Noise and Vibration Generation and Response of Mice (Mus musculus) to Routine Intrafacility Transportation Methods

Measurement of noise.

A sound meter (NSRT_mk3; Convergence Instruments, Sherbrooke, QC, Canada) was used to measure the noise generated within cages (Figure 1). The type 1 sound meter measured 19 × 42 × 160 mm and weighed 100 g. This device collected Z-weighted equivalent continuous sound pressure levels (Leq) once per second with a bandwidth of 20 Hz to 20 kHz, saturation level of 120 dBZ, and noise floor of 52 dBZ. Maximum noise levels were reported as the maximum Leq reached during a trial, and average noise levels were reported as average Leq across the duration of the trial. A manufacturer’s certificate of calibration was received along with the sound meter at the time of purchase.

Measurement of vibration.

A vibration meter (VSE mk2-8g; Convergence Instruments, Sherbrooke, QC, Canada) measuring 7.62 × 3.94 × 2.06 cm and weighing 65 g was used to measure vibration along 3 axes (x = side to side, y = forward and backward, z = up and down) once per second for the duration of the trial (Figure 1). Vibration was reported as root mean square (RMS) acceleration measured in meters per second squared (m/s²) within a dynamic range of ±78.4 m/s². Maximum vibration levels were reported as the maximum RMS acceleration reached during a trial, and average vibration levels were reported as average RMS acceleration across the duration of the trial. The vibration meter was calibrated prior to each daily use along all axes using earth’s gravity as a reference.

Cage setup.

The sound and vibration meters were positioned centrally side by side on bedding within a standard cage to best estimate the noise and vibration levels experienced by the animals. Each cage was covered with a polycarbonate filter top lid and had a metal food hopper hanging on the rear wall. An upside-down sipper-style water bottle was placed in the appropriate slot to reflect the standard intrafacility transport setup used at our institution.

Study design.

Instrumented cages without mice were placed on transport devices (1 flatbed, 1 stainless steel rack, 2 plastic carts, and 1 metal cart) or handheld and moved through a predefined route within the facility. Measurements were recorded automatically once per second. Controls were obtained in IVC housing conditions explained below. Maximum level achieved and mean level for the entire transport were compared between the transport methods. To examine simple methods to reduce noise and vibration, the experiments were repeated on each transport device using external attenuation (1 or 2 towels under the cage) or internal attenuation (brown crinkle paper within the cage).

Transportation methods.

For control conditions, data were collected by placing the instrumented cage centrally into an individually ventilated system with top-mounted ventilation units (EcoFlo; Allentown, Allentown, NJ). Three recordings of 3-min duration were taken with 2-min intervals between recordings. IVC measurements were used as the primary control comparison as this is the standard rodent housing method throughout most of our institution.

For initial comparisons of transport methods, data were measured continuously during transport. For hand carrying, the cage was held horizontally in a 2-hand hold with the cage long axis perpendicular to the direction of travel. For cart transport, the cage was placed centrally with the long axis perpendicular to the direction of travel on the top shelf of the cart. For the stainless-steel rack, the cage was placed on the central shelf. Cart details can be found in Table 1. All rack casters and those closest to the cart and flatbed handles swiveled while the remaining wheels remained fixed. All cart shelves were supported by L-shaped struts 7.6 cm wide and 0.6 cm thick on both plastic carts and 3.8 cm wide and 0.07 cm wide on the metal cart. The stainless-steel rack shelves were adjustable clip-in shelf mounts on 2.5-cm hollow square struts.

Transportation route.

The route was a standardized, 96-m loop on even epoxy resin flooring. At the halfway point, an elevator was entered and ridden up one floor, exited, and then an adjacent elevator was entered and ridden back to the original floor where the loop was completed. The route was completed in an average of 3.9 min including wait times for the elevators. One individual with an average walking speed of 4 km/h performed all of the transports. The route was repeated a total of 6 times, split between 2 different days, for each type of transport.

Attenuation methods.

