Pig Brain Research Articles

EXTH-74. COOLING GLIOBLASTOMA WITH A NEURAL IMPLANT

Abstract Survival rates for glioblastoma (GBM) patients are low due to the difficulty in eradicating all tumor cells, leading to frequent recurrence. This recurrence is frequently within 2-cm from the original site. Studies indicate that supra-total resection improves survival, and non-destructive local therapies show promise in preventing GBM recurrence. Our research demonstrates that non-freezing, ‘cytostatic’ hypothermia is effective against GBM in rats and can be scaled to pig models. This method, unlike ablative cryogenic hypothermia, safely inhibits GBM growth without damaging healthy tissue. Using in vitro culture models and in vivo rat models via a Peltier device, we tested cytostatic hypothermia. This was followed by finite-element modeling (FEM) to simulate bioheat transfer in human and pig brains. The system consists of a neural implant with a multiprobe array, a Peltier chip, a water block, and an artificial internal circulation system with a piezoelectric pump. In vitro tests identified a cytostatic window of 20–25°C. Miniature Peltier devices were fabricated to achieve this in rats to inhibit GBM growth and extend survival. Rats whose tumors received cytostatic levels of hypothermia survived their full study period. FEM simulations confirmed that multiprobe arrays could cool brain tissue to 25°C without significantly raising skin temperature. Portable systems were successfully fabricated and tested in pigs, achieving safe target temperatures with minimal EEG and OCTA changes. Our findings suggest that cytostatic hypothermia can be scaled to larger animals and made implantable and portable, offering a promising non-destructive approach to halting GBM growth and prolonging survival, moving closer to becoming a viable treatment option for patients with GBM.

Quantitative myelin water assessment for multiple sclerosis using multi-inversion magnetic resonance fingerprinting.

Multiple sclerosis (MS) is a demyelination disease. Myelin water is a biomarker of myelin and thus myelin water imaging is a vital tool to provide insight into the demyelination process. This study aimed to characterize the multiple compartments including myelin water fraction (MWF), gray matter (GM) cellular water, white matter (WM) cellular water, and cerebrospinal fluid (CSF) using multiple inversion recovery (mIR) magnetic resonance fingerprinting (MRF) on a clinical MS cohort. The Phantom experiment was conducted with tubes containing different WM and GM concentrations extracted from pig brains. For the in-vivo experiment, 23 healthy control (HC) volunteers and 18 MS patients were recruited for this study. The experiments were performed using a clinical 3T MRI. A multi-slice, fast imaging with a steady-state precession (FISP) based mIR MRF protocol was used to obtain the MWF measurements, with 6min of scan time for each volunteer. The quantification was based on the iterative non-negative least squares (NNLS) with reweighting. The brain compartments quantified were myelin water, WM cellular water, GM cellular water, and CSF. A radiologist with 6 years of experience labeled the MS lesions on FLAIR, MPRAGE, and MWF. Statistical analysis was performed by applying unpaired and paired student's t-tests to compare the MWF results in different groups and in normal-appearing white matter (NAWM) and MS lesions. The phantom result demonstrated the ability to detect MWF with various myelin concentrations. The maps derived from mIR MRF, including MWF, WM cellular water, GM cellular water, and CSF were consistent with the anatomical structures observed in FLAIR and MPRAGE. The MWF values in the NAWM of MS patients were significantly different from those in HC, with values of 0.32±0.025 and 0.25±0.036, respectively. Additionally, the MWF values in WM lesions were significantly smaller than in NAWM at 0.034±0.036. The mIR-MRF technique, using multi-compartment analysis, can simultaneously generate maps of MWF, WM cellular water, GM cellular water, and CSF with sufficient brain coverage and in a reasonably short scan time. The MWF map might provide insights into the demyelination associated with MS.

Update on a brain-penetrant cardiac glycoside that can lower cellular prion protein levels in human and guinea pig paradigms.

