Predicting Xerostomia induced by IMRT treatments: A logistic regression approach

Radiotherapy is one of the main treatments used against cancer. Radiotherapy uses radiation to destroy cancerous cells trying, at the same time, to minimize the damages in healthy tissues. The planning of a radiotherapy treatment is patient dependent, resulting in a lengthy trial and error procedure until a treatment complying as most as possible with the medical prescription is found. Intensity Modulated Radiation Therapy (IMRT) is one technique of radiation treatment that allows the achievement of a high degree of conformity between the area to be treated and the dose absorbed by healthy tissues. Nevertheless, it is still not possible to eliminate completely the potential treatments��� side-effects. In this retrospective study we use the clinical data from patients with head-and-neck cancer treated at the Portuguese Institute of Oncology of Coimbra and explore the possibility of classifying new and untreated patients according to the probability of xerostomia 12 months after the beginning of IMRT treatments by using a logistic regression approach. The results obtained show that the classifier presents a high discriminative ability in predicting the binary response ���at risk for xerostomia at 12 months���

Moisture-induced degradation of interfacial bond in FRP-strengthened masonry

Externally bonded strengthening of masonry structures using Fiber Reinforced Polymers (FRPs) has been accepted as a promising technique. Although the effectiveness of FRPs in improving the performance of masonry components has been extensively investigated, their long-term performance and durability remain poorly addressed. This paper, tackling one of the aspects related to durability of these systems, presents an experimental investigation on the effect of long-term (one year) water immersion on the performance of GFRP-strengthened bricks. The tests include materials' mechanical tests, as well as pull-off and single-lap shear bond tests, to investigate the changes in material properties and bond behavior with immersion time, respectively. The effect of mechanical surface treatment on the durability of the strengthened system as well as the reversibility of the degradation upon partial drying are also investigated. The experimental results are presented and critically discussed.

Nonsolvent induced phase separation preparation of poly(vinylidene fluoride-co-chlorotrifluoroethylene) membranes with tailored morphology, piezoelectric phase content and mechanical properties

Poly(vinylidene fluoride-co-chlorotrifluoroethylene) ��� P(VDF-CTFE) membranes are increasingly interesting for a wide range of applications, including battery separators, filtration membranes and biomedical applications. This work reports on the morphology, hydrophobicity, thermal and mechanical properties variation of P(VDF-CTFE) membranes processed by nonsolvent induced phase separation technique (NIPS) as a function of the main processing parameters. All membranes show a porous structure composed of large spherulites, (interconnected) micropores and/or microvoids depending on the processing conditions used that in turn affect their hydrophobicity and mechanical properties. The degree of crystallinity of the membranes remains approximately constant with a value of about 15 %, except for the membranes immediately immersed in ethanol, which is of about 23 %. In turn, the crystalline phases present in the copolymer is mainly affected by the temperature and nonsolvent characteristics of the coagulation bath, the ��-phase content ranging from 33 to 100 %, depending on those processing parameters. It was show that the temperature of water-based coagulation bath plays an important role in order to produce structurally uniform and homogeneous porous membranes, which is particularly important from the point of view of technological applications.

Comparing different methods to measure biofilm thickness: the techniques are: optical coherence tomography, confocal laser scanning microscopy and low load compression test

Disserta����o de mestrado integrado em Engenharia Biom��dica (��rea de especializa����o em Engenharia Cl��nica)

Transmyocardial laser revascularization. Early clinical experience

OBJECTIVE: To analyze the initial clinical experience of transmyocardial laser revascularization (TMLR) in patients with severe diffuse coronary artery disease. METHODS: Between February, 1998 and February, 1999, 20 patients were submitted to TMLR at the Heart Institute (InCor), University of S��o Paulo Medical School, Brazil, isolated or in association with conventional coronary artery bypass graft (CABG). All patients had severe diffuse coronary artery disease, with angina functional class III/IV (Canadian Cardiovascular Society score) unresponsive to medical therapy. Fourteen patients were submitted to TMLR as the sole therapy, whereas 6 underwent concomitant CABG. Fifty per cent of the patients had either been previously submitted to a CABG or to a percutaneous transluminal coronary angioplasty (PTCA). Mean age was 60 years, ranging from 45 to 74 years. RESULTS: All patients had three-vessel disease, with normal or mildly impaired left ventricular global function. Follow-up ranged from 1 to 13 months (mean 6.6 months), with no postoperative short or long term mortality. There was significant symptom improvement after the procedure, with 85% of the patients free of angina, and the remaining 15 % of the patients showing improvement in functional class, as well as in exercise tolerance. CONCLUSION: This novel technique can be considered a low risk alternative for a highly selected group of patients not suitable for conventional revascularization procedures.

