Factors influencing adverse skin responses in rats receiving repeated subcutaneous injections and potential impact on neurobehavior

Abstract

Repeated subcutaneous (s.c.) injection is a common route of administration in chronic studies of neuroactive compounds. However, in a pilot study we noted a significant incidence of skin abnormalities in adult male Long-Evans rats receiving daily s.c. injections of peanut oil (1.0 ml/kg) in the subscapular region for 21 d. Histopathological analyses of the lesions were consistent with a foreign body reaction. Subsequent studies were conducted to determine factors that influenced the incidence or severity of skin abnormalities, and whether these adverse skin reactions influenced a specific neurobehavioral outcome. Rats injected daily for 21 d with food grade peanut oil had an earlier onset and greater incidence of skin abnormalities relative to rats receiving an equal volume (1.0 ml/kg/d) of reagent grade peanut oil or triglyceride of coconut oil. Skin abnormalities in animals injected daily with peanut oil were increased in animals housed on corncob versus paper bedding. Comparison of animals obtained from different barrier facilities exposed to the same injection paradigm (reagent grade peanut oil, 1.0 ml/kg/d s.c.) revealed significant differences in the severity of skin abnormalities. However, animals from different barrier facilities did not perform differently in a Pavlovian fear conditioning task. Collectively, these data suggest that environmental factors influence the incidence and severity of skin abnormalities following repeated s.c. injections, but that these adverse skin responses do not significantly influence performance in at least one test of learning and memory.

Keywords: adverse skin reaction; foreign body reaction; learning and memory; neurobehavior; peanut oil; subcutaneous injection.

Figure 1. Representative images of skin lesions in male Long-Evans rats injected with peanut oil (1 ml/kg/d, s.c.) for up to 21 d

(A) Normal animal with no visible skin abnormalities on injection day 21. (B) Example of swelling around the injection area on injection day 21. (C) Demonstration of dermatitis on injection day 21. (D) Animal with clinically insignificant abrasion on injection day 13. (E) Illustration of a clinically significant abrasion on the neck/shoulder region on injection day 11. (F) Animal with clinically significant abrasions on the ears and neck on injection day 21.

Figure 2. Histological analyses of skin lesions from rats injected with reagent grade peanut oil (1.0 ml/kg/d s.c.) for 21 d

(A) Hematoxylin and eosin staining of the dorsal haired skin and subcutis. Deep to the dermis and skeletal muscle, the subcutaneous adipose tissue is expanded by large clear lipid vacuoles and pyogranulomatous inflammation, 20×. Bar = 200 µm. (B) Higher magnification of the subcutis demonstrating large clear lipid vacuoles surrounded by foamy uninucleate to multinucleated macrophages, 200×. Bar = 100 µm.

Figure 3. Variable incidence of skin abnormalities depending on the vehicle injected

Rats were housed on paper bedding and injected daily with food grade peanut oil, reagent grade peanut oil or Neobee M-5 (1.0 ml/kg/d s.c.) for 21 d. The percentage of rats in each treatment group exhibiting skin abnormalities (swelling, irritation and scabbing) was recorded each day. Animals injected with food grade peanut oil (open boxes, N=6) had an earlier onset and greater incidence of skin abnormalities relative to animals injected with either reagent grade peanut oil (closed boxes, N=3) or Neobee M-5 (open circles, N=3).

Figure 4. Peanut oil grade and bedding type significantly influence the incidence of skin abnormalities triggered by repeated s.c. injection with peanut oil

Rats were housed on either corn cob or paper bedding throughout the 21 day testing period and were injected with food grade peanut oil, reagent grade peanut oil, or Neobee M-5 at 1.0 ml/kg/d s.c. The incidence of skin abnormalities was recorded daily. These data were analyzed using a logistic regression model, yielding Pearson residuals that were used to generate a standardized measure of how large or small the observed skin abnormality was relative to the day’s average. For each animal, the Pearson residuals were averaged across all days to obtain an average daily Z-score for each rat. A positive Z-score indicates a high propensity to exhibit skin abnormalities whereas a negative Z-score indicates resistance to skin abnormalities relative to the average. Significant differences between treatment groups were identified using 2-way ANOVA (N=3–6 per treatment group) and are presented as a contrast comparing the mean of one factor level to the mean of another. The dot represents the point estimate of the contrast between the means of the two factors; the whiskers represent the bootstrapped 95% confidence interval. Confidence intervals that lie entirely above or below zero indicate that the contrast is significant at the 5% level.

Figure 5. Prior environmental exposures influence the development of skin abnormalities in response to injection of reagent grade peanut oil (1.0 ml/kg/d s.c.) for 21 d

Rats of the same strain, sex and weight were obtained from 2 different barrier facilities and allowed to acclimate for 7 days prior to beginning injections. The severity of skin abnormalities were scored daily according to the diameter of the lesion with 0 = no abnormality, 1 = mild abnormality (< 3 mm), 2 = moderate abnormality (3–7 mm) and 3 = marked abnormality (≥ 7 mm). These data were analyzed using a standard linear regression model, yielding Pearson residuals that were used to generate a standardized measure of how large or small the observed skin abnormality was relative to the day’s average. For each animal, the Pearson residuals were averaged across all days to obtain an average daily Z-score for each rat. A positive Z-score indicates a high propensity to exhibit skin abnormalities whereas a negative Z-score indicates resistance to skin abnormalities relative to the average. Significant differences between treatment groups were identified using 2-way ANOVA (N=6 per treatment group) and are presented as a contrast comparing the mean of one factor level to the mean of another. The dot represents the point estimate of the contrast between the means of the two factors; the whiskers represent the 95% confidence interval. Confidence intervals that lie entirely above or below zero indicate that the contrast is significant at the 5% level.

Figure 6. The barrier facility from which rats were obtained did not significantly influence Pavlovian fear conditioning

Long-Evans rats from two different barrier facilities were injected daily for 21 days with reagent grade peanut oil (1.0 ml/kg/d s.c.). At the end of the injection period, learning and memory were assessed using Pavlovian fear conditioning. There were no statistically significant differences between experimental groups in either the context test (A) or the cue test (B). Data presented as the mean ± S.E. (N=6 animals per treatment group). Data were analyzed by one-way ANOVA with significance set at p<0.05.