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.

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.

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.

Quantification and reproducibility assessment of the regional brain T2 relaxation in naïve rats at 7T.

PURPOSE: To measure the reproducibility of T2 relaxation and to determine the statistical power of T2 mapping in the rat brain as a characteristic of the baseline performance of the T2 relaxation as a potential biomarker of neurotoxicity. MATERIALS AND METHODS: Multislice multiecho spin-echo imaging was utilized to obtain the quantitative T2 maps in 138 naïve rats at 7T. Images were skull-stripped and coregistered to the common anatomical reference. A full anatomical segmentation mask, which included all major brain structures, was created using the same reference T2 map. The overall variability map was also calculated from all T2 maps and the areas with arbitrarily high variability (coefficient of variation >25%) were excluded from the full segmentation mask to produce a trimmed mask. T2 maps were segmented using both these masks and statistical power analysis was conducted in all segmented areas. RESULTS: The coefficient of variation of T2 relaxation in different brain areas varied from 5.4% (cerebrospinal fluid) to 1.2% (cortex) when using a full segmentation mask. The use of a trimmed segmentation mask decreased the coefficient of variation in many areas, which ranged between 3.2% (inferior colliculi) and 1.2% (cortex) in this case. As revealed by statistical power analysis to detect 5% change with power of 0.8, the minimum number of observations needed for different areas ranged from 3 (cortex) to 8 (inferior colliculi) in the case of use of a trimmed segmentation mask. CONCLUSION: T2 relaxation is a very reproducible MRI parameter of the rat brain with high statistical power, which allows detecting very small changes in groups consisting of a minimal number of experimental animals. LEVEL OF EVIDENCE: 1 J. Magn. Reson. Imaging 2017;45:700-709.

The use of MRI to assist the section selections for classical pathology assessment of neurotoxicity.

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.

In vivo mapping of rat brain partial white matter content using hexachlorophene-induced neurotoxicity model.

Segmentation of the white matter in MRI scans of the rat brain presents a significant challenge due to the low contrast. Existing anatomical reference maps of rat brain are usually constructed from fixed tissue, which may suffer from geometrical distortions due to fixation/processing. To significantly increase the in vivo contrast between white and gray matter in the rat brain we used a known neurotoxicant hexachlorophene, which produces selective white matter damage. This model was used to map white matter in the rat brain and estimate the partial white matter content in any given imaging voxel. Hexachlorophene was administered to rats at a dose of 30 mg/kg orally once a day over five consecutive days. A significant white matter changes were observed using quantitative T2 maps, from which the partial white matter content throughout the whole rat brain was derived. Several assumptions were made: hexachlorophene affects T2 relaxation only in the white matter; T2 of gray matter is relatively uniform in the brain; apparent T2 value in a given voxel is a combination of T2s from white and gray matter portions of that voxel, hexachlorophene affects nearly 100 % of white matter. The partial white matter map of the rat brain was constructed with the resolution of 0.2 × 0.2 × 1.0 mm per voxel. This map could be adjusted for segmentation of the brain tissue with preset threshold of the white matter content, or to establish the tissue composition in any region of interest among other applications.

Performance of the prospective T2 MRI biomarker of neurotoxicity in a trimethyltin model in rats at 7 T.

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 T2 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 T2 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 T2 changes as a predictor of neurotoxicity. TMT treatment led to a significant increase in T2 values in many brain areas. The biggest changes in T2 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. T2 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 T2 MRI with other neurotoxicants is warranted.

The effects of long-term methylphenidate administration and withdrawal on progressive ratio responding and T2 MRI in the male rhesus monkey.

