Retrospective Evaluation of Nail Trimming as a Conservative Treatment for Ulcerative Dermatitis in Laboratory Mice

Abstract

Ulcerative dermatitis (UD) is an idiopathic disease that affects C57BL/6 mice and those having a B6 background. The hallmark of UD is pruritus, which leads to self-mutilation and epidermal ulceration typically in the intrascapular region. Although several treatments for UD have been published, some involve the use of pharmacologic agents that might confound research results. In this retrospective study, we evaluated nail trimming to determine whether this conservative treatment approach improved the resolution rate of UD at our institution compared with that of untreated mice or those that received oral or topical antibiotics. Our findings show that the incidence of resolution of UD was significantly greater and that the time to resolution was shorter in mice treated with nail trimming compared with other groups. These findings support the use of nail trimming as an effective conservative treatment option for UD in B6 mice.

Ulcerative dermatitis (UD), also known as black mouse dermatitis, is an idiopathic disease that occurs in C57BL/6 mice and those with a B6 background. The strain prevalence implicates a genetic link,^3,10 with studies citing the B6 strain-specific Th1-driven immune response as a likely contributor to the pathogenesis of UD.^1,4,10 In addition, primary follicular dystrophy has been implicated in the C57BL/6J substrain.²³ Overall, the evidence supports a multifactorial etiology, with numerous risk factors—compulsive behavior,⁵ diet,^11,18,26 sex,^1,10,22 age,¹⁰ environmental factors (such as humidity and season),^10,22 and fur mite infestation³—linked to the development of UD. Clinical signs of UD include moderate to severe pruritus that results in self-trauma–induced epidermal ulceration.⁹ Lesions typically present on the dorsal intrascapular area; however, lesions may present elsewhere on the body, including the face, ears, or on top of the head. Although UD is rarely life threatening except in very severe cases, the intense pruritus, self-mutilation, and open wounds present a significant concern regarding the wellbeing of the affected mice and may necessitate euthanasia prior to study endpoints. Furthermore, untreated mild to moderate UD has the potential to confound a wide range of research studies. Inflammation from lesions associated with chronic UD can cause splenomegaly,¹³ lymphadenopathy, reactive amyloidosis, and systemic alterations in cytokines and inflammatory cell populations.¹⁹ Because pruritic mice exhibit abnormal locomotor activity and nesting behaviors,⁶ they may be poor performers in behavioral tests. Finally, pruritic behavior likely affects food and water consumption, but no study has explored this question to date.

Whereas dermatitis caused by epidermal trauma from fight wounds, excessive barbering, or ectoparasite infestations often responds to treatment of the primary cause, idiopathic UD is refractory to many treatments. Published approaches to treat or prevent UD include oral supplementation with vitamin E,^13,14 topical treatment with antibiotics or antiseptics,¹⁰ maropitant citrate,²⁵ ibuprofen,⁶ calorie restriction,^2,20,24,26 and mineral supplementation.⁸ Although the efficacy of several of these approaches appears promising, pharmacologic treatment, dietary supplements, and calorie restriction may not always be feasible in situations where such interventions might affect a research study. At our institution, casual observation suggested that the resolution rate of UD cases did not increase markedly despite treatment with topical or oral antibiotics, and, in most cases, euthanasia was elected once clinical signs began to affect the wellbeing of the mouse.

A recent epidemiologic survey of diseases in mice at a large academic research institution found that dermatitis was the most common clinical disease entity, occurring at 3.10 cases per 1000 cages each month.¹⁵ Considering the widespread use of B6 mice and the prevalence of UD, the lack of an effective, conservative treatment for UD is frustrating to both animal care professionals and investigators. Therefore, in an effort to explore conservative treatment modalities for UD, hindlimb nail trimming was performed in a number of UD cases at our institution (Figure 1), because, at the time this article was submitted, the efficacy of nail trimming as a UD treatment had not been published and had only been addressed anecdotally and in poster presentations.^17,21 Our initial observations were that nail trimming appeared to be effective in decreasing self-mutilation and seemed to increase the frequency of resolution of UD compared with other treatments used at our institution. To substantiate our observations, we retrospectively reviewed medical records from our institution to compare the outcome of UD cases treated with nail trim compared with other treatment methods, including oral antibiotics, topical antibiotics, and observation.