Evaluaci��n del efecto del l��ser y magnetoterapia en miopat��a experimental valorando biomarcadores de estr��s oxidativo, histomorfometr��a y actividad enzim��tica mitocondrial. Evaluation of the effect of laser and magnetic therapy in experimental myopathy assessing oxidative stress biomarkers, histomorphometry and mitochondrial enzyme activity

El l��ser de baja y media energ��a y la magnetoterapia son utilizados en des��rdenes osteomioarticulares por sus efectos analg��sico, antiinflamatorio y tr��fico, entre los m��s destacados. Sin embargo, son insuficientes las investigaciones sobre su mecanismo de acci��n y antecedentes cient��ficos que avalen sus efectos. Es por ello, que la determinaci��n de acontecimientos celulares y moleculares que ocurren durante la interacci��n de estos tipos de energ��a con el sistema muscular, ser��a relevante para el conocimiento y optimizaci��n de tales terapias en las ciencias biom��dicas. En las miopat��as inflamatorias idiop��ticas, se encuentra afectada la estructura, morfolog��a y bioqu��mica del tejido muscular. La energ��a que ��ste requiere para el normal funcionamiento es generada en la mitocondria. Esta organela tambi��n es la responsable de la generaci��n de especies oxidantes provocando estr��s oxidativo y el inicio de los procesos de apoptosis. Por lo antes dicho, consideramos que la determinaci��n de los biomarcadores inflamatorios asociados a estr��s oxidativo, realizando el an��lisis histomorfom��trico ultraestructural y valorando la actividad de los complejos enzim��ticos mitocondriales, permitir��a una evaluaci��n de la acci��n terap��utica del l��ser y la magnetoterapia en un modelo experimental de miopat��a. Para ello se propone evaluar el efecto de la magnetoterapia y del l��ser de baja energ��a (He-Ne y As.Ga) en miopat��a experimental determinando indicadores inflamatorios asociados a estr��s oxidativo, an��lisis histomorfom��trico y valoraci��n de la actividad enzim��tica mitocondrial. Espec��ficamente: -Determinar indicadores inflamatorios y de estr��s oxidativo: Oxido N��trico, Grupos carbonilos, L-citrulina, Fibrin��geno, Super��xido dismutasa, Glutation peroxidasa y Catalasa por espectrofotometr��a. -Identificar los cambios anatomopatol��gicos del m��sculo esquel��tico por microscop��a ��ptica (MO): cuantificaci��n del infiltrado inflamatorio; MO de alta resoluci��n (MOAR) y por microscop��a electr��nica: histomorfometr��a de la ultraestructura miofibrilar y mitocondrial. -Valorar las actividades enzim��ticas de la citrato sintasa y de los complejos: I (NADH-ubiquinona reductasa), II (succinato-ubiquinona-reductasa) III (ubiquinona-citocromo c-reductasa) y IV (citocromo c-oxidasa); en mitocondrias de tejido muscular por espectrofotometr��a. -Evaluar la actividad apopt��tica en las fibras musculares de los diferentes grupos por t��nica de T.U.N.E.L. Las mediciones mitocondriales (por ME) y de infiltrado inflamatorio (por MO) se realizar��n en un total de 5 fotos de aumentos similares en forma aleatoria por grupo estudiado (n=10). Los cambios estructurales observados se analizar��n en el programa Axiovision 4.8, para cuantificar el ��rea total ocupada, n��mero total y grado de alteraci��n de las mitocondrias y el porcentaje de infiltrado inflamatorio determinando el grado de inflamaci��n. Los resultados de los datos cuantitativos se analizar��n aplicando ANAVA (test de Fisher para comparaciones m��ltiples); y para los datos categ��ricos se utilizar�� Chi cuadrado (test de Pearson), estableci��ndose un nivel de significaci��n de p < 0.05 para todos los casos. Importancia del Proyecto: La salud y el bienestar del hombre son los logros perseguidos por las ciencias de la salud. La obtenci��n de terapias curativas o paliativas con un m��nimo de efectos colaterales para el enfermo se incluye en estos logros. Por esto y todo lo anteriormente expuesto es que consideramos de gran importancia poder esclarecer desde las ciencias b��sicas los efectos celulares y moleculares en modelos experimentales la acci��n de la terapia con l��ser y magnetoterapia para una aplicaci��n cl��nica con base cient��fica en todas las ��reas de las Ciencias M��dicas. In the idiopathic inflammatory myopathies, is affected the structure, morphology and biochemistry of muscle tissue. The mitochondria is responsible for the generation of oxidizing species leading to oxidative stress and the beginning of the process of apoptosis. As said before, we consider the determination of inflammatory biomarkers related to oxidative stress, by ultrastructural morphometric analysis and assessing the activity of mitochondrial enzyme complexes, permit an evaluation of the therapeutic action of laser and magnetic therapy in an experimental model myopathy. We propose to evaluate the effect of the treatment identifying indicators in experimental inflammatory myopathy associated with oxidative stress, histomorphometric analysis and assessment of mitochondrial enzyme activity. Specifically -determining: Nitric oxide, carbonyl groups, L-citrulline, fibrinogen, superoxide dismutase, glutathione peroxidase and catalase by spectrophotometry. -Identify the pathological changes in skeletal muscle by optical microscopy (OM): quantification of the inflammatory infiltrate, OM high resolution (MOAR) and electron microscopy, histomorphometry of myofibrillar and mitochondrial ultrastructure. -Evaluate the enzymatic activity of citrate synthase and complexes: I, II, III and IV in mitochondria muscle tissue by spectrophotometry. -Evaluate apoptotic activity in muscle fibers by TUNEL technique of Mitochondrial measurements and inflammatory infiltration (by OM) was performed in a total of 5 photos of similar increases in random by the study group (n = 10). The structural changes observed are discussed in the program Axiovision 4.8, to quantify number, degree of alteration of mitochondria and the percentage of inflammatory infiltrate determining the degree of inflammation. The results of the quantitative data were analyzed using ANOVA (Fisher test), and categorical data with Chi-square (Pearson test), establishing a significance level of p <0.05.

