WHAT’S YOUR DIAGNOSIS Thomas M. Donnelly, DVM, Column Editor

Sudden bladder distention in a female rat Genaro A. Coria-Avila, DVM, MSc, Erica Barbosa-Vargas, BA & James G. Pfaus, PhD We purchased a batch of 48 female LongEvans rats, weighing 200–250 g, from an accredited animal supplier for studies on sexual behavior. The animals lived in groups of four in large wire-mesh cages in a colony room maintained on a reversed 12:12 h light/ dark cycle at approximately 21º ± 2º C. The rats had ad libitum access to commercial rat feed and water. We anesthetized the females with a mixture of ketamine (50 mg/ml) and xylazine (4 mg/ml), mixed at a ratio of 4:3 respectively, and injected intraperitoneally in a volume of 1 ml/kg of body weight. The anesthetized females then underwent bilateral ovariectomy via a lumbar incision. We allowed the animals one week of post surgical recovery before copulatory trials, after which we induced sexual receptivity by subcutaneous injections of estradiol benzoate (10 µg) 48 hours and progesterone (500 µg) 4 hours before each test. All females underwent this hormonal treatment every four days during 10 copulatory sessions. Due to the hormonal injection schedule, we handled and checked all animals every two days. After nine copulatory sessions, we noticed one rat with an enlarged abdomen, which we determined to be caused by severe bladder distention. This condition was not present two days before. On clinical examination, the rat was dehydrated, but we found no abnormalities in gait, postural reactions, or spinal reflexes. External examination of the vagina did not reveal abnormal secretions either. Very gently, the examiner tried to void the bladder by compressing the anterior border, but it was not successful. Then, on closer examination of the vagina, the examiner found a tightly fitted sperm plug. He used a rat restrainer to hold the rat still and upside down and gently removed the plug with surgical tweezers. The exam-

FIGURE 1 | Exposed abdominal cavity of a 200– 250 g female Long-Evans rat with severe urinary bladder distention.

iner did not detect any abnormal vaginal secretions, but he did notice a foul odor. Once the plug was removed, the examiner voided the bladder very gently. He gave the rat saline subcutaneously to hydrate it and

FIGURE 3 | Detail of the abdominal organs of the rat in Figure 1. Notice the distended ureter pointed to by the tip of tweezers.

FIGURE 2 | Conical tube containing 17 ml of urine taken from the urinary bladder of the rat in Figure 1. Notice the white sediment at the bottom of the tube. Its presence is abnormal.

then returned the animal to its home cage. Two days later the same clinical signs were present (i.e., extreme bladder distention and dehydration) but this time the examiner did not find a vaginal plug After a tentative diagnosis, we decided to euthanize the rat with an overdose of sodium pentobarbital. The gross necropsy revealed a

FIGURE 4 | Photomicrograph (40X) of unstained urine sediment from Figure 2. Notice the small rounded crystals with pointed protuberances. We considered the crystals to be ammonium urate.

Center for Studies in Behavioral Neurobiology, Psychology Department, Concordia University, Montreal, Quebec, Canada. Correspondence should be addressed to G.A.C. ([email protected]).

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clean vagina with no inflammatory signs. The uterus was not affected and no secretions were detected. The remarkable sign was a greatly distended bladder (Fig. 1) with approximately 17 ml of clear urine that contained abundant white sand-like sediments and small traces of blood (Fig. 2). Kidneys were normal, but the ureters and renal pelvis of both kidneys were highly distended with

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urine (Fig. 3). We stored samples of urine and the vaginal plug in a 4˚C refrigerator. We used a pH meter strip to measure the acidity of the urine, which indicated an abnormal pH of 6.5 (normal pH is 7.3–8.5). Microscopic examination of the vaginal plug was unremarkable, but microscopic examination (40X) of the urine sediments (Fig. 4) indicated the presence of crystals resembling mainly those of

ammonium urate. They appeared as rounded crystals with pointed protuberances along the surface. Based on the clinical signs and necropsy, what do you consider is the cause of the rat’s condition? How frequent is this problem?