Lowering the levels of the cellular prion protein (PrPC) is widely considered a promising strategy for the treatment of prion diseases. Building on work that established immediate spatial proximity of PrPC and Na+, K+-ATPases (NKAs) in the brain, we recently showed that PrPC levels can be reduced by targeting NKAs with their natural cardiac glycoside (CG) inhibitors. We then introduced C4'-dehydro-oleandrin as a CG with improved pharmacological properties for this indication, showing that it reduced PrPC levels by 84% in immortalized human cells that had been differentiated to acquire neural or astrocytic characteristics. Here we report that our lead compound caused cell surface PrPC levels to drop also in other human cell models, even when the analyses of whole cell lysates suggested otherwise. Because mice are refractory to CGs, we explored guinea pigs as an alternative rodent model for the preclinical evaluation of C4'-dehydro-oleandrin. We found that guinea pig cell lines, primary cells, and brain slices were responsive to our lead compound, albeit it at 30-fold higher concentrations than human cells. Of potential significance for other PrPC lowering approaches, we observed that cells attempted to compensate for the loss of cell surface PrPC levels by increasing the expression of the prion gene, requiring daily administration of C4'-dehydro-oleandrin for a sustained PrPC lowering effect. Regrettably, when administered systemically in vivo, the levels of C4'-dehydro-oleandrin that reached the guinea pig brain remained insufficient for the PrPC lowering effect to manifest. A more suitable preclinical model is still needed to determine if C4'-dehydro-oleandrin can offer a cost-effective complementary strategy for pushing PrPC levels below a threshold required for long-term prion disease survival.

Impaired Skeletal Development by Disruption of Presenilin-1 in Pigs and Generation of Novel Pig Models for Alzheimer's Disease.

Presenilin 1 (PSEN1) is one of the genes linked to the prevalence of early onset Alzheimer's disease. In mice, inactivation of Psen1 leads to developmental defects, including vertebral malformation and neural development. However, little is known about the role of PSEN1 during the development in other species. To investigate the role of PSEN1 in vertebral development and the pathogenic mechanism of neurodegeneration using a pig model. CRISPR/Cas9 system was used to generate pigs with different mutations flanking exon 9 of PSEN1, including those with a deleted exon 9 (Δexon9). Vertebral malformations in PSEN1 mutant pigs were examined by X-ray, micro-CT and micro-MRI. Neuronal cells from the brains of PSEN1 mutant pigs were analyzed by immunoflourescence, followed by image analysis including morphometric evaluation via image J and 3D reconstruction. Pigs with a PSEN1 null mutation (Δexon9-12) died shortly after birth and had significant axial skeletal defects, whereas pigs carrying at least one Δexon9 allele developed normally and remained healthy. Effects of the null mutation on abnormal skeletal development were also observed in fetuses at day 40 of gestation. Abnormal distribution of astrocytes and microglia in the brain was detected in two PSEN1 mutant pigs examined compared to age-matched control pigs. The founder pigs were bred to establish and age PSEN1ΔE9/+ pigs to study their relevance to clinical Alzheimer's diseases. PSEN1 has a critical role for normal vertebral development and PSEN1 mutant pigs serves as novel resources to study Alzheimer's disease.

Comparing spatial distributions of ALA-PpIX and indocyanine green in a whole pig brain glioma model using 3D fluorescence cryotomography.

ALA-PpIX and second-window indocyanine green (ICG) have been studied widely for guiding the resection of high-grade gliomas. These agents have different mechanisms of action and uptake characteristics, which can affect their performance as surgical guidance agents. Elucidating these differences in animal models that approach the size and anatomy of the human brain would help guide the use of these agents. Herein, we report on the use of a new pig glioma model and fluorescence cryotomography to evaluate the 3D distributions of both agents throughout the whole brain. We aim to assess and compare the 3D spatial distributions of ALA-PpIX and second-window ICG in a glioma-bearing pig brain using fluorescence cryotomography. A glioma was induced in the brain of a transgenic Oncopig via adeno-associated virus delivery of Cre-recombinase plasmids. After tumor induction, the pro-drug 5-ALA and ICG were administered to the animal 3 and 24h prior to brain harvest, respectively. The harvested brain was imaged using fluorescence cryotomography. The fluorescence distributions of both agents were evaluated in 3D in the whole brain using various spatial distribution and contrast performance metrics. Significant differences in the spatial distributions of both agents were observed. Indocyanine green accumulated within the tumor core, whereas ALA-PpIX appeared more toward the tumor periphery. Both ALA-PpIX and second-window ICG provided elevated tumor-to-background contrast (13 and 23, respectively). This study is the first to demonstrate the use of a new glioma model and large-specimen fluorescence cryotomography to evaluate and compare imaging agent distribution at high resolution in 3D.