Methylphenidate is a frequently prescribed drug treatment for Attention-Deficit/Hyperactivity Disorder. However, methylphenidate has a mode of action similar to amphetamine and cocaine, both powerful drugs of abuse. There is lingering concern over the long-term safety of methylphenidate, especially in a pediatric population, where the drug may be used for years. We performed a long-term evaluation of the effects of chronic methylphenidate use on a behavioral measure of motivation in male rhesus monkeys. Animals were orally administered a sweetened methylphenidate solution (2.5 or 12.5 mg/kg, twice a day, Mon-Fri) or vehicle during adolescence and into adulthood. These animals were assessed on a test of motivation (progressive ratio responding), during methylphenidate treatment, and after cessation of use. Moreover, animals were evaluated with quantitative T2 MRI about one year after cessation of use. During the administration phase of the study animals treated with a clinically relevant dose of methylphenidate generally had a higher rate of responding than the control group, while the high dose group generally had a lower rate of responding. These differences were not statistically significant. In the month after cessation of methylphenidate, responding in both experimental groups dropped compared to their previous level of performance (p = 0.19 2.5 mg/kg, p = 0.06 12.5 mg/kg), and responding in the control animals was unchanged (p = 0.81). While cessation of methylphenidate was associated with an acute reduction in responding, group differences were not observed in the following months. These data suggest that methylphenidate did not have a significant impact on responding, but withdrawal from methylphenidate did cause a temporary change in motivation. No changes in T2 MRI values were detected when measured about one year after cessation of treatment. These data suggest that long-term methylphenidate use does not have a negative effect on a measure of motivation or brain function / microstructure as measured by quantitative T2 MRI. However, cessation of use might be associated with temporary cognitive changes, specifically alteration in motivation. Importantly, this study modeled use in healthy individuals, and results may differ if the same work was repeated in a model of ADHD.

Emerging technologies and their impact on regulatory science.

There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care products. Convergence of these emerging technologies is also enabling rapid advances and approaches that may impact regulatory decisions and approvals. Although the development of emerging technologies may allow rapid advances in regulatory decision making, there is concern that these new technologies have not been thoroughly evaluated to determine if they are ready for regulatory application, singularly or in combinations. The magnitude of these combined technical advances may outpace the ability to assess fit for purpose and to allow routine application of these new methods for regulatory purposes. There is a need to develop strategies to evaluate the new technologies to determine which ones are ready for regulatory use. The opportunity to apply these potentially faster, more accurate, and cost-effective approaches remains an important goal to facilitate their incorporation into regulatory use. However, without a clear strategy to evaluate emerging technologies rapidly and appropriately, the value of these efforts may go unrecognized or may take longer. It is important for the regulatory science field to keep up with the research in these technically advanced areas and to understand the science behind these new approaches. The regulatory field must understand the critical quality attributes of these novel approaches and learn from each other's experience so that workforces can be trained to prepare for emerging global regulatory challenges. Moreover, it is essential that the regulatory community must work with the technology developers to harness collective capabilities towards developing a strategy for evaluation of these new and novel assessment tools.

Sex differences in the effect of acute administration of nicotine on MRS-measured metabolic profile of the rat brain.

Women are less able to stop smoking than men. Elucidation of sex differences in the tobacco addiction could facilitate personalized treatment. Specialized brain reward systems are controlling the behavior through reinforcement using specific neuromediators. Using non-invasive magnetic resonance spectroscopy (MRS) to ascertain addiction/harm biomarkers could lead to better management of public health through advancements in regulatory and translational research. Proton MRS was used to monitor changes of specific neurometabolites in hippocampus (HC), nucleus accumbens (NAC), and anterior cingulate cortex (ACC) of rats of both sexes after single intraperitoneal injection of nicotine. At the baseline, male rats showed higher level of GABA, taurine, N-acetyl aspartate, and creatine in HC, and taurine in NAC. Also, there were stronger correlations between neurometabolites in females than in males at the baseline. Nicotine administration changed taurine, GABA, myo-inositol, choline, and N-acetyl aspartate in HC, and taurine in NAC. Significant interactions between time, treatment, and sex were detected for taurine and choline in HC. The number of inter-metabolite correlations increased significantly in ACC and decreased in NAC and HC in females after nicotine administration, while in males it was unchanged. There are distinct sex differences in neurometabolic profiles at the baseline and after acute nicotine administration. Nicotine changes inter-metabolite correlations in females more than in males.

Faster recovery of cerebral perfusion in SOD1-overexpressed rats after cardiac arrest and resuscitation.