Figure 1.

Technique used for nail trimming. (A) Materials used include a pet nail trimmer and a 50-mL conical tube. Note the air holes at the end of the tube. (B) Mouse is placed head first into the conical tube, to permit easy access to the rear toenails. (C) Once the mouse is restrained within the conical tube, the dominant hand can be used to carefully trim the sharp point of each nail.

Materials and Methods

Animals.

This study included clinical cases of UD that occurred from 2010 through 2013 in 6 vivaria at our institution. All mice were on active protocols approved by the University of Missouri–Columbia IACUC. Mice in these facilities were maintained in either conventional or barrier settings and were singly or group-housed in commercially available IVC or static cages according to IACUC-approved housing densities. Cage size varied from standard shoebox-size (29 × 19 × 13 cm) mouse cages to larger rat-sized cages (59 × 31 × 23 cm). Mice were provided with paperchip bedding, corncob bedding, or aspen shavings and standard enrichment items (cotton squares, crinkled paper, hut). All mice had unrestricted access to a standard commercially available rodent chow (LabDiet, St Louis, MO). Mice in conventional settings were provided acidified water. Mice in barrier facilities were maintained under SPF conditions, all materials (water, feed, cages, bedding, enrichments) were autoclaved or irradiated prior to entry into the animal room, and all mouse manipulations were performed in class II biologic safety cabinets (A1, A2, B1, or B2). All mice were maintained on controlled light cycles, ranging from 10 to 14 h of light.

The rodent health surveillance program at our institution primarily uses soiled-bedding sentinels (CD1, Charles River Laboratories, Wilmington, MA) to perform quarterly testing for ecto- and endoparasites and pathogenic and opportunistic enteric and respiratory bacteria (Salmonella spp., Helicobacter spp., Pseudomonas aeruginosa, Mycoplasma pulmonis, Pasteurella pneumotropica) by using microbiologic and molecular methods. Routine monitoring for ectoparasites, including fur mites of the genera Myobia, Myocoptes, and Radfordia, is performed by using postmortem direct pelage examination. Sentinel mice also are tested for antibodies against the following agents: mouse hepatitis virus, mice minute virus, mouse parvovirus, Sendai virus, Mycoplasma pulmonis, Theiler murine encephalomyelitis virus, epizootic diarrhea of infant mice virus, pneumonia virus of mice, reovirus type 3, lymphocytic choriomeningitis virus, mouse adenoviruses, ectromelia virus, and polyoma viruses. Although our institution maintains colonies that have been confirmed free of all the aforementioned agents, some colonies have known endemic infections, a recent history of eradicated infection, or are undergoing efforts to eradicate infection. To limit the transmission of infectious agents from colonies of a lower health status, colonies of similar health status routinely are housed together. In addition, at this institution, colonies at a lower health status are allowed certain opportunistic bacterial agents, specifically Helicobacter spp. and Pasteurella pneumotropica.

Because this retrospective study focused on the evaluation of UD treatment methods, identifying the health status of each mouse included was beyond the scope of the study. However, because infestation with ectoparasites might predispose mice to developing dermatitis, medical records were reviewed closely to identify dermatitis cases originating from colonies with ongoing infestations with ectoparasites, but no such cases were found. In addition, antemortem testing for ectoparasites (PCR analysis, tape testing, fur plucks) on selected cases of UD-affected mice was performed by veterinary staff. However, none of the cases included in the current study were positive for ectoparasites.

Medical record review and inclusion criteria.

We performed an initial review of all murine medical records dated 2010 through 2013 maintained by our institution to select those that listed “ulcerative dermatitis,” “UD,” “dermatitis,” or “black mouse dermatitis” as a diagnosis or differential diagnosis. A total of 330 cases meeting this criterion were entered into a spreadsheet and the following information (when noted in the record) was recorded: date of first evaluation, presumptive diagnosis, treatment implemented, description of the lesion, other concurrent disease, sex and age of the mouse affected, identifying information for the affected mouse, strain, mutation, principal investigator, housing facility, cage number, date of last observation and outcome (euthanasia or resolution of clinical signs), result of antemortem ectoparasite testing, and any additional relevant comments.