Valorization of Pig Brains for Prime Quality Oil: A Comparative Evaluation of Organic-Solvent-Based and Solvent-Free Extractions.

Pig processing industries have produced large quantities of by-products, which have either been discarded or used to make low-value products. This study aimed to provide recommendations for manufacturing edible oil from pig brains, thereby increasing the value of pork by-products. The experiment compared non-solvent extraction methods, specifically wet rendering and aqueous saline, to a standard solvent extraction method, the Bligh and Dyer method, for extracting oil from pig brains. The yield, color, fatty acid profile, a number of lipid classes, and lipid stability against lipolysis and oxidation of the pig brain oil were comprehensively compared, and the results revealed that these parameters varied depending on the extraction method. The wet rendering process provided the highest extracted oil yield (~13%), followed by the Bligh and Dyer method (~7%) and the aqueous saline method (~2.5%). The Bligh and Dyer method and wet rendering techniques produced a translucent yellow oil; however, an opaque light-brown-red oil was found in the aqueous saline method. The Bligh and Dyer method yielded the oil with the highest phospholipid, cholesterol, carotenoid, tocopherol, and free fatty acid contents (p < 0.05). Although the Bligh and Dyer method recovered the most unsaturated fatty acids, it also recovered more trans-fatty acids. Aqueous saline and wet rendering procedures yielded oil with low FFA levels (<1 g/100 g). The PV of the oil extracted using all methods was <1 meq/kg; however, the Bligh and Dyer method had a significant TBARS content (7.85 mg MDA equivalent/kg) compared to aqueous saline (1.75 mg MDA equivalent/kg) and wet rendering (1.14 mg MDA equivalent/kg) (p < 0.05). FTIR spectra of the pig brain oil revealed the presence of multiple components in varying quantities, as determined by chemical analysis experiments. Given the higher yield and lipid stability and the lower cholesterol and trans-fatty acid content, wet rendering can be regarded as a simple and environmentally friendly method for safely extracting quality edible oil from pig brains, which may play an important role in obtaining financial benefits, nutrition, the zero-waste approach, and increasing the utilization of by-products in the meat industry.

Porcine Astrovirus Infection in Brains of Pigs in Korea.

Recently, neurological diseases associated with astroviruses (AstVs) have been reported in pigs, ruminants, minks, and humans. In 2017, neuro-invasive porcine astrovirus (Ni-PAstV) 3 was detected in the central nervous system (CNS) of pigs with encephalomyelitis in Hungary and the USA. In the process of diagnosing domestic pigs exhibiting neurological signs, histopathologic lesions of non-suppurative encephalomyelitis with meningitis, neuronal vacuolation, and gliosis were detected, and PAstV was identified using reverse transcriptase PCR in CNS samples of four pigs in three farms from August to September in 2020, South Korea. Subsequently, the ORF2 region was successfully acquired from three brain samples, facilitating subsequent analysis. Four genotypes of PAstV (PAstV1, 3, 4, and 5) were detected, and coinfection of PAstV with multiple genotypes was observed in brain samples. This is the first study to report Ni-PAstV infection in pigs in South Korea.

A novel integrated extraction protocol for multi-omic studies in heavily degraded samples

The combination of multi-omic techniques, such as genomics, transcriptomics, proteomics, metabolomics and epigenomics, has revolutionised studies in medical research. These techniques are employed to support biomarker discovery, better understand molecular pathways and identify novel drug targets. Despite concerted efforts in integrating omic datasets, there is an absence of protocols that integrate all four biomolecules in a single extraction process. Here, we demonstrate for the first time a minimally destructive integrated protocol for the simultaneous extraction of artificially degraded DNA, proteins, lipids and metabolites from pig brain samples. We used an MTBE-based approach to separate lipids and metabolites, followed by subsequent isolation of DNA and proteins. We have validated this protocol against standalone extraction protocols and show comparable or higher yields of all four biomolecules. This integrated protocol is key to facilitating the preservation of irreplaceable samples while promoting downstream analyses and successful data integration by removing bias from univariate dataset noise and varied distribution characteristics.

Accessing the Porcine Brain via High-Speed Pneumatic Drill Craniectomy.

