A comprehensive visual report of urodynamic study in rats with spinal cord injury

Abstract

Objectives

Spinal cord injury (SCI) is one of the most debilitating and expensive traumatic conditions. Chronic complications after SCI have a particularly negative impact on patients’ functional independence and quality of life. Urodynamic study (UDS) provides a quantitative assessment of lower urinary tract function in these patients. In many fields, animal models are considered a precursor to clinical trials, so research using laboratory animals play a major role in knowledge acquisition.

Materials and methods

Twelve female Wistar rats (13 weeks old, 220–270 g) were divided randomly into 2 groups: sham or SCI. The sham-operated group underwent a laminectomy at T9–T10 without any spinal cord damage, while the SCI group underwent a complete transection at the T9–T10 vertebral level. We performed cystometry in all animals at the end of the fourth week. In this article, we visualize all procedures for catheter implementation and UDS in animals for the first time at Tabriz University of Medical Sciences, Iran, using a locally developed animal UDS device.

Results

The UDS results showed that the bladders in the SCI group were overactive and that peak and baseline pressures increased significantly in rats with SCI when compared with the sham group (p < 0.05 for all). Conversely, significant reductions in bladder compliance and intercontraction interval were observed in the SCI group (p < 0.05 for both).

Conclusions

This comprehensive visual report will be very useful to all researchers in the field of urology. Furthermore, the measurable variables of the UDS device have been described in this study.

1. Introduction

Spinal cord injury (SCI) is one of the most debilitating and expensive traumatic conditions.^[1] The Iranian population has a high prevalence of SCI due to a history of war, multiple accidents, occupational injuries, and natural disasters such as earthquakes. Spinal cord injury is a serious medical condition that is accompanied by functional, psychological, social, and economic disturbances, and acute or long-term secondary medical complications are common among these patients. Chronic complications have a particularly negative impact on patients’ functional independence and quality of life. Therefore, prevention, early detection, and treatment of these complications are critical to improving survival, social participation, and health-related quality of life. A neurogenic bladder is a common condition in neurological diseases.^[2,3] The goal of neurogenic bladder management in patients with spinal cord lesions is to prevent infection, protect renal function by reducing intravesical pressure, and improve quality of life.^[2] Urodynamic study (UDS) is a procedure that evaluates the function of the bladder, sphincters, and urethra.^[4] Urodynamic study is a valuable test for evaluating lower urinary tract symptoms.^[5] Animal models are widely considered the precursor to clinical trials, so research using laboratory animals plays a major role in progressing medical knowledge.^[6] Rodents, most commonly, rats, are widely used to investigate urinary storage and voiding function in both healthy animals and disease models, and the most common used methodology is cystometry.^[7] Guidelines of the International Continence Society suggest that UDS in animal models should be performed in conscious animals to avoid major bias caused by narcotics.^[8] Because of the lack of a UDS device for animal studies in Iran, the design and development of such a device are necessary. In this study, all UDS procedures are described as a visual report in sham rats and an SCI-induced neurogenic bladder model. Previously published animal studies only provided brief explanations of the UDS method and not clearly explained how do that, so researchers confront many difficulties and questions. Therefore, in this method article, we visualize all procedures for catheter implementation and UDS in an SCI-induced neurogenic bladder model and sham animals for the first time at Tabriz University of Medical Sciences, Iran, using a locally fabricated animal UDS device.

2. Materials and methods

2.1. Study design

Twelve female Wistar rats (13 weeks old, 220–270 g) were housed in a specialized animal facility with a 12-hour light-dark cycle and ad libitum access to water and standard food pellets.

All animals were divided into 2 groups (n = 6) as follows: a sham-operated group that underwent a laminectomy at T9–T10 without any spinal cord damage and an SCI group that underwent a complete transection at the T9–T10 vertebrae level. We performed cystometry in all animals at the end of the fourth week. This study was financially supported by the Student Research Committee of Tabriz University of Medical Sciences (grant no. 63880) and approved by the local ethical committee of Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC.1398.306).

2.2. Anesthesia

General anesthesia with isoflurane was used for both SCI induction and bladder catheter implantation due to its short half-life.

1. Animals were placed in a closed box and gassed with 5% isoflurane in pure oxygen (1 L/min).

2. After anesthesia induction, it was maintained at the appropriate level by a small mask (0.8–1 L/min oxygen with 1%–1.5% of isoflurane).

