A preliminary report of an ongoing study.
Abstract
The Peripheral Neuritis Trauma (PNT) model is a model for chronic neuropathic pain. Briefly, pain is induced by ligating the lateral part of the sciatic nerve with silk threads presoaked in CFA. Animal behavior is evaluated using several methodologies, including response to mechanical stimulation, behavior scoring of social behavior, and locomotor activity.
The model was running in two different vivariums: one is a standard swine facility for research, and the other is a new, state-of-the-art facility, specifically purpose-built for swine behavior studies.
The standard facility houses pigs in a corridor of rectangular pens on both sides, which are separated by opaque walls. Pigs are housed in groups of two or three, and the animals and pens are washed each morning with water. The animals are fed twice daily, and enrichments includes a choice of materials and balls. A well-trained caretaker enters the pens once daily for handling. While all tests are carried out in the pig´s home pen, locomotor activity is evaluated by gently walking the pigs to the open field arena, (sized 1.2mX2.4m). Supportive medications such as antibiotics are delivered by IM injections.
The new facility is built in a U-shape with square pens separated by bars allowing the pigs to interact with one another visually and vocally. The open side of the U-shape has a gate leading to a shared, middle space in which the pigs can enter and play. Pigs may interact freely with pigs from other pens. The pigs are held on sawdust that is replaced daily with no washing and are fed twice daily. Pens are opened twice daily for the animals to play with the caretaker (rope, balls, “feed-me”, and other tasks). All medication is administered orally after training.
Pigs are also taught to climb on to the balance weighing scale. This paper reports the preliminary results of an on-going study that looks at the effect of the different housing and handling conditions on the outcome of a PNT model.
Introduction
Across species, it is an established fact that behavior assays depend on housing environmental conditions such as light/dark cycles, temperature and humidity, noise, bedding, enrichment, and handling. In farm pigs, poor housing in barren conditions can cause damaging behaviors such as tail and ear biting. These factors lead to chronic stress and alteration of the pig’s immunity, and can affect pain behavior significantly.
Pain by definition, is a subjective experience. This makes pain evaluation in humans challenging and almost impossible to do by another person. In humans, the assessment of pain mostly relies on verbal reporting, while in animals, the assessment of pain relies on animal withdrawal from a stimulus and/or behavioral scoring. Both approaches are highly dependent, among other things, on the animal’s stressful or stress-free environment.
In this report, we examine the effect of Standard Housing Conditions (SHC) and Specifically Designed Housing Conditions (SDHC) focused on behavioral assessment on the pig outcome following PNT induction of chronic pain.
Methods
Anaesthesia and PNT surgery
An anaesthetic facemask (Stephan Akzent Color, Germany) was placed on the pig's mouth and nose, as described previously. Each animal was anaesthetised with a 3% isoflurane/100% oxygen mixture. While connected to the anaesthesia, the pig was shaved and swabbed with 70% ethanol, and then carried to the operation room. The pig was placed in the sternal position on the operating table. The area of the incision was swabbed with antiseptic liquid polidine solution (Polysept solution, Rekah Pharmaceutical Industry Ltd., Israel) and the non-operated areas were covered with sterile sheets. During anaesthesia, blood oxygen saturation was monitored (Spacelab Medical, USA) .
An incision of 8-10 cm was made through the skin and fascia on the left side of the lower back, towards the caudal end, and approximately 1.5 cm lateral and parallel to the spine line of the pig. The muscles were then retracted and the entire sciatic nerve exposed. Peripheral Neuritis Trauma (PNT) was induced by three 3-0 silk threads (Assut-UK), each 3 cm in length, which were immersed in complete Freund's adjuvant (CFA) (1 mg/ml) overnight. Following sciatic nerve exposure, the pre-soaked threads were used to create 3 loose ligations (1-2 mm apart) surrounding the lateral half of the sciatic nerve bundle.
Behaviour Tests
Approaching test to assess acclimation
The normal behavior of the pigs upon the entry of the researcher entering their housing pen is to initially move away from the intruder and then approach them. The more familiar the pigs are with the person and the more comfortable they feel, the less time it takes them to approach. The latency to approach the researcher is recorded in seconds. In this test the researcher does not approach the animals, but simply enters their cages. The approach time test can serve as a tool to assess acclimation.
von Frey testing
Mechanical sensitivity was assessed using von Frey (vF) filaments (Touch Test (Von Frey) Sensory Evaluator Kit, model 58011, Stoelting Co., Wood Dale, IL, USA). The tests were performed in the pigs’ home pen. Filaments ranging from a minimum of 0.07 g to a maximum of 15 g were used. The filaments were applied for a period of few seconds on the dorsal area of the animals’ foot. Each filament was applied three times with a 5-10 second interval between applications using the up-down methodiii, If withdrawal was not achieved, a thicker filament was applied. If withdrawal was achieved, a thinner filament was applied. By alternating the filaments, the force required to achieve a withdrawal reaction was determined. This procedure was carried out at the time points of 1 day pre-surgery (baseline), and at days 14 and 21 post-surgery.
