- Email: [email protected]
Murine bladder wall biomechanics following partial bladder obstruction
Journal of Biomechanics 46 (2013) 2752–2755
Contents lists available at ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Short communication
Murine bladder wall biomechanics following partial bladder obstruction Joseph Chen a,1, Beth A. Drzewiecki b,1, W.David Merryman a, John C. Pope IVb,n a b
Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA Department of Urologic Surgery, Division of Pediatric Urology, Vanderbilt University, Nashville, TN, USA
art ic l e i nf o
a b s t r a c t
Article history: Accepted 7 July 2013
Evaluation of bladder wall mechanical behavior is important in understanding the functional changes that occur in response to pathologic processes such as partial bladder outlet obstruction (pBOO). In the murine model, the traditional approach of cystometry to describe bladder compliance can prove difficult secondary to small bladder capacity and surgical exposure of the bladder. Here, we explore an alternative technique to characterize murine mechanical properties by applying biaxial mechanical stretch to murine bladders that had undergone pBOO. 5–6 week old female C57/Bl6 mice were ovariectomized and subjected to pBOO via an open surgical urethral ligation and sacrificed after 4 weeks (n¼12). Age matched controls (n¼ 6) were also analyzed. Bladders were separated based on phenotype of fibrotic (n¼6) or distended (n¼6) at the time of harvest. Biaxial testing was performed in modified Kreb's solution at 37 1C. Tissue was preconditioned to 10 cycles and mechanical response was evaluated by comparing axial strain at 50 kPa. The normal murine bladders exhibited anisotropy and were stiffer in the longitudinal direction. All mice showed a loss of anisotropy after 4 weeks of pBOO. The two phenotypes observed after pBOO, fibrotic and distended, exhibited less and more extensibility, respectively. These proof-of-principle data demonstrate that pBOO creates quantifiable changes in the mechanics of the murine bladder that can be effectively quantified with biaxial testing. & 2013 Published by Elsevier Ltd.
Keywords: Bladder Biomechanics
1. Introduction The function of the urinary bladder is to store urine at low pressures until it reaches capacity or receives neural input to empty. Coordinated contraction of the bladder muscle with relaxation of the external urinary sphincter should lead to complete emptying, allowing the bladder to maintain a highly compliant state. Pathologic conditions such as neurogenic bladder dysfunction, posterior urethral valves or partial bladder outlet obstruction (pBOO) can lead to decreased compliance and subsequent impairment of bladder function; however, the details of this loss of compliance are not well understood. In humans, urodynamic studies such as cystometry and uroflow are used to evaluate changes in the compliance of the bladder wall that lead to increases in intraluminal pressure at rest, during filling and voiding. Urodynamic studies evaluating the mechanical changes in the bladder wall in response to outlet obstruction or neurologic insults to the bladder are frequently performed in animal disease models such as rabbit, rats, and guinea pigs (Andersson et al., 2011; Wang et al., 2009; Nagatomi et al., 2004). However, in murine models,
n Correspondence to: Urologic Surgery and Pediatrics, 4102 Doctor's Office Tower, 2200 Children's Way, Nashville, TN 37232, USA. Tel.: +1 615 936 1060; fax: +1 615 936 1061. E-mail address: [email protected] (J.C. Pope IV). 1 These authors contributed equally.
0021-9290/$ - see front matter & 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jbiomech.2013.07.022
urodynamic tests can be difficult even in a normal mouse due to the very small bladder capacity. Additionally, conventional urodynamic tests are subject to confounding factors such as variations in bladder thickness and shape (Nagatomi et al., 2004; Gloeckner et al., 2002). Because the murine animal model offers the benefit of transgenic manipulation to more precisely evaluate specific pathways, it is an attractive animal model for research and therefore needs alternative techniques for accurate bladder wall characterization. Alternative methods such as biomechanical testing may provide an additional approach for characterizing murine bladders. Biomechanical testing is an established method of quantifying the intrinsic mechanical properties of various tissues (Fung, 1993). Furthermore, for planar soft tissues such as the bladder, biaxial mechanical testing has become the gold standard in that it is able to mimic in vivo deformations (Sacks, 2000). Biaxial mechanical testing has been used to accurately characterize many soft tissue including heart valves, myocardium, and blood vessels (Sacks, 2000; Demer and Yin, 1983; Zhou and Fung, 1997; Billiar and Sacks, 2000). Specifically in the bladder, biaxial mechanical testing has been successfully applied in a rat model (Nagatomi et al., 2004; Gloeckner et al., 2002). However, it has not to our knowledge been applied to the murine model, which is substantially smaller and offers the unique benefit of transgenic manipulation which can extend the impact of these mechanical studies to addressing mechanistic questions in a variety of bladder disorders.
