HHS Public Access Author manuscript Author Manuscript
J Diabetes Complications. Author manuscript; available in PMC 2017 July 01. Published in final edited form as: J Diabetes Complications. 2016 July ; 30(5): 778–785. doi:10.1016/j.jdiacomp.2016.03.003.
Functional and Morphological Alterations of the Urinary Bladder in Type 2 Diabetic FVBdb/db Mice Liyang Wu1,2, Xiaodong Zhang2, Nan Xiao1,3, Yexiang Huang1,4, Michael Kavran5, Rania A. Elrashidy1,6, Mingshuai Wang1,2, Firouz Daneshgari1, and Guiming Liu1 1Department
of Surgery, MetroHealth Medical Center, Case Western Reserve University, Cleveland, OH, USA
Author Manuscript
2Department
of Urology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, PRC
3Department
of Urology, Lanzhou University Second Hospital, Lanzhou, PRC
4Department
of Urology, The First Affiliated Hospital, China Medical University, Shenyang, PRC
5Department
of Urology, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, Ohio, USA
6Department
of Biochemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
Abstract Author Manuscript
Aims—Diabetic bladder dysfunction (DBD) has been extensively studied in animal models of type 1 diabetes. We aimed to examine the functional and morphological alterations of the urinary bladder in a type 2 diabetes model, FVBdb/db mice. Methods—FVBdb/db mice and age-matched FVB/NJ control mice were tested at either 12, 24 or 52 weeks of age. Body weight, blood glucose and glycated hemoglobin (HbA1c) levels were measured. Bladder function was assessed by measurement of 24-hour urination behavior and conscious cystometry. Bladder was harvested for Masson's Trichrome staining and morphometric analysis.
Author Manuscript
Results—The body weights of FVBdb/db mice were twice as those of FVB/NJ control mice. The blood glucose and HbA1c levels were higher in FVBdb/db mice at 12 and 24 weeks, but not at 52 weeks. A significant increase in the mean volume per void, but decrease in the voiding frequency, in FVBdb/db mice was observed. Cystometry evaluation showed increased bladder capacity, voided volume, and peak micturition pressure in FVBdb/db mice compared with FVB/NJ mice. Morphometric analysis revealed a significant increase in the areas of detrusor muscle and urothelium in FVBdb/db mice. In addition, some FVBdb/db mice, especially males at 12 and 24
Please send correspondence to: Guiming Liu, M.D., Ph.D., Department of Surgery, MetroHealth Medical Center, Case Western Reserve University, 2500 MetroHealth Drive, Rammelkamp Bldg, Cleveland, OH 44109, Tel: 216-778-3884, [email protected]. Conflicts of Interest: None Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Wu et al.
Page 2
Author Manuscript
weeks, showed small-volume voiding during 24-hour urination behavior measurement, and detrusor overactivity in the cystometry measurement. Conclusions—The FVBdb/db mouse, displaying DBD characterized by not only increased bladder capacity, void volume, and micturition pressure, but also bladder overactivity, is a useful model to further investigate the mechanisms of type 2 diabetes-related bladder dysfunction. Keywords Bladder; Type 2 diabetes; FVBdb/db mice; Obesity
1. Introduction Author Manuscript Author Manuscript
The prevalence of diabetes mellitus (DM) is continuously increasing, with estimates to reach 366 million cases in 2030 according to the World Health Organization (Wild et al., 2004). DM seriously affects multiple organ systems including the urinary bladder, predisposing to diabetic bladder dysfunction (DBD), one of the most common and costly complications that affects over 50% of patients (Brown et al., 2005). In 1976, Frimodt-Moller first described the classic symptoms of diabetic cystopathy, including increased bladder capacity and post voiding residual volumes, accompanied by decreased bladder sensation and contraction (Moller, 1976). Currently, DBD refers to an umbrella description for a group of clinical symptoms that encompass not only the above classic voiding problems, which are generally symptoms of later stage bladder dysfunction in DM, but also storage problems such as overactive bladder (OAB) and urge incontinence (Brown et al., 2005;Liu and Daneshgari, 2014). OAB is defined by the International Continence Society as symptoms of urgency, usually with frequency and nocturia, in the absence of causative infection or pathological conditions (Abrams et al., 2003). Detrusor overactivity (DO) is a urodynamic diagnosis frequently associated with OAB symptoms and is characterized by involuntary detrusor contractions during bladder filling (Abrams et al., 2003).
