Intracavernous Pressure Recording in a Cavernous Nerve Injury Rat Model

Summary

This protocol describes developing a stable bilateral cavernous nerve injury rat model of radical prostatectomy associated with erectile dysfunction and intracavernous pressure measurement.

Abstract

The bilateral cavernous nerve (CN) injury rat model has been extensively used to simulate clinical cavernous nerve injury associated with erectile dysfunction (ED) for evaluating the effect of clinical therapeutic methods. However, the methods of CN injury model construction are flawed and varied in the ED research field. It is CN crush injury that is the most commonly used method in recent years. This study aims to provide a detailed description of the procedure of bilateral CN injury rat model construction and measurement of intracavernous pressure (ICP) recording, providing a reliable and reproducible CN injury rat model. This work successfully developed the CN injury method of hemostat crush injury using a syringe needle as hard support and a hemostat with a rubber sleeve. Also, this method concludes that a voltage of 1.0 V, frequency of 20 Hz, and pulse-width of 5 ms are the optimized stimulation parameters for ICP recording in a bilateral CN injury rat model.

Introduction

ED is one of the common diseases in adult men. It is estimated that the number of ED patients in the world will reach 322 million by 2025¹. One multicenter extensive sample survey in China shows that the proportion of ED caused by pelvic surgery or trauma is about 8%². Despite the continuous improvement of surgical techniques and surgical instruments, the incidence of ED is still high. It has been considered that the development and progression of ED after nerve-sparing radical prostatectomy (RP) contributes to cavernous nerve injury resulting in atrophy of corpus cavernosum smooth muscle, apoptosis of endothelial cells, and pathological remodeling^3,4.

For studying the mechanism of hemodynamics and histopathology changes of CN injury associated with ED, several different types of CN injury animal models have been developed and assessed, including rodent, dog, cat, and monkey^5,6,7. Relying on the advantages in expenditure and reproducibility, the bilateral CN injury rat model has become the most common model for assessing ED after radical pelvic surgery⁸. However, various forms of nerve injury have been reported in numerous literature whose principal differences are nerve injury approaches (crush, freezing, transection, and excision)^9,10,11. Furthermore, the diversity of nerve injury approaches might lead to inconsistency in intracavernous pressure (ICP) recording parameters in the rat model, which determines the accuracy and evaluation of ICP⁸. Nevertheless, there is not a standardized method for inducing nerve injury and recording ICP of the model yet.

Therefore, this study aims to build a more reliable and reproducible bilateral CN injury rat model. This method provides a detailed description of the procedure of model construction and ICP measurement, which might be beneficial to study the mechanisms of ED and develop effective treatments in the future.

Protocol

Fifteen adult male Sprague-Dawley rats (3-month-old) weighing between 300-350 g were used in this study. All animal procedures were performed following the NIH Guidelines for the Care and Use of Laboratory Animals and with the approval of The fifth affiliated hospital of Sun Yat-Sen University Institutional Animal Care and Use Committee. Animals were housed in a comfortable facility with temperature and light controlled.

1. Preparation for surgical procedure materials

1. Prepare the following instruments: scalpel, tissue scissors, thread scissors, bending forceps, tissue forceps, microsurgery forceps, Hartman mosquito hemostatic forceps, sterile surgical sheets, a microneedle holder, rat abdominal retractors, and biological signal acquisition and processing system (see Table of Materials).
   1. Sterilize all surgical instruments before operation. Use alcohol (70% ethanol) wipes to clean the surgical area.
      NOTE: The surgical instruments should be sterilized by alcohol immersion overnight.
2. Prepare the pressure recording system
   1. Connect a 10 mL syringe containing heparin saline and a hypodermic 25 G needle to a 3-way stopcock with a tube (20 cm length). Flush the sterilized tube with sterile heparin saline (200 U/mL).
      ​NOTE: Filling the tube with heparin saline avoids introducing air bubbles into the system.
3. Lift the 25 G needle 20 cm (just the tube length) above the animal operating pad. Then examine the measurement accuracy of the pressure recording system by flushing or tapping.

2. Preparation of the animal

1. Anesthetize rats by sodium pentobarbital (60 mg/kg) intraperitoneal injection (see Table of Materials).
   NOTE: To confirm sufficient depth of anesthesia, an evaluation of spontaneous breathing rhythm and the reflexes of a rat via pinching hind paw was performed.
2. Apply ointment on bilateral eyes to avoid corneal dryness.
3. After confirming a proper anesthetization, shave the lower half of the abdomen, neck, and perineum using an electric shaver. Place the rat in the supine position on a heating pad (37 °C). Wear medical gloves to maintain sterile conditions during surgical procedures.

