Effects of Topical Anesthetics on Behavior, Plasma Corticosterone, and Blood Glucose Levels after Tail Biopsy of C57BL/6NHSD Mice (Mus musculus)

Abstract

Tail biopsy is a common procedure that is performed to obtain genetic material for determining genotype of transgenic mice. The use of anesthetics or analgesics is recommended, although identifying safe and effective drugs for this purpose has been challenging. We evaluated the effects of topical 2.5% lidocaine–2.5% prilocaine cream applied to the distal tail tip at 5 or 60 min before biopsy, immersion of the tail tip for 10 seconds in ice-cold 70% ethanol just prior to biopsy, and immersion of the tail tip in 0.5% bupivacaine for 30 s after biopsy. Mice were 7, 11, or 15 d old at the time of tail biopsy. Acute behavioral responses, plasma corticosterone, and blood glucose were measured after biopsy, and body weight and performance in elevated plus maze and open-field tests after weaning. Ice-cold ethanol prior to biopsy prevented acute behavioral responses to biopsy, and both ice-cold ethanol and bupivacaine prevented elevations in corticosterone and blood glucose after biopsy. Tail biopsy with or without anesthesia did not affect body weight or performance on elevated plus maze or open-field tests. We recommend the use of ice-cold ethanol for topical anesthesia prior to tail biopsy in mice 7 to 15 d old.

The completion of the mouse genome sequencing project³⁰ and availability of transgenic technology has revolutionized biomedical research, allowing mice to be used as research models for the study of innumerable diseases and genetic disorders. Currently, mice and rats comprise approximately 95% of research animals in the United States and 75% in the European Union.^31,34 For transgenic mice to be bred for and used in research, the genotype of the individual mouse must be known. Biopsy of the distal tail tip is the most common method of obtaining genetic material for this purpose.⁵ The US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training state that the proper use of animals includes the “avoidance or minimization of discomfort, distress, and pain” and that procedures that cause “more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia.”³² The benefit of providing anesthesia or analgesia to mice undergoing tail biopsy has been debated,² and their use is inconsistent among institutions.

Previous studies in mice (age, 17 d to adult) have documented acute behavioral and physiologic responses of mice to tail biopsy which indicate that the procedure is stressful and may be painful, although the intensity of pain is unknown.^{2,6,8,17,18,22} The behavioral responses of mice to tail biopsy include body flinch, tail flick, and vocalization.^17,18 Changes in behavior can continue for 30 to 60 min after biopsy and include grooming of the tail tip, twitching, and reduced or increased activity.^{6,8,17,18,22,26} In addition to behavioral changes, mice also develop increased heart rate and body temperature after tail biopsy.^2,8 In adult mice, restraint alone increases heart rate and body temperature to a degree similar to the response that occurs after tail biopsy.⁸ Similarly, the increases in the heart rate and body temperature of mice after inhalant anesthesia without tail biopsy last longer than those after tail biopsy without anesthesia.² Optimally, mice undergoing tail biopsy should be afforded pain relief that does not increase the overall stress of the procedure. To our knowledge, the effects of tail biopsy in preweanling mice on postweaning performance in the elevated plus maze and open-field testing paradigms have not been published.

In the current study, the effects of topical 2.5% lidocaine–2.5% prilocaine (LP) cream applied to the distal tail tip at 5 or 60 min before biopsy and of immersion of the tail tip for 10 s in ice-cold 70% ethanol prior to biopsy or in 0.5% bupivacaine for 30 s after biopsy were evaluated in preweanling mice of 3 different age groups. We hypothesized that these treatments would reduce behavioral and physiologic responses and that younger mice would have fewer pain responses to tail biopsy than would their older counterparts.

Materials and Methods

All procedures were reviewed and approved by the IACUC at Wright State University prior to initiation. Animals were housed in an AAALAC-accredited facility, and procedures were completed in accordance with federal guidelines and regulations.

Animals.

