Normal Rat Brain Research Articles

Modulatory effect of methanol extract of Annona muricata stem bark on mitochondrial membrane permeability transition pore in normal rat liver and monosodium glutamate-induced uterine hyperplasia

Uterine fibroids are benign tumors that develop in many women of reproductive age. Surgery is the main approach to treatment while other options are also associated with adverse effects. Studies have shown that certain bioactive agents present in medicinal plants elicit their anti-tumor activity by induction of mitochondrial permeability transition (mPT) opening. This research therefore aimed at investigating the effect of methanol extract of Annona muricata (MEAM) on mPT pore opening in normal and monosodium glutamate-induced uterine hyperplasia using female Wistar rats. Mitochondria, isolated from rat liver were exposed to different concentrations (20, 60, 100, 140 and 180μg/mL) of MEAM. The mPT pore opening, cytochrome c release, mitochondrial ATPase (mATPase) activity and the percentage lipid peroxidation were assessed spectrophotometrically. Histological effects of MEAM on the liver, brain and uterus of normal and MSG-treated rats were investigated. The in vitro results showed a significant induction of mPT pore opening by 2.4, 4.2 and 6.4 folds, release of cytochrome c and enhancement of mATPase activity at 100,140 and 180μg/mL, respectively. However, oral administration of MEAM did not induce mPT pore opening, neither any significant release of cytochrome c nor enhancement of mATPase activity at all the dosages used. However, histological assay revealed the presence of MSG-induced cellular damage and uterine hyperplasia which was ameliorated by MEAM co-administration. These findings suggest that MEAM contains phytochemicals that can ameliorate MSG-induced damage and uterine hyperplasia in rats; however, the mechanism might not be via upregulation of mitochondrial-mediated apoptosis.

Study on patterns and characteristics of in vivo tissue distribution of anti-inflammatory extract of Tetrastigma hemsleyanum aerial part in healthy and inflammatory pathological model rats

The concentrations of seven anti-inflammatory components in blood and tissues were determined by UPLC-MS/MS after oral administration of Tetrastigma hemsleyanum aerial part(THAA) in healthy and inflammatory pathological model rats. The determination was carried out by using positive and negative ion switching technique, and multiple reaction monitoring(MRM) mode. The tissue distributions of the seven components in different physiological states were compared, and the patterns and characteristics of the effective components of THAA were studied. The results revealed that the seven effective components have large drug-time-curve areas(AUC) in heart, brain, small intestine, and stomach in both normal rats and inflammatory pathological model rats. This suggests that the anti-inflammatory effective component groups in THAA extract can all penetrate the blood-brain barrier, and have a large distribution area in gastrointestinal tract. It is inferred that gastrointestinal reabsorption may be one of the causes of the bimodal distribution of the drug-time curve of the drug blood distribution graph. As compared to normal rats, the effective component groups in THAA extract have higher drug-time curve area(AUC) in heart, brain, small intestine, stomach, liver, spleen, lung, kidney, and muscle of inflammatory pathological model rats. Among them, the effective component groups have the largest distribution area in heart, brain, small intestine, and stomach. This suggests that the binding force of organ tissues and drugs in the body may change under pathological conditions. It is speculated that the heart, brain, small intestine, and stomach may be the target tissues of THAA to produce anti-inflammatory effect. The retention times of THAA effective component groups in various organ tissues of rats in different physiological states are all relatively short, and do not have much difference. This suggests that no effective component accumulates in body, and that the pathological state of inflammation does not affect the onset times of the effective component groups. This experiment elucidates the patterns and characteristics of the in vivo target-effecting tissue distribution of THAA anti-inflammatory extract, and provides an experimental basis for clinical treatment.

Comparative Analysis of the Expression of Chondroitin Sulfate Subtypes and Their Inhibitory Effect on Axonal Growth in the Embryonic, Adult, and Injured Rat Brains.

