Deleterious effect of sustained neuroinflammation in pediatric traumatic brain injury.

INTRODUCTION: Despite improved management of traumatic brain injury (TBI), it still leads to lifelong sequelae and disability, particularly in children. Chronic neuroinflammation (the so-called tertiary phase), in particular, microglia/macrophage and astrocyte reactivity, is among the main mechanisms suspected of playing a role in the generation of lesions associated with TBI. The role of acute neuroinflammation is now well understood, but its persistent effect and impact on the brain, particularly during development, are not. Here, we investigated the long-term effects of pediatric TBI on the brain in a mouse model. METHODS: Pediatric TBI was induced in mice on postnatal day (P) 7 by weight-drop trauma. The time course of neuroinflammation and myelination was examined in the TBI mice. They were also assessed by magnetic resonance, functional ultrasound, and behavioral tests at P45. RESULTS: TBI induced robust neuroinflammation, characterized by acute microglia/macrophage and astrocyte reactivity. The long-term consequences of pediatric TBI studied on P45 involved localized scarring astrogliosis, persistent microgliosis associated with a specific transcriptomic signature, and a long-lasting myelination defect consisting of the loss of myelinated axons, a decreased level of myelin binding protein, and severe thinning of the corpus callosum. These results were confirmed by reduced fractional anisotropy, measured by diffusion tensor imaging, and altered inter- and intra-hemispheric connectivity, measured by functional ultrasound imaging. In addition, adolescent mice with pediatric TBI showed persistent social interaction deficits and signs of anxiety and depressive behaviors. CONCLUSIONS: We show that pediatric TBI induces tertiary neuroinflammatory processes associated with white matter lesions and altered behavior. These results support our model as a model for preclinical studies for tertiary lesions following TBI.

Pharmacokinetics and pharmacodynamics of drug‒drug interactions in hospitalized older adults treated with direct oral anticoagulants.

PURPOSE: Polypharmacy is a frequent situation in older adults that increases the risk of drug-drug interactions (DDIs), both pharmacokinetic (PK) and pharmacodynamic (PD). Direct oral anticoagulants (DOACs) are frequently prescribed in older adults, mainly because of the high prevalence of atrial fibrillation (AF). DOACs are subject to cytochrome P450 3A4 (CYP3A4)- and/or P-glycoprotein (P-gp)-mediated PK DDIs and PD DDIs when co-administered with drugs that interfere with platelet function. The aim of our study was to assess the prevalence of DDIs involving DOACs in older adults and the associated risk factors at admission and discharge. METHODS: This was a cross-sectional study conducted in an acute geriatric unit between January 1, 2018 and December 31, 2022, including patients over 75 years of age treated with DOACs at admission and/or discharge, for whom a comprehensive collection of co-medications was performed. RESULTS: From 909 hospitalizations collected, the prevalence of PK DDIs involving DOACs was 16.9% at admission and 20.7% at discharge, and the prevalence of PD DDIs was 20.7% at admission and 20.2% at discharge. Factors associated with DDIs were bleeding history [adjusted odds ratio (ORa) 1.74, 95% confidence interval (CI) 1.13-2.68], number of drugs > 6 (ORa 2.54, 95% CI 1.88-3.46) and reduced dose of DOACs (ORa 0.39, 95% CI 0.28-0.54) at admission and age > 87 years (ORa 0.74, 95% CI 0.55-0.99), number of drugs > 6 (ORa 2.01, 95% CI 1.48-2.72) and reduced dose of DOACs (ORa 0.41, 95% CI 0.30-0.57) at discharge. CONCLUSION: This study provides an indication of the prevalence of DDIs as well as the profile of DDIs and patients treated with DOACs.

Importance of cytochrome 3A4 and 2D6-mediated drug-drug interactions in oxycodone consumption among older adults hospitalized for hip fracture: a cross-sectional study.

