Molecular and Cellular Biochemistry

, Volume 346, Issue 1–2, pp 179–186

The effect of in vitro ischemia/reperfusion on contraction, free fatty acid content, phospholipid content, and malondialdehyde levels of the rabbit urinary bladder

  • Florian Radu
  • Robert E. Leggett
  • Catherine Schuler
  • Robert M. Levin


To evaluate the effects of in vitro ischemia/reperfusion on contractile response to field stimulation (FS), free fatty acid (FFA) content, phospholipid (PL) content, and malondialdehyde (MDA) levels of the rabbit urinary bladder. There is significant evidence that ischemia/reperfusion injury is linked to obstructive bladder dysfunction secondary to men with benign prostatic hyperplasia (BPH). Twelve New Zealand White male rabbits were separated into two groups of six rabbits each. Each rabbit was euthanized, and the bladder was surgically removed intact for whole bladder incubation. The bladders in Group 1 received a 3-h incubation under normal oxygenated physiological conditions. These bladders received electrical field stimulation (32 Hz) after 1 and 3 h. The bladders associated with Group 2 received a 1-h incubation under normal oxygenated physiological conditions. At the end of this 1-h period, the bladders were subjected to FS. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium was changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1 h with field stimulations (32 Hz) occurring at 5-min intervals to represent overactive bladder dysfunction. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h period after which the stimulation was repeated. At the end of the experimental period, each bladder was opened longitudinally and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at −80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and supernatant (cytosol) components. PL, FFA, and MDA content were analyzed for each fraction using standard biochemical techniques. The bladder contractile responses decreased during the period of in vitro ischemia and returned to only 30% of control after reperfusion. In vitro ischemia/reperfusion showed the following: (1) There was a modest but significant decrease in the FFA content of the microsomes of the muscle and significant increases in the FFA content of the nuclei and mitochondria of the mucosa. (2) There were decreases in the PL content of the homogenate and microsomes of the muscle and decreases in the PL content of the homogenate, microsomes, and supernatant of the mucosa. (3) Significant increases were observed in the MDA levels of the homogenate, mitochondria, and microsomes of both the muscle and mucosa. The significant increases in the lipid peroxidation of the bladder smooth muscle are consistent with the marked decrease in the contractile ability of the bladder following ischemia/reperfusion. The specific increased lipid peroxidation of the mitochondrial and microsomal components is consistent with the specific dysfunctions of the mitochondria and innervations observed following I/R in earlier published studies. The marked increases in lipid peroxidation in the mucosa associated with the loss of PL and FFA from this component are consistent with the significant dysfunction in both the antiadherence and antipermeability properties of the mucosa and may play a major role in the symptomatic nature of I/R-linked diseases of the bladder.


