Spontaneous Background and Procedure-Related Microscopic Findings and Common Artifacts in Ocular Tissues of Laboratory Animals in Ocular Studies.

Identification of test article-related microscopic findings in ocular toxicology studies requires a working knowledge of the artifacts and procedure-related or background findings commonly encountered in such studies. The objective of this article is to provide a mini-atlas of the artifacts and procedure-related or spontaneous background findings commonly observed in ocular tissues from animals in toxicology studies of ocular drug candidates. Artifacts in the eye are often related to collection or fixation procedures and include swelling and vacuolation of lens fibers, separation of the neuroretina from the retinal pigment epithelium (RPE), and vacuolation of the optic nerve. Common in-life procedure-related findings include intravitreal injection needle tracks in the sclera and ciliary body pars plana and foci of RPE hypertrophy and/or hyperpigmentation at subretinal injection sites. Common background findings include corneal mineralization, uveal mononuclear cell infiltrates, and peripheral displacement of photoreceptor nuclei in the retina. A few uncommon spontaneous background findings that may be confused with test article-related findings, such as bilateral optic atrophy in macaques, are also included.

Utility of Serum miR-122, Liver Enzymes, and Hepatic Histopathology in Response to Hepatotoxicants in Sprague-Dawley Rats.

More than 80,000 chemicals are in commercial use worldwide. Hepatic metabolism to toxic intermediates is often a key mechanism leading to tissue damage and organ dysfunction. Effective treatment requires prompt detection of hepatotoxicity, ideally with rapid, minimally invasive diagnostic assays. In this study, archetypal histologic features of chemically induced hepatic injury were compared with clinical chemistries (including liver enzymes) and serum concentrations of microRNA-122 (miR-122, the processed form miR-122-5p), a biomarker of liver injury. The hepatotoxicants 4,4'-methylenedianiline (4,4'-MDA), allyl alcohol (AA), or carbon tetrachloride (CCl4) were orally administered to male Sprague-Dawley rats for 1, 5, 14, or 28 days to induce liver damage. Formalin-fixed, paraffin-embedded liver sections were evaluated histologically for inflammation, fibrosis, necrosis, and lipid accumulation. Liver enzymes were measured in serum, and serum miR-122 concentrations were assessed by quantitative polymerase chain reaction (qPCR). Histologic features of hepatic injury dose-dependently increased in both severity and frequency. Increases in liver enzymes and bilirubin were more pronounced in response to AA or 4,4'-MDA than to CCl4 at early time points. Elevated serum miR-122 levels in animals administered CCl4, AA, or 4,4'-MDA were more strongly associated with degree of hepatic histopathology than with dosage. Given this sensitive expression pattern postexposure, liver-specific miR-122 may improve the diagnostic accuracy of early hepatic injury.

An extended one-generation reproductive toxicity test of 1,2,4-Triazol-5-one (NTO) in rats.

Nitrotriazolone (1,2,4-triazol-5-one; NTO), an insensitive, energetic material used in explosive formulations, induced testicular toxicity and oligospermia in repeated-dose oral toxicity tests in rats. To evaluate whether NTO produces additional reproductive and developmental effects, a modified extended one-generation reproductive toxicity test was conducted. Rats were provided ad libitum access to NTO in drinking water at 0-, 144-, 720-, or 3600-mg/L NTO. Treatment of the parental generation began 2 (females) and 4 (males) wk premating and continued until weaning of litters. Direct dosing of offspring (F1) occurred from weaning through puberty. Pups were counted and weighed on postnatal day (PND) 0/1. Anogenital distance (AGD) was measured on PND 4 and males were examined for presence of nipples on PND 13. F1 offspring were examined daily for attainment of puberty. NTO did not markedly affect measures of fertility, including mating indices, gestation index, litter size, and sex ratio. Seminiferous tubule degeneration or atrophy was observed in P1 and F1 3600-mg/L NTO males. F1 males in the 3600 mg/L group exhibited reduced reproductive organ mass (testes, epididymides, and accessory sex organs). Nipple retention was increased in NTO exposed F1 males compared to controls. Attainment of puberty was delayed by 2.6 d in the 3600-mg/L NTO-exposed males relative to controls. Comparison of the effects of NTO with those of antiandrogens suggests absence of malformations of the genital tract in NTO-exposed males. This study supports previous findings indicating that NTO is a testicular toxicant with male developmental effects that may be secondary to testicular toxicity.

