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.

Lung injury caused by aspiration of organophosphorus insecticide and gastric contents in pigs.

INTRODUCTION: Patients who require mechanical ventilation after self-poisoning with ingested organophosphorus (OP) insecticides often die. Aspiration of stomach contents may contribute to lung injury and lethality. This study was designed to assess the severity of direct and indirect pulmonary injury created by pulmonary instillation of mixtures of OP insecticide, solvent (Solv) and porcine gastric juice (GJ) compared to controls. METHODS: Terminally anaesthetised minipigs (groups n = 5) were exposed to sham bronchoscopy or given mixtures (0.5 mL/kg) of: saline, GJ, OP insecticide and GJ (OP + GJ), or Solv and GJ (Solv + GJ), placed into the right lung, and monitored for 48 h. Lung injury was assessed through analysis of bronchoalveolar lavage fluid (BALF), computed tomography and histopathology. RESULTS: OP + GJ created a direct lung injury consisting of neutrophil infiltration, oedema and haemorrhage, as well as indirect injury to the other lung. OP + GJ directly-injured lung parenchyma had increased concentrations of BALF protein, albumin, IL-6, IL-8 and C-reactive protein (CRP) at 24 h (p < 0.05), and BALF protein, albumin and CRP at 48 h (p < 0.05), when compared with controls. Aspiration of GJ produced similar direct effects to OP + GJ but less indirect lung injury. Lung injury was less severe after Solv + GJ, for combined lung histopathology scores (vs. OP + GJ, p < 0.05) and for the proportion of directly-injured lung that was poorly/non-aerated at 48 h. CONCLUSION: Pulmonary instillation of OP + GJ created more lung damage than controls or Solv + GJ. In patients with severe OP insecticide poisoning and reduced consciousness, early airway protection is likely to reduce pulmonary damage.

In vivo application of an implantable tri-anchored methylene blue-based electrochemical pH sensor.

The development of robust implantable sensors is important in the successful advancement of personalised medicine as they have the potential to provide in situ real-time data regarding the status of health and disease and the effectiveness of treatment. Tissue pH is a key physiological parameter and herein, we report the design, fabrication, functionalisation, encapsulation and protection of a miniaturised, self-contained, electrochemical pH sensor system and characterisation of sensor performance. Notably for the first time in this environment the pH sensor was based on a methylene blue redox reporter which showed remarkable robustness, accuracy and sensitivity. This was achieved by encapsulation of a self-assembled monolayer containing methylene blue entrapped within a Nafion layer. Another powerful feature was the incorporation, within the same implanted device, of a fabricated on-chip Ag/AgCl reference electrode - vital in any electrochemical sensor, but often ignored. When utilised in vivo, the sensor allowed accurate tracking of externally induced pH changes within a naturally occurring ovine lung cancer model, and correlated well with single point laboratory measurements made on extracted arterial blood, whilst enabling in vivo time-dependent measurements. The sensors functioned robustly whilst implanted, and maintained in vitro function once extracted and together, these results demonstrate proof-of-concept of the ability to sense real-time intratumoral tissue pH changes in vivo.

Impaired neuromuscular function by conjoint actions of organophosphorus insecticide metabolites omethoate and cyclohexanol with implications for treatment of respiratory failure.

