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Johns Hopkins Bloomberg School of Public HealthCAAT

Animal Welfare Enhancement Awards - 2004 Recipients

Ultrasonic Sound Measurement as an Indicator of Pain and Distress in Laboratory Rodents
Wendy O. Williams, DVM
  Center for Research Animal Resources, Cornell University

Introduction

The assessment of pain and distress in animals is complicated by the inability of animals to verbalize their sensations. Tools to measure physiological data have limited usage in smaller animals such as laboratory rodents. Research investigators, animal care takers and veterinary personnel must often make assessments of rodent pain and discomfort based upon behavioral observations alone.

There are several well defined behaviors that are routinely used to deduce the discomfort level of rodents e.g. decreased activity and grooming, body weight loss, hunched posture, licking or chewing at incision sites. Rodents may also vocalize in response to discomfort. While some vocalizations are audible to the human ear, others are emitted at frequencies > 20 kHz which cannot be heard by humans (Jourdan et al., 1995). Those ultrasonic vocalizations at 22 kHz lasting longer than 0.3 seconds have been shown to occur during anticipation of punishment or avoidance behavior (Knutson, Burdorf and Panksepp, 2002). Such ultrasonic vocalizations may be a useful as data for clinical evaluation of laboratory rodents.

Ultrasonic vocalizations in response to both acute and chronic pain have been widely studied in the laboratory rat (Jourdon et al., 1995, 1998, 2002, Han et al., 2004). For this experiment, the mouse was chosen as the model because they are the most widely used species of laboratory rodent. Many of the mice used in research will experience some level of pain or discomfort as a direct result of research manipulations or from spontaneous health concerns. Mice are prey species that have a tendency mask their clinical signs from human observers therefore mice can present a challenge to veterinary personnel who wish to assess the level of discomfort. There is a need for more data on how mice express that they are in pain. Mice commonly undergo routine procedures such as tail snips for DNA collection and ear punching for animal identification therefore the mouse was a readily available model at our institution. These routine procedures provide an excellent opportunity to measure the vocal responses of mice to an acute insult.

Variability in behavior and physiology is reported in laboratory mice (Baumans et al., 1994). To minimize the strain differences, all recordings were taken from the same strain of mice.

The data in the cited literature on rat vocalizations was collected in a sound proof chamber. This experiment did not make use of a sound proof chamber. In attempting to assess whether an ultrasonic recording device can be used as a tool for assessing pain in laboratory mice, the normal conditions under which the mouse would be handled were maintained. Thus the mice were handled and recorded in a Biological Safety Cabinet in the same room in which the animals are housed.

Objective

The objective of this experiment is to determine whether mice vocalize at ultrasonic frequencies in response to pain and to determine whether an ultrasound recording system is useful as a clinical tool to diagnose when mice are in pain.

To investigate these questions, one must first determine whether mice vocalize when they experience a painful stimulus. The next step is to determine whether any of the vocal emissions made in response to the painful stimulus occur at frequencies that are inaudible to the human ear (i.e. ultrasonic vocalizations). The final step is to determine whether any of the ultrasonic vocal emissions occur in the absence of audible vocalizations.

Materials and Methods: Data was collected from a group of 71 laboratory mice (Mus musculus, Strain Stat 5 a/b + Stat 5b on C57BL/6 background). These mice were part of an IACUC approved research protocol. The Principal Investigator gave written consent to collect data from these animals. Both males and females were used for this experiment. No animals were purchased specifically for this experiment. The procedures used as the painful stimuli were already scheduled for this group of mice as part of the Principal Investigators research protocol. The number, age and the sex of the mice were therefore determined by the number of mice of the chosen strain that were scheduled to have tail snips and ear punches performed at weaning during the period of time available to do the recordings.

Mice were housed in micro-isolator cages on ventilated racks in the Transgenic Mouse Core Facility at an AAALAC accredited institution. (12 h light, 12 h dark cycle, with free access to food and water). None of the mice had undergone experimental manipulations prior to the sound recordings.

