Effects of Fenbendazole on Routine Immune Response Parameters of BALB/c Mice

Fenbendazole (FBZ) is an anthelmintic drug widely used to treat and prevent pinworm outbreaks in laboratory rodents. Although data in nonrodent species indicate possible effects of fenbendazole on the bone marrow and lymphocyte proliferation and function, little has been reported regarding possible effects on the rodent immune system. The purpose of the current study was to determine the effects of a therapeutic regimen of FBZ on immune parameters in BALB/c mice. Both 9-wk on–off and 5-wk continuous medicated feed protocols were assessed. No significant differences between normal and FBZ diet treated mice were observed in the following parameters: complete blood count, blood chemistry, quantitation of major T and B cell markers in spleen, quantitation of T cell markers in the thymus, spleen cell proliferation to T and B cell mitogens, bone marrow colony-forming cell assays, skin graft rejection, and primary and secondary humoral immune responses. These data indicate that FBZ treatment does not affect many standard broad measures of immune function.Abbreviation: Con A, concanavalin A; FBZ, fenbendazole; LPS, lipopolysaccharideFenbendazole (FBZ) is a broad-spectrum benzimidazole anthelmintic drug that has gained widespread use for the treatment and prophylaxis of pinworm infection.^12,20,25 FBZ typically is administered through the use of medicated feed and is often used instead of ivermectin, which can have toxic effects in young animals.²⁴ Although no toxic effects have been reported from the use of FBZ at therapeutic levels, the physiologic or biologic actions of the drug potentially might alter or interfere with ongoing research experiments.^22,25 To this point, treatment with FBZ apparently decreases fecundity but not reproduction in rats.^14,27 Furthermore, FBZ had no effect in rat behavioral studies.^3,15 Recently, FBZ reportedly enhanced lipopolysacchide-induced inflammation in rats.¹³FBZ treatment alters numerous immune parameters in nonrodent species including inducing myelosuppression and altering T and B cell responses.^{5,10,11,19,23,26} In contrast, FBZ treatment (150 ppm) for 23 wk did not alter the onset or incidence of diabetes in NOD mice.⁹ Furthermore, assays of T helper function and cytotoxicity remained unaffected in BALB/c mice treated for 2 wk with 100 ppm FBZ.²¹ In contrast, FBZ at 10mg/kg for 5 d in C57BL/6 mice resulted in increased spleen cell proliferation to mitogens.⁸ The goal of the current study was to examine BALB/c mice for changes in routine parameters and assessors of immune function during and after treatment with a commercially available FBZ-medicated (150 ppm) diet. Many treatment protocols using FBZ-medicated diet have been proposed.^12,20 This study was conducted by using both a 9-wk on–off schedule as well as 5-wk continuous application.     

Heart Rates of Male and Female Sprague�CDawley and Spontaneously Hypertensive Rats Housed Singly or in Groups

This study was conducted to confirm our previous reports that group housing lowered basal heart rate and various evoked heart-rate responses in Sprague–Dawley male and female rats and to extend these observations to spontaneously hypertensive rats. Heart rate data were collected by using radiotelemetry. Initially, group- and single-housed rats were evaluated in the same animal room at the same time. Under these conditions, group-housing did not decrease heart rate in undisturbed male and female rats of either strain compared with single-housed rats. Separate studies then were conducted to examine single-housed rats living in the room with only single-housed rats. When group-housed rats were compared with these single-housed rats, undisturbed heart rates were reduced significantly, confirming our previous reports for Sprague–Dawley rats. However, evoked heart rate responses to acute procedures were not reduced universally in group-housed rats compared with either condition of single housing. Responses to some procedures were reduced, but others were not affected or were significantly enhanced by group housing compared with one or both of the single-housing conditions. This difference may have been due, in part, to different sensory stimuli being evoked by the various procedures. In addition, the variables of sex and strain interacted with housing condition. Additional studies are needed to resolve the mechanisms by which evoked cardiovascular responses are affected by housing, sex, and strain.Group housing of rodents is recommended on grounds that it reduces stress that accompanies isolated housing and therefore improves animal wellbeing. Experimental evidence supporting this recommendation depends on methods that can monitor stress levels or wellbeing effectively. Several techniques to detect stress in rodents are currently in use. Radiotelemetry methods have allowed for minute-to-minute determination of cardiovascular and body temperature responses.^{2,4,17,20,22,23,25,27,33,34,38,39,46,47,50} Chronic blood sampling through indwelling catheters makes it possible to monitor the blood levels of stress hormones (for example, glucocorticoids, prolactin) in conscious animals,^{18,28,31,35,36,45} whereas serial collection of feces has allowed for the noninvasive monitoring of fecal corticosterone metabolites in rats.¹⁵To reduce stress and improve wellbeing, several methods that alter the animal''s environment have been studied. These include the addition of enrichment devices to the animal''s home cage,⁴¹ increased cage size,⁴³ and reduced ambient illumination³. Extending the dark phase of the photocycle has been shown to decrease blood pressure and heart rate in rats^3,51 and may prove to be yet another method to reduce stress. Finally and directly related to the current studies, social enrichment by group or colony housing has been reported to reduce cardiovascular parameters in rats,^5,14,42,44 suggesting a reduced level of stress, but this method actually can contribute to stress if the number of rats housed together produces crowding or if aggressive interaction between animals occurs.⁵The initial objectives of the current study were to confirm our previous reports of the effects of group housing on basal heart rate and evoked heart rate responses in Sprague–Dawley rats and to determine whether the effects of housing generally extend to male and female rats of other strains. We chose to evaluate the spontaneously hypertensive rat (SHR) strain because it is very susceptible to stress.^26,29,30,32 Both sexes were included for completeness of the comparisons. As the study progressed, results indicated that group- and single-housed rats living in the same room at the same time may interact with each other. Therefore, a third objective was added to determine whether our previous results could be replicated when group- and single-housed rats are in close proximity to each other.The hypotheses tested in the current studies were: that group-housing reduces heart rate in undisturbed rats; that compared with single housing, group housing reduces heart-rate responses induced by acute procedures; and that housing effects are influenced by sex and strain.     

Analgesic Activity of Tramadol and Buprenorphine after Voluntary Ingestion by Rats (Rattus norvegicus)

