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The article focuses on a study that assesses the age-dependent sensitivity to nephrotoxicant cisplatin in multi-aged rat models. It reports the cisplatin-induced nephrotoxicity in the said rats. It mentions that the level of Kim-1 tissue mRNA and urinary protein is correlated to each other and to the severity of renal lesions. It notes that the multi-age rat model can be used in demonstrating the different age-related sensitivities to renal injury.

The article presents a study which evaluates the use of cytochrome c (cyt c) as a non-invasive biomarker of drug-induced liver injury. It states that the study was undertaken to emphasize the need for additional tools in detecting human toxicity. The study used the urine of adult rats treated with hepatoxins as subject of the experiment and employed serum diagnostic tests, immunohistochemistry, and urinary analysis for cyt c. Results indicated histomorphological changes and TUNEL staining in liver coherent with hepatocellular necrosis, apoptosis, and inflammation and showed that the liver is the primary source of cyt c. It shows that cyt c is a useful indicator of hepatic injury when used as a potential urinary biomarker.

Many pharmacological and toxicological actions of drugs in children cannot be fully predicted from adult clinical experience or from standard non-clinical toxicology studies. Numerous drugs have direct or indirect pharmacological effects on the heart and are prescribed for children of all ages. Toxicity or secondary effects may be immediate or delayed for years after drug exposure has ceased. Originally, the aim of this review was to compile information on the effect of specific drugs on the post-natal development of the cardiovascular system and to examine long-term follow-up of the use of cardio-active drugs in children. The limited database of published information caused the original question to evolve into an examination of the medical literature for three areas of information: (i) whether vulnerable developmental windows have been identified that reflect the substantial functional development that the cardiovascular system undergoes after birth; (ii) what is known about pharmacological perturbation of development; and (iii) what the likelihood is of drug exposure during childhood. We examined different scenarios for exposure including random, isolated exposure, conditions historically associated with adults, primary or secondary cardiac disease, psychiatric and neurological conditions, asthma, cancer and HIV. Except for random, isolated drug exposures, each category of possible exposure contained numerous drugs known to have either primary or secondary effects on the cardiovascular system or to influence factors associated with atherosclerosis. It is likely that a significant number of children will be prescribed drugs having either direct or indirect effects upon the immature cardiovascular system. A confounding factor is the simultaneous use of over-the-counter medications and herbal or nutraceutical preparations that a patient, parent or guardian does not mention to a prescribing physician. Metabolism is also important in assessing drug effects in children. Differences in body water : body fat ratio, age-related gastrointestinal absorption, distribution, excretion, renal function and drug metabolizing capabilities make it possible for children to have a different metabolite profile for a drug compared with adults. There is little examination of drug effects on the interdependent processes of cardiac maturation and less examination of metabolite effects. It is difficult to identify delayed toxicities in children as these adverse events may take years to manifest with many patients lost to follow-up. Clearly this is an area of study where intermediate endpoints and surrogate markers would be of great benefit. Pharmacogenomics may be useful in providing markers of increased risk or susceptibility. A perspective must be kept in balancing the possibility of a problem with the very real benefits that many children experience from the use of these pharmaceuticals. [ABSTRACT FROM AUTHOR]

Abstract Background Urine from male Sprague-Dawley rats 25, 40, and 80 days old was analyzed by NMR and UPLC/MS. The effects of data normalization procedures on principal component analysis (PCA) and quantitative analysis of NMR-based metabonomics data were investigated. Additionally, the effects of age on the metabolic profiles were examined by both NMR and UPLC/MS analyses. Results The data normalization factor was shown to have a great impact on the statistical and quantitative results indicating the need to carefully consider how to best normalize the data within a particular study and when comparing different studies. PCA applied to the data obtained from both NMR and UPLC/MS platforms reveals similar age-related differences. NMR indicated many metabolites associated with the Krebs cycle decrease while citrate and 2-oxoglutarate, also associated with the Krebs cycle, increase in older rats. Conclusion This study compared four different normalization methods for the NMR-based metabonomics spectra from an age-related study. It was shown that each method of normalization has a great effect on both the statistical and quantitative analyses. Each normalization method resulted in altered relative positions of significant PCA loadings for each sample spectra but it did not alter which chemical shifts had the highest loadings. The greater the normalization factor was related to age, the greater the separation between age groups was observed in subsequent PCA analyses. The normalization factor that showed the least age dependence was total NMR intensity, which was consistent with UPLC/MS data. Normalization by total intensity attempts to make corrections due to dietary and water intake of the individual animal, which is especially useful in metabonomics evaluations of urine. Additionally, metabonomics evaluations of age-related effects showed decreased concentrations of many Krebs cycle intermediates along with increased levels of oxidized antioxidants in urine of older rats, which is consistent with current theories on aging and its association with diminishing mitochondrial function and increasing levels of reactive oxygen species. Analysis of urine by both NMR and UPLC/MS provides a comprehensive and complementary means of examining metabolic events in aging rats.