Author Manuscript
Even though type 1 diabetes mellitus (T1DM) accounts only for 5-10% of all diagnosed cases of diabetes (American Diabetes Association, 2010), most studies have focused on DBD in T1DM due to the low cost and reproducibility of streptozotocin (STZ)-induced T1DM animal models (Daneshgari et al., 2009). In such rodent models, we have characterized the full spectrum of temporal effects of T1DM on the bladder including function, morphometry, contractility, nerve innervation and vasculature (Daneshgari et al., 2006b;Xiao et al., 2013;Liu and Daneshgari, 2014). We demonstrated that the bladder undergoes temporal progression from an initial compensated phase to a later decompensated phase (>20 weeks of DM) (Daneshgari et al., 2006b;Xiao et al., 2013;Liu and Daneshgari, 2014). Polyuria causes bladder hypertrophy in the early stage of DM, whereas oxidative stress in the bladder caused by chronic hyperglycemia may play an important role in the late stage failure of bladder function (Xiao et al., 2013). The pathophysiology of DBD is multifactorial, including disturbances of the detrusor, urothelium, and nerve innervations (Liu and Daneshgari, 2014). On the other hand, type 2 diabetes mellitus (T2DM) accounts for 90–95% of all cases of diabetes, but few studies have been conducted on DBD associated with T2DM. DBD in
J Diabetes Complications. Author manuscript; available in PMC 2017 July 01.
Wu et al.
Page 3
Author Manuscript
T2DM is undoubtedly more complex than in T1DM since a large proportion of T2DM patients (30-80%) have metabolic syndrome (MetS) (Kumar et al., 2013), including obesity, elevated triglyceride levels, low high-density lipoprotein cholesterol levels, and hypertension, some or all of which may affect bladder function independently (Tai et al., 2010). The higher incidence and the distinctive complex pathophysiology of T2DM, compared with T1DM, should encourage more research to elucidate structural and functional changes of urinary bladder in T2DM, as well as the underlying molecular mechanisms, which may suggest different therapeutic targets from those for T1DM.
Author Manuscript
The db/db mouse model on the FVB/NJ background (FVBdb/db) is a well-established obese T2DM mouse model, originating from autosomal recessive mutation in the leptin receptor, resulting in impaired signaling of leptin (Chua S Jr et al., 2002;Chen and Wang, 2005). Defective leptin signaling in the hypothalamus causes persistent hyperphagia and obesity, along with severe insulin resistance and hyperglycemia (Chua S Jr et al., 2002). Chua et al. found the blood glucose levels in FVBdb/db mice increased at 1 month of age and sustained at high levels during their investigated period of 7 months (Chua S Jr et al., 2002). The circulating insulin in the FVBdb/db mice increased at 3 months of age, and continued to increase at 5 and 7 months of age. In this study, we determined the temporal functional and morphological alterations of the bladder of FVBdb/db mice up to 52 weeks of age. We chose three time points, 12, 24 and 52 weeks of age, based on the previous observation on the temporal progression of bladder dysfunction in STZ-induced T1DM rodents (Daneshgari et al., 2006a; Daneshgari et al., 2006b; Liu and Daneshgari, 2014).
2. Materials and Methods 2.1. Experimental animals and design
Author Manuscript Author Manuscript
Parent strains of FVBdb/db mice and background strain FVB/NJ mice were bought from The Jackson Laboratory (Bar Harbor, Maine). The mice for the experiments were obtained through colony breeding by laboratory personnel in the Health Sciences Animal Facility at Case Western Reserve University. Both male and female mice were tested at either 12, 24 or 52 weeks of age. Mice in each time point were divided into two sets. One set was used for bladder function assessment by 24-hour urination behavior and conscious cystometry. The other set had no surgical manipulation performed so intact bladders could be harvested for measurement of bladder weight and histological staining. Prior to euthanasia, mice were tested for non-fasting blood glucose levels using a glucometer (OneTouch SureStep, Johnson & Johnson, Milpitas, CA), and glycated hemoglobin (HbA1c) levels were measured by a commercial analyzer (in2itTM HbA1c analyzer, Bio-Rad, Hercules, CA). All protocols were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University. The number of mice used for experiments and analysis of different parameters is shown in Supplementary Table 1. 2.2. Twenty-four hour fluid consumption and urination behavior measurement Twenty-four hour urination behavior measurement was performed using mouse micturition chambers with a wire mesh bottom designed by Med Associates Inc. (St. Albans, VT) (Liu et al., 2015). The mice were placed in the micturition chambers. An electric balance with a
J Diabetes Complications. Author manuscript; available in PMC 2017 July 01.
