No description available.

No description available.

No description available.

Investigation into the molecular basis of diabetes-induced erectile dysfunction was carried out on corpus cavernosum tissue from control and streptozotocin (STZ)-induced diabetic rats. Increased expression of endothelial nitric oxide synthase (eNOS) and increased nitrosative stress was found in the diabetic penile tissue. Diabetic neuropathy is associated with a failure in axonal regeneration. <i>In vitro</i> axon growth, guidance and regeneration model systems were used to study both the intrinsic nature of diabetic DRG neurones to grow and respond to guidance cues, but also to study the supportive properties of the diabetic nerve environment for axon regeneration. Cyclic Adenosine-3’5-Monophosphate (cAMP) signalling is compromised in diabetic DRG neurones. Increasing cAMP in diabetic neurones reduced the deficit in neurite growth and reversed the aberrant response of diabetic neuronal growth cones to the cAMP-dependent trophic factor, nerve growth factor (NGF). Diabetic neurones display reduced expression of Exchange protein activated by cAMP (Epac), a downstream effector of cAMP. Specifically activating Epac also rescued the abnormal turning responses of diabetic DRG neurones. Impaired axonal regeneration in diabetes has been, in part, attributed to delayed Wallerian degeneration. Using the cryoculture bioassay, it was shown that during regeneration the peripheral nerve environment, of both control and diabetic rats are equally supportive for axon growth. In summary, this is the first study to imply that specific signalling mechanisms, involving the cAMP-Epac pathway, may be compromised in neurones from diabetic rats, which contribute towards reduced neurite growth and abnormal responses to axon guidance cues.

The object of this study was to investigate the effects of an aldose reductase inhibitor, sorbinil (Pfizer inc), in relation to some of the biochemical, structural and functional changes associated with diabetic nerves. An animal model, the streptozotocin-induced diabetic rat was used and the following studies were performed: Motor nerve conduction velocity (MNCV) was measured in the tibial nerve over a 6 month period. A group of rats were examined at the onset of the experiment (OC) to provide a baseline for comparison to all other groups. Age matched control (AMC) animals showed a 13% increase in MNCV during the first 3 months of the experiment with little increase thereafter. The diabetic animals (DC) did not significantly differ over the experimental period from the OC animals and were thus slower conducting than the corresponding AMC group. Administration of sorbinil (25 mg/Kg) to rats from the onset of diabetes had no effect on MNCV by 3 months but had normalised values by 6 months. Nerve glucose, sorbitol, fructose and myo-inositol levels were examined by GLC. Sorbinil had no effect on nerve glucose values but prevented the 10-fold increase in nerve sorbitol values observed with the DC animals. Sorbinil partially normalised nerve fructose values after 3 months of treatment and fully normalised them after 6 months. Myo-inositol (MI) levels showed a 45% reduction by 3 months of diabetes but were normal after 6 months. Sorbinil showed a tendency to restore the reduced MI values by 3 months. Morphometric profiles were examined in the tibial nerve. Axon areas demonstrated a 14% reduction at both 3 and 6 months of diabetes while myelin areas were increased by 13 and 22% respectively. Sorbinil treatment allowed normal axon growth and normalised myelin areas. MNCV was examined in the tibial and gastrocnemius nerves. As above, diabetes prevented the normal MNCV maturation in the tibial nerve. Sorbinil administration (25 mg/Kg) to rats initially diabetic for 2 months, was ineffective in restoring MNCV in the tibial nerve though a partial recovery was observed after 4 months of treatment. MNCV in the gastrocnemius nerve of the DC animals continued to fall as the experiment progressed, reaching a 33% reduction below the OC animals by 3 months. A spontaneous recovery was observed thereafter. Sorbinil partially normalised MNCV in the gastrocnemius nerve after 1 month. These changes exactly paralleled the changes in nerve MI levels. Sorbinil reversed the already elevated nerve sorbitol levels after 1 month of treatment though nerve fructose levels were only partially normalised after 4 months. A morphometric evaluation of the triceps surae nerve (containing fibres to gastrocnemius and soleus muscles) after 4 months of the experiment demonstrated an 18% increase of myelin area in the DC animals. Axon areas were unaffected by diabetes. Sorbinil treatment for 2 months partially normalised myelin areas. Sorbinil administration at doses of 7.5, 12.5 and 25 mg/Kg to rats that had been diabetic for 2 months did not normalise MNCV in the tibial nerve. However, 12.5 and 25 mg/Kg produced a significant improvement in MNCV of the gastrocnemius nerve after 1 month of treatment. 7.5 mg/Kg had no effect in this nerve. All doses of sorbinil produced a trend towards reversing the already elevated nerve sorbitol levels, though 25 mg/Kg was effective after 1 month of treatment whereas 12.5 mg/Kg required 2 months. 7.5 mg/Kg did not fully normalise nerve sorbitol levels. Nerve fructose values remained elevated, though treatment with 25 mg/Kg of sorbinil produced a reduction towards normal values. All 3 doses partially normalised MI levels. For all DC groups, sciatic nerve water content was significantly elevated after a 1 month experimental period. Sorbinil treatment, either given from the induction of diabetes or given after rats were initially diabetic for 2 months, had only a small effect on water content and values remained elevated compared with the controls.