Background: A plethora of studies has shown the utility of several chemical dyes due to their affinity to bind Aβ to enable visualization of plaques under light or fluorescence microscope, and some of them showed affinity to bind neurofibrillary tangles (NFT) as well. However, only a few of them have the propensity to bind both senile plaques (SP) and NFT simultaneously., Objective: In our current study, we aimed to modify the K114 dye and the staining procedure to substantially improve the staining of amyloid plaques in both human and rodent brains and neurofibrillary tangles in the human brain., Methods: We modified the K114 solution and the staining procedure using Sudan Black as a modifier. Additionally, to evaluate the target of the modified K114, we performed double labeling of K114 and increased Aβ against three different epitopes. We used 5 different antibodies to detect phosphorylated tau to understand the specific targets that modified K114 binds., Results: Dual labeling using hyperphosphorylated antibodies against AT8, pTau, and TNT1 revealed that more than 80% hyperphosphorylated tau colocalized with tangles that were positive for modified K114, whereas more than 70% of the hyperphosphorylated tau colocalized with modified K114. On the other hand, more than 80% of the plaques that were stained with Aβ MOAB-2 were colocalized with modified K114., Conclusion: Our modified method can label amyloid plaques within 5 min in the rat brain and within 20 min in the human brain. Our results indicated that modified K114 could be used as a valuable tool for detecting amyloid plaques and tangles with high contrast and resolution relative to other conventional fluorescence markers., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The assessment of the sensitivity and specificity of any potential biomarker against the gold standard is an important step in the process of its qualification by regulatory authorities. Such qualification is an important step towards incorporating the biomarker into the panel of tools available for drug development. In the current study we analyzed the sensitivity and specificity of T 2 MRI relaxometry to detect trimethyltin-induced neurotoxicity in rats. Seventy-five male Sprague-Dawley rats were injected with a single intraperitoneal dose of either TMT (8, 10, 11, or 12 mg/kg) or saline (2 ml/kg) and imaged with 7 T MRI before and 3, 7, 14, and 21 days after injection using a quantitative T 2 mapping. Neurohistopathology (the gold standard in the case of neurotoxicity) was performed at the end of the observation and used as an outcome qualifier in receiver-operator characteristic (ROC) curve analysis of T 2 changes as a predictor of neurotoxicity. TMT treatment led to a significant increase in T 2 values in many brain areas. The biggest changes in T 2 values were seen around the lateral ventricles, which was interpreted as ventricular dilation. The area under the ROC curve for the volume of the lateral ventricles was 0.878 with the optimal sensitivity/specificity of 0.805/0.933, respectively. T 2 MRI is a promising method for generating a non-invasive biomarkers of neurotoxicity, which shows the dose-response behavior with substantial sensitivity and specificity. While its performance was strong in the TMT model, further characterization of the sensitivity and specificity of T 2 MRI with other neurotoxicants is warranted., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Objective: Parkinson's disease (PD) is a progressive motor disease of unknown etiology. Although neuroprotective ability of endogenous bile acid, tauroursodeoxycholic acid (TUDCA), shown in various diseases, including an acute model of PD,the potential therapeutic role of TUDCA in progressive models of PD that exhibit all aspects of PD has not been elucidated. In the present study, mice were assigned to one of four treatment groups: (1) Probenecid (PROB); (2) TUDCA, (3) MPTP + PROB (MPTPp); and (3) TUDCA + MPTPp. Methods: Markers for dopaminergic function, neuroinflammation, oxidative stress and autophagy were assessed using high performance liquid chromatography (HPLC), immunohistochemistry (IHC) and western blot (WB) methods. Locomotion was measured before and after treatments. Results : MPTPp decreased the expression of dopamine transporters (DAT) and tyrosine hydroxylase (TH), indicating dopaminergic damage, and induced microglial and astroglial activation as demonstrated by IHC analysis. MPTPp also decreased DA and its metabolites as demonstrated by HPLC analysis. Further, MPTPp-induced protein oxidation; increased LAMP-1 expression indicated autophagy and the promotion of alpha-synuclein (α-SYN) aggregation. Discussion : Pretreatment with TUDCA protected against dopaminergic neuronal damage, prevented the microglial and astroglial activation, as well as the DA and DOPAC reductions caused by MPTPp. TUDCA by itself did not produce any significant change, with data similar to the negative control group. Pretreatment with TUDCA prevented protein oxidation and autophagy, in addition to inhibiting α-SYN aggregation. Although TUDCA pretreatment did not significantly affect locomotion, only acute treatment effects were measured, indicating more extensive assessments may be necessary to reveal potential therapeutic effects on behavior. Together, these results suggest that autophagy may be involved in the progression of PD and that TUDCA may attenuate these effects. The efficacy of TUDCA as a novel therapy in patients with PD clearly warrants further study.

Cerebral amyloid angiopathy or CAA is a type of vascular dementia that can cause neuroinflammation, ischemia and hemorrhage, among other complications. CAA results from the deposition of amyloid beta (Aβ) in blood vessels and is frequently observed in individuals with Alzheimer's disease (AD). One functional output of those pathological changes is measurable cognitive decline. Still not well understood, however, is the impact of gender or sex on the pathology of CAA, as well as CAA-induced cognitive decline. Here, we studied how sex impacts deposition of CAA-related pathology and the associated cognitive decline. We observed differential hippocampal pathology as far as regions of deposition, type of morphology, and total amount of pathology when assessing CAA pathology via (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB)-labeling, as well as neurodegeneration via Fluoro Jade C (FJC)-labeling, and lysosomal associated membrane protein deposition via LAMP-1 labeling. In accordance with other studies, our data suggest female TG-SwDI mice exhibit more severe pathological alterations in CAA pathology. Additionally, behavioral assessments revealed an impact of genotype that was more pronounced in TG-SwDI females. While the primary measure of learning and memory, the water maze, suggests an overall effect of genotype, effects in measures of locomotor activity and anxiety-like behavior suggest reduced habituation in females. This could be due to a lower retention for the tasks. Results of this study offer significant insight into the importance of examining effects of sex on CAA., (Published by Elsevier B.V.)

