Retraction: Effects of Sodium Lighting on Circadian Rhythms in Rats.

This retracts the article entitled, "Effects of Sodium Lighting on Circadian Rhythms in Rats" by Xian Chen, Chang-Ning Liu, and Judith E Fenyk-Melody published in the May issue vol 58, issue 3, p 311-320.1 This article is being retracted with the support of all 3 coauthors due to methodological and authorship issues.

Effects of Sodium Lighting On Circadian Rhythms in Rats.

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.

Circulating microRNA and automated motion analysis as novel methods of assessing chemotherapy-induced peripheral neuropathy in mice.

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.

Hepatic glucagon receptor binding and glucose-lowering in vivo by peptidyl and non-peptidyl glucagon receptor antagonists.

Glucagon receptor antagonists have been actively pursued as potential therapeutics for the treatment of type 2 diabetes. Peptidyl and non-peptidyl glucagon receptor antagonists have been shown to block glucagon-induced blood glucose elevation in both animals and humans. How the antagonists and the glucagon receptor interact in vivo has not been reported and is the subject of the current study. Using (125)I-labeled glucagon as a radiotracer, we developed an in vivo glucagon receptor occupancy assay in mice expressing a human glucagon receptor in place of the endogenous mouse glucagon receptor (hGCGR mice). Using this assay, we first showed that the glucagon receptor is expressed predominantly in liver, to a much lesser extent in kidney, and is below detection in several other tissues/organs in the mice. We subsequently showed that, at 2 mg/kg body weight (mg/pk) dosed intraperitoneally (i.p.), peptidyl glucagon receptor antagonist des-His-glucagon binds to approximately 78% of the hepatic glucagon receptor and blocks an exogenous glucagon-induced blood glucose elevation in the mice. Finally, we also showed that, at 10 and 30 mg/kg dosed orally (p.o.), compound A, a non-peptidyl small molecule glucagon receptor antagonist, occupied 65-70% of the hepatic glucagon receptor, and significantly diminished exogenous glucagon-induced blood glucose elevation in the mice. At 3 mg/kg, however, compound A occupied only approximately 39% of the hepatic glucagon receptor and did not affect exogenous glucagon-induced blood glucose elevation in the mice. Taken together, the results confirmed previous reports that glucagon receptors are present predominantly in the liver, and provide the first direct evidence that peptidyl and non-peptidyl glucagon receptor antagonists bind to the hepatic glucagon receptor in vivo, and that at least 60% receptor occupancy correlates with the glucose lowering efficacy by the antagonists in vivo.

Comparison of the effects of perfusion in determining brain penetration (brain-to-plasma ratios) of small molecules in rats.

In the process of drug discovery, brain and plasma measurements of new chemical entities in rodents are of interest, particularly when the target receptors are in the brain. Brain-to-plasma ratios (B/P) obtained from a rodent pharmacokinetic assay are useful in helping determine which compounds are brain penetrant. The study reported here was performed to determine whether whole-body saline perfusion for complete blood removal was required to accurately measure brain tissue compound concentrations. Diazepam was used as a positive control since it is highly brain penetrant. Compound A was used as a negative control since it had known poor brain penetration. After intravenous dosing with either diazepam or compound A, rats were anesthetized and blood was collected, then the brain was removed following no perfusion or whole-body perfusion with saline. The analytes described (compound A, diazepam, and the internal standard) were recovered from plasma or brain homogenate by use of protein precipitation, and were subsequently analyzed by use of liquid chromatography/tandem mass spectrometry (LC/MS/MS). The B/P values determined by use of LC-MS were not significantly different in perfused vs. non-perfused rats (P > or = 0.05). This approach (whole brain collected from non-perfused male rats) is an attractive alternative over brain penetration studies of perfused rats, since it has markedly reduced the technical time and potential for pain and distress required for generating B/P data due to elimination of the requirement for anesthesia and surgical preparation of animals.

Effect of quinidine on the 10-hydroxylation of R-warfarin: species differences and clearance projection.