Going GLP: Conducting Toxicology Studies in Compliance with Good Laboratory Practices.

Good laboratory practice standards are US federal regulations enacted as part of the Federal Insecticide, Fungicide, and Rodenticide Act (40 CFR Part 160), the Toxic Substance Control Act (40 CFR Part 792), and the Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58) to support protection of public health in the areas of pesticides, chemicals, and drug investigations in response to allegations of inaccurate data acquisition. Essentially, good laboratory practices (GLPs) are a system of management controls for nonclinical research studies involving animals to ensure the uniformity, consistency, reliability, reproducibility, quality, and integrity of data collected as part of chemical (including pharmaceuticals) tests, from in vitro through acute to chronic toxicity tests. The GLPs were established in the United States in 1978 as a result of the Industrial Bio-Test Laboratory scandal which led to congressional hearings and actions to prevent fraudulent data reporting and collection. Although the establishment of infrastructure for GLPs compliance is labor-intensive and time-consuming, achievement and maintenance of GLP compliance ensures the accuracy of the data collected from each study, which is critical for defending results, advancing science, and protecting human and animal health. This article describes how and why those in the US Army Medical Department responsible for protecting the public health of US Army and other military personnel made the policy decision to have its toxicology laboratory achieve complete compliance with GLP standards, the first such among US Army laboratories. The challenges faced and how they were overcome are detailed.

Infusion of solutions of pre-irradiated components in rats.

INTRODUCTION: The objective of this study was to conduct a 14-day toxicology assessment for intravenous solutions prepared from irradiated resuscitation fluid components and sterile water. METHODS: Healthy Sprague Dawley rats (7-10/group) were instrumented and randomized to receive one of the following Field IntraVenous Resuscitation (FIVR) or commercial fluids; Normal Saline (NS), Lactated Ringer's, 5% Dextrose in NS. Daily clinical observation, chemistry and hematology on days 1,7,14, and urinalysis on day 14 were evaluated for equivalence using a two sample t-test (p<0.05). A board-certified pathologist evaluated organ histopathology on day 14. RESULTS: Equivalence was established for all observation parameters, lactate, sodium, liver enzymes, creatinine, WBC and differential, and urinalysis values. Lack of equivalence for hemoglobin (p=0.055), pH (p=0.0955), glucose (p=0.0889), Alanine-Aminotransferase (p=0.1938), albumin (p=0.1311), and weight (p=0.0555, p=0.1896), was deemed not clinically relevant due to means within physiologically normal ranges. Common microscopic findings randomly distributed among animals of all groups were endocarditis/myocarditis and pulmonary lesions. DISCUSSION: These findings are consistent with complications due to long-term catheter use and suggest no clinically relevant differences in end-organ toxicity between animals infused with FIVR versus commercial fluids.

Peri-pubertal administration of 3-nitro-1,2,4-triazol-5-one (NTO) affects reproductive organ development in male but not female Sprague Dawley rats.

Nitrotriazolone (3-nitro-1,2,4-triazol-5-one; NTO) is an insensitive munition that has demonstrated effects on reproductive organs in adult male rats. NTO was administered to male (0, 250, and 500milligrams per kilogram per day (mg/kg-day)) and female (0, 500, and 1000mg/kg-day) Sprague-Dawley rats (15/sex/group) via oral gavage from weaning through post-natal day 53/54 and 42/43, respectively. Age and body mass at vaginal opening (VO) and preputial separation (PPS), as well as all measures of estrous cyclicity were not affected by treatment with NTO. Males treated with NTO exhibited reductions in testis mass associated with tubular degeneration/atrophy. Less pronounced reductions in accessory sex organ masses were also observed in the 500mg/kg-day group. Treatment with NTO did not affect thyroid hormone or testosterone levels. These findings suggest that NTO is not acting as an estrogen or thyroid active compound, but may indicate effects on steroidogenesis and/or direct testicular toxicity.

Acute and chronic plasma metabolomic and liver transcriptomic stress effects in a mouse model with features of post-traumatic stress disorder.