Although several histochemical markers for senile plaques (SP) and neurofibrillary tangles (NFTs) have been synthesized since the discovery of plaques in Alzheimer's disease (AD), only a handful of these markers stain both lesions in the human brain. Despite discovery of its ability to stain both SP and NFT over 13 years ago, the styrylbenzene derivative, (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), has only recently gained attention, primarily due to its ability to function as a contrasting agent for MRI imaging of AD pathology in vivo. The structure of the compound is a nuclide with quantized angular momentum, which explains its value as a contrast agent. In the current study, modification of the established staining procedure produced meaningful improvement in the labeling of plaques and tangles in the human brain. We utilized two rodent models of AD to show FSB's value in labeling both Aβ and tau lesions. Furthermore, our current modification allows us to detect SP in rodent brains in 15 min and both SP and NFT in human brains within 20 min. The study presents new evidence regarding potential binding targets for FSB as well as optimization protocols in which various parameters have been manipulated to show how section thickness, use of frozen versus paraffin-embedded sections, and selection of staining media can affect the intensity of the plaque and tangle staining in the brain. To determine the target FSB potentially binds, we performed double immunolabeling of FSB with mOC64 (a conformational antibody that label Aβ 1-42 ). Results indicated that all plaques in the brain colocalized with mOC64, suggesting that FSB has the potential to bind all Aβ containing plaques, making it a very sensitive detector of multiple forms of SP... All antibodies were assessed for the degree of colocalization with FSB in order to better understand potential binding targets. We found more than 90% hyperphosphorylated Tau against AT8, AT180 and S214 colocalized with FSB labeled tangles. On the other hand, more than 90% of the mOC64 containing plaques colocalized with FSB stained plaques. Our results indicate that FSB is a valuable marker that can be used to detect AD pathologies in human and rodent brains with greater fluorescence intensity relative to other conventional fluorescence markers., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background: Congo Red (CR) has been used for its binding affinity to amyloid fibrils for the better part of a century. Recently, our laboratory has demonstrated its ability to bind to tau protein as well., New Method: Here we describe a novel methodology for fast, thorough, whole-brain labeling of amyloid plaques with CR via perfusion. We tested five different variants which altered the volume of CR, the speed of perfusion, and the solution CR was solubilized in to determine the best results., Results and Conclusion: We determined that intra-cardiac perfusion of animals with 0.5 % CR in 100 ml of 50 % ethanol or perfusion with 0.5 of CR in 100 ml of 10 % neutral buffer formalin both perfused at a rate of 30 ml/min for 3.3 min resulted in the clearest CR labeling, with little to no background noise. Both variants were compatible with subsequent immunolabeling procedures for NU-1, as well as Ferritin and GFAP. Compared to traditional CR plaque labeling methodology, this new method allows for quick whole brain CR-labeling. This reduces the amount of time from days to mere minutes. It also reduces potential for variability that would result from staining slides in batches. Thus, CR-perfusion is a rapid, thorough method that can be utilized to rapidly stain amyloid in the rodent brain., (Published by Elsevier B.V.)

Although there are multiple histochemical tracers available to label plaques and tangles in the brain to evaluate neuropathology in Alzheimer disease (AD), few of them are versatile in nature and compatible with immunohistochemical procedures. Congo Red (CR) is an anisotropic organic stain discovered to label amyloid beta (Aβ) plaques in the brain. Unfortunately, its use is underappreciated due to its low resolution and brightness as stated in previous studies using bright field microscopy. Here, we modified a previous method to localize both plaques and tangles in brains from humans and a transgenic rodent model of AD for fluorescence microscopic visualization. The plaque staining affinities displayed by CR were compared with fibrillar pattern labeling seen with Thioflavin S. This study summarizes the optimization of protocols in which various parameters have been finetuned. To determine the target CR potentially binds, we have performed double labeling with different antibodies against Aβ as well as phosphorylated Tau. The plaque staining affinities exhibited by CR are compared with those associated with the diffuse pattern of labeling seen with antibodies directed against different epitopes of Aβ. Neither CP13, TNT2 or TOC1 binds all the neurofibrillary tangles as revealed by CR labeling in the human brain. Additionally, we also evaluated double labeling with AT8, AT180, and PHF1. Interestingly, PHF-1 shows 40% colocalization and AT8 shows 15% colocalization with NFT. Thus, CR is a much better marker to detect AD pathologies in human and rodent brains with higher fluorescence intensity relative to other conventional fluorescence markers.