J. Chen et al. / Journal of Biomechanics 46 (2013) 2752–2755
In the present proof-of-principle study, we utilized biaxial mechanical testing as an alternative method to measure the mechanical changes of the murine bladder wall due to pBOO. We demonstrate the feasibility of this approach and show strong correlations with conventional cystometry. In addition, we quantified the ECM changes associated with pBOO. We show here for the first time the successful application of biaxial testing on murine bladders. 2. Methods 2.1. General experimental procedure A total of 18, 5–6 week old C57/BL6 age matched female mice underwent oopherectomy to minimize variations in inflammation that may be attenuated by the presence of estrogen (Woo et al., 2011). Two weeks later, mice (n¼12) were subjected to pBOO via urethral ligation. After 4 weeks of pBOO, the mice were sacrificed and the bladders were harvested. Mice that underwent pBOO were noted at time of bladder harvest to have a silk suture present around their urethra to confirm obstruction. Bladders were separated based on a phenotypic appearance at the time of harvest—fibrotic bladder (n ¼6) or distened (n¼ 6). Control mice (n¼ 6) that did not receive pBOO or sham surgery but underwent oopherectomy were sacrificed at the same time as the pBOO mice. pBOO mice (n ¼3) and controls (n¼ 5) underwent urodynamic studies prior to sacrifice. The bladders were harvested and maintained in a modified Kreb's solution for less than 48 h and then subjected to biaxial mechanical testing (Nagatomi et al., 2004). After biaxial mechanical testing, the bladder strips were then evaluated by assay for their relative collagen and elastin content. 2.2. Partial bladder outlet obstruction pBOO was performed via a urethral ligation technique, modifying a previously reported method (Woo et al., 2011). Mice were anesthesized with 2% isoflurane and transurethrally catheterized using a 24 gauge angiocatheter. A midline suprapubic incision was made and the urethra was identified distal to the bladder neck. With minimal dissection, the urethra was ligated with a 4-zero silk suture over the catheter. The catheter was removed and the incision was closed in two layers. Mice were allowed to recover on a warming pad prior to returning to their cages. 2.3. Urodynamics Urodynamics were performed immediately prior to bladder harvest under isoflurane anesthetic, modifying a previously described method (Tanaka et al., 2009). Transurethral urodynamics were performed via a 24 gauge angiocatheter rather than the suprapubic technique. After the catheterization, the bladder was emptied and the catheter was connected to a pressure transducer (Digi-Med, Louisville, Kentucky). The bladder was instilled with sterile saline at a rate of 100 mL/min via a syringe pump (Harvard Apparatus, Holliston, Massachussets). Intravesical pressure was recorded by a pressure analyzer (Digi-Med) to a computer based data acquisition system. Infusion was continued until the bladder pressure reached greater than 55 mmHg as the transurethral catheter did not permit for accurate leak point pressures. A total of three bladder filling cycles were obtained for each mouse with a rest period of at least 5 min between cycles. Bladders were emptied prior to starting the next filling cycle. Bladder capacity was defined as volume at 55 mmHg. Bladder compliance was calculated by the change in volume
Longitudinal
Circumferential
2753
over change in pressure at 50% capacity (V50). Limited samples were successfully analyzed with urodynamics due to technical difficulties. 2.4. Biaxial mechanical testing Rectangular sections of bladder tissue were excised by removing the dome and the trigone (Fig. 1). Four fiducial markers were placed in the center of the specimen to track tissue deformation. The circumferential and longitudinal edges were mounted with two and four stainless steel hooks respectively. Polyester suture loops attached the hooks to the motor carriages. Tissue was preconditioned to 10 cycles and loaded to 50 kPa to simulate extraphysiological conditions before data acquisition; loading stress was determined using the law of Laplace as previously described (Gloeckner et al., 2002). Test protocols maintained a constant ratio of axial stress throughout cycling. Stress– strain curves were generated from data acquisition. 2.5. Collagen and elastin assay Following biaxial mechanical testing, the collagen and elastin concentrations of bladder strips from control (n¼ 5), fibrotic (n¼ 4), and distended (n¼ 2) were obtained using commercially available assays following the manufacturer's instructions (Biocolor Sircol™ Soluble Collagen assay, Biocolor Fastin™ Elastin assay, Accurate Chemical, Westbury, NY) (Nagatomi et al., 2004). Results were expressed in micrograms of collagen and elastin mass and were normalized to the wet weight of each bladder. 2.6. Statistical analysis All data for collagen and elastin assay and biaxial mechanical testing are expressed as a mean 7 SEM. Statistical significance between groups was determined via repeated measures ANOVA for biaxial data and one-way ANOVA for collagen and elastin assays, and pairwise differences were identified using post-hoc Holm–Sidak testing. A p o 0.05 was considered statistically significant.
