Abstract
Pigs are ideal animal models for airway surgical research, facilitating the successful translation of science into clinical practice. Despite their ubiquitous use, there is a paucity of information on the perioperative care of pigs, especially for major procedures. In a series of experiments to investigate laryngeal transplantation, we combined veterinary and medical experience to develop protocols for perioperative management of pigs, including high dependency care. Novel airway management methods were developed. A pain scoring system was used to direct analgesia use. Fluid balance and electrolytes were monitored closely. Recent animals received a central venous line via the femoral vein two days prior to transplantation to facilitate blood sampling and drug delivery. Intensive monitoring and airway management were required to ensure a successful outcome. Methods for optimal perioperative care are proposed. These results will help future groups wishing to use pigs in airway research, will reduce numbers of animals used and improve animal welfare.
In order to answer scientific questions about the larynx and to develop new operative techniques, large animal preclinical models are invaluable. Dogs, cats and primates have all provided important information. 1,2 However, pigs are particularly helpful, since they have similar airways to man in terms of size, anatomy 3,4 and functional characteristics. 5 A wider range of molecular probes are available than for any other non-primate large animal. 6 Pigs are physically robust and have been used widely in surgery to develop major surgical techniques. 7,8
However, perioperative care of pigs is poorly described, in medical or veterinary literature, and this is complicated further by the addition of tracheostomies in laryngeal surgery. It is a basic principle of animal experimentation that animal care be of the highest possible standard to maximize animal comfort and safety while minimizing pain. As an integral part of a programme of research into laryngeal transplantation, immunology and reinnervation, we aimed to study pig responses to anaesthesia and surgery and to develop optimal methods for perioperative care and analgesia. This paper presents observations from a subgroup of 10 laryngeal transplant recipients, during which series our care techniques significantly evolved.
The aim of this paper is to present the results of this work, and thereby to inform future groups planning to use pigs in airway research.
Methods
Preparatory and general care
Twenty cc National Institute of Health minipigs (Institute for Animal Health, Compton, Newbury, Berkshire, UK), 10 donors and 10 recipients (mean 32 kg, standard deviation [s.d.] 10; mean age 19 weeks, s.d. 4) were received at least two weeks prior to any form of intervention to reduce the confounding effects of stress that accompany transportation. 9 Straw-bedded pens were 2.5 × 2.5 m with central infrared heating. The pigs, housed in pairs, were fed twice a day with 150 g of grower pellets (Sow & Weaner, BOCM Pauls, Ipswich, UK) and visited regularly to acclimatize them to human interactions.
Preparation for transplantation
To facilitate feeding postoperatively, pig (recipient) 1 had an open gastrostomy performed at the time of transplantation, but this was not well tolerated and added to operative time. Therefore, the remaining pigs had a 20F percutaneous endoscopic gastrostomy (PEG: MILA – Direct Medical supplies Ltd, Alton, Hants, UK) placed under general anaesthesia three days before the transplant surgery to allow pigs to acclimatize. Pigs 1 to 7 had at least one indwelling peripheral venous catheter either in the pinna, cephalic or metatarsal veins. These presented problems with retention and became painful with time. Therefore, in pigs 8, 9 and 10 a central venous catheter (CVC, 7–9F, Vygon, Cirencester, Gloucestershire, UK) was placed into the caudal vena cava via the femoral vein at the time of PEG placement. After incision in the inguinal fold, the vessel was cannulated under direct vision and the catheter tunnelled subcutaneously to emerge in the left lumbar area, on the dorsum of the pig, at a position approximately 3 cm cranial to the ilial crest and 5 cm lateral to the spinous processes. A pocket was made by securing the catheter between two dressings (Primapore 10 × 20 cm, Smith and Nephew, Hull, UK) adhered to the lumbo-dorsal skin. The catheter was handled using aseptic techniques at all times subsequently, including cleaning injection ports with alcohol before use. After drug administration or blood sampling, the CVC was flushed using 2 mL of 100 iu/mL heparin (Hep-Flush, Leo Laboratories Ltd, Princes Risborough, Bucks, UK) and ports cleaned before returning to the clean dressing pocket. The PEG wound was assessed for signs of infection and pain assessments carried out formally twice a day, but additional checks and assessments (e.g. to assess response to analgesia) were carried out more frequently (as required).