2.3. Spinal cord injury induction

Spinal cord injury induction was performed by complete transection of the spinal cord after laminectomy at the T9–T10 vertebrae level using a sharp surgical blade under a surgical microscope (OMS70; Topcon, Tokyo, Japan), which was described previously.^[1] The exposed spinal cord was precisely transected by a surgical blade several times to ensure complete spinalization. Then, using dissolvable sutures, the incision site was sutured in 2 layers. At the end of the surgery, rats received ciprofloxacin (1 mg/kg) intraperitoneally to prevent urinary tract infection and ketoprofen (5 mg/kg) subcutaneously as an analgesic, both for 3 consecutive days after injury. In addition, saline was administered subcutaneously after surgery. After SCI induction, to void urine, the urinary bladders were expressed manually (Crede’s maneuver) twice a day.

To evaluate the correct site of laminectomy as well as injury site in rats, cone-beam computerized tomography, which is a radiographic imaging method that allows 3-dimensional imaging of hard tissue structures, was performed without a contrast medium by using the NewTom Vg1 Vertical Cone Beam (NewTom, VGI, KVp110, milliampere, LCD 19-inch Philips, Italy). This imaging modality is capable of providing submillimeter resolution (2 linear pairs/mm) images of higher diagnostic quality with shorter scanning time (approximately 60 seconds; Fig. 1).^[9]

Figure 1.

Cone-beam computerized tomography for the evaluation of the correct site of spinal cord injury in rats.

2.4. Urodynamic study

Four weeks after injury, the rats received general anesthesia with isoflurane as per the described protocol for anesthesia induction.

Because of the conducting conscious urodynamic in rats, the catheters were inserted into the dome of the bladder.

1. Surgical instruments were steam sterilized before use. The abdomens of the animals were shaved and disinfected with 70% ethanol before low midline laparotomy to expose the bladder was performed.

2. Under a surgical microscope, a purse-string suture was placed in the bladder dome using a nonabsorbable, monofilament suture (size 6–0).

3. Polyethylene (PE) catheters were heated to form a small cuff for fixation within the bladder.

4. After laparotomy, PE catheters were implanted in the bladder dome with a small incision, and a suture was tightened around the tube’s collar with a surgeon’s knot.

5. Saline was infused through the catheter to ensure no leakage.

6. The catheters were tunneled subcutaneously into the interscapular region using a hollow metal rod.

7. The abdomen was sutured using monofilament sutures.

8. The implanted catheters were flushed with saline to check its permeability.

9. The rats were placed for 5 to 6 hours (including 2 hours for recovery) in a restraining cage.

10. The bladder cannulas were connected to a pressure transducer and infusion pump.

11. Room-temperature saline was infused into the bladder at 10 mL/h, and intravesical pressure was recorded continuously (perfusor compact, B/BRAUN Melsungen AG. Typ 8714827; Fig. 2). Table 1 shows the materials used for UDS.

Figure 2.

Surgical procedure for the implantation of a bladder catheter. (A) Ventral abdominal midline incision. The skin layer is separated from the underlying abdominal muscle and the bladder is exposed. (B) Purse-string suture in the bladder dome with a 6/0 nonabsorbable, monofilament suture. (C) The catheter is pulled into the bladder dome with a small incision. (D, E) The suture is tightened around the tube’s collar with a surgeon’s knot. (F) Saline is infused to check for any catheter leakage. (G, F) The catheter is tunneled subcutaneously to the interscapular region using a hollow metal rod. (I) The rat is placed in a restraining cage and urodynamic setup.

Table 1.

Material used for rat urodynamic test.

Name	Manufacture	Batch number	Comment
PE tube 20 (PE 20)	Intramedic	427406	ID: 0.38 mm OD: 1.09 mm
Isuflorane	Baxter healthcare crop	1228025198	Vial 100 mL USP
Flunixin meglumine	Aburaihan pharmaceutical company
Rat	Tabriz University of Medical Sciences, Iran	N/A	Wistar
Microscissor
6/0 Nonabsorbable, monofilament suture	SUPA medical devices
3/0 Silk, monofilament suture	SUPA medical devices
Pressure transducer	Tabriz University of Medical Sciences, Iran	N/A	–
Infusion pump	(perfusor compact, B/BRAUN Melsungen AG)	Typ 8714827	N/A
Restraining cage	Tabriz University of Medical Sciences, Iran	N/A	15 × 25 cm
Transparent plexiglass funnel	Tajhiz Gostar Omid Iranian	N/A	25 × 30 cm

ID = inner diameter; N/A = not available; OD = outside diameter; PE = polyethylene; USP = United States Pharmacopeia.