Wu et al.
Page 4
Author Manuscript
data port connected to a data acquisition system was present below the bottom opening. Changes in the weight of the collection were recorded (MED-CMG, Catamount Research and Development, Saint Albans, VT). Twenty-four hours prior to micturition analysis, solid food was removed from the cages and replaced with lactose-free milk, to reduce the frequency and weight of feces generated during testing, thereby preventing feces droppings from interfering with measurement of urine output (Liu et al., 2006). The volume of milk consumed was recorded. 2.3. Suprapubic bladder catheter implantation and conscious cystometry
Author Manuscript
A suprapubic catheter was implanted 2 days prior to cystometry (Liu et al., 2015). Under anesthesia, the bladder was exposed and a circular purse string suture of 7-0 silk was used on the dome of bladder. A small incision was made and the catheter (PE-10 tubing with a flared tip) was inserted into the bladder and tightened. The catheter was tunneled subcutaneously and externalized at the back of the neck. The distal end of the tubing was sealed. Abdominal and skin incisions were closed separately.
Author Manuscript Author Manuscript
For cystometry measurement, mice were placed in individual metabolic cages. A pressure transducer (BP-100, CB Sciences, Dover, NH) and a flow pump (Kent Scientific Corporation, Torrington, CT) were connected to the implanted bladder catheter. The bladder was filled with room temperature 0.9% saline (1ml/hr for FVB/NJ mice and 2 ml/hr for FVBdb/db mice) while bladder pressure was recorded. Different infusion rates were used based on the following considerations (Liu et al., 2008): (1) the bladder capacity of mice with diabetes is much larger than that of control mice - mice with diabetes actually produce urine faster than control mice, and therefore the higher infusion rate is closer to their real situation; (2) it would take much longer to induce voiding in FVBdb/db mice if we used the same infusion rate as in control mice, and a long non-physiological infusion time might affect bladder function. Urine was collected in a beaker on a force transducer (FT-03 D, Grass Instrument Co, Quincy, MA) placed beneath each cage. The pressure and force transducers were connected to an amplifier (ETH-400, CB Sciences), and multiport controller software (MED-CMG, Catamount Research and Development. Saint Albans, VT) was used for data recording (20 samples/second). After an initial 0.5-1 hour stabilization period, the data on 4-6 representative micturition cycles were collected and the mean values were calculated. The bladder capacity was calculated by multiplying intercontraction interval by the infusion rate. Voided volume was the volume expelled at micturition. Peak micturition pressure was measured at the peak of the bladder contraction. From the cystometry pressure and void volume tracings, DO was defined as obvious increase in voiding and/or non-voiding contraction, with the latter defined as intravesical pressure rises greater than 5 cmH2O from baseline pressure without a release of fluid from the urethra (Schnegelsberg et al., 2010). 2.4. Bladder fixation and staining After mice were euthanized, their bladders were harvested and subsequently equilibrated in 37 °C Krebs' solution for 15 minutes, sectioned at the equatorial midline, and fixed in 10% neutral buffered formalin (pH 7.0) (Liu and Daneshgari, 2006). After fixation, tissues were
J Diabetes Complications. Author manuscript; available in PMC 2017 July 01.
Wu et al.
Page 5
Author Manuscript
dehydrated and embedded in paraffin. Serial 5 μm tissue sections were placed on microscope slides, and dewaxed for Masson's Trichrome staining. 2.5. Morphometric analysis Stained slides were scanned (Leica SCN400 Slide Scanner). The images were analyzed with Image Pro 6.0 image analysis software (Media Cybernetics, Silver Springs, MD) (Xiao et al., 2015). The thickness of bladder wall was measured. The whole cross-sectional tissue area and the areas occupied by smooth muscle (red), collagen (blue) and urothelium (pink) were measured. 2.6. Statistical analysis
Author Manuscript
Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). All data were expressed as mean ± SEM. For two group comparisons at 12, 24 or 52 weeks old, unpaired two-tail t-test was used. For parameters in Table 1, Fisher's exact test was used to compare the percentage of FVBdb/db group to FVB/NJ group. P
J Diabetes Complications. Author manuscript; available in PMC 2017 July 01. Published in final edited form as: J Diabetes Complications. 2016 July ; 30(5): 778–785. doi:10.1016/j.jdiacomp.2016.03.003.
