Sylvain Dukic

Modeling and quantifying biochemical changes in C6 tumor gliomas by Fourier transform infrared imaging.

The purpose of the study was to investigate molecular changes associated with glioma tissues using FT-IR microspectroscopic imaging (FT-IRM). A multivariate statistical analysis allowed one to successfully discriminate between normal, tumoral, peri-tumoral, and necrotic tissue structures. Structural changes were mainly related to qualitative and quantitative changes in lipid content, proteins, and nucleic acids that can be used as spectroscopic markers for this pathology. We have developed a spectroscopic model of glioma to quantify these chemical changes. The model constructed includes individual FT-IR spectra of normal and glioma brain constituents such as lipids, DNA, and proteins (measured on delipidized tissue). Modeling of FT-IR spectra yielded fit coefficients reflecting the chemical changes associated with a tumor. Our results demonstrate the ability of FT-IRM to assess the importance and distribution of each individual constituent and its variation in normal brain structures as well as in the different pathological states of glioma. We demonstrated that (i) cholesterol and phosphatidylethanolamine contributions are highest in corpus callosum and anterior commissure but decrease gradually towards the cortex surface as well as in the tumor, (ii) phosphatidylcholine contribution is highest in the cortex and decreases in the tumor, (iii) galactocerebroside is localized only in white, but not in gray matter, and decreases in the vital tumor region while the necrosis area shows a higher concentration of this cerebroside, (iv) DNA and oleic acid increase in the tumor as compared to gray matter. This approach could, in the future, contribute to enhance diagnostic accuracy, improve the grading, prognosis, and play a vital role in therapeutic strategy and monitoring.

Pharmacokinetics of methotrexate in the extracellular fluid of brain C6-glioma after intravenous infusion in rats.

AbstractPurpose. Establishment of the pharmacokinetic profile of methotrexate (MTX) in the extracellular fluid (ECF) of a brain C6-glioma in rats. Methods. Serial collection of plasma samples and ECF dialysates after i.v. infusion of MTX (50 or 100 mg/kg) for 4 h. HPLC assay. Results. Histological studies revealed the presence of inflammation, edema, necrosis, and hemorrhage in most animals. In vivo recovery (reverse dialysis) was 10.8 ± 5.3%. MTX concentrations in tumor ECF represented about 1−2% of the plasma concentrations. Rapid equilibration between MTX levels in brain tumor ECF and plasma. ECF concentrations almost reached steady-state by the end of the infusion (4 h), then decayed in parallel with those in plasma. Doubling of the dose did not modify MTX pharmacokinetic parameters (t1/2α, t1/2β, MRT, fb, Vd, and CLT), except for a 1.7-fold increase of AUCPlasma and a 3.8-fold increase in AUCECF which resulted in a 2.3-fold increase in penetration (AUCECF/AUCPlasma). In spite of an important interindividual variability, a relationship between MTX concentrations in plasma and tumor ECF could be established from mean pharmacokinetic parameters. Conclusions. High plasma concentrations promote the penetration of MTX into brain tissue. However, free MTX concentrations in tumor ECF remain difficult to predict consistently.

Influence of schedule of administration on methotrexate penetration in brain tumours

The influence of the administration schedule (intravenous (i.v.) bolus versus i.v. infusion) on the pharmacokinetics of methotrexate (MTX) in plasma and extracellular fluid (ECF) of a brain C6-glioma was investigated in rats. MTX concentrations were determined by high performance liquid chromatography (HPLC)-ultraviolet radiation (UV). MTX (50 mg/kg) was administered by i.v. bolus or i.v. infusion (4 h). Concentration-time profiles were fitted to a two-compartment open model. Maximum MTX concentrations ranged between 178 and 294 microgram/ml (i.v. bolus), and between 11 and 24 microgram/ml (i.v. infusion) in plasma. MTX rapidly entered the tumour tissue although its concentrations in the ECF were much lower than those observed in plasma for both modes of administration. In spite of an important interindividual variability, AUC(ECF) was approximately 5-fold higher and mean MTX penetration in tumour ECF (AUC(ECF)/AUC(Plasma)) was approximately 3-fold higher after i.v. bolus than after i.v. infusion administration. These results indicate that i.v. bolus administration schedules promote MTX delivery in brain tumour tissue.