Parkinson's disease (PD) is a progressive motor disease with clinical features emerging due to degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), which project to the caudate putamen (CPu) where they release dopamine (DA). The current study investigated whether acetyl-l-carnitine (ALC) could ameliorate the pathology seen in an in vivoin vivo chronic 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of PD. Four treatment groups were included: 1) CONTROL receiving probenecid (PROB; 250 mg/kg) only, 2) MPTP (25 mg/kg) + PROB, 3) MPTP + ALC (100 mg/kg), and 4) ALC alone. MPTP-induced losses in tyrosine hydroxylase and DA transporter immunoreactivity in the SNc and CPu were significantly reduced by ALC. HPLC data further suggests that decreases in CPu DA levels produced by MPTP were also attenuated by ALC. Additionally, microglial activation and astrocytic reactivity induced by MPTP were greatly reduced by ALC, indicating protection against neuroinflammation. Glucose transporter-1 and the tight junction proteins occludin and zonula occludins-1 were also protected from MPTP-induced down-regulation by ALC. Together, data suggest that in this model, ALC protects against MPTP-induced damage to endothelial cells and loss of DA neurons in the SNc and CPu, suggesting that ALC therapy may have the potential to slow or ameliorate the progression of PD pathology in a clinical setting., (Published by Elsevier B.V.)

Background: Alzheimer's Disease (AD) is the most common type of dementia characterized by amyloid plaques containing Amyloid Beta (Aβ) peptides and neurofibrillary tangles containing tau protein. In addition to neuronal loss, Cerebral Amyloid Angiopathy (CAA) commonly occurs in AD. CAA is characterized by Aβ deposition in brain microvessels. Recent studies have suggested that exosomes (cell-derived vesicles containing a diverse cargo) may be involved in the pathogenesis of AD., Objective: Isolate and characterize brain-derived exosomes from a transgenic mouse model of AD that presents CAA., Methods: Exosomes were isolated from serum obtained from 13-month-old wild type and AD transgenic female mice using an exosome precipitation solution. Characterization of exosomal proteins was performed by western blots and dot blots., Results: Serum exosomes were increased in transgenic mice compared to wild types as determined by increased levels of the exosome markers flotillin and alix. High levels of neuronal markers were found in exosomes, without any difference any between the 2 groups. Markers for endothelial-derived exosomes were decreased in the transgenic model, while astrocytic-derived exosomes were increased. Exosome characterization showed increased levels of oligomeric Aβ and oligomeric and monomeric forms tau on the transgenic animals. Levels of amyloid precursor protein were also increased. In addition, pathological and phosphorylated forms of tau were detected, but no difference was observed between the groups., Conclusion: These data suggest that monomeric and oligomeric forms of Aβ and tau are secreted into serum via brain exosomes, most likely derived from astrocytes in the transgenic mouse model of AD with CAA. Studies on the implication of this event in the propagation of AD are underway., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, whose hallmark is the loss of dopamine terminals in the substantia nigra pars compacta (SNpc). PD is usually diagnosed after the appearance of motor symptoms, when about 70% of neurons in the SNpc have already been lost. Because of that, it is important to search for new methods that aid in the early diagnosis of PD. In recent years, microRNAs (miRs) have emerged as potential biomarkers for a variety of diseases and hold the potential to be used to aid in the diagnosis of PD. Therefore, the aim of this study was to characterize if specific miRs are differentially expressed in serum in a mouse model of PD. To induce PD-like damage, mice were subcutaneously injected with 25 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) by administering 10 doses over a period of 5 weeks, with 3.5 days between doses. Expression of 71 different microRNAs was quantified in serum separated from blood collected at day 35, using next-generation sequencing. Histological analysis and quantification of neurotransmitters were performed to confirm dopaminergic neurodegeneration. Chronic MPTP treatment induced loss of dopaminergic terminals in the SNpc and caudate putamen, confirmed by a decrease in the number of tyrosine hydroxylase and dopamine transporter positive cells. In addition, MPTP decreased the concentration of dopamine and its metabolites in the SNpc, simulating the damage observed in PD. From the 71 miRs analyzed, only 4 were differentially expressed after MPTP treatment. Serum levels of miR19b, miR124, miR126a and miR133b were significantly decreased in MPTP-treated mice compared to control. These data suggest that specific miRs are downregulated in a pre-clinical model of PD and hold the potential to be used as biomarkers to aid in the diagnosis of this disease. Further experiments need to be conducted to validate the use of these miRs as biomarkers of PD in additional pre-clinical models as well as in samples from patients diagnosed with PD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To assess the relative performance of MRI T 2 relaxation and ADC mapping as potential biomarkers of neurotoxicity, a model of 3-nitropropionic acid (NP)-induced neurodegeneration in rats was employed. Male Sprague-Dawley rats received NP (N = 20, 16-20 mg/kg, ip or sc) or saline (N = 6, 2 ml/kg, ip) daily for 3 days. MRI was performed using a 7 T system employing quantitative T 2 and ADC mapping based on spin echo pulse sequence. All maps were skull stripped and co-registered and the changes were quantified using baseline subtraction and anatomical segmentation. Following the in vivo portion of the study, rat brains were histologically examined. Four NP-treated rats were considered responders based on their MRI and histology data. T 2 values always increased in the presence of toxicity, while ADC changes were bidirectional, decreasing in some lesion areas and increasing in others. In contrast to T 2 in some cases, ADC did not change. The effect sizes of T 2 and ADC signals suggestive of neurotoxicity were 2.64 and 1.66, respectively, and the variability of averaged T 2 values among anatomical regions was consistently lower than that for ADC. The histopathology data confirmed the presence of neurotoxicity, however, a more detailed assessment of the correlation of MRI with histology is needed. T 2 mapping provides more sensitive and specific information than ADC about changes in the rat brain thought to be associated with neurotoxicity due to a higher signal-to-noise ratio, better resolution, and unidirectional changes, and presents a better opportunity for biomarker development., (Copyright © 2018. Published by Elsevier B.V.)