Rabbit Ischemia Reperfusion Lipid peroxidation Free fatty acid Phospholipid 


  1. 1.
    Zderic SA, Levin RM, Wein AJ (1996) Voiding function and dysfunction: a relevant anatomy, physiology, and pharmacology, and molecular biology. In: Gillenwater JY, Grayhack JT, Howards SS, Duckett JD (eds) Adult and pediatric urology. Mosby Year Book Medical Publishers, Chicago, pp 1159–1219Google Scholar
  2. 2.
    Buttyan R, Chen MW, Levin RM (1997) Animal models of bladder outlet obstruction and molecular insights into the basis for the development of bladder dysfunction. Eur Urol 32(Suppl 1):32–39PubMedGoogle Scholar
  3. 3.
    Mannikarottu A, Kogan B, Levin RM (2005) Ischemic etiology of obstructive bladder dysfunction: a review. Recent Res Dev Mol Cell Biochem 2:15–34Google Scholar
  4. 4.
    Levin RM, Hypolite J, Longhurst PA, Wein AJ (1991) Comparison of the contractile and metabolic effects of muscarinic stimulation with those of KCl. Pharmacology 42:142–150CrossRefPubMedGoogle Scholar
  5. 5.
    Schlager TA, Lohr JA, Hendley JO (1993) Antibacterial activity of the bladder mucosa. Urol Res 21:313–317CrossRefPubMedGoogle Scholar
  6. 6.
    Shen Z, Ding G, Chen Z (2001) Enhancement of bacillus Calmette-Guerin attachment to the bladder wall: experimental study. Zhonghua Wai Ke Za Zhi 39:951–953PubMedGoogle Scholar
  7. 7.
    Ruggieri MR, Hanno PM, Levin RM (1985) Further characterization of bacterial adherence to urinary bladder mucosa: comparison with adherence to anion exchange resin. J Urol 134:1019–1023PubMedGoogle Scholar
  8. 8.
    Ruggieri MR, Hanno PM, Samadzadeh S, Johnson EW, Levin RM (1986) Heparin inhibition of increased bacterial adherence following overdistension, ischemia and partial outlet obstruction of the rabbit urinary bladder. J Urol 136:132–135PubMedGoogle Scholar
  9. 9.
    Shapiro E, Lepor H (1994) The pathophysiology of clinical BPH. Prog Clin Biol Res 386:253–261PubMedGoogle Scholar
  10. 10.
    Emberton M, Cornel EB, Bassi PF, Fourcade RO, Gomez JM, Castro R (2008) Benign prostatic hyperplasia as a progressive disease: a guide to the risk factors and options for medical management. Int J Clin Pract 62:1076–1086CrossRefPubMedGoogle Scholar
  11. 11.
    Shapiro E, Lepor H (1995) Pathophysiology of clinical benign prostatic hyperplasia. Urol Clin North Am 22:285–290PubMedGoogle Scholar
  12. 12.
    Schroder A, Chichester P, Kogan BA, Longhurst PA, Lieb J, Das AK, Levin RM (2001) Effect of chronic bladder outlet obstruction on blood flow of the rabbit bladder. J Urol 165:640–646CrossRefPubMedGoogle Scholar
  13. 13.
    Greenland JE, Brading AF (2001) The effect of bladder outflow obstruction on detrusor blood flow changes during the voiding cycle in conscious pigs. J Urol 165:245–248CrossRefPubMedGoogle Scholar
  14. 14.
    Greenland JE, Hvistendahl JJ, Andersen H, Jorgensen TM, McMurray G, Cortina-Borja M, Brading AF, Frokiaer J (2000) The effect of bladder outlet obstruction on tissue oxygen tension and blood flow in the pig bladder. BJU Int 85:1109–1114CrossRefPubMedGoogle Scholar
  15. 15.
    Ghafar MA, Shabsigh A, Chichester P, Anastasiadis AG, Borow A, Levin RM, Buttyan R (2002) Effects of chronic partial outlet obstruction on blood flow and oxygenation of the rat bladder. J Urol 167:1508–1512CrossRefPubMedGoogle Scholar
  16. 16.
    Azadzoi KM, Radisavljevic ZM, Golabek T, Yalla SV, Siroky MB (2010) Oxidative modification of mitochondrial integrity and nerve fiber density in the ischemic overactive bladder. J Urol 183:362–369CrossRefPubMedGoogle Scholar
  17. 17.
    Levin RM, Haugaard N, O’Connor L, Buttyan R, Das A, Dixon JS, Gosling JA (2000) Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison. Neurourol Urodyn 19:609–629CrossRefPubMedGoogle Scholar
  18. 18.
    Levin RM, Haugaard N, Mogavero L, Leggett RE, Das AK (1999) Biochemical evaluation of obstructive bladder dysfunction in men secondary to BPH: a preliminary report. Urology 53:446–450CrossRefPubMedGoogle Scholar
  19. 19.
    Levin RM, Longhurst PA, Monson FC, Kato K, Wein AJ (1990) Effect of bladder outlet obstruction on the morphology, physiology, and pharmacology of the bladder. Prostate Suppl 3:9–26CrossRefPubMedGoogle Scholar
  20. 20.
    Juan YS, Levin RM, Chuang SM, Hydery T, Li S, Kogan B, Schuler C, Huang CH, Mannikarottu A (2008) The beneficial effect of coenzyme Q10 and lipoic acid on obstructive bladder dysfunction in the rabbit. J Urol 180:2234–2240CrossRefPubMedGoogle Scholar