Acute responses to intense stressors can give rise to post-traumatic stress disorder (PTSD). PTSD diagnostic criteria include trauma exposure history and self-reported symptoms. Individuals who meet PTSD diagnostic criteria often meet criteria for additional psychiatric diagnoses. Biomarkers promise to contribute to reliable phenotypes of PTSD and comorbidities by linking biological system alterations to behavioral symptoms. Here we have analyzed unbiased plasma metabolomics and other stress effects in a mouse model with behavioral features of PTSD. In this model, C57BL/6 mice are repeatedly exposed to a trained aggressor mouse (albino SJL) using a modified, resident-intruder, social defeat paradigm. Our recent studies using this model found that aggressor-exposed mice exhibited acute stress effects including changed behaviors, body weight gain, increased body temperature, as well as inflammatory and fibrotic histopathologies and transcriptomic changes of heart tissue. Some of these acute stress effects persisted, reminiscent of PTSD. Here we report elevated proteins in plasma that function in inflammation and responses to oxidative stress and damaged tissue at 24 hrs post-stressor. Additionally at this acute time point, transcriptomic analysis indicated liver inflammation. The unbiased metabolomics analysis showed altered metabolites in plasma at 24 hrs that only partially normalized toward control levels after stress-withdrawal for 1.5 or 4 wks. In particular, gut-derived metabolites were altered at 24 hrs post-stressor and remained altered up to 4 wks after stress-withdrawal. Also at the 4 wk time point, hyperlipidemia and suppressed metabolites of amino acids and carbohydrates in plasma coincided with transcriptomic indicators of altered liver metabolism (activated xenobiotic and lipid metabolism). Collectively, these system-wide sequelae to repeated intense stress suggest that the simultaneous perturbed functioning of multiple organ systems (e.g., brain, heart, intestine and liver) can interact to produce injuries that lead to chronic metabolic changes and disorders that have been associated with PTSD.

Heat shock stress prior to hyperbaric oxygen exposure in rats.

INTRODUCTION: Heat shock proteins (HSPs) and nuclear factor-kappa B (NF-kappaB) have been established as important mediators in lung injury; however, their role in preventing pulmonary toxicity from hyperbaric oxygen (HBO) has not been evaluated. METHODS: We aimed to study the effects of heat shock (HS) injury on hyperbaric hyperoxic lung injury (HHLI) in a rat model and identify a mechanism of protection by evaluating HSP 27 and HSP 70 mRNA and protein levels, NF-kappaB p65, lung injury and oxidative parameters. By varying the times between HS and exposure to HBO, the pathways of interaction between HSPs and NF-kappaB will be further clarified. RESULTS: Our results showed that HS exposure increases the mRNA and protein levels of HSP 27 and HSP 70; HS induced 10-fold increases of HSP 27 (9.77 +/- 0.60) and HSP 70 (10.33 +/- 2.4) within the first 10 h compared to control animals. Lesion scores were higher for the first 16 h after HS, but decreased again after 31 h (N = 7 animals; 5 lesions scores). Protein nitration showed no significant differences between groups exposed to HS or HBO; similarly there was no difference with a combination of both treatments. DISCUSSION: HBO appears to attenuate the HS response by HSP 27 and HSP 70. Histopathology results suggest that HS might mitigate pathology in animals exposed to HS and HBO. No significant effect of HS on HBO-induced HHLI was observed in animals treated with both HS and HBO.

Initial evaluation of a nano-engineered hemostatic agent in a severe vascular and organ hemorrhage swine model.

OBJECTIVES: An advanced hemostatic dressing, Rapid Trauma Hemostat (RTH), was developed using nano-engineered inorganic nanofibers with hemostatic surface properties. METHODS: Yorkshire swine were treated with RTH or Combat Gauze (CBG) to stop bleeding from either an arterial puncture (G-RTH and G-CBG) or a liver lobe laceration (L-RTH and L-CBG). All animals received 500 mL of Hextend at 10 minutes after injury and were monitored for a total time of 180 minutes. RESULTS: Uncontrolled hemorrhage was similar in all animals in both models and was immediately controlled with the application of either dressing. After blood pressure was restored with fluid resuscitation, the RTH hemostatic treatment was less effective than CBG in the groin (puncture) model (rebleeding incidence, four of seven for G-RTH vs. one of seven for G-CBG; p = 0.034) but showed similar efficacy in the liver injury model (lower pressure bleeding). Interestingly, RTH exhibited a trend for higher efficacy in terms of hemostatic plug formation at the end of the experiment (no bleeding occurred after dressing removal) in the liver injury model. CONCLUSION: Overall, RTH was not as effective at stopping high-shear rate (arterial) bleeding, but it presented some advantages for intracavitary treatment with potential for long-term evacuation.