Monitoring of Biochemical Changes through the C6 Gliomas Progression and Invasion by Fourier Transform Infrared (FTIR) Imaging

We have investigated the spatial distribution of molecular changes associated with C6 glioma progression using Fourier transform infrared (FT-IR) microspectro-imaging in order to determine spectroscopic markers for early diagnosis of tumor growth. Our results showed that at day 7 after tumor implantation, FTIR investigations displayed a very small abnormal zone associated with the proliferation of C6 cells in the caudate putamen. From this day, rats developed solid and well-circumscribed tumors and invasive areas. The volume of peritumoral areas increased rapidly until day 19. The maturation of the tumor was accompanied by a diminution in its proliferative and invasive area. The presence of necrotic areas was visible from day 15. A non-negative least-squares algorithm was used to quantify spatial distribution of molecular changes in tissues (lipids, nucleic acids, and proteins) associated with glioma progression. Compared to those in normal brain, statistical tests on fit coefficients showed that the concentrations of sphingomyelin (SMY), nucleic acids, phosphatidylserine (PS), and galactocerebroside (GalC) were significantly affected during C6 glioma development. These constituents can be used as spectroscopic markers for C6 glioma progression. Indeed, the concentration of DNA decreased significantly from tumor to invasion, to normal brain tissues, the necrotic area has higher concentrations of the Galc than other areas. The PS content was significantly higher in the peritumoral zone and decreased in the tumor zones matter.

Screening of biochemical/histological changes associated to C6 glioma tumor development by FTIR/PCA imaging

Principal components analysis (PCA) combined to Fourier transform infrared (FTIR) microspectroscopic imaging was used to screen biochemical changes associated to C6 glioma tumor from 7 to 19 days growth. Normal brain and tumor obtained at 7, 12, 19 days after C6 cell injection were used to develop a diagnostic model of brain glioma based on PCA analysis. This classification model was validated using extra-measurements on normal and tumor at 9 and 15 days post-implantation. The spatial and biochemical information obtained from FTIR/PCA maps can be used to improve the discrimination between normal and grading human glioma. The first 4 PCs which account for more than 93.6% of total spectral variance were used to construct pseudo-color scores maps and compared each map to the corresponding hematoxylin and eosin (H&E) staining. Our results reported that by correlating pseudocolor map scores with H&E staining it was possible to screen histological changes associated with tissue transformation. In fact, PC1 and PC4 were associated to the tumor, surrounding tumor and necrosis. Indeed, at day 7 after tumor implantation, FTIR investigations displayed a very small abnormal zone associated with the proliferation of C6 cells in the caudate putamen (CP). PC2 and PC3 described normal brain structures such as white matter (corpus callosum (CC) and commissura anterior (CA)) and some cortex layers (grey matter). After delipidation of the tissues, we were still able to differentiate between different tissue features based on nucleic acid and protein content. By comparing the patterns of the PC loads with the spectra of lipids extracted from white and gray matters, and DNA, we have identified some biochemical changes associated with tissue transformation. This work demonstrated that our classification model provides a successful histological classification of different brain structures.

Determination of Free Extracellular Levels of Methotrexate by Microdialysis in Muscle and Solid Tumor of the Rabbit