Pain was assessed by a combination of wound palpation, behaviour and locomotion scoring. Behavioural indices included interaction with carers (e.g. grunting, playfulness), nesting behaviour and appetite. Locomotion was assessed by observing willingness to approach for food and gait when moving within the pen. Wound tenderness (of the PEG and CVC wounds) was assessed by gently moving the hand near the wound and gently increasing pressure until a response was noted. Responses included turning away or staccato grunting. If the wound was very sensitive to pressure, the pig would generally turn towards the assessor to defend the area of discomfort. A numerical score was allocated, based on behavioural indices and wound tenderness, between 0 (no pain) and 10 (worst pain imaginable).
Analgesic therapy using non-steroidal anti-inflammatory drugs (NSAIDs) was instigated using 3 mg/kg ketoprofen (Ketofen 10%, Merial Animal Health, Harlow, UK) or 0.4 mg/kg meloxicam (Metacam 20 mg/mL, Boehringer Ingelheim, Bracknell, Berkshire, UK) once daily intramuscularly and supplemented with opioids using 0.01 mg/kg buprenorphine (Vetergesic, Alstoe, Sheriff Hutton, Yorkshire, UK) intramuscularly, up to twice daily if required before transplantation surgery.
The pigs were fed 150 g pellets (Sow & Weaner, BOCM Pauls, Ipswich, UK) twice a day. Additional observations were carried out during this period. Supplementary twice daily nutritional support was provided with milk (Piggy Milk, Parnutts Foods Ltd, Sleaford, Lincolnshire, UK). Basic metabolic requirements were calculated as 2.621 × weight (kg)0.63 MJ/day. 10 This amount was provided in each of solid feed and milk to supply a total of twice the calculated requirement. Water (22.5 mL/kg/day) was added to the milk powder representing half the estimated normal water intake for pigs of this size. The pigs had ad libitum access to clean water preoperatively.
During transplantation surgery, the operating table was tilted to provide an approximate 5° head-up angle, in order to reduce cranial congestion.
Postoperative care (after transplantation)
Environment
A confined recovery pen (0.7 × 1.3 m) on wheels, with removable and fold down sides (35 cm high) to permit access was developed for early recovery. A prewarmed covered foam mattress was placed in the pen with synthetic fleece (VetBed, Keith Rushfirth, Wakefield, Yorkshire, UK) over the mattress. When the pigs were able to stand the mattress was removed and replaced by additional synthetic fleece. For 12 h, three team members shared care, and 24 h care was continued with a minimum of two members of the care team present at any time. Ambient temperature was 18–24°C, humidity was not measured but if air-conditioning caused air drying, humidification was employed. Natural light was available during daylight hours. At night, a small desk lamp was available for note taking and observation but allowing a darker area for the pig to sleep. Environmental enrichment was provided using novel objects for investigation (such as cardboard boxes, mirrors and strips of rubber) interaction with carers and radio sound.
