Urogenital reconstructive surgery - phd-project

Phd-protocol:Urogenital Reconstructive Surgery -in Animal Models of Congenital Malformations BackgroundMalformations of the urinary tract can be found in 0.5% of all newborn children. Of these 25% are so severe that they will have important consequences for the health and quality of life of the child, and surgical intervention is needed.1 Children with malformations of the lower urinary tract, such as urethral valves (complete or partial closure of the urethra), or neurogenic bladder dysfunction (as in myelomeningocele) often have functional micturition disturbances and high pressures within the urinary tract. The problems may manifest already at the time of birth or during the first year of life. This may cause increasing negative effect on renal function, eventually ending with renal failure and kidney transplantation if treatment is not instituted.2 Early intervention, where the system is unloaded and free passage of urine from the bladder is created through a vesicocutaneostomy (opening of the bladder to the skin), suprapubic catheter (catheter through the skin to the bladder), removal of urethral valves or intermittent catheterization (emptying the bladder via a transurethral catheter), is therefore very important.3 Later on a more long-lasting reconstruction and a manageable outlet from the bladder can be offered by creation of a “low pressure system” by bladder augmentation and also drainage from this system. Doing so, the remaining renal function will be preserved and micturition improved.4Several studies have been published on either conservative or surgical treatment of the obstructed bladder (a small bladder with high pressure). 2, 5 The conservative treatment consists of bladder emptying at regular intervals through a transurethral catheter (clean intermittent catheterization = CIC) with or without additional medical (pharmacological) treatment, and will change the lower urinary tract from a constant to an intermittent high pressure system. With surgical treatment, a continuous drainage from the bladder is created by a vesicocutaneostomy, which secures a constant low pressure system. Surgical removal of a urethral valve, in contrast, will result in an intermittent high pressure system. With all approaches, the pressure in the bladder is reduced, and thereby pressure in the whole system, and renal function and micturition will be improved.6 However, it is unclear if the intermittent high pressure system will secure sufficient unloading of the bladder, and it is important to further study advantages and disadvantages of the different approaches to reduce bladder pressure. Studies on the morphological changes that occur in the different parts of the bladder (mucosa, smooth muscle) after different unloading procedures have not been performed, but may give important information that can have translational impact.One of the bladder structures that can be changed by intravesical pressure changes is the mucosa, which, among other important factors, contain aquaporins (AQPs). Particularly AQP3 is expressed in the bladder mucosa. Previous research in rodents have shown that AQPs are of importance for reabsorption of water in the kidney,10 and in animal models of outflow obstruction, a significant downregulation of AQP2 in the kidney was demonstrated.10 The presence of these membrane proteins in the bladder urothelium has only recently been demonstrated, and their role here is still unclear.7-10 In the human urothelium AQPs are suggested to provide a mechanism for modifying urine composition, and thus to have a modulatory role in water and salt homeostasis.26 This makes AQPs an interesting and important parameter that might reflect obstruction induced changes in the bladder mucosa. However, systematic studies of the AQPs in the pig bladder mucosa do not seem to have been performed previously. In addition, information on how high intravesical pressures affect the mucosal barrier function proteins is scarce or lacking.Lack of sufficient amounts of tissue limits urogenital reconstructive surgery, and the possibilities of regenerative medicine are increasingly explored.11-15 Currently gut tissue is most commonly used for reconstruction, but there is a number of complications involved, e.g. disturbances in the patient ́s acid-base, salt and fluid balance,16 insufficient uptake of vitamins (gut resection), generation of mucus, stone formation and infections. Tissue engineering is built on the principle that it is possible to use the body ́s own cells to influence regeneration and the maintenance of normal function in a tissue or an organ. 