Abstract

Objective: The aim of this work was to investigate urethral sphincter resistance in female rats under normal conditions as well as with chemically induced cystitis.
Materials and Methods: Female rats were anesthetized with urethane (1.2 g/kg, subcutaneously) and submitted to a gradual increase of the intravesical pressure with saline. The effect of increased cumulative doses of NG-L-Arginine methyl ester and L-Arginine, injected via intrathecal and intracerebroventricular was evaluated in these animals.
Results: We verified that the injection through intrathecal of NG-L-Arginine methyl ester and L-Arginine did not affect the normal rats. But rats treated with a single dose of cyclophosphamide (150 mg/kg, intraperitoneally), 20-22 hours following the experiment, and in normal or treated rats when injections of NG-L-Arginine methyl ester (or L-Arginine) were applied in increased cumulative doses via i.c.v. there was a significant increase (decrease) of the magnitude of the dose-dependent responses of the external urethral sphincter, respectively.
Conclusions: Our results suggest that nitric oxide participates in the EUS function, and is regulated by the supraspinal area. Furthermore, the results also indicate that the participation of nitric oxide in the lumbosacral area only occurs in cystitis conditions.

Introduction

The storage and elimination of urine require coordinated function between activities of the smooth muscles of the bladder and the external urethral sphincter (EUS). This activity is integrated in regulating centers found in the supraspinal nuclei, in the spinal cord nuclei and in the peripheral ganglia. These centers regulatethree groups of peripheral fibers: sympathetic fibers that release noradrenaline in the smooth muscle causing contraction in the bladder neck and in the EUS, as well as relaxationof the detrusor; parasympathetic fibers that cause nitrergic inhibitory reaction in the urethral smooth muscle and contractile reaction in the bladder muscle; and lumbosacral somatic fibers, which cause contractile action in the striated muscle of the EUS1. In this way, the relationship between action in both the bladder and urethra is reciprocal during urine filling/storage or voiding/micturition, i.e. during the storage of urine the bladder tonus is reduced, while the EUS tonus is elevated. However, at micturition, the smooth muscle of the bladder contractsand the striated external urethral sphincter relaxes1.

In neuroanatomy studies with rats, various authors have shown that neurons from the micturition pontine center (PMC) ventrolateral area have direct projection on the Onuf’s nuclei. It has been verified that the electrical or chemical stimulation of these nuclei evokes EUS contractions2-5. Furthermore, the striated muscle of the EUS is innervated by the pudendum nerve that arises in the dorsolateral nuclei (DL) of the dorsal horn (Onuf’s nucleus)6 and its long dendrites project dorsolaterally towards the intermediumlateraliscell group (IML), ventromedially towards the central canal and towards the medial parts of the lamina VIII and dorsal commissural L5-S1 of the spinal cord7,8.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), an indicator of the presence of neuronal nitric oxide synthase (nNOS), has been detected in sympathetic and parasympathetic preganglionic neurons, in laminae V-VI, and parasympathetic postganglionic neurons that innervate the urethra and bladder9-14. This fact suggests that the urethral smooth muscle is regulated by sympathetic and parasympathetic preganglionic neurons. Using retrograde axonal tracing studies with pseudorabiae virus in the urethral smooth muscle, labeled cells were identified in the sacral parasympathetic nuclei, in the dorsal commissure, around the central canal of the spinal cord and in theintermediumlateralis area of the L6-S1 segments6,15,16.

The EUS muscles are innervated by somatic cholinergic fibers and non-adrenergic and non-cholinergic fibers associated to neurotransmitter/neuromo-dulator nitric oxide (NO) which induces relaxation of both the smooth and striated muscles of the urethra17,18. Pharmacologically, it has been reported that the N-Nitro-L-Arginine (L-NOARG), a nitric oxide synthase (NOS) inhibitor, reduced the magnitude and duration of the urethral reflex during micturition. This effect was decreased or inhibited after L-Arginine (L-ARG) administration, suggesting the involvement of NO18,19. In addition, the NO as a mediator of relaxation of the smooth muscle and of the urethral striated muscle has been reported in different experimental animals20-22.

Considering thatboth spinal cord (lumbosacral) and supraspinal nuclei have active participation in the EUS function, the purpose of the present research was to determine if NO is involved at spinal cord and supraspinal levels in the EUS resistance in both normal and pathological female ratswhich underwent augmented intravesical pressure.

