Implementation of an Alternative Training Method for Cardiac Blood Collection in Mice.

Training personnel to work with animals presents a variety of challenges, both logistically and with regard to animal welfare. These issues make training an ideal opportunity to evaluate practices and to implement the 3R principles (refinement, replacement, and reduction). Cardiac blood collection from mice is a procedure that can compromise the 3Rs by requiring repeated practice and animal euthanasia. The development of a non-animal training model would promote the 3R principles. Our goals for the development of a new training model for cardiac blood collection from mice were to reduce the number of mice needed to achieve competency, improve our culture of care, and refine the training approach by improving competency. The training model was developed using commonly available materials. The total cost of the model was less than $15 USD per model. Two training curricula were conducted concurrently over a 5-mo period: 1) a curriculum in which trainees used the model before progressing to live mice and 2) the traditional curriculum, which used euthanized mice throughout. The measured variables included the total number of mice used, proportions of trainees who reached competency, the time needed to reach competency, method comprehension, quality of skill performance, trainer and trainee feedback, and training costs. The alternative group used at least 10 fewer mice per technician as compared with the traditionally trained group. The alternative group had a higher competency rate, with 82% (9 of 11 trainees) reaching competency compared with 60% (3 of 5 trainees) in the traditional group. Skill comprehension and quality were superior in the alternative group, as evidenced by fewer gross lesions at necropsy. Overall, personnel in the alternative group provided positive feedback with regard to the use of fewer mice, acquisition of both skill and confidence, and benefits for compassion fatigue. The use of this model is now our standard approach for training personnel in cardiac blood collection in mice. Our results demonstrate that the use of models in training curricula can enhance skill development and reduce the use of mice.

Humane Intervention Points: Refining endpoint terminology to incorporate non-euthanasia intervention options to improve animal welfare and preserve experimental outcomes.

Consideration of The Three R's (Replacement, Reduction, and Refinement) is essential when setting Humane Endpoints; however, a common interpretation assumes that Humane Endpoints are timepoints to perform euthanasia. This interpretation is not always consistent with the three Rs. There are many available intervention options that - when used to respond to pain, discomfort, or distress - facilitate application of the three Rs while achieving experimental goals. At our institution, the term 'Humane Endpoints' was replaced with 'Humane Intervention Points', to ensure responses beyond euthanasia are given priority by laboratory animal professionals and researchers. Identification of Humane Intervention Points in the research protocol provides us with a more accurate reflection of the measures used to alleviate pain and distress in animals used for research, testing, and teaching - an outcome easily envisioned elsewhere.Points d'intervention humains (PIH): Affinage de la terminologie d'évaluation finale pour intégrer des options d'intervention sans recours à l'euthanasie afin d'améliorer le bien-être des animaux et de préserver les résultats expérimentaux Résumé.

Influence of the position of charcoal air-filtration canisters on the efficacy of waste isoflurane scavenging as assessed in a randomized experiment.

OBJECTIVE To determine whether the position or elevation of charcoal air-filtration canisters would impact efficacy of waste anesthetic gas (WAG) scavenging. DESIGN Randomized experiment. SAMPLE 2 types of bottom-vented and 1 type of top-vented charcoal air-filtration canisters (n = 8 of each canister type/evaluation session). PROCEDURES Canisters were evaluated in a vertical or horizontal position at both low and high isoflurane gas flow rates in a modified Bain nonrebreathing circuit. Waste anesthetic gas concentrations were measured 2.54 cm from canister exhaust ports with an ambient air analyzer every 30 seconds for a maximum of 15 min/experimental condition. One type of bottom-vented canister was tested in a vertical position elevated above or suspended below the vaporizer at a high isoflurane flow rate and then a standard maintenance flow rate. RESULTS Position had no significant effect on WAG emission by any canister type at low isoflurane flow rates. Horizontally positioned bottom-vented canisters at the high isoflurane flow rate emitted significantly more WAG than vertically positioned canisters. Horizontally positioned top-vented canisters at high flow rates emitted significantly more WAG than vertically positioned canisters at the final 15-minute time point only. Cannister types differed significantly in intercanister variability. Canister elevation relative to the vaporizer had no impact on WAG scavenging efficacy. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that charcoal air-filtration canisters should be used in a vertical position when anesthetizing animals with the anesthetic delivery system used in this study.

