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.

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.

The use of Wistar Han (WH) rats in regulatory toxicology studies, including juvenile animal studies, is increasing. The current study was performed to characterize femur development in neonatal and juvenile WH rats. Beginning on postnatal day (PND) 4 through PND 50, groups of WH rats ( n = 5/sex) were evaluated for the development and ossification of the femur by Alizarin Red S staining, micro‐computed tomography (micro‐CT) imaging, microscopic examination, and Collagen X immunostaining. The focus of these evaluations was limited to the proximal femur including the head, neck, greater trochanter, lesser trochanter, and shaft. Through the multiple methods utilized, it was determined that the femoral neck is the first structure of the proximal femur to ossify, which occurs between PND 15 and 20. The femoral head and greater trochanter begin to separate on PND 15 with separation completing on PND 25 (both still composed of cartilage). Evidence of mineralization and ossification of these structures is apparent at PND 25, with a progressive increase with age, reaching adult‐like morphology by PND 40. This work has provided a thorough characterization of postnatal femur development in WH rats to help inform bone and femur effects in juvenile toxicity studies as well as the design of investigative studies.

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.

In oncology research, while xenograft tumor models are easily visualized and humane endpoints can be clearly defined, metastatic tumor models are often based on more subjective clinical observations as endpoints. This study aimed at identifying objective non-invasive criteria for predicting imminent distress and mortality in metastatic lung tumor-bearing mice. BALB/c and C57BL/6 mice were inoculated with CT26 or B16F10 cells, respectively. The mice were housed in Vium smart cages to continuously monitor and stream respiratory rate and locomotion for up to 28 days until scheduled euthanasia or humane endpoint criteria were met. Body weight and body temperature were measured during the study. On days 11, 14, 17 and 28, lungs of subsets of animals were microCT imaged in vivo to assess lung metastasis progression and then euthanized for lung microscopic evaluations. Beginning at day 21, most tumor-bearing animals developed increased respiratory rates followed by decreased locomotion 1-2 days later, compared with the baseline values. Increases in respiratory rate did not correlate to surface tumor nodule counts or lung weight. Body weight measurement did not show significant changes from days 14-28 in either tumor-bearing or control animals. We propose that increases in respiratory rate (1.3-1.5 X) can be used to provide an objective benchmark to signal the need for increased clinical observations or euthanasia. Adoption of this novel humane endpoint criterion would allow investigators time to collect tissue samples prior to spontaneous morbidity or death and significantly reduce the distress of mice in the terminal stages of these metastatic lung tumor models.

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.

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.

Clinical use of the chemotherapeutic agent vincristine (VCR) is limited by chemotherapy-induced peripheral neuropathy (CiPN). A new formulation of VCR encapsulated by nanoparticles has been proposed and developed to alleviate CiPN. We hypothesized in nonclinical animals that the nanoparticle drug would be less neurotoxic due to different absorption and distribution properties to the peripheral nerve from the unencapsulated free drug. Here, we assessed whether VCR encapsulation in nanoparticles alleviates CiPN using behavioral gait analysis (CatWalk), histopathologic and molecular biological (RT-qPCR) approaches. Adult male C57BL/6 mice were assigned to 3 groups (empty nanoparticle, nano-VCR, solution-based VCR, each n = 8). After 15 days of dosing, animals were euthanized for tissue collection. It was shown that intraperitoneal administration of nano-VCR (0.15 mg/kg, every other day) and the empty nanoparticle resulted in no changes in gait parameters; whereas, injection of solution-based VCR resulted in decreased run speed and increased step cycle and stance (P < 0.05). There were no differences in incidence and severity of degeneration in the sciatic nerves between the nano-VCR-dosed and solution-based VCR-dosed animals. Likewise, decreased levels of a nervous tissue-enriched microRNA-183 in circulating blood did not show a significant difference between the nano- and solution-based VCR groups (P > 0.05). Empty nanoparticle administration did not cause any behavioral, microRNA, or structural changes. In conclusion, this study suggests that the nano-VCR formulation may alleviate behavioral changes in CiPN, but it does not improve the structural changes of CiPN in peripheral nerve. Nanoparticle properties may need to be optimized to improve biological observations.

