Hypothesis / aims of study
Diabetes Mellitus type 2 is a prevalent metabolic disease affecting multiple organ systems, including the urinary tract system, leading to diabetic bladder dysfunction (DBD). A temporal theory suggests that hyperglycemia-related polyuria initially causes a compensatory hypertrophic bladder with overactive features; however, over time, decompensation of bladder function occurs along with myogenic and neurogenic changes, leading to an underactive bladder with increased bladder capacity and poor emptying.
Establishing a suitable animal model for Diabetes Mellitus type 2 is essential for the development of new therapies for DBD. Animal models provide a better understanding of the pathophysiology behind this complex disease. Most diabetic mouse models are induced chemically, leading to type 1 diabetes. We know however that this pathophysiology differs from the metabolic syndrome involved in type 2 diabetes. The Tally Ho mouse model is a polygenic mouse model for type 2 diabetes characterized by obesity, hyperinsulinemia, impaired glucose tolerance and hyperglycemia. We aim to portray the bladder function of the diabetic type 2 mouse model, Tally Ho, as it progresses from an early overactive to a decompensated underactive state. This is the first study looking at this mouse model in DBD. This characterization will open up avenues for future studies related to therapy and treatment of diabetes type 2.
Study design, materials and methods
Tally Ho (TH) mice were used as our diabetic type 2 mouse model. The control group were SWR/J mice. We studied mice at different age groups in order to characterize DBD voiding patterns throughout time. 6 mice per strain were assessed at 10, 14 and 18 weeks of age.
General characteristics such as body weight, glycemia and bladder weight were collected at each time point. Voiding spot assays, performed over a 4-hour timeframe, evaluated total urine volume, number of voids and average volume per void for each mice at the preset age intervals.
Suprapubic catheters were implanted surgically via a midline abdominal incision. This was connected to a pressure transducer and microinjection pump to perform in vivo urodynamic studies. Maximal voiding pressure, basal vesical pressure, intermicturition pressure, intermicturition time, micturition volume, bladder capacity, bladder compliance and power-void residual volume were obtained.
Ex vivo contractility studies were performed using detrusor smooth muscle strips. Two detrusor muscle strips were taken from each mice. They were placed within organ baths containing Krebs solution with a gas mixture of 95% oxygen and 5% carbon dioxide. Each strip was then stimulated sequentially with KCl 60mM, electrical field stimulation at 1, 2, 4, 8, 16 and 32 Hz, and increasing carbachol concentrations from 3nM to 100µM to assess contractility.
Gene expression levels were assessed using the remaining bladder from each mice. RNA from bladder samples were extracted and transcribed to cDNA. This was then amplified by polymerase chain reaction to assess for expression of P2X1, SCLC, myosin, GADPH, calcium channel, muscarinic 2 and muscarinic 3 receptor and beta actin.
Statistical analysis was performed using the Student’s T-Test in order to compare age and sex equivalent Tally Ho mice with SWR/J mice.
Results
Our results show that Tally Ho mice had a significantly larger body weight than SWR/J mice at 10 weeks of age and this persisted at 14 and 18 weeks of age. There was no difference in glycemia and bladder weight at 10 weeks of age for either sexes. The male Tally Ho mice developed significantly higher glycemias than male SWR/J mice at 14 and 18 weeks of age and all Tally Ho mice had significantly larger bladder weights than their SWR/J counterparts.
The Tally Ho mice consistently voided larger total volumes with more frequency than the SWR/J mice throughout all age groups. Interestingly though, the Tally Ho mice initially voided less volume per void than the SWR/J mice at 10 weeks of age, however, by 18 weeks of age, the volume per void was significantly increased in the Tally Ho mice.
The Tally Ho male mice initially had lower maximal bladder pressures with similar spontaneous activity at 10 weeks of age compared to the SWR/J mice, however by 18 weeks of age, this shifted to increased bladder pressures and more spontaneously activities. The residual volume and bladder compliance were also elevated in the 18 week old Tally Ho male mice.
In terms of contractility, there was no difference between the Tally Ho and the SWR/J mice at 10 weeks of age to any stimulants. At 14 weeks, there was a clear increase in contractility to KCl, EFS and Carbachol for Tally Ho male mice compared to its SWR/J counterparts. This difference remained at 18 weeks of age.
Gene expression levels of P2X1, SCLC, myosin, calcium channel, muscarinic 2 and muscarinic 3 receptors were significantly lower in the 18 weeks old male Tally Ho mice compared to the SWR/J mice. Prior, there was no difference in gene expression levels between the two strains.
Interpretation of results
The Tally Ho mice strain is a polygenic mouse model that has been previously validated for metabolic syndromes including diabetes type 2 in the male phenotype. Our results demonstrate that the Tally Ho species expresses the characteristics seen in DBD. At 10 weeks of age, the Tally Ho mice are at a pre-diabetic stage where they do not demonstrate differences in glycemia and weight compared to the control SWR/J strain. However, changes in the bladder physiology are already starting to be seen with signs of polyuria and frequency. At 14 weeks of age, clear overactivity was seen with increased contractility. By 18 weeks of age, the Tally Ho male mice exhibited decompensation with increased bladder compliance, increased bladder capacities, increased post-void residuals and changes in genetic expression related to decreased bladder function.