Intrahepatic Injection of Sodium Pentobarbital as an Alternative to Intraperitoneal Injection for the Euthanasia of Rats (Rattus norvegicus).

The most commonly accepted method of rat euthanasia in North America is intraperitoneal injection of sodium pentobarbital (PB). However, misinjection can occur, and intraperitoneal PB may cause pain and distress. The objective of this study was to test an alternative method of euthanasia: intrahepatic injection of PB. A pilot study was conducted to develop a method of intrahepatic injections (evaluated using CT scans and test injections), followed by a full study comparing intraperitoneal (n = 14) and intrahepatic PB injections (n = 66) in adult rats. Full study outcomes were: 1) time from injection to loss of right- ing reflex (LORR), 2) time from injection to cessation of heartbeat (CHB), 3) number of failed euthanasia attempts, and 4) confirmation of successful intrahepatic injection or misinjection via necropsy. All injections were performed by a veterinary student. CT revealed that intrahepatic injections were feasible. Times (median [range]) to LORR and CHB were faster after successful intrahepatic injections (LORR, 3 s [1 to 5 s]; CHB, 8 s [2 to 242 s]) than after intraperitoneal injections (LORR, 89.5 s [73 to 110 s], CHB: 284.5 s [237 to 423 s]). The misinjection rate was higher with intrahepatic injections (59%) than with intraperitoneal injections (29%), but intrahepatic misinjection still resulted in fast and successful euthanasia (LORR, 29 s [1 to 96 s]; CHB, 216 s [12 to 330 s]), with the injectate distributed between the intraperitoneal and intrahepatic locations. The number of failed euthanasia attempts with intrahepatic injections was low (n = 2). Intrahepatic injections show potential as an alternative to intraperitoneal injections for rat euthanasia.

Development and Testing of a Sedation Scale for Use in Rabbits (Oryctolagus cuniculus).

In biomedical research, rabbits are commonly sedated to facilitate a variety of procedures. Developing a sedation assessment scale enables standardization of levels of sedation and comparisons of sedation protocols, and may help in predicting sedation level requirements for different procedures. The goal of this study was to develop a rabbit sedation assessment scale using a psychometric approach. We hypothesized that the sedation scale would have construct validity, good internal consistency, and reliability. In a prospective, randomized, blinded study design, 15 (8 females, 7 males) healthy 1-y-old New Zealand white rabbits received 3 intramuscular treatments: midazolam (0.5 mg/kg; n = 6); midazolam (1.5 mg/kg)-ketamine (5 mg/kg; n = 7); and alfaxalone (4 mg/kg)-dexmedetomidine (0.1 mg/kg)-midazolam (0.2 mg/kg; n = 3). One rabbit received 2 treatments. A sedation scale was developed by using psychometric methods, with assessment performed by 6 independent raters who were blind to treatment. Final sedation scale items included posture, palpebral reflex, orbital tightening, lateral recumbency, loss of righting reflex, supraglottic airway device placement, toe pinch, and general appearance. The scale showed construct validity, good to very good interrater reliability for individual items (6 raters; intraclass correlation coefficient, 0.671 to 0.940), very good intrarater reliability (5 raters; intraclass correlation coefficient, 0.951 to 0.987), and excellent internal consistency (Cronbach α, 0.947). The sedation scale performed well under the conditions tested, suggesting that it can be applied in a wider range of settings (different populations, raters, sedation protocols).

Hypothermia During General Anesthesia Interferes with Pain Assessment in Laboratory Rats (Rattus norvegicus).

Accurate pain assessment methods are necessary to ensure animal welfare and reliable data collection in animal research. The Rat Grimace Scale (RGS), a facial expression pain scale, allows effective identification of pain. However, the potential confounds of this method remain mostly unexplored. General anesthesia, which is used in many laboratory procedures, suppresses thermoregulation and results in hypothermia. We investigated the effects of isoflurane-induced hypothermia on RGS scores. Twenty (10 male and 10 female) Sprague-Dawley rats each received 30 min of anesthesia, followed by 30 min of observation after the return of sternal recumbency. Rats were randomized to receive warming with an electric heating pad or no warming during both periods. Unwarmed rats became hypothermic within 15 min after isoflurane exposure began and returned to normothermia within 15 min after returning to sternal recumbency. Warmed rats did not deviate from the normothermic range. The RGS scores of unwarmed rats were significantly higher than baseline levels for 3 h after anesthesia and were higher than those of warmed rats at 5 and 180 min after anesthesia. Hypothermia resulted in a larger proportion of rats crossing a predetermined analgesic intervention threshold. Our findings show that hypothermia induced by isoflurane anesthesia presents a confound to accurate RGS scoring. These results emphasize the importance of maintaining normothermia to avoid inflated pain scores and to obtain accurate pain assessment.

