Tell-tale TINT: Does the Time to Incorporate into Nest Test Evaluate Postsurgical Pain or Welfare in Mice?

Identifying early indicators of distress in mice is difficult using either periodic monitoring or current technology. Likewise, poor pain identification remains a barrier to providing appropriate pain relief in many mouse models. The Time to Incorporate to Nest Test (TINT), a binary measure of the presence or absence of nesting behavior, was developed as a species-specific method of identifying moderate to severe distress and pain in mice. The current study was designed to evaluate alterations in nesting behavior after routine surgery and to validate the TINT's ability to measure pain-related behavioral changes. CD1 mice undergoing carotid artery catheterization as part of a commercial surgical cohort were randomly assigned various nesting, surgery, and analgesia conditions. To provide context for the TINT outcomes, we measured other variables affected by pain, such as weight loss, food consumption, and scores derived from the Mouse Grimace Scale (MGS). Mice that had surgery were more likely to have a negative TINT score as compared with controls. All mice were more likely to fail the TINT after receiving postoperative buprenorphine, suggesting that buprenorphine may have contributed to the failures. The TINT, MGS live scoring, and scoring MGS images all loaded strongly on a single component in a principal component analysis, indicating strong convergent validity between these measures. These data indicate that the TINT can provide a quick, objective indicator of altered welfare in mice, with the potential for a wide range of uses.

Psychogenic Stress in Hospitalized Dogs: Cross Species Comparisons, Implications for Health Care, and the Challenges of Evaluation.

Evidence to support the existence of health consequences of psychogenic stress has been documented across a range of domestic species. A general understanding of methods of recognition and means of mitigation of psychogenic stress in hospitalized animals is arguably an important feature of the continuing efforts of clinicians to improve the well-being and health of dogs and other veterinary patients. The intent of this review is to describe, in a variety of species: the physiology of the stress syndrome, with particular attention to the hypothalamic-pituitary-adrenal axis; causes and characteristics of psychogenic stress; mechanisms and sequelae of stress-induced immune dysfunction; and other adverse effects of stress on health outcomes. Following that, we describe general aspects of the measurement of stress and the role of physiological measures and behavioral signals that may predict stress in hospitalized animals, specifically focusing on dogs.

Pain-Suppressed Behaviors in the Red-tailed Hawk (Buteo jamaicensis).

Our ability to provide analgesia in wild and exotic patients is hampered by a lack of species-specific information on effective drugs and protocols. One contributing factor is the difficulty of applying data from traditional laboratory tests of nociception to clinical conditions frequently involving combinations of inflammatory, mechanical, and neuropathic pain. Pain-suppressed behaviors have become a valuable predictor of clinical utility in other species; in this study we extend this framework to red -tailed hawks in a wildlife hospital, in an attempt to develop a new, humane testing method for birds of prey. We scored six behaviors in hawks hospitalized either for orthopedic trauma or for non-painful conditions. These behaviors included: movement about the cage, grooming, head motions, foot shifts, beak clacks, and rouse. Movement, head motions, and beak clacks were all significantly reduced in hawks with recent orthopedic injury, but not in hawks with healed or minor injuries (P<0.05 for all behaviors). However, it should be noted that due to stringent admission criteria, and the difficulties inherent in studying naturally-occuring injury in wild patients, this study only included -subjects in four experimental groups, and this limited our ability to fully investigate confounds within our data. A follow-up experiment was conducted to determine potential effects of buprenorphine, a mu opioid agonist, on the behaviors listed above. Buprenorphine in the absence of pain caused minor, non-significant decreases in most behaviors, and had no effect on head movement frequency. This suggests that head movements in particular may be sensitive to pain but not to sedative side-effects of buprenorphine. Overall, red -tailed hawks with recent orthopedic trauma show consistent and marked red uctions in several normal maintenance behaviors. Head movements, reported for the first time in this study as a potential marker of pain in birds, in particular seem to be insensitive to sedative side effects of buprenorphine, while being a sensitive measu re of affective state in hawks with painful injuries. These behaviors can be scored humanely and with minimal expense, and should be considered for further research on pain and analgesia in avian species.