Chondroitin sulfate glycosaminoglycans (CS-GAGs) are the primary inhibitory GAGs for neuronal growth after central nervous system (CNS) injury. However, the inhibitory or permissive activity of CS-GAG subtypes is controversial and depends on the physiological needs of CNS tissues. In this study, we investigated the characteristics and effects of CS-GAGs on axonal growth, which was isolated from the brain cortices of normal rat embryo at E18, normal adult rat brain and injured adult rat brain. Isolated CS-GAGs from embryo, normal adult, and injured adult rat brains were used for analyzing their effect on attachment and axonal growth using modified spot assay with dorsal root ganglion (DRG) explants and cerebellar granule neurons (CGNs). CS-GAGs were separated using high performance liquid chromatography (HPLC), and the subtypes of CS-GAGs were analyzed. CS-GAGs of all three groups inhibited CGN attachment and axonal growth of DRGs. However, CS-GAGs of normal adult rat brain exhibited higher inhibitory activity than those of the other groups in both assays. When subtypes of CS-GAGs were analyzed using HPLC, CS-A (4S) was the most abundant in all three groups and found in largest amount in normal adult rat brain. In contrast, unsulfated CS (CS0) and CS-C (6S) were more abundant by 3-4-folds in E18 group than in the two adult groups. When compared with the normal adult rat brain, injured rat brain showed relatively similar patterns to that of embryonic rat brain at E18 in the expression of CS subtypes and their inhibitory effect on axonal growth. This phenomenon could be due to differential expression of CS-GAGs subtypes causing decrease in the amount of CS-A and mature-type CS proteoglycan core proteins.

MTORC1 and mTORC2 expression in inner retinal neurons and glial cells

The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.

Synthesis, biological evaluation and preclinical study of a novel 99mTc-peptide: A targeting probe of amyloid-β plaques as a possible diagnostic agent for Alzheimer's disease.

With respect to the main role of amyloid-β (Aβ) plaques as one of the pathological hallmarks in the brain of Alzheimer's patients, the development of new imaging probes for targeted detection of Aβ plaques has attracted considerable interests. In this study, a novel cyclopentadienyl tricarbonyl Technetium-99m (99mTc) agent with peptide scaffold, 99mTc-Cp-GABA-D-(FPLIAIMA)-NH2, for binding to the Aβ plaques was designed and successfully synthesized using the Fmoc solid-phase peptide synthesis method. This radiopeptide revealed a good affinity for Aβ42 aggregations (Kd=20µM) in binding affinity study and this result was confirmed by binding to Aβ plaques in brain sections of human Alzheimer's disease (AD) and rat models using in vitro autoradiography, fluorescent staining, and planar scintigraphy. Biodistribution studies of radiopeptide in AD and normal rats demonstrated a moderate initial brain uptake about 0.38 and 0.35% (ID/g) 2min post-injection, respectively. Whereas, AD rats showed a notable retention time in the brain (0.23% ID/g at 30min) in comparison with fast clearance in normal rat brains. Normal rats following treatment with cyclosporine A as a p-glycoprotein inhibitor showed a significant increase in the radiopeptide brain accumulation compared to non-treated ones. There was a good correlation between data gathered from single-photon emission computed tomography/computed tomography (SPECT/CT) imaging and biodistribution studies. Therefore, these findings showed that this novel radiopeptide could be a potential SPECT imaging agent for early detection of Aβ plaques in the brain of patients with AD.

Protective effects of anthocyanins against brain ischemic damage.

Anthocyanins are considered as bioactive components of plant-based diets that provide protection against ischemic cardiovascular pathologies by mechanisms dependent on their antioxidant and reductive capacities. However, it is not clear whether similar anthocyanin-mediated mechanisms can provide protection against ischemia-induced brain mitochondrial injury and cell death. In this study, we compared effects of three cyanidin-3-glycosides - glucoside (Cy3G), galactoside (Cy3Gal) and rutinoside (Cy3R), with pelargonxidin-3-glucoside (Pg3G) and found that at 10-20μM concentrations they have no direct effect on respiratory functions of mitochondria isolated from normal or ischemia-damaged rat brain slices. However, intravenous injection of Cy3Gal and Cy3G (0,025mg/kg or 0,05mg/kg what matches 10μM or 20μM respectively) but not Cy3R in rats protected against ischemia-induced caspase activation and necrotic cell death, and reduced infarct size in cerebral cortex and cerebellum. These effects correlated with cytochrome c reducing capacity of cyanidin-3-glycosides. In contrast, intravenous injection of 0,025mg/kg Pg3G which has the lowest cytochrome c reducing capacity among investigated anthocyanins, had no effect on ischemia-induced caspase activation and necrosis but reduced brain infarct size whereas intravenous injection of 0,05mg/kg of Pg3G slightly promoted necrosis in the brain. Our data suggest that reductive rather than antioxidant capacities of anthocyanins may be important components in providing protection against ischemic brain damage.