  21. 21.
    Juan YS, Li S, Levin RM, Kogan BA, Schuler C, Leggett RE, Huang CH, Mannikarottu A (2009) The effect of ischemia/reperfusion on rabbit bladder—role of Rho-kinase and smooth muscle regulatory proteins. Urology 73:1126–1130CrossRefPubMedGoogle Scholar
  22. 22.
    Juan YS, Lin WY, Kalorin C, Kogan BA, Levin RM, Mannikarottu A (2007) The effect of partial bladder outlet obstruction on carbonyl and nitrotyrosine distribution in rabbit bladder. Urology 70:1249–1253CrossRefPubMedGoogle Scholar
  23. 23.
    Kalorin CM, Mannikarottu A, Neumann P, Leggett R, Weisbrot J, Johnson A, Kogan BA, Levin RM (2008) Protein oxidation as a novel biomarker of bladder decompensation. BJU Int 102:495–499CrossRefPubMedGoogle Scholar
  24. 24.
    Hass MA, Levin RM (2003) The role of lipids and lipid metabolites in urinary bladder dysfunction induced by partial outlet obstruction. Adv Exp Med Biol 539:217–237PubMedGoogle Scholar
  25. 25.
    O’Connor LJ, Goldner CW, Lau ST, Hass MA, Levin RM (1999) Effect of partial outflow obstruction on the distribution of free fatty acids and phospholipids in the rabbit bladder. World J Urol 17:261–265CrossRefPubMedGoogle Scholar
  26. 26.
    Fruhwirth GO, Hermetter A (2008) Mediation of apoptosis by oxidized phospholipids. Subcell Biochem 49:351–367CrossRefPubMedGoogle Scholar
  27. 27.
    Hubel CA, Davidge ST, McLaughlin MK (1993) Lipid hydroperoxides potentiate mesenteric artery vasoconstrictor responses. Free Radic Biol Med 14:397–407CrossRefPubMedGoogle Scholar
  28. 28.
    Lin WY, Radu F, Schuler C, Leggett RE, Mannikarottu A, Levin RM (2008) The effect of ovariectomy and oestrogen therapy on the free fatty acid content, endogenous lipase activity, and phospholipid content of the rabbit urinary bladder. BJU Int 102:885–889CrossRefPubMedGoogle Scholar
  29. 29.
    Wade CR, van Rij AM (1988) Plasma thiobarbituric acid reactivity: reaction conditions and the role of iron, antioxidants and lipid peroxy radicals on the quantitation of plasma lipid peroxides. Life Sci 43:1085–1093CrossRefPubMedGoogle Scholar
  30. 30.
    Lin AT, Yang CH, Chen KK, Chang LS (2005) Detrusor mitochondrial lipid peroxidation and superoxide dismutase activity in partial bladder outlet obstruction of rabbits. Neurourol Urodyn 24:282–287CrossRefPubMedGoogle Scholar
  31. 31.
    Walker JM (1994) The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol 32:5–8PubMedGoogle Scholar
  32. 32.
    Verhamme KM, Dieleman JP, Bleumink GS, van der Lei J, Sturkenboom MC, Artibani W, Begaud B, Berges R, Borkowski A, Chappel CR, Costello A, Dobronski P, Farmer RD, Jimenez Cruz F, Jonas U, MacRae K, Pientka L, Rutten FF, van Schayck CP, Speakman MJ, Tiellac P, Tubaro A, Vallencien G, Vela Navarrete R (2002) Incidence and prevalence of lower urinary tract symptoms suggestive of benign prostatic hyperplasia in primary care—the Triumph project. Eur Urol 42:323–328CrossRefPubMedGoogle Scholar
  33. 33.
    Epstein RS, Lydick E, deLabry L, Vokonas PS (1991) Age-related differences in risk factors for prostatectomy for benign prostatic hyperplasia: the VA Normative Aging Study. Urology 38:9–12CrossRefPubMedGoogle Scholar
  34. 34.
    Garraway WM, Collins GN, Lee RJ (1991) High prevalence of benign prostatic hypertrophy in the community. Lancet 338:469–471CrossRefPubMedGoogle Scholar
  35. 35.
    McConnell JD (2004) Bladder responses to obstruction. In: Kirby RS, McConnell JD, Fitzpatrick JM, Roehrborn CG, Boyle P (eds) Textbook of benign prostatic hyperplasia. Informa Healthcare, New York, pp 105–109Google Scholar
  36. 36.
    Monson FC, Sun L, Wein AJ, Levin RM (1995) Hyperplasia in the rabbit bladder urothelium following partial outlet obstruction. Autoradiographic evidence. Mol Cell Biochem 152:167–173CrossRefPubMedGoogle Scholar
  37. 37.
    Monson FC, Wein AJ, Eika B, Murphy M, Levin RM (1994) Stimulation of DNA synthesis in rabbit bladder wall after partial outlet obstruction and acute overdistension. Neurourol Urodyn 13:51–61CrossRefPubMedGoogle Scholar
  38. 38.
    Kato K, Monson FC, Longhurst PA, Wein AJ, Haugaard N, Levin RM (1990) The functional effects of long-term outlet obstruction on the rabbit urinary bladder. J Urol 143:600–606PubMedGoogle Scholar
  39. 39.
    Levin RM, Longhurst PA, Monson FC, Haugaard N, Wein AJ (1993) Experimental studies on bladder outlet obstruction. In: Lepor H, Lawson RK (eds) Prostate diseases. W.B. Saunders, Philadelphia, pp 119–130Google Scholar