Hip fracture is a common injury and represents a major health problem with an increasing incidence. In older adults, opioids such as oxycodone are often preferred to other analgesics such as tramadol because of a lower risk of delirium. Different parameters, such as inhibition of cytochrome P450 (CYP450) 2D6 and/or 3A4, can potentially lead to pharmacokinetic variations of oxycodone representing a risk of adverse drugs effects or lack of drug response. There is a risk of drug-drug interactions involving CYP450 in older adults due to the high prevalence of polypharmacy. This study sought to identify patient characteristics that influence oxycodone administration. A single-center observational study included 355 patients with a hip fracture hospitalized in a geriatric postoperative unit. Composite endpoint based on form, duration, and timing to intake separated patients into three groups: "no oxycodone", "low oxycodone ", and "high oxycodone ". CYP450 interactions were studied based on a composite variable defining the most involved CYP450 pathways between CYP2D6 and CYP3A4. CYP450 interactions with CYP2D6 pathway involved were associated with the risk of "high oxycodone" [odds ratio adjusted on age and the type of hip fracture (OR*) 4.52, 95% confidence interval (CI) 1.39-16.83, p = 0.02)], as well as serum albumin levels (OR* 1.09, 95% CI 1.02-1.17, p = 0.01). Cognitive impairment was negatively associated with the risk of "high oxycodone" (OR* 0.38, 95% CI 0.18-0.77, p = 0.02). This study showed an association between CYP2D6 interactions and higher oxycodone consumption indirectly reflecting the existence of uncontrolled postoperative pain.

Overcoming matrix effects in quantitative liquid chromatography-mass spectrometry analysis of steroid hormones in surface waters.

RATIONALE: Accurate and reliable measurements are mandatory in the field of environmental monitoring. Matrix effects are often depicted as the Achilles' heel of liquid chromatography-mass spectrometry analysis since they may be prejudicial for analytical performances such as detection capability and accuracy, if not documented or compensated. Here a methodology for the evaluation and compensation of matrix effects is described. METHODS: Natural and synthetic representative water samples were used for the evaluation of matrix effects with the post-extraction addition technique. Samples were analysed using ultra-performance liquid chromatography separation coupled to tandem mass spectrometry and electrospray ionization. Isotopic dilution was investigated as a way to allow compensation of signal alteration and therefore satisfactory quantification. When this approach was not possible, a methodology was conducted for choosing the most appropriate internal standard. RESULTS: The matrix effects were dependent on both matrix composition and nature of analyte. They ranged from total signal suppression to signal enhancement of +27% but were independent of compound concentration. The correction of matrix effects by internal standards was satisfactory, particularly for compounds benefiting from isotope dilution leading to acceptable quantification performances. CONCLUSIONS: Even if no exhaustive or agreed criteria exist for the final interpretation of matrix effects, this study highlights the interest in isotope dilution for reducing their inherent prejudicial effects in quantification and the need to conduct this type of study for representative matrices. Moreover, a methodological approach is proposed for choosing the most appropriate available internal standard when isotope dilution is not possible.

Bisphenol A Impairs Lipid Remodeling Accompanying Cell Differentiation in the Oligodendroglial Cell Line Oli-Neu.

In the central nervous system, the process of myelination involves oligodendrocytes that wrap myelin around axons. Myelin sheaths are mainly composed of lipids and ensure efficient conduction of action potentials. Oligodendrocyte differentiation is an essential preliminary step to myelination which, in turn, is a key event of neurodevelopment. Bisphenol A (BPA), a ubiquitous endocrine disruptor, is suspected to disrupt this developmental process and may, thus, contribute to several neurodevelopmental disorders. In this study, we assessed the effect of BPA on oligodendrocyte differentiation through a comprehensive analysis of cell lipidome by UHPLC-HRMS. For this purpose, we exposed the oligodendroglial cell line Oli-neu to several BPA concentrations for 72 h of proliferation and another 72 h of differentiation. In unexposed cells, significant changes occurred in lipid distribution during Oli-neu differentiation, including an increase in characteristic myelin lipids, sulfatides, and ethanolamine plasmalogens, and a marked remodeling of phospholipid subclasses and fatty acid contents. Moreover, BPA induced a decrease in sulfatide and phosphatidylinositol plasmalogen contents and modified monounsaturated/polyunsaturated fatty acid relative contents in phospholipids. These effects counteracted the lipid remodeling accompanying differentiation and were confirmed by gene expression changes. Altogether, our results suggest that BPA disrupts lipid remodeling accompanying early oligodendrocyte differentiation.

Deepening of lipidome annotation by associating cross-metathesis reaction with mass spectrometry: application to an in vitro model of corneal toxicity.