Rodent studies often must be conducted during an animal's active phase (that is, in darkness) yet also during a typical day shift for staff. Low-pressure sodium lighting (LPSL), to which human retinas are more sensitive than rodents' at low intensity, has been used to facilitate study conduct in dark phase. The assumption was that LPSL would be equivalent to total darkness due to low rodent retinal sensitivity but provide enough lighting for safe technical manipulations due to higher human retinal sensitivity. Unlike other light sources, LPSL has been tested for effects on circadian rhythm specific to locomotive activities in albino mice. Whether LPSL affects circadian rhythms in rats is unknown. In this study, circadian endpoints were derived from body temperature and locomotor activity via telemeters in 8 adult male Wistar rats. When moved from a 12:12-h white-light (that is, cold white fluorescent light):dark (LD) cycle to a 12:12-h white-light:sodium-light cycle, rats demonstrated free-running and disrupted circadian rhythms (that is, lengthened circadian period and reduced circadian robustness and amplitude). Body temperature and locomotor activity were significantly lower in the LPSL phase as compared with dark phase under the baseline condition. When exposed to a 12:12 h sodium-light:dark (SD) cycle, rats entrained with a circadian period similar to 12:12-h white-light:dark (LD), but significantly different from the period under constant darkness (DD). Circadian onset and acrophase were delayed under SD compared with LD. When illuminated with a LPSL pulse under DD, rats showed phase shifts similar to white-light pulse effects, consistent with the phase response curve. To determine whether the image-forming photoreceptors are involved in this process, we used electroretinography. Compared with white light, 589-nm light generated during electroretinography elicited rod photoreceptors responses with longer latency and cone photoreceptor responses with lower amplitude. These results indicate that LPSL is a weaker zeitgeber than white light and may alter the circadian system in rats. Furthermore, because LPSL appeared to be visible to rats, it may not be an appropriate substitute for actual darkness.

The potential for neurogenesis in the cranial (superior) cervical ganglia (SCG) of the sympathetic nervous system was evaluated. Eleven consecutive daily doses of guanethidine (100 mg/kg/d) were administered intraperitoneally to rats in order to destroy postganglionic sympathetic neurons in SCG. Following the last dose, animals were allowed to recover 1, 3, or 6 months. Right and left SCG from guanethidine-treated and age-matched, vehicle-treated control rats were harvested for histopathologic, morphometric, and stereologic evaluations. Both morphometric and stereologic evaluations confirmed neuron loss following guanethidine treatment. Morphometric analysis revealed a 50% to 60% lower number of tyrosine hydroxylase (TH)-positive neurons per unit area of SCG at both 3 and 6 months of recovery, compared to ganglia of age-matched controls, with no evidence of restoration of neuron density between 3 and 6 months. Reductions in TH-positive neurons following guanethidine treatment were corroborated by unbiased stereology of total hematoxylin and eosin-stained neuron numbers in SCG. Stereologic analyses revealed that total neuron counts were lower by 37% at 3 months of recovery when compared to age-matched vehicle controls, again with no obvious restoration between 3 and 6 months. Thus, no evidence was found that postganglionic neurons of the sympathetic nervous system in the adult rat have a neurogenic capacity.

In drug discovery and development, X-ray micro-computed tomography (micro-CT) has gained increasing importance over the past decades. In recent years, micro-CT imaging of soft tissues has become popular due to the introduction of a variety of radiopaque contrast agents. More recently, nanoparticle-based ExiTron nano 12,000 has become commercially available for the nonclinical micro-CT imaging of soft tissues in rodents. Phagocytosis and accumulation of the contrast agent by Kupffer cells in the liver, as well as macrophages in the spleen, increase the soft tissue X-ray attenuation for up to 6 months. Therefore, it is essential to understand the potential toxicity of this nanomaterial in micro-CT imaging prior to its application in pharmacology and/or toxicology studies. Herein, we describe the time-course and distribution of the contrast in the liver, spleen and blood after a single intravenous injection (IV) of this nanoparticle contrast agent at 0.1 ml/mouse. Thoracic images of male adult C57BL/6 mice were acquired using a Bruker SkyScan 1276 micro-CT over a period of 29 days. The stability of X-ray attenuation enhancement in the above tissues was also tested after a single dose of Kupffer cell toxicant gadolinium chloride (GdCl3) at 15 mg/kg on day 2. The liver, spleen and kidney were examined microscopically on days 15 and 29 post treatment. Serum and liver cytokines (IL-1β, IL-2, IL-6, IL-10, IL-12p70, IFN-γ, IP-10, MIP1-α, MIP1-β and TNF-α) were quantified on days 15 and 29 as indicators of a pro-inflammatory response to treatment. This study determined that there was an accumulation of amphophilic granular material in the cells of the mononuclear phagocyte system in the liver and spleen following a single dose of ExiTron nano 12,000 and a second dose of GdCl3 or its vehicle. However, ExiTron nano12000 contrast administration did not cause any hepatotoxicity in the liver, nor did pro-inflammatory cytokines release in the liver or serum. Similarly, there were no adverse pathologies in the spleen or kidneys. In summary, ExiTron nano12000 contrast agent-enhanced micro-CT could be used as a safe method in up to 29-day longitudinal efficacy and toxicology mouse studies for the non-invasive assessment of the liver and spleen.