A Description of a Noninvasive Surgical Training Pathway Using Translational Tools to Teach Intracoelomic Implantation of Acoustic Transmitters in Fish.

Researchers engaged in surgical implantation of acoustic transmitters into fish must receive adequate and appropriate training to ensure the welfare of their subjects and the quality of the data collected. Increasingly, researchers are being encouraged to partner with veterinarians to improve training and to consider the principles of animal welfare in training. Here, we describe a five-stage training pathway, including implementation of new training tools (the Translational Training Tools and field certification), that was developed collaboratively by researchers and veterinarians and addresses the "three Rs" of animal welfare in the context of surgical training. The three Rs include animal replacement, reduction of the number of animals used, and refinement of techniques to decrease or eliminate pain or distress. The Translational Training Tools, described in the context of the training pathway, use tools as replacement models during training to reduce the number of animals used and allow for refinement of surgical skills prior to working on live animals. The purpose of this paper is to document the Translational Training Tools and the training pathway, which will be useful in developing de novo protocols for review by Institutional Animal Care and Use Committees and similar bodies.

Comparing Phlebotomy by Tail Tip Amputation, Facial Vein Puncture, and Tail Vein Incision in C57BL/6 Mice by Using Physiologic and Behavioral Metrics of Pain and Distress.

Tail tip amputation with minimal restraint is not widely used for mouse phlebotomy. In part, this infrequency may reflect policies influenced by tail tip amputation procedures for genotyping, which involve greater handling and tissue removal. To assess tail tip amputation with minimal restraint as a phlebotomy technique, we compared it with 2 more common methods: scruffing with facial vein puncture and lateral tail vein incision with minimal restraint. Blood glucose levels, audible and ultrasonic vocalizations, postphlebotomy activity and grooming behavior, open field and elevated plus maze behaviors, nest-building scores, and histologic changes at the phlebotomy site were evaluated. Mice in the facial vein phlebotomy group produced more audible vocalizations, exhibited lower postphlebotomy activity in the open field, and had more severe histologic changes than did mice in the tail incision and tail tip amputation groups. Facial vein phlebotomy did not affect grooming behavior relative to sham groups, whereas tail vein incision-but not tail tip amputation-increased tail grooming compared with that in control mice. Blood glucose levels, nest-building scores, and elevated plus maze behavior did not differ between groups, and no mice in any group produced ultrasonic vocalizations. Tail tip amputation mice did not perform differently than sham mice in any metric analyzed, indicating that this technique is a potentially superior method of blood collection in mice in terms of animal wellbeing.

Dual modality endomicroscope with optical zoom capability.

We present a miniature endomicroscope that combines large field-of-view (FOV) (1.15 mm) reflectance imaging with high-resolution (~0.5 µm) multiphoton intrinsic fluorescence imaging. We acquired in vivo and ex vivo images of unstained normal and tumor-laden tissues by using the large-FOV mode to navigate to the site of interest and then switching to the high-resolution modality to resolve cellular details.

Multiphoton tomographic imaging: a potential optical biopsy tool for detecting gastrointestinal inflammation and neoplasia.

Endoscopy is widely used to detect and remove premalignant lesions with the goal of preventing gastrointestinal (GI) cancers. Because current endoscopes do not provide cellular resolution, all suspicious lesions are biopsied and subjected to histologic evaluation. Technologies that facilitate directed biopsies should decrease both procedure-related morbidity and cost. Here we explore the use of multiphoton microscopy (MPM), an optical biopsy tool that relies on intrinsic tissue emissions, to evaluate pathology in both experimental and human GI specimens, using hematoxylin and eosin (H&E)-stained sections from these tissues for comparison. After evaluating the entire normal mouse GI tract, MPM was used to investigate disease progression in mouse models of colitis and colorectal carcinogenesis. MPM provided sufficient histologic detail to identify all relevant substructures in ex vivo normal GI tissue, visualize both acute and resolving stages of colitis, and show the progression of colorectal carcinogenesis. Next, ex vivo specimens from human subjects with celiac sprue, inflammatory bowel disease, and colorectal neoplasia were imaged by MPM. Finally, colonic mucosa in live anesthetized rats was imaged in vivo using a flexible endoscope prototype. In both animal models and human specimens, MPM images showed a striking similarity to the results of H&E staining, as shown by the 100% concordance achieved by the study pathologists' diagnoses. In summary, MPM is a promising technique that accurately visualizes histology in fresh, unstained tissues. Our findings support the continued development of MPM as a technology to enhance the early detection of GI pathologies including premalignant lesions.

