Qualitative and quantitative assessment of cartilage degeneration using full-field optical coherence tomography ex vivo.

OBJECTIVE: The purpose of this study was to investigate the ability of full-field optical coherence tomography (FFOCT) to qualitatively and quantitatively evaluate cartilage degeneration using the qualitative evaluation of histology sections as the reference. DESIGN: Thirty-three human knee cartilage samples of variable degeneration were included in the study. A closely matching histology and FFOCT image was acquired for each sample. The cartilage degeneration was qualitatively evaluated by assigning a grade to each histology and FFOCT image. The relevance of the performed grading was assessed by calculating the intra- and inter-observer reproducibility and calculating the concordance between the histology and FFOCT grades. A near-automatic algorithm was developed to quantitatively characterize the cartilage surface in each image. The correlation between the quantitative results and the reference qualitative histology was calculated. RESULTS: An almost perfect agreement was achieved for both the intra- and inter-reproducibility of the histology and FFOCT qualitative grading (κ ≥ 0.91). A high and statistically significant level of agreement was measured between the histology and FFOCT grades (W = 0.95, P < 0.05). Strong and statistically significant correlations were measured between the quantitative results and the reference qualitative histology grades (ρ ≥ 0.75, P < 0.05). CONCLUSIONS: We have demonstrated that FFOCT is an alternative approach to conventional optical coherence tomography (OCT) that is as well adapted for the qualitative and quantitative assessment of human cartilage as the reference gold standard - histology. This study constitutes the first promising results towards developing a new diagnostic tool in the field of osteoarthritis.

Acute and long-term transcriptional responses in sulfur mustard-exposed SKH-1 hairless mouse skin.

Sulfur mustard (HD) ranks among the alkylating chemical warfare agents. Skin contact with HD produces an inflammatory response that evolves into separation at the epidermal-dermal junction conducting to blistering and epidermis necrosis. Up to now, current treatment strategies of HD burns have solely consisted in symptomatic management of skin damage. Therapeutic efficacy studies are still being conducted; classically using appropriate animal skin toxicity models. In order to substantiate the use of SKH-1 hairless mouse as an appropriate model for HD-induced skin lesions, we investigate the time-dependent quantitative gene expression of various selected transcripts associated to the dorsal skin exposure to HD saturated vapors. Using quantitative real time polymerase chain reaction (RT-qPCR), the expression of interleukins (IL-1ß and IL-6), tumor necrosis factor (TNF)-α, macrophage inflammatory proteins (MIP)-2α (also called Cxcl2) and MIP-1αR (also called Ccr1), matrix metalloproteases (MMP-9 and MMP-2), laminin γ2 monomer (Lamc2) and keratin (K)1 was determined up to 21 days after HD challenge in order to allow enough time for wound repair to begin. Specific transcript RT-qPCR analysis demonstrated that IL-6, IL-1ß, Ccr1, Cxcl2 mRNA levels increased as early as 6 h in HD-exposed skins and remained up-regulated over a 14-day period. Topical application of HD also significantly up-regulated MMP-9, TNF-α, and Lamc2 expression at specific time points. In contrast, MMP-2 mRNA levels remained unaffected by HD over the time-period considered, whereas that long-term study revealed that K1 mRNA level significantly increased only 21 days after HD challenge. Our study hereby provides first-hand evidence to substantiate a long period variation expression in the inflammatory cytokine, MMPs and structural components following cutaneous HD exposure in hairless mouse SKH-1. Our data credit the use of SKH-1 for investigating mechanisms of HD-induced skin toxicity and for the development of pharmacological countermeasures.

Percutaneous penetration and distribution of VX using in vitro pig or human excised skin validation of demeton-S-methyl as adequate simulant for VX skin permeation investigations.