Effective pain management for rats and mice is crucial due to the continuing increase in the use of these species in biomedical research. Here we used a recently validated operant orofacial pain assay to determine dose–response curves for buprenorphine and tramadol when mixed in nut paste and administered to male and female rats. Statistically significant analgesic doses of tramadol in nut paste included doses of 20, 30, and 40 mg/kg for female rats but only 40 mg/kg for male rats. For male rats receiving buprenorphine mixed in nut paste, a significant analgesic response was observed at 0.5 and 0.6 mg/kg. None of the doses tested produced a significant analgesic response in female rats. Our results indicate that at the doses tested, tramadol and buprenorphine produced an analgesic response in male rats. In female rats, tramadol shows a higher analgesic effect than buprenorphine. The analgesic effects observed 60 min after administration of the statistically significant oral doses of both drugs were similar to the analgesic effects of 0.03 mg/kg subcutaneous buprenorphine 30 min after administration. The method of voluntary ingestion could be effective, is easy to use, and would minimize stress to the rats during the immediate postoperative period.Abbreviations: VI, voluntary ingestion; LFR, lick:facial contact ratio; OPAD, orofacial pain assessment deviceEffective pain management for rats and mice is crucial due to the continuing increase in the use of these species in biomedical research. It is necessary not only to satisfy ethical and legal standards, but providing effective pain management may also reduce distress, decrease mortality, and overall eliminate many of the negative postsurgical physiologic consequences that may be confounding factors in research.⁵² Effective pain management entails providing analgesics at the optimal dosing regimen (dose, frequency, and duration). Continual refinement of the optimal analgesic dosing regimen is possible due to the availability of new methods for evaluating pain (that is, mouse and rat grimace scales),^47,69 the increasing knowledge of pain mechanisms and pathways,^56,71 and the development of new analgesic formulations.^14,68Tramadol is a centrally acting analgesic with both opioid and nonopioid mechanisms of action.⁶⁰ Its analgesic activity is due to a high affinity for μ-opioid receptors and both serotonin and norepinephrine reuptake inhibition.² In vitro, tramadol has been shown to inhibit the activity of voltage-operated Na⁺ channels, delayed rectifier K⁺ channels, N-methyl-d-aspartate receptors, and substance P receptors.^27,28,49,75 Tramadol also exhibits relatively few of the adverse effects typically associated with classic opioids, including respiratory depression and ileus.⁶⁰ The most commonly reported tramadol-associated adverse events in humans include nausea, dizziness, and drowsiness.²⁶ In the United States, oral formulations of tramadol are widely used in both human medicine and in companion animals.^59,64 Despite the potential benefits, very few studies have evaluated the efficacy of tramadol after oral administration in laboratory rats.⁵⁵In comparison, buprenorphine is one of the most common analgesics used for mild to moderate pain in rats, and its use is considered a standard of care for postoperative pain.¹⁴ Buprenorphine acts as a partial μ-opioid receptor agonist, and its slow dissociation kinetics allows for a longer duration of action compared with that of classic μ-opioid agonists, such as morphine.^45,76,79 Additional benefits of buprenorphine include a ceiling effect on respiratory depression and a lack of immunosuppression at doses relevant for analgesia.^30,57,61 Side effects in rats are usually limited but include sedation, cardiovascular depression, decreased appetite, and gastrointestinal distress, which may or may not be accompanied by pica.^15,19,63 Administration of buprenorphine by the oral route in rats is limited by a lack of information regarding its pharmacokinetics and conflicting reports of its efficacy.^{1,5,23,24,36,46,74}Providing analgesics mixed in the food or water of rats and mice is one of the least stressful methods of administration. This method eliminates postoperative manual restraint and parenteral injections, which have been shown to induce stress-like responses in mice and rats.^6,66,67 Providing analgesics by this method has several drawbacks. First, the neophobic behavior of rats and mice may lead to significant underdosing when a period of habituation to the drug is not observed.⁷⁰ Second, some drugs are unpalatable and so may not be consumed in sufficient quantities to provide analgesia.³³ Third, animals undergoing surgery typically have reduced food and water intake during the immediate postoperative period, and this behavior may limit the dose administered.²⁹ Fourth, overdosing may occur when analgesics are provided with a palatable vehicle, such as a cherry-flavored solution.⁸ Finally, when opioids are administered in the food or water, tolerance may develop, leading to a decrease in analgesic efficacy.³⁵To mitigate these issues, sufficient dosages of analgesics can be offered for voluntary ingestion (VI) by mixing the drug in a highly palatable vehicle without needing to add it to food or water. Several recent studies using a sweet nut paste for the administration of buprenorphine have shown its promise as an effective vehicle for administration.^1,23,24 Providing analgesics in this manner allows for a fixed dosage of the drug to be administered consistently, with the added assurance that the animals are consuming an effective dose and are not under- or overdosed.The goal of the current study was to evaluate the analgesic effects of a range of oral doses of tramadol or buprenorphine mixed with nut paste and provided to rats by VI. Rats were evaluated by using a recently described, nontraumatic, reversible, operant-based, thermal orofacial pain assessment device (OPAD).^53,62 In this model, a low concentration of capsaicin is applied topically to the test area for 5 min and then is removed. Capsaicin sensitizes the transient receptor potential vanilloid 1 receptor to heat.⁵¹ This receptor is a key channel for signaling and modulating heat and inflammatory pain,^12,13,58 and previous studies in mice have documented the importance of the receptor in the development of incisional pain.^40,58 Heat is then applied to the sensitized test area, thereby activating these receptors and eliminating the need for surgical procedures. Our hypothesis was that OPAD evaluation would be effective at establishing clinically relevant doses for tramadol and buprenorphine in both male and female rats.     

Eliminating Animal Facility Light-at-Night Contamination and Its Effect on Circadian Regulation of Rodent Physiology, Tumor Growth, and Metabolism: A Challenge in the Relocation of a Cancer Research Laboratory

Appropriate laboratory animal facility lighting and lighting protocols are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations. Our recent experience in relocating to a new laboratory facility illustrates the importance of these considerations. Previous studies in our laboratory demonstrated that animal room contamination with light-at-night (LAN) of as little as 0.2 lx at rodent eye level during an otherwise normal dark-phase disrupted host circadian rhythms and stimulated the metabolism and proliferation of human cancer xenografts in rats. Here we examined how simple improvements in facility design at our new location completely eliminated dark-phase LAN contamination and restored normal circadian rhythms in nontumor-bearing rats and normal tumor metabolism and growth in host rats bearing tissue-isolated MCF7(SR^–) human breast tumor xenografts or 7288CTC rodent hepatomas. Reducing LAN contamination in the animal quarters from 24.5 ± 2.5 lx to nondetectable levels (complete darkness) restored normal circadian regulation of rodent arterial blood melatonin, glucose, total fatty and linoleic acid concentrations, tumor uptake of O₂, glucose, total fatty acid and CO₂ production and tumor levels of cAMP, triglycerides, free fatty acids, phospholipids, and cholesterol esters, as well as extracellular-signal-regulated kinase, mitogen-activated protein kinase, serine–threonine protein kinase, glycogen synthase kinase 3β, γ-histone 2AX, and proliferating cell nuclear antigen.Abbreviation: 13-HODE, 13-hydroxyoctadecadienoic acid; γH2AX, histone 2AX; AKT, serine–threonine protein kinase; ERK1/2, extracellular signal-regulated kinase p44/46; GSK3β, glycogen synthase kinase 3β: LAN light at night; MEK, mitogen-activated protein kinase kinase, PCNA, proliferating cell nuclear antigen; SR^–, steroid-receptor–negativeRelocating laboratory animal research from one institution to another can be a daunting task for both scientists and animal care personnel with regard to control of lighting and elimination of light-at-night (LAN) contamination. Appropriate facility lighting and lighting protocols, as outlined in the Guide for the Care and Use of Laboratory Animals,³⁰ are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations.^16-18,22 The profound effect of light on circadian behavior and physiology is well established.^{2,3,5,9,11,12,16-18,22,29,32,44,46,49,52,55-58,64}Minor alterations in light intensity,¹¹ spectral quality,¹² and duration⁹ at any given time of day can alter or disrupt chronobiologic rhythms markedly in all mammals.^{6,17,26,44,55-59} Light information, which initially is detected by a small group of intrinsically photosensitive retinal ganglion cells containing the blue light-sensitive photopigment melanopsin,^6,26 is transmitted through the retinohypothalamic tract⁵⁹ to a central molecular clock located in the suprachiasmatic nucleus of the hypothalamus.^32,57 The suprachiasmatic nucleus, the activity of which is entrained by the light:dark cycle,^32,57 sends projections over a polysynaptic pathway to the pineal gland driving a series of molecular events leading to the production of the pineal neurohormone melatonin (N-acetyl-5 methoxytryptamine), primarily during the night.^29,46 The daily rhythmic melatonin signal contributes to the temporal coordination of normal behavioral and physiologic functions including the sleep–wake^23,46,66 and reproductive cycles,^51,55 immune function,^38,41,56 hormone levels,^{19,31,45,47,68} temperature regulation,²³ electrolyte balance,⁶⁹ neural protein synthesis,⁶⁵ and redox states.^24,53Dark-phase LAN exposure suppresses endogenous melatonin concentrations and may lead to various disease states,^42,58 including carcinogenesis,^7,8,16,18,33 and metabolic syndrome.^{17,34-37,39,70} Previous in vivo studies in our former laboratory (at the Bassett Research Institute, Cooperstown, NY) demonstrated that animal room LAN of as little as 0.2 lx (0.08 µW/cm²; rodent eye level) during an otherwise normal dark-phase suppressed normal physiologic nighttime melatonin levels, leading to markedly disrupted circadian regulation of physiology and metabolism in nontumor-bearing host animals^16,18 and a stimulation in metabolism and proliferation of both tissue-isolated MCF7 steroid-receptor–negative (SR^–) human breast cancer xenografts and syngeneic grafts of rodent hepatoma 7288CTC in rats.^7,17 This effect was mediated by melatonin receptor-mediated suppression of cAMP, leading to inhibition of tumor linoleic acid uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). These events culminated in downregulation of epidermal growth factor and insulin-like growth factor 1 pathways.^7,8,16-19,62Exposure to LAN is likely to exert pervasive and problematic effects on mammalian behavior and physiology in laboratory animal facilities around the world. During the past decade, improved facility design and better adherence to animal room lighting protocols certainly has helped to reduce the problem. In moving to our laboratory animal facility at Tulane University School of Medicine (New Orleans, LA), we discovered considerable preexisting LAN contamination that had to be eliminated before we could resume our human cancer research.The current study was performed to monitor the effects of the elimination of animal room LAN contamination over time on animal health and wellbeing, tumor growth, and metabolic profiles by assessing well-established circadian parameters in physiology and metabolism.^7,8,16-18 We measured light-induced suppression of melatonin, an accepted and sensitive marker of the effects of light on the circadian system in all mammals,^{2,3,5,9,11,12,15,16,18,20,21,29,44,46,49,52,55-58,64} before and after tumor implantation and growth. Tissue-isolated MCF7(SR^–) human breast cancer xenografts and 7288CTC rat hepatomas have been well-characterized over the years in our former light-tight facilities^7,8,16-18 and provided us with unique markers and measures of the extent to which LAN contaminated our new animal quarters. In tumor-bearing animals exposed to even minimal LAN, the latency-to-onset of tumor growth and proliferation rates of these tumors increase markedly in direct proportion to LAN intensity. As improvements were made to eliminate LAN contamination in the new location over the course of more than 20 generations of tumor passages, we measured the changes and reestablishment of normal rat and host–tumor circadian regulation. The information from this study may assist investigators and animal care personnel in addressing this important influence on the health and wellbeing of laboratory animals and consequent effects on the outcome of scientific investigations.     