Male and female C57Bl/6NHsd breeder mice (Harlan Laboratories, Indianapolis, IN) were housed in polypropylene cages (35 cm × 20 cm × 13 cm; Bryan Research Equipment, Bryan, TX) with Sani-Chip bedding (Harlan Laboratories). Cotton squares (Ancare, Bellmore, NY) and cardboard tubes (Jonesville Paper Tube, Jonesville, MI) were provided as enrichment. Mice were housed as breeding trios, with 1 male and 2 female mice per cage. Pregnant female mice were removed from the cage several days prior to parturition and housed singly until birth. Mice were fed a commercial rodent chow (Harlan Teklad 8640, Madison, WI) free choice, and tap water in water bottles was available at all times. Lighting was maintained on a 12:12-h light:dark cycle, and the ambient temperature maintained at 74 ± 4° F (23.3 ± 2.2° C). At 3 to 5 d after birth, each mouse was tattooed in a unique identification pattern on the palmar and plantar foot pads, and at 7 d, litters were culled to a maximum of 8 pups. Several litters were born with 3 or fewer pups; all pups in these litters were used in terminal experiments at the age of 15 d and were not used for body weight or behavioral observations. Body weights were recorded at 7, 14, 21, and 24 d of age. Weaning occurred at day 20, with all pups in the litter housed together after weaning until study completion, approximately 1 wk later. Moistened rodent chow was provided daily for the first 3 d after weaning.

The health of the colony was monitored by using bedding-contact sentinel mice, which were euthanized and tested quarterly. Parasitology tests were completed in house and confirmed the absence of fur mites and pinworms (Syphacia spp. and Aspiculuris spp., by fecal flotation, tape testing, and direct cecal content exam). Serology by an outside contract laboratory (IDEXX BioResearch) evaluated for antibodies to pathogens including Mycoplasma pulmonis, mouse parvovirus (MPV), minute virus of mice, murine norovirus, mouse hepatitis virus, Theiler murine encephalomyelitis virus, epizootic diarrhea of infant mice virus, and Sendai virus. Serologic evidence of MPV was detected shortly after the study began. The colony was depopulated and a new colony established; however the data collected from 35 mice prior to depopulation are included. Statistical analysis of the parameters deemed most likely to be affected by MPV (blood glucose and plasma corticosterone) was completed with and without including the affected mice, and no differences were found. Therefore data from all mice are included in the results.

Experimental groups.

A total of 250 mouse pups were randomly assigned to 25 experimental groups (Table 1) after the tattoo identification procedure was completed. Each group contained 10 mice (5 female, 5 male). Tail biopsies were collected when mice were 7, 11, or 15 d of age in groups designated for biopsy. Each litter provided mice for multiple experimental groups (with a maximum of 3 mice per litter assigned to the same experimental group), and tail biopsies were obtained on as many as 3 different days for each litter. Tail biopsy anesthesia methods evaluated included topical LP cream (Akorn, Lake Forest, IL) applied 5 min (BxLP₅) or 60 min (BxLP₆₀) prior to biopsy, distal tail dipped in ice-cold 70% ethanol prior to biopsy (BxE), and transected tail tip dipped in 0.5% bupivacaine (Hospira, Lake Forest, IL) after biopsy (BxB).

Table 1.

Identification numbers of experimental groups (n = 10 per group)

Group	Tail biopsy?	Treatment	Age at treatment (d)			Corticosterone and blood glucose levels on day 15	Behavioral testinga
		7	11	15
BxC	Yes	None	1	2	3	4	1–3
BxLP5	Yes	Topical 2.5% lidocaine–2.5% prilocaine at 5 min before biopsy	5	6	7	8	5–7
BxLP60	Yes	Topical 2.5% lidocaine–2.5% prilocaine at 60 min before biopsy	9	10	11	12	9–11
BxE	Yes	Ice-cold ethanol: 10-s dip before biopsy	13	14	15	16	13–15
BxB	Yes	0.5%Bupivacaine: 30-s dip after biopsy	17	18	19	20	17–19
B	No	0.5% Bupivacaine: 30-s dip after mock biopsy			21	22	21
C	No	No handling				24	23
M	No	Maternal separation for 1 h				25

^aTested at 24 to 28 d of age

Mice assigned to the LP experimental groups had LP cream generously applied to the distal 7 to 10 mm of the tail for the required time period. Although 60 min of dermal contact with LP is the manufacturer's recommendation for minor dermatologic procedures, we also evaluated a 5-min contact time in an attempt to maintain economic efficiency in our animal facility. To prevent the topical LP from being removed through grooming by the dam, treated pups in the BxLP₆₀ group were placed in a clean cage with half of the nest and 1 or 2 siblings. For mice that received ice-cold ethanol, ethanol was chilled in a microcentrifuge tube stored in a container of ice chips for at least 5 min prior to use. Mice in the bupivacaine groups were exposed to room-temperature 0.5% bupivacaine in a microcentrifuge tube. This treatment was applied after the biopsy because bupivacaine is not formulated for transdermal delivery. Control groups included biopsy without topical anesthesia (BxC), mock biopsy followed by 30-s bupivacaine dip (B), and no biopsy or handling (C). In addition, a control group of mice that underwent 1 h maternal separation without biopsy (M) was evaluated; mice in this group were marked for identification on the dorsum with a light-colored permanent marker and placed in a clean cage with half of the litter's nest and 1 or 2 siblings (Table 1).

