Neuroanatomy-based matrix-guided trimming protocol for the rat brain.

Brain trimming through defined neuroanatomical landmarks is recommended to obtain consistent sections in rat toxicity studies. In this article, we describe a matrix-guided trimming protocol that uses channels to reproduce coronal levels of anatomical landmarks. Both setup phase and validation study were performed on Han Wistar male rats (Crl:WI(Han)), 10-week-old, with bodyweight of 298 ± 29 (SD) g, using a matrix (ASI-Instruments(®), Houston, TX) fitted for brains of rats with 200 to 400 g bodyweight. In the setup phase, we identified eight channels, that is, 6, 8, 10, 12, 14, 16, 19, and 21, matching the recommended landmarks midway to the optic chiasm, frontal pole, optic chiasm, infundibulum, mamillary bodies, midbrain, middle cerebellum, and posterior cerebellum, respectively. In the validation study, we trimmed the immersion-fixed brains of 60 rats using the selected channels to determine how consistently the channels reproduced anatomical landmarks. Percentage of success (i.e., presence of expected targets for each level) ranged from 89 to 100%. Where 100% success was not achieved, it was noted that the shift in brain trimming was toward the caudal pole. In conclusion, we developed and validated a trimming protocol for the rat brain that allow comparable extensiveness, homology, and relevance of coronal sections as the landmark-guided trimming with the advantage of being quickly learned by technicians.

Hyaline droplet accumulation in kidney of rats treated with hexachloro-1:3-butadiene: influence of age, dose and time-course.

The present research investigates the occurrence of hyaline droplet (HD) accumulation related to age, dose and time after treatment in male Wistar rats given a single i.p. injection of hexachloro-1:3-butadiene (HCBD). In the study on age, rats from 1 to 12 months of age were treated with 100 mg kg(-1) body weight (b.w.) HCBD dose. Rats treated at 2 months of age showed a greater accumulation of HD than the other age groups; HD accumulation was not observed in 1-month-old rats. In the dose-response study, the treatment with 25, 50 and 100 mg kg(-1) b.w. at 2 months of age caused HD accumulation in the proximal convoluted tubule at all doses, with the 100 mg kg(-1) b.w. group slightly more affected. Finally, in the time-course study, rats treated with a 100 mg kg(-1) b.w. dose at 2 months of age and sacrificed at 6, 12, 24, 48, 72 and 96 h post-dosing showed a time-related HD accumulation in terms of incidence and severity, after 6 h, with a peak at 24 and 48 h and decreasing at 72 and 96 h. The present results show that HD accumulation is an early finding, and is unrelated to dose level and particularly evident in rats of 2 month of age. These findings in male rats treated with HCBD emphasize the importance of considering the age of rats at the start of a study. The more sensitive model was used in the detection of nephrotoxic effects of chemicals.

A novel and integrated approach for the identification and characterization of drug-induced cardiac toxicity in the dog.

Cardiovascular toxicity represents one of the major reasons for the termination of the development of drugs, even in late development phases. This growing issue is often not restricted to specific therapeutic areas, and it is gaining critical importance, in particular for chronically administered drugs, highlighting the limitations in terms of sensitivity of the current investigational paradigms. Furthermore, drug-related changes may become evident after long-term administration for different reasons, including accumulation of the drug in the heart. This article describes how the integrated use of investigational tools represents a powerful approach for the early identification and characterization of cardiotoxicity in preclinical development. Cardiac changes were observed in the dog after long-term oral administration of casopitant, a neurokinin 1 receptor antagonist, developed for the treatment of depression and anxiety. Different approaches and sensitive biomarkers were used in a time-course study to investigate the onset, progression, and reversibility of the lesion. The integrated evaluation of cardiovascular parameters, electron microscopy, troponin I, and natriuretic peptide results highlighted any minimal early changes, allowing the full and deep characterization of the lesion. The outcome of this study was the driver for drug development decision making on casopitant and backup drugs.

Mouse pneumonia model by Acinetobacter baumannii multidrug resistant strains: Comparison between intranasal inoculation, intratracheal instillation and oropharyngeal aspiration techniques.