Empleo de ��ptica adaptativa y fluorescencia en un oftalmoscopio laser de barrido para el registro de c��lulas

Estudio elaborado a partir de una estancia en la Universidad de Rochester, Estados Unidos, de octubre del 2006 a enero del 2007. La estancia realizada en la Universidad de Rochester estuvo orientada al aprendizaje en profundidad del oftalmoscopio l��ser de barrido. El oftalmoscopio l��ser de barrido emplea una t��cnica confocal con la finalidad de visualizar diferentes estructuras retinianas en seres vivos. El instrumento dise��ado y desarrollado en el Centro de Ciencias de la Visi��n incorpora un sistema de ��ptica adaptativa y fluorescencia. La ��ptica adaptativa aplicada en este oftalmoscopio tiene como objetivo corregir las aberraciones existentes en el ojo y as�� permitir observar detalles de la retina que de otra forma se ver��an emborronados. De esta forma se consigue alcanzar valores de resoluci��n muy cercanos a los impuestos por difracci��n. Por otro lado el uso de fluorescencia tiene por objetivo el permitir la visualizaci��n de c��lulas y estructuras que, de no ser te��idas, son transparentes a la luz y visible. Esta t��cnica se ha estado utilizando principalmente en primates y ratas, aunque actualmente tambi��n se est��n llevando a cabo medidas de c��lulas de epitelio pigmentario en seres humanos ya que el pigmento contenido en estas c��lulas permite la aplicaci��n de la fluorescencia sin necesidad de utilizar tinci��n.

Terrestrial laser scanning for landslides deformation monitoring

Within last few years a new type of instruments called Terrestrial Laser Scanners (TLS) entered to the commercial market. These devices brought a possibility to obtain completely new type of spatial, three dimensional data describing the object of interest. TLS instruments are generating a type of data that needs a special treatment. Appearance of this technique made possible to monitor deformations of very large objects, like investigated here landslides, with new quality level. This change is visible especially with relation to the size and number of the details that can be observed with this new method. Taking into account this context presented here work is oriented on recognition and characterization of raw data received from the TLS instruments as well as processing phases, tools and techniques to do them. Main objective are definition and recognition of the problems related with usage of the TLS data, characterization of the quality single point generated by TLS, description and investigation of the TLS processing approach for landslides deformation measurements allowing to obtain 3D deformation characteristic and finally validation of the obtained results. The above objectives are based on the bibliography studies and research work followed by several experiments that will prove the conclusions.

Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. In the present study, we prospectively compared MALDI-TOF MS to the conventional phenotypic method for the identification of routine isolates. Colonies were analyzed by MALDI-TOF MS either by direct deposition on the target plate or after a formic acid-acetonitrile extraction step if no valid result was initially obtained. Among 1,371 isolates identified by conventional methods, 1,278 (93.2%) were putatively identified to the species level by MALDI-TOF MS and 73 (5.3%) were identified to the genus level, but no reliable identification was obtained for 20 (1.5%). Among the 1,278 isolates identified to the species level by MALDI-TOF MS, 63 (4.9%) discordant results were initially identified. Most discordant results (42/63) were due to systematic database-related taxonomical differences, 14 were explained by poor discrimination of the MALDI-TOF MS spectra obtained, and 7 were due to errors in the initial conventional identification. An extraction step was required to obtain a valid MALDI-TOF MS identification for 25.6% of the 1,278 valid isolates. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional phenotypic identification for most bacterial strains routinely isolated in clinical microbiology laboratories.

Low-Dose Vascular Photodynamic Therapy Decreases Tumor Interstitial Fluid Pressure, which Promotes Liposomal Doxorubicin Distribution in a Murine Sarcoma Metastasis Model.

INTRODUCTION: Solid tumors are known to have an abnormal vasculature that limits the distribution of chemotherapy. We have recently shown that tumor vessel modulation by low-dose photodynamic therapy (L-PDT) could improve the uptake of macromolecular chemotherapeutic agents such as liposomal doxorubicin (Liporubicin) administered subsequently. However, how this occurs is unknown. Convection, the main mechanism for drug transport between the intravascular and extravascular spaces, is mostly related to interstitial fluid pressure (IFP) and tumor blood flow (TBF). Here, we determined the changes of tumor and surrounding lung IFP and TBF before, during, and after vascular L-PDT. We also evaluated the effect of these changes on the distribution of Liporubicin administered intravenously (IV) in a lung sarcoma metastasis model. MATERIALS AND METHODS: A syngeneic methylcholanthrene-induced sarcoma cell line was implanted subpleurally in the lung of Fischer rats. Tumor/surrounding lung IFP and TBF changes induced by L-PDT were determined using the wick-in-needle technique and laser Doppler flowmetry, respectively. The spatial distribution of Liporubicin in tumor and lung tissues following IV drug administration was then assessed in L-PDT-pretreated animals and controls (no L-PDT) by epifluorescence microscopy. RESULTS: L-PDT significantly decreased tumor but not lung IFP compared to controls (no L-PDT) without affecting TBF. These conditions were associated with a significant improvement in Liporubicin distribution in tumor tissues compared to controls (P &lt; .05). DISCUSSION: L-PDT specifically enhanced convection in blood vessels of tumor but not of normal lung tissue, which was associated with a significant improvement of Liporubicin distribution in tumors compared to controls.

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

Sistemes lidar (radar laser) d'ona cont��nua i baixa pot��ncia per a la detecci�� i mesura de concentraci�� de gasos amb resoluci�� espacial

The present project has performed the study and development of a new technique for the detection of gases with range resolution. This technique called FMCW-lidar is a technique that evolves from the FMCW-radar technique to be applied to lidar systems. Moreover, it takes advantage of the appearance of spectral absorption lines because of the interaction between light and gases to tune the light wavelength of a laser emitter with one of this spectral lines and then detects the backscattered light and analyzes it in order to obtain gas concentration measurements. The first part of the project consisted in the analysis of the WMS technique which is a technique for the in-situ measurement of gases. A complete theoretical analysis has been performed and some experiments have been carried out in order to test the technique and to validate its application to an FMCW-modulated system for the detection of gases. The second part of the project consisted in the analysis of the lidar FMCW technique for solid target detection and its extension to continuous media. The classical form of this technique has been analyzed for a distributed medium and a filtering effect has been found which prevents the accurate acquisition of the medium response. A modification of the technique has been proposed and a validation via simulations and some experiments has been carried on. After performing these tests, a novel system is proposed to be developed and tested in order to perform the indicated gas detection with range resolution.