Nous avons effectué une étude bibliographique intitulée “The diabetic foot microbiota: A review”. Cette revue a permis une réactualisation des données connues sur le microbiote du pied diabétique. Nous avons discuté le rôle des bactéries dans la pathogénèse de l’ulcère du pied diabétique et la différenciation entre l’infection et la colonisation bactérienne au niveau de la plaie ainsi que la superposition de l’approche moléculaire et la culture. Nous avons étudié le microbiote du pied diabétique infecté par culturomic. Il s’agit de varier les conditions de culture, puis identifier les colonies par MALDI-TOF ou par amplification et séquençage ARNr 16S. Cette technique s’est montrée efficace dans l’élargissement de l’éventail des bactéries identifiées. 53 espèces bactériennes différentes ont été identifiées à partir des échantillons de 43 patients. L’hétérogénéité et la richesse des plaies se sont montrées importantes. Staphylococcus aureus était l’espèce dominante. Nous nous sommes servis de tests statistiques pour évaluer l’influence que les facteurs cliniques chez les patients pourraient avoir sur la composition de cette flore et l’évolution de la plaie. On s’est orienté vers des souches spéciales « ExPEC » isolées des pieds diabétiques reconnues par leur virulence et impliquées dans des infections extra intestinales sévères. Nous avons disposé d’une collection de souches d’E Coli isolées des pieds diabétiques afin de mener une étude génétique. Nous sommes parvenus à amplifier les gènes de virulence, classer les souches par phylogroupes et les assembler dans des clones. En plus, nous avons réalisé un typage des souches ainsi qu’une exploitation des gènes de résistance. / We conducted a bibliographic study entitled "The Diabetic Foot Microbiota: a review". This review has offered an update of the data concerning the diabetic foot microbiota. We discussed the role of bacteria in the pathogenesis of diabetic foot ulcer, the differentiation between infection and bacterial colonization of the wound and the importance of superposing culture and molecular Tools.We studied the diabetic foot microbiota using the culturomics. It consists in varying the culture conditions and then to identify the colonies by MALDI-TOF or 16s rRNA amplification and sequencing. We decided to practice this technique on the diabetic foot microbiota because it has proven to be effective in broadening the range of identified bacteria. 53 different bacterial species were identified from the samples of 43 patients. The heterogeneity and richness of the wounds were elevated. Staphylococcus aureus was the dominant species. We used statistical tests to evaluate the influence that clinical factors in patients might have on the composition of this flora and the evolution of the wound. We targeted special strains « ExPEC »isolated from diabetic feet known to their virulance and involved in severe extra intestinal infections. We disposed of a collection of E Coli strains isolated from diabetic wounds to conduct a genetic study. We have managed to amplify the virulence genes, classify the strains by phylogroupes and assemble them in clones. In addition, we performed a strain typing as well as an exploitation of the resistance genes.