BOT-01 BLOOD-BRAIN BARRIER OPENING USING 220-KHZ TRANSCRANIAL MRI-GUIDED FOCUSED ULTRASOUND AND MICROBUBBLES IN MOUSE AND RAT

OBJECTIVEIn neuro-oncology, it is believed that one major obstacle to effective chemotherapy is the high vascularity and heterogenous permeability of brain tumors. Focused ultrasound (FUS) exposure with the microbubbles has been shown to transiently open the blood-brain barrier (BBB) without depositing thermal energy, and thus may enhance the delivery of various therapeutic drugs into brain tumors. The aim of this study was to evaluate the BBB opening using 220-kHz transcranial MRI-guided FUS (TcMRgFUS) device and microbubbles in mouse and rat.METHODSThe experiments were performed with the 220-kHz ExAblate Neuro TcMRgFUS system (InSightec) and novel lipid bubbles (LB, Teikyo Univ.). Normal mouse and rat brains were irradiated with TcMRgFUS (output power, 5W; duration of irradiation, 30 s; duty cycle 100%) following intravenous injection of 6x107 LB per mouse and rat, respectively. On irradiation, target temperature rise & cavitation signal were monitored by MR thermometry and cavitation receiver, respectively. Immediately after irradiation, BBB opening and complications were detected based on T1, T2, T2*, and Gadolinium (Gd) enhanced T1-weighted images.RESULTSThe maximum temperature of brain tissue was under 42 C. There were no risky-cavitation signals causing hemorrhage. The FUS-LB exposure induced successful BBB opening effect in both mouse and rat, confirmed by Gd enhancement in the target region, lateral ventricles, and sulcus. In addition, there were no complications such as edema, coagulation, and hemorrhage.CONCLUSIONSAlthough there remain many conditions to be optimized, BBB opening using a 220-kHz TcMRgFUS device and LB can offer a non-invasive and feasible drug delivery for brain malignancies.

Protein Expression and Functional Relevance of Efflux and Uptake Drug Transporters at the Blood-Brain Barrier of Human Brain and Glioblastoma.

The knowledge of transporter protein expression and function at the human blood-brain barrier (BBB) is critical to prediction of drug BBB penetration and design of strategies for improving drugdelivery to the brain orbrain tumor. This study determined absolute transporter protein abundances in isolated microvessels of human normal brain (N=30), glioblastoma (N=47), rat (N=10) and mouse brain (N=10), and cell membranes of MDCKII cell lines, using targeted proteomics. In glioblastoma microvessels, efflux transporters (ABCB1and ABCG2), monocarboxylate transporter 1 (MCT1), glucose transporter 1 (GLUT1), sodium-potassiumpump (Na/K ATPase), and Claudin-5 protein levels were significantly reduced, whilelarge neutral amino acid transporter 1 (LAT1) was increased and GLU3 remained the same, as compared with human normal brain microvessels. ABCC4, OATP1A2, OATP2B1, and OAT3 were undetectable in microvessels of both human brain and glioblastoma. Species difference in BBB transporter abundances was noted. Cellular permeability experiments and modeling simulations suggested that not a single apical uptake transporter but a vectorial transport system consisting of an apical uptake transporter and basolateral efflux mechanism was required for efficient delivery of poor transmembrane permeability drugs from the blood to brain.

Vitamin B5 (d-pantothenic acid) localizes in myelinated structures of the rat brain: Potential role for cerebral vitamin B5 stores in local myelin homeostasis