BACKGROUND: Ingestion of agricultural organophosphorus insecticides is a significant cause of death in rural Asia. Patients often show acute respiratory failure and/or delayed, unexplained signs of neuromuscular paralysis, sometimes diagnosed as "Intermediate Syndrome". We tested the hypothesis that omethoate and cyclohexanol, circulating metabolites of one agricultural formulation, cause muscle weakness and paralysis. METHODS: Acetylcholinesterase activity of insecticide components and metabolites was measured using purified enzyme from eel electroplaque or muscle homogenates. Mechanomyographic recording of pelvic limb responses to nerve stimulation was made in anaesthetized pigs and isometric force was recorded from isolated nerve-muscle preparations from mice. Omethoate and cyclohexanol were administered intravenously or added to physiological saline bathing isolated muscle. We also assessed the effect of MgSO4 and cooling on neuromuscular function. RESULTS: Omethoate caused tetanic fade in pig muscles and long-lasting contractions of the motor innervation zone in mouse muscle. Both effects were mitigated, either by i.v. administration of MgSO4 in vivo or by adding 5 mM Mg2+ to the medium bathing isolated preparations. Combination of omethoate and cyclohexanol initially potentiated muscle contractions but then rapidly blocked them. Cyclohexanol alone caused fade and block of muscle contractions in pigs and in isolated preparations. Similar effects were observed ex vivo with cyclohexanone and xylene. Cyclohexanol-induced neuromuscular block was temperature-sensitive and rapidly reversible. CONCLUSIONS: The data indicate a crucial role for organophosphorus and solvent metabolites in muscle weakness following ingestion of agricultural OP insecticide formulations. The metabolites omethoate and cyclohexanol acted conjointly to impair neuromuscular function but their effects were mitigated by elevating extracellular Mg2+ and decreasing core temperature, respectively. Clinical studies of MgSO4 therapy and targeted temperature management in insecticide-poisoned patients are required to determine whether they may be effective adjuncts to treatment.

Development of a histopathology scoring system for the pulmonary complications of organophosphorus insecticide poisoning in a pig model.

Organophosphorus (OP) insecticide self-poisoning causes over 100,000 global deaths annually. Around a third of patients are intubated and up to half of these can die. Post-mortem analysis of OP poisoned patients' lungs reveals consolidation, edema and hemorrhage, suggesting that direct or indirect lung damage may contribute to mortality. The lung injury caused by these formulated agricultural preparations is poorly characterised in humans, and a valid histopathology scoring system is needed in a relevant animal model to further investigate the disease and potential treatments. We conducted two pilot studies in anesthetized minipigs, which are commonly used for toxicological studies. In the first, pigs were given 2.5 mL/kg of either OP (n = 4) or saline (n = 2) by gavage and compared with positive controls (iv oleic acid n = 2). The second study simulated ingestion followed by gastric content aspiration: mixtures of OP (n = 3) or saline (n = 2) (0.63-0.71mL/kg) were placed in the stomach, and then small volumes of the gastric content were placed in the lung. At post-mortem examination, lungs were removed and inflation-fixed with 10% neutral buffered formalin. Samples (n = 62) were taken from cranial and caudal regions of both lungs. Two experienced lung histopathologists separately scored these samples using 8 proposed features of damage and their scores related (Kendall rank order). Two elements had small and inconsistent scores. When these were removed, the correlation increased from 0.74 to 0.78. Eight months later, a subset of samples (n = 35) was re-scored using the modified system by one of the previous histopathologists, with a correlation of 0.88. We have developed a reproducible pulmonary histopathology scoring system for OP poisoning in pigs which will assist future toxicological research and improve understanding and treatment of human OP poisoning.

Comparative anatomy of the mammalian neuromuscular junction.

The neuromuscular junction (NMJ)-a synapse formed between lower motor neuron and skeletal muscle fibre-represents a major focus of both basic neuroscience research and clinical neuroscience research. Although the NMJ is known to play an important role in many neurodegenerative conditions affecting humans, the vast majority of anatomical and physiological data concerning the NMJ come from lower mammalian (e.g. rodent) animal models. However, recent findings have demonstrated major differences between the cellular anatomy and molecular anatomy of human and rodent NMJs. Therefore, we undertook a comparative morphometric analysis of the NMJ across several larger mammalian species in order to generate baseline inter-species anatomical reference data for the NMJ and to identify animal models that better represent the morphology of the human NMJ in vivo. Using a standardized morphometric platform ('NMJ-morph'), we analysed 5,385 individual NMJs from lower/pelvic limb muscles (EDL, soleus and peronei) of 6 mammalian species (mouse, cat, dog, sheep, pig and human). There was marked heterogeneity of NMJ morphology both within and between species, with no overall relationship found between NMJ morphology and muscle fibre diameter or body size. Mice had the largest NMJs on the smallest muscle fibres; cats had the smallest NMJs on the largest muscle fibres. Of all the species examined, the sheep NMJ had the most closely matched morphology to that found in humans. Taken together, we present a series of comprehensive baseline morphometric data for the mammalian NMJ and suggest that ovine models are likely to best represent the human NMJ in health and disease.

