Anchorage of Twisted Testis: A Rat Model of Ischemia-Reperfusion Injury of the Testis

Summary

This article describes a step-by-step procedure for creating a model of testicular ischemia/reperfusion for the production of a reproducible model of testicular torsion in the study of the pathological mechanisms of ischemia-reperfusion injury in the testis.

Abstract

Ischemia-reperfusion (IR) injuries are associated with several pathologies, including testicular injury. Earlier animal models of testicular IR, which involves twisting the spermatic cord to achieve ischemia, are fraught with many technical difficulties that result in the inability to reproduce the model consistently. Hence, we present a simple method for inducing testicular IR in a rat model that can easily be replicated.

This method involves the separation of the gubernaculum from the testis, creation of a scrotal pouch, twisting of the testis 720° clockwise at its lower pole, and anchorage of the testis to the base of the scrotum. This model effectively constricts testicular vessels and induces testicular ischemia. After 1 h of ischemia, the anchoring stitch is removed and the testis untwisted. The cord and its accompanying vessels are inspected to ensure patency. After IR, the testis is collected and sectioned for histopathological evaluation.

Testicular histology was examined for focal hemorrhagic lesions, vascular congestion, widened interstitial space, and infiltration of inflammatory cells. Furthermore, markers of oxidative stress (malondialdehyde, reduced glutathione, catalase, and superoxide dismutase), inflammation (MPO, TNF-α, IL-1β), and apoptosis (caspase 3 expression) were assayed. This method provides a simpler and easily reproducible model to study the pathophysiology of testicular IR injury. More so, this model opens a window for exploring potential therapeutic agents in the management of testicular IR injury.

Introduction

Torsion of the testis (TT) is an acute urological emergency in which the spermatic cord, along with its accompanying vessels, twists, resulting in ischemia of the testis. It is characterized by severe abdominal pain, nausea, and vomiting¹. The ischemia, if unresolved by timely detorsion, results in necrosis and loss of the testis concerned². However, timely surgical intervention by untwisting and restoration of blood flow to the testis results in ischemia-reperfusion injury (IRI), which results in histological and biochemical disruption of the testis and its functions^3,4.

Animal models of TT have been used extensively to investigate the mechanisms of IRI, which invariably results from attempts to prevent TT from progressing to necrosis^5,6. There are two models of TT, spermatic cord clamping and cord twisting. Both are performed under sterile conditions and adequate anesthesia. Common anesthetic agents used include ketamine alone or in combination with xylazine⁷. Other commonly used anesthetic agents are urethane and thiopental sodium, usually administered intraperitoneally⁷. Nonetheless, both models have their limitations.

While direct clamping of the testicular artery with microvascular clips^8,9 has been used as a means of inducing ischemia for a determined length of time (usually 1 h) before induction of reperfusion by removal of the clamps, the method does not accurately mimic the clinical appearance of torsion of the testis¹⁰. Further, though surgical twisting of the spermatic cord to achieve ischemia^11,12 is more representative of the clinical presentation, it is fraught with many technical difficulties that have not been addressed by the short descriptions encountered in the methods section of articles on the subject.

A clear technical difficulty often encountered has been that of returning and anchoring the testis to the floor of the scrotum to prevent untwisting during the period of ischemia. While some investigators do not mention the anchoring of the testis to the scrotal wall^13,14, others prescribe anchoring the testis to the scrotal wall by stitching the tunica albuginea to it^15,16,17. The descriptions of the process of anchoring the testis to the scrotal wall have, by and large, been very brief and non-explicit^7,18. Attempts at duplicating the procedures described in methods sections of previous articles have thus been met with serious practical difficulties, such as injury to the testis by perforating the tunica albuginea and being unable to return the testis to the scrotum without exerting undue pressure. Moreover, in practical terms, the return of the testis to the scrotal sac after inducing torsion has been a major challenge in our attempts to surgically induce experimental TT. Such technical difficulties have thus resulted in the inability to consistently reproduce the model among different investigators.

Hence, the procedure described in this study for creating a model of TT in rats identifies the technical difficulties encountered in previous models and offers solutions for them. This model provides a simple technique that is easily reproducible and mimics human experience. The model is, therefore, a reliable and reproducible starting point in the study of the pathological mechanisms arising from IRI.