Stimulation by quinidine of warfarin metabolism in vitro was first demonstrated with liver microsomal preparations. We report herein that this drug interaction is reproducible in an animal model but that it exhibits profound species differences. Thus, using rabbit liver microsomes and a kinetic model incorporating two binding sites, the hepatic intrinsic clearance of R-warfarin via the 10-hydroxylation pathway (CL(int)(W)) was projected to be 6 +/- 1 and 128 +/- 51 microl/min/g liver, respectively, in the absence and presence of 21 microM unbound quinidine. These estimates were consistent with the results from studies in which rabbit livers (n = 5) were perfused in situ with R-warfarin or R-warfarin plus quinidine. The CL(int)(W) increased from 7 +/- 3 to 156 +/- 106 microl/min/g liver after increasing the hepatic exposure of unbound quinidine from 0 to 21 microM. In contrast, when liver microsomes or intact livers from rats were examined, R-warfarin metabolism was inhibited by quinidine, the CL(int)(W) decreasing to 26% of the control value after exposure of perfused rat livers (n = 5) to 22 microM unbound quinidine. The third example involved monkey liver microsomes, in which the rate of 10-hydroxylation of R-warfarin was little affected in the presence of quinidine (<2-fold increase). In all three species, the 10-hydroxylation of R-warfarin was catalyzed primarily by members of CYP3A, based on immuno- and chemical inhibition analyses. These findings not only highlight the variability of drug interactions among different species but also suggest that changes in hepatic clearance resulting from stimulation of cytochrome P450 activity may be projected based on estimates generated from corresponding liver microsomal preparations.

Genetic control of autoimmunity: protection from diabetes, but spontaneous autoimmune biliary disease in a nonobese diabetic congenic strain.

At least 20 insulin-dependent diabetes (Idd) loci modify the progression of autoimmune diabetes in the NOD mouse, an animal model of human type 1 diabetes. The NOD.c3c4 congenic mouse, which has multiple B6- and B10-derived Idd-resistant alleles on chromosomes 3 and 4, respectively, is completely protected from autoimmune diabetes. We demonstrate in this study, however, that NOD.c3c4 mice develop a novel spontaneous and fatal autoimmune polycystic biliary tract disease, with lymphocytic peribiliary infiltrates and autoantibodies. Strains having a subset of the Idd-resistant alleles present in the NOD.c3c4 strain show component phenotypes of the liver disease: NOD mice with B6 resistance alleles only on chromosome 3 have lymphocytic liver infiltration without autoantibody formation, while NOD mice with B10 resistance alleles only on chromosome 4 show autoantibody formation without liver infiltration. The liver disease is transferable to naive NOD.c3c4 recipients using splenocytes from affected NOD.c3c4 mice, demonstrating an autoimmune etiology. Thus, substitution of non-NOD genetic intervals into the NOD strain can prevent diabetes, but in turn cause an entirely different autoimmune syndrome, a finding consistent with a generalized failure of self-tolerance in the NOD genetic background. The complex clinical phenotypes in human autoimmune conditions may be similarly resolved into largely overlapping biochemical pathways that are then modified, potentially by alleles at a few key chromosomal regions, to produce specific autoimmune syndromes.

The role of dipeptidyl peptidase IV in the cleavage of glucagon family peptides: in vivo metabolism of pituitary adenylate cyclase activating polypeptide-(1-38).

Dipeptidyl peptidase IV (DP-IV) is a cell surface serine dipeptidase that is involved in the regulation of the incretin hormones, glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). There is accumulating evidence that other members of the glucagon family of peptides are also endogenous substrates for this enzyme. To identify candidate substrates for DP-IV, a mass spectrometry-based protease assay was developed that measures cleavage efficiencies (kcat/Km) of polypeptides in a mixture, using only a few picomoles of each substrate and physiological amounts of enzyme in a single kinetic experiment. Oxyntomodulin and the growth hormone-(1-43) fragment were identified as new candidate in vivo substrates. Pituitary adenylate cyclase-activating polypeptide-(1-38) (PACAP38), a critical mediator of lipid and carbohydrate metabolism, was also determined to be efficiently processed by DP-IV in vitro. The catabolism of exogenously administered PACAP38 in wild type and DP-IV-deficient C57Bl/6 mice was monitored by tandem mass spectrometry. Animals lacking DP-IV exhibited a significantly slower clearance of the circulating peptide with virtually complete suppression of the inactive DP-IV metabolite, PACAP-(3-38). These in vivo results suggest that DP-IV plays a major role in the degradation of circulating PACAP38.