Assessment of Routine Procedure Effect on Breathing Parameters in Mice by Using Whole-Body Plethysmography

We used whole-body plethysmography to investigate the effect of restraint, ear marking, tail vein and retroorbital blood sampling, and tail clipping on respiration in Balb/c × TCR-HA^+/– F1 hybrid mice (F1h). Baseline values of breathing parameters were determined. During the experiment, mice experienced a procedure and then plethysmographic recordings were obtained immediately and at 4, 24, and 48 h afterward. Baseline breathing parameters showed significant differences between sexes. Restraint affected minute volume differently than did handling in male mice and to a lesser extent in female mice. Ear marking significantly changed minute volume compared with handling but not restraint in male mice and in the opposite manner in female mice. Tail vein blood sampling changed minute volume in a significant manner compared with restraint but not compared with handling in both sexes. Retroorbital blood sampling significantly changed minute volume compared with values for both handling and restraint in male mice but only compared with handling in female mice. Tail clipping modified minute volume significantly compared with handling in male mice and compared with restraint in both sexes. Analysis of data showed that routine procedures affect minute volume in mice depending on invasiveness of maneuver and in a sex-biased manner for as long as 24 h after the procedure. Our experiment shows that procedures performed on laboratory mice can change respiratory parameters and can be investigated by plethysmography.Abbreviation: F1h, Balb/c × TCR-HA^+/– F1 hybrid miceHandling, restraint, identification methods, and blood or tissue sampling are regarded as routine procedures in animal experiments. This aspect is particularly important when working with transgenic animals, where the need for genotyping demands the frequent use of these procedures, which can readily yield DNA samples.⁵ The effect of routine procedures on physiologic parameters varies depending on method, frequency, and duration, but these procedures generally are considered to be acute stressors.^2,5,26,27 A large body of research has focused on the effect of routine procedures on animals’ physiology. These effects were assessed by studying changes in behavior,^1,23,36,37 body weight,^29,37 food and water intake,^29,37 body temperature³⁵ and heart rate by radiotelemetry,^{5,10,19,26,27} corticosterone^{11,12,24,25,29} and glucose levels,³³ and blood variables.^29,36 Several differences in the response of laboratory animals to stress have been correlated to breed,^2,33 strain,^11,14,37 age,²⁴ and sex.^12,23,24 Some authors²⁴ have noted that female mice show a lower overall stress level due to the modest effect of social competition. Another study¹² showed a different, sex-specific response to hypoxic ventilation in rats, which was higher in male than female rats. In addition, these authors¹² suggested that female ovarian hormones are prime candidates for stress regulation.Whole-body plethysmography is a noninvasive, indirect method of studying respiratory function in conscious, unrestrained animals. This method has been used particularly in pharmacologic and toxicologic studies in diverse animal species including mice,^3,9,14,15,38 rats,^8,20 cats,²² dogs,^16,34 and pigs.¹³ The present study was designed to investigate the extent to which routine procedures affect breathing parameters in a transgenic mouse colony. Male and female mice were exposed to 4 routine procedures that are used for DNA sampling and identification of transgenic mice (ear marking, tail clipping, and tail vein and retroorbital blood sampling). Respiratory parameters were recorded by whole-body plethysmography immediately and at 4, 24, and 48 h after the procedure. Handling and restraint were used as control procedures.     

Multiple Extrauterine Pregnancy with Early and Near Full-Term Mummified Fetuses in a New Zealand White Rabbit (Oryctolagus cuniculus)

Extrauterine pregnancy (EP) is infrequent in mammalian species and occurs when fertilized ova implant and develop outside the uterus. A common outcome is abdominal pregnancy resulting in mummified fetuses (lithopedia). Here we describe an unusual case of abdominal pregnancy with early and near full-term lithopedia. Macroscopic findings supported the diagnosis of lithopedia with distinct age differences and facilitated further characterization of primary ectopia and risk factors leading to this occurrence.Abbreviation: EP, extrauterine pregnancyExtrauterine pregnancy (EP) occurs infrequently in most mammalian species.¹² The term derives from the Latin prefix meaning ‘outside’ or ‘beyond’ and refers to the implantation of a fertilized ovum outside the uterine cavity. Extrauterine pregnancy was first recognized more than 900 y ago² among other discoveries with a hereditary nature.¹³ Early reports compared EP in women, cats, dogs, and rabbits⁷ and described the presence of mummified fetuses in laboratory rabbits.^16,35EP is a serious obstetric complication that occurs asymptomatically in most cases.¹⁷ There are 4 classifications of EP: tubal, ovarian, abdominal–peritoneal, and cervical. The fallopian tube is the most common location and leads to tubal pregnancy. When gestation occurs in the abdominal–peritoneal cavity, abdominal pregnancy results and is subdivided as primary, when fertilization occurs outside the uterus after an oocyte is accidentally released from the fimbria, and secondary, when an oocyte is released due to direct tubal trauma.⁴⁵ A rare form of EP associated with high maternal morbidity and fetal mortality is called heterotopic (or combined) pregnancy, which occurs when 2 fertilized eggs coexist, one outside the uterus and the other inside.^18,33,44Undetected EP is frequently associated with fatal outcomes to the dam and offspring, including the formation of mummified fetuses, which may eventually become calcified and are called lithopedia (from the Greek: lithos, stone; paidion, child).^11,51,53 The condition is infrequent, and the factors that influence the unexpected outcomes of this pathology are not well understood.^26,34,35 Epizootiologic investigations are few,¹² although a recent report outlined the prevalence of EP in large NZW rabbit breeding colonies.⁵¹Examples of EP have been documented in dogs,¹⁷ cats,^14,39,42,49 rabbits,^20,29,45,51 hamsters,^9,46 rats,²⁶ mice,^8,12 guinea pigs,^3,30 lambs,⁴⁰ nonhuman primates,^10,34,38,50 and other species including humans.^11,12 However, despite the number of documented species, the majority of reports failed to note detailed clinical symptoms that interfered with reproduction even in instances that led to the formation of lithopedions.⁴²Experimentally, mouse embryos have successfully been transferred to a variety of sites including the peritoneal cavity, kidney, spleen, muscles, testis, and the anterior chamber of the eye.^1,6,21,31 The aim of the current report is to describe a rare case of abdominal pregnancy in which lithopedia developed clinically silently and coexisted with multiple pregnancies in a healthy doe rabbit.     

Progression of Ulcerative Dermatitis Lesions in C57BL/6Crl Mice and the Development of a Scoring System for Dermatitis Lesions