Protocol

The study was approved by the Ethics Review Committee of the Faculty of Basic Medical Sciences, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria (ERCFBMSLAUTECH:033/05/2024). Twenty male albino Wistar rats, aged 10-12 weeks, and weighing 180 ± 10 g were used for this study. They were acclimated for 2 weeks and then, randomly allotted into either the sham-operated group (SO) or the torsion/detorsion group (T/D) (n = 10 rats per group). The SO rats underwent a similar surgery, just like the T/D rats, but the testes were not twisted or anchored to induce T/D.

1. Preoperative preparation and anesthesia

1. Expose the animals (SO and T/D groups) to an overnight 12 h fast before surgery.
2. On the day of surgery, anesthetize the animals (SO and T/D groups) with a combination of ketamine 50 mg/kg and xylazine 10 mg/kg intraperitoneally, and confirm the adequacy and depth of anesthesia by observing the lack of response of the animals to paw pad pressure and light stimulation of the whiskers.
3. Shave the left scrotal, perineal, and inguinal areas of the animals (SO and T/D groups) and then clean them with methylated spirit and povidone antiseptic solution or follow local institutional guidelines on disinfection.

2. Induction of ischemia of the testis

1. Mobilization of the testis
   1. Gently exert downward pressure on the abdomen to push the left testis (of both the SO andT/D groups) into the scrotum.
   2. Firmly grasp the left testis (of both the SO andT/D groups) and make a high scrotal incision, ~1.5 cm in length, to expose the underlying testis within the tunica vaginalis (TV).
   3. Locate the lower pole of the testis (of both the SO andT/D groups), specifically the cauda epididymis, and make a nick in the overlying TV with small dissecting scissors.
   4. Place the scissors under the TV and widen them slightly to show the two edges of the TV, and then grasp the edges on either side with curved mosquito artery forceps. This applies to both the SO andT/D groups.
      NOTE: This step is very important as it establishes a tissue plane that will enable the easy return of the testis to the scrotum after induction of torsion.
   5. Extend the incision (of both the SO andT/D groups) in the TV proximally by ~0.5 cm.
   6. Exert gentle pressure on the lower abdomen to exteriorize the testis (of both the SO and T/D groups).
   7. For only the T/D group, mobilize the testis and identify the gubernaculum testis, then cut it to detach the testis from the floor of the scrotum.
      NOTE: This step is important as it allows the testis to be twisted. The gubernaculum is identified as the ligament that extends from the caudal end of the testis to the inguinal canal and ends in a knob-like expansion^19,20.
   8. Grasp the testis (of the T/D animals) at its lower pole (where the cauda epididymis is located), and twist it 720° clockwise and record the time (marking the onset of ischemia).
2. Anchoring the ischemic testis
   1. Create a scrotal pouch by holding the twisted testis (of the T/D animals) in place and pushing long dissecting scissors downwards along the tissue plane under the TV into the scrotum to form a pouch.
   2. For only the T/D animals, insert the anchoring stitch by holding the scissors in place and inserting an anchoring stitch using chromic 2-0 suture from the outside of the lowest point of the scrotal pouch created earlier. Insert the stitch into the connective tissue on the lower pole of the testis and bring it back down into the pouch and outside it next to the initial point of entry.
   3. Anchor the testis to the base of the scrotum by pulling the torsed testis (of the T/D animals) into the scrotum and gently exerting downward traction on the two ends of the suture, and then tie the two ends of the suture firmly on the scrotal skin.
   4. Close the scrotal incision (of the SO and T/D animals) with a continuous purse-string suture using chromic 2-0.
      NOTE: This is recommended for easy removal when reperfusion is to be done.

3. Biochemical and histopathological investigations

NOTE: Ensure that the investigators carrying out the biochemical assays and histopathological examinations are blinded to the study protocol.

