Optimization of Detection of Gadodiamide Brain Retention in Rats Using Quantitative T2 Mapping and Intraperitoneal Administration.

BACKGROUND: Currently, the gadolinium retention in the brain after the use of contrast agents is studied by T1 -weighted magnetic resonance imaging (MRI) (T1 w) and T1 mapping. The former does not provide easily quantifiable data and the latter requires prolonged scanning and is sensitive to motion. T2 mapping may provide an alternative approach. Animal studies of gadolinium retention are complicated by repeated intravenous (IV) dosing, whereas intraperitoneal (IP) injections might be sufficient. HYPOTHESIS: T2 mapping will detect the changes in the rat brain due to gadolinium retention, and IP administration is equivalent to IV for long-term studies. STUDY TYPE: Prospective longitudinal. ANIMAL MODEL: A total of 31 Sprague-Dawley rats administered gadodiamide IV (N = 8) or IP (N = 8), or saline IV (N = 6) or IP (N = 9) 4 days per week for 5 weeks. FIELD STRENGTH/SEQUENCES: A 7 T, T1 w, and T2 mapping. ASSESSMENT: T2 relaxation and image intensities in the deep cerebellar nuclei were measured pre-treatment and weekly for 5 weeks. Then brains were assessed for neuropathology (N = 4) or gadolinium content using inductively coupled plasma mass spectrometry (ICP-MS, N = 12). STATISTICAL TESTS: Repeated measures analysis of variance with post hoc Student-Newman-Keuls tests and Hedges' effect size. RESULTS: Gadolinium was detected by both approaches; however, T2 mapping was more sensitive (effect size 2.32 for T2 vs. 0.95 for T1 w), and earlier detection (week 3 for T2 vs. week 4 for T1 w). ICP-MS confirmed the presence of gadolinium (3.076 ± 0.909 nmol/g in the IV group and 3.948 ± 0.806 nmol/g in the IP group). There was no significant difference between IP and IV groups (ICP-MS, P = 0.109; MRI, P = 0.696). No histopathological abnormalities were detected in any studied animal. CONCLUSION: T2 relaxometry detects gadolinium retention in the rat brain after multiple doses of gadodiamide irrespective of the route of administration. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 1.

Quantitative neurotoxicology: Potential role of artificial intelligence/deep learning approach.

Neurotoxicity studies are important in the preclinical stages of drug development process, because exposure to certain compounds that may enter the brain across a permeable blood brain barrier damages neurons and other supporting cells such as astrocytes. This could, in turn, lead to various neurological disorders such as Parkinson's or Huntington's disease as well as various dementias. Toxicity assessment is often done by pathologists after these exposures by qualitatively or semiquantitatively grading the severity of neurotoxicity in histopathology slides. Quantification of the extent of neurotoxicity supports qualitative histopathological analysis and provides a better understanding of the global extent of brain damage. Stereological techniques such as the utilization of an optical fractionator provide an unbiased quantification of the neuronal damage; however, the process is time-consuming. Advent of whole slide imaging (WSI) introduced digital image analysis which made quantification of neurotoxicity automated, faster and with reduced bias, making statistical comparisons possible. Although automated to a certain level, simple digital image analysis requires manual efforts of experts which is time-consuming and limits analysis of large datasets. Digital image analysis coupled with a deep learning artificial intelligence model provides a good alternative solution to time-consuming stereological and simple digital analysis. Deep learning models could be trained to identify damaged or dead neurons in an automated fashion. This review has focused on and discusses studies demonstrating the role of deep learning in segmentation of brain regions, toxicity detection and quantification of degenerated neurons as well as the estimation of area/volume of degeneration.

Global Neurotoxicity: Quantitative Analysis of Rat Brain Toxicity Following Exposure to Trimethyltin.

