Noninvasive Detection of Ischemic Vascular Damage in a Pig Model of Liver Donation After Circulatory Death.

BACKGROUND AND AIMS: Liver graft quality is evaluated by visual inspection prior to transplantation, a process highly dependent on the surgeon's experience. We present an objective, noninvasive, quantitative way of assessing liver quality in real time using Raman spectroscopy, a laser-based tool for analyzing biomolecular composition. APPROACH AND RESULTS: A porcine model of donation after circulatory death (DCD) with normothermic regional perfusion (NRP) allowed assessment of liver quality premortem, during warm ischemia (WI) and post-NRP. Ten percent of circulating blood volume was removed in half of experiments to simulate blood recovery for DCD heart removal. Left median lobe biopsies were obtained before circulatory arrest, after 45 minutes of WI, and after 2 hours of NRP and analyzed using spontaneous Raman spectroscopy, stimulated Raman spectroscopy (SRS), and staining. Measurements were also taken in situ from the porcine liver using a handheld Raman spectrometer at these time points from left median and right lateral lobes. Raman microspectroscopy detected congestion during WI by measurement of the intrinsic Raman signal of hemoglobin in red blood cells (RBCs), eliminating the need for exogenous labels. Critically, this microvascular damage was not observed during WI when 10% of circulating blood was removed before cardiac arrest. Two hours of NRP effectively cleared RBCs from congested livers. Intact RBCs were visualized rapidly at high resolution using SRS. Optical properties of ischemic livers were significantly different from preischemic and post-NRP livers as measured using a handheld Raman spectrometer. CONCLUSIONS: Raman spectroscopy is an effective tool for detecting microvascular damage which could assist the decision to use marginal livers for transplantation. Reducing the volume of circulating blood before circulatory arrest in DCD may help reduce microvascular damage.

Transcranial bioimpedance measurement in horses: a pilot study.

OBJECTIVE: This pilot study aimed to evaluate the feasibility of transcranial bioimpedance (TCBI) measurement and variability of TCBI values in healthy conscious horses and to study effects of body position and time on TCBI in anaesthetized horses. STUDY DESIGN: Prospective, observational study. ANIMALS: A total of four research horses and 16 client-owned horses presented for surgery. METHODS: After establishing optimal electrode position using computed tomography scans of cadaver heads, TCBI [described using impedance at zero frequency, R0, (Ω)] was measured in four conscious, resting horses to investigate the feasibility and changes in TCBI over time (80 minutes). Data were compared using a paired t test. TCBI was then measured throughout anaesthesia (duration 92 ± 28 minutes) in 16 horses in dorsal and lateral recumbency. Data were analysed using a general linear model; gamma regression was chosen as a model of characteristic impedance [Zc; (Ω)] against time. Data are presented as mean ± standard deviation. RESULTS: No change in R0 was seen in conscious horses (age = 15.3 ± 7.3 years, body mass = 512 ± 38 kg) over 80 minutes. The technique was well tolerated and caused no apparent adverse effects. In 16 horses (age = 7.4 ± 4.7 years; body mass = 479 ± 134 kg) anaesthetized for 92 ± 28 minutes, Zc fell during anaesthesia, decreasing more in horses in lateral recumbency than in horses in dorsal recumbency (p = 0.008). There was no relationship between Zc and body mass or age. CONCLUSIONS AND CLINICAL RELEVANCE: TCBI is readily measured in horses. TCBI did not change with time in conscious horses, but decreased with time in anaesthetized horses; this change was greater in horses in lateral recumbency, indicating that TCBI changes in anaesthetized horses may be related to the effects of recumbency, general anaesthesia, surgery or a combination of these factors.

Tracking Ovine Pulmonary Adenocarcinoma Development Using an Experimental Jaagsiekte Sheep Retrovirus Infection Model.