Vitamin B5 (d-pantothenic acid; pantothenate) is an essential trace nutrient that functions as the obligate precursor of coenzyme A (CoA), through which it plays key roles in myriad biological processes, including many that regulate carbohydrate, lipid, protein, and nucleic acid metabolism. In the brain, acetyl-CoA is necessary for synthesis of the complex fatty-acyl chains of myelin, and of the neurotransmitter acetylcholine. We recently found that cerebral pantothenate is markedly lowered, averaging ∼55% of control values in cases of Huntington’s disease (HD) including those who are pre-symptomatic, and that regions where pantothenate is lowered correspond to those which are more severely damaged. Here we sought to determine the previously unknown distribution of pantothenate in the normal-rat brain, and whether the diabetic rat might be useful as a model for altered cerebral pantothenate metabolism. We employed histological staining (Nissl) to identify brain structures; immunohistochemistry with anti-pantothenate antibodies to determine the distribution of pantothenate in caudate putamen and cerebellum; and gas-chromatography/mass-spectrometry to quantitate levels of pantothenate and other metabolites in normal- and diabetic-rat brain. Remarkably, cerebral pantothenate was almost entirely localized to myelin-containing structures in both experimental groups. Diabetes did not modify levels or disposition of cerebral pantothenate. These findings are consistent with physiological localization of pantothenate in myelinated white-matter structures, where it could serve to support myelin synthesis. Further investigation of cerebral pantothenate is warranted in neurodegenerative diseases such as HD and Alzheimer’s disease, where myelin loss is a known characteristic of pathogenesis.

CBMT-02. UP-REGULATION OF Γ-GLUTAMYL-TRANSFERASE CAN BE USED TO IMAGE GLIOBLASTOMA USING HYPERPOLARIZED Γ-GLUTAMYL-[1-13C]GLYCINE MRS

Abstract γ-glutamyl-transferase (GGT) is a key enzyme in the γ-glutamyl cycle, which regulates glutathione homeostasis. The enzyme is localized on the outer cell membrane and cleaves glutathione to glutamate and cysteinylglycine. As such, it facilitates uptake of the amino acids essential for intracellular synthesis of glutathione (GSH), which is the major thiol anti-oxidant. GGT is upregulated in glioblastoma, but remains low in normal brain. It is therefore an attractive molecular imaging target for specific detection of glioblastoma. The goal of our study was therefore to assess for the first time the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine (γ-Glu-[1-13C]Gly) for imaging glioblastoma in orthotopic tumor-bearing rats. Athymic nude rats with U87 glioblastoma tumors or tumor-free controls were investigated. First, we confirmed that GGT expression was significantly higher in U87 tumors compared to normal rat brain tissue. GSH levels were also higher. Imaging studies were then performed by injecting HP γ-Glu-[1-13C]Gly and acquiring dynamic 13C MR spectra using a flyback spectral-spatial slab sequence on a preclinical Bruker 3T MR system. The dynamic data were analyzed by measuring the signal-to-noise (SNR) ratios of the substrate (γ-Glu-[1-13C]Gly) and the product ([1-13C]Glycine). Comparison of control and tumor-bearing rats showed no statistically significant difference in the SNR of the substrate. In contrast, the SNR of the product demonstrated a significantly higher level of HP [1-13C]Glycine in the tumor-bearing rats compared to controls. Consistent with the higher levels of HP [1-13C]Glycine, the [1-13C]Gly-to-γ-Glu-[1-13C]Glycine ratio was also significantly higher in tumor-bearing animals relative to controls (0.046±0.004 vs 0.021±0.008 respectively; p=0.03). Further studies are needed to assess the generality of our findings. Nonetheless, this study demonstrates for the first time the feasibility of using γ-Glu-[1-13C]Gly to monitor GGT activity and thus could serve as a new approach for monitoring the presence of tumor.

Fast correction of B0 field inhomogeneity for pH-specific magnetization transfer and relaxation normalized amide proton transfer imaging of acute ischemic stroke without Z-spectrum.

The magnetization transfer and relaxation normalized amide proton transfer (MRAPT) analysis is promising to provide a highly pH-specific mapping of tissue acidosis, complementing commonly used CEST asymmetry analysis. We aimed to develop a fast B0 inhomogeneity correction algorithm for acute stroke magnetization transfer and relaxation normalized amide proton transfer imaging without Z-spectral interpolation. The proposed fast field inhomogeneity correction describes B0 artifacts with linear regression. We compared the new algorithm with the routine interpolation correction approach in CEST imaging of a dual-pH phantom. The fast B0 correction was further evaluated in amide proton transfer imaging of normal and acute stroke rats. Our phantom data showed that the proposed fast B0 inhomogeneity correction significantly improved pH MRI contrast, recovering over 80% of the pH MRI contrast-to-noise-ratio difference between the raw magnetization transfer ratio asymmetry and that using the routine interpolation-based B0 correction approach. In normal rat brains, the proposed fast B0 correction improved pH-specific MRI uniformity across the intact tissue, with the ratio of magnetization transfer and relaxation normalized amide proton transfer ratio being 10% of that without B0 inhomogeneity correction. In acute stroke rats, fast B0 inhomogeneity-corrected pH MRI reveals substantially improved pH lesion conspicuity, particularly in regions with nonnegligible B0 inhomogeneity. The pH MRI contrast-to-noise ratio between the ipsilateral diffusion lesion and contralateral normal tissue improved significantly with fast B0 correction (from 1.88 ± 0.48 to 2.20 ± 0.44, P < .01). Our study established an expedient B0 inhomogeneity correction algorithm for fast pH imaging of acute ischemia.