Prewarming Followed by Active Warming is Superior to Passive Warming in Preventing Hypothermia for Short Procedures in Adult Rats (Rattus norvegicus) Under Isoflurane Anesthesia.

General anesthesia is a common procedure in laboratory rats; however, it impairs thermoregulation, rapidly leading to hypothermia as warm core blood is distributed to the cooler periphery. The protective strategy of prewarming before the onset of anesthesia delays hypothermia, but only for a short period. This prospective, randomized, cross-over, experimental study in adult male and female SD rats (n = 8) was designed to compare passive (fleece blanket) and active (temperature controlled heating pad) warming. Initial treatment order was randomized, with a cross-over after a minimum 5 d washout period. Both groups underwent a period of prewarming in a warming box to increase core temperature by 1% (median 0.4 °C). At completion of prewarming, general anesthesia was induced and maintained for 30 min with isoflurane carried in oxygen. Core temperature was monitored for a further 30 min after anesthesia. Active warming resulted in higher core temperatures during anesthesia. During passive warming, hypothermia occurred after approximately 30 min of anesthesia and continued into recovery. In contrast, active warming prevented hypothermia. Prewarming followed by passive warming delayed hypothermia for approximately 30min, but active warming was more effective at maintaining normothermia both during and after general anesthesia.

Evaluating Intrahepatic and Intraperitoneal Sodium Pentobarbital or Ethanol for Mouse Euthanasia.

Intraperitoneal (IP) injection of sodium pentobarbital (PB) is an accepted method of euthanasia for mice. However, this method has important drawbacks, including the potential for pain or misinjection. The objective of this prospective, randomized, blinded study was to determine whether intrahepatic (IH) injection of PB is more effective than IP delivery for mouse euthanasia. Secondary objectives were to: 1) determine whether IP ethanol (ET) is a suitable alternative to PB and 2) study the effect of isoflurane anesthesia on euthanasia with either PB or ET. Eighty adult CD1 mice were randomly assigned to 6 different treatment groups, were euthanized by using IP or IH injections of either PB or ET, and were either anesthetized or conscious before injection. Variables of interest were: 1) misinjection rates (based on necropsy evaluation), 2) time from injection to apnea and 3) time to cessation of heartbeat (CHB). The misinjection rate for IH injections was 93% (28/30). Two successful IH injections resulted in death within 4 s, but this method cannot be recommended due to the possibility for intrathoracic injection ( n = 4). In nonanesthetized mice, time to apnea and CHB was significantly shorter with IP ET (apnea: 72.5 s [median], CHB: 115 s) than with IP PB (apnea: 136 s, CHB: 176 s). Anesthesia at time of injection was associated with a shorter CHB time for IP PB. These data show the difficulty in achieving successful IH injections in mice, but confirm that IP ET is a viable and potentially superior alternative to IP PB. Lastly, anesthesia can shorten time to death after IP injection of PB.

Influence of Rater Training on Inter- and Intrarater Reliability When Using the Rat Grimace Scale.

Rodent grimace scales facilitate assessment of ongoing pain. Reported rater training using these scales varies considerably and may contribute to the observed variability in interrater reliability. This study evaluated the effect of training on interrater reliability with the Rat Grimace Scale (RGS). Two training sets (42 and 150 images) were prepared from acute pain models. Four trainee raters progressed through 2 rounds of training, scoring 42 images (set 1) followed by 150 images (set 2a). After each round, trainees reviewed the RGS and any problematic images with an experienced rater. The 150 images were then rescored (set 2b). Four years later, trainees rescored the 150 images (set 2c). A second group of raters (no-training group) scored the same image sets without review with the experienced rater. Inter- and intrarater reliability were evaluated by using the intraclass correlation coefficient (ICC), and ICC values were compared by using the Feldt test. In the trainee group, interrater reliability increased from moderate to very good between sets 1 and 2b and increased between sets 2a and 2b. Action units with the highest and lowest ICC at set 2b were orbital tightening and whiskers, respectively. In comparison to an experienced rater, the ICC for all trainees improved, ranging from 0.88 to 0.91 at set 2b. Four years later, very good interrater reliability was retained, and intrarater reliability was good or very good). The interrater reliability of the no-training group was moderate and did not improve from set 1 to set 2b. Training improved interrater reliability, with an associated reduction in 95%CI. In addition, training improved interrater reliability with an experienced rater, and performance was retained.