post-void residual volume. Relative to the control, SCI bladders were heavier and thicker. The developed urodynamics protocol allows for repetitive evaluation of lower urinary tract function in pigs at different time points post-SCI. This technique manifests the potential for using the pig as an intermediary, large animal model for translational studies in NLUTD.

Net release and uptake of xenometabolites across intestinal, hepatic, muscle, and renal tissue beds in healthy conscious pigs.

Xenometabolites from microbial and plant sources are thought to confer beneficial as well as deleterious effects on host physiology. Studies determining absorption and tissue uptake of xenometabolites are limited. We utilized a conscious catheterized pig model to evaluate interorgan flux of annotated known and suspected xenometabolites, derivatives, and bile acids. Female pigs (n = 12, 2-3 mo old, 25.6 ± 2.2 kg) had surgically implanted catheters across portal-drained viscera (PDV), splanchnic compartment (SPL), liver, kidney, and hindquarter muscle. Overnight-fasted arterial and venous plasma was collected simultaneously in a conscious state and stored at -80°C. Thawed samples were analyzed by liquid chromatography-mass spectrometry. Plasma flow was determined with para-aminohippuric acid dilution technology and used to calculate net organ balance for each metabolite. Significant organ uptake or release was determined if net balance differed from zero. A total of 48 metabolites were identified in plasma, and 31 of these had at least one tissue with a significant net release or uptake. All bile acids, indole-3-acetic acid, indole-3-arylic acid, and hydrocinnamic acid were released from the intestine and taken up by the liver. Indole-3-carboxaldehyde, p-cresol glucuronide, 4-hydroxyphenyllactic acid, dodecanendioic acid, and phenylacetylglycine were also released from the intestines. Liver or kidney uptake was noted for indole-3-acetylglycine, p-cresol glucuronide, atrolactic acid, and dodecanedioic acid. Indole-3-carboxaldehyde, atrolactic acid, and dodecanedioic acids showed net release from skeletal muscle. The results confirm gastrointestinal origins for several known xenometabolites in an in vivo overnight-fasted conscious pig model, whereas nongut net release of other putative xenometabolites suggests a more complex metabolism.NEW & NOTEWORTHY Xenometabolites from microbe origins influence host health and disease, but absorption and tissue uptake of these metabolites remain speculative. Results herein are the first to demonstrate in vivo organ uptake and release of these metabolites. We used a conscious catheterized pig model to confirm gastrointestinal origins for several xenometabolites (e.g., indolic compounds, 4-hydroxyphenyllactic acid, dodecanendioic acid, and phenylacetylgycine). Liver and kidney were major sites for xenometabolite uptake, likely highlighting liver conjugation metabolism and renal excretion.

Intestinal, Hepatic, Muscle, and Renal Xenometabolite Release and Uptake in Healthy Conscious Pigs

Abstract Objectives To characterize the intestinal, hepatic, muscle, and renal release and uptake of xenometabolites (non-host derived metabolites) and endogenously modified xenometabolites by measuring their across-organ net release and uptake in a post-absorptive conscious pig model. Methods Twelve 2–3 months old female pigs (25.6 ± 2.2 kg) had surgically-implanted catheters across portal drained viscera (PDV), splanchnic area (SPL), liver, kidney, and hindquarter muscle. Ten days after catheter implantation, overnight fasted arterial and venous plasma was collected in a conscious state and stored at -80°C. Thawed samples were analyzed by liquid chromatography-mass spectrometry. Xenometabolites were identified by retention time and MS2 spectra from in-house authentic standards and expressed as peak areas. Plasma flow measurements determined with para-aminohippuric acid dilution technology. Net organ balance rate (peak area/kg body weight/min) was calculated. Significant (P &amp;lt; 0.05) release or uptake was determined if tissue net balance differed from zero, using the Wilcoxon Signed Rank Test. Results Forty-eight xenometabolites were structurally identified in this model. Of these, 31 had at least one tissue with a significant net release or uptake. Metabolites suggestive of intestinal origin (i.e., significant PDV net release) included 2-hydroxybenzoic acid, 4-hydroxyphenyllactic acid, acetic acid, atrolactic acid, dodecanedioic acid, hydroxycinnamic acid, p-cresol sulfate, p-cresol glucuronide, stercobilin, and several bile acids and indole derivatives. Of these, atrolactic acid, hydrocinnamic acid, indole-3-acetic acid, stercobilin, and all of the bile acids were taken up by the liver; p-cresol glucuronide was taken up by the kidneys. No xenometabolite released from the gut was taken up by muscle, but 4-hydroxyphenyllactic acid, dodecanedioic acid, and indole-3-carboxaldehyde were also released by muscle tissue. Conclusions Our study confirm gastrointestinal origins for a number of known xenometabolites. Liver and kidney are key organs for xenometabolite uptake. Other putative xenometabolites appear to have either non-gut origins or released by other tissues. Funding Sources Funding provided by the US Department of Agriculture-Agricultural Research Service.