AbstractPurpose. To determine of the pharmacokinetic profile of methotrexate (MTX) in blood and extracellular fluid (ECF) of VX2 tumor and muscle in rabbits. Methods. Microdialysis probes were inserted into VX2 tumor and in muscle tissue. Following intravenous administration of MTX (30 mg/ kg), serial collection of arterial blood samples and dialysates of muscle and tumor ECF for 4 h was carried out. Quantitation of MTX and determination of free plasma concentrations was performed by fluorescence polarization immunoassay and ultrafiltration, respectively. Correlations were established between the unbound plasma and ECF MTX concentrations. Results. Total and free plasma concentrations exhibited a parallel three exponential decay in both healthy and tumorigenic animals. Total clearance (8.9 vs 6.5 ml−1.min−1.kg−1) and volume of distribution (4.0 vs 2.9 1.kg−1), however, tended to decrease in the tumor-bearing group. The ECF/plasma AUC ratio equaled 14.2 ± 8.8% in muscle and 23.9 ± 15.9% in tumor. The concentration-time profile of muscle ECF MTX was parallel and highly correlated (r = 0.97) to that determined in plasma. In contrast, free MTX plasma levels were not correlated with tumor ECF concentrations (r = 0.564). Conclusions. In addition to the well-known pharmacological variability in the concentration-effect relationship, the important inter-individual variability in tumor exposure to MTX may partly explain that studies in patients with solid tumors have often failed to demonstrate firm correlations between MTX blood pharmacokinetics and the chemotherapeutic response.

Extracellular NAADP affords cardioprotection against ischemia and reperfusion injury and involves the P2Y11-like receptor

BACKGROUND AND PURPOSE Extracellular nucleotides may play important regulatory roles within the cardiovascular system and notably in cardioprotection. We aimed to look for a possible pharmacological preconditioning effect of extracellular NAADP ([NAADP]e) against ischemia/reperfusion injury. [NAADP]e has been recently reported to be a full agonist of the P2Y11 receptor. Therefore, we characterized the involvement of the P2Y11-like receptor in mediating ischemic/reperfusion tolerance induced by [NAADP]e. EXPERIMENTAL APPROACH The cardioprotective effects of [NAADP]e were evaluated in a model of ischemia/reperfusion carried out on Langendorff perfused rat hearts. This model was also instrumented with a microdialysis probe. Furthermore, using isolated cardiomyocytes, we assessed cAMP, inositol phosphate accumulation and prosurvival protein kinases activation induced by [NAADP]e pretreatement. RESULTS Pretreatment with 1μM [NAADP]e induced cardioprotective effects with regards to functional recovery, necrosis and arrhythmogenesis (p<0.05). These effects were completely suppressed with NF157, an antagonist of the P2Y11 receptor. Moreover, global ischemia induced a time-dependent increase in interstitial concentration of adenosine, NAADP and UTP. In cardiomyocyte cultures, NF157 suppressed cAMP and inositol phosphate accumulation induced by [NAADP]e. [NAADP]e induced phosphorylation of ERK 1/2, AKT and its downstream target GSK-3β (p<0.05). These activations were also suppressed by NF157. CONCLUSIONS Evidence suggests that NAADP signalling at the P2Y11-like receptor affords significant cardioprotection against ischemia/reperfusion injury. Besides adenosine and UTP, microdialysis study supports a potential endogenous role of [NAADP]e.

Influence of C6 and CNS1 brain tumors on methotrexate pharmacokinetics in plasma and brain tissue

AbstractPurpose: Comparison of the influence of two different brain tumors (C6 and CNS1 glioma) on methotrexate (MTX) disposition in plasma, brain, and tumor tissue extracellular fluid (ECF). Methods: Serial collection of plasma samples and brain ECF dialysates after i.v. bolus administration of MTX (50 mg kg-1) for 4 h. Quantitation of MTX concentrations by HPLC-UV. Results: Histological studies revealed a 3-fold higher number of blood vessels in CNS1 than in C6 tumor tissue. In vivo recoveries (reverse dialysis) were significantly different in tumor tissue (C6: 8.0 ± 3.8%; CNS1: 4.9 ± 2.5%), and in the contralateral hemisphere (C6: 6.0 ± 4.0%; CNS1: 3.9 ± 2.5%) between the two tumors. Area under the concentration–time curve (AUC) in plasma was 30% higher in CNS1 than in C6 due to a lower systemic clearance. Maximum MTX levels in brain tumor ECF were significantly higher in CNS1 than in C6, and decreased faster in CNS1 than in C6 tumor-bearing rats. Penetration in tumor ECF (AUCECF/AUCPlasma ratio) was similar in CNS1 and C6. MTX concentrations in contralateral hemisphere were significantly lower than in tumor tissue and dependent on tumor model. Conclusion: C6 and CNS1 brain tumors have a distinct yet highly variable impact on MTX penetration in brain and brain tumor ECF.