Procedure for tail biopsy.

The home cage housing the dam and litter was moved to an adjacent procedure room for the duration of the biopsy procedure and behavioral observations. All tail biopsies were performed between 1230 and 1600 daily to minimize the effects of circadian rhythm. The mouse scheduled for biopsy or mock biopsy was identified by its tattoo, and a temporary identification mark placed on the dorsum by using a light-colored permanent marker so that the mouse could easily be identified after biopsy without additional handling. The selected anesthesia technique was applied and the mouse gently held on a plastic board, which was premeasured and marked to facilitate precise removal of the distal 5 mm of the tail. The tail was briefly wiped with 70% ethanol and transected 5 mm from the tip by using a no. 10 scalpel blade. The blade was cleaned with ethanol between mice and a new blade used for each litter. After biopsy, bleeding was controlled with direct pressure as needed, after which the mouse was returned to its home cage for behavioral observations.

Behavioral observations.

During the biopsy procedure, episodes of vocalization, movement, urination, and defecation were recorded. Behavior also was recorded at 5, 10, 15, 30, 60, and 120 min after biopsy. At each time point, the mouse was observed for 1 min and its primary behavior recorded as sleeping, nursing, mobile, or grooming tail. In cases where multiple behaviors occurred, secondary and tertiary behaviors were recorded also. In addition, if the dam interacted with the pup during the 1-min observation period, the pup was noted as having received maternal attention, but the specific maternal behavior was not recorded. Dam behaviors toward the pups included grooming, sniffing, carrying, and nursing.

Blood collection and analysis.

Blood glucose and plasma corticosterone were measured only in 15-d-old mice. Mice underwent tail biopsy or mock biopsy, and their behavior was observed for 15 min afterward. After the 15-min observation period, the mice were transferred into an induction chamber, transported to a separate room, narcotized with CO₂ inhalation, and then quickly decapitated with sharp scissors. Trunk blood was collected in a heparinized collection tube and chilled, and blood glucose was measured within 1 h by using a point-of-care glucometer (AlphaTrak2, Abbott Animal Health, Abbott Park, IL). The time from removal of the mouse from the cage to sample collection was recorded. The time (mean ± 1 SD) required for sample collection was 102 ± 23 s; all samples were collected in less than 4 min. The heparinized samples were centrifuged at 385 x g for 15 min, and the plasma was stored at –18 °C until corticosterone analysis. Corticosterone levels were determined in duplicate by using a standard enzyme immunoassay kit (Arbor Assays, Ann Arbor, MI) according to the manufacturer's instructions. The interassay coefficient of variation was calculated to be 10.2%, and the intraassay coefficient of variation was 10.6%.

Postweaning behavior testing.

Mice (age, 24 to 28 d) were evaluated by using the elevated plus maze and open-field tests (Motor Monitor version 3.11, Hamilton Kinder, Poway, CA), as previously described.³⁹ Recent evidence of the effect of human male olfactory stimuli on mouse performance in the open-field test led us to limit testing personnel to women only.⁴² Mice were transported in the home cage to an adjacent testing room and allowed to acclimate for 1 h undisturbed. After acclimation, a single test was administered, with the remaining test completed the following day. Mice were tested for 5 min in the elevated plus maze and for 10 min in the open field. Time spent in the closed arms of the elevated plus maze was evaluated statistically among experimental groups. Time spent in the periphery of the open field and the ratio of distance traveled in the periphery to the total distance were evaluated statistically. Testing occurred between 1300 and 1600 to minimize any potential effects caused by variations in circadian rhythm.

Statistical analysis.