  40. 40.
    Agartan CA, Leggett RE, Kogan BA, Levin RM (2007) Effect of age on the response to in vitro ischemia and reperfusion of the rabbit bladder. Urol Int 78:155–159CrossRefPubMedGoogle Scholar
  41. 41.
    Lin WY, Rehfuss A, Schuler C, Levin RM (2008) Effect of co-enzyme Q10 and alpha-lipoic acid on response of rabbit urinary bladder to repetitive stimulation and in vitro ischemia. Urology 72:214–219CrossRefPubMedGoogle Scholar
  42. 42.
    Monson FC, Wein AJ, McKenna BA, Whitmore K, Levin RM (1991) Indigocarmine as a quantitative indicator of urothelial integrity. J Urol 145:842–845PubMedGoogle Scholar
  43. 43.
    Parsons CL, Boychuk D, Jones S, Hurst R, Callahan H (1990) Bladder surface glycosaminoglycans: an epithelial permeability barrier. J Urol 143:139–142PubMedGoogle Scholar
  44. 44.
    Levin RM, Saito M, Wein AJ, Packard D, Cohen A, Haugaard N (1993) Effect of partial outlet obstruction on choline acetyltransferase activity in the rat and rabbit. Neurourol Urodyn 12:255–261CrossRefPubMedGoogle Scholar
  45. 45.
    Gosling JA, Kung LS, Dixon JS, Horan P, Whitbeck C, Levin RM (2000) Correlation between the structure and function of the rabbit urinary bladder following partial outlet obstruction. J Urol 163:1349–1356CrossRefPubMedGoogle Scholar
  46. 46.
    Haugaard N, Potter L, Wein AJ, Levin RM (1992) Effect of partial obstruction of the rabbit urinary bladder on malate dehydrogenase and citrate synthase activity. J Urol 147:1391–1393PubMedGoogle Scholar
  47. 47.
    Wang Z, Wu X, Levin RM, Hudson AP (2001) Loss of mitochondrial DNA in rabbit bladder smooth muscle following partial outlet obstruction results from lack of organellar DNA replication. Mol Urol 5:99–104CrossRefPubMedGoogle Scholar
  48. 48.
    Wu X, Wang Z, Levin RM, Hudson AP (2002) Cloning of COX4 cDNA from the NZ white rabbit and expression of this gene in bladder smooth muscle following partial outlet obstruction. World J Urol 20:255–259PubMedGoogle Scholar
  49. 49.
    Zhao Y, Levin RM, Levin SS, Nevel CA, Haugaard N, Hsu TH, Hudson AP (1994) Partial outlet obstruction of the rabbit bladder results in changes in the mitochondrial genetic system. Mol Cell Biochem 141:47–55CrossRefPubMedGoogle Scholar
  50. 50.
    Nevel-McGarvey CA, Levin RM, Hudson AP (1997) Transcription of mitochondrial and mitochondria-related nuclear genes in rabbit bladder following partial outlet obstruction. Mol Cell Biochem 173:95–102CrossRefPubMedGoogle Scholar
  51. 51.
    Agartan CA, Whitbeck C, Sokol R, Chichester P, Levin RM (2004) Protection of urinary bladder function by grape suspension. Phytother Res 18:1013–1018CrossRefPubMedGoogle Scholar
  52. 52.
    Lin AD, Mannikarottu A, Chaudhry A, Whitbeck C, Kogan BA, Chichester P, Levin RM (2005) Protective effects of grape suspension on in vivo ischaemia/reperfusion of the rabbit bladder. BJU Int 96:1397–1402CrossRefPubMedGoogle Scholar
  53. 53.
    Levin RM, Kawashima Y, Leggett RE, Whitbeck C, Horan P, Mizutani K (2002) Effect of oral Kohki tea on bladder dysfunction induced by severe partial outlet obstruction. J Urol 167:2260–2266CrossRefPubMedGoogle Scholar
  54. 54.
    Levin RM, Leggett RE, Whitbeck C, Matsumoto S, Ohto N, Ikeda T, Mizutani K, Higurashi A (2008) Kohki tea protects the rabbit bladder from ischemia/reperfusion-induced contractile dysfunction. Urol Int 80:425–430CrossRefPubMedGoogle Scholar
  55. 55.
    Levin RM, Leggett RE, Whitbeck C, Murakami T, Kambara T, Aikawa K (2004) Oral Kohki Tea and its protective effect against in vitro ischemic damage to the bladder. Neurourol Urodyn 23:355–360CrossRefPubMedGoogle Scholar
  56. 56.
    Juan YS, Hydery T, Mannikarottu A, Kogan B, Schuler C, Leggett RE, Lin WY, Huang CH, Levin RM (2008) Coenzyme Q10 protect against ischemia/reperfusion induced biochemical and functional changes in rabbit urinary bladder. Mol Cell Biochem 311:73–80CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Florian Radu
    • 1
  • Robert E. Leggett
    • 1
    • 2
  • Catherine Schuler
    • 2
  • Robert M. Levin
    • 1
    • 2
    • 3
  1. 1.Albany College of Pharmacy and Health SciencesAlbanyUSA
  2. 2.Stratton Veterans Administration Medical CenterAlbanyUSA
  3. 3.Division of UrologyAlbany Medical CenterAlbanyUSA

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