Quantitative Differences in Interorgan Kinetics of Short-Chain Fatty Acids (SCFA)

Abstract Objectives The short-chain fatty acids (SCFA) acetate (C2), propionate (C3), butyrate (C4), isovalerate (Ci5), and valerate (C5) are endproducts of anaerobic fermentation by the intestinal microbiota and known to play a crucial role as energy source for colonocytes (C2 and C4) and muscle (C2), and as mediators in signaling pathways. To unravel the interorgan kinetics of SCFA, we measured their net balances across organs in a translational pig model. Methods In multi-catheterized conscious pigs (n = 11, 25.6 ± 2.2 kg), net balances across portal drained viscera (PDV), liver, kidney and hindquarter (HQ, muscle compartment) were measured in the postabsorptive state. Arterial and venous plasma (in μM) was collected and concentrations were measured by GC-MS. Data are mean (SE). Net balance in nmol/kg BW/min. Significance (P &amp;lt; 0.05) from zero was tested by Wilcoxon Signed Rank Test. Results In the postabsorptive state, arterial concentrations were C2: 189.1 (17.1), C3: 4.8 (0.3), C4: 7.1 (0.9), Ci5: 0.45 (0.09) and C5: 0.15 (0.03). A net release of C2 by PDV (8764 (527.5)) and liver resulted in a high splanchnic release (10,969 (1205)). PDV release of C3 was 2893 (353.9), of C4 1081 (175.9), of Ci5 133.7 (11.1), and of C5 271.2 (44.7). In contrast to C2, after extensive hepatic uptake, splanchnic release of C3, C4, Ci5, and C5 was only 6.5 (0.5)%, 39.3 (8.3)%, 26.3 (6.0)%, and 3.7 (0.9)% of the PDV release. All SCFA were taken up by HQ and kidney. However, while only 62% of C2 released by splanchnic compartment were taken up by muscle (hindquarter and forequarter) and kidneys, net uptake of C3, C4, Ci5 and C5 by muscle and kidney was comparable to splanchnic release. Conclusions Interorgan kinetics differ between C2 and the other SCFA. Due to differences in hepatic SCFA metabolism, acetate is the main SCFA in the systemic circulation possibly acting as energy source to muscle tissue, kidneys and organs or tissues not analyzed in this study. The liver controls the systemic concentrations of all other SCFA. Funding Sources Huffines Institute for Sports Medicine and Human Performance.

The KCa2 Channel Inhibitor AP30663 Selectively Increases Atrial Refractoriness, Converts Vernakalant-Resistant Atrial Fibrillation and Prevents Its Reinduction in Conscious Pigs.