All analyses were conducted in SPSS (version 22, IBM, Armonk, NY). Significance was set as a P level of less than 0.05. Categorical behavioral outcome data were analyzed for each time point with part of the family of χ² tests. The Kendall τ-b test was used to determine whether blood glucose levels were dependent or independent of behavior during biopsy. Correlations between blood glucose levels and body weight were assessed by using standard Pearson r tests. Maternal attention was analyzed by using logistic regression, because the outcome variable was binary. Corticosteroid and blood glucose levels were tested by using one-way ANOVA and, when necessary, Tukey posthoc tests were used to clarify differences. Assumptions of normality were checked; all assumptions for performing ANOVA were met. Results for the postweaning behavior tests underwent ANOVA and posthoc tests when appropriate.

Results

Behavior during biopsy.

Acute behaviors recorded at the time of tail biopsy in mice included movement, vocalization, urination, and defecation. No mouse urinated or defecated at the time of biopsy. When mice in different age groups were compared (Figure 1A), 7-d-old pups vocalized more often than did either 11- or 15-d-old mice (P = 0.001 for both comparisons). In addition, 7-d-old mice moved in response to tail biopsy more often (P = 0.037) than did 11-d-old pups. When various anesthesia groups were compared (Figure 1B), significant differences were also observed for both vocalization and movement. Regardless of age, mice treated with ice-cold ethanol before biopsy were 85% less likely to vocalize (P = 0.015) and 80% less likely to move during biopsy (P = 0.005) than were mice that did not receive anesthesia; no significant differences in vocalization or movement emerged when mice in other anesthesia groups were compared with those that did not receive anesthesia before biopsy (Figure 1B).

Figure 1.

(A) Acute behavioral response to tail biopsy. Mice that were 7 d old moved more often in response to biopsy than did 11-d-old mice (*, P = 0.037) and vocalized more often during tail biopsy than did 11- and 15-d-old mice (†, P < 0.001 and P = 0.001, respectively). (B) Acute behavioral response to tail biopsy in mice treated with different anesthetics compared with those that underwent biopsy without anesthesia. Treatment with ice-cold ethanol prior to biopsy significantly reduced movement (*, P = 0.005), and vocalization (†, P = 0.015) during biopsy when compared with mice undergoing tail biopsy without anesthesia.

Behavior at 5 to 120 min after tail biopsy.

After biopsy, recorded behaviors included sleeping, nursing, moving, and tail grooming. No significant differences in behavior at any time point emerged among 15-d-old mice that experienced biopsy or mock biopsy and different types of anesthesia. However, when mice of different ages and receiving different types of anesthesia were compared, significant differences in behavior occurred during the first 30 min but not at 60 or 120 min after biopsy (Table 2).

Table 2.

Differences in behavior after biopsy of mice compared with all other mice

Age at biopsy (d)	5 min (P = 0.001)	10 min (P = 0.001)	15 min (P < 0.001)	30 min (P = 0.004)
7	BxLP60 ↑ N	BxLP5 ↑ S	BxLP5 ↑ S	No differences
	BxE ↑ S	BxE ↑ S	BxB ↑ S, ↓ N
11	BxC ↑ G	BxB ↑ M	BxLP60 ↑ N	BxLP60 ↑ N
	BxLP60 ↑ S, ↓ M			BxB ↑ S
15	BxLP5 ↑ N	BxC ↓ S	BxLP60 ↑ N	No differences
	BxLP60 ↑ G	BxB ↓ S

↑, significant increase in activity; ↓, significant decrease in activity; S = sleeping, N = nursing, M = mobile, G = tail grooming.

P values for all differences are listed in the column header. See Table 1 for definitions of group designators.

Maternal care.

To determine whether mice were more likely to receive attention if they underwent tail biopsy at a particular age or type of anesthesia, maternal attention was recorded. Age at the time of biopsy was not a significant predictor of maternal attention, but significant differences among anesthesia groups were found at 10 min (P = 0.009) and 15 min (P < 0.001) after biopsy. Mice in the BxLP₆₀ group were more likely to receive attention at 10 min after biopsy than were mice in the BxLP₅ or BxE groups. In addition, the BxLP₅ and BxLP₆₀ groups were more likely to receive maternal attention at 15 min after biopsy compared with mice in which anesthesia was withheld. No other differences were present at 10 or 15 min after biopsy or in any group at 5, 30, 60, or 120 min after biopsy. No incidents of aggressive maternal behavior occurred after biopsy.

Corticosterone and glucose analyses.