P11.57 A 3D brain organoid coculture system delineates the invasive cell components in glioblastoma

Abstract BACKGROUND Glioblastoma (GBM) cell infiltration into the surrounding normal brain tissue where the blood brain barrier is intact, represents a major problem for clinical management and therapy. There is a vital need to understand the molecular mechanism that drives tumor cell invasion into the surrounding brain. We have previously developed a 3D coculture model where mature brain organoids are confronted with patient-derived glioblastoma stem-like cells (GSCs). In such a coculture system, single cell invasion into the normal brain tissue can be studied in detail. Here, we first describe in detail, by RNA-seq and proteomics, the differentiation of various neural cell lineages into mature brain organoids as well as their cellular organization. By real-time confocal microscopy and imaging analyses we also determine the speed of tumor cell invasion into the brain. Finally, we used this coculture system to delineate in detail the cellular heterogeneity within the invasive compartment and their gene expression. MATERIAL AND METHODS Immunohistochemistry and immunofluorescence were used to determine the expression and distribution of mature neurons, astrocytes, oligodendrocytes, and microglia within the brain organoids. Proteomics and RNA-seq were used to determine brain development ex-vivo. To assess the clonal composition of the GBM-invasive compartment, we used cellular (RGB) barcoding technology. By advanced imaging, we tracked in real time the invasion of barcoded cells into the brain organoids. Finally, we isolated invasive cells and non-invasive cells from our coculture system and used single cell sequencing to analyze their gene expression profiles and molecular phenotypes. RESULTS Immunohistochemistry and immunofluorescence showed that brain organoids, after 21 days of differentiation, display a highly cellular and structural organization. RNA-seq and proteomics, performed at different time points of organoid differentiation, revealed that the brain organoids develop into mature brain structures after 21 days as verified by a comparative analysis to normal rat brain development in vivo. Imaging analyses showed that multiple clones within the GBMs have the capacity to invade into the brain tissue with an average speed of ~ 20 μm/h. RNA-sec analysis of the invasive compartment revealed a strong up-regulation of genes and pathways associated with anaerobic respiration (glycolysis). CONCLUSION We describe a highly standardized brain organoid coculture system that can be used to delineate GBM invasion ex-vivo. We demonstrate that this platform can be used to unravel the mechanisms that drive GBM invasion into the normal brain.

Evaluation of the Effects of Fullerene C60 Nanoparticles on Oxidative Stress Parameters at Liver and Brain of Normal Rats

Evaluation of the Effects of Fullerene C60 Nanoparticles on Oxidative Stress Parameters at Liver and Brain of Normal Rats

Volume entropy and maturation of normal rat brain

Volume entropy and maturation of normal rat brain

In vivo monitoring of superoxide anion from Alzheimer's rat brains with functionalized ionic liquid polymer decorated microsensor

The superoxide anion (O2•−) is an important reactive oxygen species (ROS) in the brain system, which has been associated with the development of many neurological diseases, including Alzheimer's disease (AD). Herein, we introduced a carbon fiber microelectrode (CFME) based in vivo technique for specific and sensitive monitoring of the O2•− radical in the living brains of both normal and AD model rats. Compared with other reported superoxide dismutase (SOD) electrochemical biosensors, the microsensor presented in our work was featured in the coating of a functionalized ionic liquid polymer (PIL) onto PB nanoparticles (PBNPs) and carbon nanotubes (CNT). It was demonstrated that the cationic and carboxyl-rich PILs provided abundant interaction sites with SOD to prevent enzyme leakage from sensor, which was beneficial for the enhancement of sensitivity. Additionally, CCK-8 assay and autoxidation of pyrogallol tests showed that MCF-7 cells maintained a high viability after incubated with PIL and most of the SOD bioactivity was retained in the presence of PIL, which implied the PIL itself possessed an excellent biocompatibility. These properties allow the sensor to track the fluctuation of O2•− levels in vivo between normal and AD rats. This is the first report on application of functionalized PIL to reveal the O2•− related pathological process of AD.

