Expression and response of acid-sensing ion channels in urinary bladder to cyclophosphamide-induced cystitis

The expression of acid-sensing ion channel (ASIC) isoforms, ASIC1, ASIC2a, and ASIC3, was examined in the urinary bladder after cyclophosphamide (CYP)-induced cystitis of varying duration (4 h, 48 h, and chronic). Immunohistochemical, Western blot, and quantitative PCR approaches were used to evaluate channel expression and effects of CYP-induced cystitis in whole urinary bladder and split-bladder preparations from control (no inflammation) and CYP-treated rats. Quantitative PCR demonstrated significant ( P ≤ 0.01) increases in ASIC2a and ASIC3 transcripts with CYP-induced cystitis (48 h and chronic) in the urothelium but no changes (e.g., ASIC3) or modest changes (e.g., ASIC2a) in detrusor smooth muscle. ASIC1 mRNA expression in the urothelium or detrusor was not affected by CYP-induced cystitis. Immunohistochemistry for ASIC2a and ASIC3 protein expression revealed significant ( P ≤ 0.01) increases in ASIC immunoreactivity in the urothelium and suburothelial plexus with CYP-induced cystitis at all time points examined. Western blotting for ASIC2a and ASIC3 protein expression was complementary and revealed significant ( P ≤ 0.01) increases in ASIC immunoreactivity. For the first time, these studies demonstrate that CYP-induced cystitis alters ASIC2a and ASIC3 expression in the urinary bladder; ASIC1 transcript expression is not altered by CYP-induced cystitis. Future studies are necessary to determine ASIC isoform contributions to micturition reflexes in control and inflamed urinary bladder.

Differential effects of urethane and isoflurane on external urethral sphincter electromyography and cystometry in rats

Urethane is a common and often preferred anesthetic agent for urodynamic recordings in rats, but its use is often restricted to terminal procedures because of a prolonged duration of action and potentially toxic effects. When urodynamic recordings are part of survival procedures in rodent experimental models, inhalation anesthetics, such as isoflurane, are frequently used and generally well tolerated. In this study, we compared the effects of urethane and isoflurane on lower urinary tract function. For this purpose, adult female rats were anesthetized by subcutaneous administration of urethane ( n = 6) or by inhalation of isoflurane ( n = 5). Micturition reflexes were assessed by concurrent cystometrogram and external urethral sphincter (EUS) electromyography (EMG) recordings to determine bladder contractile properties, EUS activation patterns, and the coordination between bladder contractions and EUS activation. Compared with urethane, isoflurane reduced frequency of bursts, firing frequency, and amplitude of EUS EMG activity during voiding as well as the EUS EMG amplitude during the bladder filling phase. Isoflurane also prolonged the bladder intercontractile intervals. Other several key functional aspects of the bladder contractile properties as well as the coordination between bladder contractions were not different between the two experimental groups. We conclude that micturition reflexes were differentially affected by isoflurane and urethane. Specifically, isoflurane exhibited a significant suppression of the EUS EMG activity and prolonged the bladder intercontractile intervals compared with urethane. We suggest that these anesthetic properties be taken into consideration during the experimental design and interpretation of urodynamic recordings in rodent models.

Expression of corticotropin-releasing factor and CRF receptors in micturition pathways after cyclophosphamide-induced cystitis

Corticotropin-releasing factor (CRF) is a prominent neuropeptide involved in micturition reflexes, and different roles in these reflexes have been suggested. These studies examined the expression of CRF in the urinary bladder and lumbosacral sacral parasympathetic nucleus (SPN) in response to cyclophosphamide (CYP)-induced cystitis (4 h, 48 h, or chronic) in rats. The expression of CRF receptors, CRF1 and CRF2, was examined in urinary bladder from control and CYP-treated rats. Urinary bladder and lumbosacral spinal cord were harvested from rats killed by isoflurane (4%) and thoracotomy. CRF protein expression in whole urinary bladders significantly ( P ≤ 0.01) increased with 48 h or chronic CYP treatment. CRF immunoreactivity (IR) was increased significantly ( P ≤ 0.01) in the urothelium and SPN after CYP treatment. CRF IR nerve fibers increased in density in the suburothelial plexus and detrusor smooth muscle whole mounts with CYP-induced cystitis. CRF2 receptor transcript was expressed in the urothelium or detrusor smooth muscle, and CRF2 receptor expression increased in whole bladder with CYP-treatment, whereas no CRF1 receptor transcript was expressed in either urothelium or detrusor. Immunohistochemical studies demonstrated CRF2 IR in urinary bladder nerve fibers and urothelial cells from control animals, whereas no CRF1 IR was observed. These studies demonstrated changes in the expression of CRF in urinary bladder and SPN region with CYP-induced cystitis and CRF receptor (CRF2) expression in nerve fibers and urothelium in control rats. CRF may contribute to urinary bladder overactivity and altered sensory processing with CYP-induced cystitis.