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BackgroundTarget organ toxicity is often a reason for attritions in nonclinical and clinical drug development. Leveraging emerging safety biomarkers in nonclinical studies provides an opportunity to monitor such toxicities early and efficiently, potentially translating to early clinical trials. As a part of the European Union’s Innovative Medicines Initiative (IMI), two projects have focused on evaluating safety biomarkers of nervous system (NS) toxicity: Translational Safety Biomarker Pipeline (TransBioLine) and Neurotoxicity De-Risking in Preclinical Drug Discovery (NeuroDeRisk).MethodsPerformance of fluid-based NS injury biomarker candidates neurofilament light chain (NF-L), glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE) and total Tau in plasma and cerebrospinal fluid (CSF) was evaluated in 15 rat in vivo studies. Model nervous system toxicants as well as other compounds were used to evaluate sensitivity and specificity. Histopathologic assessments of nervous tissues and behavioral observations were conducted to detect and characterize NS injuries. Receiver operator characteristic (ROC) curves were generated to compare the relative performance of the biomarkers in their ability to detect NS injury.ResultsNF-L was the best performer in detecting both peripheral nervous system (PNS) and CNS injury in plasma, (AUC of 0.97–0.99; respectively). In CSF, Tau correlated the best with CNS (AUC 0.97), but not PNS injury. NSE and GFAP were suitable for monitoring CNS injury, but with lesser sensitivity. In summary, NF-L is a sensitive and specific biomarker in rats for detecting compound-induced central and peripheral NS injuries. While NF-L measurement alone cannot inform the site of the injury, addition of biomarkers like Tau and NSE and analysis in both blood and CSF can provide additional information about the origin of the NS injury.ConclusionThese results demonstrate the utility of emerging safety biomarkers of drug-induced NS injury in rats and provide additional supporting evidence for biomarker translation across species and potential use in clinical settings to monitor drug-induced NS injury in patients.

Introduction: Wistar Han rats are a preferred strain of rodents for general toxicology and safety pharmacology studies in drug development. In some of these studies, visual functional tests that assess for retinal toxicity are included as an additional endpoint. Although the influence of gender on human retinal function has been documented for more than 6 decades, preclinically it is still uncertain if there are differences in retinal function between naïve male and female Wistar Han rats.Methods: In this study, sex-related differences in the retinal function were quantified by analyzing electroretinography (ERG) in 7-9-week-old (n = 52 males and 51 females) and 21–23-week-old Wistar Han rats (n = 48 males and 51 females). Optokinetic tracking response, brainstem auditory evoked potential, ultrasonic vocalization and histology were tested and evaluated in a subset of animals to investigate the potential compensation mechanisms of spontaneous blindness.Results/Discussion: Absence of scotopic and photopic ERG responses was found in 13% of 7-9-week-old (7/52) and 19% of 21–23-week-old males (9/48), but none of female rats (0/51). The averaged amplitudes of rod- and cone-mediated ERG b-wave responses obtained from males were significantly smaller than the amplitudes of the same responses from age-matched females (−43% and −26%, respectively) at 7–9 weeks of age. There was no difference in the retinal and brain morphology, brainstem auditory responses, or ultrasonic vocalizations between the animals with normal and abnormal ERGs at 21–23 weeks of age. In summary, male Wistar Han rats had altered retinal responses, including a complete lack of responses to test flash stimuli (i.e., blindness), when compared with female rats at 7–9 and 21–23 weeks of age. Therefore, sex differences should be considered when using Wistar Han rats in toxicity and safety pharmacology studies with regards to data interpretation of retinal functional assessments.

Abstract Background CBA/J mice are standard experimental animals in auditory studies, and age-related changes in auditory pathways are well documented. However, changes in locomotion-related brain regions have not been systematically explored. Results We showed an increase in immunoreactivity for glial fibrillary acidic protein (GFAP) in the cerebellar molecular layer associated with Purkinje cells in mice at 24 weeks of age but not in the younger mice. Increased GFAP immunoreactivity appeared in the form of clusters and distributed multifocally consistent with hyperplasia of astrocytes that were occasionally associated with Purkinje cell degeneration. Three out of 12 animals at 16 and 24 weeks of age exhibited pre-convulsive clinical signs. Two of these 3 animals also showed increased GFAP immunoreactivity in the cerebellum. Rotarod behavioral assessments indicated decreased performance at 24 weeks of age. Conclusions These results suggest minimal to mild reactive astrocytosis likely associated with Purkinje cell degeneration in the cerebellum at 24 weeks of age in CBA/J mice. These findings should be taken into consideration prior to using this mouse strain for studying neuroinflammation or aging.

Rodents emit ultrasonic vocalizations (USVs) above the human hearing threshold of ~ 20 kHz to communicate emotional states and to coordinate their social interactive behavior. Twenty-two kHz USVs emitted by adult rats have been reported in a variety of aversive social and behavioral situations. They occur not only under painful or restraining conditions but can also be evoked by gentle cutaneous touch or airflow. This study aimed to test if placement of a human hand in a cage can evoke 22-kHz USVs. It was found that 36% of the adult male Sprague-Dawley and 13% of the adult male Wistar Han rats emitted 22-kHz USVs when a gloved hand was introduced into the cages. Average vocalization onset latencies were 5.0 ± 4.4 s (Sprague-Dawley) and 7.4 ± 4.0 s (Wistar Han) and the USVs had a stable frequency (22 kHz) across the calls, ranging from 0.1 to 2.3 seconds in duration. Surprisingly, no 22-kHz USVs were found in any female Wistar Han rats tested. To further explore the mechanisms underlying this observation, we compared retinal function, basal serum corticosterone, and testosterone levels between the 22-kHz USV responders and non-responders. None of these parameters or endpoints showed any significant differences between the two cohorts. The results suggest that the introduction of a gloved-hand inside the cage can trigger adult male albino rats to emit 22-kHz ultrasonic vocalizations. This response should be considered in USV studies and animal welfare.