Pain measurement and management
Dexamethasone (Dexadreson, Intervet UK Ltd, Milton Keynes, Bucks, UK) 0.06 mg/kg was administered intravenously during anaesthesia for transplantation to minimize inflammation postoperatively. From the day after surgery, NSAID treatment was continued as described above, i.e. ketoprofen or meloxicam every 24 h. In addition, morphine (Morphine sulphate injection BP, Martindale Pharmaceuticals, Romford, Essex, UK) was administered intravenously or intramuscularly, initially at 0.2 mg/kg every 4 h. The dose was gradually reduced if pain assessment was satisfactory over approximately 5–6 days and then stopped. Pain assessments were carried out as described above; in addition, the willingness of the pigs to lie down or to stretch the neck out was observed. Wound tenderness was assessed by using gentle pressure with flat pads of several fingers and manipulating from the mandible under the jawline to the manubrium. Assessments were carried out at hourly intervals and a score assigned (as before). Sedation was also scored, on a numerical rating scale (0 = fully aware, 5 = unrouseable); similar scales have been used in dogs. 11
Airway management
For pig 1 a double-barrelled stoma was created, but led to excessive wound tension and was felt to be less physiological than later methods. For pig 2 an adjustable flange tracheostomy tube (Smiths Medical International, Hythe, Kent, UK) was used, but proved to be easily dislodged. Therefore, for pigs 3 and 4 a modified T-tube airway device (with limbs adjusted to appropriate angles for the porcine neck) was used. This served as both stent and airway. A heat and moisture exchanger (HME, Hydrotrach-T HME, Intersurgical Ltd, Wokingham, Hants, UK) was fitted at all times. The design of this device, with lateral side ports, is ideal for pigs, since their preferred resting position is prone. For pigs 3–7, a separate tracheostomy with adjustable flange tracheostomy tube was used during anaesthesia and removed in recovery. Due to blockage of the T-tube with secretions and in one case a flap of mucosa, for pigs 8–10, a liner was used. This consisted of a 5.5 mm cuffed endotracheal tube inserted into the T-tube for ventilation intraoperatively, which was changed to a 5 mm uncuffed endotracheal tube postoperatively. This obviated the need for a separate tracheostomy. The liner tube was trimmed to protrude approximately 3 mm from the distal arm of the T-tube. The liner was changed at regular intervals (at least every 12 h but more frequently, e.g. every 2–4 h in the first 48 h after surgery).
Suction of the T-tube was performed using a 10Fg catheter (Smiths Medical International) hourly with nebulization (Medic-aid, Bognor Regis, West Sussex, UK), lavage with saline and deep breathing exercises/physiotherapy at four-hourly intervals or more frequently if required. The latter were achieved by blowing onto the pig's snout, opening and closing the mouth (mimicking pig behaviour). Pigs respond by taking large tidal volumes and forcing expiration in attempted grunting. Crust formation was treated by lavage and suction of the T-tube and trachea in conjunction with deep breathing exercises. Endoscopy was performed daily using a flexible fibreoptic ureteroscope (Karl Storz UK, Slough, Bucks, UK), to assess mucosal colour/perfusion/condition and to detect and manage crusting or chest infection. Helium–gas mixture (21% oxygen) was available to permit low-density gas administration if partial airway obstruction was detected.
General postoperative care and monitoring
Postoperatively, pulse rate, respiratory rate, oxygen saturation (SpO2) (Datascope Passport 11 with gas module, Datascope Corp, Paramus, NJ, USA) character of respiration and rectal temperature were monitored. Oxygen was administered via the HME at 6 L/min initially and then titrated to maintain SpO2 above 95% until saturations were maintained in room air alone.
Oxygen saturation was measured hourly using an adult SpO2 finger probe (LNOPDC1 adult re-usable sensor, Datascope Medical Services, Huntingdon, Cambridgeshire, UK) or adhesive types (LNOP adt, Masimo Corp, Irvine, CA, USA) placed across the tail or the anterior margin of the pinna. If SpO2 fell below 95%, airway patency was checked, airway and lung fields auscultated and treatment instigated using supplemental oxygen with nebulization and suction as required. Alternative sites for SpO2 measurement (e.g. pinna, tail) would be used if artefactual readings were suspected. Pulse rate was detected by the oximetry probe and recorded.
Respiratory rate were recorded, noting the rate and degree of thoracic and abdominal movement. Abnormal respiratory movement such as paradoxical ventilation or abnormality in the ratio of inspiratory to expiratory phases was noted. Additionally, capnography was used to assess ventilatory efficacy or as an indication of partial airway obstruction (as required) using a sidestream analysis from the gas port of the HME.
