Revised guides for organ sampling and trimming in rats and mice – Part 3

STP Position Paper

“…Globes are fixed for 18 to 48 hr (depending on their size) and then transferred to 70% ethanol until trimming. Fixation in NBF has been reported to be acceptable for ocular preservation in long-term rodent studies (Morawietz et al 2004), but the experience of Working Group members is that NBF is less suited for ocular fixation both because its relatively poor penetration power permits autolysis of the retina and tissues (especially the lens) often are so brittle that acquisition of intact sections is difficult.…”

“…The eye with the optic nerve is the only special sense organ that commonly is evaluated during nonclinical general toxicity studies (Morawietz et al 2004). The best preservation is achieved if the globe and attached optic nerve are gently removed from the orbit as soon as possible at necropsy, followed by removal of extraocular fat and skeletal muscle; glands associated with the eye (e.g., Harderian) may be left in place.…”

“…A potential advantage of this approach is that the CNS/PNS interface (including dorsal root ganglia [DRG]) may remain intact in microscopic sections. The main pitfalls of this method are that neural structures may be distorted by incomplete fixation and/or extended decalcification (Morawietz et al 2004). Spinal cord evaluation in situ typically is undertaken for non-GLP projects rather than for GLP-type general toxicity studies.…”

“…However, substantial variation exists across regulatory agencies (Bolon et al 2011a) regarding preferred neuropathology practices for registering new compounds. The recommendations vary by species (non-rodents [e.g., dog, nonhuman primate vs. rodents; Krinke 1989;Morawietz 2004]); by age (e.g., developing rodents [U.S. Environmental Protection Agency {EPA} 1998b; Organisation for Economic Co-operation and Development {OECD} 2007;Garman et al 2001;Bolon et al 2006Bolon et al , 2011b vs. adult rodents [Broxup 1991;EPA 1998a;OECD, 1997]); by the type of study (general toxicity screen vs. dedicated neurotoxicity bioassay; Bolon et al 2011a); by the kind of industry (agrochemical firms vs. pharmaceutical companies, for which potential exposure levels and, therefore, risk-to-benefit assessments will vary); and according to whether the study was conducted by Good Laboratory Practices (GLP) standards to support product registration. Regulatory guidelines for conducting the neuropathology analysis of GLP-type general toxicity studies (i.e., screening or ''Tier I'' surveys) provide wide-ranging advice for designing experiments that will evaluate many disparate organs and systems.…”

“…The routine practice at many institutions for rodent brain sampling evaluates three or four brain levels (typically rostral forebrain [cerebral cortex and basal nuclei], caudal forebrain [cerebral cortex and hippocampus with either diencephalon or rostral midbrain], and hindbrain [usually cerebellum with pons and/ or cerebellum with medulla oblongata]; Morawietz et al 2004). In contrast, a recent discourse on comprehensive brain sampling (Switzer, Lowry-Franssen, and Benkovic 2011) reported that small structural lesions elicited by half of the 14 known chemical and pharmaceutical neurotoxicants reviewed might not (N ¼ 3; domoic acid, kainic acid, and methamphetamine) or would not (N ¼ 4; alcohol, carbonyl sulfide, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP], and 2-amino-MPTP) be detected by conventional 3-level sampling of rodent brain.…”