Absence of functional deficits in rats following systemic administration of an AAV9 vector despite moderate peripheral nerve and dorsal root ganglia findings: A clinically silent peripheral neuropathy.

Adeno-associated virus (AAV)-based vectors are commonly used for delivering transgenes in gene therapy studies, but they are also known to cause dorsal root ganglia (DRG) and peripheral nerve toxicities in animals. However, the functional implications of these pathologic findings and their time course remain unclear. At 2, 4, 6, and 8 weeks following a single dose of an AAV9 vector carrying human frataxin transgene in rats, non-standard functional assessments, including von Frey filament, electrophysiology, and Rotarod tests, were conducted longitudinally to measure allodynia, nerve conduction velocity, and coordination, respectively. Additionally, DRGs, peripheral nerves, brain and spinal cord were evaluated histologically and circulating neurofilament light chain (NfL) was quantified at 1, 2, 4, and 8 weeks, respectively. At 2 and 4 weeks after dosing, minimal-to-moderate nerve fiber degeneration and neuronal degeneration were observed in the DRGs in some of the AAV9 vector-dosed animals. At 8 weeks, nerve fiber degeneration was observed in DRGs, with or without neuronal degeneration, and in sciatic nerves of all AAV9 vector-dosed animals. NfL values were higher in AAV9 vector-treated animals at weeks 4 and 8 compared with controls. However, there were no significant differences in the three functional endpoints evaluated between the AAV9 vector- and vehicle-dosed animals, or in a longitudinal comparison between baseline (predose), 4, and 8 week values in the AAV9 vector-dose animals. These findings demonstrate that there is no detectable functional consequence to the minimal-to-moderate neurodegeneration observed with our AAV9 vector treatment in rats, suggesting a functional tolerance or reserve for loss of DRG neurons after systemic administration of AAV9 vector.

Sex-related differences in retinal function in Wistar rats: implications for toxicity and safety studies.

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.

A comparison of activity data generated from cardiovascular telemetry studies - With quantitative open field locomotor studies in Wistar Han rats.

Compound-mediated locomotion changes, conducted via open field infrared photobeam breaks, are an important common component of neurological assessments conducted in safety pharmacology studies. In addition to open field locomotor activity assessments, activity data (derived from changes in signal strength) from cardiovascular (CV) telemetry studies can also be an alternative method potentially used to assess locomotor effects. However, comparisons of these two methods have not been extensively characterized. The goal of this work was to compare these two methodologies to assess activity in rats using reference compounds known to have central nervous system (CNS)-stimulant (preladenant) or CNS-depressant (chlorpromazine) effects. Open field activity was conducted using the Kinder Scientific Motor Monitor system and data were collected for 30 min at each drug's expected time of maximum plasma exposure (Tmax). Telemetry-based CV assessment data were continuously acquired using DSI radiotelemetry instrumented animals for 24 h postdose (HPD). Drugs were administered during the lights-on period for both study types. Administration of preladenant caused increases in activity within 0.5-2 HPD for both methods. While administration of chlorpromazine caused decreases in activity in the infrared beam-based open field assessment (1.0-1.5 HPD), there was no effect on telemetry-derived activity during a similar time period. However, telemetry-derived decreases in activity were observed during the lights-off period (16-20 HPD), suggesting CNS-depressant compounds may be mischaracterized if the optimal dose administration time is not selected based on the light/dark cycle and pharmacokinetics. Overall, these results suggest that telemetry-based activity assessment is capable of detecting CNS-stimulant effects of compounds.

Performance of biomarkers NF-L, NSE, Tau and GFAP in blood and cerebrospinal fluid in rat for the detection of nervous system injury.

Background: Target 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). Methods: Performance 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. Results: NF-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. Conclusion: These 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 of gloved hand to cage induces 22-kHz ultrasonic vocalizations in male albino rats.

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.

Increases in GFAP immunoreactive astrocytes in the cerebellar molecular layer of young adult CBA/J mice.

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.

Novel in vivo and ex vivo hybrid in vivo imaging system (IVIS) imaging offers a convenient and precise way to measure the glomerular filtration rate in conscious mice.

INTRODUCTION: In pharmacology and toxicology studies, the glomerular filtration rate (GFR) is the gold standard for the assessment of renal function, and the renal clearance of inulin in blood measured by photometers is known as a filtration marker for the determination of GFR. Preclinically, a non-invasive GFR measurement method was recently developed in which near-infrared fluorescently labelled inulin (GFR-Vivo 680) was scanned with fluorescence molecular tomography (FMT). However, measurement of GFR using FMT has major disadvantages and technical challenges, such as requiring experienced skills in animal handling and rapid and precise time management. Additionally, fur and skin pigmentation may severely compromise imaging due to tissue fluorescence absorption. To overcome these drawbacks of FMT imaging, we have developed an in- and ex vivo hybrid method for measuring GFR using the in vivo imaging system (IVIS). METHODS: An IVIS-based imaging method was tested to determine the clearance kinetics of plasma GFR-Vivo 680 after a single bolus injection in conscious C57BL/6 mice administered vehicle or cyclosporine A (CsA, 80 mg/kg) for 14 days. RESULTS: Based on a two-compartment model fitting, the estimated GFR was 235 ± 53 and 189 ± 19 µL/min in vehicle-treated and CsA-treated male mice, respectively (p < 0.01). Our assay revealed the decreased GFR, similar to the sensitivity of FMT imaging, which yielded comparable GFR values (229 ± 61 and 151 ± 35 µL/min in vehicle-treated and CsA-treated mice, respectively, p < 0.01), and to those previously reported in the literature. DISCUSSION: These studies demonstrate the feasibility of IVIS imaging measurement of inulin clearance in untreated, vehicle-treated and cyclosporine A-treated mice. We propose this new method as an alternative, simple, and versatile way to measure GFR in vivo and ex vivo in pharmacological and toxicological studies.

Physiological approaches to assess diminished sympathetic activity in the conscious rat.

The purpose of this study was to evaluate functional measures of diminished sympathetic activity after postganglionic neuronal loss in the conscious rat. To produce variable degrees of sympathetic postganglionic neuronal loss, adult rats were treated daily with toxic doses of guanethidine (100mg/kg) for either 5days or 11days, followed by a recovery period of at least 18days. Heart rate, blood pressure, cardiac baroreflex responsiveness, urinalysis (for catecholamine metabolite, 3-methoxy-4-hydroxyphenylethylenglycol; MHPG), and pupillometry were performed during the recovery period. At the end of the recovery period stereology of superior cervical ganglia (SCG) was performed to determine the degree of neuronal loss. Total number of SCG neurons was correlated to physiological outcomes using regression analysis. Whereas guanethidine treatment for 11days caused significant reduction in the number of neurons (15,646±1460 vs. 31,958±1588), guanethidine treatment for 5days caused variable levels of neuronal depletion (26,009±3518). Regression analysis showed that only changes in urinary MHPG levels and systolic blood pressure significantly correlated with reduction of SCG neurons (r2=0.45 and 0.19, both p<0.05). Although cardiac baroreflex-induced reflex tachycardia (345.7±19.6 vs. 449.7±20.3) and pupil/iris ratio (0.50±0.03% vs. 0.61±0.02%) were significantly attenuated in the 11-day guanethidine treated rats there was no significant relationship between these measurements and the number of remaining SCG neurons after treatment (p>0.05). These data suggest that basal systolic blood pressure and urinary MHPG levels predict drug-induced depletion of sympathetic activity in vivo.