BACKGROUND AND PURPOSE: Protracted hypoperfusion is one of the hallmarks of secondary cerebral derangement after cardiac arrest and resuscitation (CAR), and reactive oxygen species have been implicated in reperfusion abnormalities. METHODS: Using transgenic (Tg) rats overexpressing copper zinc superoxide dismutase (SOD1), we investigated the role of this intrinsic antioxidant in the restoration of cerebral blood flow (CBF) after CAR. Nine Tg and 11 wild-type (WT) rats were subjected to a nominal 15-minute cardiac arrest, and CBF was measured using the noninvasive arterial spin labeling MRI method before and during cardiac arrest, and 0 to 2 hours and 1 to 5 days after resuscitation. RESULTS: The SOD1-Tg rats showed rapid normalization of CBF 1 day after the insult, whereas CBF in WT animals remained abnormal for at least 5 days, showing a progressive increase in CBF from hypo- to hyperperfusion on postresuscitation days 1 to 5. The long-term outcome, as measured by survival time, change in body weight, and mapping of apparent diffusion coefficient (ADC) for ion/water homeostasis, was significantly better in the SOD1-Tg rats. CONCLUSIONS: Our results support the notion that reactive oxygen species are at least partially responsible for microvascular reperfusion disorders.

The T- and L-type calcium channel blocker (CCB) mibefradil attenuates leg edema induced by the L-type CCB nifedipine in the spontaneously hypertensive rat: a novel differentiating assay.

Among the L-type calcium channel blockers (CCBs), particularly dihydropyridines like nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], a common adverse effect is vasodilatory edema. Newer CCBs, such as the T- and L-type CCB, mibefradil [(1S,2S)-2-[2[[3-(2-benzimidazolylpropyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl methoxyacetate dihydrochloride hydrate], demonstrate antihypertensive efficacy similar to that of their predecessors but seem to have a reduced propensity to cause edema. Using a magnetic resonance imaging (MRI) T(2) mapping technique, we investigated the ability of mibefradil to reduce extracellular water accumulation caused by the L-type CCB, nifedipine, in the hindleg skeletal muscle of the spontaneously hypertensive rat. Mibefradil (10 mg/kg i.v.) and nifedipine (1 mg/kg i.v.) lowered mean arterial blood pressure by 97 +/- 5 and 77 +/- 4 mm Hg, respectively. MRI edema index (expressed as percentage increase of integral T(2) over predrug control) was significantly higher with nifedipine (2606 +/- 86%; p < 0.05) than with mibefradil (981 +/- 171%) measured 30 to 60 min after the start of drug infusion. The hindleg edema caused by nifedipine was dose dependently decreased by coadministration of mibefradil (0, 0.3, or 3 mg/kg). The hindleg edema formation was not due to albumin leakage into the interstitial space based on immunostaining. However, a 4.2-fold increase in the arterial L-/T-type CC mRNA expression ratio was observed compared with the venous L/T ratio as shown by quantitative reverse transcription polymerase chain reaction. These results demonstrate the novel utility of MRI to measure extravascular water after acute exposure to CCBs and indicate that T-type CCB activity may reduce L-type CCB-induced vasodilatory edema in the skeletal muscle vasculature, possibly by a differential effect on arteriole and venule dilatation.

Comparison of quantitative T2 and ADC mapping in the assessment of 3-nitropropionic acid-induced neurotoxicity in rats.

To assess the relative performance of MRI T2 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 T2 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. T2 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 T2 in some cases, ADC did not change. The effect sizes of T2 and ADC signals suggestive of neurotoxicity were 2.64 and 1.66, respectively, and the variability of averaged T2 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. T2 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.