Introduction: Parkinson's Disease (PD) is a debilitating, age-related disorder characterized by selective degeneration of dopaminergic neurons in the midbrain substantia nigra (SNc). Dopaminergic neurons originating in the midbrain project to the striatum (Caudate-putamen-CPU). Although studies have suggested that the extracellular signal-regulated kinase ½ (ERK ½) in the brain is activated after 1-Methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP) exposure, to our knowledge no study has yet been done to demonstrate whether such activation occurs in neurons or in glia., Material and Methods: In the current study, we utilized both an acute and a repeat dose mouse model of PD using the neurotoxicant MPTP as the causative agent. Immunohistochemical studies using phospho ERK ½ antibody suggested that ERK ½ activation takes place in the striatum (CPU) and SNc of both animal models. Moreover, double immunolabeling studies using phospho ERK ½ and the microglial marker, CD11b or the astrocyte marker, Glial Fibrillary Acidic Protein (GFAP) suggested that the phospho ERK ½ was present exclusively in the microglia and not in the astrocytes., Results: Western Blot results suggested that there were no alterations in ERK in either MPTPtreated animals or in control animals; however, phospho ERK ½ was found to be significantly increased in the striatum and SNc in both acute chronic mouse PD models. Tyrosine Hydroxylase (TH) immunolabeling revealed significant decreases in dopaminergic neurons in the SNc in both animal models' concomitant with activation of microglia and ERK activation., Conclusion: These observations suggest that ERK activation takes place following MPTP treatment and that activation of ERK occurs primarily in the microglia. The data provided also suggest that ERK activation may be involved in transcriptional activation of microglia following neurotoxicant insults., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The aim of this study was to assess quantitative changes in T2 relaxation using magnetic resonance imaging approaches in rats exposed to kainic acid to assess the utility of such endpoints as biomarkers of neurotoxicity. Quantitative T2 mapping was performed in 21 rats before and 2, 24, and 48 h after a single ip injection of 10 mg/kg of kainic acid. Three methods of quantifying T2 changes were explored: (1) Thresholding: all voxels exhibiting T2 ≤ 72 ms were designated normal tissue, whereas voxels exhibiting T2 > 72 ms were designated as lesioned tissue; (2) Statistical mapping: T2 maps obtained after treatment were statistically compared with averaged "baseline" maps, voxel-by-voxel; (3) Within-subject difference from baseline: for each individual the baseline T2 map was subtracted from the T2 map obtained after treatment. Based on the follow-up histopathological response there were 9 responders, 7 nonresponders, and 5 animals were not classified due to early sacrifice at 2 h which was too soon after treatment to detect any morphological evidence. The "thresholding" method (1) detected differences between groups only at the later time point of 48 h, the "statistical mapping" approach (2) detected differences 24 and 48 h after treatment, and the "within-subject difference from baseline" method (3) detected statistically significant differences between groups at each time point (2, 24, and 48 h). T2 mapping provides an easily quantifiable biomarker and the quantification method employing the use of the same animal as its own control provides the most sensitive metrics., (Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.)

MRI was utilized to probe T2 changes in living brain following exposure of rats to one of ten classical neurotoxicants. Brains were subsequently perfused for classical neuropathology examination. This approach was predicated on the assumption that the T2 changes represent loci of neurotoxicity encompassing those seen using neuropathology techniques. The traditional neurotoxicologic approach of selecting a few arbitrary brain sections is dramatically improved by MRI targeting that can indicate the location(s) at which to collect "smart sections" for subsequent workup. MRI scans can provide the equivalent of 64 coronal sections; the number estimated for full coverage of the rat brain if only traditional neuropathology is utilized. Use of MRI allows each animal to serve as its own control as well as longitudinal observations of the life cycle of the neurotoxic lesion(s) (inception, apex and regression). Optimization of time of sacrifice and selection of an appropriate stain based on MRI-identified brain areas could be greatly enhanced should this approach prove successful. The application of full brain MRI imaging that informs neuropathology offers the potential to dramatically improve detection of neurotoxicity produced by new drugs and facilitate new drug development, review and approval processes, and to qualify an imaging biomarker of neuropathology., (Published by Elsevier Inc.)