Optical coefficients as tools for increasing the optical coherence tomography contrast for normal brain visualization and glioblastoma detection

The methods used for digital processing of optical coherence tomography (OCT) and crosspolarization (CP) OCT images are focused on improving the contrast ratio of native structural OCT images. Such advances are particularly important for the intraoperative detection of glioma margins where the visual assessment of OCT images can be difficult and lead to errors. The aim of the study was to investigate the application of optical coefficients obtained from CP OCT data for the differentiation of glial tumorous tissue from a normal brain. Pseudocolor en-face OCT maps based on two optical coefficients (the commonly used rate of attenuation in the cochannel, and in addition, the interchannel attenuation difference) were constructed for normal rat brain coronal cross sections and for brains with a 101.8 rat glioblastoma model. It was shown that the use of optical coefficients significantly increased the available information from the OCT data in comparison with unprocessed images. As a result, this allowed contrasting of the white matter from the gray matter and tumorous tissue ex vivo, and for this purpose, the interchannel attenuation difference worked better. The interchannel attenuation difference values of white matter were at least seven and two times higher than corresponding values of the cortex and tumorous tissue, whereas the same parameter for cochannel attenuation coefficient values of white matter are about 4 and 1.4. However, quantitative analysis shows that both coefficients are suitable for the purpose of glioblastoma detection from normal brain tissue regardless of whether a necrotic component was present (in all compared groups ).

High-sensitivity quantification of glycosphingolipid glycans by ESI-MS utilizing ozonolysis-based release and isotopic Girard's reagent labeling

Quantitative analysis of glycosphingolipids (GSLs) has been hindered by the lack of chromogenic groups for spectral detection or active functional groups for specific derivatization. In this study, a highly sensitive method based on ozonolysis-induced release and isotopic Girard's reagent P labeling of GSL glycans coupled with mass spectrometric detection for the quantification of animal tissue GSLs is developed. First, different approaches for the release of glycans from GSLs were compared with each other and the ozonolysis-based method was found to have the highest glycan yield under relative mild reaction conditions. Then a relative quantification method of ozonolysis-released GSL glycans based on stable isotope labeling using nondeuterated (d0-) and 2,3,4,5,6-pentadeuterated (d5-) Girard's reagent P (GP) was established, and its good linearity, accuracy and reproducibility were statistically verified. Finally, the new method was successfully applied to revealing the difference between porcine brain and liver as well as between the brain of normal and aging rats in GSL glycome by online hydrophilic interaction liquid chromatography coupling with ultraviolet detection and tandem mass spectrometry (HILIC-UV-MS/MS). This novel method is versatile and sensitive, enabling accurate quantitative analysis of tissue GSLs and showing great significance for glycomic studies.

Abstract 4296: Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells

Abstract Background: Although interfering with the actions of microtubule-associated proteins (MAP’s) is successful against ovarian epithelial carcinoma (OVCA), knowledge of the MAP’s in OVCA cells is lacking. We sought to identify cancer-specific MAP’s in OVCA cells.Methods: Four human OVCA cell lines, primary OVCA tissue, ascitic cells and metastases from 9 patients and normal ovarian tissue from six women were compared to control human and rat brain tissue, uterine myomas (benign tumors) and rat and human non-neural organs. Modified Western blotting using panels of anti-microtubule-associated protein monoclonal antibodies were used. An in-vitro brain and OVCA mixing and recovery experiment was carried out to assess possible degradation of MAP1a by OVCA. A paclitaxel-driven tubulin assembly assay was performed to determine whether the OVCA protein has microtubule-associated protein properties. The transcriptome of CSP-80 was sequenced using primer extension sequencing. Results: (1) In contradistinction to the 350 kDa MAP1a present in control brain extracts, only an 80 kDa molecule with MAP1a immune-reactivity was found in OVCA cell lines and tissues. No immune-reactivity was present in the negative controls. (2)Following the mixing experiments there was no increase of CSP-80; CSP-80 is not a degradation artifact of MAP1a. (3) The paclitaxel-driven tubulin assembly assay showed that CSP-80 bound to microtubules during tubulin polymerization. (4) No CSP-80 was identified in human or rat non-malignant tissues including normal human and rat brain or normal human ovary, placenta or uterine myomas, or normal rat liver, spleen or lung. (5) cDNA sequencing shows a 2603 bp fragment at a 5’ terminal of p80 to be homologous with the analogous segment of MAP1a. (6) Further testing showed that CSP-80 was present in other reproductive and non-reproductive carcinomas. Again, no 350 kDa MAP1a was present. Studies are underway to determine whether CSP-80 is a mutated protein or the product of post-transcriptional modification. Conclusion: CSP-80 is a novel cancer-related protein that appears to be limited to an 80 kDa sequence of the MAP1a gene, or a post-transcriptionally modified product. Since CSP-80 exhibits tubulin-binding activity, this may explain the efficacy of taxane derivatives. CSP-80 may disclose some aspects of carcinogenesis, be a valuable marker of cancer and therapeutic efficacy, and offer new avenues for cancer treatment. Citation Format: Ahmed Fadiel, Viswam S. Nair, Peter Aceto, Frederick Naftolin. Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4296.