Blood glucose and plasma corticosterone levels were compared between groups of 15-d-old mice that underwent tail biopsy and those that did not (Figure 2). Blood glucose levels at the 15-min point were higher in the mice that underwent biopsy without anesthesia (P = 0.02) or with LP for 5 min (P = 0.034) or 60 min (P = 0.004) than in those that received mock biopsy and bupivacaine only. In addition, biopsied mice treated with LP for 60 min had higher (P = 0.036) blood glucose levels than did unmanipulated control mice. The plasma corticosterone levels of BxLP₅ and BxLP₆₀ mice were higher than those in mice that underwent mock biopsy with bupivacaine treatment (P = 0.02 and P = 0.011) and in unmanipulated control mice (P = 0.02 and P = 0.014), respectively. There were no other significant differences in corticosterone or glucose levels between experimental and control groups (Figure 2).

Figure 2.

Plasma corticosterone and blood glucose levels (mean ± 1 SD) at 15 min after biopsy compared with those in mice that received bupivacaine after mock biopsy and in unmanipulated mice. Experimental groups denoted with the same symbol (*, †, α, and β) are significantly different (P < 0.05) than nonbiopsied control groups, but this does not infer significance between the experimental groups that received biopsies. For definitions of experimental groups, see Table 1.

To determine the influence of the 1-hour period of maternal separation experienced by pups in the BxLP₆₀ groups, we included a control group comprising mice that were separated from their dams for 1 h and then underwent immediate blood collection (that is, no tail biopsy). Mice that underwent maternal separation without biopsy showed differences in both blood glucose (P = 0.004) and corticosterone (P = 0.004) compared with pups that underwent maternal separation followed by biopsy (BxLP₆₀). However, these parameters did not differ between pups that underwent maternal separation only and mice that did not receive a tail biopsy (Figure 3).

Figure 3.

Plasma corticosterone and blood glucose levels (mean ± 1 SD) 15 min after biopsy or maternal separation. *, †, α, and β indicate significant differences (P = 0.004, 0.004, 0.036, and 0.014, respectively) between groups identified with the same symbol. For definitions of experimental groups, see Table 1.

As noted previously, vocalization was the only behavior that differed significantly according to both mouse age and type of anesthesia used. The mean blood glucose for all mice in the study was 162 ± 30 mg/dL. Using a χ² test, we found that the blood glucose levels of mice that vocalized during biopsy were significantly (P = 0.014) different. Specifically, mice whose blood glucose concentration was between 125 and 150 mg/dL vocalized less often, whereas pups with a blood glucose of 176 to 200 mg/dL vocalized more often (Figure 4).

Figure 4.

Vocalization response during biopsy compared with blood glucose level in 15-d-old mice. Fewer mice with blood glucose levels of 125 to 150 mg/dL and more of those with blood glucose levels of 176 to 200 mg/dL vocalized during tail biopsy (*, P = 0.014).

Body weight and postweaning behavior tests.

Body weight analysis of mice between 7 and 24 d of age revealed no significant difference in growth in any experimental group (P = 0.75). Mice that underwent tail biopsy with or without anesthesia grew at the same rate as did mice that were not biopsied. Similar to the results of body weight analysis, performance in the open-field test or elevated plus maze did not differ among any experimental groups (P ≥ 0.43). Mice spent the majority of their time in the closed arms of the elevated plus maze (85%) and along the periphery of the open field chamber (89%), with no differences among groups. In addition, there were no intergroup differences in the ratio of the distance traveled along the periphery of the open field to the total distance traveled (data not shown).

Discussion

Tail biopsy is a procedure commonly performed on mice. Previous studies have evaluated the behavioral and physiologic responses of mice after tail biopsy and explored the effects of various anesthetic techniques.^2,6,17,22,26 Several studies document the lack of benefit of inhalant anesthetics such as isoflurane and methoxyflurane when used for tail biopsy or the presence of prolonged behavioral effects after the use of inhalant anesthetics for this purpose.^2,6,17,22 In addition, topical vapocoolants such as ethyl chloride spray are reported to cause swelling, bleeding, and erythema when applied prior to toe amputation³³ and to have inflammatory effects in the dermis and subcutis when used prior to tail biopsy.⁶ We evaluated the ability of topical anesthetics to reduce pain and stress of tail biopsy without causing physical trauma or long-term effects. When a topical anesthetic was found to be ineffective, it was beyond the scope of this study to investigate the underlying cause of this failure. Topical anesthetics can be removed after application by the mouse or through environmental contact, and this removal may have contributed to the ineffectiveness of certain agents.