The time-to-integrate-to-nest test as an indicator of wellbeing in laboratory mice.

Minimizing and alleviating pain and distress in laboratory mice without compromising the methodologic integrity of research is a crucial goal. However, current methods for welfare assessment in mice are not well suited to cageside checks. In the present study, we developed a simple assessment tool-the time-to-integrate-to-nest test (TINT)-and evaluated its ability to identify mice with compromised welfare. To conduct the TINT, a nominal amount of nesting material is added to a mouse cage, and the nesting behaviors that occur immediately thereafter are observed. The TINT yields a positive result when a mouse integrates the new nesting material into the main nest site within 10 min; failure to interact with the nesting material is defined as a negative TINT. Our first experiment examined whether genetic background and sex are associated with differences in the likelihood of a positive TINT in unmanipulated mice. A significant effect related to mouse strain was found: C3H/HeNCrl had the lowest positive TINT rate among the 10 strains evaluated. A second experiment assessed whether results of the TINT would be altered after a painful surgical procedure, such as carotid artery injury. Despite all mice having received buprenorphine as analgesia at the time of surgery, significantly more mice had a negative TINT for 2 d after surgery than before surgery. Based on the results of the current study, additional work is needed to specifically validate the TINT in injured and noninjured subjects.

Nest building as an indicator of health and welfare in laboratory mice.

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

Salivary cortisol concentrations and behavior in a population of healthy dogs hospitalized for elective procedures.

Identification of severe stress in hospitalized veterinary patients may improve treatment outcomes and welfare. To assess stress levels, in Study 1, we collected salivary cortisol samples and behavioral parameters in 28 healthy dogs hospitalized prior to elective procedures. Dogs were categorized into two groups; low cortisol (LC) and high cortisol (HC), based on the distribution of cortisol concentrations (< or ≥ 0.6 µg/dL). We constructed a stress research tool (SRT) based on three behaviors, (head resting, panting and lip licking) that were most strongly related to salivary cortisol concentrations. In Study 2, we collected salivary cortisol samples from 39 additional dogs, evaluated behavior/cortisol relationships, assigned each dog to an LC or HC group, and tested the ability of the SRT to predict salivary cortisol. Median (interquartile range) salivary cortisol concentrations were not different between Study 1 (0.43 µg/dL, 0.33 to 1.00 µg/dL) and Study 2 dogs (0.41 µg/dL, 0.28 to 0.52 µg/dL). The median salivary cortisol concentration was significantly lower (P ≤ 0.001) in LC versus HC dogs in each study; (Study 1 LC: 0.38 µg/dL, (0.19 to 0.44), n = 19, HC: 2.0 µg/dL, (1.0 to 2.8), n = 9, and Study 2 LC: 0.35 µg/dL, (0.25 to 0.48), n = 28, HC: 0.89 µg/dL, (0.66 to 1.4), n = 7). In Study 1, three behaviors were found to be associated with salivary cortisol concentrations. Duration of head resting was negatively associated with salivary cortisol (ρ = -0.60, P = 0.001), panting and lip licking were positively associated with cortisol (ρ = 0.39, P = 0.04, and 0.30, P = 0.05, respectively), Head resting (p = 0.001) and panting (p = 0.003) were also associated with LC/HC group assignment. In Study 2 dogs, the three behaviors correlated (but not significantly) with salivary cortisol concentration; of the three, only head resting was significantly associated with LC/HC group assignment (P = 0.03). The SRT derived from Study 1 was effective at prediction of salivary cortisol concentrations when applied to 20 min but not 2 min of behavioral data from Study 2. Additionally, we note that dexmedetomidine and butorphanol sedation more than 6 h prior to measurement was found to be significantly (P = 0.05) associated with lower salivary cortisol concentrations when compared to unsedated dogs. Our work offers support for eventual construction of a rating tool that utilizes the presence or absence of specific behaviors to identify higher salivary cortisol concentrations in dogs subjected to hospitalization, which may be tied to greater psychogenic stress levels. Future work to investigate the effects of stress on dogs and its mitigation in clinical situations may be approached by studying a combination o f parameters, and should consider the possible beneficial effects of sedatives.