To further refine the search, these initial 330 cases were evaluated by a reviewer blinded to treatment and final outcome to exclude cases containing the following in the medical record: noted absence of pruritus (determined clinically as a lack of scratching behavior observed during cage-side examination or lack of evidence of excoriation) during initial case assessment (n = 11), euthanasia due to an unrelated condition (n = 14), and spontaneous death (n = 6). In addition, because we sought to determine whether nail trim was effective compared with other stand-alone treatments, we excluded cases in which multiple treatments were used (n = 9). Finally, only mice that could be followed for at least 3 d after treatment initiation were included, given that this time frame afforded reasonable assessment of treatment efficacy; mice that were euthanized before 3 d had elapsed were excluded from the study (n = 56).

A total of 234 UD cases met the final inclusion criteria for the study. All of these cases involved weaned B6 mice or those with a B6 background. Each case represents one mouse affected with UD.

Veterinary care.

At our institution, mice requiring veterinary care are identified by animal care or research personnel, and the facility veterinarian is notified. The veterinarian creates a medical record in the traditional SOAP format, recording subjective and objective findings, assessment (diagnosis or differential diagnoses), treatment and diagnostic plans, and treatments administered. A date for follow-up and reassessment of the case is selected at this time. Generally, rechecks are scheduled to occur at least weekly. However, at the discretion of the veterinarian managing the case, recheck examinations may occur more or less frequently, depending on condition severity or due to factors unrelated to the condition. A new entry following the SOAP format is made in the medical record at the time of each follow-up examination.

In 26% (60 of 234) of UD cases in this study, a fur pluck or tape impression of the affected area was collected for microscopic examination to rule out ectoparasites, specifically fur mites. One mouse was swabbed for fur mite testing (Myobia, Myocoptes, and Radfordia) by PCR analysis at a diagnostic laboratory (IDEXX BioResearch, Columbia, MO). These antemortem diagnostic samples typically were collected during the initial case assessment. None of the cases in this study tested positive for ectoparasites.

Treatment groups.

UD cases meeting the inclusion criteria underwent observation or treatment with oral antibiotic, topical antibiotic, or nail trimming; cases under observation received no treatment other than monitoring by a veterinarian until resolution or euthanasia. Prior to the initiation of antibiotic treatments, principal investigators were contacted to confirm that antibiotic therapy would not interfere with their research goals. Enrofloxacin-medicated (Baytril, 100 mg/mL, Bayer HealthCare Animal Health Division, Shawnee Mission, KS) drinking water was prepared to a final concentration of approximately 0.20 to 0.25 mg/mL water (that is, approximately 60 to 70 mg/kg, assuming a daily consumption of 6 to 7 mL of water). This treated water was replaced with a freshly prepared solution at least every 5 d until resolution or euthanasia. Cases treated with topical antibiotic received an application of a generic triple-antibiotic ointment to the UD lesion once daily until resolution or euthanasia. Mice treated with nail trimming underwent nail trimming by a veterinarian using iris scissors or pet nail trimmers, which were used to clip the point of each hindlimb nail distal to the quick while the animal was restrained within a 50-mL conical tube (Figure 1). Once nail trimming was initiated, subsequent nail trimming was at the discretion of the veterinarian managing the case, who evaluated the animal at each recheck examination to determine whether additional nail trims were necessary in light of the clinical presentation and the sharpness of the hindlimb nails.

Outcomes.

In this study, the outcome for each case was categorized as either resolution or euthanasia. Cases were declared as resolved only after assessment by a veterinarian; the declaration of a case as resolved was left to the discretion of the veterinarian. For euthanized cases, an IACUC-approved method of euthanasia, according to then-current American Veterinary Medical Association guidelines, was performed by trained animal care personnel after recommendation by a veterinarian or at the request of the principal investigator. However, research staff frequently euthanized affected mice prior to veterinary recommendation, usually due to a perceived lack of clinical improvement, poor prognosis, or loss of suitability of affected mice for the primary research study. For the purposes of the current study, all euthanized cases were grouped together, regardless of whether the mice were euthanized by veterinary or animal care staff or by research personnel.