Similar to previous studies,^17,18 we found vocalization and body movement to be the most common behavioral responses to tail biopsy. To explore whether vocalization was an indicator of pain and stress or simply a reflexive response, we compared the blood glucose levels measured 15 min after biopsy with this behavior. Mice that vocalized at the time of biopsy were more likely to have higher blood glucose levels 15 min after biopsy than were mice that did not vocalize. This finding supports vocalization during tail biopsy as a possible indicator of pain or stress in mice. We considered the possibility that the act of vocalization (and not pain associated with tail biopsy) was the stimulus for the elevations in blood glucose. Glucose is regulated by glucocorticoids through a variety of stimulatory, permissive, and inhibitory actions.²⁴ Glucocorticoids are secreted in response to stress and have the overall function of maintaining adequate blood glucose for the organism to respond appropriately to the stressor.²⁴ We feel it is most likely that blood glucose elevations occurred as a response to the tail-biopsy procedure and not as a response to vocalization. By exposing the tail tip to ice-cold ethanol prior to biopsy, we significantly reduced vocalization and movement responses. This decrease in behaviors was the best evidence that dipping the tail tip in ice-cold ethanol was most efficacious at reducing the acute pain of tail biopsy.

Several changes in behavior occurred after the biopsy procedure, but the importance of each is a matter of debate. For example, the increased tail grooming observed 5 min after biopsy in 11-d-old mice that were biopsied without anesthesia likely indicates pain. In contrast, we suspect that other changes, such as increased nursing in mice biopsied after 60 min exposure to LP cream, may not be due to pain. These mice experienced a 1-h maternal separation after LP was applied to the tail tip, which we deemed necessary to prevent the dam from removing and ingesting the anesthetic cream after application. Increased maternal care after separation has been documented as a mechanism of attenuating the stress associated with maternal separation in preweanling mice,²⁷ and the increase in nursing behavior may have been maternally driven. Missed nursing opportunities and a potential reduction in pup body temperature during separation may also have influenced this behavior.

We observed changes in mobility, nursing, and sleeping behavior as long as 30 min after tail biopsy in several experimental groups. Decreased mobility is considered a sign of pain in adult mice.^9,23,35 Previous publications report that 20-d-old mice showed reduced climbing or general locomotor behavior at 30 min after tail biopsy,⁴¹ and adult mice also showed reductions in locomotor activity.¹⁷ However, studies using telemetry units report increased mobility in adult mice after tail biopsy.^2,8 In those studies, activity after tail biopsy was increased compared with baseline activity, but when compared with that in mice that experienced restraint or anesthesia alone, activity was indeed lower. In the current study, the behavior of 15-d-old mice that underwent mock biopsy generally transitioned from mobile to sleeping or nursing within 30 min of the procedure, and many groups of P7 mice showed increased sleeping behavior during the first 15 min after biopsy. We cannot preclude the possibility that systemic absorption of some local anesthetic agents contributed to the observed increases in sleeping behavior. The significance of these changes is unknown, and additional research is warranted in this area.

Previous studies have examined the physiologic stress response after tail biopsy in mice.^2,8 This response involves concurrent activation of the sympathetic–adrenal–medullary axis and the hypothalamic–pituitary–adrenal (HPA) axis; activators are nonspecific in nature and may be psychologic or physical.^16,38 Physiologic changes reported after tail biopsy in male HanIbm:NMRI mice included elevations in heart rate and body temperature,^2,8 which can both occur during stress. Restraint and tail biopsy both caused significant elevations in heart rate that persisted for 1 h.⁸ A related study found that elevations in heart rate and body temperature persisted for as long as 4 h in mice undergoing inhalant anesthesia without tail biopsy.² The heart rate elevations observed in those studies likely reflect concurrent activation of the sympathetic–adrenal–medullary axis and withdrawal of parasympathetic influence.⁷ Activation of the HPA axis in rodents causes the secretion of the glucocorticoid corticosterone from the adrenal cortex.¹¹ Corticosterone levels have thus far not been reported after tail biopsy in mice, but elevations in corticosterone have been well documented after surgery and tail bleeding.^13-15,43,44 Surgical procedures including laparotomy and catheterization cause acute elevations in corticosterone, and these elevations can be ameliorated through the administration of analgesics, such as buprenorphine.^13-15,43 This effect suggests that pain is a stressor that activates the HPA axis during and after surgery and likely after tail biopsy.