The organotin, trimethyltin (TMT), is a highly toxic compound. In this study, silver-stained rat brain sections were qualitatively and quantitatively evaluated for degeneration after systemic treatment with TMT. Degenerated neurons were counted using image analysis methods available in the HALO image analysis software. Specific brain areas including the cortex, inferior and superior colliculus, and thalamus were quantitatively analyzed. Our results indicate extensive and widespread damage to the rat brain after systemic administration of TMT. Qualitative results suggest severe TMT-induced toxicity 3 and 7 days after the administration of TMT. Trimethyltin toxicity was greatest in the hippocampus, olfactory area, cerebellum, pons, mammillary nucleus, inferior and superior colliculus, hypoglossal nucleus, thalamus, and cerebellar Purkinje cells. Quantification showed that the optic layer of the superior colliculus exhibited significantly more degeneration compared to layers above and below. The inferior colliculus showed greater degeneration in the dorsal area relative to the central area. Similarly, in cortical layers, there was greater neurodegeneration in deeper layers compared to superficial layers. Quantification of damage in various thalamic nuclei showed that the greatest degeneration occurred in midline and intralaminar nuclei. These results suggest selective neuronal network vulnerability to TMT-related toxicity in the rat brain.

Hepatic Transcript Profiles of Cytochrome P450 Genes Predict Sex Differences in Drug Metabolism.

Safety assessments of new drug candidates are an important part of the drug development and approval process. Often, possible sex-associated susceptibilities are not adequately addressed, and better assessment tools are needed. We hypothesized that hepatic transcript profiles of cytochrome P450 (P450) enzymes can be used to predict sex-associated differences in drug metabolism and possible adverse events. Comprehensive hepatic transcript profiles were generated for F344 rats of both sexes at nine ages, from 2 weeks (preweaning) to 104 weeks (elderly). Large differences in the transcript profiles of 29 drug metabolizing enzymes and transporters were found between adult males and females (8-52 weeks). Using the PharmaPendium data base, 41 drugs were found to be metabolized by one or two P450 enzymes encoded by sexually dimorphic mRNAs and thus were candidates for evaluation of possible sexually dimorphic metabolism and/or toxicities. Suspension cultures of primary hepatocytes from three male and three female adult rats (10-13 weeks old) were used to evaluate the metabolism of 11 drugs predicted to have sexually dimorphic metabolism. The pharmacokinetics of the drug or its metabolite was analyzed by liquid chromatography/tandem mass spectrometry using multiple reaction monitoring. Of those drugs with adequate metabolism, the predicted significant sex-different metabolism was found for six of seven drugs, with half-lives 37%-400% longer in female hepatocytes than in male hepatocytes. Thus, in this rat model, transcript profiles may allow identification of potential sex-related differences in drug metabolism. SIGNIFICANCE STATEMENT: The present study showed that sex-different expression of genes coding for drug metabolizing enzymes, specifically cytochrome P450s, could be used to predict sex-different drug metabolism and, thus, provide a new tool for protecting susceptible subpopulations from possible adverse drug events.

Quantitative Neurotoxicology: An Assessment of the Neurotoxic Profile of Kainic Acid in Sprague Dawley Rats.

This study consisted of a qualitative and quantitative assessment of neuropathological changes in kainic acid (KA)-treated adult male rats. Rats were administered a single 10 mg/kg intraperitoneal injection of KA or the same volume of saline and sacrificed 24 or 48 hours posttreatment. Brains were collected, sectioned coronally (∼ 81 slices), and stained with amino cupric silver to reveal degenerative changes. For qualitative assessment of neural degeneration, sectioned material was evaluated by a board-certified pathologist, and the level of degeneration was graded based upon a 4-point scale. For measurement of quantitative neural degeneration in response to KA treatment, the HALO digital image analysis software tool was used. Quantitative measurements of specific regions within the brain were obtained from silver-stained tissue sections with quantitation based on stain color and optical density. This quantitative evaluation method identified degeneration primarily in the cerebral cortex, septal nuclei, amygdala, olfactory bulb, hippocampus, thalamus, and hypothalamus. The KA-produced neuronal degeneration in the cortex was primarily in the piriform, insular, rhinal, and cingulate areas. In the hippocampus, the dentate gyrus was found to be the most affected area. Our findings indicate global neurotoxicity due to KA treatment. Certain brain structures exhibited more degeneration than others, reflecting differential sensitivity or vulnerability of neurons to KA.

Lipid profiling as an effective approach for identifying biomarkers/adverse events associated with pediatric anesthesia.