3. Results Urodynamic studies were performed on control (n¼5), fibrotic (n¼2), and distended (n¼1) mice prior to sacrifice and bladder harvest (Table 1). The median compliance7SD of control mice was 9.1 mL/cmH2O and for fibrotic mice was 7.12 mL/cmH2O. The distended bladder had a compliance of 18.06 mL/cmH2O. Median bladder capacity in the control and fibrotic groups was 210 mL and 170.5 mL respectively; the bladder capacity of the distended bladder was 363.4 mL. Due to the limited data on experimental mice, direct statistical comparison of UDS results between the three groups could not be performed. However fibrotic bladders trended towards having smaller capacity and less compliant bladders than controls, and the single distended bladder had almost twice the compliance of control bladders. Control (n¼6), fibrotic (n¼ 6) and distended (n¼4) bladders were evaluated under biaxial stretch (Fig. 1). Bladder mechanical response was evaluated by comparing axial strain at 50 kPa (Fig. 2A–C). Control bladders exhibited anisotropy, with lesser strains in the longitudinal direction (4.2170.825) than circumferential (15.4073.17). After 4 weeks of pBOO, all mice exhibited a loss of anisotropy. The fibrotic phenotype became less compliant in the circumferential direction (6.3970.861) while becoming more compliant in the longitudinal direction (7.0470.671). The distended phenotype became more compliant in both the circumferential (19.4775.05) and the longitudinal direction (21.2373.76). Additionally, urodynamic plots reveal similar trends with the fibrotic bladder exhibiting lower
Table 1 Median compliance of murine bladders on urodynamics.
Fig. 1. Schematic of murine bladder preparation for biaxial testing. Bladders were cut along the urachus and rectangular samples were cut out along the longitudinal and circumferential directions for biaxial testing.
Group
Compliance (lL/cmH2O)
V50 (lL)
Bladder capacity (lL)
Control (n¼ 5) Fibrotic (n¼ 2) Distended (n¼ 1)
9.1 7.12 18.06
105 85.25 181.7
210 170.5 363.4
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J. Chen et al. / Journal of Biomechanics 46 (2013) 2752–2755
Fig. 2. Biaxial stress–strain curves (A), (B), and (C) and urodynamic plots (D), (E), and (F) of murine bladder. Biaxial stress–strain curves were generated for control (A), fibrotic (B) and distended (C) phenotypes. *p≤0.05 between fibrotic and control in the circumferential direction and #p≤0.05 between distended and control in the longitudinal direction. Urodynamic plots were conducted for control (D), fibrotic (E), and distended (F) phenotypes.
4. Discussion
Table 2 Collagen and elastin assay results. ECM protein
Control (n¼ 5)
Fibrotic (n¼3)
Distended (n ¼2)
Collagen Elastin
38.9 74.9 13.3 74.1
51.5 7 9.4 23.2 7 7.2
44.6 7 8.0 17.4 7 6.3
bladder capacity and the distended bladder exhibiting higher bladder capacity when compared to controls (Fig. 2D–F, Table 1). There was a significant difference in circumferential compliance in the fibrotic phenotype compared with controls (po0.05). The change in longitudinal compliance was significantly different from controls for the distended phenotype only (po0.001). There was also a significant difference between both the circumferential and longitudinal compliance between the distended and the fibrotic phenotypes (p¼0.008, po0.001). Standard curves were generated for the collagen and elastin assays as per protocol with R2 values of 0.9886 and 0.9893 respectively. Data was available for control (n ¼5), fibrotic (n ¼4) and distended (n ¼2) mice (Table 2). Compared to control bladders, the collagen and elastin content of the fibrotic bladders were significantly higher by 32% and 74% respectively (p o0.02, p o0.02). Distended bladders also had higher collagen and elastin content by 15% and 31% respectively, although these did not reach statistical significance.