Methods

Ethical approval
The protocol applied in this experiment was approved by the Committee of Ethics of the Ribeirão Preto Medical School– University of S. Paulo. This current study was developed in the Laboratory of Neurourology – Division of Urology,Ribeirão Preto MedicalSchool – University of S. Paulo. All efforts were made to minimize suffering and reduce the number of animals used in this experimental protocol.The animals were maintained under standard laboratory conditions (at a temperature of 22±2 ºC, in a 12:12 h, light/dark cycle), with free access to food pellets and tap water.

Experimental procedure
Twenty female Wistar rats, weighing 230-260 g, were anesthetized with urethane (1.2 g/kg, subcutaneously).After a midline incision in the lower abdomen (~1.0 cm), the bladder was exposed and a polyethylenecatheter(PE-50; id=0.58 mm, od: 0.96 mm)containing a small collar created with a fire-flared tip was inserted into the bladderdomeand tied with 4-0 silk for bladder filling and pressure recording simultaneously.Thereupon the animal was placed on a special table for this kind of procedure in rats (B. Braun Melsungen AG, type 876030) which permitted a bipedal position (similar to the human position), without provoking muscular stretching. The catheter inserted into the bladder was connected via a three-way stopcock to a pressure transducer (WPI BPcable,Instruments Inc., Akron, OH, USA) to monitor bladder pressure during cystometry. Areceptacle containing 0.9% saline (37 ºC) should be moved slowly along a shaft in perpendicular position which will slowly infuse the bladder by gravity, thus gradually determining the volume/pressure. When the first fluid dropwas expelled, via urethra (leak point pressure), evoked by distension of the bladder, the pressure valuecorresponding to maximum resistance of the EUSwas defined. The correspondent recording was repeated three times at each step of the experiment. The recordeddata were collected and stored in the computer using the Windaqsoftware (DATAQ-DI-720 Instruments Inc., Akron, OH, USA). When the experiments were finished, the animals were euthanatized by exsanguination.

Implantation of the Intrathecal Catheter
For intrathecal (i.t.)catheter implantation, the animals were anesthetized with ethyl ether inhalation; after that a surgical incision (~1.0 cm) was made in the lower lumbar region of the vertebral column, preserving the muscular layer. A special needle was inserted in the intervertebral space between L5-L6. The correct position of the needle in the subarachnoid space was confirmed when the animal showed a tail-flick reflex. Subsequently, a polyethylene-10catheter (PE-10; od = 0.61 mm, id = 0.28 mm) with 10% heparin was inserted through the needle, and its tip was placed in the adjacent site of the spinal cord (L6 through S1); the catheter was tied with 4-0 silk to the subcutaneous tissue. The needle was removed and the external tip of the catheter was sealed by electrocautery. This catheter was placedtemporarily in the subcutaneous space. The skin was then running sutured with 6-0 mononylon thread. The experiment recordings of bladder pressure were performed 4-6 days after implantation of the i.t. catheter. The surgical evaluation of the accurate position of the catheter tip in the spinal cord of the animals was confirmed with dye (methylene blue) administration via i.t. immediately after the experimental recording.
Implantation of the Intracerebroventricular Cannula

For intracerebroventricular (i.c.v.) cannula implantation, the animals were anesthetized with 2.5% tribromoethanol (1.0 μL/100 g of the body weight, intraperitoneally). Additional doses of the anesthetic were administered when necessary. The animals were placed in the stereotaxic frame (David Kopf Instruments, USA) for the implantation of the cannula in the right lateral ventricle (coordinates of 1.0 mm caudal to bregma, 1.6 mm lateral from midline, 3.2 mm ventral from dura) in accordance with Paxinos& Watson Atlas23. The cannula was fixed into the skull with a small screw placed as an anchor. An occlusive metallic mandrill was placed in the cannula to avoid cerebroventricular fluid infection or drainage. Procaine and potassicbenzilpenicillin (8.000 UI, i.m.) was administered immediately after the surgery ended. These animals were maintained in special boxes in the facility with a synchronized light/dark cycle of 12/12 hour, at temperatures of 24 ± 2.0 ºC, with free access to food and water. The experimental recordings of bladder pressure were performed 4-6 days after the i.c.v. cannula implantation.