A multi-center preclinical study of gadoxetate DCE-MRI in rats as a biomarker of drug induced inhibition of liver transporter function.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and transplantation. DILI can be the result of impaired hepatobiliary transporters, with altered bile formation, flow, and subsequent cholestasis. We used gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), combined with pharmacokinetic modelling, to measure hepatobiliary transporter function in vivo in rats. The sensitivity and robustness of the method was tested by evaluating the effect of a clinical dose of the antibiotic rifampicin in four different preclinical imaging centers. The mean gadoxetate uptake rate constant for the vehicle groups at all centers was 39.3 +/- 3.4 s-1 (n = 23) and 11.7 +/- 1.3 s-1 (n = 20) for the rifampicin groups. The mean gadoxetate efflux rate constant for the vehicle groups was 1.53 +/- 0.08 s-1 (n = 23) and for the rifampicin treated groups was 0.94 +/- 0.08 s-1 (n = 20). Both the uptake and excretion transporters of gadoxetate were statistically significantly inhibited by the clinical dose of rifampicin at all centers and the size of this treatment group effect was consistent across the centers. Gadoxetate is a clinically approved MRI contrast agent, so this method is readily transferable to the clinic. CONCLUSION: Rate constants of gadoxetate uptake and excretion are sensitive and robust biomarkers to detect early changes in hepatobiliary transporter function in vivo in rats prior to established biomarkers of liver toxicity.

Changes in the metabolome and microRNA levels in biological fluids might represent biomarkers of neurotoxicity: A trimethyltin study.

Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.

The Pathophysiological Sequence of Glucocorticoid-Induced Osteonecrosis of the Femoral Head in Male Mice.

In search of the sequence of pathogenic events leading to glucocorticoid-induced osteonecrosis, we determined the molecular, biomechanical, cellular, and vascular changes in the femur of C57BL/6 mice receiving prednisolone for 14, 28, or 42 days. The femoral head, but not the distal femur, of mice treated for 14 days showed a decrease in the expression of the hypoxia-inducible factor (Hif)-1α and vascular endothelial growth factor (VEGF), the number of osteoblasts, and bone formation rate and strength and showed an increase in osteoclasts. These changes were accompanied by conversion of the normal dendritic vasculature to pools of edema as detected by magnetic resonance imaging, providing robust diagnostic evidence of early osteonecrosis. At that time point, there were no detectable changes in bone density, cortical or cancellous bone architecture, midshaft or distal cancellous bone, or osteocyte apoptosis. In mice treated for 28 days, femoral head cancellous density, cortical width, and trabecular thickness decreased, and by 42 days the femoral heads had full-depth cortical penetrations and cancellous tissue osteonecrosis. These results indicate that the femoral head is a particularly sensitive anatomical site to the adverse effects of glucocorticoid excess on bone and that decreases of Hif-1α and VEGF expression, bone vascularity, and strength precede the loss of bone mass and microarchitectural deterioration, thus rendering the femoral head vulnerable to collapse.

High resolution imaging of the mitral valve in the natural state with 7 Tesla MRI.

Imaging techniques of the mitral valve have improved tremendously during the last decade, but challenges persist. The delicate changes in annulus shape and papillary muscle position throughout the cardiac cycle have significant impact on the stress distribution in the leaflets and chords, thus preservation of anatomically accurate positioning is critical. The aim of this study was to develop an in vitro method and apparatus for obtaining high-resolution 3D MRI images of porcine mitral valves in both the diastolic and systolic configurations with physiologically appropriate annular shape, papillary muscle positions and orientations, specific to the heart from which the valve was harvested. Positioning and mounting was achieved through novel, customized mounting hardware consisting of papillary muscle and annulus holders with geometries determined via pre-mortem ultrasonic intra-valve measurements. A semi-automatic process was developed and employed to tailor Computer Aided Design models of the holders used to mount the valve. All valve mounting hardware was 3D printed using a stereolithographic printer, and the material of all fasteners used were brass for MRI compatibility. The mounted valves were placed within a clear acrylic case, capable of holding a zero-pressure and pressurized liquid bath of a MRI-compatible fluid. Obtaining images from the valve submerged in liquid fluid mimics the natural environment surrounding the valve, avoiding artefacts due to tissue surface tension mismatch and gravitational impact on tissue shape when not neutrally buoyant. Fluid pressure was supplied by reservoirs held at differing elevations and monitored and controlled to within ±1mmHg to ensure that the valves remained steady. The valves were scanned in a 7 Tesla MRI system providing a voxel resolution of at least 80µm. The systematic approach produced 3D datasets of high quality which, when combined with physiologically accurate positioning by the apparatus, can serve as an important input for validated computational models.

Magnetic resonance spectroscopic analysis of neurometabolite changes in the developing rat brain at 7T.