Ovine pulmonary adenocarcinoma (OPA) is an infectious, neoplastic lung disease of sheep that causes significant animal welfare and economic issues throughout the world. Understanding OPA pathogenesis is key to developing tools to control its impact. Central to this need is the availability of model systems that can monitor and track events after Jaagsiekte sheep retrovirus (JSRV) infection. Here, we report the development of an experimentally induced OPA model intended for this purpose. Using three different viral dose groups (low, intermediate and high), localised OPA tumour development was induced by bronchoscopic JSRV instillation into the segmental bronchus of the right cardiac lung lobe. Pre-clinical OPA diagnosis and tumour progression were monitored by monthly computed tomography (CT) imaging and trans-thoracic ultrasound scanning. Post mortem examination and immunohistochemistry confirmed OPA development in 89% of the JSRV-instilled animals. All three viral doses produced a range of OPA lesion types, including microscopic disease and gross tumours; however, larger lesions were more frequently identified in the low and intermediate viral groups. Overall, 31% of JSRV-infected sheep developed localised advanced lesions. Of the sheep that developed localised advanced lesions, tumour volume doubling times (calculated using thoracic CT 3D reconstructions) were 14.8 ± 2.1 days. The ability of ultrasound to track tumour development was compared against CT; the results indicated a strong significant association between paired CT and ultrasound measurements at each time point (R2 = 0.799, p < 0.0001). We believe that the range of OPA lesion types induced by this model replicates aspects of naturally occurring disease and will improve OPA research by providing novel insights into JSRV infectivity and OPA disease progression.

The unique contribution of manual chest compression-vibrations to airflow during physiotherapy in sedated, fully ventilated children.

OBJECTIVE: This study aimed to quantify the specific effects of manual lung inflations with chest compression-vibrations, commonly used to assist airway clearance in ventilated patients. The hypothesis was that force applied during the compressions made a significant additional contribution to increases in peak expiratory flow and expiratory to inspiratory flow ratio over and above that resulting from accompanying increases in inflation volume. DESIGN: Prospective observational study. SETTING: Cardiac and general pediatric intensive care. PATIENTS: Sedated, fully ventilated children. INTERVENTIONS: Customized force-sensing mats and a commercial respiratory monitor recorded force and respiration during physiotherapy. MEASUREMENTS: Percentage changes in peak expiratory flow, peak expiratory to inspiratory flow ratios, inflation volume, and peak inflation pressure between baseline and manual inflations with and without compression-vibrations were calculated. Analysis of covariance determined the relative contribution of changes in pressure, volume, and force to influence changes in peak expiratory flow and peak expiratory to inspiratory flow ratio. MEASUREMENTS AND MAIN RESULTS: Data from 105 children were analyzed (median age, 1.3 yrs; range, 1 wk to 15.9 yrs). Force during compressions ranged from 15 to 179 N (median, 46 N). Peak expiratory flow increased on average by 76% during compressions compared with baseline ventilation. Increases in peak expiratory flow were significantly related to increases in inflation volume, peak inflation pressure, and force with peak expiratory flow increasing by, on average, 4% for every 10% increase in inflation volume (p < .001), 5% for every 10% increase in peak inflation pressure (p = .005), and 3% for each 10 N of applied force (p < .001). By contrast, increase in peak expiratory to inspiratory flow ratio was only related to applied force with a 4% increase for each 10 N of force (p < .001). CONCLUSION: These results provide evidence of the unique contribution of compression forces in increasing peak expiratory flow and peak expiratory to inspiratory flow ratio bias over and above that related to accompanying changes from manual hyperinflations. Force generated during compression-vibrations was the single significant factor in multivariable analysis to explain the increases in expiratory flow bias. Such increases in the expiratory bias provide theoretically optimal physiological conditions for cephalad mucus movement in fully ventilated children.

Near-fatal misuse of medical tape around an endotracheal tube connector during inhalation anesthesia in a horse.