A model for clinical evaluation of perioperative analgesia in rabbits (Oryctolagus cuniculus).

Assessment of pain in rabbits is challenging, and studies of effective surgical analgesia are lacking for this species. Seeking potential indicators of postoperative pain, we performed ovariohysterectomy and telemeter placement as a form of moderate surgical injury in 20 female rabbits. Rabbits were assigned to 1 of 4 treatment groups (5 per group): buprenorphine (0.02 mg/kg SC every 12 h for 3 d); fentanyl (25-µg patch placed 24 h preoperatively); ketoprofen (1 mg/kg SC every 24 h for 3 d), and control (no treatment given). Various physiologic and behavioral variables were recorded by blinded observers, including food and water consumption, fecal output, and remotely recorded behaviors during daily exercise in 1.2 × 1.8 m floor pens. Compared with preoperative values, significant declines occurred in: food consumption (days 1 to 7), water consumption (days 1 to 4), fecal output (days 1 to 2), mean travel distance, and rearing (days 1 to 3 and day 7). No single treatment proved significantly better than another. Our results demonstrate substantial inappetance and reduction of normal activity levels in rabbits after surgery. Although results from rabbits treated with empirical doses (those typically recommended) of analgesics did not appear substantially better than those from the untreated control group, comparison of other doses and multimodal analgesic techniques by using these behavioral monitoring strategies may prove useful in future studies aimed at optimizing postoperative analgesia in rabbits.

Use of wound soaker catheters for the administration of local anesthetic for post-operative analgesia: 56 cases.

OBJECTIVE: To describe the administration of local anesthetic through wound soaker catheters for post-operative veterinary patients and to characterize complications. STUDY DESIGN: Retrospective study of hospital records. ANIMALS: Records of patients in which a wound soaker catheter was placed post-operatively between November 1, 2004 and July 1, 2006 at a veterinary teaching hospital. Records in which a limb amputation was performed between January 1, 2002 and August 1, 2007 and in which a wound soaker catheter was not placed were reviewed for historic control. RESULTS: A total of 56 cases were identified in which a wound soaker catheter was placed post-operatively including 52 dogs, 2 cats, and 2 goats. Twenty canine cases were identified in which limb amputation was performed and no wound soaker catheter was placed. The majority of surgical procedures for which a wound soaker catheter was placed included thoracic limb amputation (46.4%) and pelvic limb amputation (35.7%). Wound soaker catheters remained in place for an average of 1.6 +/- 0.5 days. Feline and caprine patients received intermittent bupivacaine boluses every 6 hours. Canine patients received continuous lidocaine infusions. Complications included disconnection of the catheter from the infusion (7.7%), one seroma, and one suspected lidocaine neurotoxicity. Incisional infections were noted in 3/56 (5.3%) limb amputations with wound soaker catheters placed which was not higher than the incisional infection rate found in the historic control cases 3/20 (15%). CONCLUSION AND CLINICAL RELEVANCE: Use of the wound soaker catheter was a viable means of providing local analgesia in post-operative veterinary patients. Studies are needed to evaluate efficacy of pain management, and to further investigate techniques for catheter placement and maintenance which may help to optimize the analgesia achieved using this technique.

Barriers to assessment and treatment of pain in laboratory animals.

Pain is an undesirable potential consequence of many of the procedures conducted on animals in the course of scientific research, and in most cases it is unnecessary. The US Congress, the public, and laboratory animal medical professionals have indicated that pain should be prevented or minimized in laboratory animals, yet there is ample evidence to suggest that unalleviated pain is still a problem for some laboratory animals. This evidence is circumstantial to some extent but has its basis in problematic issues of pain control in both veterinary and human medicine. The author attempts to identify specific barriers to reduction of pain in laboratory animals. She then seeks to determine the relative importance of each obstacle and to develop approaches to overcoming each obstacle.