Functional and Morphological Alterations of the Urinary Bladder in Type 2 Diabetic FVBdb/db Mice Liyang Wu1,2, Xiaodong Zhang2, Nan Xiao1,3, Yexiang Huang1,4, Michael Kavran5, Rania A. Elrashidy1,6, Mingshuai Wang1,2, Firouz Daneshgari1, and Guiming Liu1 1Department
of Surgery, MetroHealth Medical Center, Case Western Reserve University, Cleveland, OH, USA
Author Manuscript
2Department
of Urology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, PRC
3Department
of Urology, Lanzhou University Second Hospital, Lanzhou, PRC
4Department
of Urology, The First Affiliated Hospital, China Medical University, Shenyang, PRC
5Department
of Urology, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, Ohio, USA
6Department
of Biochemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
Abstract Author Manuscript
Aims—Diabetic bladder dysfunction (DBD) has been extensively studied in animal models of type 1 diabetes. We aimed to examine the functional and morphological alterations of the urinary bladder in a type 2 diabetes model, FVBdb/db mice. Methods—FVBdb/db mice and age-matched FVB/NJ control mice were tested at either 12, 24 or 52 weeks of age. Body weight, blood glucose and glycated hemoglobin (HbA1c) levels were measured. Bladder function was assessed by measurement of 24-hour urination behavior and conscious cystometry. Bladder was harvested for Masson's Trichrome staining and morphometric analysis.
Author Manuscript
Results—The body weights of FVBdb/db mice were twice as those of FVB/NJ control mice. The blood glucose and HbA1c levels were higher in FVBdb/db mice at 12 and 24 weeks, but not at 52 weeks. A significant increase in the mean volume per void, but decrease in the voiding frequency, in FVBdb/db mice was observed. Cystometry evaluation showed increased bladder capacity, voided volume, and peak micturition pressure in FVBdb/db mice compared with FVB/NJ mice. Morphometric analysis revealed a significant increase in the areas of detrusor muscle and urothelium in FVBdb/db mice. In addition, some FVBdb/db mice, especially males at 12 and 24
Please send correspondence to: Guiming Liu, M.D., Ph.D., Department of Surgery, MetroHealth Medical Center, Case Western Reserve University, 2500 MetroHealth Drive, Rammelkamp Bldg, Cleveland, OH 44109, Tel: 216-778-3884, [email protected]. Conflicts of Interest: None Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Wu et al.
Page 2
Author Manuscript
weeks, showed small-volume voiding during 24-hour urination behavior measurement, and detrusor overactivity in the cystometry measurement. Conclusions—The FVBdb/db mouse, displaying DBD characterized by not only increased bladder capacity, void volume, and micturition pressure, but also bladder overactivity, is a useful model to further investigate the mechanisms of type 2 diabetes-related bladder dysfunction. Keywords Bladder; Type 2 diabetes; FVBdb/db mice; Obesity
1. Introduction Author Manuscript Author Manuscript
The prevalence of diabetes mellitus (DM) is continuously increasing, with estimates to reach 366 million cases in 2030 according to the World Health Organization (Wild et al., 2004). DM seriously affects multiple organ systems including the urinary bladder, predisposing to diabetic bladder dysfunction (DBD), one of the most common and costly complications that affects over 50% of patients (Brown et al., 2005). In 1976, Frimodt-Moller first described the classic symptoms of diabetic cystopathy, including increased bladder capacity and post voiding residual volumes, accompanied by decreased bladder sensation and contraction (Moller, 1976). Currently, DBD refers to an umbrella description for a group of clinical symptoms that encompass not only the above classic voiding problems, which are generally symptoms of later stage bladder dysfunction in DM, but also storage problems such as overactive bladder (OAB) and urge incontinence (Brown et al., 2005;Liu and Daneshgari, 2014). OAB is defined by the International Continence Society as symptoms of urgency, usually with frequency and nocturia, in the absence of causative infection or pathological conditions (Abrams et al., 2003). Detrusor overactivity (DO) is a urodynamic diagnosis frequently associated with OAB symptoms and is characterized by involuntary detrusor contractions during bladder filling (Abrams et al., 2003).