In addition, 43 cases were classified as lost to follow-up, in light of unknown outcomes (that is, mouse not found in cage, outcome not noted in medical record).

Statistical analysis.

All statistical analyses were performed by using Prism 5 (GraphPad, San Diego, CA). Time-to-event analysis, using resolution as the event of interest, was performed by using the log-rank (Mantel–Cox) test for overall and pairwise comparisons of data curves. Cases that were lost to follow-up or were euthanized, either for experimental reasons or for reasons related to UD, were censored. Data are reported as mean ± SEM. Statistical significance was defined as a P value of less than 0.05.

Results

A total of 234 UD cases fulfilled the inclusion criteria for this study: 74 observation cases, 125 cases treated with nail trimming, 23 treated with oral antibiotics, and 12 treated with a topical antibiotic. Of these cases, resolution was seen in 74 of the 125 nail trim cases, 10 of the 74 observation cases, 3 of the 23 oral antibiotic cases, and 3 of the 12 topical antibiotic cases.

Resolution differed significantly (P < 0.0001, log-rank test) among treatment groups (Figure 2). Pairwise comparisons showed that resolution was significantly greater in the nail trim treatment group when compared with observed cases (χ² = 20.51, P < 0.0001), oral antibiotics (χ² = 13.02, P = 0.0003), and topical antibiotics (χ² = 9.78, P = 0.0018). Resolution did not differ between cases treated with oral antibiotics compared with observation only (χ² = 0.59, P = 0.4437) or topical antibiotics (χ² = 0.04, P = 0.8517) or between topical antibiotics compared with observation (χ² = 1.93, P = 0.1651).

Figure 2.

Kaplan–Meier estimates of resolution of ulcerative dermatitis in mice treated by using 1 of 4 methods: nail trim (n = 125), observation (n = 74), oral antibiotic (n = 23), and topical antibiotic (n = 12). Censored subjects are indicated on each curve by using the corresponding tick mark. Time to resolution (log-rank test) was significantly greater in the nail trim treatment group when compared with observed cases (×, P < 0.0001), oral antibiotics (+, P = 0.0003), and topical antibiotic (#, P = 0.0018).

The median resolution time (time at which 50% of resolved cases had reached resolution) was 17 d for nail trim cases and 36 d for observation cases (Figure 2). The earliest resolution was seen by 3 d in the nail trim treatment group, 9 d in the oral antibiotic treatment group, 14 d in the observation treatment group, and 21 d in the topical antibiotic treatment group.

The typical progression of UD in a mouse treated with nail trim is shown in Figure 3. Of the 74 UD cases that resolved after nail trimming, more than half (66%; 49 of 74 cases) resolved after receiving 1 nail trim (mean time to resolution, 12.06 ± 0.75 d). Less than 25% of cases received 2 nail trims before resolution (mean time to resolution, 16.72 ± 1.43 d), and approximately 7% of cases (5 of 74) resolved after 3 nail trims (mean time to resolution, 24.0 ± 2.50 d), 1% after 4 trims (1 of 74; resolved at 25 d), and 1% after 5 trims (resolved at 46 d). When multiple nail trims were administered, they were repeated at an interval of about 5 to 7 d.

Figure 3.

Resolution of ulcerative dermatitis in a C57BL/6 mouse treated with nail triming. All images are of the same mouse. On day 1, the mouse presented with a pruritic, ulcerated intrascapular lesion; hindlimb nail trimming was performed. By day 4, the mouse was no longer pruritic, and the skin showed signs of healing. By day 7, the ulcerated skin appeared to be healed, and hair regrowth was noticeable. On day 16, hair regrowth was nearly complete.

Grouping cases of UD according to sex showed that 35% (82 of 234) of affected mice were male and 57% (133 of 234) were female; sex was not noted for 8% (19 of 234) of cases.

Discussion

UD is a disease that occurs commonly in B6 mice and mice with a B6 background. This retrospective study revealed that at our institution, UD cases treated with nail trimming had a greater resolution rate and more rapid resolution time compared with all other treatment measures (observation, and oral and topical antibiotics). These findings support the use of nail trimming as an effective, nonpharmacologic treatment for UD that improves animal wellbeing by more rapidly alleviating clinical signs in affected mice.