No description available.

Diabetic nephropathy is the leading cause of end-stage renal disease, yet the mechanisms responsible for hyperglycemia-induced kidney injury have not been fully elucidated. Activation of the renin-angiotensin system and NADPH oxidase-dependent generation of reactive oxygen species are important mediators of chronic kidney disease. I first studied the effect of ACE2, an important enzyme in the renin-angiotensin system, in diabetic kidney injury in the Akita mouse and related the effect to angiotensin peptide and NADPH oxidase. I then demonstrated the interaction between Angiotensin II, the main substrate, and angiotensin-(1-7), the main product of ACE2, respectively, on cell signaling in mesangial cells to better understand the in vitro effect of ACE2. Finally I studied the effect of deletion of p47phox, a regulatory subunit of the NADPH oxidase, on initiation and progression of diabetic nephropathy in the Akita mouse and mesangial cell. Administration of human recombinant ACE2 decreased angiotensin II levels, increased angiotensin-(1-7) levels, normalized NADPH oxidase activity in the Akita mice, and ameliorated diabetes-induced kidney injury. In vitro, hrACE2 attenuated both high glucose and ANG II–induced oxidative stress and NADPH oxidase activity in mesangial cells. Ang-(1–7)-induced ERK1/2 phosphorylation in mesangial cells in a mas receptor-cAMP-PKA-dependent manner. This effect of ang-(1-7) on ERK1/2 phosphorylation is not mediated by AT1R, AT2R, epidermal growth factor or NADPH oxidase. Pre-treatment with Ang-(1-7) attenuated Ang II-induced NADPH oxidase activity and ERK1/2 activation also in a cAMP-PKA-dependent manner. Deletion of p47phox not only reduced diabetes-induced kidney injury but also reduced hyperglycemia by increasing pancreatic and circulating insulin concentrations. p47phox-/- mice exhibited improved glucose tolerance but modestly decreased insulin sensitivity. Deletion of p47phox attenuated high glucose-induced activation of NADPH oxidase and pro-fibrotic gene expression in mesangial cells. There was a positive correlation between p47phox and collagen Iα1 mRNA levels in renal biopsy samples from control subjects and subjects with diabetic nephropathy. The data generated in this thesis strongly suggest a protective role of ACE2, via Ang-(1-7), and a deleterious role of p47phox in diabetic nephropathy. Future therapeutic strategies should include enhancing ACE2 activity in the kidney and inhibiting p47phox-dependent activation of NADPH oxidase in both the kidney and the pancreas.