Infectious pneumonia induced by multidrug resistant (MDR) Acinetobacter baumannii strains is among the most common and deadly forms of healthcare acquired infections. Over the years, different strategies have been put in place to increase host susceptibility to MDR A. baumannii, since only a self-limiting pneumonia with no or limited local bacterial replication was frequently obtained in mouse models. Direct instillation into the trachea or intranasal inoculation of the bacterial suspension are the techniques used to induce the infection in most of the preclinical models of pneumonia developed to date. More recently, the oropharyngeal aspiration procedure has been widely described in the literature for a variety of purposes including pathogens administration. Aim of this study was to compare the oropharyngeal aspiration technique to the intranasal inoculation and intratracheal instillation in the ability of inducing a consistent lung infection with two MDR A. baumannii clinical isolates in immunocompromised mice. Moreover, pneumonia obtained by bacteria administration with two out of three techniques, intratracheal and oropharyngeal, was characterised in terms of histopathology of pulmonary lesions, biomarkers of inflammation level and leukocytes cells infiltration extent after mice treatment with either vehicle or the antibiotic tigecycline. The data generated clearly showed that both strains were not able to colonize the lungs when inoculated by intranasal route. By contrast, the bacterial load in lungs of mice intratracheally or oropharyngeally infected significantly increased during 26 hours of monitoring, thus highlighting the ability of these strains to generate the infection when directly instilled into the lower respiratory airways. Furthermore, the intragroup variability of mice was significantly reduced with respect to those intranasally administered. Tigecycline was efficacious in lung bacterial load and cytokines release reduction. Findings were supported by semi-quantitative histopathological evaluation of the pulmonary lesions and by inflammatory biomarkers analysis. To conclude, both intratracheal instillation and oropharyngeal aspiration techniques showed to be suitable methods for inducing a robust and consistent pneumonia infection in mice when difficult MDR A. baumannii clinical isolates were used. Noteworthy, oropharyngeal aspiration not requiring specific technical skills and dedicated equipment, was proven to be a safer, easier and faster technique in comparison to the intratracheal instillation.

Kidney injury molecule-1 expression in rat proximal tubule after treatment with segment-specific nephrotoxicants: a tool for early screening of potential kidney toxicity.

Dose-response expression of kidney injury molecule-1 (KIM-1) gene in kidney cortex and its correlation with morphology and traditional biomarkers of nephrotoxicity (plasma creatinine and blood urea nitrogen, BUN) or segment-specific marker of proximal tubule injury (kidney glutamine synthetase, GSK) were studied in male rats treated with proximal tubule segment-specific nephrotoxicants. These included hexachloro-1:3-butadiene (HCBD, S(3) segment-specific), potassium dichromate (chromate, S(1)-S(2) segment-specific), and cephaloridine (Cph, S(2) segment-specific). Rats were treated with a single intraperitoneal (ip) injection of HCBD 25, 50, and 100 mg/kg, subcutaneous (sc) injection of chromate 8, 12.5, and 25 mg/kg; or ip injection of Cph 250, 500, and 1,000 mg/kg. KIM-1 gene showed a dose-dependent up-regulation induced by all segment-specific nephrotoxicants. Interestingly, magnitude of the up-regulation reflected the severity of microscopic tubular changes (degeneration, necrosis, and regeneration). Even low-severity microscopic observations were evidenced by significant gene expression changes. Furthermore, KIM-1 showed significant up-regulation even in the absence of morphological changes. In contrast, traditional and specific markers demonstrated low sensitivity or specificity. In conclusion, this study suggested KIM-1 as a sensitive molecular marker of different levels of tubular injury, and it is likely to represent a potential tool for early screening of nephrotoxicants.

Evaluation of aging influence on renal toxicity caused by segment-specific nephrotoxicants of the proximal tubule in rat.