Previously we have utilized urodynamic studies to evaluate the changes in bladder compliance after pBOO (Woo et al., 2011; Tanaka et al., 2009; Anumanthan et al., 2009). However, urodynamic analysis in mice can be technically challenging due to the small size of their bladder capacity as well as the small size of the tubing needed to catheterize them (Andersson et al., 2011). Cystometic parameters and micturition patterns in rodents also differ from humans, creating obvious translational limitations (Andersson et al., 2011). Rodents, and mice especially, are useful experimental models as they allow for transgenic manipulation, and therefore we sought to identify other methodologies to investigate the changes in the compliance of the mouse bladder after pBOO. Biaxial mechanical testing to evaluate the mechanical properties of the urinary bladder wall has been well described in animal models such as the rat and pig (Nagatomi et al., 2004; Gloeckner et al., 2002; Gilbert et al., 2008; Freytes et al., 2008). Biaxial testing is important for realistic characterization of the in vivo two dimensional stretching that occurs during bladder filling (Wang et al., 2009). In this study, we successfully applied biaxial testing to quantify murine bladders mechanics. With biaxial testing, we identified two distinct phenotypes after pBOO which we labeled distended and fibrotic. These changes correspond to end results commonly seen by urologists from human bladders after being subjected to partial outlet obstruction such as benign prostatic hypertrophy or posterior uretheral valves. Both
J. Chen et al. / Journal of Biomechanics 46 (2013) 2752–2755
phenotypes reveal a loss of anisotropy with distended bladders becoming more extensible in the longitudinal direction and fibrotic becoming stiffer in the circumferential direction. This alteration in anisotropic behavior due to pBOO corresponds well with current literature which documents anisotropic changes due to bladder dysfunction (Toosi et al., 2008). Additionally, we show that the biaxial mechanical responses observed correspond well with conventional urodynamic analysis demonstrating the effectiveness of biaxial testing as a tool for describing murine bladders mechanics. In addition to overcoming the technical challenges of urodynamic tests, biaxial testing provides intrinsic mechanical information free from confounding effects such as variations in bladder shape and size (Nagatomi et al., 2004; Gloeckner et al., 2002). Biaxial testing also offers users more flexibility in experimental design in that it can control loading patterns which may allow for deeper investigations into ECM organization and composition (Gloeckner et al., 2002). These types of tests may address questions regarding the role of ECM remodeling due to bladder pathology, which is a major inquiry in this field (Jiang et al., 2012). Lastly, biomechanical information can be utilized to develop constitutive models that can simulate bladder responses in a variety of loading conditions (Nagatomi et al., 2004, 2008). To our knowledge this is the first quantification of biaxial mechanical properties of the murine bladder. We demonstrate that biaxial testing is a useful tool in assessing mechanical changes associated with bladder dysfunction following pBOO. The advantages of biaxial testing may allow for a new avenue of investigation that can generate deeper insights into mechanistic questions associated with bladder disorders.
Conflict of interest statement The authors declare that we have no conflict of interest for our submission.
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References Andersson, K.E., Soler, R., Fullhase, C., 2011. Rodent models for urodynamic investigation. Neurourology and Urodynamics 30 (5), 636–646. Anumanthan, G., Tanaka, S.T., Adams, C.M., et al., 2009. Bladder stromal loss of transforming growth factor receptor II decreases fibrosis after bladder obstruction. Journal of Urology 182 (Suppl 4), 1775–1780. Billiar, K.L., Sacks, M.S., 2000. Biaxial mechanical properties of the natural and glutaraldehyde treated aortic valve cusp—part I: experimental results. Journal of Biomechanical Engineering 122 (1), 23–30. Demer, L.L., Yin, F.C., 1983. Passive biaxial mechanical properties of isolated canine myocardium. Journal of Physiology 339, 615–630. Fung, Y.C. (Ed.), 1993. Biomechanics: Mechanical Properties of Living Tissues, 2nd edition Springer. Freytes, D.O., Stoner, R.M., Badylak, S.F., 2008. Uniaxial and biaxial properties of terminally sterilized porcine urinary bladder matrix scaffolds. Journal of Biomedical Materials Research Part B Applied Biomaterials 84 (2), 408–414. Gilbert, T.W., Wognum, S., Joyce, E.M., Freytes, D.O., Sacks, M.S., Badylak, S.F., 2008. Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix. Biomaterials 29 (36), 4775–4782. Gloeckner, D.C., Sacks, M.S., Fraser, M.O., Somogyi, G.T., de Groat, W.C., Chancellor, M.B., 2002. Passive biaxial mechanical properties of the rat bladder wall after spinal cord injury. Journal of Urology 167 (5), 2247–2252. Jiang, X., Luttrell, I., Li, D.Y., Yang, C.C., Chitaley, K., 2012. Altered bladder function in elastin-deficient mice at baseline and in response to partial bladder outlet obstruction. BJU International 110 (3), 413–419. Nagatomi, J., Gloeckner, D.C., Chancellor, M.B., DeGroat, W.C., Sacks, M.S., 2004. Changes in the biaxial viscoelastic response of the urinary bladder following spinal cord injury. Annals of Biomedical Engineering 32 (10), 1409–1419. Nagatomi, J., Toosi, K.K., Chancellor, M.B., Sacks, M.S., 2008. Contribution of the extracellular matrix to the viscoelastic behavior of the urinary bladder wall. Biomechanics and Modeling in Mechanobiology 7 (5), 395–404. Sacks, M.S., 2000. Biaxial mechanical evaluation of planar biological materials. Journal of Elasticity 61 (1–3), 199–246. Tanaka, S.T., Martinez-Ferrer, M., Makari, J.H., et al., 2009. Recruitment of bone marrow derived cells to the bladder after bladder outlet obstruction. Journal of Urology 182 (Suppl 4), 1769–1774. Toosi, K.K., Nagatomi, J., Chancellor, M.B., Sacks, M.S., 2008. The effects of long-term spinal cord injury on mechanical properties of the rat urinary bladder. Annals of Biomedical Engineering 36 (9), 1470–1480. Wang, C.C., Nagatomi, J., Toosi, K.K., et al., 2009. Diabetes-induced alternations in biomechanical properties of urinary bladder wall in rats. Urology 73 (4), 911–915. Woo, L.L., Tanaka, S.T., Anumanthan, G., et al., 2011. Mesenchymal stem cell recruitment and improved bladder function after bladder outlet obstruction: preliminary data. Journal of Urology 185 (3), 1132–1138. Zhou, J., Fung, Y.C., 1997. The degree of nonlinearity and anisotropy of blood vessel elasticity. Proceedings of the National Academy of Sciences of the United States of America 94 (26), 14255–14260.
Contents lists available at ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Short communication
Murine bladder wall biomechanics following partial bladder obstruction Joseph Chen a,1, Beth A. Drzewiecki b,1, W.David Merryman a, John C. Pope IVb,n a b
Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA Department of Urologic Surgery, Division of Pediatric Urology, Vanderbilt University, Nashville, TN, USA
art ic l e i nf o
a b s t r a c t
Article history: Accepted 7 July 2013
Evaluation of bladder wall mechanical behavior is important in understanding the functional changes that occur in response to pathologic processes such as partial bladder outlet obstruction (pBOO). In the murine model, the traditional approach of cystometry to describe bladder compliance can prove difficult secondary to small bladder capacity and surgical exposure of the bladder. Here, we explore an alternative technique to characterize murine mechanical properties by applying biaxial mechanical stretch to murine bladders that had undergone pBOO. 5–6 week old female C57/Bl6 mice were ovariectomized and subjected to pBOO via an open surgical urethral ligation and sacrificed after 4 weeks (n¼12). Age matched controls (n¼ 6) were also analyzed. Bladders were separated based on phenotype of fibrotic (n¼6) or distended (n¼6) at the time of harvest. Biaxial testing was performed in modified Kreb's solution at 37 1C. Tissue was preconditioned to 10 cycles and mechanical response was evaluated by comparing axial strain at 50 kPa. The normal murine bladders exhibited anisotropy and were stiffer in the longitudinal direction. All mice showed a loss of anisotropy after 4 weeks of pBOO. The two phenotypes observed after pBOO, fibrotic and distended, exhibited less and more extensibility, respectively. These proof-of-principle data demonstrate that pBOO creates quantifiable changes in the mechanics of the murine bladder that can be effectively quantified with biaxial testing. & 2013 Published by Elsevier Ltd.
Keywords: Bladder Biomechanics
1. Introduction The function of the urinary bladder is to store urine at low pressures until it reaches capacity or receives neural input to empty. Coordinated contraction of the bladder muscle with relaxation of the external urinary sphincter should lead to complete emptying, allowing the bladder to maintain a highly compliant state. Pathologic conditions such as neurogenic bladder dysfunction, posterior urethral valves or partial bladder outlet obstruction (pBOO) can lead to decreased compliance and subsequent impairment of bladder function; however, the details of this loss of compliance are not well understood. In humans, urodynamic studies such as cystometry and uroflow are used to evaluate changes in the compliance of the bladder wall that lead to increases in intraluminal pressure at rest, during filling and voiding. Urodynamic studies evaluating the mechanical changes in the bladder wall in response to outlet obstruction or neurologic insults to the bladder are frequently performed in animal disease models such as rabbit, rats, and guinea pigs (Andersson et al., 2011; Wang et al., 2009; Nagatomi et al., 2004). However, in murine models,
n Correspondence to: Urologic Surgery and Pediatrics, 4102 Doctor's Office Tower, 2200 Children's Way, Nashville, TN 37232, USA. Tel.: +1 615 936 1060; fax: +1 615 936 1061. E-mail address: [email protected] (J.C. Pope IV). 1 These authors contributed equally.
