Littérature scientifique sur le sujet « Laser-assisted microdissection »

Introduction Dysphonia is caused by voice misuse and various environmental factors. It is manifested as varied pathological lesions of the vocal cords. Surgical excision of these lesions is mainly by conventional cold steel or laser assisted microsurgical techniques. Both modalities have seen extensive advancements and refinement in technologies in the recent past. In this study we have compared the microdissection microlaryngeal treatment and microspot superpulsed beam carbon dioxide (CO2) laser assisted surgical techniques for the management of benign and precancerous lesions of the vocal cords. Material and Methods A total of 36 cases of benign lesions of vocal cord, were divided randomly to undergo surgery either by microdissection or CO2laser assisted techniques. The groups were assessed through vocal cord morphological observation and subjective voice assessment parameters GRBAS score and VHI10 index. Results Peroperative bleeding was observed to be significantly reduced in the laser excision group. However, operating time was significantly increased in this group. Patients recovered remarkably well following both the techniques as denoted by voice parameters. No difference was observed in duration of hospital stay. Conclusion Both surgical techniques give satisfactory results in their management of benign and precancerous lesions of the vocal cords. Both techniques have their advantages and disadvantages. After a learning curve, laser surgery with its precision and cleaner surgical fields will be more effective in the management of such cases.

For focal events such as myocardial infarction, it is important to dissect infarction-induced biological responses as a function of space with respect to the infarct core. Laser microdissection pressure catapulting (LMPC) represents a recent variant of laser capture microdissection that enables robot-assisted rapid capture of catapulted tissue without direct user contact. This work represents the maiden effort to apply laser capture microdissection to study spatially resolved biological responses in myocardial infarction. Infarcted areas of the surviving ischemic-reperfused murine heart were identified using a standardized hematoxylin QS staining procedure. Standard staining techniques fail to preserve tissue RNA. Exposure of the tissue to an aqueous medium (typically used during standard immunohistochemical staining), with or without RNase inhibitors, resulted in a rapid degradation of genes, with ∼80% loss in the 1st h. Tissue elements (1 × 104–4 × 106 μm2) captured from infarcted and noninfarcted sites with micrometer-level surgical precision were collected in a chaotropic RNA lysis solution. Isolated RNA was analyzed for quality by microfluidics technology and reverse transcribed to generate high-quality cDNA. Real-time PCR analysis of the cDNA showed marked (200- and 400-fold, respectively) induction of collagen Ia and IIIa at the infarcted site compared with the noninfarcted site. This work reports a sophisticated yet rapid approach to measurement of relative gene expressions from tissue elements captured from spatially resolved microscopic regions in the heart with micrometer-level precision.