Programmed Cell Death in Procyclic Form Trypanosoma brucei rhodesiense - Identification of Differentially Expressed Genes during Con A Induced Death

Trypanosoma brucei rhodesiense can be induced to undergo apoptosis after stimulation with Con A. As cell death in these parasites is associated with de novo gene expression we have applied a differential display technique, Randomly Amplified Differential Expressed Sequence-Polymerase Chain Reaction (RADES-PCR) to the study of gene expression during Con A induced cell death in these organisms. Twenty-two differentially displayed products have been cloned and sequenced. These represent the first endogenous genes to be identified as implicated in cellular death in trypanosomatids (the most primitive eukaryote in which apoptosis has been described). Evidence for an ancestral death machinery, `proto-apoptosis' in single celled organisms is discussed.

Gene expression mapping in neuropathic pain : molecular plasticity in nociceptors and superficial dorsal horn

RESUME : La douleur neuropathique est le r��sultat d'une l��sion ou d'un dysfonctionnement du syst��me nerveux. Les sympt��mes qui suivent la douleur neuropathique sont s��v��res et leur traitement inefficace. Une meilleure approche th��rapeutique peut ��tre propos��e en se basant sur les m��canismes pathologiques de la douleur neuropathique. Lors d'une l��sion p��riph��rique une douleur neuropathique peut se d��velopper et affecter le territoire des nerfs l��s��s mais aussi les territoires adjacents des nerfs non-l��s��s. Une hyperexcitabilit�� des neurones appara��t au niveau des ganglions spinaux (DRG) et de la corne dorsale (DH) de la moelle ��pini��re. Le but de ce travail consiste �� mettre en ��vidence les modifications mol��culaires associ��es aux nocicepteurs l��s��s et non-l��s��s au niveau des DRG et des laminae I et II de la corne dorsale, l�� o�� l'information nociceptive est int��gr��e. Pour ��tudier les changements mol��culaires li��s �� la douleur neuropathique nous utilisons le mod��le animal d'��pargne du nerf sural (spared nerve injury model, SNI) une semaine apr��s la l��sion. Pour la s��lection du tissu d'int��r��t nous avons employ�� la technique de la microdissection au laser, afin de s��lectionner une sous-population sp��cifique de cellules (notamment les nocicepteurs l��s��s ou non-l��s��s) mais ��galement de pr��lever le tissu correspondant dans les laminae superficielles. Ce travail est coupl�� �� l'analyse �� large spectre du transcriptome par puce ADN (microarray). Par ailleurs, nous avons ��tudi�� les courants ��lectriques et les propri��t��s biophysiques des canaux sodiques (Na,,ls) dans les neurones l��s��s et non-l��s��s des DRG. Aussi bien dans le syst��me nerveux p��riph��rique, entre les neurones l��s��s et non-l��s��s, qu'au niveau central avec les aires recevant les projections des nocicepteurs l��s��s ou non-l��s��s, l'analyse du transcriptome montre des diff��rences de profil d'expression. En effet, nous avons constat�� des changements transcriptionnels importants dans les nocicepteurs l��s��s (1561 g��nes, &gt; 1.5x et pairwise comparaison &gt; 77%) ainsi que dans les laminae correspondantes (618 g��nes), alors que ces modifications transcriptionelles sont mineures au niveau des nocicepteurs non-l��s��s (60 g��nes), mais important dans leurs laminae de projection (459 g��nes). Au niveau des nocicepteurs, en utilisant la classification par groupes fonctionnels (Gene Ontology), nous avons observ�� que plusieurs processus biologiques sont modifi��s. Ainsi des fonctions telles que la traduction des signaux cellulaires, l'organisation du cytosquelette ainsi que les m��canismes de r��ponse au stress sont affect��s. Par contre dans les neurones non-l��s��s seuls les processus biologiques li��s au m��tabolisme et au d��veloppement sont modifi��s. Au niveau de la corne dorsale de la moelle, nous avons observ�� des modifications importantes des processus immuno-inflammatoires dans l'aire affect��e par les nerfs l��s��s et des changements associ��s �� l'organisation et la transmission synaptique au niveau de l'aire des nerfs non-l��s��s. L'analyse approfondie des canaux sodiques a d��montr�� plusieurs changements d'expression, principalement dans les neurones l��s��s. Les analyses fonctionnelles n'indiquent aucune diff��rence entre les densit��s de courant t��trodotoxine-sensible (TTX-S) dans les neurones l��s��s et non-l��s��s m��me si les niveaux d'expression des ARNm des sous-unit��s TTX-S sont modifi��s dans les neurones l��s��s. L'inactivation