0021-9290/$ - see front matter & 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jbiomech.2013.07.022
urodynamic tests can be difficult even in a normal mouse due to the very small bladder capacity. Additionally, conventional urodynamic tests are subject to confounding factors such as variations in bladder thickness and shape (Nagatomi et al., 2004; Gloeckner et al., 2002). Because the murine animal model offers the benefit of transgenic manipulation to more precisely evaluate specific pathways, it is an attractive animal model for research and therefore needs alternative techniques for accurate bladder wall characterization. Alternative methods such as biomechanical testing may provide an additional approach for characterizing murine bladders. Biomechanical testing is an established method of quantifying the intrinsic mechanical properties of various tissues (Fung, 1993). Furthermore, for planar soft tissues such as the bladder, biaxial mechanical testing has become the gold standard in that it is able to mimic in vivo deformations (Sacks, 2000). Biaxial mechanical testing has been used to accurately characterize many soft tissue including heart valves, myocardium, and blood vessels (Sacks, 2000; Demer and Yin, 1983; Zhou and Fung, 1997; Billiar and Sacks, 2000). Specifically in the bladder, biaxial mechanical testing has been successfully applied in a rat model (Nagatomi et al., 2004; Gloeckner et al., 2002). However, it has not to our knowledge been applied to the murine model, which is substantially smaller and offers the unique benefit of transgenic manipulation which can extend the impact of these mechanical studies to addressing mechanistic questions in a variety of bladder disorders.
J. Chen et al. / Journal of Biomechanics 46 (2013) 2752–2755
In the present proof-of-principle study, we utilized biaxial mechanical testing as an alternative method to measure the mechanical changes of the murine bladder wall due to pBOO. We demonstrate the feasibility of this approach and show strong correlations with conventional cystometry. In addition, we quantified the ECM changes associated with pBOO. We show here for the first time the successful application of biaxial testing on murine bladders. 2. Methods 2.1. General experimental procedure A total of 18, 5–6 week old C57/BL6 age matched female mice underwent oopherectomy to minimize variations in inflammation that may be attenuated by the presence of estrogen (Woo et al., 2011). Two weeks later, mice (n¼12) were subjected to pBOO via urethral ligation. After 4 weeks of pBOO, the mice were sacrificed and the bladders were harvested. Mice that underwent pBOO were noted at time of bladder harvest to have a silk suture present around their urethra to confirm obstruction. Bladders were separated based on a phenotypic appearance at the time of harvest—fibrotic bladder (n ¼6) or distened (n¼ 6). Control mice (n¼ 6) that did not receive pBOO or sham surgery but underwent oopherectomy were sacrificed at the same time as the pBOO mice. pBOO mice (n ¼3) and controls (n¼ 5) underwent urodynamic studies prior to sacrifice. The bladders were harvested and maintained in a modified Kreb's solution for less than 48 h and then subjected to biaxial mechanical testing (Nagatomi et al., 2004). After biaxial mechanical testing, the bladder strips were then evaluated by assay for their relative collagen and elastin content. 2.2. Partial bladder outlet obstruction pBOO was performed via a urethral ligation technique, modifying a previously reported method (Woo et al., 2011). Mice were anesthesized with 2% isoflurane and transurethrally catheterized using a 24 gauge angiocatheter. A midline suprapubic incision was made and the urethra was identified distal to the bladder neck. With minimal dissection, the urethra was ligated with a 4-zero silk suture over the catheter. The catheter was removed and the incision was closed in two layers. Mice were allowed to recover on a warming pad prior to returning to their cages. 2.3. Urodynamics Urodynamics were performed immediately prior to bladder harvest under isoflurane anesthetic, modifying a previously described method (Tanaka et al., 2009). Transurethral urodynamics were performed via a 24 gauge angiocatheter rather than the suprapubic technique. After the catheterization, the bladder was emptied and the catheter was connected to a pressure transducer (Digi-Med, Louisville, Kentucky). The bladder was instilled with sterile saline at a rate of 100 mL/min via a syringe pump (Harvard Apparatus, Holliston, Massachussets). Intravesical pressure was recorded by a pressure analyzer (Digi-Med) to a computer based data acquisition system. Infusion was continued until the bladder pressure reached greater than 55 mmHg as the transurethral catheter did not permit for accurate leak point pressures. A total of three bladder filling cycles were obtained for each mouse with a rest period of at least 5 min between cycles. Bladders were emptied prior to starting the next filling cycle. Bladder capacity was defined as volume at 55 mmHg. Bladder compliance was calculated by the change in volume