The Impact of the Ketogenic Diet on Glial Cells Morphology. A Quantitative Morphological Analysis

Ketogenic diet is reported to protect against cognitive decline, drug-resistant epilepsy, Alzheimer's Disease, damaging effect of ischemic stroke and many neurological diseases. Despite mounting evidence that this dietary treatment works, the exact mechanism of its protective activity is largely unknown. Ketogenic diet acts systemically, not only changing GABA signaling in neurons, but also influencing the reliance on mitochondrial respiration, known to be disrupted in many neurological diseases. Normally, human body is driven by glucose while ketogenic diet mimics starvation and energy required for proper functioning comes from fatty acids oxidation. In the brain astrocytes are believed to be the sole neural cells capable of fatty oxidation. Here we try to explain that not exclusively neurons, but also morphological changes of astroglia and/or microglia due to different metabolic state are important for the mechanism underlying the protective role of ketogenic diet. By quantifying different parameters describing cellular morphology like ramification index or fractal dimension and using Principal Component Analysis to discover the regularities between them, we demonstrate that in normal adult rat brain, ketogenic diet itself is able to change glial morphology, indicating an important role of these underappreciated cells in the brain metabolism.

Conventional Anti-glioblastoma Chemotherapy Affects Proteoglycan Composition of Brain Extracellular Matrix in Rat Experimental Model in vivo.

Temozolomide (TMZ) is a conventional chemotherapy drug for adjuvant treatment of glioblastoma multiforme (GBM), often accompanied by dexamethasone (DXM) to prevent brain oedema and alleviate clinical side effects. Here, we aimed to investigate an ability of the drugs to affect normal brain tissue in terms of proteoglycan (PG) composition/content in experimental rat model in vivo. Age- and brain zone-specific transcriptional patterns of PGs were demonstrated for 8, 60, and 120 days old rats, and syndecan-1, glypican-1, decorin, biglycan, and lumican were identified as the most expressed PGs. DXM treatment affected both PG core proteins expression (mainly syndecan-1, glypican-1, decorin, biglycan, lumican, versican, brevican, and NG2) and heparan sulphate (HS)/chondroitin sulphate (CS) content in organotypic brain slice culture ex vivo and experimental animals in vivo in a dose-dependent manner. TMZ treatment did not result in the significant changes in PG core proteins expression both in normal rat brain hippocampus and cortex in vivo (although generics did), but demonstrated significant effects onto polysaccharide HS/CS content in the brain tissue. The effects were age- and brain zone-specific and similar with the age-related PGs expression changes in rat brain. Combination of TMZ with DXM resulted in the most profound deterioration in PGs composition and content in the brain tissue both at core protein and glycosaminoglycan levels. Taken together, the obtained results demonstrate that conventional anti-glioblastoma therapy affects proteoglycan structure and composition in normal brain tissue, potentially resulting in deterioration of brain extracellular matrix and formation of the favourable tumorigenic niche for the expansion of the residual glioma cells. During the TMZ chemotherapy, dose and regimen of DXM treatment matter, and repetitive low DXM doses seem to be more sparing treatment compared with high DXM dose(s), which should be avoided where possible, especially in combination with TMZ.