The success of nail trimming is noteworthy, considering the broad range of research models that use the B6 mouse. Although it is unclear how nail trimming contributes to the resolution of UD, we speculate that this technique decreases the self-induced trauma caused by continuous scratching and breaks the scratch–itch cycle. Before implementation of nail trimming at our institution, UD cases that were not euthanized shortly after diagnosis were monitored but typically left untreated because of concerns that treatment with therapeutic agents, such as systemic antibiotics, ibuprofen,⁶ and maropitant citrate,²⁵ could potentially confound research studies. The current study confirmed our observation that the few cases that were treated with either oral or topical antibiotics did not fare better than untreated cases. Although nail trimming may temporarily affect animal wellbeing by preventing mice from effectively scratching their pruritic lesions, this study provides evidence that nail trimming results in improved overall animal wellbeing by preventing self-trauma and allowing for quicker resolution of UD.

A few UD cases in this study were treated with antibiotics. A recent study has found that antibiotic dosing in the drinking water of mice does not result in plasma antibiotic concentrations high enough to be effective against most bacterial pathogens.¹⁶ This result suggests that even in UD cases with a bacterial component, treatment failure may have played a role in the lack of significant clinical improvement in those cases treated with oral antibiotics. In cases treated topically, antibiotic contact time may have been insufficient as mice were observed quickly grooming the ointment off shortly after application. Given the growing evidence that changes in microbiota can alter model phenotypes, the use of antibiotics to treat experimental animals may decline further.

Although our study was limited to comparing nail trimming with other UD treatments typically used at our institution, we are confident that animal care professionals at other institutions will find nail trimming to be both cost- and time-effective when compared with other UD treatments. Furthermore, nail trimming is a simple procedure that can be taught to animal care technicians or research staff (Figure 1). The materials needed to perform this technique are readily available and inexpensive. With the appropriate tools and technique, an experienced person can perform the entire procedure in less than 2 min.

This retrospective study has been informative; however, future prospective studies are needed to further evaluate nail trimming. Although we attempted to evaluate recurrence rates associated with each treatment method, we were unable to consistently track individual mice over time. Unique identifying information for every mouse in this study was not always available, either due to loss of identifying markers (that is, ear tags) or a lack of an established identification system. In addition, future studies are needed to identify why a female predilection appears to exist: in the current study, more than half (57%; 133 of 234) of UD cases occurred in female mice. Although we were unable to evaluate this trend statistically because census information for our rodent population was not available, our observations concur with previous reports that found an increased prevalence of UD in female B6 mice.^1,10,22 One study also has identified an increased incidence of UD that coincides with increasing age,¹⁰ but we were unable to evaluate an age-effect in our study due to the large number of medical records that did not contain detailed date of birth information. Interestingly, there is evidence that nail growth in mice is accelerated in aged animals¹² and is affected by environmental factors, such as ambient temperature and diet.⁷ Although nail growth and UD may not be directly related, nail growth may be an important factor to consider in UD studies evaluating nail trimming.

In addition, future studies should clarify whether mice presenting with both early and more advanced UD respond similarly to nail trimming. In this study, more than 66% of resolved UD cases treated with nail trims resolved, on average, within 12 d after a single nail trim. Cases that received 3 or more nail trims were associated with a longer time to resolution (24 d or more). The extended time to resolution seems to suggest that cases treated with 3 or more nail trims represent mice with more advanced UD than that of animals requiring only one nail trim. A quantitative UD scoring system, such as those published previously^6,9 was not in use at the institution at the time of this study but would be beneficial in future studies, especially at institutions where multiple people attend clinical cases. In addition to providing a system for objective evaluation of treatment responses associated with various treatment methods, a scoring system could be used to standardize criteria for UD outcomes (resolution or euthanasia) and to create a schedule for reassessment of ongoing UD cases on the basis of lesion severity.

Because we evaluated nail trimming as a treatment for UD only at our own institution, we recognize that the effectiveness of nail trimming might vary at other institutions due to many factors, including (but not limited to) husbandry practices; experimental, environmental, or breeding stresses; and host-dependent factors. We hope that this report will provide animal care professionals and rodent researchers with an effective and economical treatment option for UD that improves animal wellbeing in B6 mice and enables better use of research resources.