Ulcerative dermatitis (UD) is a common, spontaneous condition in mice with a C57BL/6 background. Although initial lesions may be mild, UD is a progressive disease that often results in ulcerations or debilitating fibrotic contractures. In addition, lesions typically are unresponsive to treatment. Euthanasia is often warranted in severe cases, thereby affecting study outcomes through the loss of research subjects. Because the clinical assessment of UD can be subjective, a quantitative scoring method and documentation of the likely time-frame of progression may be helpful in predicting when animals that develop dermatitis should be removed from a study. Such a system may also be helpful in quantitatively assessing success of various treatment strategies and be valuable to clinical laboratory animal veterinarians. In this 1.5-y, prospective cohort study, we followed 200 mice to monitor the development and course of UD. Mice were examined every 2 wk. A clinical sign (alopecia, pruritus, or peripheral lymphadenopathy) was not identified that predicted development of UD lesions in the subsequent 2-wk period. Once UD developed, pruritus, the character of the lesion (single or multiple crust, coalescing crust, erosion, or ulceration), and the size of the lesion were the only parameters that changed (increased) over the course of the disease. Pruritus was a factor in the rapid progression of UD lesions. We used these findings to develop a quantitative scoring system for the severity of UD. This enhanced understanding of the progression of UD and the quantitative scoring system will enhance the monitoring of UD.Abbreviation: UD, ulcerative dermatitis; S number, scratching number; COL, character of lesionsUlcerative dermatitis (UD) is an idiopathic, spontaneous, debilitating syndrome of laboratory mice that is typically a disease of aged^1,19,43,46 C57BL/6 mice or genetically engineered mice on a C57BL/6 background.^1,19,43,44 Some reports discuss a similar condition in young, weanling mice that presents initially as alopecia.^24,42,44,45 Prevalence rates of UD between 4.1% to 21% have been reported.^1,6,19 Although no etiology has been identified, environmental factors,^{6,19,41,42,44} diet,^{5,29,41,42,46} season,^19,41,43,44 age at weaning,⁴² alopecia,^24,42,44,45 sex,^19,39,41,43 immune complex vasculitis,¹ follicular dysplasia,⁴⁴ lesion location,²⁰ and deficiencies in vitamin A metabolism⁴⁴ have all been implicated as predisposing factors for disease development. In addition, oronasal pain and chronic inflammation may lead to self-mutilation as a result of, initiating an “itch” response.¹⁰ UD is diagnosed by ruling out other causes of dermatitis in laboratory mice, such as fur mites,⁹ infections, fight wounds,¹⁷ strain phenotype,^15,35,40,49 and experimentally induced dermatitis.^4,50 Other diagnostic criteria are based on professional judgment and may include strain (C57BL/6 background),^1,19,44 lesion location (head and dorsal thorax),^1,19,43,44 intense pruritus,^1,19,44 peripheral lymphadenopathy,^6,19,39 and failure to respond to treatment.¹⁹ The rapid progression of UD lesions results in significant morbidity in laboratory mice.^6,19,44 Typically, the lesions progress to large, irregularly shaped, confluent ulcerations on the dorsal cervical and thoracic region.^1,19,39,44 As the lesions heal, contracted scar tissue forms, which can impair species-typical behaviors and mobility.^39,41,43,44 The presence of large dermal ulcerations or debilitating contractures affect animal welfare and typically necessitate euthanasia of affected mice. Although reports on the later stages of UD have been consistent,^{1,19,39,41,43,44} information on the initiation and progression of UD lesions is conflicting. Pruritus,^1,19,44 pain,¹⁰ and genetic predisposition^1,19,43,44 have been implicated as initiators of the disease. Alopecia, pruritus, erythema, and single or multiple(s) crust have all been reported as early signs of the disease.^{1,19,39,42-44} However, the majority of this information has been collected retrospectively, at timed necropsies, or based on anecdotal reports.Scoring systems are useful tools to evaluate clinical diseases in laboratory animals. For example, scoring systems have been published for tumors,^14,28 body condition,^14,28,47 and neurologic phenotype¹³ in mice to aid in assessment of clinical disease severity. Even though the progressive and severe nature of UD typically warrants eventual euthanasia, determining the severity of disease has typically been based on professional judgment,^1,39 subjective scoring,^{12,15,19,40,43,48,49} or postmortem histology.^40,44 A quantitative scoring system for UD in live mice has not been described and could greatly aid laboratory animal veterinarians and researchers in determining the severity of the disease and response to treatment.The purpose of the current study is to investigate clinical parameters that reflect the progression of UD to facilitate management and veterinary care of mice with UD. We followed 200 mice from 3 wk of age until the development of UD to determine the initial signs and progression of UD lesions. We hypothesized that mice will first develop signs of pruritus prior to any clinical lesion. From there, we predicted that clinical lesions will progress stepwise though the following stages of severity, with or without alopecia: (1) excoriations; (2) a single, small punctuate crust; (3) multiple, small punctuate crusts; (4) coalescing crust; (5) erosion; and (6) ulceration. In addition, we hypothesized that a quantitative, validated, and reliable UD scoring system can be created that is based on physical examination parameters that do, in fact, predict development and progression of UD. Having a more thorough understanding of the initiation and progression of ulcerative lesions likely will enhance our ability to predict the outcome for a given mouse and develop earlier end-points for that mouse. Furthermore, use of this scoring system will enable accurate monitoring of UD lesions.     

Environmental-Enrichment�CRelated Variations in Behavioral, Biochemical, and Physiologic Responses of Sprague�CDawley and Long Evans Rats

The behavioral, biochemical, and physiologic consequences of 6 wk of environmental enrichment were evaluated in male Long Evans and Sprague–Dawley rats and compared with those of rats in standard single-housing conditions. Standard housing provided little or no social or physical stimulation whereas environmental enrichment comprised group housing for 8 h daily in a 3-story cage equipped with novel stimuli. Dependent measures included performance in the forced swim test, thresholds for brain-stimulation reward, sucrose intake and preference, determination of corticosterone levels before and after brief restraint stress, and rate of weight gain. In forced swimming tests, active behaviors (diving, swimming with struggling, and climbing) tended to dominate over passive behaviors (sinking, floating) in both groups and outbred rat stocks (especially in enriched groups) on the first day. These behaviors were replaced with maintenance behaviors such as grooming and swimming without struggling on the second exposure, with enriched Long Evans rats showing the largest decline in activity. Baseline plasma corticosterone levels were elevated in both rat stocks after 6 wk of enrichment. After restraint stress, hormone levels in enriched animals tended to peak earlier and approach or exceed baseline values more quickly than was observed in the comparable control groups. Rate of body weight gain was greater in enriched Long Evans rats than Sprague–Dawley or control rats. Our observations indicate that stock- and group-associated differences in several indices occur in association with enrichment. The data support the claim that environmental enrichment may render animals more resilient to challenges.Abbreviation: BSR, brain-stimulation reward; EE, environmental enrichment; FST, forced swim testEnvironmental enrichment (EE) paradigms are designed to enhance laboratory animals’ surroundings to encourage natural behaviors. Some enrichment paradigms also include a social component, based on the social interactions typical of the genus and species. For example, wild mice and rats generally live in colonies, whereas hamsters are known to be social with unfamiliar animals only during mating.²¹Adverse environmental conditions have been shown to affect the susceptibility of animals exposed to diverse stress regimes, reflected in their behavioral,^7,34 physiologic,^{8,25,29,36,56} and biochemical^6,8,16 responses in a strain-dependent manner.^7,8,16 Therefore, a diverse environment might be expected to alter their response to such stressors. A review of the literature reveals few behavioral investigations of the effects of EE on response to a stressor, and the results of biochemical studies in this context have generally been inconsistent. For example, some laboratories have reported no difference in corticosterone levels between EE- and standard-housed animals after exposure to a stressor,^22,33,46 whereas others have observed a reduction in the corticosterone levels of Sprague–Dawley rats⁴ or even elevated levels of plasma corticosterone in enriched Wistar rats.³² These differences may be due to length of EE exposure or in-strain responsivity to stress. Therefore, the first aim of the present set of experiments was to investigate whether rat strain influences the behavioral and physiologic measures typically used to assess stress responses.Behaviors observed during the forced swim test (FST) and sucrose intake values are known to be affected by environmental conditions.^7,15,28,37 Historically, the FST has been used to assess behavioral despair, as indexed by the degree of immobility in an inescapable environment. After antidepressant administration, immobility typically is replaced by more active behaviors.^41-44 EE attenuated behavioral despair in male Sprague–Dawley rats during the FST.⁹ In our hands, exposure to 5 min of FST generally results in more active or escape behaviors (mostly frantic swimming with struggling and climbing) and in the second 5-min test, less vigorous swimming (without struggling). This pattern is not as prominent in stressed male rats.⁷ In the present study, we evaluated the effects of EE on behaviors exhibited in the FST, hypothesizing that animals with experience in an enriched environment would demonstrate less swimming with struggling in the second test compared with rats living in standard housing conditions.In the context of stress research, the presence of an anhedonic state typically is evaluated by using behavioral measures such as thresholds for brain-stimulation reward^2,7,34 and sucrose intake and preference.^28,37,55 The recent finding that EE also alters the behavioral profile of animals with respect to sucrose intake⁹ prompted us to include this measure in our study to determine the general hedonic status of animals in an enriched environment. We also evaluated rate of weight gain and corticosterone levels after 6 wk of EE, because these 2 measures are used frequently as indices of environmental challenges.^3,10,58 In chronic mild stress studies, 3 to 6 wk of administration is a fairly standard regime.^{7,8,19,26,28,50}In summary, we conducted 2 studies using male Sprague–Dawley and Long Evans rats. In the first, we assessed weight gain and plasma corticosterone levels after 6 wk of EE. In addition to these physiologic measures, we administered weekly sucrose intake and preference tests. In the second study, thresholds for brain-stimulation reward were collected biweekly, and exposure to the FST was evaluated after 6 wk of EE.     