Distribution of Gacyclidine Enantiomers in Spinal Cord Extracellular Fluid

AbstractPurpose. Determination of the pharmacokinetics of gacyclidineenantiomers, a non-competitive NMDA antagonist, in plasma and spinal cordextracellular fluid (ECF) of rats. Methods. Implantation of microdialysis probes in spinal cord (T9).Serial collection of plasma samples and ECF dialysates over 5 hoursafter IV bolus administration of (±)-gacyclidine (2.5 mg/kg). Plasmaprotein binding determined in vivo by equilibrium dialysis. ChiralGC/MS assay. Results. Plasma concentrations of (+)-gacyclidine were ∼25% higherthan those of (−)-gacyclidine over the duration of the experiment inall animals. Plasma concentrations decayed in parallel in a biphasicmanner (t1/2α ∼9 min; t1/2β ∼90 min) with no significant differencebetween enantiomers. Clearance and volume of distribution of(−)-gacyclidine were approximately 20% higher than those of its opticalantipode (CL: 248 vs 197 ml.kg−1.min−1;Vdβ: 31.6 vs 23.5 l/kg).Protein binding (∼90%) was not stereoselective. Both gacyclidineenantiomers were quantifiable in spinal cord ECF 10 min after drugadministration and remained stable over the duration of the experimentin spite of changing blood concentrations. Penetration of(−)-gacyclidine was significantly higher (∼40%) than that of (+)-gacyclidine inall animals. Yet, exposure of spinal cord ECF was similar for bothenantiomers, and not correlated with plasma AUCs. Conclusions. The disposition of gacyclidine enantiomers isstereoselective. Both enantiomers exhibit a high affinity for spinal cord tissueand their distribution may involve a stereoselective and active transportsystem. This hypothesis could also explain the discrepancy betweendrug concentrations in plasma and spinal cord ECF.

Distribution of gacyclidine enantiomers after experimental spinal cord injury in rats: possible involvement of an active transport system.

The pharmacokinetics of gacyclidine enantiomers, a noncompetitive N-methyl-D-aspartate (NMDA) antagonist, were studied in plasma and spinal cord extracellular fluid (ECF) after experimental spinal cord injury in rats. Spinal cord trauma was produced by introducing an inflatable balloon in the dorsal subdural space. Upon implantation of microdialysis probes in spinal cord (T9) and intravenous (iv) bolus administration of (+/-)-gacyclidine (2.5 mg/kg), concentrations in plasma and ECF were monitored over 5 h and analyzed by a stereospecific gas chromatography-mass spectrometry (GC-MS) assay. In plasma, concentrations of (+)-gacyclidine were approximately 25% higher than those of (-)-gacyclidine over the duration of the experiment and decayed in parallel (t(1/2 alpha) approximately 7 min; t(1/2 beta) approximately 90 min) with no significant difference between the two enantiomers. Clearance (CL) and volume of distribution (Vd) of (-)-gacyclidine were approximately 20% higher than those of its optical antipode (CL: 285 versus 236 mL. kg(-1). min(-1); Vd(beta): 39.3 versus 31.2 l/kg). Protein binding (approximately 91%) was not stereoselective. In spinal cord ECF, both enantiomers were quantifiable within 10 min after drug administration, and their concentration remained stable over the duration of the experiment in spite of changing blood concentrations. Repeated iv bolus injections of gacyclidine did not modify these profiles. Areas under the curves (AUCs) of concentration in ECF versus time were similar for both enantiomers and not correlated with AUCs in plasma. Penetration of (-)-gacyclidine was, however, significantly higher (approximately 30%) than that of (+)-gacyclidine. In summary, the disposition of gacyclidine enantiomers is stereoselective. Both enantiomers exhibit a high affinity for spinal cord tissue, and the drug exchange between plasma and spinal cord ECF involves an active transport system. These findings contribute to the explanation of the discrepancy between drug concentrations in plasma and spinal cord ECF.