Murine model of repeated exposures to conspecific trained aggressors simulates features of post-traumatic stress disorder.

We evaluated repeated exposures of mice to a trained aggressor mouse as a model (adapted from "social stress" models of traumatic stress) for aspects of post-traumatic stress disorder (PTSD). Using a "cage-within-cage resident-intruder" protocol, subject C57BL/6J mice were exposed to aggressors for 6 h daily for 5 or 10 days. At one to three random times during each 6-h session, subjects were exposed directly to aggressor for 1 min or 10 bites, whichever came first. Behavioral, physiological, and histological changes associated with aggressor-exposure were assessed for up to 6 weeks. During aggressor exposure, subjects displayed less territorial behavior, gained weight, and increased body temperature. One day after the last aggressor exposure, inflammatory cardiac histopathologies were prevalent; after 10 days, only mild myocardial degeneration with fibrosis or fibroplasias was evident, while controls showed almost no cardiac abnormalities at any time. After 4 weeks, the medial prefrontal cortex of control mice showed increased dendritic spine density, but aggressor-exposed mice showed no increase. Behaviors affected by aggressor exposure were evaluated in a partition test wherein the subject mouse is separated from the aggressor by a fenestrated partition that permits sensory cues to pass but prevents direct physical interaction. For up to 4-6 weeks after the last aggressor exposure, subjects showed prolonged grooming, freezing, retarded locomotion and no tail rattling. PTSD and its co-morbidities are often consequent to repeated aggravated "social" assaults (e.g., combat) and manifest socially over time, suggesting the relevance of this repeated aggressor-exposure model to clinical aspects of PTSD.

Pathogenesis of Escherichia coli O157:H7 strain 86-24 following oral infection of BALB/c mice with an intact commensal flora.

Escherichia coli O157:H7 is a food-borne pathogen that can cause hemorrhagic colitis and, occasionally, hemolytic uremic syndrome, a sequela of infection that can result in renal failure and death. Here we sought to model the pathogenesis of orally-administered E. coli O157:H7 in BALB/c mice with an intact intestinal flora. First, we defined the optimal dose that permitted sustained fecal shedding of E. coli O157:H7 over 7 days ( approximately 10(9) colony forming units). Next, we monitored the load of E. coli O157:H7 in intestinal sections over time and observed that the cecum was consistently the tissue with the highest E. coli O157:H7 recovery. We then followed the expression of two key E. coli O157:H7 virulence factors, the adhesin intimin and Shiga toxin type 2, and detected both proteins early in infection when bacterial burdens were highest. Additionally, we noted that during infection, animals lost weight and approximately 30% died. Moribund animals also exhibited elevated levels of blood urea nitrogen, and, on necropsy, showed evidence of renal tubular damage. We conclude that conventional mice inoculated orally with high doses of E. coli O157:H7 can be used to model both intestinal colonization and subsequent development of certain extraintestinal manifestations of E. coli O157:H7 disease.

Altered gene expression in asymptomatic SHIV-infected rhesus macaques (Macacca mulatta).

Simian-Human immunodeficiency virus is a chimeric virus which, in rhesus macaques (Macacca mulatta) closely imitates immunodeficiency virus infection in human (HIV). A relatively new way to study pathogenesis of viral infection is to study alterations in host gene expression induced by the virus. SHIV infection with certain strains does not result in clinical signs. We hypothesized that alterations in gene expression relating to the immune system would be present in SHIV-infected animals despite the lack of clinical signs. Splenic tissue from four adult male Indian-origin Rhesus monkeys serologically positive for non-pathogenic SHIV 89.6 was processed by cDNA microarray analysis. Results were compared with the corresponding outcome using splenic tissues from four unexposed adult male Rhesus monkeys. Subsequent gene analysis confirmed statistically significant variations between control and infected samples. Interestingly, SHIV-infected monkeys exhibited altered expression in genes related to apoptosis, signal transduction, T and B lymphocyte activation and importantly, to immune regulation. Although infected animals appeared asymptomatic, our study demonstrated that SHIV-infected monkeys cannot reliably be used in studies of other infectious agents as their baseline gene expression differs from that of normal Rhesus monkeys. The gene expression differences in SHIV-infected animals relative to uninfected animals offer additional clues to the pathogenesis of altered immune function in response to secondary infection.