AimsTo describe the effects of the KCa2 channel inhibitor AP30663 in pigs regarding tolerability, cardiac electrophysiology, pharmacokinetics, atrial functional selectivity, effectiveness in cardioversion of tachy-pacing induced vernakalant-resistant atrial fibrillation (AF), and prevention of reinduction of AF.Methods and ResultsSix healthy pigs with implanted pacemakers and equipped with a Holter monitor were used to compare the effects of increasing doses (0, 5, 10, 15, 20, and 25 mg/kg) of AP30663 on the right atrial effective refractory period (AERP) and on various ECG parameters, including the QT interval. Ten pigs with implanted neurostimulators were long-term atrially tachypaced (A-TP) until sustained vernakalant-resistant AF was present. 20 mg/kg AP30663 was tested to discover if it could successfully convert vernakalant-resistant AF to sinus rhythm (SR) and protect against reinduction of AF. Seven anesthetized pigs were used for pharmacokinetic experiments. Two pigs received an infusion of 20 mg/kg AP30663 over 60 min while five pigs received 5 mg/kg AP30663 over 30 min. Blood samples were collected before, during, and after infusion on AP30663. AP30663 was well-tolerated and prominently increased the AERP in pigs with little effect on ventricular repolarization. Furthermore, it converted A-TP induced AF that had become unresponsive to vernakalant, and it prevented reinduction of AF in pigs. Both a >30 ms increase of the AERP and conversion of AF occurred in different pigs at a free plasma concentration level of around 1.0–1.4 µM of AP30663, which was achieved at a dose level of 5 mg/kg.ConclusionAP30663 has shown properties in animals that would be of clinical interest in man.

P700The SK channel inhibitor AP30663, converts vernakalant-resistant persistent AF and prevents its reinduction in pigs

Abstract Background Small conductance Ca2+-activated K+-channels (SK-channels) are a promising new atrial selective target for treatment of atrial fibrillation (AF). AP30663 is a small molecule inhibitor of SK-channels that is currently undergoing clinical trials for treatment of AF. Here we present preclinical data from conscious pigs with persistent AF treated with AP30663. Purpose To examine the pharmakokintetics (PK) of AP30663 in anaesthetized pigs and to test whether AP30663 could cardiovert AF that was resistant to treatment by 4 mg/kg vernakalant in pigs. Methods A total of 12 Danish landrace pigs (gilts) were used for the experiments (2 for PK and 10 for cardioversion of AF). Ten conscious pigs with implanted neurostimulators were tachypaced in the right atrium for 17±5 days until persistent AF that did not respond to treatment with 4 mg/kg vernakalant was obtained. After 3±2 days of vernakalant-resistant AF, the pigs received an infusion of 20 mg/kg AP30663 over 60 minutes. During the infusion the pigs remained conscious and the ECG was monitored. If AF reverted within this period, burst pacing with 50 Hz was applied thrice. Cardioversion was considered successful if sustained AF was reverted. Protection against reinduction of AF was considered successful if no episodes of AF lasting for more than 10 minutes could be reinduced by burst pacing. Results Six out of ten pigs with vernakalant-resistant AF cardioverted during infusion of 20 mg/kg AP30663 over 60 minutes. Four out of six pigs were protected against re-induction of AF by burst pacing. The average time to cardioversion was 29±18 minutes corresponding to calculated free plasma concentrations of 1.0–1.4 μM AP30663. In all the conscious pigs AP30663 was well tolerated with no adverse events. The maximal plasma concentration (Cmax) of AP30663 observed at the end of infusion was 4532±844 ng/ml corresponding to an unbound concentration of 1.54±0.29 μM. The plasma concentrations during infusion were described well by first-order kinetics with a half-time of 5.2 minutes, whereas the plasma concentrations after infusion followed Michaeilis-Menten kinetics with a fast half-life of 6.8 minutes and a slow half-life of 93.7 minutes. Thus, it seems that there is a fast distribution to other tissues and an elimination half-life of approximately 90 minutes. The plasma concentration during infusion reached 90% of the steady state concentration after 17 minutes. Conclusion In an advanced pig model of persistent AF where clinically relevant doses of vernakalant could no longer convert the AF to SR, AP30663 able to convert the pigs to sinus rhythm and protect against reinduction of AF. No signs of adverse events were observed during or after infusion of AP30663. The SK channel inhibitor AP30663 had a fast distribution and an elimination half-life of approximately 90 minutes. The results support the development of AP30663 for treatment of AF in man.