We measured plasma corticosterone 15 min after tail biopsy and found significantly higher levels in mice undergoing tail biopsy at 5 min or 60 min after the application of LP to the tail than in mice that did not receive a biopsy or those that underwent mock biopsy. In contrast, there was no difference in corticosterone levels of mice that were biopsied and treated with ice-cold ethanol or bupivacaine when compared with mice that received mock biopsies or that were not biopsied. These results indicate that the use of LP prior to biopsy did not prevent activation of the HPA axis; however dipping the tail in ice-cold ethanol before biopsy or providing bupivacaine after biopsy clearly did. This finding is consistent with our behavioral observation that ice-cold ethanol prevented vocalization and movement during biopsy. Although dipping the tail tip in bupivacaine after biopsy did not prevent the acute behavioral response, in general, mice ceased struggling once the transected tail tip was placed in bupivacaine. The sustained anesthetic effects of bupivacaine may have contributed to the lack of HPA activation. Our findings that ice cold ethanol and bupivacaine prevent corticosterone elevations after tail biopsy strongly support the probability that tail biopsy in preweanling mice is a painful procedure.

The acute physiologic effects of corticosterone promote survival of the individual; enhanced production of glucose by the liver is one such effect.¹¹ Elevated blood glucose in a stressful situation ensures an adequate energy source for muscles in case an altercation or rapid retreat becomes necessary. Elevations in blood glucose of 4-wk-old male and female C57BL/6J mice at 15 min after tail biopsy have previously been documented, although a control group for determining the effects of handling or restraint without biopsy was not included.²⁶ In our study, handling during mock biopsy did not affect blood glucose levels. Dipping the tail tip in ice-cold ethanol prior to biopsy or in bupivacaine after biopsy were the only treatments evaluated that prevented elevations in blood glucose. The apparent benefit of bupivacaine is consistent with a previous study that found immersion of the tail tip in bupivacaine reduced tail-grooming behavior in preweanling mice after biopsy,²² however, to our knowledge, the current report is the first to describe the benefits of using topical ice-cold ethanol prior to tail biopsy.

Long-term effects of tail biopsy in mice have been explored.^29,41,46 A significant difference in thermal nociception in the hindpaws was present in 129S6, but not C57BL/6J, mice 3 wk after tail biopsy performed at weaning, although there was no difference in other tests of nociception.²⁹ The mice in that study²⁹ did not receive anesthetics or analgesics at the time of biopsy, and whether their provision would have influenced results is unknown. Long-term hyperalgesia in the hindpaw and tail was noted for as long as 5 wk after removal of the distal 2.5 cm of the tail in adult C57BL/6J mice in another study;⁴⁶ the amount of tissue collected in that study is a significantly larger proportion of tail than is typically removed during tail biopsy for genotyping or research purposes, including the research reported in the present manuscript. A third study measured body weight, anxiety behavior, and balance in mice several weeks after tail biopsy in 12- to 20-d-old mice and did not find any effects.⁴¹ The present study is the first to measure the effect of preweaning tail biopsy with topical anesthesia on postweaning performance in the elevated plus maze and open field test paradigms, and we found no significant differences among experimental groups. Finally, we measured the body weight of mice during a period of rapid growth (days 7 through 24) and again found no differences among experimental groups. Mice undergoing tail biopsy with or without topical anesthetics gained weight similarly to mice that did not receive a tail biopsy.

The International Association for the Study of Pain defines pain as an “unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.”²¹ Pain occurs when a noxious insult stimulates nociceptor neurons and is transmitted to the CNS for processing.¹⁰ Mineralized bone is innervated with a rich network of sensory and sympathetic fibers, which allow nociception to readily occur.²⁵ The provision of anesthesia or analgesia has been suggested in mice undergoing tail biopsy, beginning at the age of 17 d when vertebral maturity of the distal tail is likely .¹⁸ However, another study found nociceptor fibers present in the bones of the distal tail in mice as young as 7 d, making nociception possible much earlier.⁴⁰ Our finding that 7-d-old mice vocalized significantly more often than did 11-d-old or 15-d-old mice at the time of tail biopsy supports the possibility of pain perception as young as 7 d. However, a review of neonatal pain perception development in rats suggests that CNS processing of nociceptive signals in the brain does not develop until 12 to 14 d of age.^3,4,12,40 It is perplexing that vocalization in response to tail biopsy occurred most often in 7-d-old pups. This finding may be due to the larger proportion of tail that was removed during biopsy from these mice when compared with that removed from the older mice. Interestingly, similar findings occurred in another study in which 75% of 7-d-old mice and none of the 17-d-old mice vocalized in response to toe clipping without anesthesia.³³ Neonatal hypersensitivity is recognized in human infants,¹ and a potential physiologic basis has been identified in mice.⁴⁵ The increased vocalizations at the time of biopsy in 7-d-old mice may represent hypersensitivity to pain at this age, although further investigations are warranted.