The organophosphorus (OP) chemical warfare V agent O-ethyl-S-[2(di-isopropylamino)ethyl] methyl phosphonothioate (VX), is a highly toxic compound which mainly penetrates the body via percutaneous pathways. Hence, the following prerequisite: to ascertain compound absorption and percutaneous profile distribution with a view to further assessing the efficacy of topical skin protectants. We first selected the most appropriate receptor fluid to carry out in vitro VX absorption experiments, namely: Hanks's Balanced Salt Solution (HBSS). After a 24-h topical exposure time lapse, we measured altogether the percentage of applied dose unabsorbed and absorbed, penetration rate, lag time, permeability coefficient (K(p)), and dose of VXeq present in skin. To such an end, we used full-thickness and split-thickness pig-ear or human abdominal skin membranes. Further, we scrutinised the potential use of two specific molecules as suitable surrogates for VX percutaneous penetration analyses: thus, we compared the present VX toxicokinetic parameters to earlier findings from our research unit, with respect to OP insecticides demethon-S-methyl (DSM) and paraoxon (POX). Within the framework of our study, we wish to highlight the following evidence: (a) pig-ear skin proves a relevant model to predict in vitro human abdominal skin, taking into account a 2-fold higher skin permeability to VXeq; (b) both full or split-thickness skin membranes may be used indiscriminately to gauge penetration rate and absorbed dose; (c) DSM applied on full-thickness pig-ear skin is the most relevant model to mimic the in vitro VX absorption through full-thickness skin model.

In vitro percutaneous penetration of organophosphorus compounds using full-thickness and split-thickness pig and human skin.

Organophosphorus compounds (OPs), such as pesticides and chemical warfare agents like sarin (GB), soman (GD) and VX, are highly toxic compounds. The OP vapours and their liquid forms are readily absorbed through the skin, therefore, protecting the skin of people who are potentially exposed to these agents is crucial. The development of effective countermeasures relies on a better knowledge of the percutaneous penetration of such molecules. The purpose of this present study is to determine the in vitro percutaneous penetration parameters of two pesticides DSM and DFP, as potential simulants of V and G agents, respectively, using four in vitro systems: full-thickness and split-thickness human abdominal and pig-ear skin membranes mounted on static diffusion cells. Based on the toxicokinetic parameters of the percutaneous penetration of DSM and DFP, we demonstrated that (a) pig-ear skin is a relevant model to predict the in vitro human skin permeability taking into account a 2-fold difference between these two species (b) both full and split-thickness skin membranes could be used indiscriminately, (c) DSM and DFP would be appropriate surrogates for V and G agents to perform skin permeation studies.

[Topical enzyme-based formulations]. / Les formulations topiques à base d'enzymes.

Research studies aimed at improving the enzyme catalytic efficiency and operational stability are considered as a recurrent challenge with topical enzyme-based formulations. A large number of plant and bacterial proteolytic enzymes have been studied as an early substitutive replacement for the mechanical debridement of partial-thickness burns. In addition, progress in protein engineering and vectorisation of drugs provide improvements in the approach of topical and photoprotective enzyme therapy.

Cutaneous challenge with chemical warfare agents in the SKH-1 hairless mouse. (I) Development of a model for screening studies in skin decontamination and protection.

Exposure to lethal chemical warfare agents (CWAs) is no longer only a military issue due to the terrorist threat. Among the CWAs of concern are the organophosphorus nerve agent O-ethyl-S-(2[di-isopropylamino]ethyl)methyl-phosphonothioate (VX) and the vesicant sulfur mustard (SM). Although efficient means of decontamination are available, most of them lose their efficacy when decontamination is delayed after exposure of the bare skin. Alternatively, CWA skin penetration can be prevented by topical skin protectants. Active research in skin protection and decontamination is thus paramount. In vivo screening of decontaminants or skin protectants is usually time consuming and may be expensive depending on the animal species used. We were thus looking for a suitable, scientifically sound and cost-effective model, which is easy to handle. The euthymic hairless mouse Crl: SKH-1 (hr/hr) BR is widely used in some skin studies and has previously been described to be suitable for some experiments involving SM or SM analogs. To evaluate the response of this species, we studied the consequences of exposing male anaesthetized SKH-1 mice to either liquid VX or to SM, the latter being used in liquid form or as saturated vapours. Long-term effects of SM burn were also evaluated. The model was then used in the companion paper (Taysse et al.(1)).