basale d��pendante du voltage des canaux t��trodotoxine-insensible (TTX-R) est d��plac��e vers des potentiels positifs dans les cellules l��s��es et non-l��s��es. En revanche la vitesse de r��cup��ration des courants TTX-S et TTX-R apr��s inactivation est acc��l��r��e dans les neurones l��s��s. Ces changements pourraient ��tre �� l'origine de l'alt��ration de l'activit�� ��lectrique des neurones sensoriels dans le contexte des douleurs neuropathiques. En r��sum��, ces r��sultats sugg��rent l'existence de m��canismes diff��renci��s affectant les neurones l��s��s et les neurones adjacents non-l��s��s lors de la mise en place la douleur neuropathique. De plus, les changements centraux au niveau de la moelle ��pini��re qui surviennent apr��s l��sion sont probablement int��gr��s diff��remment selon la perception de signaux des neurones p��riph��riques l��s��s ou non-l��s��s. En conclusion, ces modulations complexes et distinctes sont probablement des acteurs essentiels impliqu��s dans la gen��se et la persistance des douleurs neuropathiques. ABSTRACT : Neuropathic pain (NP) results from damage or dysfunction of the peripheral or central nervous system. Symptoms associated with NP are severe and difficult to treat. Targeting NP mechanisms and their translation into symptoms may offer a better therapeutic approach.Hyperexcitability of the peripheral and central nervous system occurs in the dorsal root ganglia (DRG) and the dorsal horn (DH) of the spinal cord. We aimed to identify transcriptional variations in injured and in adjacent non-injured nociceptors as well as in corresponding laminae I and II of DH receiving their inputs.We investigated changes one week after the injury induced by the spared nerve injury model of NP. We employed the laser capture microdissection (LCM) for the procurement of specific cell-types (enrichment in nociceptors of injured/non-injured neurons) and laminae in combination with transcriptional analysis by microarray. In addition, we studied function��l properties and currents of sodium channels (Nav1s) in injured and neighboring non-injured DRG neurons.Microarray analysis at the periphery between injured and non-injured DRG neurons and centrally between the area of central projections from injured and non-injured neurons show significant and differential expression patterns. We reported changes in injured nociceptors (1561 genes, &gt; 1.5 fold, &gt;77% pairwise comparison) and in corresponding DH laminae (618 genes), while less modifications occurred in non-injured nociceptors (60 genes) and in corresponding DH laminae (459 genes). At the periphery, we observed by Gene Ontology the involvement of multiple biological processes in injured neurons such as signal transduction, cytoskeleton organization or stress responses. On contrast, functional overrepresentations in non-injured neurons were noted only in metabolic or developmentally related mechanisms. At the level of superficial laminae of the dorsal horn, we reported changes of immune and inflammatory processes in injured-related DH and changes associated with synaptic organization and transmission in DH corresponding to non-injured neurons. Further transcriptional analysis of Nav1s indicated several changes in injured neurons. Functional analyses of Nav1s have established no difference in tetrodotoxin-sensitive (TTX-S) current densities in both injured and non-injured neurons, despite changes in TTX-S Nav1s subunit mRNA levels. The tetrodotoxin-resistant (TTX-R) voltage dependence of steady state inactivation was shifted to more positive potentials in both injured and non-injured neurons, and the rate of recovery from inactivation of TTX-S and TTX-R currents was accelerated in injured neurons. These changes may lead to alterations in neuronal electrogenesis. Taken together, these findings suggest different mechanisms occurring in the injured neurons and the adjacent non-injured ones. Moreover, central changes after injury are probably driven in a different manner if they receive inputs from injured or non-injured neurons. Together, these distinct and complex modulations may contribute to NP.

Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy.

BACKGROUND: Second Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet's membrane, in the posterior cornea. METHODOLOGY/PRINCIPAL FINDINGS: We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet's membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats. CONCLUSIONS/SIGNIFICANCE: Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.