The transportation process was repeated with each cart using 3 different attenuation methods: a double-folded towel (approximately 20 mm thick) placed under the cage, 2 double-folded towels (40 mm thick) placed under the cage, or 6 g of brown crinkle paper within the cage but under the sensors.

Study design.

The transport methods generating the lowest level of noise and vibration (pneumatic cart) and the highest levels (metal cart) were used in subsequent animal testing. Transport stress was assessed with behavior testing or blood biomarkers in separate experiments. In the behavioral study groups, mice (n = 10/group) were implanted with a temperature transponder one week prior to experimentation and group housed 2 to 3 mice per cage. Each mouse underwent transport followed by an open-field test (OFT) directly after transport and an elevated plus maze (EPM) test 24 h later. Body temperature readings were obtained immediately before and after transport. Control mice were not transported prior to testing aside from transfer from housing to the behavior testing room prior to acclimation to the testing room. After the testing, the group-housed mice remained in standard housing for one month. The mice were then singly housed for one week followed by repetition of the transport and behavioral studies. Figure 2 summarizes the timeline of the behavior measurements.

For evaluation of blood biomarkers, mice were catheterized for automated serial blood collection and singly housed after surgery. After 3 d of recovery, a baseline blood sample was collected. Mice were disconnected from the automated collection device and transported on their assigned transport device (metal cart or plastic pneumatic cart). After transport, automatic blood collection was resumed immediately and at predetermined intervals for 48 h. Control mice were disconnected from the automatic collection device after baseline sampling, held in the same room in a clean shoebox cage for 15 min, and reconnected for sampling. Blood samples were processed upon completion of sampling and analyzed for corticosterone levels. Figure 3 summarizes the timeline of the biomarker measurements. Experimental groups for animal studies are summarized in Table 2.

Animals.

Male (10 to 12 wk of age) C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were used in all animal experiments. Male mice were selected to limit variability in biomarker and behavioral responses as a result of estrus cyclicity.^9,11,13,20 Mice were housed in a 18- × 29- × 12.5-cm polycarbonate mouse cage (Allentown, Allentown, NJ) on approximately 300  mL of a 50/50 blend of ¼- and ⅛-in. irradiated corncob bedding (Anderson’s Bed-o’Cobs; Frontier Distributing, Maumee, OH) with 6 g of brown crinkle paper encased in a white tea bag material (EnviroPak; Shepherd Specialty Papers, Watertown, TN). Individually housed mice were also provided with a clear red plastic mouse igloo (Bio-Serv, Flemington, NJ) or a 2- × 2-in. compressed cotton square (Ancare, Bellmore, NY) if cannulated for blood collection. Prior to experimental use, mice were acclimated for at least 7 d in ventilated microisolation cages in a temperature-controlled room (22.2  ±  1.1 °C and relative humidity of 60%  ±  10%). Mice were group housed by experimental condition assignment with up to 3 mice per cage. Mice that underwent surgical cannulation were individually housed following surgery to protect their exterior catheter site. Housing was in a SPF barrier facility with a 12:12-h dark:light cycle. Mice were monitored for pathogens via serology and PCR analysis of soiled-bedding sentinel mice and by an exhaust air duct PCR assay and were negative for the following pathogens: mouse hepatitis virus, minute virus of mice, mouse parvovirus, epizootic diarrhea of infant mice virus, ectromelia virus, Sendai virus, pneumonia virus of mice, Theiler murine encephalomyelitis virus, reovirus, lymphocytic choriomeningitis virus, mouse adenovirus, polyomavirus, Mycoplasma pulmonis, and pinworms. The Jackson Laboratory routinely tests free of mouse norovirus and Helicobacter spp., although mouse norovirus, Helicobacter spp., and other bacterial pathogens are not tested for routinely at our institution. All mice were apparently healthy and free of any known pathogens, excluded or otherwise. Mice had ad libitum access to food (Laboratory Rodent Diet 5001, PMI; LabDiet, St. Louis, MO) and water. All procedures involving animals were approved by the University of Michigan Animal Care and Use Committee prior to implementation. All animals were housed in an AAALAC-accredited facility, and all procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.¹⁶

Transportation methods.