Observation and assessment were continuous, but values were recorded at one-hourly intervals or more frequently if there were unusual or adverse findings. Rectal temperature was measured at four-hourly intervals using a digital thermometer (Digitemp, Sevropax, 3S, Royal Portbury, Bristol, UK). If body temperature was greater than approximately 40ºC or there was an otherwise unexplained increase in respiratory rate, corrective measures such as altering room temperature and sponging the skin with tepid water were used. A blood sample was taken daily via the CVC to assess acid base and electrolyte status (348 blood gas analyser, Bayer Diagnostics Newbury, Berks, UK), blood glucose, packed cell volume and ‘total solids’ (using a refractometer).
Fluid balance and nutrition
Hourly cumulative totals of fluid intake (water and saline via the PEG tube and intravenous fluids) and output (urine, faeces and contents of surgical drains) were recorded. A cumulative running total of fluid balance was maintained, with adjustments made to the water intake (content of the milk) as required. Cumulative fluid balances were carried over to the next day's records for continuity. Positive fluid balance was maintained to allow for insensible losses.
Pigs were fed at four hour intervals using milk feed as above, administered via the PEG. The amount of milk powder used supplied four times the calculated metabolic requirement of the pig (as above 10 ) mixed into a total of 45 mL/kg/day water (initially, adjusted as required). The PEG tube was flushed after feeds with water or hypertonic saline (if normovolaemic hyponatraemia was detected) to flush the tube.
Results
Five pigs were maintained to seven days. Euthanasia was performed using intravenous pentobarbitone. A summary of survival data is illustrated in Table 1. Pig 1 was euthanized at day 2 since failure of the surgically inserted gastrostomy prevented adequate nutrition and fluid balance. The subsequent use of PEG obviated this problem. The tracheostomy of pig 2 dislodged causing acute airway obstruction leading to death. Subsequent use of a dedicated T-tube device prevented this problem. Tables 2 and 3 summarize physiological variables, sedation and pain scores for all pigs in the postoperative period. Data from four pigs demonstrated weight loss over the seven-day postoperative period ranging from 0% to 4.8% (median 2.2%).
Survival outcome seven days after laryngeal transplantation in National Institute of Health minipigs
Values for the physiological variables, sedation and pain scores measured during the postoperative period
n represents the number of measurements for each variable
Values for the venous acid–base status, electrolytes, total solids, glucose and PCV measured during the postoperative period
n represents the number of measurements for each variable. PCV: packed cell volume
In the initial recovery period (2 days) there was a small but persistent coating of exudates on the liner tube requiring frequent changes. Using the liner tube improved the removal of secretions which would otherwise have built up on the inner surface of the T-tube. Any small degree of blockage, however, could cause respiratory distress as the tube was narrow proportional to tracheal size. Generally, crusting was more common on the outside of the inner tube, so an airway was maintained. Liner tubes could occasionally narrow or kink at the maximum curvature, especially if tubes were cleaned and reused excessively.
A persistent sitting posture and reluctance to lie down when tired was observed to be a potential indicator of discomfort but was also observed during partial airway obstruction by crusts of dried exudate. Aggression, only observed if pigs were uncomfortable, was quickly relieved by additional morphine. If ambient room temperature rose above 22°C, some pigs exhibited an altered respiratory pattern especially increased respiratory rate in response to rising body temperature. Lowering room temperature to 18–20°C and tepid sponging corrected this.
Discussion
Pigs are ideal animal models for airway research, but their use raises important concerns regarding welfare and postoperative care, especially in relation to the airway itself. In the course of a project designed to study the immune response to laryngeal transplantation, we developed protocols and devices that optimized long-term care of pigs undergoing major airway surgery. Thorough assessment of the animals and intensive care protocols appeared necessary to maintain a suitable airway. With careful management, it was possible to maintain adequate analgesia and wellbeing of the animals despite the complexity of the intervention. This work is of importance to any groups planning airway reconstruction experiments in pigs.