125-LB: An Ingestible Capsule to Deliver Parenteral Pharmaceuticals into the Jejunal Wall

Aim: We developed a platform to painlessly deliver by mouth drugs that cannot be administered orally. This ingestible, capsule-like device deploys inside the small intestine and injects into its wall a biotherapeutic payload sealed inside a dissolvable microneedle. Results: We tested the delivery and examined the pharmacokinetics of several biopharmaceuticals, including recombinant human insulin, human immunoglobulin G, octreotide and teriparatide in anesthetized and conscious pigs and dogs. The bioavailability of these compounds delivered by the capsules was similar to that after subcutaneous delivery. For instance, the mean peak serum concentration of insulin after capsule delivery in 8 anesthetized pigs was 517 ± 109 pmol/l, versus 342 ± 50 pmol/l after subcutaneous injection in 9 other, sham-operated pigs. In vivo safety studies were conducted in 23 conscious beagles, which received between 2 and 18 capsules. All devices transited painlessly through the GI tract and were excreted within 96 h. The mean gastric residence time (GRT) of the capsules was 93 min, and subsequent intestinal deployment time (IDT) was 28 minutes. A pilot study in 10 fasting and 10 postprandial human volunteers examined the tolerability and safety of the capsule devoid of microneedle and payload. The mean GRT of the capsule was 217 ± 36 min in the postprandial and 100 ± 79 min in the fasting state, while the IDT were closely similar (100 ± 40 vs. 97 ± 30 min) in both groups. No subject perceived the device transit, deployment or excretion. Conclusions: The studies completed thus far in anesthetized or conscious animals have confirmed that this ingestible device can deliver drugs in sufficient amounts and rate to obtain therapeutic blood concentrations. Its oral administration was well tolerated by the human subjects. First-in-man studies are being planned to test the ability of this ingestible device to deliver pharmaceuticals in healthy human volunteers and patients. Disclosure R. Ruffy: Employee; Self; RANI Therapeutics. M. Hashim: Board Member; Self; Rani Therapeutics. Employee; Self; InCube Labs LLC. A. Dhalla: Employee; Self; Rani Therapeutics, LLC. R. Korupolu: None. P. Karamchedu: Employee; Self; Incube labaoratories, Rani Therapeutics. B. Syed: None. R. Abdul Wahab: Employee; Self; Rani Therapeutics. S. Beraki: Employee; Self; InCube Laboratories, RANI Therapeutics. A. Yamaguchi: Employee; Self; Rani Therapeutics. L. Fusaro: None. A. Garapaty: None. A. Dasari: None. L. Fung: Employee; Self; Rani Therapeutics. Z.T. Al-Shamsie: Employee; Spouse/Partner; Google. Employee; Self; Rani Therapeutics, InCube Labs LLC. A. Toledo: Employee; Self; InCube Labs LLC, Rani Therapeutics. V. Salgotra: Employee; Self; Incubelabs, Rani Therapeutics. S. Sharma: None. E. Liang: None. D. Gratta: Employee; Self; InCube Labs, Rani Theraputics. M. Imran: Other Relationship; Self; InCube Labs, InCube Ventures, Rani Therapeutics.