The metal teacart and plastic pneumatic cart were tested since they produced the largest differences in sound and vibration during the initial transport studies. Cages containing mice were transported one at a time alongside an unoccupied cage that was instrumented to record noise and vibration (Figure 1). The 2 cages were placed side by side centrally, approximately 5 cm apart, with the long axis perpendicular to the direction of travel on the top shelf of the cart. The transported cages were covered with a lightweight polypropylene gown (Medline Industries, Northfield, IL) per institutional policy.

Transportation route.

The transport route was extended to 612 m to simulate transportation of mice between 2 housing areas within the facility. The route was a moderately trafficked loop on even epoxy resin flooring with utilization of the adjacent elevators described for the initial testing. The route was completed by the same individual in an average of 15 min. Cage components and setup remained consistent for all conditions and transport sessions.

Behavioral assays.

Mice were transferred in their home cage on a metal cart across a hallway from the housing room to the testing room (< 10 m transport distance) and acclimated for at least 1 h prior to transport. Immediately following testing, an OFT began with each mouse placed in the center of a square open-field chamber measuring 43 × 43 × 30 cm³ made of dark gray plastic. Video recording began 3 s after the mouse was detected in the arena and continued for 30 min. Four mice (of 3 groups in 4 separated chambers) were tested at a time. The mice were removed and returned to their home cage and housing room upon completion of the test. Twenty-four hours following the OFT, mice were again acclimated to the testing room for 1 h prior to undergoing EPM testing. The EPM apparatus was constructed of white painted wood board consisting of 2 open arms (34 × 7.5 × 0.5 cm³) and 2 closed (with walls) arms (34 × 7.5 × 15 cm³), crossing perpendicularly in the middle with a small center area. The entire platform was raised approximately one meter above the floor. The test began by placing the mouse in the center zone facing the same open arm. Video recording began 2 s after the mouse was detected in the arena and continued for 5 min. When the test was finished, the mice were returned to their home cage and housing. EthoVision XT (Noldus, Leesburg, VA) software was used to record, track, and analyze the videos from both tests, calculating total distance traveled, average velocity, time spent in each zone, overall movement, and rearing. Testing chambers and objects were wiped down after each trial with Sani-Cloth wipes (PDI Healthcare, Woodcliff Lake, NJ) and cleaned with water to eliminate olfactory cues from previous mice. Lighting intensity was set at 300 lx for OFT and 20 lx for EPM using a ceiling bounce light generated by LED floor lamps. Room temperature, humidity, and background noise were kept consistent throughout the experiments.

Temperature measurement.

Mice assigned to serial behavioral testing were anesthetized with isoflurane (VetOne, Boise, ID) for aseptic surgical implantation with an intraperitoneal temperature transponder (12 mm long and 2 mm in diameter) (IPTT-300; Bio Medic Data Systems, Seaford, DE). They were allowed to recover for at least a week prior to the study. A handheld reader (DAS-8007; Bio Medic Data Systems, Seaford, DE) was used to record body temperature directly before and after transport.

Automated blood sampling.