The in-depth knowledge of lipid biological functions needs a comprehensive structural annotation including a method to locate fatty acid unsaturations, which remains a thorny problem. For this purpose, we have associated Grubbs' cross-metathesis reaction and liquid chromatography hyphenated to tandem mass spectrometry to locate double bond positions in lipid species. The pretreatment of lipid-containing samples by Grubbs' catalyst and an appropriate alkene generates substituted lipids through cross-metathesis reaction under mild, chemoselective, and reproducible conditions. A systematic LC-MS/MS analysis of the reaction mixture allows locating unambiguously the double bonds in fatty acid side chains of phospholipids, glycerolipids, and sphingolipids. This method has been successfully applied at a nanomole scale to commercial standard mixtures consisting of 10 lipid subclasses as well as in lipid extracts of human corneal epithelial (HCE) cell line allowing to pinpoint double bond of more than 90 species. This method has also been useful to investigate the lipid homeostasis alteration in an in vitro model of corneal toxicity, i.e., HCE cells incubated with benzalkonium chloride. The association of cross-metathesis and tandem mass spectrometry appears suitable to locate double bond positions in lipids involved in relevant biological processes.

A comprehensive population-based study comparing the phenotype and genotype in a pretherapeutic screen of dihydropyrimidine dehydrogenase deficiency.

BACKGROUND: Pretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended or required prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD-deficient patients remains elusive. METHODS: Among a nationwide cohort of 5886 phenotyped patients with cancer who were screened for DPD deficiency over a 3 years period, we assessed the characteristics of both DPD phenotypes and DPYD genotypes in a subgroup of 3680 patients who had completed the two tests. The extent to which defective allelic variants of DPYD predict DPD activity as estimated by the plasma concentrations of uracil [U] and its product dihydrouracil [UH2] was evaluated. RESULTS: When [U] was used to monitor DPD activity, 6.8% of the patients were classified as having DPD deficiency ([U] > 16 ng/ml), while the [UH2]:[U] ratio identified 11.5% of the patients as having DPD deficiency (UH2]:[U] < 10). [U] classified two patients (0.05%) with complete DPD deficiency (> 150 ng/ml), and [UH2]:[U] < 1 identified three patients (0.08%) with a complete DPD deficiency. A defective DPYD variant was present in 4.5% of the patients, and two patients (0.05%) carrying 2 defective variants of DPYD were predicted to have low metabolism. The mutation status of DPYD displayed a very low positive predictive value in identifying individuals with DPD deficiency, although a higher predictive value was observed when [UH2]:[U] was used to measure DPD activity. Whole exon sequencing of the DPYD gene in 111 patients with DPD deficiency and a "wild-type" genotype (based on the four most common variants) identified seven heterozygous carriers of a defective allelic variant. CONCLUSIONS: Frequent genetic DPYD variants have low performances in predicting partial DPD deficiency when evaluated by [U] alone, and [UH2]:[U] might better reflect the impact of genetic variants on DPD activity. A clinical trial comparing toxicity rates after dose adjustment according to the results of genotyping or phenotyping testing to detect DPD deficiency will provide critical information on the best strategy to identify DPD deficiency.

Cytotoxic and Anti-Inflammatory Effects of Ent-Kaurane Derivatives Isolated from the Alpine Plant Sideritis hyssopifolia.

This paper reports the isolation and structural characterization of four new ent-kaurane derivatives from the Lamiaceae plant Sideritis hyssopifolia. Planar structures and relative configurations were determined using both mass spectrometry and nuclear magnetic resonance (1D and 2D). Absolute configurations were determined by comparing experimental and theoretical electronic circular dichroism spectra. The cytotoxic and microbial activities of all new compounds were tested. Compounds that were non-cytotoxic were further evaluated for anti-inflammatory activity.

Design, synthesis and biological evaluation of novel 1,2,3-triazole linked coumarinopyrazole conjugates as potent anticholinesterase, anti-5-lipoxygenase, anti-tyrosinase and anti-cancer agents.

A series of new 1,2,3-triazole linked coumarinopyrazole conjugates 4a-e and 5a-e have been synthesized via the Copper(I)-catalysed Alkyne-Azide Cycloaddition (CuAAC). Going through the reaction of compound 2 with the 3-propargyl bromide gave a mixture of propargylated regioisomers 3 + 3' used as a dipolarophile to access to triazoles 4a-e and 5a-e. The structures of the prepared cycloadducts were determined by 1H, 13C and 2D-NMR techniques and by HRMS analysis. All the synthesized derivatives have been evaluated for their anticholinesterase, anti-5-lipoxygenase, anti-tyrosinase, and cytotoxic activities.

MS/MS-Guided Isolation of Clarinoside, a New Anti-Inflammatory Pentalogin Derivative.