The procedures were performed in a Biological Safety Cabinet (BSC) as part of the normal routine. Prior to starting the experiment, recordings were taken in the empty BSC to determine what type of background noise might interfere with ultrasonic vocalizations from the mice. Measurements were also taken of common manipulations done in the BSC i.e. spraying gloves with disinfectant, rubbing gloved fingers together, snipping the scissor in the absence of mice and pressing the ear puncher in the absence of mice. In addition, a small, inexpensive bat detector was used to check for background noise. This bat detector had a small range of frequencies so its use was limited but effective for some immediate feedback about ultrasound emissions in the surroundings.

Recordings were taken of vocal emissions made by recently weaned mice during two routinely performed but potentially painful procedures; ear notching and tail snipping. Recordings were performed with an Avisoft UltraSoundGate USG 116-200 and Avisoft-RECORDER USG. This kit comes with a sensitive solid-state dielectric condenser microphone that can detect ultrasounds up to about 200 kHz.

Mice were randomly assigned by a coin toss as either real or placebo. Real subjects were recorded during actual ear notch and tail snip procedures. Placebo subjects were recorded while the ear notch instrument was punched close to but not touching the ear such that the mouse was exposed to the sound of the ear puncher without receiving direct physical stimulation. Similarly, the placebo tail snips measurements were taken by placing the scissors beside the tail and snipping the scissors closed without touching the tail.

The microphone was pointed directly at the head of the subject. The microphone was placed at a distance of 10 cm from the subject being recorded. The recording frequency was set to record maximum frequencies of 125,000 Hz to 190,000 Hz. Manipulations were performed by the same animal handler for all subjects. Recordings were performed on three different days approximately one month apart. The recordings were identified by saving them on a lap top computer file labeled by the date of the recording and a number assigned sequentially to each mouse.

Mice were restrained for 3 seconds prior to performing the procedure, allowing enough time for the animal to cease any vocalizations that occurred in response to restraint. Any vocalizations that occurred during the initial restraint were not included in the data. The handler performed the procedures three seconds apart from one another to allow the vocalizations from the first stimulus to cease before initiating the second stimulus.

The first group (22 mice) was randomized into real or placebo stimuli groups. The ear punch was performed first for the entire group of mice followed by the tail snip. The second group (23 mice) was randomized into real or placebo stimuli groups. The tail snip was performed first for the entire group of mice followed by the ear punch. The third group (26 mice) was randomized into real or placebo groups. The order of the tail snip and ear punch was also randomized for each mouse.

To determine whether mice made audible vocalizations in response to the painful procedures, each recording was played back to a listener who was blind to whether the recorded subject received a real or placebo procedure. Documentation was kept on which mice made audible vocalizations and which mice did not. The number of vocalizations made in response to each painful stimulus was also documented. A spectrogram analysis was performed on each individual's recording to determine by visual inspection, whether any ultrasonic vocalizations occurred in response to the painful stimuli. Documentation was kept on which animals made ultrasonic vocalizations. The number of audible and ultrasonic vocalizations in response to each painful stimulus was also documented.

The spectrogram data was compared to the audible data to first determine whether any of the vocal emissions made in response to the painful procedures occurred at ultrasonic frequencies and secondly whether any of those ultrasonic vocalizations occurred in the absence of audible vocalizations.

Results

The number of ultrasonic and audible vocalizations that each mouse made in response to the painful stimulus was documented however, there were too many biases to determine whether a group of sounds were separate vocalizations rather than one staccato vocalization. Statistical analysis was therefore based on whether the mouse produced a vocalization in response to the stimulus or not rather than on the number of vocalizations produced per noxious stimulus.

There were no audible or ultrasonic vocalizations made in response to placebo ear punches or placebo tail snips. The audible and ultrasonic vocalizations produced in response to real ear punches and tail snips were consistently emitted immediately after the stimulus was applied. Several of the mice produced audible vocalizations when restrained. These vocalizations had ceased before proceeding with the tail snip or ear punches. No other sounds were made before the painful stimulus was applied.