Electroencephalographic responses of anaesthetised pigs to intraperitoneal injection of sodium pentobarbital

AbstractSmall laboratory animals are commonly euthanased via intraperitoneal (IP) injection of sodium pentobarbital. However, there is concern that animals may experience pain prior to loss of consciousness with this delivery route. The present study investigated electroencephalographic (EEG) nociceptive responses of anaesthetised pigs to IP sodium pentobarbital injection using an established minimal anaesthesia model. Thirty commercial white line entire male pigs aged 10-15 days were minimally anaesthetised with halothane in oxygen. Following 10 min of baseline EEG data collection, pigs had their tails docked using side-cutters and, after a further 5-min interval, were euthanased via IP injection of sodium pentobarbital (250 mg kg-1). The summary variables median frequency (F50), 95% spectral edge frequency (F95) and total power (PTOT) were derived from the EEG data. For each variable in each pig, means were calculated for the following 60-s periods: immediately prior to tail-docking (baseline 1); immediately prior to pentobarbital injection (at least 4 min after docking; baseline 2); and for two consecutive 60-s periods immediately following pentobarbital injection (P1 and P2). Statistical analyses revealed no differences between the two baseline periods, indicating that transient EEG changes induced by tail-docking had resolved prior to pentobarbital injection. IP pentobarbital injection induced a significant increase in F50 and decrease in PTOTof the EEG during P1. This response is characteristic of acute nociception, indicating that conscious pigs likely perceive IP sodium pentobarbital as painful in the period prior to loss of consciousness.

P1713Novel negative, allosteric modulator inhibits small conductance Ca2+-activated K+-channels and selectively prolongs atrial refractoriness in conscious pigs

P1713Novel negative, allosteric modulator inhibits small conductance Ca2+-activated K+-channels and selectively prolongs atrial refractoriness in conscious pigs

Effects of chronic abdominal vagal stimulation of small-diameter neurons on brain metabolism and food intake

BackgroundAbdominal bilateral vagal stimulation reduces food intake in animals. However, the classical square wave, mA range current generator is poorly effective to evoke action potentials on A∂ and C neurons that represent the majority of vagal neurons at the abdominal level. Objective/Hypothesis(i)To ascertain the capability of very high-frequency stimulation schemes (pulsons) to trigger action potentials in abdominal vagal neurons in anaesthetized pigs. (ii) To compare these stimulation schemes with classical ones using PET imaging of brain metabolism and food intake behaviour in conscious pigs. MethodsThe current thresholds for pulsons (S2 & S3) and millisecond pulses (S1) required to trigger action potentials were calculated in 5 anaesthetized pigs using single fibre recording. Similar stimulation protocols were compared chronically to sham stimulation in 24 pigs. After two weeks of chronic stimulation, food intake and brain metabolism were investigated. The electrical characteristics and histology of the vagus nerve were also studied. ResultsS3 stimulation required a lower amount of charges to trigger an action potential. Chronically applied S2 & S3 activated the dorsal vagal complex and increased the metabolism of its afferent cortical structures. They also reduced energy intake together with a reduced ingestion of high fat and high sugar diets. All these effects were not observed for the S1 group. The vagal histology for the S1, S2 and S3 groups was not different from that of the sham. ConclusionsThese findings demonstrate that pulsons applied bilaterally on the abdominal vagus reduced food intake as a consequence of the activation of the brainstem and higher-order brain areas.

A Translational Study of a New Therapeutic Approach for Acute Myocardial Infarction: Nanoparticle-Mediated Delivery of Pitavastatin into Reperfused Myocardium Reduces Ischemia-Reperfusion Injury in a Preclinical Porcine Model