Re-investigation of the chemical composition of the annual plant Mitracarpus scaber Zucc. led to the identification of clarinoside, a new pentalogin derivative containing a rare quinovose moiety, and the known compound harounoside. While the planar structure was fully determined using tandem mass spectrometry (MS) and quantum mechanics (QM) calculations, the tridimensional structure was unravelled after isolation and NMR analysis. The absolute configuration was assigned by comparison of experimental and theoretical synchrotron radiation circular dichroism spectra. Both compounds were tested for anti-inflammatory activity, and compound 1 showed the ability to inhibit the production of interleukin-8 (Il-8) with an IC 50 value of 9.17 µ M.

Cholesterol 24-hydroxylase defect is implicated in memory impairments associated with Alzheimer-like Tau pathology.

Alzheimer's disease (AD) is characterized by both amyloid and Tau pathologies. The amyloid component and altered cholesterol metabolism are closely linked, but the relationship between Tau pathology and cholesterol is currently unclear. Brain cholesterol is synthesized in situ and cannot cross the blood-brain barrier: to be exported from the central nervous system into the blood circuit, excess cholesterol must be converted to 24S-hydroxycholesterol by the cholesterol 24-hydroxylase encoded by the CYP46A1 gene. In AD patients, the concentration of 24S-hydroxycholesterol in the plasma and the cerebrospinal fluid are lower than in healthy controls. The THY-Tau22 mouse is a model of AD-like Tau pathology without amyloid pathology. We used this model to investigate the potential association between Tau pathology and CYP46A1 modulation. The amounts of CYP46A1 and 24S-hydroxycholesterol in the hippocampus were lower in THY-Tau22 than control mice. We used an adeno-associated virus (AAV) gene transfer strategy to increase CYP46A1 expression in order to investigate the consequences on THY-Tau22 mouse phenotype. Injection of the AAV-CYP46A1 vector into the hippocampus of THY-Tau22 mice led to CYP46A1 and 24S-hydroxycholesterol content normalization. The cognitive deficits, impaired long-term depression and spine defects that characterize the THY-Tau22 model were completely rescued, whereas Tau hyperphosphorylation and associated gliosis were unaffected. These results argue for a causal link between CYP46A1 protein content and memory impairments that result from Tau pathology. Therefore, CYP46A1 may be a relevant therapeutic target for Tauopathies and especially for AD.

CYP46A1 inhibition, brain cholesterol accumulation and neurodegeneration pave the way for Alzheimer's disease.

Abnormalities in neuronal cholesterol homeostasis have been suspected or observed in several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and Huntington's disease. However, it has not been demonstrated whether an increased abundance of cholesterol in neurons in vivo contributes to neurodegeneration. To address this issue, we used RNA interference methodology to inhibit the expression of cholesterol 24-hydroxylase, encoded by the Cyp46a1 gene, in the hippocampus of normal mice. Cholesterol 24-hydroxylase controls cholesterol efflux from the brain and thereby plays a major role in regulating brain cholesterol homeostasis. We used an adeno-associated virus vector encoding short hairpin RNA directed against the mouse Cyp46a1 mRNA to decrease the expression of the Cyp46a1 gene in hippocampal neurons of normal mice. This increased the cholesterol concentration in neurons, followed by cognitive deficits and hippocampal atrophy due to apoptotic neuronal death. Prior to neuronal death, the recruitment of the amyloid protein precursor to lipid rafts was enhanced leading to the production of ß-C-terminal fragment and amyloid-ß peptides. Abnormal phosphorylation of tau and endoplasmic reticulum stress were also observed. In the APP23 mouse model of Alzheimer's disease, the abundance of amyloid-ß peptides increased following inhibition of Cyp46a1 expression, and neuronal death was more widespread than in normal mice. Altogether, these results suggest that increased amounts of neuronal cholesterol within the brain may contribute to inducing and/or aggravating Alzheimer's disease.

Mass spectrometry imaging of biological tissue: an approach for multicenter studies.