What’s your diagnosis?

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Diagnosis | Urolithiasis The rat had urolithiasis, or bladder stones, that obstructed the urethra, preventing urination. Urolithiasis is a heterogeneous disorder, with varying chemical composition and pathophysiologic background1. Multiple predisposing factors may also initiate urolithiasis 1. In humans, calculi develop from a variety of metabolic or environmental disturbances, including varying forms of hypercalciuria, hypocitraturia, undue urinary acidity, hyperuricosuria, hyperoxaluria, infection with urease-producing organisms, and cystinuria. While spontaneous urinary calculi are common in dogs, cats, rabbits and guinea pigs, they are rare in rats. Diet and the predisposition of certain inbred strains (e.g., DA and ACI strains) are associated with spontaneous urolithiasis in rats2–6. Experimentally, unusual dietary supplements, a large number of chemicals, and inherited metabolic defects in inbred strains (e.g., genetic hypercalciuric stoneforming (GHS) rat7) are associated with urolithiasis in rats8–10. We considered two possible mechanisms to explain the unusual clinical presentation in this rat. The first possibility is that the bladder distention was caused by an acute obstruction of the bladder exit, due to a physical barrier made up of sand-like bladder stones that precipitated by gravity. This event prevented micturition, which usually helps to rinse out the vagina after copulation, including those remnants of copulatory plugs not dislodged by male intromissions11. This explanation suggests that the copulatory plug found in the vagina of the rat was secondary to obstructive urolithiasis.

The second possibility is that a urinary tract infection contributed to urolithiasis formation. Urinary tract infection is a known risk factor in spontaneously occurring urolithiasis in cats, dogs, rabbits, and guinea pigs, and in experimentally induced urolithiasis in rats12–15. If the female rat received a sperm plug that was not properly dislodged from the vagina, it could become a focus of infection and this may have contributed to formation of urolithiasis. During the procedure of vaginal plug removal, the rat was also held still with the pelvic area directed upwards, which we assumed helped remove the bladder stones from the urethral sphincter and facilitate manual voiding. Sediments may have blocked the exit immediately after, since two days later the bladder was distended again. Urolithiasis in rats has also been reported to be induced by autologous or homologous sperm experimentally deposited in the bladder neck of males6. We considered that following several ejaculations, some sperm might have reached the bladder, initiating the formation of stones. Researchers have observed a significant negative correlation between urinary oxalate and plasma estradiol/testosterone ratio16–19. In one study, estradiol-implanted rats, whether male or female, intact or castrated, developed kidney crystal deposits18. Yet in the same study, testosterone-implanted rats had a 43–88% rate of kidney calcium oxalate crystal deposition. These results indicate that androgens increase and estrogens decrease urinary oxalate excretion, plasma oxalate concentration, and kidney calcium oxalate

crystal deposition. However, Matsushita20 found the administration of 1 mg estradiol every other week for 12 weeks to female rats resulted in a 50% incidence of struvite bladder stones. Matsushita suggested there was a predisposition to urinary tract infection due to metaplasia of the transitional bladder epithelium by estrogen to a stratified or pseudostratified columnar epithelium20. Enterobacteria, staphylococcus, and streptococcus were identified as infecting organisms. Acute urolithiasis and urethral obstruction in rats can be accompanied by difficulty in urinating or complete absence of urination, with bladder distention, foul odor, and some behavioral signs, like constant licking of the anogenital area. Complete bladder obstruction can cause uremia, leading to depression, anorexia, dehydration, and sometimes coma and death. The spontaneous formation of bladder stones in domestic animals appears to be associated with hypercalciuria, urine retention, and bladder infection. Calculi do not appear to form unless the concentration of urine components is sufficiently high. Additionally, the transit of crystals within the urinary tract has to be slowed down and urine pH modified to facilitate stone formation1. 1.