BackgroundThere is an unmet need to develop an innovative cardioprotective modality for acute myocardial infarction, for which interventional reperfusion therapy is hampered by ischemia-reperfusion (IR) injury. We recently reported that bioabsorbable poly(lactic acid/glycolic acid) (PLGA) nanoparticle-mediated treatment with pitavastatin (pitavastatin-NP) exerts a cardioprotective effect in a rat IR injury model by activating the PI3K-Akt pathway and inhibiting inflammation. To obtain preclinical proof-of-concept evidence, in this study, we examined the effect of pitavastatin-NP on myocardial IR injury in conscious and anesthetized pig models.Methods and ResultsEighty-four Bama mini-pigs were surgically implanted with a pneumatic cuff occluder at the left circumflex coronary artery (LCx) and telemetry transmitters to continuously monitor electrocardiogram as well as to monitor arterial blood pressure and heart rate. The LCx was occluded for 60 minutes, followed by 24 hours of reperfusion under conscious conditions. Intravenous administration of pitavastatin-NP containing ≥ 8 mg/body of pitavastatin 5 minutes before reperfusion significantly reduced infarct size; by contrast, pitavastatin alone (8 mg/body) showed no therapeutic effects. Pitavastatin-NP produced anti-apoptotic effects on cultured cardiomyocytes in vitro. Cardiac magnetic resonance imaging performed 4 weeks after IR injury revealed that pitavastatin-NP reduced the extent of left ventricle remodeling. Importantly, pitavastatin-NP exerted no significant effects on blood pressure, heart rate, or serum biochemistry. Exploratory examinations in anesthetized pigs showed pharmacokinetic analysis and the effects of pitavastatin-NP on no-reflow phenomenon.ConclusionsNP-mediated delivery of pitavastatin to IR-injured myocardium exerts cardioprotective effects on IR injury without apparent adverse side effects in a preclinical conscious pig model. Thus, pitavastatin-NP represents a novel therapeutic modality for IR injury in acute myocardial infarction.

A Food and Drug Administration-Approved Antiviral Agent that Inhibits Adenylyl Cyclase Type 5 Protects the Ischemic Heart Even When Administered after Reperfusion.

A Food and Drug Administration-approved antiviral agent, known as vidarabine or adenine 9-β-D-arabinofuranoside (AraA), has features of inhibiting adenylyl cyclase type 5 (AC5) and protects against chronic coronary artery occlusion (CAO). The goal of this investigation was to determine whether AraA protects against myocardial ischemia, even when delivered after coronary artery reperfusion (CAR). AraA, delivered after CAR in wild-type mice, reduced infarct size by 55% compared with vehicle-treated controls, whereas an equal dose of adenosine reduced infarct size only when administered before CAR. A 5-fold greater dose of adenosine was required to reduce infarct size when delivered after CAR, which also reduced arterial pressure by 15%, whereas AraA did not affect pressure. The reduction in infarct size with AraA was prevented by a MEK/extracellular signal-regulated kinase blocker, a pathway also involved in the mechanism of protection of the AC5 knockout (KO) model. Infarct size was also reduced in cardiac-specific AC5 KO mice similarly in the presence and absence of AraA, further suggesting that AraA protection involves the AC5 pathway. AraA reduced infarct size in chronically instrumented conscious pigs when delivered after CAR, and in this model, it also reduced post-CAR coronary hyperemia, which could be another mechanism for cardioprotection (i.e., by reducing oxidative stress during CAR). Thus, AraA inhibits AC5 and exhibits unique cardioprotection when delivered after CAR, which is critical for clinical translation.

Modelling severe Staphylococcus aureus sepsis in conscious pigs: are implications for animal welfare justified?

BackgroundA porcine model of haematogenous Staphylococcus aureus sepsis has previously been established in our research group. In these studies, pigs developed severe sepsis including liver dysfunction during a 48 h study period. As pigs were awake during the study, animal welfare was challenged by the severity of induced disease, which in some cases necessitated humane euthanasia. A pilot study was therefore performed in order to establish the sufficient inoculum concentration and application protocol needed to produce signs of liver dysfunction within limits of our pre-defined humane endpoints.MethodsFour pigs received 1 × 108 cfu/kg BW of S. aureus, and two controls were sham inoculated with saline. A fixed infusion rate of 3 mL/min was used, while the inoculum concentration, i.e., the dose volume, was changed between the pigs. The following dose volumes were used: 10 mL (n = 1), 20 mL (n = 2), and 30 mL (n = 1), corresponding to infusion durations of 3.33, 6.66, and 10 min at dose rates of 3 × 107, 1.5 × 107, and 1 × 107 cfu/min/kg BW, respectively. Blood samples were drawn for complete blood count, clinical chemistry, and inflammatory markers before and every 6 h after inoculation. Prior to euthanasia, a galactose elimination capacity test was performed to assess liver function. Pigs were euthanised 48 h post inoculation for necropsy and histopathological evaluation.ResultsWhile infusion times of 6.66 min, and higher, did not induce liver dysfunction (n = 3), the infusion time of 3.33 min (n = 1) caused alterations in parameters similar to what had been seen in our previous studies, i.e., increasing bilirubin and aspartate aminotransferase, as well as histopathological occurrence of intravascular fibrin split products in the liver. This pig was however euthanised after 30 h, according to humane endpoints.ConclusionsA usable balance between scientific purpose and animal welfare could not be achieved, and we therefore find it hard to justify further use of this conscious porcine sepsis model. In order to make a model of translational relevance for human sepsis, we suggest that future model versions should use long-term anaesthesia.