Open field test
The open field apparatus size was either 2.5m wide by 4.8 meters long (smaller open field), or 4 meters wide by 4 meters long (new open field). The walls of the small open-field were smooth and 1.6m high. The new open field was a separate room. The animals were introduced to the open field for 5 minutes. The walking pattern of the animals was recorded using a CCTV camera connected to the AnyMaze data acquisition software. After each open field session, the total walking distance was presented, as well as the walking pattern.
Results
Approach time as assessment of acclimation
The first reaction of the animal when entering their pen is to move away from the intruder followed by slowly approaching the intruder. The time for the animals to approach the intruder was measured in the two facilities during the acclimation period. The data collected in both facilities show that the time for the animals to habituate to the researcher’s entry was shorter in the SDF than in the SHF. It shows that the approaching time 4 days post animals’ arrival to the SHF was reduced by nearly 60% vs. the first day of their housing. In comparison, 4 days of housing in the new SDF facility resulted in faster acclimation as expressed by the reduction in nearly 100% in the approaching time (p<0.001 vs. traditional facility).
Withdrawal force
The withdrawal response to mechanical stimulation was assessed using the von Frey methodology. The withdrawal force at baseline was not different between animals housed in the SHC faclity and those housed using the behavior-specific design SDHC housing approach. In both cases, the highest withdrawal force ranged between 10g to 15g.
Fourteen days following PNT operation, the withdrawal force recorded was reduced significantly in both groups. A lower mean group withdrawal force was expressed in the animals housed in the SHC facility (2.09 ±0.29 vs. 0.84±0.15g p<0.01). Twenty-one days post PNT, the withdrawal force of the animals housed in the SHC facility was increased (1.92±0.39g; p<0.05 vs the withdrawal force measured on day 14.). No change was observed in the withdrawal force of the animals housed in the SDHC facility. Two weeks later, on study day 35, the withdrawal force of the animals housed in the SDHC facility was unchanged.
The withdrawal force of the animals housed in the SDC facility was significantly lower (Figure 2 ).
Open field assay
Previously, we have shown that following PNT operation, the walking pattern detected in the open field arena was altered in castrated young male domestic pigsv.
In this study, we are comparing the effect of the open field size and the housing environment on the walking pattern of adult female GMP. The data show that at baseline- before PNT operation- animals introduced to the small open field walked less than the animals introduced to the large open field.
Walking pattern
Fourteen days post PNT operation, the animals that were introduced to the old open field tended not to enter the central zone and preferred to walk next to the open field arena walls. This phenomenon was observed also 21 days post PNT but to a lesser extent.
The animals that were introduced to the large open field arena, showed similar behavior at baseline, meaning the animals covered the entire area without a specific preference. Fourteen days post PNT the animals tended to stay closer to the entry door. Three weeks post PNT, this tendency strengthened and some of the animals developed what appears to be a repetitive behavior, meaning they went back and forth to the entrance door. This phenomenon was not observed in the small open field arena (figure 3 page 14).
Walking distance
At baseline, before the injury, the animals that were exposed to the larger open-field arena in the SDHC facility walked significantly more than the animals exposed to the smaller open-field arena in the SHC facility (figure 4) (56.33±21.13 meters vs. 73.57±35.72).
Following PNT the animals exposed to the smaller open field arena, at the SHC facility, showed a transient decrease in locomotor activity. Animals exposed to the large open field arena showed no change in the locomotor activity and no reduction in walking distance.
Discussions
In the last six months, we have been operating from a new ani-mal facility that was specifically designed for behavior assessment of large swine studies (60 animals at all times). The move created an opportunity to re-validate specific pain models and investigate an improved facility design. In this study, we report on an ongoing re-validation of the chronic pain model in GMP. Although this is an interim report, some differences have already been observed and can be discussed.
Acclimation
The acclimation period is very critical in behavior studies, if it is carried out mainly at the animal’s housing space involving the presence of investigators. The normal behavior of pigs is to move away from a person entering their pen, followed by their approach to the investigator. The latency time for the pigs to approach the intruder reflects their calm or distressed behavior. Under distressed conditions, pigs do not tend to approach the intruder. In this study, we show that the habituation of the pigs to a repeat intruder in their home pen was more rapid in the SDHC facility than in the SHC facility. This was shown by the relatively shorter approach time measured in the SDHC facility compared to that of the SHC facility. This also suggests that the pigs were more calmer and far less stressed, which shows that the SDHC facility potentially offers a better solution for behaviour assays in pigs.