Improved biomarkers of acute nephrotoxicity are coveted by the drug development industry, regulatory agencies, and clinicians. In an effort to identify such biomarkers, urinary peptide profiles of rats treated with two different nephrotoxins were investigated. 493 marker candidates were defined that showed a significant response to cis-platin comparing a cis-platin treated cohort to controls. Next, urine samples from rats that received three consecutive daily doses of 150 or 300 mg/kg gentamicin were examined. 557 potential biomarkers were initially identified; 108 of these gentamicin-response markers showed a clear temporal response to treatment. 39 of the cisplatin-response markers also displayed a clear response to gentamicin. Of the combined 147 peptides, 101 were similarly regulated by gentamicin or cis-platin and 54 could be identified by tandem mass spectrometry. Most were collagen type I and type III fragments up-regulated in response to gentamicin treatment. Based on these peptides, classification models were generated and validated in a longitudinal study. In agreement with histopathology, the observed changes in classification scores were transient, initiated after the first dose, and generally persistent over a period of 10-20 days before returning to control levels. The data support the hypothesis that gentamicin-induced renal toxicity up-regulates protease activity, resulting in an increase in several specific urinary collagen fragments. Urinary proteomic biomarkers identified here, especially those common to both nephrotoxins, may serve as a valuable tool to investigate potential new drug candidates for the risk of nephrotoxicity.

Repeated administration of phencyclidine (PCP), an N-methyl-d-aspartate (NMDA) receptor antagonist, during development, may result in neuronal damage that leads to behavioral deficits in adulthood. The present study examined the potential neurotoxic effects of PCP exposure (10mg/kg) in rats on postnatal days (PNDs) 7, 9 and 11 and the possible underlying mechanism(s) for neurotoxicity. Brain tissue was harvested for RNA extraction and morphological assessments. RNA was collected from the frontal cortex for DNA microarray analysis and quantitative RT-PCR. Gene expression profiling was determined using Illumina Rat Ref-12 Expression BeadChips containing 22,226 probes. Based on criteria of a fold-change greater than 1.4 and a P-value less than 0.05, 19 genes including NMDAR1 (N-methyl-d-aspartate receptor) and four pro-apoptotic genes were up-regulated, and 25 genes including four anti-apoptotic genes were down-regulated, in the PCP-treated group. In addition, the schizophrenia-relevant genes, Bdnf (Brain-derived neurotrophic factor) and Bhlhb2 (basic helix-loop-helix domain containing, class B, 2), were significantly different between the PCP and the control groups. Quantitative RT-PCR confirmed the microarray results. Elevated neuronal cell death was further confirmed using Fluoro-Jade C staining. These findings support the hypothesis that neurodegeneration caused by PCP occurs, at least in part, through the up-regulation of NMDA receptors, which makes neurons possessing these receptors more vulnerable to endogenous glutamate. The changes in schizophrenia-relevant genes after repeated PCP exposure during development may provide important information concerning the validation of an animal model for this disorder., (Published by Elsevier Ltd.)

Previously our laboratory has shown that ketamine exposure (24h of clinically relevant anesthesia) causes significant increases in neuronal cell death in perinatal rhesus monkeys. Sensitivity to this ketamine-induced neurotoxicity was observed on gestational days 120-123 (in utero exposure via maternal anesthesia) and on postnatal days (PNDs) 5-6, but not on PNDs 35-37. In the present study, six monkeys were exposed on PND 5 or 6 to intravenous ketamine anesthesia to maintain a light surgical plane for 24h and six control animals were unexposed. At 7 months of age all animals were weaned and began training to perform a series of cognitive function tasks as part of the National Center for Toxicological Research (NCTR) Operant Test Battery (OTB). The OTB tasks used here included those for assessing aspects of learning, motivation, color discrimination, and short-term memory. Subjects responded for banana-flavored food pellets by pressing response levers and press-plates during daily (M-F) test sessions (50 min) and were assigned training scores based upon their individual performance. As reported earlier (Paule et al., 2009) beginning around 10 months of age, control animals significantly outperformed (had higher training scores than) ketamine-exposed animals for approximately the next 10 months. For animals now over 3 and one-half years of age, the cognitive impairments continue to manifest in the ketamine-exposed group as poorer performance in the OTB learning and color and position discrimination tasks, as deficits in accuracy of task performance, but also in response speed. There are also apparent differences in the motivation of these animals which may be impacting OTB performance. These observations demonstrate that a single 24-h episode of ketamine anesthesia, occurring during a sensitive period of brain development, results in very long-lasting deficits in brain function in primates and provide proof-of-concept that general anesthesia during critical periods of brain development can result in subsequent functional deficits. Supported by NICHD, CDER/FDA and NCTR/FDA., (Published by Elsevier Inc.)

Exposure of the vasculature to vasodilators, pharmaceuticals and industrial chemicals may lead to injury of the blood vessel wall in animals. Vascular injury may begin with changes in the permeability of vascular endothelial cell and vessels, resulting in possible hemorrhage and edema leading subsequently to immune cell infiltration. The present study was undertaken to determine if the direct exposure of the Sprague Dawley rat mesenteric vasculature through the perfusion of aminophylline, fenoldopam, compound 48/80, histamine or serotonin has any such effects on the blood vessels, and if the two vital dyes Monastral blue B and Evans blue can be used to enhance the visualization of the vascular damage. Microscopic visualization was enhanced by the use of dyes and a variety of alterations of the perfused mesenteric vessels were detected, including varying degrees of mast cell degranulation, microvascular vasodilatation and increased vascular permeability. Macroscopic evidence of vascular damage was minimal. This study demonstrates that in situ perfusion of the rat mesentery is a simple and useful method to eliminate the influence of a variety of physiologic influences or homeostatic responses and can be used to further investigate drug-induced vascular damage., (Copyright 2010 Prous Science, S.A.U. or its licensors. All rights reserved.)