3. Results

Cystometric data were collected 4 weeks after SCI in rats. The data were compared between 2 experimental groups. The UDS results showed that the bladders in the SCI group were overactive. The UDS results showed that peak and baseline pressures increased significantly in rats with SCI when compared with the sham group (p < 0.05 for both). Conversely, reductions in bladder compliance and intercontraction interval (ICI) were observed in the SCI group (p < 0.05 for both; Table 2).

Table 2.

Urodynamic parameters (cystometry) in the experimental groups.

UDS parameters/groups, mean ± SD	Peak pressure, cmH2O	Baseline pressure, cmH2O	Bladder compliance, mL/cmH2O	Intercontraction interval, s	Amplitude of contraction, cmH2O
Sham	12.00 ± 2.60	4.05 ± 1.44	0.09 ± 0.007	258.16 ± 23.82	10.04 ± 2.35
SCI	40.00 ± 19.22	24.54 ± 12.76	0.013 ± 0.004	12.65 ± 2.38	25.29 ± 9.94
p	0.008	0.001	0.012	<0.0001	<0.01

Values are presented as the mean ± SD.

SCI = spinal cord injury; SD = standard deviation; UDS = urodynamic study.

4. Discussion

The present study visualized the induction of SCI, the development of a new UDS device at Tabriz University of Medical Sciences, and the results of UDS parameters in a rodent model of SCI-induced neurogenic bladder.

Previous studies reported 2 different UDS methods in rats. In one of them, a cystostomy “Y” catheter (PE50), which is connected to a continuous infusion system and polygraph,^[10] determines the behavior of bladder pressure during the filling and emptying phases as well as the maximum urethral closure pressure. The second method^[11] is done using a “Y” catheter with a 0.64-mm external diameter and 0.5-mm internal diameter, with 2 orifices, that are introduced via the urethra, up to the bladder and connected to the continuous infusion pump system and polygraph. However, in the current study, we used 2 separate PE catheters (PE20 and PE60) in the rat bladder via the dome region after purse-string suture at this site. To perform UDS, the catheter can be inserted in the urethra or bladder. In conscious cystometry, the catheter is inserted in the bladder dome and then fastened by string sutures.^[12] Different sizes of PE tubing have been used in different animal studies.^[7]

The bladder catheter is connected to an infusion pump and pressure transducer through a 3-way stopcock or T tube. The whole system is filled with normal saline. One head of the catheter is connected to a transducer amplifier and another head to a data acquisition system, which in turn connects to a computer during the investigation. Data are stored and further analyzed using specific software. Bladder pressure and urine volume are continuously and synchronously recorded.^[12] Micturition control differs between rodents and humans. Hence, cystometric measurements in rodents have only limited translational value.^[13] Spinal cord injury–induced disabilities in the bladder are the most common and serious problem in patients with neurogenic bladder.^[14] In the study by Frias et al.^[15] on the role of brain-derived neurotrophic factor in SCI-induced neurogenic bladder, detrusor overactivity was evident after 4 weeks in rats with SCI. In addition, significant elevations were reported in the peak pressure, baseline pressure, frequency, and amplitude of urinary function.^[15] In the current study, a comparison of cystometric data between 2 experimental groups showed overactive bladder dysfunction in the SCI group. Furthermore, the baseline and peak pressures were significantly elevated, while bladder compliance and ICI were significantly diminished.

Cystometry in rodents can be conducted in both anesthetized or conscious animals, so subjects can be restrained or freely moving.^[7] There may be obvious differences in UDS parameters among these 2 settings.^[12] Cystometry in anesthetized rats may affect bladder dynamic and may change synaptic transmission dynamics.^[16] In the current study, we used conscious cystometry to reduce and eliminate any potential errors.

5. Conclusions

The UDS results showed that the bladders in the SCI group were overactive. Furthermore, peak and baseline pressures increased significantly in rats with SCI when compared with the sham group. Conversely, reductions in bladder compliance and ICI were observed in the SCI group. All these differences were statistically significant (p < 0.05). In this study, we illustrated all steps of rodent UDS that was conducted for the first time using an animal UDS device that was designed and fabricated at Tabriz University of Medical Sciences.

Conflict of interest statement

The authors declare that they have no conflicts of interest.