In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope.

We use a compact and flexible multiphoton microendoscope (MPME) to acquire in vivo images of unstained liver, kidney, and colon from an anesthetized rat. The device delivers femtosecond pulsed 800 nm light from the core of a raster-scanned dual-clad fiber (DCF), which is focused by a miniaturized gradient-index lens assembly into tissue. Intrinsic fluorescence and second-harmonic generation signal from the tissue is epi-collected through the core and inner clad of the same DCF. The MPME has a rigid distal tip of 3 mm in outer diameter and 4 cm in length. The image field-of-view measures 115 µm by 115 µm and was acquired at 4.1 frames/s with 75 mW illumination power at the sample. Organs were imaged after anesthetizing Sprague-Dawley rats with isofluorane gas, accessing tissues via a ventral-midline abdominal incision, and isolating the organs with tongue depressors. In vivo multiphoton images acquired from liver, kidney, and colon using this device show features similar to that of conventional histology slides, without motion artifact, in ~75% of imaged frames. To the best of our knowledge, this is the first demonstration of multiphoton imaging of unstained tissue from a live subject using a compact and flexible MPME device.

Sedation or inhalant anesthesia before euthanasia with CO2 does not reduce behavioral or physiologic signs of pain and stress in mice.

CO(2) administration is a common euthanasia method for research mice, yet questions remain regarding whether CO(2) euthanasia is associated with pain and stress. Here we assessed whether premedication with acepromazine, midazolam, or anesthetic induction with isoflurane altered behavioral and physiologic parameters that may reflect pain or stress during CO(2) euthanasia. Mice were assigned to 1 of 6 euthanasia groups: CO(2) only at a flow rate of 1.2 L/min which displaces 20% of the cage volume per minute (V/min; control group); premedication with acepromazine (5 mg/kg), midazolam (5 mg/kg), or saline followed by 20% V/min CO(2); induction with 5% isoflurane followed by greater than 100% V/min CO(2) (>6L/min); and 100% V/min CO(2) only (6 L/min). Measures included ultrasonic sound recordings, behavioral analysis of video record- ings, plasma ACTH and corticosterone levels immediately after euthanasia, and quantification of c-fos from brain tissue. Compared with 20% V/min CO(2) alone, premedication with acepromazine or midazolam did not significantly alter behavior but did induce significantly higher c-fos expression in the brain. Furthermore, the use of isoflurane induction prior to CO(2) euthanasia significantly increased both behavioral and neuromolecular signs of stress. The data indicate that compared with other modalities, 20% V/min CO(2) alone resulted in the least evidence of stress in mice and therefore was the most humane euthanasia method identified in the current study.

Ultrasonic sound as an indicator of acute pain in laboratory mice.

In response to pain, mice may vocalize at frequencies above the range of human hearing (greater than 20 kHz). To determine whether an ultrasonic recording system is a reliable tool for assessing acute pain, we measured audible and ultrasonic vocalization in mice subjected to either nonpainful or potentially painful procedures performed routinely in animal facilities. Data were collected from 109 weanling mice (Mus musculus; B6, 129S6-Stab 5b) scheduled for 2 potentially painful procedures: DNA testing by tail snip and identification by ear notching. The mice each were assigned randomly to 1 of 4 groups: 1) actual tail snip, 2) sham tail snip, 3) actual ear notch, or 4) sham ear notch. Vocalizations during the treatments were recorded with an ultrasonic recorder. Most mice (65%; n = 55) demonstrated no vocal response to the potentially painful procedures. More mice that received actual tail snips produced audible sounds (11 of 29 mice) than did those that underwent sham tail snips (0 of 30 mice). In addition, audible vocalizations occurred more frequently during ear notch procedures (8 of 26 mice) than during sham ear-notch manipulations (2 of 24 mice). For all 20 of the mice that produced ultrasonic vocalizations, these calls were accompanied by simultaneous audible components. We conclude that ultrasonic vocalizations do not provide any more information than do audible vocalizations for assessing responses to potentially painful procedures. In addition, because many mice made no sound at all after a potentially painful stimulus, vocalizations generally are not good metrics of acute pain in laboratory mice. Alternatively, the lack of vocalizations in many of the mice may suggest that tail snipping and ear notching are not particularly painful procedures for most of these mice.