Sex-Associated Effects on Hematologic and Serum Chemistry Analytes in Sand Rats (Psammomys obesus)

We sought to determine whether sex had a significant effect on the hematologic and serum chemistry analytes in adult sand rats (Psammomys obesus) maintained under normal laboratory conditions. According to the few data available for this species, we hypothesized that levels of hematologic and serum chemistry analytes would not differ significantly between clinically normal male and female sand rats. Data analysis revealed several significant differences in hematologic parameters between male and female sand rats but none for serum biochemistry analytes. The following hematologic parameters were greater in male than in female sand rats: RBC count, hemoglobin, hematocrit, red cell hemoglobin content, and percentage monocytes. Red cell distribution width, hemoglobin distribution width, mean platelet volume, and percentage lymphocytes were greater in female than in male sand rats. The sex of adult sand rats is a source of variation that must be considered in terms of clinical and research data. The data presented here likely will prove useful in the veterinary medical management of sand rat colonies and provide baseline hematologic and serum chemistry analyte information for researchers wishing to use this species.Psammomys obesus, commonly called the sand rat or fat sand rat, is a diurnal desert animal belonging to the family Muridae and subfamily Gerbillinae. These terrestrial mammals naturally inhabit the salty desert areas of North Africa and regions to the east across the Arabian Peninsula.^5,28,29,37Sand rats have been useful animal models for numerous human diseases, but because of the special nutritional requirements and low reproductive performance of this species, they are difficult to breed and maintain in captivity, averaging 2.5 pups per litter.^30,37 Sand rats are prone to develop hyperinsulinemia, hyperglycemia, and obesity when fed a high-energy diet.^1,22 The diabetic state that develops under high-energy conditions can be prevented by feeding the rats a high-fiber, low-carbohydrate diet.^21,22 Researchers at the Hebrew University (Jerusalem, Israel) generated an animal model for type 2 diabetes mellitus by isolating 2 separate lines of sand rats: a diabetes-prone line and a diabetes-resistant line.^2,21,22 Sands rats have also been used as models for the study of otic cholesteatoma, spondylosis, renal function, and numerous diabetes-related disorders.^3,12,27 Sand rats are naturally susceptible to infection with Leishmania major and have been identified as the main reservoir host that maintains and transmits leishmaniasis to sand flies in southern and central Israel and southern Iraq.^26,34,35 In addition, sand rats are the most important reservoir host of zoonotic cutaneous leishmaniasis²⁶ in the Middle East and North Africa.^28,34,35 Most recently, sand rats have been used in behavioral studies and are considered an appropriate model to study circadian mechanisms that are involved in mood and anxiety disorders in humans.^4,10Sand rats currently are not raised at any commercial rodent breeding farms in the United States. Instead, they typically must be bred inhouse or shipped from the Hebrew University– Hadassah Medical Center. Our institution, the Walter Reed Army Institute of Research (Silver Spring, MD), maintains one of the 2 sand rat breeding colonies in the United States. This breeding colony originated from the colony at Wake Forest University (Winston Salem, NC), which itself was established from the well-characterized colony at Hebrew University–Hadassah Medical Center. The colony at the Walter Reed Army Institute of Research is maintained for educational and research purposes.We performed the present study to determine whether hematologic and serum chemistry analytes differed between male and female adult sand rats. Only limited hematologic, glucose, and insulin values for the diabetic-prone sand rat have been available previously.^{1,2,14,21,22,39} Given the admittedly limited data available for diabetes-prone sand rats and the inconsistent published data regarding sex-associated hematologic differences for most of the common laboratory animals,¹¹ we hypothesized that hematologic and serum chemistry analytes would not differ significantly between clinically normal adult male and female sand rats.^1,22     

The Influence of Red Light Exposure at Night on Circadian Metabolism and Physiology in Sprague–Dawley Rats

Early studies on rodents showed that short-term exposure to high-intensity light (> 70 lx) above 600 nm (red-appearing) influences circadian neuroendocrine and metabolic physiology. Here we addressed the hypothesis that long-term, low-intensity red light exposure at night (rLEN) from a ‘safelight’ emitting no light below approximately 620 nm disrupts the nocturnal circadian melatonin signal as well as circadian rhythms in circulating metabolites, related regulatory hormones, and physiologic parameters. Male Sprague–Dawley rats (n = 12 per group) were maintained on control 12:12-h light:dark (300 lx; lights on, 0600) or experimental 12:12 rLEN (8.1 lx) lighting regimens. After 1 wk, rats underwent 6 low-volume blood draws via cardiocentesis (0400, 0800, 1200, 1600, 2000, and 2400) over a 4-wk period to assess arterial plasma melatonin, total fatty acid, glucose, lactic acid, pO₂, pCO₂, insulin, leptin and corticosterone concentrations. Results revealed plasma melatonin levels (mean ± 1 SD) were high in the dark phase (197.5 ± 4.6 pg/mL) and low in the light phase (2.6 ± 1.2 pg/mL) of control conditions and significantly lower than controls under experimental conditions throughout the 24-h period (P < 0.001). Prominent circadian rhythms of plasma levels of total fatty acid, glucose, lactic acid, pO₂, pCO₂, insulin, leptin, and corticosterone were significantly (P < 0.05) disrupted under experimental conditions as compared with the corresponding entrained rhythms under control conditions. Therefore, chronic use of low-intensity rLEN from a common safelight disrupts the circadian organization of neuroendocrine, metabolic, and physiologic parameters indicative of animal health and wellbeing.Abbreviation: rLEN, red light exposure at night; SCN, suprachiasmatic nuclei; TFA, total fatty acidLight is a powerful biologic force that entrains circadian rhythms of behavior, physiology and metabolism for all mammals.^{1-3,4,7-12,26,27,29,56,63} Alterations in intensity, duration, and spectral transmittance (that is, wavelength; perceived as color) of light at an inappropriate time of the day can induce the disruption of many circadian rhythms, including those of locomotor activity,^2,3,21,44,54 the sleep–wake cycle,^2,3,21 dietary and water intake,^2,3,21,38 metabolism,^{5,6,13-18,38,39,44,60} and neurologic functions,⁴³ among others. Circadian disruption is defined here as a significant change in the phasing, periodicity, amplitude, or duration of a circadian rhythm from its usual entrained pattern under 12:12-h light:dark conditions. Our early work provided the first in vivo experimental evidence demonstrating that the increased risk of breast cancer in humans, as occurs in the night-shift worker population, may be mediated by light exposure at night.^19,31,57 Our previous studies^4,5,13-18,64 also showed that adherence to appropriate lighting and lighting protocols, as outlined in The Guide for the Care and Use of Laboratory Animals,³⁷ is not only beneficial for but essential to the health and wellbeing of laboratory animals and experimental outcomes. For example, we found that a small light leak of as little as 0.2 lx (0.08 μW/cm²) intensity during an otherwise normal dark phase (12 h) in our animal rooms elicited a disruption in circadian rhythms of plasma measures of endocrine physiology and metabolism in rats.^13-18,64 In a process termed phototransduction, photic information from the eyes is transmitted via the retinohypothalamic tract, which projects to the suprachiasmatic nuclei (SCN), or master biologic clock; in turn, signals from the SCN to the pineal gland via a polysynaptic pathway regulate the nocturnal production of the hormone melatonin (N-acetyl-5-methoxytryptamine).^36,48,53,54 More recent studies^17,18,64 revealed that the spectral transmittance of light (blue-, amber-, or red-appearing) through laboratory rodent cages dramatically influences the temporal coordination of circadian patterns of plasma melatonin, total fatty acid (TFA), glucose, lactic acid, corticosterone, pO₂ and pCO₂ levels in both pigmented and nonpigmented rats.^17,18,64For many years, it has been common practice to make use of red ‘safe lamps’ (wavelengths above 600 nm) during dark-phase laboratory animal exposure or to cover animal room rodent racks, lights, and observation windows with red-tinted film primarily for nighttime observation.^{24,44,45,62,65} It was assumed light with wavelengths above 600 nm (red) had little or no effect on neurodendocrine or circadian systems, given that several nonpigmented species (felines, canines, ungulates, and most species of marine mammals) cannot visually perceive red light via the primary optic tract,^43,47 despite a few early studies on nonpigmented rodents showing otherwise.^43-45,60 Both polychromatic and analytic action spectra studies in humans subsequently showed that these responses to red-appearing light were weak.^8,28,29 At similar irradiances, the acute melatonin-suppressive effects of full-spectrum light were not observed with red light in most mammalian species.^7,38,53 This finding led to the use of red-tinted observation windows and red safety lights for nighttime laboratory animal observation.Recent studies in both rats and humans have shown, however, that high-intensity red light^8,30,51,59 over a short duration (that is, minutes) elicits marked suppression of the nocturnal melatonin signal. Long-wavelength light in the spectral region above 550 nm (perceived as yellow to red) of sufficiently high intensity and duration can acutely suppress melatonin and cause phase shifts or entrain circadian rhythms primarily by means of the nonvisual neural pathway of the retinohypothalamic tract.^{30,34,45-47,50} Input to the retinohypothalamic tract differs from the classic visual rod-and-cone system of the primary optic tract, relying rather on a small subset of retinal ganglion cells called the intrinsically photosensitive retinal ganglion cells.^{3,26,32,33,43,52,60} The neurophysiology of this newly detailed sensory pathway has important implications for consideration in the measurement, production, and application of light in all settings, including those of both human and laboratory animal environments.⁴³ Many investigators and animal care personnel, however, still assume that the use of light above 610 nm (orange- to red-appearing) for observation purposes, particularly at nighttime, results in little or no circadian disruptive effect on normal circadian physiology and behavior.^45,65 Arguably, sufficient scientific justification for the chronic use of red-tinted observation windows or red safelights, for examining potential neuroendocrine, neurobehavioral, metabolic, and physiologic parameters in laboratory animals is unavailable.In this study we examined the hypothesis that chronic, low-intensity red light exposure throughout an otherwise normal dark phase (that is, red light exposure at night, rLEN) disrupts circadian melatonin production and the temporal coordination of normal metabolic and physiologic activities in the nonpigmented rat species commonly used for the preclinical study of human physiology or pathophysiology. Our approach was to expose experimental rats, for several weeks and under highly controlled conditions, to a low-intensity safelight (1A Red Safelight, Eastman Kodak, Rochester, NY) during the daily 12-h dark phase and compare their responses with those of control animals maintained under total dark-phase conditions. Use of the safelight eliminated potentially confounding factors in spectral transmittance variability of the many types of red-tinted observation windows and coverings in common use today by ensuring that experimental animals were exposed only to red light in the visible spectrum of wavelengths above 620 nm. Light intensity, duration, and wavelength during light phase were the same for all animals.     