Author Manuscript
Even though type 1 diabetes mellitus (T1DM) accounts only for 5-10% of all diagnosed cases of diabetes (American Diabetes Association, 2010), most studies have focused on DBD in T1DM due to the low cost and reproducibility of streptozotocin (STZ)-induced T1DM animal models (Daneshgari et al., 2009). In such rodent models, we have characterized the full spectrum of temporal effects of T1DM on the bladder including function, morphometry, contractility, nerve innervation and vasculature (Daneshgari et al., 2006b;Xiao et al., 2013;Liu and Daneshgari, 2014). We demonstrated that the bladder undergoes temporal progression from an initial compensated phase to a later decompensated phase (>20 weeks of DM) (Daneshgari et al., 2006b;Xiao et al., 2013;Liu and Daneshgari, 2014). Polyuria causes bladder hypertrophy in the early stage of DM, whereas oxidative stress in the bladder caused by chronic hyperglycemia may play an important role in the late stage failure of bladder function (Xiao et al., 2013). The pathophysiology of DBD is multifactorial, including disturbances of the detrusor, urothelium, and nerve innervations (Liu and Daneshgari, 2014). On the other hand, type 2 diabetes mellitus (T2DM) accounts for 90–95% of all cases of diabetes, but few studies have been conducted on DBD associated with T2DM. DBD in
J Diabetes Complications. Author manuscript; available in PMC 2017 July 01.
Wu et al.
Page 3
Author Manuscript
T2DM is undoubtedly more complex than in T1DM since a large proportion of T2DM patients (30-80%) have metabolic syndrome (MetS) (Kumar et al., 2013), including obesity, elevated triglyceride levels, low high-density lipoprotein cholesterol levels, and hypertension, some or all of which may affect bladder function independently (Tai et al., 2010). The higher incidence and the distinctive complex pathophysiology of T2DM, compared with T1DM, should encourage more research to elucidate structural and functional changes of urinary bladder in T2DM, as well as the underlying molecular mechanisms, which may suggest different therapeutic targets from those for T1DM.
Author Manuscript
The db/db mouse model on the FVB/NJ background (FVBdb/db) is a well-established obese T2DM mouse model, originating from autosomal recessive mutation in the leptin receptor, resulting in impaired signaling of leptin (Chua S Jr et al., 2002;Chen and Wang, 2005). Defective leptin signaling in the hypothalamus causes persistent hyperphagia and obesity, along with severe insulin resistance and hyperglycemia (Chua S Jr et al., 2002). Chua et al. found the blood glucose levels in FVBdb/db mice increased at 1 month of age and sustained at high levels during their investigated period of 7 months (Chua S Jr et al., 2002). The circulating insulin in the FVBdb/db mice increased at 3 months of age, and continued to increase at 5 and 7 months of age. In this study, we determined the temporal functional and morphological alterations of the bladder of FVBdb/db mice up to 52 weeks of age. We chose three time points, 12, 24 and 52 weeks of age, based on the previous observation on the temporal progression of bladder dysfunction in STZ-induced T1DM rodents (Daneshgari et al., 2006a; Daneshgari et al., 2006b; Liu and Daneshgari, 2014).
2. Materials and Methods 2.1. Experimental animals and design
Author Manuscript Author Manuscript
Parent strains of FVBdb/db mice and background strain FVB/NJ mice were bought from The Jackson Laboratory (Bar Harbor, Maine). The mice for the experiments were obtained through colony breeding by laboratory personnel in the Health Sciences Animal Facility at Case Western Reserve University. Both male and female mice were tested at either 12, 24 or 52 weeks of age. Mice in each time point were divided into two sets. One set was used for bladder function assessment by 24-hour urination behavior and conscious cystometry. The other set had no surgical manipulation performed so intact bladders could be harvested for measurement of bladder weight and histological staining. Prior to euthanasia, mice were tested for non-fasting blood glucose levels using a glucometer (OneTouch SureStep, Johnson & Johnson, Milpitas, CA), and glycated hemoglobin (HbA1c) levels were measured by a commercial analyzer (in2itTM HbA1c analyzer, Bio-Rad, Hercules, CA). All protocols were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University. The number of mice used for experiments and analysis of different parameters is shown in Supplementary Table 1. 2.2. Twenty-four hour fluid consumption and urination behavior measurement Twenty-four hour urination behavior measurement was performed using mouse micturition chambers with a wire mesh bottom designed by Med Associates Inc. (St. Albans, VT) (Liu et al., 2015). The mice were placed in the micturition chambers. An electric balance with a
J Diabetes Complications. Author manuscript; available in PMC 2017 July 01.
