Primary cell cultures for understanding rat epididymal inflammation.

Treatment-induced epididymal inflammation and granuloma formation is only an occasional problem in preclinical drug development, but it can effectively terminate the development of that candidate molecule. Screening for backup molecules without that toxicity must be performed in animals (generally rats) that requires at least 2 to 3 weeks of in vivo exposure, a great deal of specially synthesized candidate compound, and histologic examination of the target tissues. We instead hypothesized that these treatments induced proinflammatory gene expression, and so used mixed-cell cultures from the rat epididymal tubule to monitor the induction of proinflammatory cytokines. Cells were exposed for 24 hr and then cytotoxicity was evaluated with the MTS assay and mRNA levels of Interleukin-6 (IL-6) and growth-related oncogene (GRO) were measured. We found that compounds that were more toxic in vivo stimulated a greater induction of IL-6 and GRO mRNA levels in vitro. By relating effective concentrations in vitro with the predicted C(eff), we could rank compounds by their propensity to induce inflammation in rats in vivo. This method allowed the identification of several compounds with very low inflammatory induction in vitro. When tested in rats, the compounds produced small degrees of inflammation at an acceptable margin (approximately 20×), and have progressed into further development.

Comparative assessment of the timing of sexual maturation in male Wistar Han and Sprague-Dawley rats.

Given the increasing use of Wistar Han (WH) rats in regulatory toxicology studies, these studies were performed to characterize the onset of sexual maturation in maturing WH rats as compared to Sprague-Dawley (SD) rats. Beginning on postnatal day (PND) 38 through PND 91 groups (n=8) of untreated WH rats were evaluated for maturation of the male reproductive system. Testicular spermatid head counts increased beginning on PND 42 until PND 70. Sperm were detected in the caput, corpus, and cauda epididymis on PND 45, 49, and 49, respectively, and counts increased through PND 91. Sperm motility was at adult levels by PND 63. The morphology of the testis/epididymis of all animals at day 70 or older was consistent with qualitative sexual maturity. Based on these endpoints, WH rats were determined to be sexually mature at PND 70, and many of these endpoints evaluated in SD rats exhibited nearly identical trends.

Isoflurane reduces motile sperm counts in the Sprague-Dawley rat.

Animal and care use practices are constantly evolving. These can have unexpected consequences on the data collected from such procedures. One example is the recent change in our animal facility, based on recommendations from the Newcastle Consensus Meeting on Carbon Dioxide Euthanasia of Laboratory Animals, from CO(2) to isoflurane for anesthesia. The current study was conducted to determine the effects of isoflurane on sperm motility, as compared to two different CO(2) euthanasia procedures. Sperm motility was evaluated after euthanasia by a standard 5-minute CO(2) euthanasia procedure, an extended 10-minute CO(2) euthanasia procedure, or by isoflurane anesthesia followed by exsanguination (iso/exsanguination). The 5-minute CO(2) procedure produced sperm motility of 94.3 ± 1.7% motile sperm with 65.6 ± 16.8 sperm/field. By comparison, iso/exsanguination reduced that count to 3.3 ± 2.3 sperm/field and only 60.7 ± 32.0% motile sperm. The reduction in sperm motility after iso/exsanguination appeared to have been due primarily to the reduction in the number of sperm expelled from the vas deferens (3.3), compared to that after 5-minute CO(2) (65.6). This reduction in number of sperm available for evaluation, in the presence of a constant level of background debris, which was counted by the computer optics system as nonmotile sperm, resulted in an apparent reduction in motility. Using the extended 10-minute CO(2) procedure produced sperm data in between the other two extremes: 77.6 ± 36.1% motile sperm with 34.6 ± 28.3 sperm/field. The results of this study support the hypothesis that isoflurane inhibits contraction of the smooth muscle of the vas deferens, resulting in a decreased number of expelled sperm. Given these findings, it is important that careful consideration be taken to select an appropriate anesthesia/euthanasia method.