The use of pigs as an experimental animal model is especially relevant in neuroscience research, as the porcine and human central nervous systems (CNS) share many important functional and architectural properties. Consequently, pigs are expected to have an increasingly important role in future research on various neurological diseases. Here, a method to perform an anterior craniectomy through the porcine frontal bone is described. After a midline incision and subsequent exposure of the porcine frontal bone, anatomical landmarks are used to ensure the optimal location of the craniectomy. By careful and gradual thinning of the frontal bone with a rounded drill, a rectangular opening to the dura mater and underlying cerebral hemispheres is achieved. The presented method requires certain surgical materials, including a pneumatic high-speed drill, and some degree of surgical experience. Potential complications include unintended lesions of the dura mater or dorsal sagittal sinus. However, the method is simple, time-efficient, and offers a high degree of reproducibility for researchers. If performed correctly, the technique exposes a large portion of the unaffected pig brain for various neuromonitoring or analyses.

Chemical composition of wild and domestic animals’ organism in areas of geophagy distribution in Altai Republic

Relevance. The need to study the elemental composition of the organism of wild and domestic animals in natural environments with different levels of chemical elements. Aim. To reveal features of the elemental composition of the organism of the Altai red deer (Cervus elaphus sibiricus Severtzov, 1873), wild boar (Sus scrofa scrofa Linnaeus, 1758) and domestic pigs (Sus scrofa domesticus Erxleben, 1777) in Ongudai district, Altai Republic, including the area with signs of active geophagy. Objects. Organs and tissues of wild and domestic animals. Methods. Mass spectrometry with inductively coupled plasma; atomic-emission spectrometry; ion chromatography, analytical electron microscope with energy dispersion spectrometer. Results. The median values of chemical elements concentrations in the organisms of the Altai red deer, the wild boar and the domestic pig have been determined. S, Cl and K showed the highest values in the composition of macroelements and La, Ce, Nd and Th among rare-earth and radioactive elements. Among the elemental composition features of the studied animals, an increased accumulation of Hg and Pb in the Altai red deer; metals of the Fe-group, W, as well as rare-earth and radioactive elements – in the boar; platinoids – in the domestic pig. In comparison with the results of previous studies, increased concentrations of Hg in red deer kidneys, W in boar bronchi and Pb in the brain of domestic pigs were recorded. Assumptions were made about the factors influencing the formation of the elemental composition of the body of animals. For red deer and wild boar these are the factors of nutrition (including geophagy) and features of accumulation of elements in bone tissue; for domestic pigs – features of forage and local metallogeny, including composition of natural waters.

MRI-only based material mass density and relative stopping power estimation via deep learning for proton therapy: a preliminary study

Magnetic Resonance Imaging (MRI) is increasingly being used in treatment planning due to its superior soft tissue contrast, which is useful for tumor and soft tissue delineation compared to computed tomography (CT). However, MRI cannot directly provide mass density or relative stopping power (RSP) maps, which are required for calculating proton radiotherapy doses. Therefore, the integration of artificial intelligence (AI) into MRI-based treatment planning to estimate mass density and RSP directly from MRI has generated significant interest. A deep learning (DL) based framework was developed to establish a voxel-wise correlation between MR images and mass density as well as RSP. To facilitate the study, five tissue substitute phantoms were created, representing different tissues such as skin, muscle, adipose tissue, 45% hydroxyapatite (HA), and spongiosa bone. The composition of these phantoms was based on information from ICRP reports. Additionally, two animal tissue phantoms, simulating pig brain and liver, were prepared for DL training purposes. The phantom study involved the development of two DL models. The first model utilized clinical T1 and T2 MRI scans as input, while the second model incorporated zero echo time (ZTE) MRI scans. In the patient application study, two more DL models were trained: one using T1 and T2 MRI scans as input, and another model incorporating synthetic dual-energy computed tomography (sDECT) images to provide accurate bone tissue information. The DECT empirical model was used as a reference to evaluate the proposed models in both phantom and patient application studies. The DECT empirical model was selected as the reference for evaluating the proposed models in both phantom and patient application studies. In the phantom study, the DL model based on T1, and T2 MRI scans demonstrated higher accuracy in estimating mass density and RSP for skin, muscle, adipose tissue, brain, and liver. The mean absolute percentage errors (MAPE) were 0.42%, 0.14%, 0.19%, 0.78%, and 0.26% for mass density, and 0.30%, 0.11%, 0.16%, 0.61%, and 0.23% for RSP, respectively. The DL model incorporating ZTE MRI further improved the accuracy of mass density and RSP estimation for 45% HA and spongiosa bone, with MAPE values of 0.23% and 0.09% for mass density, and 0.19% and 0.07% for RSP, respectively. These results demonstrate the feasibility of using an MRI-only approach combined with DL methods for mass density and RSP estimation in proton therapy treatment planning. By employing this approach, it is possible to obtain the necessary information for proton radiotherapy directly from MRI scans, eliminating the need for additional imaging modalities.