Seventy five percent of the animals that produced audible vocalizations in response to ear punching (n=8) also produced US vocalizations (n=6). One hundred percent of the animals that made audible vocalizations in response to tail snips (n=8) also made ultrasonic vocalizations (n=8). All of the mice that produced ultrasonic vocalizations in response to real ear punches (n=6) made accompanying calls in the audible range. All of the mice that produced ultrasonic vocalizations in response to real tail snips (n=8), also made accompanying vocalizations in the audible range.

Ultrasonic cries usually occurred in those subjects that made multiple vocalizations in response to the painful procedures. The first few audible cries were accompanied by ultrasonic calls. The audible calls that had accompanying ultrasonic calls were more intense i.e. louder than the other audible calls.

The same kind of data resulted, regardless of the order of the ear and tail snip procedures therefore the data were lumped together for statistical analysis to determine whether there was a significant difference in vocalizations. The ultrasonic data did not provide any additional information to that of the audible data therefore, the following statistical analysis was performed on the audible vocalizations regardless of whether they were accompanied by ultrasonic vocalizations or not: Mice vocalized more often during real ear punches than during placebo ear punches (chi-square test, P=0.0031). Mice vocalized more often during real tail snips than during placebo tail snips (chi-square test, P=0.0031).

Conclusions

As a means to measure a response to discomfort in a laboratory mouse, we investigated whether the visual inspection of ultrasonic vocalizations from mice exposed to noxious stimuli would generate information beyond that which we are able to hear. Measurement of ultrasonic vocalizations did not give any additional information beyond what was learned from the audible vocalizations. Although the mice did sometimes produce ultrasonic vocalizations in response to the painful stimuli, these ultrasonic vocalizations did not occur in the absence of audible vocalizations therefore from this experiment we cannot conclude that recording ultrasound is an effective means to determine whether a mouse is in pain.

We would like to do similar experiments to measure ultrasonic vocalizations in mice that are exposed to other routine procedures that potentially cause acute pain e.g. subcutaneous or intraperitoneal injections, toe tattooing and ear tagging. In addition, we are interested in looking at chronic pain models e.g. post surgical cases and tumor models in existing approved research protocols as well as animals that have spontaneous health concerns such as abscesses, ulcerative dermatitis, fight wounds. A continuation of the ear punch and tail snip data has been proposed in which we record the mice at specific time intervals post procedure to determine whether there is a delayed response to the pain which may be picked up by ultrasound.

A topic for further investigation is the characterization of mouse calls from the spectrogram to determine if pure ultrasonic vocalizations are produced. In the cited literature, rat vocalizations were characterized into three types of sounds i.e. peeps, chatters and pure ultrasound (Jourdan et al. 1995). Based on a preliminary review of the literature, the mouse vocalizations seen in our data look similar to what Jourdan et. al. described as a peep i.e. a mixture of audible and ultrasonic waves. According to this reference, the peeps are not considered pure ultrasound however pure ultrasonic sounds were produce by some rats during an acute noxious insult.

We would like to learn more about the appearance of the ultrasonic cries. From the data collected in this experiment and future work, we will review summary statistics such as the highest frequency emitted, lowest frequency emitted, frequency with greatest energy, length of the call. These characteristics of the sounds may have significance in deciphering the types of vocalizations that are made in response to painful stimuli.

References

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  • Han, J.S., Bird, G.C., Wiedong, L., Jones, J. and Neugebauer, V., Computerized analysis of audible and ultrasonic vocalizations of rats as a standardized measure of pain-related behavior, Journal of Neuroscience Methods, Volume 141, Issue 2, February 2005, pp. 261-269.
  • Jourdan, D., Ardid, D., Chapuy, E., Le Bars, D., and Eshcalier, A., Effect of analgesics on audible and ultrasonic pain-induced vocalization in the rat. Life Sciences, Volume 63, Number 20,1998, Pages 1761-1768, 1998.
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  • Knutson, B., Burdorf, J., and Panksepp, J., Ultrasonic Vocalizations as Indices of Affective States in Rats,Pyschological Bulletin, Volume 128, Number 6, Pages 961-977.