13-15,17 It is possible to culture a person ́s own cells outside the body (in vitro) and generate an autologous tissue, i.e. the body ́s own, which can be transplanted back into the patient.18 By using tissue engineering for reconstruction of the urinary bladder many of the disadvantages of using gut tissue can be avoided, 19 and resection of parts of the gut is no longer necessary. It has been shown that engineered bladder tissue can be used to augment the normal bladder,16 but there is still a need to develop the methodology for clinical use. 12,20,21 A large part of this research is carried out on normal bladders from small animals.To increase the translational impact of animal data it is important to study the approaches described above in large animal models where the diseased human bladder is mimicked. The present project aims at exploring (I) Functional and morphological changes after establishing a high pressure bladder system in the pig, and the optimal surgical treatment. After gathering such information (II) regenerative medicine approaches will be applied.(I) Questions1.Can an obstructive pig model be developed, mimicking the obstructed human bladder? 2.Which is the best method for unloading the high pressure obstructed bladder -vesicocutaneostomy or
intermittent emptying? 3.What functional (urodynamic) and morphological (urothelial permeability proteins, collagen 
deposition, smooth muscle hypertrophy, denervation) changes occur in the obstructed bladder and how are these changes affected by the different methods for pressure reduction? (II) Questions1. Can autologous tissue material for bladder augmentation be constructed as a useful alternative to gut tissue?General Aims of the ProjectThe overarching aim of the project is to develop a large animal model of obstruction of the lower urinary tract with a high pressure system that mimics what is found in human disease. Using this model, the possible differences in urodynamic, biomechanical and morphological changes occurring when relieving pressure constantly via a vesicocutaneostomy, or intermittently (corresponding to removal of a urethral valve and/or instituting CIC) can be studied. As a second part of the project the possibilities to develop a regenerative medicine approach (tissue engineering) for bladder augmentation will be explored.Specific (I) Aims/Approaches1.Development of a pig model of chronic infravesical obstruction (high pressure, small bladder capacity) 2.Unloading intervention a) constant low pressure system (vesicocutaneostomy), 
b) intermittent high pressure system (mimicking removal of a urethral valve) 3.Urodynamic evaluation of bladder function before, during, and after relief of obstruction 4.In vitro evaluation of bladder smooth muscle contractility in normal bladders and in obstructed 
bladders before and after relief of obstruction 5.Histological and immunohistological evaluation of the bladder before, during and after relief of 
obstruction, including assessment of AQP changes Specific (II) Aims/Approaches1. Use of tissue engineering techniques (cell culture, scaffolds) for construction of autologous bladder tissue suitable for bladder augmentationMethodsProject (I): Study design and proceduresThe general study design is illustrated in Figure 1. Four groups of animals (n=6) will be investigated. In groups 2-4 outflow obstruction will be created. The animals are anesthetized and the abdomen is opened under sterile conditions. Two pressure sensors are placed in the bladder and abdominal cavity enabling continuous recording of detrusor pressure by telemetry. A port catheter will also be placed in the bladder with the port at the back of the pig. This will serve as volume-control both for the urodynamics and for assessment of residual-urine and bladder emptying. After a week of recordings, the animals are anesthetized again, the urethra is isolated and an expanding ring is placed around the urethra to create obstruction. A gradual obstruction will develop as described previously. 22,23 After 4 weeks, all animals are anesthetized again and bladder biopsy will be performed. In group 3 the obstruction is removed, and bladder emptying in the remaining period will be conducted in a way similar to CIC, via the port catheter. In group 4 a vesicocutaneostomy is created. Group 1 serves as a sham-operated control and group 2 is the control of the obstructed animals. Before the study, and at 4 and 8 weeks, urodynamic assessment will be performed in all groups. The telemetric pressure measurements will provide the pressure data for this assessment, and the pigs will be trained to void in a flow-meter, either in a metabolic cage or in a sling, to provide the flow-data. After 8 weeks the animals are again anesthetized and euthanized, the bladder is removed and weighed, and tissue samples taken for biomechanical and pharmacological