Efficacy of Various Analgesics on Shoulder Function and Rotator Cuff Tendon-to-Bone Healing in a Rat (Rattus norvegicus) Model

Although relief of postoperative pain is an imperative aspect of animal welfare, analgesics that do not interfere with the scientific goals of the study must be used. Here we compared the efficacy of different analgesic agents by using an established rat model of supraspinatus tendon healing and a novel gait-analysis system. We hypothesized that different analgesic agents would all provide pain relief in this model but would cause differences in tendon-to-bone healing and gait parameters. Buprenorphine, ibuprofen, tramadol–gabapentin, and acetaminophen were compared with a no-analgesia control group. Gait measures (stride length and vertical force) on the operative forelimb differed between the control group and both the buprenorphine (2 and 4 d postsurgery) and ibuprofen (2 d postsurgery) groups. Step length was different in the control group as compared with the tramadol–gabapentin (2 d after surgery), buprenorphine (2 and 4 d after surgery), and ibuprofen (2 d after surgery) groups. Regarding tendon-to-bone healing, the ibuprofen group showed less stiffness at the insertion site; no other differences in tendon-to-bone healing were detected. In summary, the analgesics evaluated were associated with differences in both animal gait and tendon-to-bone healing. This information will be useful for improving the management of postsurgical pain without adversely affecting tissue healing. Given its ability to improve gait without impeding healing, we recommend use of buprenorphine for postsurgical pain management in rats. In addition, our gait-analysis system can be used to evaluate new analgesics.The relief of postprocedural pain and distress is an imperative aspect of animal welfare. However adequate analgesia must be achieved without adverse effects on the goals of the study. Therefore, the detection and management of pain in animal research models is continually being studied and refined. Postprocedural pain is a complex process that involves hypersensitivity and hyperalgesia to several stimuli.^8,9,19,32,68 Furthermore, surgical procedures can cause pain through inflammation and the manipulation and damage of tissues.¹ Several methods for evaluating postprocedural pain in rodents involve variably subjective scoring systems and assessment of in-cage locomotor and behavior activity, hypersensitivity to stimuli, or observing food and water intake.^{37,39,43,53-55,61,63} An objective functional assessment test may provide a more reliable and quantifiable way to measure postoperative pain.Several rodent models to study musculoskeletal injuries are currently being used in biomedical research,^6,29,34 including a well-established rat model for rotator cuff injury.^49,51,56,60 This surgical model involves considerable injury to and manipulation of both bone and soft tissues. Because “it should be considered that procedures that cause pain in humans may also cause pain in vertebrate species,” it is clear that this model would also serve as a good model for significant postprocedural pain.^27,40,65 The objective of the current study was to compare the efficacy of different analgesic agents by using an established rat model for supraspinatus tendon healing and a novel gait-analysis system.⁵⁶We assessed different classes of analgesics, which we chose to represent common recommendations for postprocedural care. Buprenorphine is one of the most commonly used analgesics in laboratory animal medicine due to its proven analgesic qualities in rodents and other species.^{13,17,25,26,58} However, its status as a controlled substance may limit its use, and other options may be desirable. NSAID are often chosen for the management of postprocedural analgesia in both rodents and humans.^16,25,26,38 Because ibuprofen is used frequently after tendon repair in human medicine, we selected it for analysis in the current study. Due to the ease of administration, putting analgesics like acetaminophen in the drinking water of rodents has been a popular suggestion recently.^4,17,62 However, numerous studies have found variation in analgesic efficacy in rodents using acetaminophen in the drinking water.^{11,33,45,50,64} Finally, a tramadol–gabapentin combination was recently reported to have some analgesic effects in rats, but additional research is required.⁴⁴A secondary goal of the current study was to determine whether these commonly used analgesics affect tendon-to-bone healing. NSAID may have adverse effects on tendon healing,^10,14 but these are far less studied than are their effects on the healing of bone.^21,41,59 In addition, pain may influence cage activity levels, which consequently could change with the application of analgesics.^35,39,53 Increased activity may alter loads on the healing tissue as well as joint mobility, thus affecting tendon healing.^7,24,47,69In the current study, we used various spatial, temporal, and force parameters to analyze gait in the rat model of rotator cuff healing. In other species, pain in a forelimb decreases stride length, limb speed, and gait forces.^28,31,36,46 We expected to find similar changes in the gait of rats after surgery when analgesia is inadequate. Our custom gait-analysis system allowed us to measure several parameters, which were compared between treated and control groups to determine whether significant differences occurred. We used biomechanical testing procedures to determine how changes in tendon-to-bone healing after repair differed among the various analgesics. The weakest point of the tendon is the healing site, because of the development of new immature tissue, and changes in the mechanical properties of the repaired tendon indicate alterations in healing. Therefore, poor healing leads to decreases in the mechanical properties of the repaired tendon.^22,23 We hypothesized that the different analgesics evaluated all would provide pain relief in this model but would demonstrated differences in tendon-to-bone healing and in gait parameters compared with those of a no-analgesia control group.     

Effects of a Complex Housing Environment on Heart Rate and Blood Pressure of Rats at Rest and after Stressful Challenges

