Korean J Urol KJU Korean Journal of Urology 2005-6737 2005-6745 The Korean Urological Association 10.4111/kju.2009.50.8.797 Original Article Basic and Translational Research Protective Effect of Hyaluronic Acid on Cyclophosphamide-Induced Cystitis in Rats Lee Jung Won Kim Youn Wha 1 Huh Jung Sik 2 Lee Sun-Ju Department of Urology, School of Medicine, Kyung Hee University, Seoul, Korea. Department of Pathology, School of Medicine, Kyung Hee University, Seoul, Korea. Department of Urology, School of Medicine, Cheju National University, Jeju, Korea. Correspondence to: Sun-Ju Lee. Department of Urology, School of Medicine, Kyung Hee University, 1, Hoegi-dong, Dongdaemun-gu, Seoul 130-702, Korea. TEL: 02-958-8535, FAX: 02-959-6048, sjlee@khu.ac.kr 08 2009 19 08 2009 50 8 797 804 02 04 2009 17 07 2009 Copyright © The Korean Urological Association, 2009 2009 Purpose

We investigated the effect and duration of hyaluronic acid (HA) on the bladder of rats with cyclophosphamide (CYP)-induced cystitis.

 Materials and Methods

CYP (250 mg/kg) was injected intraperitoneally in female Sprague-Dawley rats to induce cystitis. HA (0.5%) was instilled intravesically. First, the rats were separated into 2 groups for urodynamic study (CYP group, CYP+HA group), and we assessed the inter-contraction interval, maximal voiding pressure (MVP), and pressure threshold of the 2 groups. Second, the rats were separated into 3 groups to assess the duration of effect of HA (CYP group; HA-1 group: HA was instilled 1 day before injection of CYP; HA-3 group: HA was instilled 3 days before injection of CYP), and we assessed the voiding interval for 7 days after confining the rats in metabolic cages.

 Results

The inter-contraction interval of the CYP+HA group (236.0±18.3 min) was significantly longer than that of the CYP group (178.0±13.4 min; p<0.03), but the MVPs of both groups did not differ significantly (32.3±1.2 vs. 34.0±2.4 cmH2O, respectively; p=0.56). The pressure threshold of the CYP+HA group (9.9±0.5 cmH2O) was significantly longer than that of the CYP group (13.2±0.7 cmH2O; p=0.002). The voiding interval decreased in each of the 3 groups according to the time after cystitis had been induced. The voiding interval was maximally decreased on the second day and returned to the pre-CYP injection level on the fifth day in each group. There was a significant difference in voiding interval among the 3 groups on the second day (p<0.05). There was no significant difference between the CYP+HA-1 and CYP+HA-3 groups on the second day (p>0.05).

 Conclusions

HA has an effect of increasing the voiding interval in rats with CYP-induced cystitis, and the duration of effect is concluded to be 5 days.

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(A) Inter-contraction interval (ICI) after instillation of cyclophosphamide (CYP). ICI of CYP+hyaluronic acid intravesical instillation (HA) group was significantly longer than the ICI of CYP group (p=0.03)a. (B) Maximal voiding pressure (MVP) after instillation of cyclophosphamide. There was no significant difference of MVP between CYP group and HA+CYP group (p=0.56)b. (C) Pressure threshold (PT) after instillation of cyclophosphamide. There was significant difference of PT between the HA+CYP group and the CYP group (p=0.002)c. No. of each animal group (CYP, CYP+HA)=11, NS: not significant.

Voiding interval (VI) changes after intraperitoneal injection of cyclophosphamide (CYP), checked in the metabolic cage. Voiding interval between pre-CYP injection and each 0-8, 9-24, 48, 72 hour group was decreased significantly (ap<0.05), however, 96, 120 hour group was not decreased significantly (bp>0.05, paired t-test) in CYP, hyaluronic acid intravesical instillation (HA)-1, and HA-3 group. On the second day after CYP injection, the VI of the CYP group was significantly shorter than HA-1 group (n=6) and HA-3 group (n=6) (cp<0.05), however, there was no difference between HA-1 and HA-3 group in VI. There was no significant difference in VI between the HA-3 group and the CYP group after the third day (p>0.05). HA-1: hyaluronic acid intravesical instillation a day before CYP intraperitoneal injection, HA-3: hyaluronic acid intravesical instillation 3 days before CYP intraperitoneal injection.

(A, B) Control group: normal urothelium in rat bladder. (C, D) Cyclophosphamide (CYP) group (n=11): bladder 1 day after injection of CYP. Totally ulcerated urothelium with marked edema in the lamina propria. (E, F) CYP+hyaluronic acid intravesical instillation (HA) group (n=11): bladder 1 day after injection of cyclophosphamide. Focally denuded urothelium with focal edema and hemorrhage in the lamina propria (H&E stain, A, C, E ×40; B, D, F ×200).

Photomicrographs of bladder 7 days after injection of cyclophosphamide (CYP). After injection, voiding intervals of rats were checked for 5 days in the metabolic cage. One week after injection, changes in the bladders were confirmed by biopsy. (A, B) CYP group (n=6): hyperplastic changes in the urothelium and severe hemorrhages in the lamina propria and muscle layer. (C, D) hyaluronic acid intravesical instillation (HA)-1 group (n=6): hyperplastic changes in the urothelium and focal infiltration of neutrophils (less than 10/HPF) and lymphocytes (less than 10/HPF) in the lamina propria. (E, F) HA-3 group (n=6): hyperplastic changes in the urothelium and diffuse neutrophilic (more than 50/HPF) and lymphocytic (more than 100/HPF) infiltration in the lamina propria (H&E stain, A, C, E ×40; B, D, F ×200). HPF: high power field (count under ×400).

Changes in the voiding interval (VI) after intraperitoneal injection of cyclophosphamide in the 3 groups (CYP, HA-1, HA-3)

CYP: cyclophosphamide, HA-1: hyaluronic acid intravesical instillation a day before CYP intraperitoneal injection, HA-3: hyaluronic acid intravesicalinstillation 3 days before CYP intraperitoneal injection, PRE: VI before CYP intraperitoneal injection as a control. Each VI represents Mean±SE, a: performed by Duncan multivariated analysis (CYP vs. HA-1 and CYP vs. HA-2 have a significant difference, however, HA-1 vs. HA-3 has no significant difference)