Neuropathy is a major complication that affects nearly half of all patients with diabetes, greatly decreasing their quality of life. Patients experience a wide range of symptoms including pain, numbness, weakness and other morbidities. While its pathogenesis has been the focus of extensive research, there are still few effective treatment options available for this disease. The discovery of novel molecular targets underlying this diabetic neuropathy may lead to the development of new, more effective therapeutics. DLC2, a Rho GTPase-activating protein with specific activity for RhoA, was shown to be involved in pain signaling. Mice deficient for this protein (DLC2-/-) have increased RhoA activity in their peripheral nerves, and have heightened pain responses compared to wild type (DLC2+/+) in acute pain tests, displaying increased sensitivity to noxious thermal and inflammatory stimuli. DLC2-/- mice also show elevated blood glucose levels, lower body weight and increased sensitivity to blood glucose compared to wild type. Because of the hyperalgesia to acute pain displayed by DLC2-/- mice compared to wild type, and since the RhoA pathway is known to be involved in the pathogeneses and maintenance of diabetes and its complications, these mice were used to investigate more clinically relevant, chronic pain in a model of diabetic neuropathy. Streptozotocin (STZ), given in multiple low doses over five days (MLDS treatment), was used to induce diabetes in DLC2+/+ and DLC2-/- mice, and their pain responses were tested 8 weeks later. Diabetic DLC2-/- mice (DLC2-/--STZ) were hyperalgesic to thermal stimuli from the hot plate test compared to diabetic DLC2 wild type mice (DLC2+/+-STZ) and vehicle-treated controls of both genotypes (DLC2-/--Veh and DLC2+/+-Veh. Similar responses were seen from the von Frey filament test, where the DLC2-/--STZ group exhibited mechanical allodynia compared to the DLC2+/+-STZ group and both control groups. Dorsal root ganglia (DRG) were dissected from these four groups of mice for qPCR screening and protein analysis. DLC2-/--STZ mice showed significantly higher gene expression of the voltage-gated sodium channel Nav 1.9 compared to DLC2+/+-STZ mice, while there was a strong trend of increased levels in the DLC2-/--STZ group compared to both non-diabetic groups. Western blot analysis of the DRG from these mice shows increased levels of COX-2 expression of DLC2-/--STZ mice compared to DLC2+/+-Veh, and elevated levels of phosphorylated ERK (pERK) in DLC2-/--Veh and both diabetic groups compared to DLC2+/+-Veh. Overall, diabetic DLC2-/- mice have more severe painful diabetic neuropathy, with thermal hyperalgesia and mechanical allodynia. Increased RhoA activity and pERK, which are known to be involved in regulation, transcription and trafficking of sodium channels, may lead to increased Nav1.9 mRNA levels and activation. Localized mainly to nociceptors of the DRG, Nav1.9 is known to play a role in sensitizing neurons through lowering the threshold for action potentials, possibly leading to the observed heightened pain response. Additionally, elevated COX-2 levels in DLC2-/--STZ mice may lead to further deficits through activation of inflammatory responses. Future studies will further investigate how these mechanisms are involved in the altered pain response from diabetes. / published_or_final_version / Anatomy / Master / Master of Philosophy

The importance of soft tissue structures in the pathogenesis of diabetic complications in the hand and particularly the forefoot due to nerve damage or neuropathy has long been overlooked. This is partly due to the inability of most clinical techniques to examine tissue structures in vivo. Magnetic Resonance Imaging (MRI) provides a unique opportunity to study these tissues in detail. Through the use of a specially designed "targetted" radiofrequency coil, high-resolution imaging of the forefoot anatomy has been achieved. In addition to anatomical imaging, MRI has been used to give functional information about water dynamics in various tissue types, e. g. the fat pad and the muscles in the foot. The potential of this type of information to provide an insight into the severity and progression of diabetes has been investigated during this research work. In detail two patient groups with and without symptoms of neuropathy have been investigated. Deterioration of foot joints with a collapsed arch and intrinsic muscle wasting due to nerve damage were observed but more importantly several as yet unknown and unobserved phenomenon were also demonstrated. (i) At sites of maximum pressure resulting from neuropathic changes evidence of microhemorrhaging (or localised hemosiderin deposits) was observed through signal voids when using a gradient echo sequence. It is postulated that the occurrence of these voids is due to the paramagnetic iron core of the hemosiderin aggregate and therefore the signal from tissue in its proximity may be recovered with a spin echo sequence. (ii) The use of a Magnetisation Transfer (MT) sequence to provide functional information revealed a distinct change in MT activity deep within the fat pad of the foot at sites of maximum pressure. This is in contrast to the normal fat pad core which is MT inactive since its hydrophobic nature prevents the adsorption of free water. The fat pads of patients suffering from neuropathy showed various degrees of MT activity which hasbeen taken to indicate the presence of fibrotic material as a replacement for fat. This is consistent with current theories of the formation of neuropathic ulcers. The fibrotic tissue is likely to be the result of repetitive trauma to fat tissues leading to fibrosis where the condition of diabetes serves to catalyse its production via glycation and cross-linking of collagen. Histopathological examination of the fat pad tissues of similar patients, confirmed the presence of fibrosis. This is the first time that such a change in plantar fat pad tissue has been demonstrated in vivo. Further it was possible to correlate the extent of MT activity with disease severity. (iii) In an additional study of Limited Joint Mobility in the metacarpophalangeal (MCP) joints of the hand it was found that the cross-sectional area of the joint capsule reduced linearly with age for patients and controls and that there was no significant thickening relative to controls as postulated from work by others using plain radiographs