Housing enrichment for rodents continues to be a discussion topic within the animal care community. The objective of this study was to determine the extent to which a complex housing environment affects heart rate, blood pressure, and activity of rats when undisturbed and after exposure to stressful challenges and whether autonomic controls of heart rate would be affected. Male and female Sprague–Dawley and Wistar rats with radiotelemetry transmitters were evaluated under nonenriched single-housing conditions and after acclimation to a complex environment of dim light and cohabitation with 3 conspecifics in large cages with hiding, food foraging, and nesting items. Telemetry data were collected when rats were undisturbed, after acute challenges (cage change, intraperitoneal injections, restraint), during a forced running protocol, and after cholinergic or adrenergic blockade. The complex environment reduced heart rate and increased activity in undisturbed rats but did not affect blood pressure. Heart rate responses to challenges were unaffected, decreased, or increased by complex housing, depending on the stock and sex of rats. Forced running was either unaffected or decreased, depending on the stock and sex of rats. Heart rate responses to cholinergic or β1-adrenergic blockade were not affected. We conclude that the complex housing did not reduce indices of stress (for example, heart rate) as compared with simpler housing. However, the possibility that some environmental elements interact negatively with each other must be considered in future studies.The inclusion of environmental enrichment for individually housed rats is generally supported by a sizable volume of literature that shows positive effects on behavior, cognitive function, and recovery from induced neural deficits^{5,7,10,12,13,17-20,22-25,31,32} and on some parameters of wellbeing and various stress responses.^1,6,9,14 A recent review²¹ summarized the effects of enrichment and physical activity on cognitive function.One problem encountered when comparing the results in the various reports on environmental enrichment is that its specific elements are inconsistent across studies, and its effects have been variable depending on the type, onset, and duration of the enrichment; the age, strain, and sex of the animals; and the physiologic parameters examined. These issues have been reviewed recently.²⁹ Such inconsistencies of design and variable results complicate the decision of whether enrichment should be adopted and, if so, which type of enrichment is most effective. One approach is to adopt or recommend a simple program to minimize possible confounding effects on experimental outcomes. An alternative approach is to select a complex enrichment plan in the attempt to achieve maximal effect.The objective of the current study was to determine the extent to which a complex housing environment affects the heart rate, blood pressure, and activity of rats when undisturbed and the heart rate after exposure to stressful challenges and whether autonomic controls of heart rate would be affected. The underlying premise for these experiments was that rats housed individually without any form of environmental enrichment are believed to experience chronic stress, leading to increased heart rate and blood pressure due to altered autonomic drive to the heart and blood vessels, and that a complex environment can reduce or alleviate this chronic stress. Many of the elements that we combined to form the complex environment (10 lx room lighting,³ group housing,^2,27,28 and addition of inanimate hiding, food foraging, and nesting items^4,26) are those that we have shown previously to significantly reduce heart rate. The hypotheses were that the combined program would have larger effects than those previously reported for the separate environmental elements and would increase parasympathetic or decrease sympathetic input to the heart.     

Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

Nitric oxide (NO) is a ubiquitous mediator of inflammation and immunity, involved in the pathogenesis and control of infectious diseases, autoimmunity, and cancer. We observed that the expression of nitric oxide synthase-2 (NOS2/iNOS) positively correlates with Th17 responses in patients with ovarian cancer (OvCa). Although high concentrations of exogenous NO indiscriminately suppress the proliferation and differentiation of Th1, Th2, and Th17 cells, the physiological NO concentrations produced by patients’ myeloid-derived suppressor cells (MDSCs) support the development of RORγt(Rorc)⁺IL-23R⁺IL-17⁺ Th17 cells. Moreover, the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4⁺ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase (cGK) pathway of NO signaling within CD4⁺ T cells. Inhibition of NOS2 or cGMP–cGK signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. Our data indicate that, apart from its previously recognized role as an effector mediator of Th17-associated inflammation, NO is also critically required for the induction and stability of human Th17 responses, providing new targets to manipulate Th17 responses in cancer, autoimmunity, and inflammatory diseases.Nitric oxide (NO; a product of nitrite reduction or the NO synthases NOS1, NOS2, and NOS3; Culotta and Koshland, 1992), is a pleiotropic regulator of neurotransmission, inflammation, and autoimmunity (Culotta and Koshland, 1992; Bogdan, 1998, 2001; Kolb and Kolb-Bachofen, 1998) implicated both in cancer progression and its immune-mediated elimination (Culotta and Koshland, 1992; Coussens and Werb, 2002; Hussain et al., 2003; Mantovani et al., 2008). In different mouse models, NO has been paradoxically shown to both promote inflammation (Farrell et al., 1992; Boughton-Smith et al., 1993; McCartney-Francis et al., 1993; Weinberg et al., 1994; Hooper et al., 1997) and to suppress autoimmune tissue damage through nonselective suppression of immune cell activation (Bogdan, 2001; Bogdan, 2011), especially at high concentrations (Mahidhara et al., 2003; Thomas et al., 2004; Niedbala et al., 2011). Although previous studies demonstrated a positive impact of NO on the induction of Th1 cells (Niedbala et al., 2002) and forkhead box P3–positive (FoxP3⁺) regulatory T (T reg) cells (Feng et al., 2008) in murine models, the regulation and function of the NO synthase (NOS)–NO system have shown profound differences between mice and humans (Schneemann and Schoedon, 2002, Schneemann and Schoedon, 2007; Fang, 2004), complicating the translation of these findings from mouse models to human disease.In cancer, NOS2-derived NO plays both cytotoxic and immunoregulatory functions (Bogdan, 2001). It can exert distinct effects on different subsets of tumor-infiltrating T cells (TILs), capable of blocking the development of cytotoxic T lymphocytes (CTLs; Bronte et al., 2003), suppressing Th1 and Th2 cytokine production, and modulating the development of FoxP3⁺ T reg cells (Brahmachari and Pahan, 2010; Lee et al., 2011). NOS2-driven NO production is a prominent feature of cancer-associated myeloid-derived suppressor cells (MDSCs; Mazzoni et al., 2002; Kusmartsev et al., 2004; Vuk-Pavlović et al., 2010; Bronte and Zanovello, 2005), which in the human system are characterized by a CD11b⁺CD33⁺HLA-DR^low/neg phenotype consisting of CD14⁺ monocytic (Serafini et al., 2006; Filipazzi et al., 2007; Hoechst et al., 2008; Obermajer et al., 2011) and CD15⁺ granulocytic (Zea et al., 2005; Mandruzzato et al., 2009; Rodriguez et al., 2009) subsets (Dolcetti et al., 2010; Nagaraj and Gabrilovich, 2010).Production of NO in chronic inflammation is supported by IFN-γ and IL-17 (Mazzoni et al., 2002; Miljkovic and Trajkovic, 2004), the cytokines produced by human Th17 cells (Veldhoen et al., 2006; Acosta-Rodriguez et al., 2007a,b; van Beelen et al., 2007; Wilson et al., 2007). Human Th17 cells secrete varying levels of IFN-γ (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; Kryczek et al., 2009; Miyahara et al., 2008; van Beelen et al., 2007; Wilson et al., 2007) and have been implicated both in tumor surveillance and tumor progression (Miyahara et al., 2008; Kryczek et al., 2009; Martin-Orozco and Dong, 2009). Induction of Th17 cells typically involves IL-1β, IL-6, and IL-23 (Bettelli et al., 2006; Acosta-Rodriguez et al., 2007a,b; Ivanov et al., 2006; van Beelen et al., 2007; Veldhoen et al., 2006; Wilson et al., 2007; Zhou et al., 2007), with the additional involvement of TGF-β in most mouse models (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007; Ghoreschi et al., 2010), but not in the human system (Acosta-Rodriguez et al., 2007a; Wilson et al., 2007). IL-1β₁, IL-6, and IL-23 production by monocytes and DCs, and the resulting development of human Th17 cells, can be induced by bacterial products, such as LPS or peptidoglycan (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; van Beelen et al., 2007). However, the mechanisms driving Th17 responses in noninfectious settings, such as autoimmunity or cancer, remain unclear.Here, we report that the development of human Th17 cells from naive, effector, and memory CD4⁺ T cell precursors induced by the previously identified Th17-driving cytokines (IL-1β, IL-6, and IL-23) or by IL-1β/IL-6/IL-23-producing MDSCs, is promoted by exogenous NO (or NO produced by human MDSCs) and critically depends on the induction of endogenous NOS2 in differentiating CD4⁺ T cells.     

Assessment of Immune Activation in Mice before and after Eradication of Mite Infestation