Pharmacological modulation of ureteric peristalsis in a chronically instrumented conscious pig model: effect of adrenergic and nitrergic modulation.

To evaluate the role of adrenergic and nitrergic signaling on ureteric peristaltic frequency and contraction force in vivo using a large animal model. Twelve female pigs (72±4kg) were chronically instrumented with an electronic pressure-monitoring catheter in the right ureter. Nephrostomy, cystostomy, and arterial and venous catheters were left in situ. Ureteral peristalsis was recorded before and after the administration of propranolol, isoprenaline, doxazosin, urapidil, phenylephrine, LNNA (Nω-nitro-L-arginine), and L-arginine. α1-Adrenergic receptor stimulation resulted in an increased P max and peristaltic frequency. However, α1-inhibition decreased P max alone. Similarly, β-adrenergic stimulation decreased P max and peristaltic frequency, whereas β-inhibition increased only P max. LNNA administration increased P max in the distal ureter and hydrostatic pressure in the pyelocalyceal system. L-Arginine did not affect P max or frequency, but resulted in a significantly higher diuresis. Either agonist or antagonist of NO did not affect peristaltic frequency and length of contraction. Activation of α- and β-adrenergic receptors, respectively, stimulates and inhibits ureteric peristalsis. The biological effect of NO on ureteric motility is regionally determined and corresponds to the distribution of NOS-positive nerves. Inhibition of NOS activity increases P max in the distal ureter and tonic activity of the ureteric muscle resulting in higher hydrostatic pressure in the renal pelvis.

Shock wave lithotripsy does not impair renal function in a Swine model of metabolic syndrome.

To determine whether shock wave lithotripsy (SWL) may be a risk factor for renal functional impairment in a swine model of metabolic syndrome (MetS). Nine-month-old female Ossabaw pigs were fed an excess calorie atherogenic diet to induce MetS. At 15 months of age, the MetS pigs were treated with 2000 SWs or an overtreatment dose of 4000 SWs targeted at the upper pole calyx of the left kidney (24 kV at 120 SWs/min using the unmodified Dornier HM3 lithotripter; n=5-6 per treatment group). Serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured in conscious pigs before and ∼60 days after SWL to provide a qualitative assessment of how well both kidneys were filtering (glomerular filtration rate [GFR]). Bilateral renal function was assessed at ∼65 days post-SWL in anesthetized pigs with GFR and effective renal plasma flow (ERPF) quantified by the renal clearance of inulin and para-amino hippurate, respectively. Cr and BUN values were within normal limits before SWL and remained unchanged after lithotripsy in both the 2000 SW- and 4000 SW-treated pigs. GFR and ERPF of kidneys treated with SWL at either SW dose were similar to the contralateral nontreated kidney. Chronic histological changes in the SW-treated pole of the kidney included interstitial fibrosis, sclerotic glomeruli, and dilated and atrophic tubules. Our results are consistent with the view that a single SWL session does not result in renal impairment, even in the presence of MetS.