Several challenges were encountered during the course of this study. An outbreak of MPV in the colony housing room resulted in culling and replacement of the colony. At the time of disease outbreak, approximately 70 mice had undergone experimental procedures. We attempted to replace these study animals by increasing breeding efforts in the new colony room but were successful in replacing only half. Data collected from mice potentially infected with MPV did not include performance on the elevated plus maze or open-field tests. Statistical analysis of corticosterone and blood glucose data with and without the infected mice did not reveal a difference; therefore we do not believe that MPV affected our study. The second challenge we encountered involved normal HPA physiology of neonatal mice. During the perinatal period, rodents experience reduced sensitivity of the HPA axis to even mild stressors.^36,37 In mice, this stress-hyporesponsive period occurs from birth to approximately day 12 and is characterized by very low basal levels of corticosterone and a failure to release corticosterone in response to stressors.³⁷ This characteristic restricted our ability to use corticosterone as a physiologic measure of stress in mice undergoing tail biopsy to our oldest age group, 15-d-old mice.

Our final challenge involved application of LP cream to tails for 60 min prior to biopsy. These mice were isolated from the dam for 60 min prior to biopsy. We were concerned that this maternal separation would alter levels of plasma corticosterone, blood glucose, or both. Although maternal separation during the neonatal period is a validated model of stress in some rodents,^16,19 even 3-h periods of daily maternal separation did not induce long-term anxiety or depressive behaviors in mice.^27,28 A previous study documented increased maternal care after separation, which may have attenuated the stress response.²⁷ The presence of littermates has been shown to buffer the corticosterone response to stressors in preweanling rats.²⁰ When maternal isolation was necessary in the present study, isolated pups were always maintained with at least one littermate. Our study design included a control group of mice that underwent a 1-h period of maternal separation followed by immediate blood collection for the measurement of corticosterone and glucose; there were no significant differences in corticosterone or glucose of mice that were separated from the dam for 1 h and did not undergo biopsy when compared with mice that remained with the dam and did not receive a biopsy. However, the blood glucose and corticosterone levels of mice in the BxLP₆₀ group, in which a 1-h period of maternal separation occurred prior to biopsy, were significantly higher than those in the group which underwent a period of 1 hr maternal separation without biopsy, and the group which remained with the dam but did not receive a biopsy. Maternal separation did not have a significant influence on glucose and corticosterone concentrations; we suspect that this result was due to the social buffering effect of the littermate(s). We recorded maternal care at each time point after biopsy and found that mice that experienced maternal separation followed by biopsy did indeed receive significantly higher levels of maternal care at 10 and 15 min after biopsy, which may have further prevented increases in corticosterone.

In conclusion, the elevations in corticosterone and blood glucose that we noted after tail biopsy confirm that the procedure is stressful to preweanling mice. Attenuation of corticosterone and glucose elevations through topical treatment with ice-cold ethanol or bupivacaine at the time of biopsy, along with behavioral observations, support the inference that tail biopsy without anesthesia is painful. Identifying a suitable anesthetic or analgesic that effectively reduces the pain of tail biopsy without causing tissue trauma or prolonged behavioral or physiologic responses is challenging. Although bupivacaine was effective at preventing HPA activation, its use is suboptimal because it does not reduce the acute pain response to the procedure. We found that dipping the tail tip in ice-cold ethanol for 10 s prior to tail biopsy reduced the acute behavioral and physiologic responses to biopsy without causing long-term alterations in growth or anxiety behavior. We recommend the use of ice-cold ethanol prior to tail biopsy in mice 7 through 15 d of age. Investigators and IACUC should be aware that this range includes ages at which neonatal hypersensitivity may occur and at which nociceptive processing likely occurs, underscoring the importance of anesthesia for tail-biopsy procedures in preweanling mice.