Adverse effects related to central nervous system (CNS) function in pediatric populations may, at times, be difficult, if not impossible to evaluate. Prolonged anesthetic exposure affects brain excitability and anesthesia during the most sensitive developmental stages and has been associated with mitochondrial dysfunction, aberrant lipid metabolism and synaptogenesis, subsequent neuronal damage, as well as long-term behavioral deficits. There has been limited research evaluating whether and how anesthetic agents affect cellular lipids, the most abundant components of the brain other than water. Therefore, this review discusses: (1) whether the observed anesthetic-induced changes in lipid profiles seen in preclinical studies represents early signs of neurotoxicity; (2) the potential mechanisms underlying anesthetic-induced brain injury; and (3) whether lipid biomarker(s) identified in preclinical studies can serve as markers for the early clinical detection of anesthetic-induced neurotoxicity.

Corticosterone and exogenous glucose alter blood glucose levels, neurotoxicity, and vascular toxicity produced by methamphetamine.

Our previous studies have raised the possibility that altered blood glucose levels may influence and/or be predictive of methamphetamine (METH) neurotoxicity. This study evaluated the effects of exogenous glucose and corticosterone (CORT) pretreatment alone or in combination with METH on blood glucose levels and the neural and vascular toxicity produced. METH exposure consisted of four sequential injections of 5, 7.5, 10, and 10 mg/kg (2 h between injections) D-METH. The three groups given METH in combination with saline, glucose (METH+Glucose), or CORT (METH+CORT) had significantly higher glucose levels compared to the corresponding treatment groups without METH except at 3 h after the last injection. At this last time point, the METH and METH+Glucose groups had lower levels than the non-METH groups, while the METH+CORT group did not. CORT alone or glucose alone did not significantly increase blood glucose. Mortality rates for the METH+CORT (40%) and METH+Glucose (44%) groups were substantially higher than the METH (< 10%) group. Additionally, METH+CORT significantly increased neurodegeneration above the other three METH treatment groups (≈ 2.5-fold in the parietal cortex). Thus, maintaining elevated levels of glucose during METH exposure increases lethality and may exacerbate neurodegeneration. Neuroinflammation, specifically microglial activation, was associated with degenerating neurons in the parietal cortex and thalamus after METH exposure. The activated microglia in the parietal cortex were surrounding vasculature in most cases and the extent of microglial activation was exacerbated by CORT pretreatment. Our findings show that acute CORT exposure and elevated blood glucose levels can exacerbate METH-induced vascular damage, neuroinflammation, neurodegeneration and lethality. Cover Image for this issue: doi. 10.1111/jnc.13819.

Vascular-directed responses of microglia produced by methamphetamine exposure: indirect evidence that microglia are involved in vascular repair?

BACKGROUND: Brain microglial activations and damage responses are most commonly associated with neurodegeneration or systemic innate immune system activation. Here, we used histological methods to focus on microglial responses that are directed towards brain vasculature, previously undescribed, after a neurotoxic exposure to methamphetamine. METHODS: Male rats were given doses of methamphetamine that produce pronounced hyperthermia, hypertension, and toxicity. Identification of microglia and microglia-like cells (pericytes and possibly perivascular cells) was done using immunoreactivity to allograft inflammatory factor 1 (Aif1 a.k.a Iba1) and alpha M integrin (Itgam a.k.a. Cd11b) while vasculature endothelium was identified using rat endothelial cell antigen 1 (RECA-1). Regions of neuronal, axonal, and nerve terminal degeneration were determined using Fluoro-Jade C. RESULTS: Dual labeling of vasculature (RECA-1) and microglia (Iba1) showed a strong association of hypertrophied cells surrounding and juxtaposed to vasculature in the septum, medial dorsal hippocampus, piriform cortex, and thalamus. The Iba1 labeling was more pronounced in the cell body while Cd11b more so in the processes of activated microglia. These regions have been previously identified to have vascular leakage after neurotoxic methamphetamine exposure. Dual labeling with Fluoro-Jade C and Iba1 indicated that there was minimal or no evidence of neuronal damage in the septum and hippocampus where many hypertrophied Iba1-labeled cells were found to be associated with vasculature. Although microglial activation around the prominent neurodegeneration was found in the thalamus, there were also many examples of activated microglia associated with vasculature. CONCLUSIONS: The data implicate microglia, and possibly related cell types, in playing a major role in responding to methamphetamine-induced vascular damage, and possibly repair, in the absence of neurodegeneration. Identifying brain regions with hypertrophied/activated microglial-like cells associated with vasculature has the potential for identifying regions of more subtle examples of vascular damage and BBB compromise.