3. CN isolation and injury procedure

1. Use a scalpel to make a 4 cm incision through the skin at the lower, midline abdominal. To fully expose the bladder and the prostate, use tissue scissors and tissue forceps to make a proper length incision through the subcutaneous fascia, the muscular tissue, and the peritoneum.
2. Use a rat abdominal retractor to enlarge the operative field map. Use absorbent cotton swabs to separate the prostate from the adjacent tissues, such as ligaments.
   NOTE: Major pelvic ganglion (MPG) and CN could be found at one of two the dorsolateral areas of the prostate.
3. Use angled micro scissors to incise the fascia overlying CN 1-6 mm distal to MPG. Then slide a 9-0 suture under the CN with the use of microsurgery forceps.
4. Place a syringe needle (25 G) underneath the CN, 5 mm distal to MPG. Then put the hemostat in the light of the "hemostat tip-syringe needle-nerve-hemostat tip" sandwich structure (Figure 1 and Figure 2).
   NOTE: The syringe needle needs to be ground flat.
5. Apply the hemostat with full tip closure at 5 mm distal from the ganglion for 1 min, then withdraw the hemostat and the syringe needle (Figure 2).
6. Uplift the nerve slightly via a 9-0 suture, and place the hooks of the bipolar electrode (see Table of Materials) around the CN 2-4 mm distal to MPG (Figure 3).
   ​NOTE: Two pairs of MPG and CN wereoperated in the same way.

4. Catheterization of the corpus cavernosum and stimulation of the CN for ICP measurement

1. Flush the tube with sterile heparin saline (200 U/mL) before introducing it into the corpus cavernosum.
2. Hold the 25 G needle and keep the insert direction parallel with the course of the corpus cavernosum (Figure 3).
   NOTE: The tunica albuginea should be stretched to facilitate the insertion.
3. Push the 25 G needle 6 mm into the corpus cavernosum (Figure 3). Flush the tube and press the corpus cavernosum lightly to evaluate the sensitivity of the transducer (Figure 4). To prevent accidental falling off, fix the pipe on the worktable with adhesive tape.
4. Use the following parameters for CN stimulation: voltage at 1.0 V, frequency at 20 Hz, pulse width at 5 ms. Apply 1 min of stimulation with 5 min of rest between the following stimulation.
   ​NOTE: Turn the 3-way stopcock to the pressure transducer channel when starting the measurement.

5. Postoperative Care

1. Place the rats on a warmed pad (37 °C) and monitor them carefully for anesthesia recovery.
2. For postoperative pain control, provide non-steroidal anti-inflammatory drugs (such as Carprofen, 0.5 mg/kg, subcutaneous injection) (see Table of Materials) when the rats fully recover.
3. Move rats to the aseptic cage and monitor them 2 days to evaluate the incisional wound's nourishment state, mental state, and infection.

Results

The surgery procedure produced a typical ICP response curve using this protocol with the recommended stimulation settings. The ICP response curve rises instantly when stimulating the nerve and drops when the stimulation is withdrawn (Figure 5). It is essential to examine the intracavernous pressure line before measuring the ICP, which affects the evaluation of increased ICP values (Figure 4).

As illustrated in Fig...

Discussion

ED is a severe complication of pelvic surgery or trauma. Although undergoing a nerve-sparing operation, the incidence rate of ED is approximately 14-90% in radical prostatectomy (RP)¹². Due to the problematic regeneration of injury CN, the clinical curative effect is less than satisfactory. Thus, a stable CN injury animal model for exploring treatments of ED is essential. Quinlan et al. first reported the CN injury rat model for the study of RP-associated ED¹³. Several stud...

Materials

Name	Company	Catalog Number	Comments
25 G needle	BD Bioscience	367391
Abdominal retractor	RWD Life Science	R22009-01
Animal operating pad	Provided by Guangdong Provincial Key Laboratory of Biomedical Imaging	NA
Bending forceps	RWD Life Science	F12011-10
Biological signal acquisition and processing system	Techman Software	BL-420S
Bipolar electrode	Techman Software	AC0047
Carprofen	Sigma-Aldrich	MFCD00079028
HARTMAN mosquito hemostatic forceps	RWD Life Science	F22002-10
Heparin	Shanghai Aladdin Biochemical Technology	2608411
Micro needle holder	RWD Life Science	F31047-12
Microsurgery forceps	RWD Life Science	F11001-11
Scalpel	RWD Life Science	S32003-12
Sodium pentobarbital	Guangdong Provincial Key Laboratory of Biomedical Imaging	NA
Sprague–Dawley rat	Guangdong Medical Laboratory Animal Center	GDMLAC-035
Thread scissors	RWD Life Science	S15001-11
Tissue forceps	RWD Life Science	F13019-12
Tissue scissors	RWD Life Science	S13029-14