We utilized proton magnetic resonance spectroscopy to evaluate the metabolic profile of the hippocampus and anterior cingulate cortex of the developing rat brain from postnatal days 14-70. Measured metabolite concentrations were modeled using linear, exponential, or logarithmic functions and the time point at which the data reached plateau (i.e. when the portion of the data could be fit to horizontal line) was estimated and was interpreted as the time when the brain has reached maturity with respect to that metabolite. N-acetyl-aspartate and myo-inositol increased within the observed period. Gluthathione did not vary significantly, while taurine decreased initially and then stabilized. Phosphocreatine and total creatine had a tendency to increase towards the end of the experiment. Some differences between our data and the published literature were observed in the concentrations and dynamics of phosphocreatine, myo-inositol, and GABA in the hippocampus and creatine, GABA, glutamine, choline and N-acetyl-aspartate in the cortex. Such differences may be attributed to experimental conditions, analysis approaches and animal species. The latter is supported by differences between in-house rat colony and rats from Charles River Labs. Spectroscopy provides a valuable tool for non-invasive brain neurochemical profiling for use in developmental neurobiology research. Special attention needs to be paid to important sources of variation like animal strain and commercial source.

Brain endothelial dysfunction following pyrithiamine induced thiamine deficiency in the rat.

Prolonged vitamin B1 (thiamine) deficiency can lead to neurological disorders such as Wernicke's encephalopathy and Wernicke-Korsakoff Syndrome (WKS) in humans. These thiamine deficiency disorders have been attributed to vascular leakage, blood-brain barrier breakdown and neuronal loss in the diencephalon and brain stem. However, endothelial dysfunction following thiamine deficiency and its relationship to the phenomenon of neurodegeneration has not been clearly elucidated. The present study sought to begin to address this issue by evaluating vascular morphology and integrity in a pyrithiamine (PT)-induced rat model of thiamine deficiency. Adjacent brain sections were used to either assess vascular integrity through immunohistochemical localization of rat endothelial cell antigen (RECA-1) and endothelial brain barrier antigen (EBA-1) or neurodegeneration using the de Olmos cupric silver method. GFAP and CD11b immunolabeling was used to evaluate astrocytic and microglial/macrophagic changes. Extensive neurodegeneration occurred concomitant with both vascular damage (thinning and breakage) and microglial activation in the inferior olive, medial thalamic area, and medial geniculate nuclei of pyrithiamine treated rats. Likewise, glucose transporter-1 (Glut-1), which is mostly expressed in endothelial cells, was also severely decreased in this pyrithiamine induced thiamine deficient rat model. MRI scans of these animals prior to sacrifice show that the pyrithiamine induced thiamine deficient animals have abnormal T2 relaxation values, which are commensurate with, and possibly predictive of, the neurodegeneration and/or endothelial dysfunction subsequently observed histologically in these same animals.

Longitudinal diffusion tensor imaging of the rat brain after hexachlorophene exposure.

Longitudinal MRI employing diffusion tensor imaging and T2 mapping approaches has been applied to investigate the mechanisms of white matter damage caused by acute hexachlorophene neurotoxicity in rats in vivo. Male Sprague-Dawley rats were administered hexachlorophene orally once a day for five consecutive days at a dose of 30mg/kg and were monitored in 7T MRI scanner at days 0 (baseline), 3, 6, 13, and 20 following the first hexachlorophene dose. Quantitative T2 maps as well as a number of diffusion tensor parameters (fractional anisotropy, radial and axial diffusivity, apparent diffusion coefficient, and trace) were calculated from corresponding MR images. T2, as well as all diffusion tensor derived parameters (except fractional anisotropy) showed significant changes during the course of neurotoxicity development. These changes peaked at 6days after the first dose of hexachlorophene (one day after the last dose) and recovered to practically baseline levels at the end of observation (20days from the first dose). While such changes in diffusivity and T2 relaxation clearly demonstrate myelin perturbations consistent with edema, the lack of changes of fractional anisotropy suggests that the structure of the myelin sheath was not disrupted significantly by hexachlorophene in this study. This is also confirmed by the rapid recovery of all observed MRI parameters after cessation of hexachlorophene exposure.