Intelligent magnetic manipulation for gastrointestinal ultrasound.

Diagnostic endoscopy in the gastrointestinal tract has remained largely unchanged for decades and is limited to the visualization of the tissue surface, the collection of biopsy samples for diagnoses, and minor interventions such as clipping or tissue removal. In this work, we present the autonomous servoing of a magnetic capsule robot for in-situ, subsurface diagnostics of microanatomy. We investigated and showed the feasibility of closed-loop magnetic control using digitized microultrasound (µUS) feedback; this is crucial for obtaining robust imaging in an unknown and unconstrained environment. We demonstrated the functionality of an autonomous servoing algorithm that uses µUS feedback, both on benchtop trials as well as in-vivo in a porcine model. We have validated this magnetic-µUS servoing in instances of autonomous linear probe motion and were able to locate markers in an agar phantom with 1.0 ± 0.9 mm position accuracy using a fusion of robot localization and µUS image information. This work demonstrates the feasibility of closed-loop robotic µUS imaging in the bowel without the need for either a rigid physical link between the transducer and extracorporeal tools or complex manual manipulation.

Ovine Pulmonary Adenocarcinoma: A Unique Model to Improve Lung Cancer Research.

Lung cancer represents a major worldwide health concern; although advances in patient management have improved outcomes for some patients, overall 5-year survival rates are only around 15%. In vitro studies and mouse models are commonly used to study lung cancer and their use has increased the molecular understanding of the disease. Unfortunately, mouse models are poor predictors of clinical outcome and seldom mimic advanced stages of the human disease. Animal models that more accurately reflect human disease are required for progress to be made in improving treatment outcomes and prognosis. Similarities in pulmonary anatomy and physiology potentially make sheep better models for studying human lung function and disease. Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring lung cancer that is caused by the jaagsiekte sheep retrovirus. The disease is endemic in many countries throughout the world and has several features in common with human lung adenocarcinomas, including histological classification and activation of common cellular signaling pathways. Here we discuss the in vivo and in vitro OPA models that are currently available and describe the advantages of using pre-clinical naturally occurring OPA cases as a translational animal model for human lung adenocarcinoma. The challenges and options for obtaining these OPA cases for research purposes, along with their use in developing novel techniques for the evaluation of chemotherapeutic agents or for monitoring the tumor microenvironment in response to treatment, are also discussed.

In Vivo Characterization of a Wireless Telemetry Module for a Capsule Endoscopy System Utilizing a Conformal Antenna.

This paper describes the design, fabrication, packaging, and performance characterization of a conformal helix antenna created on the outside of a capsule endoscope designed to operate at a carrier frequency of 433 MHz within human tissue. Wireless data transfer was established between the integrated capsule system and an external receiver. The telemetry system was tested within a tissue phantom and in vivo porcine models. Two different types of transmission modes were tested. The first mode, replicating normal operating conditions, used data packets at a steady power level of 0 dBm, while the capsule was being withdrawn at a steady rate from the small intestine. The second mode, replicating the worst-case clinical scenario of capsule retention within the small bowel, sent data with stepwise increasing power levels of -10, 0, 6, and 10 dBm, with the capsule fixed in position. The temperature of the tissue surrounding the external antenna was monitored at all times using thermistors embedded within the capsule shell to observe potential safety issues. The recorded data showed, for both modes of operation, a low error transmission of 10-3 packet error rate and 10-5 bit error rate and no temperature increase of the tissue according to IEEE standards.

Thermography as an early predictive measurement for evaluating epidural and femoral-sciatic block success in dogs.