In Vivo Monitoring of Sevoflurane-induced Adverse Effects in Neonatal Nonhuman Primates Using Small-animal Positron Emission Tomography.

BACKGROUND: Animals exposed to sevoflurane during development sustain neuronal cell death in their developing brains. In vivo micro-positron emission tomography (PET)/computed tomography imaging has been utilized as a minimally invasive method to detect anesthetic-induced neuronal adverse effects in animal studies. METHODS: Neonatal rhesus monkeys (postnatal day 5 or 6, 3 to 6 per group) were exposed for 8 h to 2.5% sevoflurane with or without acetyl-L-carnitine (ALC). Control monkeys were exposed to room air with or without ALC. Physiologic status was monitored throughout exposures. Depth of anesthesia was monitored using quantitative electroencephalography. After the exposure, microPET/computed tomography scans using F-labeled fluoroethoxybenzyl-N-(4-phenoxypyridin-3-yl) acetamide (FEPPA) were performed repeatedly on day 1, 1 and 3 weeks, and 2 and 6 months after exposure. RESULTS: Critical physiologic metrics in neonatal monkeys remained within the normal range during anesthetic exposures. The uptake of [F]-FEPPA in the frontal and temporal lobes was increased significantly 1 day or 1 week after exposure, respectively. Analyses of microPET images recorded 1 day after exposure showed that sevoflurane exposure increased [F]-FEPPA uptake in the frontal lobe from 0.927 ± 0.04 to 1.146 ± 0.04, and in the temporal lobe from 0.859 ± 0.05 to 1.046 ± 0.04 (mean ± SE, P < 0.05). Coadministration of ALC effectively blocked the increase in FEPPA uptake. Sevoflurane-induced adverse effects were confirmed by histopathologic evidence as well. CONCLUSIONS: Sevoflurane-induced general anesthesia during development increases glial activation, which may serve as a surrogate for neurotoxicity in the nonhuman primate brain. ALC is a potential protective agent against some of the adverse effects associated with such exposures.

Computational identification of a phospholipidosis toxicophore using (13)C and (15)N NMR-distance based fingerprints.

Modified 3D-SDAR fingerprints combining (13)C and (15)N NMR chemical shifts augmented with inter-atomic distances were used to model the potential of chemicals to induce phospholipidosis (PLD). A curated dataset of 328 compounds (some of which were cationic amphiphilic drugs) was used to generate 3D-QSDAR models based on tessellations of the 3D-SDAR space with grids of different density. Composite PLS models averaging the aggregated predictions from 100 fully randomized individual models were generated. On each of the 100 runs, the activities of an external blind test set comprised of 294 proprietary chemicals were predicted and averaged to provide composite estimates of their PLD-inducing potentials (PLD+ if PLD is observed, otherwise PLD-). The best performing 3D-QSDAR model utilized a grid with a density of 8ppm×8ppm in the C-C region, 8ppm×20ppm in the C-N region and 20ppm×20ppm in the N-N region. The classification predictive performance parameters of this model evaluated on the basis of the external test set were as follows: accuracy=0.70, sensitivity=0.73 and specificity=0.66. A projection of the most frequently occurring bins on the standard coordinate space suggested a toxicophore composed of an aromatic ring with a centroid 3.5-7.5Å distant from an amino-group. The presence of a second aromatic ring separated by a 4-5Å spacer from the first ring and at a distance of between 5.5Å and 7Å from the amino-group was also associated with a PLD+ effect. These models provide comparable predictive performance to previously reported models for PLD with the added benefit of being based entirely on non-confidential, publicly available training data and with good predictive performance when tested in a rigorous, external validation exercise.

Comparison of the global gene expression of choroid plexus and meninges and associated vasculature under control conditions and after pronounced hyperthermia or amphetamine toxicity.