Different oil sources impacting brain lipid and transcriptome profiles of pigs

Lipids are fundamental to the structure and function of the brain, and their fatty acids (FA) composition is rich in polyunsaturated fatty acids (PUFA), which have protective effects and modulate gene transcription. For nutrigenomics studies, pigs (Sus scrofa) have been widely used as a biomedical model. Thus, the aimed to investigate whether different dietary oil sources modify the pig brain's lipid and transcriptomic profile. A 98-day study was performed using fifty-four male pigs. Treatments consisted of corn-soybean meal diets containing 3 % of soybean oil (SOY), canola oil (CO), or fish oil (FO). Total mRNA was extracted for sequencing. As a result, feeding diets with different oil sources affected the percentage of some FA. Palmitic acid showed a greater concentration in diets containing SOY with 27.037 %. Oleic acid and eicosenoic acid, showed a greater concentration in diets containing SOY, with 30.968 % and 2.096 %, respectively; and, total PUFA showed a better concentration in diets containing SOY and FO, with 11.685 % and 12.150 %, respectively. After quality control, considering the total reads obtained for the three groups, 94.87% were mapped against the reference genome SScrofa11.1. A comparison of gene expression between the groups of pigs was carried out by using the DESeq2 statistical package (R/Bioconductor). From SOY vs CO comparison, five differentially expressed genes (DEG, FDR < 0.05) were identified, from SOY vs FO forty-four DEG were verified, and from CO vs FO thirty-nine DEG were found. The functional enrichment analysis resulted in pathway maps (P < 0.05) related to apoptosis and cell proliferation, obesity and type 2 diabetes, neurophysiological process, and inflammation. The networks were associated with signal transduction, calcium transport, and oxidative stress. Overall, the results showed that diets with different oil sources could affect some brain tissue parameters and may help guide future research on the availability of dietary FA in the brain.

Comprehensive mapping of histamine H1 receptor mRNA in the mouse brain.

Histamine H1 receptor (H1R) in the central nervous system plays an important role in various functions, including learning and memory, aggression, feeding behaviors, and wakefulness, as evidenced by studies utilizing H1R knockout mice and pharmacological interventions. Although previous studies have reported the widespread distribution of H1R in the brains of rats, guinea pigs, monkeys, and humans, the detailed distribution in the mouse brain remains unclear. This study provides a comprehensive description of the distribution of H1R mRNA in the mouse brain using two recently developed techniques: RNAscope and in situ hybridization chain reaction, both of which offer enhanced sensitivity and resolution compared to traditional methodologies such as radioisotope labeling, which were used in previous studies. The H1R mRNA expression was observed throughout the entire brain, including key regions implicated in sleep-wake regulatory functions, such as the pedunculopontine tegmental nucleus and dorsal raphe. Additionally, strong H1R mRNA signals were identified in the paraventricular hypothalamus and ventromedial hypothalamus, which may explain the potential mechanisms underlying histamine-mediated feeding regulation. Notably, we identified strong H1R mRNA expression in previously unreported cerebral regions, such as the dorsal endopiriform nucleus, bed nucleus of the accessory olfactory tract, and postsubiculum. These findings significantly contribute to our understanding of the multifaceted roles of H1R in diverse brain functions.

Pure-Shift-Based Proton Magnetic Resonance Spectroscopy for High-Resolution Studies of Biological Samples.