Ketamine, a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, is associated with accelerated neuronal apoptosis in the developing rodent brain. In this study, postnatal day (PND) 7 rats were treated with 20 mg/kg ketamine or saline in six successive doses (s.c.) at 2-h intervals. Brain frontal cortical areas were collected 6 h after the last dose and RNA isolated and hybridized to Illumina Rat Ref-12 Expression BeadChips containing 22,226 probes. Many of the differentially expressed genes were associated with cell death or differentiation and receptor activity. Ingenuity Pathway Analysis software identified perturbations in NMDA-type glutamate, GABA and dopamine receptor signaling. Quantitative polymerase chain reaction (Q-PCR) confirmed that NMDA receptor subunits were significantly up-regulated. Up-regulation of NMDA receptor mRNA signaling was further confirmed by in situ hybridization. These observations support our working hypothesis that prolonged ketamine exposure produces up-regulation of NMDA receptors and subsequent over-stimulation of the glutamatergic system by endogenous glutamate, triggering enhanced apoptosis in developing neurons., (Published by Elsevier Ltd.)

We have investigated urine obtained from Sprague Dawley rats before and after administration of cis-Platin, aiming at the definition of biomarkers for drug-induced cytotoxicity. Rats were treated with 3 or 6 mg/kg cis-Platin (i.p., single injection) and urine samples were collected before and after drug or saline treatment. Analysis of the low molecular weight proteome (<20 kDa) using capillary-electrophoresis coupled mass spectrometry allowed us to tentatively identify 34 urinary peptides that show significant differences between control and treated animals, and hence may serve as a potential biomarker for cis-Platin-induced nephrotoxicity. These biomarkers were confirmed in a blinded assessment of additional samples. The blinded data also revealed time-dependency of induced changes. Some of the potential biomarkers could be sequenced. This information revealed great similarity between cis-Platin-induced changes and significant changes in the urinary proteome of patients suffering from tubular injury (Fanconi syndrome). Our study strongly suggests that (drug-induced) nephrotoxicity can be detected with high accuracy in laboratory rodents using urinary proteome analysis. The effects observed are very similar to those seen in corresponding human diseases and similar approaches may be very helpful in evaluating drug-induced organ damage in preclinical animal models. This study aiming at the definition of biomarkers for drug-induced cytotoxicity may serve as a proof-of-principle for the use of urinary proteomics in assessment of drug-induced nephrotoxicity., (Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Drug-associated vascular injury can be caused by phosphodiesterase (PDE) IV inhibitors and drugs from several other classes. The pathogenesis is poorly understood, but it appears to include vascular and innate immunological components. This research was undertaken to identify changes in peripheral blood associated with vascular injury caused by PDE IV inhibitors. We evaluated twelve proteins, serum nitrite, and leukocyte populations in peripheral blood of rats treated with experimental PDE IV inhibitors. We found that these compounds produced histological microvascular injury in a dose- and time-dependent manner. Measurement of these serum proteins showed changes in eight of the twelve examined. Changes were seen in the levels of: tissue inhibitor of metalloproteinase-1, alpha1-acid glycoprotein, GRO/CINC-1, vascular endothelial growth factor, C-reactive protein, haptoglobin, thrombomodulin, and interleukin-6. No changes were seen in levels of tumor necrosis factor-alpha, hepatocyte growth factor, nerve growth factor, and granulocyte-monocyte colony stimulating factor. Serum levels of nitrite were also increased. Circulating granulocyte numbers were increased, and lymphocyte numbers were decreased. The changes in these parameters showed both a dose- and time-dependent association with histopathologic changes. These biomarkers could provide an additional tool for the nonclinical and clinical evaluation of investigational compounds.

The anesthetic gas nitrous oxide (N2O) and the volatile anesthetic isoflurane (ISO) are commonly used in surgical procedures for human infants and in veterinary and laboratory animal practice to produce loss of consciousness and analgesia. Recent reports indicate that exposure of the developing brain to general anesthetics that block N-methyl-D-aspartate (NMDA) glutamate receptors or potentiate GABA(A) receptors can trigger widespread apoptotic neurodegeneration. In the present study, the question arises whether a relatively low dose of ISO alone or its combination with N2O entails significant risk of inducing enhanced apoptosis. In addition, the role of L-carnitine to attenuate these effects was also examined. Postnatal day 7 (PND-7) rat pups were exposed to N2O (75%) or a low dose of ISO (0.55%) alone, or N2O plus ISO for 2, 4, 6 or 8 h with or without L-carnitine. The neurotoxic effects were evaluated 6 h after completion of anesthetic administration. No significant neurotoxic effects were observed for the animals exposed to N2O or ISO alone. However, enhanced apoptotic cell death was apparent when N2O was combined with ISO at exposure durations of 6 h or more. Co-administration of L-carnitine (300 or 500 mg/kg, i.p.) effectively protected neurons from the anesthetic-induced damage. These data indicate that 6 h or more of inhaled anesthetic exposure consisting of a combination of N2O and ISO results in enhanced neuronal apoptosis, and L-carnitine effectively blocks the neuronal apoptosis caused by inhalation anesthetics in the developing rat brain.