A 7-year-old gelded Irish sports horse weighing 650 kg was anesthetized on 2 consecutive days for lavage of a septic right radio-carpal joint. On both occasions the endotracheal tube connector, which had been bound in medical tape to produce an airtight seal, functioned as a unidirectional valve during mechanical ventilation, retarding expiration, imposing positive end expiratory pressure (PEEP), and probably continuous positive airway pressure (CPAP). The equipment dysfunction was not identified on either occasion despite close inspection prompted by progressive increases in airway pressure and thoracic distension. Whilst the PEEP and CPAP exerted unexpectedly modest cardiovascular effects and the horse recovered uneventfully on both occasions, the improvisation may have proven fatal in a higher-risk subject.A 7-year-old gelded Irish sports horse weighing 650 kg was anesthetized on 2 consecutive days for lavage of a septic right radio-carpal joint. On both occasions the endotracheal tube connector, which had been bound in medical tape to produce an airtight seal, functioned as a unidirectional valve during mechanical ventilation, retarding expiration, imposing positive end expiratory pressure (PEEP), and probably continuous positive airway pressure (CPAP). The equipment dysfunction was not identified on either occasion despite close inspection prompted by progressive increases in airway pressure and thoracic distension. Whilst the PEEP and CPAP exerted unexpectedly modest cardiovascular effects and the horse recovered uneventfully on both occasions, the improvisation may have proven fatal in a higher-risk subject.

RésuméMauvais usage quasi-mortel de ruban adhésif médical autour d'un raccord de tube endotrachéal durant l'anesthésie par inhalation chez un cheval. Un hongre de sport Irlandais âgé de 7 ans pesant 650 kg a été anesthésié deux journées consécutives pour le lavage d'une articulation radio-carpienne droite septique. Lors des deux instances, le raccord du tube endotrachéal, qui avait été attaché à l'aide de ruban adhésif médical pour produire une fermeture hermétique, a servi de valve unidirectionnelle durant la ventilation mécanique, retardant l'expiration, imposant une pression positive en fin d'expiration et probablement une ventilation spontanée en pression positive continue. La défaillance de l'équipement n'a pas été identifiée dans l'une ou l'autre de ces instances, malgré une inspection minutieuse suscitée par des hausses progressives de la pression des voies aériennes et un gonflement thoracique. Même si la pression positive en fin d'expiration et la ventilation spontanée en pression positive continue ont eu des effets cardiovasculaires modestes inattendus et que le cheval s'est rétabli sans incident lors des deux instances, l'improvisation aurait pu s'avérée mortelle chez un sujet présentant des risques plus élevés.(Traduit par Isabelle Vallières).

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.

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.

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.

Cross-species efficacy of enzyme replacement therapy for CLN1 disease in mice and sheep.

CLN1 disease, also called infantile neuronal ceroid lipofuscinosis (NCL) or infantile Batten disease, is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by administering monthly infusions of recombinant human PPT1 (rhPPT1) to PPT1-deficient mice (Cln1-/-) and CLN1R151X sheep to assess how to potentially scale up for translation. In Cln1-/- mice, intracerebrovascular (i.c.v.) rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1-treated mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1R151X sheep, i.c.v. infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural MRI and neuropathology. This study demonstrates the feasibility and therapeutic efficacy of i.c.v. rhPPT1 ERT. These findings represent a key step toward clinical testing of ERT in children with CLN1 disease and highlight the importance of a cross-species approach to developing a successful treatment strategy.

Feeding management before gastrointestinal studies in pigs.

Pigs are used to model humans in gastrointestinal (GI) studies because of their comparable size, physiology and behaviour: both are monogastric omnivores. A porcine surgical model for testing novel, tethered ultrasound capsule endoscopes (USCE) requires a clean, motile small intestine. Recommendations for human GI tract preparation before the mechanically similar process of video capsule endoscopy describe using oral purgatives, while high-carbohydrate drinks are recommended before colorectal surgery. Reports of the GI preparation of pigs exist but lack technical details, that is, administration, efficacy and side effects. This report details feeding a high-energy liquid diet to 11 female pigs undergoing surgery and USCE which was readily accepted and easily administered, and which produced a clean, motile small intestine and caused no detectable physiological/behavioural abnormalities.