Surgical cannulation and automated sampling (Culex; BASi Research Products, West Lafayette, IN) allowed for serial blood collection in conscious, free-moving, and undisturbed mice.³⁰ Mice assigned to serial blood collection underwent surgical catheterization of the carotid artery. Anesthesia was induced with 5% isoflurane (VetOne, Boise, ID) and maintained on 1% to 3% isoflurane to effect on a nose cone. Lubricating eye ointment (Puralube; Dechra Pharmaceuticals, Northwich, UK) was applied. The ventral and dorsal neck were prepared for aseptic surgery and a small incision was made superior to the clavicle, exposing the carotid artery. A catheter made of MicroRenathane tubing (0.025-in. outer diameter × 0.012-in. inner diameter, Braintree Scientific) was inserted into the artery and ligated in place with 7-0 silk suture (Fine Science Tools, Foster City, CA). The catheter was tunneled subcutaneously and exteriorized at the back of the neck via a stainless steel tubing connector (made of 25-gauge needle and silicon). The skin was closed with sutures. The exteriorized catheter was filled with heparinized saline and tightly plugged with stainless steel surgical wire. Carprofen (5 mg/kg, SC) (Rimadyl; Zoetis, Parsippany, NJ) was given preemptively and once daily for 2 d after surgery. Mice were individually housed and allowed to recover for 72 h following surgery in the housing room. Following recovery, mice were transferred with their home cage bedding to the caging system and the exteriorized catheter was attached to a tether. The platform of the cage rotated automatically in an opposite direction to mouse movement allowing free movement of the mouse without tension or tangling of the sampling line. The system was programmed to flush a small volume (5 to 10 µL) of heparinized saline (10 U/mL) once every 15 min to maintain catheter patency. Mice were acclimated for 24 h and then 50 µL of blood was collected for baseline controls. Mice assigned to transport conditions were then untethered and transferred to a clean transport cage and underwent their transport route. Control mice were untethered and transferred to a clean cage that remained stationary for 15 min. After transport or allotted time in a clean cage, mice were returned to their infusion cage and automated serial blood draws (50 µL) were conducted immediately, then at 1, 2, 6, 12, 18-, 24, 30, 36, 42, and 48 h. An equal volume of saline was administered for fluid replacement at each time point. All blood samples were diluted in equal parts 50% heparinized (Sagent Pharmaceuticals, Schaumburg, IL) saline (Baxter, Deerfield, IL) (10 U/mL) and held in a refrigerated carousel until completion of the last sampling time point. The samples were centrifuged (14,000 × g, 30 s) and plasma stored at −80 °C until analyzed.

Stress biomarker assays.

Plasma corticosterone concentrations were measured with a commercially available ELISA kit (K014-H5W; Arbor Assays, Ann Arbor, MI) according to the manufacturer’s instructions. Corticosterone samples were diluted to a final concentration of 1:100 in the provided assay buffer. Optical density was determined by a microplate spectrophotometer (EPOCH2; BioTek, Broadview, IL) at 450 nm, and output was processed by corresponding imager software (Gen5, BioTek, Broadview, IL). All samples and standards were run in duplicate and averaged for final concentration. Any readings below the limit of detection were assigned half the lower limit of detection.

Euthanasia.

At the conclusion of experiments, mice were euthanized via 40% CO₂ inhalation with cervical dislocation performed after breathing had ceased.

Statistical analyses.

Group size was determined via a prior power analysis using G*Power version 3.1.9.7 (Heinrich Heine University Düsseldorf, Düsseldorf, Germany) with an α of 0.05 and power of 0.8. All analytics were performed on GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA). Data normality was verified using Kolmogorov–Smirnov or Shapiro–Wilk tests depending on sample size and expressed as mean and SE. P values were determined using one-way and 2-way ANOVA with P < 0.05 considered significant. Corrections were applied for multiple comparisons of noise and vibration levels and repeated measures for behavior and biomarker data. Post hoc Tukey–Kramer and Dunnett tests were applied for analysis of multiple groups. All reported P values are based on post hoc testing pursued after indication of a significant main effect.

Common anxiety-sensitive behavioral assays are minimally influenced by brief intrafacility transport.

In group and singly housed mice, an OFT conducted directly after transport showed no significant differences in mouse movement, distance and velocity traveled, or time within the central, intermediate, and outer zones between the mice transported on the 2 different carts and compared with nontransported, control mice (Table 3, Figure 7A, C). Similarly, no significant differences in time or entries on the open arms or distance traveled on the EPM were noted in either group or singly housed mice (Table 3, Figure 7B, D). However, transported mice tended to spend more time in the open arms compared with mice at a level that approached significance in group-housed mice (P = 0.09) and to a lesser degree in singly housed mice (P = 0.16) (Table 3, Figure 7B, D). Singly housed mice in general spent significantly (P < 0.05) less time on the open arms and traveled farther on the EPM than did group-housed mice (Figure 7B, D).