Longitudinal
Circumferential
2753
over change in pressure at 50% capacity (V50). Limited samples were successfully analyzed with urodynamics due to technical difficulties. 2.4. Biaxial mechanical testing Rectangular sections of bladder tissue were excised by removing the dome and the trigone (Fig. 1). Four fiducial markers were placed in the center of the specimen to track tissue deformation. The circumferential and longitudinal edges were mounted with two and four stainless steel hooks respectively. Polyester suture loops attached the hooks to the motor carriages. Tissue was preconditioned to 10 cycles and loaded to 50 kPa to simulate extraphysiological conditions before data acquisition; loading stress was determined using the law of Laplace as previously described (Gloeckner et al., 2002). Test protocols maintained a constant ratio of axial stress throughout cycling. Stress– strain curves were generated from data acquisition. 2.5. Collagen and elastin assay Following biaxial mechanical testing, the collagen and elastin concentrations of bladder strips from control (n¼ 5), fibrotic (n¼ 4), and distended (n¼ 2) were obtained using commercially available assays following the manufacturer's instructions (Biocolor Sircol™ Soluble Collagen assay, Biocolor Fastin™ Elastin assay, Accurate Chemical, Westbury, NY) (Nagatomi et al., 2004). Results were expressed in micrograms of collagen and elastin mass and were normalized to the wet weight of each bladder. 2.6. Statistical analysis All data for collagen and elastin assay and biaxial mechanical testing are expressed as a mean 7 SEM. Statistical significance between groups was determined via repeated measures ANOVA for biaxial data and one-way ANOVA for collagen and elastin assays, and pairwise differences were identified using post-hoc Holm–Sidak testing. A p o 0.05 was considered statistically significant.
3. Results Urodynamic studies were performed on control (n¼5), fibrotic (n¼2), and distended (n¼1) mice prior to sacrifice and bladder harvest (Table 1). The median compliance7SD of control mice was 9.1 mL/cmH2O and for fibrotic mice was 7.12 mL/cmH2O. The distended bladder had a compliance of 18.06 mL/cmH2O. Median bladder capacity in the control and fibrotic groups was 210 mL and 170.5 mL respectively; the bladder capacity of the distended bladder was 363.4 mL. Due to the limited data on experimental mice, direct statistical comparison of UDS results between the three groups could not be performed. However fibrotic bladders trended towards having smaller capacity and less compliant bladders than controls, and the single distended bladder had almost twice the compliance of control bladders. Control (n¼6), fibrotic (n¼ 6) and distended (n¼4) bladders were evaluated under biaxial stretch (Fig. 1). Bladder mechanical response was evaluated by comparing axial strain at 50 kPa (Fig. 2A–C). Control bladders exhibited anisotropy, with lesser strains in the longitudinal direction (4.2170.825) than circumferential (15.4073.17). After 4 weeks of pBOO, all mice exhibited a loss of anisotropy. The fibrotic phenotype became less compliant in the circumferential direction (6.3970.861) while becoming more compliant in the longitudinal direction (7.0470.671). The distended phenotype became more compliant in both the circumferential (19.4775.05) and the longitudinal direction (21.2373.76). Additionally, urodynamic plots reveal similar trends with the fibrotic bladder exhibiting lower
Table 1 Median compliance of murine bladders on urodynamics.
Fig. 1. Schematic of murine bladder preparation for biaxial testing. Bladders were cut along the urachus and rectangular samples were cut out along the longitudinal and circumferential directions for biaxial testing.
Group
Compliance (lL/cmH2O)
V50 (lL)
Bladder capacity (lL)
Control (n¼ 5) Fibrotic (n¼ 2) Distended (n¼ 1)
9.1 7.12 18.06
105 85.25 181.7
210 170.5 363.4
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J. Chen et al. / Journal of Biomechanics 46 (2013) 2752–2755
Fig. 2. Biaxial stress–strain curves (A), (B), and (C) and urodynamic plots (D), (E), and (F) of murine bladder. Biaxial stress–strain curves were generated for control (A), fibrotic (B) and distended (C) phenotypes. *p≤0.05 between fibrotic and control in the circumferential direction and #p≤0.05 between distended and control in the longitudinal direction. Urodynamic plots were conducted for control (D), fibrotic (E), and distended (F) phenotypes.