Mechanical Allodynia testing
This study shows that following PNT operation all animals developed a robust and significant decrease in the withdrawal force regardless of the housing environment. However, 14 and 35 days following PNT operation, a clear difference was found between the withdrawal force of the animals housed in the SDHC facility and the animals housed in the SHC facility. Animals in the SDHC facility had higher sensitivity compared to the animals housed in the SHC facility. This difference was not noticed at 21 days post PNT operation as the mean group withdrawal force on the animals housed in the SCH facility was slightly increased. The relatively higher withdrawal force obtained in animals housed in the SDHC facility is possibly due to increased animal mobility fact that the animals were able to move more. In humans, it is shown that moderate regular exercise decreases excitability and improves inhibition in both the central nervous system (brainstem inhibitory/facilitatory sites) and the immune system. In rodents, it was shown that moderate daily exercise results in a slight increase in withdrawal threshold 3 weeks after spared nerve injury model . The SDHC facility provides pigs more opportunities for physical activity during their housing period. They walk more, can freely access the shared space, and are intrigued by different tasks.
Another explanation for the slight increase in the withdrawal force 3 and 5 weeks after PNT is that the animals were calmer. It is possible that the animals housed in the SHC facility were more sensitive to mechanical stimulation post PNT due to exposure to daily persisting stress, which resulted from daily washing. Persistent stress from daily washing can increase sensitivity to pain and pain behavior. The results obtained from the SDHC facility may have resulted from the stress-free conditions experienced by the animals. Further observations and studies are being carried out to better understand this point.
Locomotor activity
The animals were introduced to a small open field (SHC) or a large open field (SDHC). The animals that were introduced to the large open field (SDHC facility) walked more before the PNT operation. In both cases, the animals covered the entire open field arena without a specific location preference or a specific pattern. This is in line with other studies run on domestic pigs, suggesting that the behavior of the pigs is to cover the entire space when first introduced to the arena. The longer walking distances of animals housed in the SDCH open field facility may be ascribed to more opportunities for activity at their home-pen; these animals are able to be more active. Post PNT, the animals housed in the SHC facility had a transient decrease in walking distance while no change was observed in animals housed in the SDHC open-field facility.
An interesting difference was found in the walking pattern after PNT operation between the animals housed in the SHC facility and those housed in SDHC facility. The animals housed in the SHC facility tended to walk in circles, avoiding the central zone. This behavior suggests distress, and is in line with what we have previously reported in young castrated domestic pigs. This might further support the cross-talk between light chronic stress and chronic pain that is expressed in the animals housed in the SHC facility. Animals housed in the SDHC facility showed no change in the distance but a dramatic change in the walking pattern. They focused almost exclusively near the entrance door, suggesting a very fast learning behavior that was not affected by the painful condition. After one time, the animals learned that the open door allows them to freely walk back to their home pen, a safe and comfort zone. Stress has a complex effect on learning and memory, some papers suggest that acute stress can enhance learning and memory while others show that it interfers with learning and memory. However, most researchers believe that chronic stress can impair the learning and memory process.
Economic aspects
The housing of 14-21 pigs in one room with 2-3 animals per pen require 15 minutes handling per day, at least twice daily. This means that it can take 3.5 hours per day (15 minutes per pen, 2 animals twice daily) for 14 animals. Handling the pigs using the shared space approach requires 0.5 hour to 1 hour depending on the program of handling a reduction of up to 70% in handling time. Changing the sawdust while the pigs are playing in the shared space takes about 0.5 hours for 2 people per room. The savings in the water cost and waste per day is about 80% since there is no daily pen washing.
The new SDHC facility allows housing, handling, and medicating of 50-60 pigs at any given time, which can be achieved with a staff of only 2 Full Time Employee (FTE) positions. Similar studies that run in an SHC facility typically require 5- 6 FTE to fulfill the same mission.
The time saving for a full-screen veterinarian check-up
Traditional housing requires the veterinarian to stand in front of or inside of each pen every morning to check the animals, observe whether animals are standing or walking, and look for abnormalities. This requires about 10-30 minutes per pen. In a room with 7 pens, the morning checkup can take 1-3 hours. The new pig housing approach allows the veterinarian to enter the shared space. All the animals will approach the pen’s gate upon the veterinarian’s entry. This allows an immediate screen of all the animals as well as an immediate observation of any abnormalities. This approach allows the veterinarian to screen the pigs and focus on the abnormalities within minutes, which is a more efficient use of time.
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