OBJECTIVE: To evaluate skin temperature increase as an early predictive measure for evaluating epidural and femoral-sciatic block success in dogs. STUDY DESIGN: Prospective clinical trial. ANIMALS: A total of 29 dogs undergoing orthopaedic surgery on one hindlimb. METHODS: Dogs were anaesthetized and placed into lateral recumbency with the affected limb uppermost and the coat was clipped. Baseline infrared thermographic images (T0) of the affected limb, of the paw pad of the affected leg and of the ipsilateral paw pad were taken. Subsequently, dogs were administered either an epidural (EPI; n=11) or a femoral-sciatic block (FS; n=18) using bupivacaine 1 mg kg-1. Then, 2 minutes after placement of the block, thermographic images were obtained every 3 minutes for a total of four measurements (T1-T4) and surgery was commenced. Rescue analgesia consisting of fentanyl 1 µg kg-1 was administered if needed. A regional block was considered successful if the dose of fentanyl administered was less than the lower 95% confidence interval of the geometric mean of the total fentanyl used in each group. A ≥ 1 °C increase of skin temperature was considered as the minimum increase required for detection of a successful block. RESULTS: A total of 12 out of 18 blocks in the FS and eight of 11 in the EPI group were considered successful based on fentanyl consumption. Out of these, only four of 12 in the FS and one of eight in the EPI group developed an increase in temperature of ≥ 1 °C. Contrarily, four of six of the nonsuccessful cases in the FS and three of three in the EPI group developed an increase in temperature of ≥ 1 °C. CONCLUSIONS AND CLINICAL RELEVANCE: Contrary to reports in humans, thermography did not indicate regional block success prior to surgery in dogs. However further studies under more controlled conditions are needed to determine whether thermography can be used to indicate failure of regional blockade.

Development of an invasively monitored porcine model of acetaminophen-induced acute liver failure.

BACKGROUND: The development of effective therapies for acute liver failure (ALF) is limited by our knowledge of the pathophysiology of this condition, and the lack of suitable large animal models of acetaminophen toxicity. Our aim was to develop a reproducible invasively-monitored porcine model of acetaminophen-induced ALF. METHOD: 35kg pigs were maintained under general anaesthesia and invasively monitored. Control pigs received a saline infusion, whereas ALF pigs received acetaminophen intravenously for 12 hours to maintain blood concentrations between 200-300 mg/l. Animals surviving 28 hours were euthanased. RESULTS: Cytochrome p450 levels in phenobarbital pre-treated animals were significantly higher than non pre-treated animals (300 vs 100 pmol/mg protein). Control pigs (n = 4) survived 28-hour anaesthesia without incident. Of nine pigs that received acetaminophen, four survived 20 hours and two survived 28 hours. Injured animals developed hypotension (mean arterial pressure; 40.8 +/- 5.9 vs 59 +/- 2.0 mmHg), increased cardiac output (7.26 +/- 1.86 vs 3.30 +/- 0.40 l/min) and decreased systemic vascular resistance (8.48 +/- 2.75 vs 16.2 +/- 1.76 mPa/s/m3). Dyspnoea developed as liver injury progressed and the increased pulmonary vascular resistance (636 +/- 95 vs 301 +/- 26.9 mPa/s/m3) observed may reflect the development of respiratory distress syndrome.Liver damage was confirmed by deterioration in pH (7.23 +/- 0.05 vs 7.45 +/- 0.02) and prothrombin time (36 +/- 2 vs 8.9 +/- 0.3 seconds) compared with controls. Factor V and VII levels were reduced to 9.3 and 15.5% of starting values in injured animals. A marked increase in serum AST (471.5 +/- 210 vs 42 +/- 8.14) coincided with a marked reduction in serum albumin (11.5 +/- 1.71 vs 25 +/- 1 g/dL) in injured animals. Animals displayed evidence of renal impairment; mean creatinine levels 280.2 +/- 36.5 vs 131.6 +/- 9.33 mumol/l. Liver histology revealed evidence of severe centrilobular necrosis with coagulative necrosis. Marked renal tubular necrosis was also seen. Methaemoglobin levels did not rise >5%. Intracranial hypertension was not seen (ICP monitoring), but there was biochemical evidence of encephalopathy by the reduction of Fischer's ratio from 5.6 +/- 1.1 to 0.45 +/- 0.06. CONCLUSION: We have developed a reproducible large animal model of acetaminophen-induced liver failure, which allows in-depth investigation of the pathophysiological basis of this condition. Furthermore, this represents an important large animal model for testing artificial liver support systems.