BACKGROUND: The meninges (arachnoid and pial membranes) and associated vasculature (MAV) and choroid plexus are important in maintaining cerebrospinal fluid (CSF) generation and flow. MAV vasculature was previously observed to be adversely affected by environmentally-induced hyperthermia (EIH) and more so by a neurotoxic amphetamine (AMPH) exposure. Herein, microarray and RT-PCR analysis was used to compare the gene expression profiles between choroid plexus and MAV under control conditions and at 3 hours and 1 day after EIH or AMPH exposure. Since AMPH and EIH are so disruptive to vasculature, genes related to vasculature integrity and function were of interest. RESULTS: Our data shows that, under control conditions, many of the genes with relatively high expression in both the MAV and choroid plexus are also abundant in many epithelial tissues. These genes function in transport of water, ions, and solutes, and likely play a role in CSF regulation. Most genes that help form the blood-brain barrier (BBB) and tight junctions were also highly expressed in MAV but not in choroid plexus. In MAV, exposure to EIH and more so to AMPH decreased the expression of BBB-related genes such as Sox18, Ocln, and Cldn5, but they were much less affected in the choroid plexus. There was a correlation between the genes related to reactive oxidative stress and damage that were significantly altered in the MAV and choroid plexus after either EIH or AMPH. However, AMPH (at 3 hr) significantly affected about 5 times as many genes as EIH in the MAV, while in the choroid plexus EIH affected more genes than AMPH. Several unique genes that are not specifically related to vascular damage increased to a much greater extent after AMPH compared to EIH in the MAV (Lbp, Reg3a, Reg3b, Slc15a1, Sct and Fst) and choroid plexus (Bmp4, Dio2 and Lbp). CONCLUSIONS: Our study indicates that the disruption of choroid plexus function and damage produced by AMPH and EIH is significant, but the changes may not be as pronounced as they are in the MAV, particularly for AMPH. Expression profiles in the MAV and choroid plexus differed to some extent and differences were not restricted to vascular related genes.

Establishment of neural stem cells from fetal monkey brain for neurotoxicity testing.

Neurotoxicity assessments are generally performed using laboratory animals. However, as in vitro neurotoxicity models are continuously refined to reach adequate predicative concordance with in vivo responses, they are increasingly used for some endpoints of neurotoxicity. In this study, gestational day 80 fetal rhesus monkey brain tissue was obtained for neural stem cells (NSCs) isolation. Cells from the entire hippocampus were harvested, mechanically dissociated, and cultured for proliferation and differentiation. Immunocytochemical staining and biological assays demonstrated that the harvested hippocampal cells exhibited typical NSC phenotypes in vitro: (1) cells proliferated vigorously and expressed NSC markers nestin and sex-determining region Y-box 2 (SOX2) and (2) cells differentiated into neurons, astrocytes, and oligodendrocytes, as confirmed by positive staining with class III ß-tubulin, glial fibrillary acidic protein, and galactocerebroside, respectively. The NSC produced detectable responses following neurotoxicant exposures (e.g. trimethyltin and 3-nitropropionic acid). Our results indicated that non-human primate NSCs may be a practical tool to study the biology of neural cells and to evaluate the neurotoxicity of chemicals in vitro, thereby providing data that are translatable to humans and may also reduce the number of animals needed for developmental neurotoxicological studies.

Endoplasmic reticulum stress responses differ in meninges and associated vasculature, striatum, and parietal cortex after a neurotoxic amphetamine exposure.

Amphetamine (AMPH) is used to treat attention deficit and hyperactivity disorders, but it can produce neurotoxicity and adverse vascular effects at high doses. The endoplasmic reticulum (ER) stress response (ERSR) entails the unfolded protein response, which helps to avoid or minimize ER dysfunction. ERSR is often associated with toxicities resulting from the accumulation of unfolded or misfolded proteins and has been associated with methamphetamine toxicity in the striatum. The present study evaluates the effect of AMPH on several ERSR elements in meninges and associated vasculature (MAV), parietal cortex, and striatum. Adult, male Sprague-Dawley rats were exposed to saline, environmentally induced hyperthermia (EIH) or four consecutive doses of AMPH that produce hyperthermia. Expression changes (mRNA and protein levels) of key ERSR-related genes in MAV, striatum, and parietal cortex at 3 h or 1 day postdosing were monitored. AMPH increased the expression of some ERSR-related genes in all tissues. Atf4 (activating transcription factor 4, an indicator of Perk pathway activation), Hspa5/Grp78 (Glucose regulated protein 78, master regulator of ERSR), Pdia4 (protein disulfide isomerase, protein-folding enzyme), and Nfkb1 (nuclear factor of kappa b, ERSR sensor) mRNA increased significantly in MAV and parietal cortex 3 h after AMPH. In striatum, Atf4 and Hspa5/Grp78 mRNA significantly increased 3 h after AMPH, but Pdia4 and Nfkb11 did not. Thus, AMPH caused a robust activation of the Perk pathway in all tissues, but significant Ire1 pathway activation occurred only after AMPH treatment in the parietal cortex and striatum. Ddit3/Chop, a downstream effector of the ERSR pathway related to the neurotoxicity, was only increased in striatum and parietal cortex. Conversely, Pdia4, an enzyme protective in the ERSR, was only increased in MAV. The overall ERSR manifestation varied significantly between MAV, striatum, and parietal cortex after a neurotoxic exposure to AMPH.