Surgical evaluation of the accurate position of the catheter tip in animals was confirmed with dye (methylene blue) administration viai.c.v. immediately after the experimental recording.
Intrathecal and Intracerebroventricular Injections
The drugs were injected directly into the subarachnoid space (L6-S1) or into the right lateral ventricle witha volume of 5.0 μL, followed by flushing of 5.0 μL of physiological saline, using a Hamilton’smicrosyringe (10.0 μL) attached to the i.t.catheter or to the i.c.v.cannula, by means of a PE-10 catheter.

Induction of Cystitis
Cystitis was induced by cyclophosphamide (CYP) (150 mg/kg, intraperitoneally). This drug is metabolized to acroleinand excreted in the urine, inducing bladder a hypersensitivity process. The experiments were performed after 45-50 hours of drug administration.

Drug Administration
To evaluate the NO involvement on the EUS of female rats in normal conditions and with cystitis, (a) NG-Nitro-L-Arginine Methyl Ester (L-NAME), a potent non-selective NOS inhibitor, was administered via i.t. or i.c.v., using cumulative doses of 1.0, 3.0 and 10.0 mmol; (b) L-Arginine (L-ARG) drug, a NO precursor, was also administered via i.t. or i.c.v., using cumulative doses of 5.0 and 10.0 mmol. Drugs were dissolved in Milli-Q water. Stock solutions were made and kept at -20 ºC. Subsequent dilutions were made with 0,9% saline. Drug administration was performed slowly (5.0 μL/min), at intervals of 25-30 min. Catheter (PE-10) volume attached to the Hamilton’s microsyringe was approximately 7 μL.
Four groups were created: G1 (control, n=6); G2 (rats treated with CYP, n=4); in both groups (G1 and G2) the drugs were administered via intrathecal; G3 (control, n=4); G4 (rats treated with CYP, n=6): in both groups (G3 and G4) the drugs were administered viaintracerebroventricular.

Drugs
NG-Nitro-L-Arginine Methyl Ester, L-Arginine and Urethane (Lab. Tocris, Ellisville, MO, USA), Cyclophosphamide and Tribromoethanol (Lab. Sigma Chemical, Co. St. Louis, MO, USA), Ethyl Ether (Lab. CAQ, SP, Brazil), Procaine and Potassic Benzilpenicillin (Lab. EMS Indústria,SP, Brazil)were drugs used in the current study.

Methods of Analysis
All data values were expressed as mean plus or minus standard error of the mean, and results were calculated at a percentage of the maximal effect (mean of three recordings) obtained after the drug application.The significance between groups was determined using Student’s t test. Values of P Results We observed that rats treated with CYP showed a thick, tumescent, friable, hemorrhagic bladder wall, and the weight of the bladder was apparently greater than that of untreated rats. There was no significant difference in body weight of these animals before and after catheter (i.t.) or cannula (i.c.v.) implantation. After 60 min of anesthetic administration, the animals were placed in an orthostatic position on the Braun Melsungen table; warm physiological saline solution (37 ºC) placed in a small bottle started to slowly infuse the bladder. The first sign of fluid loss that occurred via urethra was considered as leak point pressure.

Figure 1

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Graphic representation of the intravesical pressure that expresses urethral sphincter resistance values of a female rat after CYP admi- nistration, following the injection of L-NAME and L-ARG via i.t, with the addition of cumulative doses which increase and decrease the urethral sphincter resistance, respectively. Three recordings were performed during the experimental evaluation, 25-30 min after the administration of each drug dose. The top of the recording corres- ponds to the maximum intravesical pressure of the leak point of the fluid via urethra. Represents the beginning of fluid infusion into the bladder and the correspondent increase of the intravesical pressure. Graphic representation of the intravesical pressure that expresses urethral sphincter resistance values of a female rat after CYP admi- nistration, following the injection of L-NAME and L-ARG via i.t, with the addition of cumulative doses which increase and decrease the urethral sphincter resistance, respectively. Three recordings were performed during the experimental evaluation, 25-30 min after the administration of each drug dose. The top of the recording corres- ponds to the maximum intravesical pressure of the leak point of the fluid via urethra. Represents the beginning of fluid infusion into the bladder and the correspondent increase of the intravesical pressure.