Background: Urine from male Sprague-Dawley rats 25, 40, and 80 days old was analyzed by NMR and UPLC/MS. The effects of data normalization procedures on principal component analysis (PCA) and quantitative analysis of NMR-based metabonomics data were investigated. Additionally, the effects of age on the metabolic profiles were examined by both NMR and UPLC/MS analyses., Results: The data normalization factor was shown to have a great impact on the statistical and quantitative results indicating the need to carefully consider how to best normalize the data within a particular study and when comparing different studies. PCA applied to the data obtained from both NMR and UPLC/MS platforms reveals similar age-related differences. NMR indicated many metabolites associated with the Krebs cycle decrease while citrate and 2-oxoglutarate, also associated with the Krebs cycle, increase in older rats., Conclusion: This study compared four different normalization methods for the NMR-based metabonomics spectra from an age-related study. It was shown that each method of normalization has a great effect on both the statistical and quantitative analyses. Each normalization method resulted in altered relative positions of significant PCA loadings for each sample spectra but it did not alter which chemical shifts had the highest loadings. The greater the normalization factor was related to age, the greater the separation between age groups was observed in subsequent PCA analyses. The normalization factor that showed the least age dependence was total NMR intensity, which was consistent with UPLC/MS data. Normalization by total intensity attempts to make corrections due to dietary and water intake of the individual animal, which is especially useful in metabonomics evaluations of urine. Additionally, metabonomics evaluations of age-related effects showed decreased concentrations of many Krebs cycle intermediates along with increased levels of oxidized antioxidants in urine of older rats, which is consistent with current theories on aging and its association with diminishing mitochondrial function and increasing levels of reactive oxygen species. Analysis of urine by both NMR and UPLC/MS provides a comprehensive and complementary means of examining metabolic events in aging rats.

A multi-age rat model was used to identify potential age-related differences in renal injury following exposure to gentamicin (GM). In this study, 10-, 25-, 40-, and 80-day-old Sprague-Dawley rats were dosed with GM at 0, 50, or 100 mg kg(-1) body weight per day (mkd) sc for 6 or 14 days. Urine samples were collected up to 72 h after initial dosing. The maximum tolerated dose was lower in 10-day-old rats than for other ages (none survived 11 days of treatment). Eighty-day-old rats given the highest dose showed a diminished rate of growth and an increase in serum creatinine, blood urea nitrogen (BUN), urinary kidney injury molecule-1 (Kim-1), and renal pathology. Ten- and 40-day-old rats given 100 mkd of GM for 6- or 14 days also had increased levels of serum BUN and Cr and renal pathology, whereas only mild renal alterations were found in 25-day-old rats. After 6 days of treatment with 100 mkd GM, significant increases in Havcr-1 (Kim-1) gene expression were detected only in 10- and 80-day-old rats. In urine samples, nuclear magnetic resonance and ultra performance liquid chromatography/mass spectrometry analysis detected changes related to GM efficacy (e.g., hippurate) and increases in metabolites related to antioxidant activity, which was greatest in the 80-day-old rats. The magnitude of the genomic, metabonomic, and serum chemistry changes appeared to correlate with the degree of nephropathy. These findings indicate that an experimental animal model that includes several developmental stages can detect age-related differences in drug-induced organ toxicities and may be a useful predictor of pediatric drug safety in preclinical studies.

Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is used as a general pediatric anesthetic. Recent data suggest that anesthetic drugs may cause neurodegeneration during development. The purpose of this study was to determine the robustness of ketamine-induced developmental neurotoxicity using rhesus monkey frontal cortical cultures and also to determine if dysregulation of NMDA receptor subunits promotes ketamine-induced cell death. Frontal cortical cells collected from the neonatal monkey were incubated for 24 h with 1, 10, or 20 microM ketamine alone or with ketamine plus either NR1 antisense oligonucleotides or the nuclear factor kB translocation inhibitor, SN-50. Ketamine caused a marked reduction in the neuronal marker polysialic acid neural cell adhesion molecule and mitochondrial metabolism, as well as an increase in DNA fragmentation and release of lactate dehydrogenase. Ketamine-induced effects were blocked by NR1 antisenses and SN-50. These data suggest that NR1 antisenses and SN-50 offer neuroprotection from the enhanced degeneration induced by ketamine in vitro.

The noise level in an animal facility is an important environmental variable that can adversely affect animal welfare, as well as experimental data. The authors describe the strategy they used to record, evaluate, and control excess noise and vibration during a period of renovation, while maintaining the operation of a research facility.

Influence of protein deficiency on the neurobehavioral toxicity of styrene during gestation and early infancy was studied in rats. Eye opening and fur growth were delayed in rat pups born to dams receiving a low protein diet. These pups also showed a delay in the development of surface and air righting reflexes and cliff avoidance response and a marginal increase in the levels of dopamine and serotonin receptors in comparison to those born to dams receiving a normal protein diet. Alterations in these parameters were more marked in pups born to dams exposed to styrene and receiving a low protein diet. In addition, these pups also showed a significant decrease in the activity of monoamine oxidase, Na+, K(+)-ATPase and succinic dehydrogenase as well as significant increases in motor activity and receptor sensitivity when compared to rat pups born to dams receiving a low protein diet. No significant alterations in behavioral and biochemical parameters were observed in the pups born to dams exposed to styrene and receiving a normal protein diet at this dose level. These results suggest that protein deficiency during early life renders the animals more susceptible to styrene.