4. Discussion
Table 2 Collagen and elastin assay results. ECM protein
Control (n¼ 5)
Fibrotic (n¼3)
Distended (n ¼2)
Collagen Elastin
38.9 74.9 13.3 74.1
51.5 7 9.4 23.2 7 7.2
44.6 7 8.0 17.4 7 6.3
bladder capacity and the distended bladder exhibiting higher bladder capacity when compared to controls (Fig. 2D–F, Table 1). There was a significant difference in circumferential compliance in the fibrotic phenotype compared with controls (po0.05). The change in longitudinal compliance was significantly different from controls for the distended phenotype only (po0.001). There was also a significant difference between both the circumferential and longitudinal compliance between the distended and the fibrotic phenotypes (p¼0.008, po0.001). Standard curves were generated for the collagen and elastin assays as per protocol with R2 values of 0.9886 and 0.9893 respectively. Data was available for control (n ¼5), fibrotic (n ¼4) and distended (n ¼2) mice (Table 2). Compared to control bladders, the collagen and elastin content of the fibrotic bladders were significantly higher by 32% and 74% respectively (p o0.02, p o0.02). Distended bladders also had higher collagen and elastin content by 15% and 31% respectively, although these did not reach statistical significance.
Previously we have utilized urodynamic studies to evaluate the changes in bladder compliance after pBOO (Woo et al., 2011; Tanaka et al., 2009; Anumanthan et al., 2009). However, urodynamic analysis in mice can be technically challenging due to the small size of their bladder capacity as well as the small size of the tubing needed to catheterize them (Andersson et al., 2011). Cystometic parameters and micturition patterns in rodents also differ from humans, creating obvious translational limitations (Andersson et al., 2011). Rodents, and mice especially, are useful experimental models as they allow for transgenic manipulation, and therefore we sought to identify other methodologies to investigate the changes in the compliance of the mouse bladder after pBOO. Biaxial mechanical testing to evaluate the mechanical properties of the urinary bladder wall has been well described in animal models such as the rat and pig (Nagatomi et al., 2004; Gloeckner et al., 2002; Gilbert et al., 2008; Freytes et al., 2008). Biaxial testing is important for realistic characterization of the in vivo two dimensional stretching that occurs during bladder filling (Wang et al., 2009). In this study, we successfully applied biaxial testing to quantify murine bladders mechanics. With biaxial testing, we identified two distinct phenotypes after pBOO which we labeled distended and fibrotic. These changes correspond to end results commonly seen by urologists from human bladders after being subjected to partial outlet obstruction such as benign prostatic hypertrophy or posterior uretheral valves. Both
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phenotypes reveal a loss of anisotropy with distended bladders becoming more extensible in the longitudinal direction and fibrotic becoming stiffer in the circumferential direction. This alteration in anisotropic behavior due to pBOO corresponds well with current literature which documents anisotropic changes due to bladder dysfunction (Toosi et al., 2008). Additionally, we show that the biaxial mechanical responses observed correspond well with conventional urodynamic analysis demonstrating the effectiveness of biaxial testing as a tool for describing murine bladders mechanics. In addition to overcoming the technical challenges of urodynamic tests, biaxial testing provides intrinsic mechanical information free from confounding effects such as variations in bladder shape and size (Nagatomi et al., 2004; Gloeckner et al., 2002). Biaxial testing also offers users more flexibility in experimental design in that it can control loading patterns which may allow for deeper investigations into ECM organization and composition (Gloeckner et al., 2002). These types of tests may address questions regarding the role of ECM remodeling due to bladder pathology, which is a major inquiry in this field (Jiang et al., 2012). Lastly, biomechanical information can be utilized to develop constitutive models that can simulate bladder responses in a variety of loading conditions (Nagatomi et al., 2004, 2008). To our knowledge this is the first quantification of biaxial mechanical properties of the murine bladder. We demonstrate that biaxial testing is a useful tool in assessing mechanical changes associated with bladder dysfunction following pBOO. The advantages of biaxial testing may allow for a new avenue of investigation that can generate deeper insights into mechanistic questions associated with bladder disorders.
Conflict of interest statement The authors declare that we have no conflict of interest for our submission.
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