Effect of caudal epidural xylazine on intraoperative distress and post-operative pain in Holstein heifers.

OBJECTIVE: To compare the effects of caudal epidural xylazine versus saline on tolerance of paravertebral nerve block and flank surgery and on post-operative pain in heifers used for a veterinary student training laboratory. STUDY DESIGN: Randomized controlled prospective study. ANIMALS: Fourteen one-year-old, nongravid, healthy Holstein heifers, weighing 360 +/- 5 kg. METHODS: Xylazine (0.05 mg kg(-1)) or 0.9% saline (5 mL) was injected using a caudal epidural technique to seven heifers undergoing a flank surgery. Nerve block of the right paravertebral fossa was performed using equal parts of lidocaine 2% and bupivacaine 0.5%. Heart and respiratory rates, rectal temperature, rumination frequency, and appetite were recorded before and at 4, 8, and 24 hours after surgery. Scores were recorded for: tolerance of local anesthesia injections (pre-operatively), sedation, ataxia and distress (intraoperatively, every 30 minutes), and pain (4, 8, and 24 hours post-operatively). RESULTS: The animals reaction to local anesthetic injection was judged to be less in the xylazine group by both an experienced observer (p<0.001) and student surgeons (p<0.01). The xylazine group required less local anesthetic (82.9 +/- 13.8 mL) versus the saline group (108.4 +/- 19.6 mL, p=0.035). Intraoperatively, xylazine heifers were more sedated at all times (p-values from <0.001 to 0.017), were more ataxic for the first 1.5 hours (p-values from <0.001 to 0.026), and lower in distress at all times (p-values from <0.001 to 0.007). No difference in post-operative pain or physiologic variables was found, except immediately post-operatively, rectal temperature was higher in the xylazine group (39.5 +/- 0.3 degrees C) than in the saline group (38.6 +/- 0.2 degrees C, p<0.001). CONCLUSION AND CLINICAL RELEVANCE: Compared with epidural saline, caudal epidural xylazine reduced distress of anesthetic injection and surgical manipulation in heifers and an improvement in animal well-being was apparent. This effect may have been as a result of sedation. Pre-operative epidural xylazine did not appear to improve post-surgical analgesia in our study.

Use of rocuronium for endotracheal intubation of North American Gulf Coast box turtles.

OBJECTIVE: To determine whether rocuronium, a reversible neuromuscular blocking agent, would provide safe, short-term immobilization to facilitate endotracheal intubation in turtles. DESIGN: Prospective study. ANIMALS: 30 healthy adult Gulf Coast box turtles. PROCEDURE: Turtles were given rocuronium, and responses were recorded every 3 minutes. Times to onset of effects, intubation, and recovery were recorded and analyzed for associations with dose and patient characteristics to determine an optimal dose range. Neostigmine and glycopyrrolate were given to augment recovery from neuromuscular blockade. RESULTS: Rocuronium administered at a dose of 0.25 to 0.5 mg/kg (0.11 to 0.23 mg/lb), IM, permitted intubation; lower doses were not effective. Mean +/- SD time to loss of the palpebral reflex was 6.4 +/- 4.0 minutes, and mean time to intubation was 9.2 +/- 6.4 minutes. Mean time to return of the palpebral reflex was 44 +/- 13.2 minutes, and mean time to walking was 55 +/- 16.6 minutes. Time to onset of effects was not associated with dose, but recovery times were prolonged with higher doses of rocuronium. Cardiac arrhythmias were observed in 13 (43%) turtles. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of rocuronium at a dose of 0.25 to 0.5 mg/kg is a safe and effective adjunct to general anesthesia in Gulf Coast box turtles. Because rocuronium does not provide any analgesic or sedative effects, the duration of neuromuscular blockade without anesthesia should be minimized to avoid undue distress.