[Methods and models for percutaneous absorption studies of organophosphates]. / Méthodes et modèles d'étude de l'absorption percutanée des composés organophosphorés.

Organophosphates are highly toxic compounds, mainly penetrating the organism through the percutaneous route. In this context, having topical skin protectant capable of limitating their percutaneous penetration is of crucial importance for exposed people. Knowledge of the toxicokinetic parameters corresponding to the percutaneous penetration of these compounds is a key step for the development of these protective formulations. The different experimental approaches, in vivo and ex vivo, used to predict percutaneous penetration of these toxics are presented. The relevance and reliability of cutaneous experimental models are analysed.

Percutaneous penetration and absorption of parathion using human and pig skin models in vitro and human skin grafted onto nude mouse skin model in vivo.

This study determined and compared the percutaneous penetration and absorption of an organophosphorus (OP) pesticide, parathion (PA), using three experimental skin models: namely the human abdominal- and pig-ear skin in vitro models and the Human Skin grafted onto a nude mouse (HuSki) in vivo model. The percentage of topically applied dose absorbed and the doses present in the stratum corneum and skin were systematically determined at 24 h under similar experimental conditions. The three experimental skin models were first compared. Then, the advantages of the HuSki model for in vivo PA skin absorption studies were evaluated compared with the pig in vivo model previously used by others. Lastly, the relevance of each skin model to predict the permeability of human skin to PA in vivo was assessed by comparing our results with previously published in vivo human volunteer values. It was demonstrated that (a) pig-ear skin is relevant for predicting the in vitro human abdominal skin absorption taking into account a 2-3 times higher skin permeability to PA, (b) using ethanol as the vehicle, the absorption of PA was 4-5 times higher in the HuSki model than in the pig model but supports the usefulness of the HuSki model to easy mass balance studies, (c) both human in vitro and HuSki models closely predict the in vivo human volunteer absorption at 24 h when acetone is used as a vehicle but the HuSki model overcomes the known limitations of in vitro models for studying the fate of PA in the different skin layers after topical application.

In vivo and in vitro percutaneous absorption of [(14)C]di-N-butylphthalate in rat.

This study evaluated the toxicokinetics of [(14)C]di-n-butylphthalate ([(14)C]DBP) after an intravenous administration (1 and 10 mg/kg, in Cremophor) or a topical application (10 microl/cm(2); 10 cm(2), neat) in haired male Sprague-Dawley rats. Additional in vivo and in vitro percutaneous penetration studies of [(14)C]DBP were conducted on male and female haired rats and male hairless rats. After intravenous administration, unchanged DBP disappeared rapidly from the plasma, following a two-exponential function (T1/2beta = 5-7 min). The peak levels of monobutylphthalate (MBP) and its glucuronide conjugate (MBP-Gluc) occurred 1 to 2 and 20 to 30 min after administration, respectively. These metabolites were intensively and rapidly excreted in urine (57% of the dose). However, about 35% of the dose recovered in urine was primarily excreted in bile (mainly as MBP-Gluc) and underwent hepatobiliary recycling. Unchanged DBP was barely detectable in excreta. DBP rapidly penetrated the skin, which constituted a reservoir. The absorption flux determined for 0.5 to 8 and 8 to 48 h of exposure were 43 and 156 microg/cm(2)/h, respectively. The higher flux may be due to radial diffusion of DBP in the stratum and/or epidermis. The in vivo and in vitro experiments revealed that DBP was intensively metabolized into the skin. In vivo percutaneous absorption flux was very similar in male and female haired rats. In contrast, the percutaneous absorption determined in vivo and in vitro was higher in hairless than in haired male rats. Absorption flux was accurately estimated from urinary excretion rate of MBP or MBP-Gluc.