A gastrostomy tube was placed, as pharyngeal surgery precluded oral feeding for one week. 12,13 Nasogastric tubes could cause distress due to the sensitivity of a pig's snout, and also carry a risk of sinusitis and reflux. 14,15 Enteral nutrition is preferred to parenteral nutrition. 16 The high calorific intake was based on experience with veterinary and human patients undergoing major surgery, and was justified by the minimal or no weight loss observed.
Peripheral venous catheters were found to be difficult to place, care for and use, also access involved distressing restraint. Placing a CVC provided continuous venous access, while the double lumen allowed the separation of sampling and drug administration. Although femoral CVC carries an infection risk in human studies, 17 no clinical or postmortem signs of infection were found, possibly due to rigorous asepsis. Jugular placement was avoided due to the site of surgery. Lumbar placement permitted normal movement and behaviour, and access without disturbing sleep.
Despite the frequent use of pigs in research models, few studies have examined analgesia and pain in pigs. In other species, use of physiological measurements such as heart rate and respiratory rate may not lead to accurate pain assessments. 18,19 The use of multifactorial pain scales has been reported for pain assessment in pigs. 20,21 Feeding behaviour and food consumption have been used as part of pain assessment, 20 although this was not suitable for use in pigs after laryngeal transplantation. Activity levels have been recognized to change postoperatively, 22 decreased activity does not appear to be related to opioid sedation. 23 Opioid-related sedation was difficult to assess in this study as many other factors (such as the room temperature, time period after surgery, room lighting, airway patency and previous amount of active movement) appeared to affect sleep patterns. For pain assessment, we developed a numerical rating scale using psychological and tactile components and based on scales from other veterinary settings, 19 which is similar to the global pain scale used in pigs by Reyes et al. 21 Changes in human–animal interaction and playfulness have been noted as signs of discomfort in other mammals. 24,25 We could not reproduce published observations that pigs tolerate handling more readily when in pain than when not in pain. 26 However, we cannot exclude the possibility that farm pigs may react differently to those accustomed to close attention. Unwillingness to lie down was an important marker. To improve accuracy, scores were confirmed by a second observer. 27 At the dose rates used in this case series, no opioid-related respiratory depression was noted.
Sequelae and complications following tracheostomy were similar to those reported in man following laryngectomy. 13 Plugging of bronchi and T-tube by dried secretions was prevented by two-hourly nebulization in the first few days. Stimulating the pig to breathe deeply (‘physiotherapy’) improved expectoration. This improves gas exchange. 28 Flexible tracheoscopy for crust removal was well tolerated.
We found that the use of an inner liner tube substantially improved airway management, and we incorporated this in the design of a novel T-tube (under development). The inner tube also obviated the need for a separate tracheostomy for airway management during anaesthesia, which was another source of potential airway complications (e.g. damaged mucosa). A reduction in aspiration pneumonia by the use of liners has been reported. 29 As HMEs may not hold enough water in a dry atmosphere, high environmental humidity is important. We recognize that the proposed regimen of airway care is time and resource intensive. However, any attempt to reduce the intensity of observations and management in the first few days always resulted in crusting and partial airway obstruction.
High pulse and respiratory rates were observed in hyperthermic pigs or if ambient temperature was high. It was important to distinguish this effect from other causes of rapid shallow respiration (e.g. partial airway obstruction).
Environmental enrichment and interaction with carers were considered important as pigs are social animals unused to being kept without other pigs. Interactions such as using novel objects (cardboard boxes, plastic containers, etc.) for investigation by the pigs and interactive grooming treatments such as skin sponging were used to encourage mobilization. Such management subjectively improved compliance with airway and other interventions as well as benefiting the welfare of the animals.
Our recommendations for the perioperative care of pigs undergoing experimental airway surgery include considerable investments in time and personnel; however, the results are improved physiological and welfare outcomes. This work may also be of value to groups using pigs in other forms of surgical research.
Footnotes
ACKNOWLEDGEMENTS
Martin Birchall, some equipment and consumable costs were supported by a Wellcome Trust Clinical Research Leave Fellowship (GR061125); some equipment was kindly provided by Karl Storz UK, Ltd.