Mass spectrometry imaging has become a popular tool for probing the chemical complexity of biological surfaces. This led to the development of a wide range of instrumentation and preparation protocols. It is thus desirable to evaluate and compare the data output from different methodologies and mass spectrometers. Here, we present an approach for the comparison of mass spectrometry imaging data from different laboratories (often referred to as multicenter studies). This is exemplified by the analysis of mouse brain sections in five laboratories in Europe and the USA. The instrumentation includes matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF), MALDI-QTOF, MALDI-Fourier transform ion cyclotron resonance (FTICR), atmospheric-pressure (AP)-MALDI-Orbitrap, and cluster TOF-secondary ion mass spectrometry (SIMS). Experimental parameters such as measurement speed, imaging bin width, and mass spectrometric parameters are discussed. All datasets were converted to the standard data format imzML and displayed in a common open-source software with identical parameters for visualization, which facilitates direct comparison of MS images. The imzML conversion also allowed exchange of fully functional MS imaging datasets between the different laboratories. The experiments ranged from overview measurements of the full mouse brain to detailed analysis of smaller features (depending on spatial resolution settings), but common histological features such as the corpus callosum were visible in all measurements. High spatial resolution measurements of AP-MALDI-Orbitrap and TOF-SIMS showed comparable structures in the low-micrometer range. We discuss general considerations for planning and performing multicenter studies in mass spectrometry imaging. This includes details on the selection, distribution, and preparation of tissue samples as well as on data handling. Such multicenter studies in combination with ongoing activities for reporting guidelines, a common data format (imzML) and a public data repository can contribute to more reliability and transparency of MS imaging studies.

Ceramides and sphingomyelinases in senile plaques.

The senile plaque is a hallmark lesion of Alzheimer disease (AD). We compared, without a priori, the lipidome of the senile plaques and of the adjacent plaque-free neuropil. The analysis by liquid chromatography coupled with electrospray ionization mass spectrometry revealed that laser microdissected senile plaques were enriched in saturated ceramides Cer(d18:1/18:0) and Cer(d18:1/20:0) by 33 and 78% respectively with respect to the surrounding neuropil. This accumulation of ceramides was not explained by their affinity for Aß deposits: no interaction between ceramide-liposomes and Aß fibrils was observed in vitro by surface plasmon resonance and fluorescent ceramide-liposomes showed no affinity for the senile plaques in AD brain tissue. Accumulation of ceramides could be, at least partially, the result of a local production by acid and neutral sphingomyelinases that we found to be present in the corona of the senile plaques.

Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples.

Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost.

Are empty methadone bottles empty? An analytic study.

BACKGROUND: Methadone maintenance treatment is the most widely prescribed treatment for opiate dependence with proven benefits for patients. In naïve users or in case of recreational misuse, methadone can be a source of potentially lethal intoxications, resulting in fatal overdoses. A few cases of infantile intoxications have been described in the literature, some of which resulted in death. Nowadays, more than 50,000 bottles are used every day in France, most of which are thrown away in the bin. Relatives at home, especially children, can have access to these empty bottles. This study aims to determine whether the residual quantity of methadone in the bottles is associated with a risk of intoxication for someone who has a low tolerance to opiates, such as a child. METHODS: The methadone dosage left in a sample of 175 bottles recapped after use by the patients taking their maintenance treatment in an addiction treatment program centre was analysed during a 2-week period in March 2013. RESULTS: The mean residual quantity of methadone left in each bottle after use is 1.9 ± 1.8 mg and 3.3 ± 2.4 mg in the sample of 60 mg bottles. CONCLUSIONS: There is a potential danger of accidental overdose with empty bottles of methadone syrup, especially for children. To take into account this hazard, several harm reduction strategies can be proposed, such as favouring the taking of the treatment within the delivery centres rather than the 'take home' doses, asking methadone users to bring back their used bottles, and raising patients' awareness of the intoxication risks and the necessary everyday precautions. For stable patients with take home methadone, the use of capsules could be considered.

Prevalence and factors associated with inappropriate dosing of apixaban and rivaroxaban in hospitalized older adults with atrial fibrillation: a cross-sectional study.

INTRODUCTION: Atrial fibrillation (AF) is a common condition among older adults, requiring anticoagulation therapy to prevent thromboembolic events. Direct oral anticoagulants (DOACs) are now recommended as first-line therapy for this purpose. Apixaban and rivaroxaban are two direct-factor Xa inhibitors whose dosing is based on various factors (age, weight, creatinine, and creatinine clearance) that can affect the pharmacokinetics of the medication. This study aimed to evaluate factors associated with inappropriate dosing of apixaban or rivaroxaban based on the summary of product characteristics. METHODS: A retrospective, single-center study included 777 hospitalizations of patients treated with apixaban or rivaroxaban for AF between 1 January 2018 and 31 December 2022. Primary endpoint assessed whether the dose of apixaban or rivaroxaban was within the summary of product characteristics used by European Medicine Agency (EMA). RESULTS: Inappropriate dosing of apixaban or rivaroxaban is noted for approximately 30% of hospitalizations mostly underdosing. Factors associated with the risk of inappropriate dosing were the presence of cognitive impairment [adjusted odds ratio (OR*) 1.65, 95% confidence interval (CI) 1.19-2.29, p value (p) = 0.002], weight per kilogram increase (OR* 1.03, 95% CI 1.01-1.04, p < 0.0001), and history of bleeding under apixaban or rivaroxaban (OR* 1.94, 95% CI 1.24-3.03, p = 0.003). CONCLUSION: This study highlighted the high prevalence of inappropriate apixaban or rivaroxaban doses in older adults, particularly underdosing, which increases the risk of thromboembolism.