Little is known concerning the sensitivity of aged rats to xenobiotics inducing kidney damage. To increase this knowledge, the age-dependent response of the kidney to hexachloro-1 : 3-butadiene (HCBD) or potassium dichromate (chromate) was investigated. Rats were treated at different ages with a single dose of segment-specific nephrotoxicants of the proximal tubule, chosen on the basis of their specificity for S(3) and for S(1)-S(2) segments, respectively. The toxicological impact of these xenobiotics has been evaluated through biochemical and genomic markers, and histopathological investigation of kidney samples. HCBD treatment induced tubular necrosis of the S(3) segment of the proximal tubule associated with changes of toxicological markers unrelated to the age. In contrast, chromate treatment induced an increased kidney damage related to the rat age. In fact, histopathological investigation revealed that at 1 month of age tubular vacuolar degeneration was seen affecting S(1)-S(2) segments of the proximal tubule, whereas at 3 months of age tubular necrosis occurred in the same segments associated with tubular dilation of the distal portions. Consistently, biochemical analysis confirmed a direct correlation among genomic and biochemical marker variability and animal age. Altogether, the results show that during aging there is an increased sensitivity of kidney to chromate but not to HCBD-induced damage and evidence differential age-related selectivity of rats for nephrotoxic compounds. Significance for human risk assessment is discussed.

Regucalcin down-regulation in rat kidney tissue after treatment with nephrotoxicants.

Gene expression of regucalcin (Rgn), a calcium-binding protein, was investigated in kidney of male Wistar rats treated with proximal tubule segment-specific nephrotoxicants, namely hexachloro-1:3-butadiene (HCBD), specific for S(3) segment (pars recta) and potassium dichromate (chromate) specific for S(1)-S(2) segments (pars convoluta), according to age of animals and dose of chemicals. In the age-dependent study, male Wistar rats were treated with a single injection of HCBD (100mg/kg b.w. i.p.) or chromate (25 mg/kg b.w. s.c.) at 5 weeks or 12 weeks of age; in dose-response study, rats were treated with a single injection of three doses of HCBD (25, 50, and 100 mg/kg b.w. i.p.) or chromate (8, 12.5, and 25mg/kg b.w. s.c.) at 8 weeks of age. Forty-eight hours after treatment, Rgn and glutamine synthetase (GS) activity in kidney cortex, blood urea nitrogen (BUN) and plasma creatinine were measured; light microscopy was performed also. The results show that young rats are less susceptible to chromate (severe necrosis is evident only in adult rats), whereas age does not influence HCBD nephrotoxicity. Rgn is down regulated by HCBD at both age points, but not by chromate at 5 weeks of age. In addition, HCBD causes down-regulation of Rgn from the low dose in 8-week-old rats, whereas chromate causes the same effect at the high dose only. GS activity in kidney cortex shows a similar behavior, even if sensitive to low doses of chromate also, whereas BUN and creatinine increase after the high dose of both chemicals only. Accordingly, light microscopy shows a segment-specific, dose-dependent increase of severity of damage caused by the chemicals. Rgn gene expression appears a sensitive genomic marker to evaluate the renal impairment caused by chemicals and its down-regulation seems to be related to damage, early or already established, to S(3) segment of the proximal tubule.

Early onset of spontaneous renal preneoplastic and neoplastic lesions in young conventional rats in toxicity studies.

Although occurring in aged laboratory rodents, spontaneous renal tumour are unknown in animals younger than 18 weeks. A survey on renal preneoplastic and neoplastic lesions has been performed on Sprague-Dawley rats from general toxicology studies over the period January 2004 - May 2006. Data from 2249 rats necropsied and 1206 rats (688 males and 518 females) examined microscopically from 52 studies, are reported. The age range at necropsy was between 12 and 18 weeks and all the animals were obtained from the same supplier. Three cases of tubular carcinoma, 1 tubular adenoma, and 4 cases of atypical tubular hyperplasia were observed in 5 females and 3 males from both control and treated groups from 6 studies with unrelated test compounds. In treated rats, the lesions were considered spontaneous in nature, rather than related to treatment, because of: (1) their sporadic incidence, (2) the short duration of the studies, and (3) the absence of similar lesions in other rats given the same test compound. These lesions are considered a recently occurring spontaneous finding, and the similarities with the familial renal cancer models, namely the Eker and the Nihon models, strongly suggest genetic factors as responsible for the lesions.