Proton magnetic resonance spectroscopy (1H MRS) presents a powerful tool for revealing molecular-level metabolite information, complementary to the anatomical insight delivered by magnetic resonance imaging (MRI), thus playing a significant role in in vivo/in vitro biological studies. However, its further applications are generally confined by spectral congestion caused by numerous biological metabolites contained within the limited proton frequency range. Herein, we propose a pure-shift-based 1H localized MRS method as a proof of concept for high-resolution studies of biological samples. Benefitting from the spectral simplification from multiplets to singlet peaks, this method addresses the challenge of spectral congestion encountered in conventional MRS experiments and facilitates metabolite analysis from crowded NMR resonances. The performance of the proposed pure-shift 1H MRS method is demonstrated on different kinds of samples, including brain metabolite phantom and in vitro biological samples of intact pig brain tissue and grape tissue, using a 7.0 T animal MRI scanner. This proposed MRS method is readily implemented in common commercial NMR/MRI instruments because of its generally adopted pulse-sequence modules. Therefore, this study takes a meaningful step for MRS studies toward potential applications in metabolite analysis and disease diagnosis.

Affinity of structural white matter tracts between infant and adult pig

BackgroundThe piglet brain has been increasingly used as an excellent surrogate for investigation of pediatric neurodevelopment, nutrition, and traumatic brain injuries. This study intends to establish a piglet brain’s structural connectivity model and compare it with the adult pig, enhancing its application for structurally guided functional analysis. MethodsIn this study, diffusion-weighted (DW)-MRI data from piglets (n=11, 3-week-old) was used to establish piglet model and compare with adult pigs. We employed a data-driven independent component analysis (ICA) method to derive piglet-specific tracts. Pearson correlations and Kullback-Leibler (KL) divergences was employed to identify common tracts and unique tracts for piglet. Common tracts were then used in a blueprint connectome study to highlight differences in regions of interest (ROI). ResultsThe data-driven approach applied to piglet brains revealed 17 common tracts, showing high similarity with adult pigs' white matter (WM) tracts, and identified 3 tracts unique to piglets and 10 negative marker tracts. Additionally, the study highlighted notable differences in 3 ROIs associated with blueprint connectome. Comparing with existing methodsThis study marks a significant shift from surface-based to voxel-based methodologies in analyzing pig brain structural connectivity and generating connectome blueprints. Additionally, it sheds light on the use of the piglet model for developmental studies, offering new perspectives in this area. ConclusionThis study established a piglet brain tract model and conducts a comparative analysis of adult pig’s and piglet’s structural connectivity. These findings underscore the potential use of the piglet brain model in employing piglet model for developmental studies.

Characterizing the binding of TC-5619 and encenicline on the alpha7 nicotinic acetylcholine receptor using PET imaging in the pig.

The alpha7 nicotinic acetylcholine receptor (α7-nAChR) has has long been considered a promising therapeutic target for addressing cognitive impairments associated with a spectrum of neurological and psychiatric disorders, including Alzheimer's disease and schizophrenia. However, despite this potential, clinical trials employing α7-nAChR (partial) agonists such as TC-5619 and encenicline (EVP-6124) have fallen short in demonstrating sufficient efficacy. We here investigate the target engagement of TC-5619 and encenicline in the pig brain by use of the α7-nAChR radioligand 11C-NS14492 to characterize binding both with in vitro autoradiography and in vivo occupancy using positron emission tomography (PET). In vitro autoradiography demonstrates significant concentration-dependent binding of 11C-NS14492, and both TC-5619 and encenicline can block this binding. Of particular significance, our in vivo investigations demonstrate that TC-5619 achieves substantial α7-nAChR occupancy, effectively blocking approximately 40% of α7-nAChR binding, whereas encenicline exhibits more limited α7-nAChR occupancy. This study underscores the importance of preclinical PET imaging and target engagement analysis in informing clinical trial strategies, including dosing decisions.

Identification of New Ketamine Metabolites and Their Detailed Distribution in the Mammalian Brain.

Ketamine is a common anesthetic used in human and veterinary medicine. This drug has recently received increased medical and scientific attention due to its indications for neurological diseases. Despite being applied for decades, ketamine's entire metabolism and pharmacological profile have not been elucidated yet. Therefore, insights into the metabolism and brain distribution are important toward identification of neurological effects. Herein, we have investigated ketamine and its metabolites in the pig brain, cerebrospinal fluid, and plasma using mass spectrometric and metabolomics analysis. We discovered previously unknown metabolites and validated their chemical structures. Our comprehensive analysis of the brain distribution of ketamine and 30 metabolites describes significant regional differences detected mainly for phase II metabolites. Elevated levels of these metabolites were identified in brain regions linked to clearance through the cerebrospinal fluid. This study provides the foundation for multidisciplinary studies of ketamine metabolism and the elucidation of neurological effects by ketamine.