[P-glycoprotein activity in vivo in older adults: physiological, pathophysiological and pharmacokinetic interplay at the blood-brain barrier]. / Activité de la p-glycoprotéine in vivo chez la personne âgée : intrications physiologique, physiopathologique et pharmacocinétique au niveau de la barrière hémato-encéphalique.

p-glycoprotein (P-gp) is an efflux transporter of xenobiotic and endogenous compounds across the blood-brain barrier (BBB). P-gp plays an essential role by limiting passage of these compounds into the brain tissue. It is susceptible to drug-drug interactions when interactors drugs are co-administrated. The efficiency of P-gp may be affected by the aging process and the development of neurodegenerative diseases. Studying this protein in older adults is therefore highly relevant for all these reasons. Understanding P-gp activity in vivo is essential when considering the physiological, pathophysiological, and pharmacokinetic perspectives, as these aspects seem to be interconnected to some extent. In vivo exploration in humans is based on neuroimaging techniques, which have been improving over the last years. The advancement of exploration and diagnostic tools is opening up new prospects for understanding P-gp activity at the BBB.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging reveals cholesterol overload in the cerebral cortex of Alzheimer disease patients.

Although cholesterol has been involved in the pathophysiology of Alzheimer disease (AD), its distribution in the cerebral cortex over the course of AD is unknown. We describe an original method to quantify cholesterol distribution using time-of-flight secondary ion mass spectrometry imaging. Cholesterol was unevenly distributed along the cortical thickness, being more abundant close to the white matter, in both control and AD cases. However, the mean cholesterol signal was significantly higher in the lower half of the cortex in AD samples compared to controls. This increase, when converted into cortical layers, was statistically significant for layers III and IV and did not reach significance in layers V + VI, the variability being too high at the interface between grey and white matter. The density of neurofibrillary tangles and of senile plaques was not statistically linked to the abundance of cholesterol. Cholesterol overload thus appears a new and independent alteration of AD cerebral cortex. The structure in which cholesterol accumulates and the mechanism of this accumulation remain to be elucidated.

Localisation and quantification of benzalkonium chloride in eye tissue by TOF-SIMS imaging and liquid chromatography mass spectrometry.

Benzalkonium (BAK) chloride is the most commonly used preservative in eye drops. It is generally composed of benzyldimethyldodecylammonium C12 and benzyldimethyltetradecylammonium C14 and is supposed to increase penetration of active compounds. However, numerous studies have reported its toxic effect to ocular surface especially in long-term treatments like against glaucoma, a sight-threatening disease. Albino rabbits were treated with a hyperosmolar solution and a high concentration of BAK solution for 1 month. Enucleated eyes were cryo-sectioned and analysed by mass spectrometry. Mass spectrometry imaging using time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to characterize the spatial distribution and to determine the relative quantity of BAK at the surface of rabbit eye sections. Liquid chromatography coupled with mass spectrometry (LC-MS) using a hybrid linear ion trap-Orbitrap® mass spectrometer was used to obtain relative quantification of BAK at the sample surface. TOF-SIMS images of BAK ions indicated a distribution at the ocular surface and in deeper structures. Didecyldimethylammonium (DDMAC), which is used in hospitals as a substitute for BAK, was also detected and showed an accumulation around the eyes. After extraction with acetonitrile and chromatographic separation using a Gemini C18 column and an original elution gradient, the relative quantities of BAK and DDMAC present in the whole eye section surface were determined. This LC-MS method was validated in terms of limits of quantification, linearity, repeatability and reproducibility and its feasibility was evaluated in surgically obtained human samples. Specimens of iris, lens capsule or trabecular meshwork were found with significant levels of BAK and DDMAC, thus confirming the penetration of BAK in deep ocular structures, with potential deleterious effects induced by this cytotoxic compound. The analytical method developed here could therefore be of primary interest in the field of pharmaco-toxicology in order to localise, identify and quantify drugs or xenobiotic compounds present at biological sample surfaces.