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LAB ANIMAL welcomes reader contributions to “What’s Your Diagnosis” in case history/ diagnosis format. Submissions should include two to five illustrations or photos. Please email manuscripts to [email protected]. Selections are made on the basis of relevance and interest to readers. Please refer to the Guide to Authors for information on submission of digital figures.

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Kahn, C.M. & Line, S. in The Merck Veterinary Manual (Online book). (2003). http:// www.merckvetmanual.com/mvm/index. jsp?cfile=htm/bc/130412.htm. Klurfeld, D.M. Kidney and bladder stones in rodents fed purified diets. J. Nutr. 132(12), 3784 (2002). Kuhlmann, E.T. & Longnecker, D.S. Urinary calculi in Lewis and Wistar rats. Lab. Anim. Sci. 34(3), 299–302 (1984). Kunstyr, I., Naumann, S. & Werner, S. Urinary bladder stones in some inbred and hybrid strains of “SPF” rats. Z. Versuchstierkd. 23(3), 181 (1981). Kunstyr, I., Naumann, S. & Werner, J. Urolithiasis in female inbred SPF rats. Possible predisposition of DA and ACI strains. Z. Versuchstierkd. 24(4), 214–218 (1982). Paterson, M. Urolithiasis in the SpragueDawley rat. Lab. Anim. 13(1), 17–20 (1979).

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Bushinsky, D.A. Genetic hypercalciuric stoneforming rats. Curr. Opin. Nephrol. Hypertens. 8(4), 479–488 (1999). Khan, S.R. & Hackett, R.L. Calcium oxalate urolithiasis in the rat: is it a model for human stone disease? A review of recent literature. Scan. Electron Microsc. Pt 2,: 759–774 (1985). Linnemann, U., Kuch, P. & Schwille, P.O. Ammonium urate urolithiasis in the rat with portocaval shunt--some aspects of mineral metabolism and urine composition. Urol. Res. 14(6), 319–322 (1986). Khan, S.R. Animal models of kidney stone formation: an analysis. World J. Urol. 15(4), 236–243 (1997). Wallach, S.J. & Hart, B.L. The role of the striated penile muscles of the male rat in seminal plug dislodgement and deposition. Physiol. Behav. 31(6), 815–821 (1983). Ruutu, M. & Lehtonen, T. Urinary tract complications in spinal cord injury patients. Ann. Chir. Gynaecol. 73(6), 325–330 (1984). Morrisey, J.K. Urinary tract disorders in rabbits and guinea pigs. Exotic Pet Pract. 2(1), 1–2 (1997). Osborne, C.A. & Lulich, J.P. Risk and protective factors for urolithiasis. What do they mean? Vet. Clin. North Am. Small Anim. Pract. 29(1), 39–43 (1999). Linsenmeyer, T.A. & Ottenweller, J. Bladder stones following SCI in the Sprague-Dawley rat. J. Spinal Cord Med. 26(1), 65–68 (2003). Lee, Y.H. et al. Determinant role of testosterone in the pathogenesis of urolithiasis in rats. J. Urol. 147(4), 1134–1138 (1992). Terada, S., Suzuki, N., Uchide, K., Akasofu, K. & Nishida, E. Effect of testosterone on the development of bladder tumors and calculi in female rats. Gyneco. Obstet. Invest. 34(2), 105–110 (1992). Fan, J., Chandhoke, P.S. & Grampsas, S.A. Role of sex hormones in experimental calcium oxalate nephrolithiasis. J. Am. Soc. Nephrol. 10 (Suppl 14), S376–S380 (1999). Iguchi, M., Takamura, C., Umekawa, T., Kurita, T. & Kohri, K. Inhibitory effects of female sex hormones on urinary stone formation in rats. Kidney Int. 56(2), 479–485 (1999). Matsushita, K. Effect of estrogen on the formation of struvite calculi in female rats. Urol. Int. 39(5), 303–307 (1984).

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