Wu et al.
Page 4
Author Manuscript
data port connected to a data acquisition system was present below the bottom opening. Changes in the weight of the collection were recorded (MED-CMG, Catamount Research and Development, Saint Albans, VT). Twenty-four hours prior to micturition analysis, solid food was removed from the cages and replaced with lactose-free milk, to reduce the frequency and weight of feces generated during testing, thereby preventing feces droppings from interfering with measurement of urine output (Liu et al., 2006). The volume of milk consumed was recorded. 2.3. Suprapubic bladder catheter implantation and conscious cystometry
Author Manuscript
A suprapubic catheter was implanted 2 days prior to cystometry (Liu et al., 2015). Under anesthesia, the bladder was exposed and a circular purse string suture of 7-0 silk was used on the dome of bladder. A small incision was made and the catheter (PE-10 tubing with a flared tip) was inserted into the bladder and tightened. The catheter was tunneled subcutaneously and externalized at the back of the neck. The distal end of the tubing was sealed. Abdominal and skin incisions were closed separately.
Author Manuscript Author Manuscript
For cystometry measurement, mice were placed in individual metabolic cages. A pressure transducer (BP-100, CB Sciences, Dover, NH) and a flow pump (Kent Scientific Corporation, Torrington, CT) were connected to the implanted bladder catheter. The bladder was filled with room temperature 0.9% saline (1ml/hr for FVB/NJ mice and 2 ml/hr for FVBdb/db mice) while bladder pressure was recorded. Different infusion rates were used based on the following considerations (Liu et al., 2008): (1) the bladder capacity of mice with diabetes is much larger than that of control mice - mice with diabetes actually produce urine faster than control mice, and therefore the higher infusion rate is closer to their real situation; (2) it would take much longer to induce voiding in FVBdb/db mice if we used the same infusion rate as in control mice, and a long non-physiological infusion time might affect bladder function. Urine was collected in a beaker on a force transducer (FT-03 D, Grass Instrument Co, Quincy, MA) placed beneath each cage. The pressure and force transducers were connected to an amplifier (ETH-400, CB Sciences), and multiport controller software (MED-CMG, Catamount Research and Development. Saint Albans, VT) was used for data recording (20 samples/second). After an initial 0.5-1 hour stabilization period, the data on 4-6 representative micturition cycles were collected and the mean values were calculated. The bladder capacity was calculated by multiplying intercontraction interval by the infusion rate. Voided volume was the volume expelled at micturition. Peak micturition pressure was measured at the peak of the bladder contraction. From the cystometry pressure and void volume tracings, DO was defined as obvious increase in voiding and/or non-voiding contraction, with the latter defined as intravesical pressure rises greater than 5 cmH2O from baseline pressure without a release of fluid from the urethra (Schnegelsberg et al., 2010). 2.4. Bladder fixation and staining After mice were euthanized, their bladders were harvested and subsequently equilibrated in 37 °C Krebs' solution for 15 minutes, sectioned at the equatorial midline, and fixed in 10% neutral buffered formalin (pH 7.0) (Liu and Daneshgari, 2006). After fixation, tissues were
J Diabetes Complications. Author manuscript; available in PMC 2017 July 01.
Wu et al.
Page 5
Author Manuscript
dehydrated and embedded in paraffin. Serial 5 μm tissue sections were placed on microscope slides, and dewaxed for Masson's Trichrome staining. 2.5. Morphometric analysis Stained slides were scanned (Leica SCN400 Slide Scanner). The images were analyzed with Image Pro 6.0 image analysis software (Media Cybernetics, Silver Springs, MD) (Xiao et al., 2015). The thickness of bladder wall was measured. The whole cross-sectional tissue area and the areas occupied by smooth muscle (red), collagen (blue) and urothelium (pink) were measured. 2.6. Statistical analysis
Author Manuscript
Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). All data were expressed as mean ± SEM. For two group comparisons at 12, 24 or 52 weeks old, unpaired two-tail t-test was used. For parameters in Table 1, Fisher's exact test was used to compare the percentage of FVBdb/db group to FVB/NJ group. P