Aging and brain free cholesterol concentration on amyloid-β peptide accumulation in guinea pigs.

Alzheimer's disease is a complex multifactorial neurodegenerative disorder wherein age is a major risk factor. The appropriateness of the Hartley guinea pig (GP), which displays high sequence homologies of its amyloid-β (Aβ40 and Aβ42) peptides, Mdr1 and APP(amyloid precursor protein) and similarity in lipid handling to humans, was appraised among 9-40weeks old guinea pigs. Protein expression levels of P-gp (Abcb1) and Cyp46a1 (24(S)-hydroxylase) for Aβ40, and Aβ42 efflux and cholesterol metabolism, respectively, were decreased with age, whereas those for Lrp1 (low-density lipoprotein receptor related protein 1), Rage (receptor for advanced glycation endproducts) for Aβ efflux and influx, respectively, and Abca1 (the ATP binding cassette subfamily A member 1) for cholesterol efflux, were unchanged among the ages examined. There was a strong, negative correlation of the brain Aβ peptide concentrations and Abca1 protein expression levels with free cholesterol. The correlation of Aβ peptide concentrations with Cyp46a1 was, however, not significant, and concentrations of the 24(S)-hydroxycholesterol metabolite revealed a decreasing trend from 20weeks old toward 40weeks old guinea pigs. The composite data suggest a role for free cholesterol on brain Aβ accumulation. The decreases in P-gp and Lrp1 protein levels should further exacerbate the accumulation of Aβ peptides in guinea pig brain.

Comparing fluorescent contrast agents for fluorescence guided surgery using 3-D cryo-imaging.

Fluorescence cryo-imaging is a high-resolution optical imaging technique that produces 3-D whole-body biodistributions of fluorescent molecules within an animal specimen. To accomplish this, animal specimens are administered a fluorescent molecule or reporter and are frozen to be autonomously sectioned and imaged at a temperature of -20°C or below. Thus, to apply this technique effectively, administered fluorescent molecules should be relatively invariant to low temperature conditions for cryo-imaging and ideally the fluorescence intensity should be stable and consistent in both physiological and cryo-imaging conditions. Herein, we assessed the mean fluorescence intensity of 11 fluorescent contrast agents as they are frozen in a tissue-simulating phantom experiment and show an example of a tested fluorescent contrast agent in a cryo-imaged whole pig brain. Most fluorescent contrast agents were stable within ~25% except for FITC and PEGylated FITC derivatives, which showed a dramatic decrease in fluorescence intensity when frozen.

Radiosynthesis and In Vitro Evaluation of [11C]tozadenant as Adenosine A2A Receptor Radioligand.

Tozadenant (4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide) is a highly selective adenosine A2A receptor (A2AR) antagonist and a promising lead structure for the development of A2AR-selective positron emission tomography (PET) probes. Although several 18F-labelled tozadenant derivatives showed favorable in vitro properties, recent in vivo PET studies observed poor brain penetration and lower specific binding than anticipated from the in vitro data. While these findings might be attributable to the structural modification associated with 18F-labelling, they could also reflect inherent properties of the parent compound. However, PET studies with radioisotopologues of tozadenant to evaluate its cerebral pharmacokinetics and brain distribution are still lacking. In the present work, we applied N-Boc-O-desmethyltozadenant as a suitable precursor for the preparation of [O-methyl-11C]tozadenant ([11C]tozadenant) by O-methylation with [11C]methyl iodide followed by acidic deprotection. This approach afforded [11C]tozadenant in radiochemical yields of 18 ± 2%, with molar activities of 50-60 GBq/µmol (1300-1600 mCi/µmol) and radiochemical purities of 95 ± 3%. In addition, in vitro autoradiography in pig and rat brain slices demonstrated the expected striatal accumulation pattern and confirmed the A2AR specificity of the radioligand, making it a promising tool for in vivo PET studies on the cerebral pharmacokinetics and brain distribution of tozadenant.