Biomarkers of endothelial cell activation serve as potential surrogate markers for drug-induced vascular injury.

Drug-induced vascular injury (DIVI) is a nonclinical finding that often confounds the toxicological evaluation of investigational drugs, but there is an absence of qualified biomarkers that can be used to detect and monitor its appearance in animals and patients during drug development and clinical use. It is well known that endothelial cell (EC) activation plays a key role in the expression and evolution of DIVI, and the various immunological and inflammatory factors involved in its expression may serve as potential biomarker candidates. Activated ECs change their morphology and gene expression, generating endothelial adhesion molecules, pro-coagulant molecules, cytokines, chemokines, vasodilators, nitric oxide, and acute-phase reactants. This review provides a brief historical background of EC activation and the search for biomarkers of early EC activation for monitoring DIVI. At present, no biomarkers of EC activation have been qualified to predict DIVI in the nonclinical or clinical context, and a robust pathologic foundation for their use is still lacking. We propose three categories of EC activation biomarkers: recommended surrogate markers, potentially useful markers, and emerging candidate markers. This review alerts pharmaceutical companies, research institutions, and regulatory agencies to the continuing need for reliable biomarkers of EC activation in drug development.

Application of microRNA profiling to understand sevoflurane-induced adverse effects on developing monkey brain.

We have described that prolonged sevoflurane exposure at a clinically-relevant concentration of 2.5 % caused neuronal cell death in the developing monkey brain. Postnatal day 5 or 6 rhesus monkeys (n = 3) were exposed to 2.5 % sevoflurane for 8 h. Monkeys kept at environmental conditions in the procedure room served as controls (n = 3). Brain tissues were harvested four hours after sevoflurane exposure for histological analysis, and RNA or protein extraction. MicroRNA (miRNA) profiling on the frontal cortex of monkey brains was performed using next-generation sequencing. 417 miRNAs were identified in the frontal cortex, where most neuronal cell death was observed. 7 miRNAs were differentially expressed in frontal cortex after sevoflurane exposure. Five of these were expressed at significantly lower levels than controls and the other two miRNAs were expressed significantly higher. These differentially expressed miRNAs (DEMs) were then loaded to the Ingenuity Pathway Analysis database for pathway analysis, in which targeting information was available for 5 DEMs. The 5 DEMs target 2,919 mRNAs which are involved in pathways that contribute to various cellular functions. Of note, 78 genes that are related to axon guidance signaling were targeted, suggesting that development of the neural circuit may be affected after sevoflurane exposure. Such changes may have long-term effects on brain development and function. These findings are supplementary to our previous observations and provide more evidence for better understanding the adverse effects of sevoflurane on the developing brain after an 8 -h exposure.

Microglial activation and responses to vasculature that result from an acute LPS exposure.