Chemotherapy-induced peripheral neuropathy (CiPN) is a serious adverse effect in the clinic, but nonclinical assessment methods in animal studies are limited to labor intensive behavioral tests or semi-quantitative microscopic evaluation. Hence, microRNA (miRNA) biomarkers and automated in-life behavioral tracking were assessed for their utility as non-invasive methods. To address the lack of diagnostic biomarkers, we explored miR-124, miR-183 and miR-338 in a CiPN model induced by paclitaxel, a well-known neurotoxic agent. In addition, conventional and Vium's innovative Digital Vivarium technology-based in-life behavioral tests and postmortem microscopic examination of the dorsal root ganglion (DRG) and the sciatic nerve were performed. Terminal blood was collected on days 8 or 16, after 20 mg/kg paclitaxel was administered every other day for total of 4 or 7 doses, respectively, for plasma miRNA quantification by RT-qPCR. DRG and sciatic nerve samples were collected from mice sacrificed on day 16 for miRNA quantification. Among the three miRNAs analyzed, only miR-124 was statistically significantly increased (5 fold and 10 fold on day 8 and day 16, respectively). The increase in circulating miR-124 correlated with cold allodynia and axonal degeneration in both DRG and sciatic nerve. Automated home cage motion analysis revealed for the first time that nighttime motion was significantly decreased (P < 0.05) in paclitaxel-dosed animals. Although both increase in circulating miR-124 and decrease in nighttime motion are compelling, our results provide positive evidence warranting further testing using additional peripheral nerve toxicants and diverse experimental CiPN models.

Paclitaxel is an antineoplastic drug, and its usage is often limited by severe peripheral polyneuropathy characterized by symptoms in remote extremities. It is suggested that subpopulations of dorsal root ganglion (DRG) neurons are impacted functionally and/or structurally. In this study, the neurotoxic effect of this drug in cultured rat DRG neurons was analyzed by measuring their neurite length and electrophysiological properties. It was observed that exposure of the cultured DRG neurons to 3, 10 and 30 μM paclitaxel for 24 or 48h caused a dose-dependent shortening of length or loss of neurite, typically in the “dying back” pattern. Eight of 28 large DRG neuron populations with extensive neurites showed less sensitivity or were entirely resistant to paclitaxel treatment. Whole-cell patch clamp experiments revealed that these paclitaxel-resistant neurons may fall in the category of Aδ or Aβ neurons. Deeper understanding as to why this particular subclass of neurons is resistant could reveal novel approaches to protect the primary sensory neurons from chemotherapy agent-induced toxicity and screening methods to identify chemical agents that will not induce peripheral neuropathy in cancer patients.

Chemotherapy-induced peripheral neuropathy (CiPN) is a frequent adverse effect in patients and a leading safety consideration in oncology drug development. Although behavioral assessment and microscopic examination of the nerves and dorsal root ganglia can be incorporated into toxicity studies to assess CiPN risk, more sensitive and less labor-intensive endpoints are often lacking. In this study, rats and mice administered vincristine (75g kg(-1)day(-1), i.p., for 10days in rats and 100g kg(-1)day(-1), i.p., for 11days in mice, respectively) were employed as the CiPN models. Behavioral changes were assessed during the dosing phase. At necropsy, the sural or sciatic nerve was harvested from the rats and mice, respectively, and assessed for mechanical and histopathological endpoints. It was found that the maximal load and the load/extension ratio were significantly decreased in the nerves collected from the animals dosed with vincristine compared with the vehicle-treated animals (P<0.05). Additionally, the gait analysis revealed that the paw print areas were significantly increased in mice (P<0.01), but not in rats following vincristine administration. Light microscopic histopathology of the nerves and dorsal root ganglia were unaffected by vincristine administration. We concluded that ex vivo mechanical properties of the nerves is a sensitive endpoint, providing a new method to predict CiPN in rodent. Gait analysis may also be a useful tool in these pre-clinical animal models. Rats and mice administered vincristine were employed as the chemotherapy-induced peripheral neuropathy models. Behavior and ex vivo sural (rats) or sciatic nerve (mice) were assessed. It was found that the load and the load/extension ratio were decreased in the nerves collected from the animals dosed with vincristine. We concluded that mechanical properties of the nerves are a sensitive endpoint for chemotherapy-induced peripheral neuropathy prediction.

There is emerging evidence showing that lncRNAs can be involved in various critical biological processes. Zebrafish is a fully developed model system being used in a variety of basic research and biomedical studies. Hence, it is an ideal model organism to study the functions and mechanisms of lncRNAs. Here, we constructed ZFLNC-a comprehensive database of zebrafish lncRNA that is dedicated to providing a zebrafishbased platform for deep exploration of zebrafish lncRNAs and their mammalian counterparts to the relevant academic communities. The main data resources of lncRNAs in this database come from the NCBI, Ensembl, NONCODE, zflncRNApedia and literature. We also obtained lncRNAs as a supplement by analysing RNA-Seq datasets from SRA database. With these IncRNAs, we further carried out expression profiling, co-expression network prediction, Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG)/Online Mendelian Inheritance in Man (OMIM) annotation and conservation analysis. As far as we know, ZFLNC is the most comprehensive and well-annotated database for zebrafish lncRNA.