Le vieillissement de la population fait naître des problématiques de santé publique telles que l’augmentation du nombre de fractures dues à la fragilisation des os et la nécessité d’adapter les traitements. Aujourd’hui, un certain nombre de stratégies sont adoptées pour prévenir ou ralentir la perte de masse osseuse et pour traiter les fractures. Elles présentent toutes des limitations qui poussent la recherche vers l’identification de nouvelles cibles de traitements. Les ostéocytes représentent 95 % des cellules de l’os et vivent plusieurs dizaines d’années à l’intérieur de la matrice minéralisée. Ils ont une forme caractéristique avec des prolongements partant du corps cellulaire vers les autres ostéocytes, les cellules à la surface de l’os et les vaisseaux sanguins. Pendant longtemps, ils ont été considérés comme passifs parce qu’emmurés au cœur de la matrice. Cependant, le développement d’outils in vitro et in vivo a permis d’identifier leur rôle central dans le maintien de la masse osseuse. En effet, les ostéocytes sont les cellules de l’os les plus sensibles aux variations de sollicitations mécaniques provoquées par l’exercice physique ou l’immobilisation. En réponse, ils envoient des signaux aux ostéoblastes et ostéoclastes pour renforcer la matrice ou l’éliminer. Le vieillissement provoque des changements métaboliques et hormonaux systémiques qui affectent le réseau ostéocytaire. Cependant, comme il est aujourd’hui encore difficile d’étudier les ostéocytes dans leur environnement d’origine, beaucoup reste à explorer. Notamment, leur rapport au réseau vasculaire qui subit également des changements lors du vieillissement ainsi que l’impact de ce dernier sur leur métabolisme énergétique. De plus, ils ont été très peu considérés en tant qu’acteurs de la régénération osseuse pouvant potentiellement avoir un impact sur la qualité de la réparation. Les fractures problématiques, qui ne se réparent pas spontanément sont appelées défauts critiques. Pour les réparer, des solutions sous forme de substituts osseux sont depuis des années en développement. Parmi ceux-ci, les biocéramiques bénéficient d’un intérêt particulier car elles sont capables de libérer des ions Ca2+ et PO43- dans leur environnement. Leur effet sur les ostéocytes a été très peu étudié bien qu’ils régulent le métabolisme du calcium et du phosphate. Pour aborder ces différents aspects, le projet de thèse a d’abord consisté en l’optimisation d’une méthode de microdissection laser pour spécifiquement collecter les ostéocytes dans leur environnement. Ensuite, cette méthode a été utilisée pour analyser leur expression génique dans différents contextes : d'une part la maturation et le vieillissement physiologique, d'autre part la réparation d’un défaut critique avec ou sans biocéramique chez la souris mâle. Lors de la première phase du projet, en supplément des données d’expression génique pour les ostéocytes, l’évolution morphologique des réseaux ostéocytaire et vasculaire a été décrite lors de la maturation et du vieillissement grâce à des techniques d’imagerie en fluorescence. Les changements opposés observés sur la morphologie de l’os étaient accompagnés de changements spécifiques à chaque réseau, sans lien apparent. L’objectif de la deuxième phase (effectuée dans un laboratoire aux Etats-Unis) était d’établir des techniques d’analyse du métabolisme énergétique des ostéocytes en s’intéressant aux acides gras à longue chaîne comme source d’énergie. Cela a mené à l’établissement de protocoles de mesure bioénergétique in vitro et d’imagerie ex vivo. Finalement, l’étude de l’expression génique des ostéocytes dans les phases précoces du processus de réparation a suggéré une contribution de leur part et un effet des biocéramiques via les gènes Il6 et Dmp1. Les outils mis au point et les résultats produits serviront de base pour initier des études visant à mieux comprendre le fonctionnement des ostéocytes dans des contextes encore peu explorés
Populations live longer raising public health concerns related to aging, such as the increase in fracture number due to bone frailty and the necessity to adapt treatments. Nowadays, multiple strategies are followed to prevent or slow down the loss of bone mass, and to treat fractures. They all present limitations forcing researchers to look for new treatment targets. Osteocytes represent 95 % of the cells in bone and live decades embedded inside their mineralized matrix. They have a specific shape with dendrites extending from their body towards other osteocytes, cells at the bone surface, and towards blood vessels. For a long time, they have been considered passive because of their location. However, the development of in vitro and in vivo tools enabled to identify their central role in bone mass maintenance. This is due to the fact that osteocytes are the most mechanosensitive cells in bone, meaning that they react to variations in mechanical loading coming from exercise or disuse. They are able to send signals to osteoblasts and osteoclasts to form and resorb the matrix where it is needed. Aging causes systemic hormonal and metabolic changes affecting the osteocyte network. However, a lot remains to be explored because it is still difficult to study them in their environment. In particular, the nature of their interactions with the vascular network and the changes in energy metabolism with aging need to be investigated. Moreover, very few studies considered osteocytes as having a role in the bone healing process, or an impact on the quality of the repair. Difficult fractures do not repair spontaneously and are called critical. To repair them, bone substitutes have been under development for years. Among them, bioceramics benefit from a specific interest because they are able to release Ca2+ et PO43- in their environment. Their impact on osteocytes has not been well studied, although these cells regulate calcium and phosphate metabolism. To address these different aspects, the first task of the Ph.D. work was to optimize a laser-assisted microdissection protocol to specifically collect osteocytes in their environment. Then, this method was applied to the analysis of osteocyte gene expression during maturation, aging, and during the repair of a critical-size defect in male mice. For the first part of the project, in addition to the osteocyte gene expression analysis, the evolution of the osteocyte and blood vessel network morphologies was described during maturation and aging, with the help of fluorescent imaging techniques. The opposite changes in bone morphology observed during maturation and aging were characterized by distinct, network-specific changes. The second part of the project was elaborated within a lab in the USA, the goal was to establish different techniques to analyze osteocyte energy metabolism using long-chain fatty acids as a fuel source. This led to the optimization and use of in vitro bioenergetics assays and ex vivo imaging. In the last part of the project, the osteocyte gene expression during the early phases of bone repair was analyzed. Among the genes tested, a contribution of osteocytes was identified through the genes Il6 and Dmp1, as well as an impact of the presence of the bioceramics. The different tools and techniques optimized, and the results produced during this PhD project will enable the initiation of new research studies to better understand osteocyte function in contexts still underexplored