Bacterial cell wall endotoxins, i.e. lipopolysaccharides (LPS), are some of the original compounds shown to evoke the classic signs of systemic inflammation/innate immune response and neuroinflammation. The term neuroinflammation often is used to infer the elaboration of proinflammatory mediators by microglia elicited by neuronal targeted activity. However, it also is possible that the microglia are responding to vasculature through several signaling mechanisms. Microglial activation relative to the vasculature in the hippocampus and parietal cortex was determined after an acute exposure of a single subcutaneous injection of 2 mg/kg LPS. Antibodies to allograft inflammatory factor (Aif1, a.k.a. Iba1) were used to track and quantify morphological changes in microglia. Immunostaining of platelet/endothelial cell adhesion molecule 1 (Pecam1, a.k.a. Cd31) was used to visualize vasculature in the forebrain and glial acidic fibrillary protein (GFAP) to visualize astrocytes. Neuroinflammation and other aspects of neurotoxicity were evaluated histologically at 3 h, 6 h, 12 h, 24 h, 3 d and 14 d following LPS exposure. LPS did not cause neurodegeneration as determined by Fluoro Jade C labeling. Also, there were no signs of mouse IgG leakage from brain vasculature due to LPS. Some changes in microglia size occurred at 6 h, but by 12 h microglial activation had begun with the combined soma and proximal processes size increasing significantly (1.5-fold). At 24 h, almost all the microglia soma and proximal processes in the hippocampus, parietal cortex, and thalamus were closely associated with the vasculature and had increased almost 2.0-fold in size. In many areas where microglia were juxtaposed to vasculature, astrocytic endfeet appeared to be displaced. The microglial activation had subsided slightly by 3 d with microglial size 1.6-fold that of control. We hypothesize that acute LPS activation can result in vascular mediated microglial responses through several mechanisms: 1) binding to Cd14 and Tlr4 receptors on microglia processes residing on vasculature; 2) damaging vasculature and causing the release of cytokines; and 3) possibly astrocytic endfeet damage resulting in cytokine release. These acute responses may serve as an adaptive mechanism to exposure to circulating LPS where the microglia surround the vasculature. This could further prevent the pathogen(s) circulating in blood from entering the brain. However, diverting microglial interactions away from synaptic remodeling and other types of microglial interactions with neurons may have adverse effects on neuronal function.

Effects of Xenon-Based Anesthetic Exposure on the Expression Levels of Polysialic Acid Neural Cell Adhesion Molecule (PSA-NCAM) on Human Neural Stem Cell-Derived Neurons.

Numerous studies suggest a long duration of anesthesia during the late gestation period and infancy is associated with an increased risk of neuronal damage and neurocognitive impairment. The noble gas xenon is an anesthetic that is reported to have neuroprotective effects in some circumstances at certain concentrations. Currently, the effects of xenon on the brain and its potential neuroprotective properties, and/or the effects of xenon used in combination with other anesthetics, are not clearly understood and some reported data appear contradictory. In the present study, human neural stem cells were employed as a human-relevant model to evaluate the effects of xenon when it was co-administered with propofol, a frequently used anesthetic in pediatric anesthesia, and to understand the mechanism(s). The expression of polysialic acid (PSA) neural cell adhesion molecule (NCAM) on human neural stem cell-differentiated neurons was investigated as a key target molecule. PSA is a specific marker of developing neurons. It is essential for neuronal viability and plasticity. Human neural stem cells were maintained in neural differentiation medium and directed to differentiate into neuronal and glial lineages, and were exposed to propofol (50 µM) for 16 h in the presence or absence of xenon (33%). The neural stem cell-derived neurons were characterized by labelling cells with PSA-NCAM, after 5 days of differentiation. Propofol- and/or xenon-induced neurotoxicities were determined by measuring PSA immunoreactivity. A time course study showed that neuronal cell surface PSA was clearly cleaved off from NCAM by endoneuraminidase N (Endo-N), and eliminated PSA immunostaining was not re-expressed 4, 8, or 16 h after Endo-N washout. However, in the presence of 33% xenon, intense PSA staining on neuronal cell surface and processes was evident 16 h after Endo-N washout. In addition, prolonged (16 h) propofol exposure significantly decreased the positive rate of PSA-labeled neurons. When combined with xenon, propofol's adverse effects on neurons were attenuated. This work, conducted on the human neural stem cell-derived models, has provided evidence of the beneficiary effects of xenon on neurons and helps develop xenon-based anesthesia regimens in the pediatric population.

Protective Effects of Xenon on Propofol-Induced Neurotoxicity in Human Neural Stem Cell-Derived Models.