Mite infestation of mice remains a persistent problem for many institutions, leading to numerous health problems and creating unknown and unwanted variables for research. In this study, mice with mite infestation demonstrated significantly higher levels of inflammatory cytokines, both at draining lymph nodes (axillary) and systemically, as compared with mice without mites. In addition, histologic evaluation revealed significant inflammation in mite-infested mice. Inflammatory changes were still present in the skin of mice at 6 to 8 wk after treatment, despite absence of detectable infestation at that time. Because these significant and lasting local and systemic changes have the potential to alter research findings, eradication of mites infestations should be an important goal for all institutions.Abbreviation: KC, keratinocyte-derived chemokine; MIP, macrophage inflammatory proteinLaboratory mice can harbor several species of acarids (fur mites), including Myobia musculi, Radfordia affinis, Myocoptes musculinus, and Psorergates simplex.^{11,14,29,40,45} Fur mites are an excluded pathogen in most research facilities, particularly within barrier suites, and in order to control or avoid mite infestations, many facilities, including those with ongoing infestations, will not accept infested animals from outside sources. Such policies can prevent or halt collaborative research between investigators in different institutions because mite infestation is a sporadic or endemic problem in many facilities that house mice under conventional conditions, despite attempts at eradication.^{12,22,25,43,62,69}Mite infestations cause several health problems in mice, including ulcerative dermatitis, amyloidosis, and other immune system alterations.^{2,12,22,27,29-31,37,44,45,61} For example, mite infestations are associated with increased serum concentrations of IgE and IgG in mice.^30,44,48 Alterations in immune responses could alter research data and thereby perhaps alter the associated conclusions.^36,65,66,70 Mice with mite infections often develop dermatitis, which can lead to bacterial infection and additional changes in immune status.^{15,30,31,45,61} Because any pathogenic infection can cause variability and alter basal measures of immune function, clinical chemistry, and behavior in mice, maintaining laboratory rodents in a disease-free state is crucial to their use for the collection of valid research data.⁵¹The eradication of external parasites is a difficult process. Many reports have been published that attempt mite eradication using various drug treatments,^{5-7,17,18,23,24,35,39,41-43,46,47,49,50,57,59,67} with each method having distinct advantages and disadvantages. Some, but not all, of these treatment regimens have been compared directly.¹⁰ The mite life cycle complicates treatment, because eggs and larvae can be less susceptible to drugs than are adult parasites.^2,19,20,55 In addition, mite eggs can contaminate the environment, providing a source for re-infection of treated animals.^20,63,64 Some drugs (for example, ivermectin) have been associated with toxicity and death in mice, especially among specific transgenic lines.^{8,12,28,53,55,69} Other drugs may require frequent or repeated treatment of the mice. Furthermore, the drugs themselves may have properties that alter physiology or immune function in animals.^2,13,60 The development of new veterinary drugs for treatment of parasites has increased the available therapies for rodent acariasis. Compounds such as fipronil and selamectin provide good efficacy against external parasites with limited side effects in mammals.^9,22,68Our facility housed a large colony of mice that occupied several rooms and were infested with Myocoptes musculinus and Myobia musculi. Although the majority of mite-infested mice had mild or no dermatitis, some infected mice had severe dermatitis. The goal of this study was to evaluate the local and systemic immune response in mice infested with mites. To our knowledge, this study is the first to comprehensively compare cytokine levels and histologic findings in mite-infested, treated, and mite-negative mice. We hypothesized that the immune response would be altered in mite-infested mice as demonstrated by significantly elevated cytokine levels in the draining lymph nodes or spleen as compared with mice that had never been infested with mites. In addition, we hypothesized that significant pathologic changes in the epidermis, dermis, and subcutaneous tissues would be present in response to mite infestation.     

Soiled Bedding Sentinels for the Detection of Fur Mites in Mice

Identification and eradication of murine fur mite infestations are ongoing challenges faced by many research institutions. Infestations with Myobia musculi and Myocoptes musculinus can lead to animal health problems and may impose unwanted research variables by affecting the immune and physiologic functions of mice. The purpose of this study was to evaluate the utility and efficacy of soiled bedding sentinels in the detection of fur mite infestations in colony mice. Female young-adult CRL:CD1(ICR) mice (n = 140) were exposed over a 12-wk period to various volume percentages of soiled bedding (11%, 20%, 50%, and 100%) from fur-mite–infested animals. Mice were tested every 2 wk with the cellophane tape test to identify the presence of fur mite adults and eggs. At the end of 12 wk, all mice exposed to 11%, 20%, and 50% soiled bedding tested negative for fur mites. One of the 35 mice (3%) receiving 100% soiled bedding tested positive for fur mites at the end of the 12-wk follow-up period. These findings suggest that the use of soiled bedding sentinels for the detection of fur mite infestations in colony mice is unreliable.Ectoparasite infestations present an ongoing threat to barrier facilities. Murine acariasis in laboratory mice frequently is caused by Myobia musculi, Myocoptes musculinus, and Radfordia affinis.^{1,13,17,40,41} These infestations can be challenging to identify and control and often lead to animal health problems and research complications. For this reason, many institutions strive to exclude these parasites from their barrier facilities.^1,17,18,41 Infestations can further compromise ongoing research by disrupting collaboration with institutions affected by sporadic or endemic mite infestations in their facilities.¹⁸Myocoptes musculinus is the most common fur mite identified among laboratory mice, although mixed infections with Myobia musculi are common.¹⁷ The life cycles of Myocoptes and Myobia are 14 and 23 d, respectively.^2,17 Myobia mites most frequently are found to inhabit the head and neck of mice, whereas Myocoptes are reported to have a predilection for the back, ventral abdomen, and inguinal regions.^2,17 Mite infestations in live animals are often diagnosed by using cellophane tape tests.^5,14,25 A clear piece of cellophane tape is pressed against the fur of the mouse, affixed to a slide, and examined microscopically for the presence of eggs or adult mites. Pelage collection and examination and skin scraping are 2 other common diagnostic methods. These tests have been shown to have increased sensitivity when compared with the tape test, but they have the disadvantage of requiring an anesthetized or recently euthanized animal.^2,5,17Fur mites feed on the superficial skin tissues and secretions of the animals they infest.^1,2,17 Mite infestations in mice have been associated with numerous health problems. Common clinical manifestations of acariasis include alopecia, pruritis, and scruffiness.^{1,2,10,15,17-20,22,26,31,42,44} Severe health problems including ulcerative dermatitis, hypersensitivity dermatitis, and pyoderma can develop also.^1,2,10,17,41 Infested mice may also be prone to secondary infections, reduced life span, and decreased body weight.^2,17,42 Several studies have analyzed the potential research complications associated with murine acariasis.^{10,15,18-20,22,26,31,42,44} Mite infestations have been shown to cause elevations in IgE, IgG, and IgA levels; mast cell degranulation; increased levels of inflammatory cytokines; and lymphocytopenia.^{18-20,22,26,31,44} The changes in the immunologic function of affected mice can persist even after mite eradication.¹⁸Multiple chemical treatment modalities have been proposed for the eradication of fur mites in infested animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} Conflicting information exists regarding the success of many of these treatment regimes. In addition, several of the proposed treatments have been associated with toxicity, adverse health effects in mice, and alterations in the physiologic or immune function of the animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} The complications associated with identifying an effective treatment for murine acariasis while minimizing toxicity and the introduction of unknown research variables highlight the importance of rapid and effective detection of mite infestations in barrier facilities.Many institutions rely on soiled bedding sentinels for their primary source of information on colony health status.^9,21,33,35 Several studies have demonstrated the efficacy of soiled bedding sentinels to detect common murine pathogens such as mouse hepatitis virus, mouse norovirus, Helicobacter spp., and pinworms.^{4,7,24,28,37,38} However, not all pathogens are easily transmitted through soiled bedding exposure. Agents that are not routinely identified through soiled bedding sentinels include those that are shed in low numbers, are susceptible to environmental factors, or are not easily transmitted through the fecal–oral route.^6,21,33 Examples of pathogens that are not easily transmitted or detected through soiled bedding exposure include mouse Sendai virus, Pasteurella pneumotropica, lymphocytic choriomeningitis virus, and cilia-associated respiratory bacillus.^7,9,11,16,35 In addition, the sensitivity of soiled bedding sentinel programs varies with the number of animals affected within the colony.^27,38In 2008, our institution faced a fur-mite outbreak that affected more than 25 rooms in a single barrier facility. Animals positive for Myobia musculi, Myocoptes musculinus, or both were identified through either health check requests for itching and scratching animals and by testing of animals scheduled for export to other institutions. Despite the extent of this outbreak, the soiled bedding sentinels in all mite-positive rooms consistently tested negative on cellophane tape tests for fur mites.To our knowledge, only one study has specifically examined the efficacy of soiled bedding sentinels in the detection of fur mites in mice.³⁴ A separate study, examining the transmission of mouse hepatitis virus to soiled bedding sentinels,³⁸ demonstrated that 75% of cages (3 of 4) exposed to soiled bedding from colony animals tested positive for fur mites after 19 wk of exposure. That previous study used 8 cages of 12 mice each; 4 cages received soiled bedding from colony animals, whereas the other 4 cages received clean nonsoiled bedding. In that study,³⁸ 56.3% of colony mice were known to be mite-positive. Other literature suggests that spread of mites to naïve animals requires direct contact and that soiled bedding does not serve as an effective mechanism for transmission.^1,17,23,39 However, we were unable to identify any research or experiments that substantiated these conclusions.The purpose of the present study was to evaluate whether CRL:CD1(ICR) mice housed in static microisolation caging on soiled bedding from mice with Myobia and Myocoptes infestations can be used as sentinels for the detection of fur mites and to determine how the efficacy of these soiled bedding sentinels for fur-mite detection varies with the prevalence of fur-mite infestation among colony animals.