investigations (organ bath studies) and for histology and immunohistochemistry. Blood and urine samples are taken before the study and at 4 and 8 weeks.Project (II)Urothelial and muscle cells taken by biopsy will be cultured and seeded on scaffolds which are then implanted in normal and obstructed bladders. The long-term goal is to augment the bladder to increase bladder capacity and reduce bladder pressure in obstructed animals.Experimental animalsThe experiments will be performed on pigs (Danish Landrace) weighing approximately 5-10 kg, corresponding to an age of 4-6 weeks, which is the normal time of weaning. The animals will receive all necessary care. All investigations will be done under sterile conditions.Anesthesia and medicationThe operations will be performed under general anesthesia and sterile conditions. The sedation is started with dormicum (midazolam). A venflon is placed in an ear vein and the animal is intubated after injection of hypnomidate (etomidate) and afterwards continuously sedated with propofol. Artificial respiration (respirator) is initiated and blood pressure, pulse and temperature are monitored. The animal is given fentanyl for pain relief and kept hydrated by glucose infusion during the operation. Local anesthesia with bupivacaine/adrenaline is applied around the incision. Postoperatively, pain relief is given by intramuscular or oral Flunixin vet once daily for 3-5 days, then as needed. As infection prophylaxis Tribrissen vet (trimethoprim-sulfa) is given daily for 5 days after the operation. A weekly dose of ammoniumchloride is given to prevent clotting of the catheters. The animals are euthanized by an overdose of phenobarbital while still anesthetized.Organ Bath StudiesAfter euthanasia, the bladders will be removed and immediately placed into ice-cold Krebs/Ringer buffer. Bladder strips (3mm x10mm) will be prepared. The strips (with intact urothelium) will be attached to tissue holders at one end and force transducers at the other in an organ bath system (Danish Myo Technology) containing 15mL of Krebs buffer aerated with 95% O2 / 5% CO2 at 37 ̊C. Bladder strips will be subjected to a resting tension of 1gm and allowed to stabilize for at least 30min. Contractions will be recorded as changes in tension from baseline in response to 60mM KCl, carbachol, and electrical field stimulation (EFS). Carbachol dose-response curves will be generated by adding increasing concentrations of carbachol at 0.5 log increments starting at 3nM up to 100μM. For EFS, strips will be placed between two platinum electrodes in the organ chamber, and electrical pulses (0.1 ms pulse width, 20 V in the bath) delivered, lasting 10sec at increasing frequency (1, 2, 4, 8, 16 and 32Hz), using an S88 stimulator (Grass Instruments). All tissue responses will be normalized to gm tissue weight.Histological ExaminationThe bladders from each group will be immersed in 10% neutral buffered formalin, embedded in paraffin and cut into 6 μm sections. Slides cleared in xylene and dehydrated will be used for staining. Staining with standard hematoxylin and eosin (H&E), and Masson’s trichrome stain will be performed. Computer assisted histomorphometric analysis of H&E stained and Masson’s trichrome stained bladder tissues will be performed using image analysis software and a microscope.Immunohistochemistry and western blottingAnalysis using standard methodology will be performed for cholinergic (vesicular acetyl choline transporter; VACht ), adrenergic (tyrosine hydroxylase) and peptidergic (substance P, calcitonin gene-related peptide) nerves, AQPs , and urothelial cellular proteins that may be involved in maintaining urothelial barrier functions such as uroplakins, connexins, and tight junction proteins8. Cell cultureUrothelial and smooth muscle cells will be cultured according to published protocols12,18,24,25 . Muscle fragments will be plated on culture dishes with explant techniques, and cells expanded in Dulbeccos Modifi ed Eagles Medium supplemented with 10% fetal bovine serum. Urothelial cultures will be expanded with keratinocyte growth medium (Gibco). The cells will be maintained in a humidified 5% CO2 incubator at 37°C until seeding. Biodegradable scaffolds such as polyglycolic acid (PGA) or poly(lactic-co-glycolic) acid (PLGA) will be used. The procedures used for implanting the cultured cells are not yet described as the applicant will achieve knowledge of these during visits to the collaborators at Karolinska University Hospital.StatisticsData will be presented as medians (range). ANOVA and t-tests will be used as appropriate. Kruskall-Wallis test will be used for non-parametric comparisons, and Pearson's rho will be used for calculation of pair-wise correlations. Significance level will be set a p