Early life exposure to general anesthetics can have neurotoxic consequences. Evidence indicates that xenon, a rare noble gas with anesthetic properties, may lessen neuronal damage under certain conditions. However, its potential neuroprotective properties, when used alone or in combination with other anesthetics, remain largely unknown. While it is difficult to verify the adverse effects of long duration anesthetic exposure in infants and children, the utilization of relevant non-clinical models (i.e., human-derived neural stem cells) may serve as a "bridging" model for evaluating the vulnerability of the nervous system. Neural stem cells, purchased from PhoenixSongs Biologicals, Inc., were guided to differentiate into neurons, astrocytes, and oligodendrocytes, which were then exposed to propofol (50 µM) for 16 h in the presence or absence of xenon (33%). Differentiation into cells of the neural lineage was confirmed by labelling with cell-specific markers, ß-tubulin for neurons, glial fibrillary acidic protein (GFAP) for astrocytes, and galactocerebroside (GALC) for oligodendrocytes after 5 days of differentiation. The presence and severity of neural damage induced by anesthetic exposures were assessed by several methods, including the TUNEL assay, and immuno-histochemical measurements. Our data demonstrate that prolonged exposure to propofol results in a significant increase in the number of TUNEL-positive cells, indicating increased neural apoptosis. No significant changes were detected in the number of GFAP-positive astrocytes or GALC-positive oligodendrocytes. However, the number of ß-tubulin-positive neurons was substantially reduced in the propofol-exposed cultures. Co-administration of xenon effectively blocked the propofol-induced neuronal damage/loss. No significant effects were observed when xenon was administered alone. The data indicate that prolonged exposure to propofol during development produces elevated levels of neuronal apoptosis in a human neural stem cell-derived model. However, sub-clinical, non-anesthetic concentrations of xenon, when used in combination with propofol, can prevent or ameliorate the toxic effects associated with prolonged anesthetic exposure. This is important as a more complete understanding of the neurotoxic mechanisms associated with a variety of clinically relevant anesthetic combinations becomes available. Protective approaches are critical for developing sound guidance on best practices for the use of these agents in the pediatric setting.

Differences in immunolocalization of Kim-1, RPA-1, and RPA-2 in kidneys of gentamicin-, cisplatin-, and valproic acid-treated rats: potential role of iNOS and nitrotyrosine.

The present study compared the immunolocalization of Kim-1, renal papillary antigen (RPA)-1, and RPA-2 with that of inducible nitric oxide synthase (iNOS) and nitrotyrosine in kidneys of gentamicin sulfate (Gen)- and cisplatin (Cis)-treated rats. The specificity of acute kidney injury (AKI) biomarkers, iNOS, and nitrotyrosine was evaluated by dosing rats with valproic acid (VPA). Sprague-Dawley (SD) rats were injected subcutaneously (sc) with 100 mg/kg/day of Gen for six or fourteen days; a single intraperitoneal (ip) dose of 1, 3, or 6 mg/kg of Cis; or 650 mg/kg/day of VPA (ip) for four days. In Gen-treated rats, Kim-1 was expressed in the epithelial cells, mainly in the S1/S2 segments but less so in the S3 segment, and RPA-1 was increased in the epithelial cells of collecting ducts (CD) in the cortex. Spatial expression of iNOS or nitrotyrosine with Kim-1 or RPA-1 was detected. In Cis-treated rats, Kim-1 was expressed only in the S3 segment cells, and RPA-1 and RPA-2 were increased in the epithelial cells of medullary CD or medullary loop of Henle (LH), respectively. Spatial expression of iNOS or nitrotyrosine with RPA-1 or RPA-2 was also identified. These findings suggest that peroxynitrite formation may be involved in the pathogenesis of Gen and Cis nephrotoxicity and that Kim-1, RPA-1, and RPA-2 have the potential to serve as site-specific biomarkers for Gen or Cis AKI.

Early life exposure to extended general anesthesia with isoflurane and nitrous oxide reduces responsivity on a cognitive test battery in the nonhuman primate.

Despite the widespread use of general anesthesia, a growing body of research suggests that anesthesia exposure early in life may be associated with acute neurotoxicity and lasting behavioral changes. To better evaluate the risk posed by early life anesthesia on cognitive development, infant rhesus monkeys were exposed to an anesthesia regimen previously shown to be neurotoxic and their cognitive development was subsequently measured using a translational operant test battery. On postnatal day 5 or 6, animals were exposed to 8 h of isoflurane (n = 6, 1% isoflurane in a vehicle gas of 70% nitrous oxide and 30% oxygen) or a control condition (n = 8). Starting at 7 months of age, the monkeys were continuously trained and assessed on the NCTR Operant Test Battery (OTB). The OTB consists of cognitive tests which also exist in near identical forms for use in rats and humans, and includes tests of learning, memory, color discrimination, and motivation. Monkeys previously exposed to anesthesia showed a clear decrease in responding in a measure of motivation, as well as a lower response rate in a learning task. These data further support the hypothesis that prolonged anesthesia early in life may increase the risk of developing cognitive impairments later in life.