1.  At the end of the experimental period, animals should be culled by euthanasia after a 12 h fast overnight (50 mg/kg of ketamine and 10 mg/kg of xylazine i.p.) to remove the testes.
2. Remove and separate the left testes from adhering structures.
3. Homogenize five out of 10 testicular tissues from each group in homogenization buffer (0.25 M sucrose, 0.5 mM EDTA, 5 mM histidine, and PI dissolved in PBS, pH 7.4), and fix the remaining five testicular tissues in Bouin's solution for histological evaluation.
4. Spin the homogenates at 10,000 × g for 15 min at 4 °C in a cold centrifuge to obtain the supernatant for biochemical analyses.
5. Determine testicular xanthine oxidase (XO) activity by an enzymatic colorimetric assay using standard laboratory kits following the manufacturer's guidelines.
6. Assay testicular malondialdehyde (MDA) content by colorimetry²¹.
   1. Lipid peroxidation concentration determination using the thiobarbituric acid reactive substances (TBARS) produced during lipid peroxidation
      NOTE: This method is based on the reaction between 2-thiobarbituric acid (TBA) and malondialdehyde, an end product of lipid peroxidation. Heating at acidic pH gives a pink chromogen complex ([TBS] 2-malondialdehyde adduct) whose absorbance at 532 nm is measured.
      1. Deproteinize 200 µL of the sample with 500 µL of Trichloroacetic acid (TCA) and centrifuge at 3,000 rpm for 10 min.
      2. Add 1 mL of 0.75% TBA to 0.1 mL of the supernatant, boil in a water bath for 20 min at 100 ˚C, and cool with ice water.
      3. Read the absorbance of the sample/standard at 532 nm using a spectrophotometer against the blank. Determine the concentration of TBARS generated by extrapolating from the standard curve.
7. Assay testicular activities of superoxide dismutase (SOD)²², catalase²³, and glutathione peroxidase (GPx)²⁴ using established protocols as previously reported.
   1. SOD determination
      1. Dilute 1 mL of the sample in 9 mL of distilled water to make a 1 in 10 dilution. Add 0.2 mL of this diluted sample to 2.5 mL of 0.05 M carbonate buffer (pH 10.2) for spectrophotometer equilibration.
      2. Start the reaction by the addition of 0.3 mL of freshly prepared 0.3 mM adrenaline to the mixture and quickly mix by inversion.
      3. To the reference cuvette, add 2.5 mL of buffer, 0.3 mL of the substrate (adrenaline), and 0.2 mL of water.
      4. Monitor the increase in absorbance every 30 s for 150 s at 480 nm.
   2. Catalase determination
      1. Mix 1 mL of the supernatant of the testicular homogenate with 19 mL of diluted water to give a 1:29 dilution of the sample.
      2. Take 4 mL of H₂O₂ solution (800 µmoles) and 5 mL of phosphate buffer in a 10 mL flat-bottom flask.
      3. Mix 1 mL of the properly diluted enzyme preparation (step 4.6.3.1) with the reaction mixture (step 4.6.3.2) using a gentle swirling motion at 37 ˚C. Withdraw 1 mL of the reaction mixture and add it to 2 mL of dichromate/acetic acid reagent at 60 s intervals.
      4. Determine catalase levels in the sample by comparing the absorbance at 653 nm to that of a certified catalase standard.
   3. GPx assay
      1. Incubate the reaction mixture containing the sample at 37 ˚C for 3 min, add 0.5 mL of 10% trichloroacetic acid (TCA), and centrifuge at 3,000 rpm for 5 min.
      2. To 1 mL of the supernatant, add 2 mL of phosphate buffer and 1 mL of 5'-5'- dithiobis-(2-dinitrobenzoic acid (DTNB) solution, and read the absorbance at 412 nm against a blank.
      3. Determine glutathione peroxidase activity by plotting the standard curve and extrapolating the concentration of the remaining GSH from the curve.
8. MPO and tumor necrosis factor-alfa (TNFα) assay
   NOTE: This assay involves the oxidation of guaiacol to oxidized guaiacol in the presence of hydrogen peroxide. Guaiacol in its oxidised form has a brown color, which is measured photometrically at 470 nm.
   1. Use standard ELISA kits to determine testicular myeloperoxidase (MPO) activity^21,25 and tumor necrosis factor-alpha (TNF-α) levels.
9. Histopathological examination using hematoxylin and eosin (H&E) stain^26,27 and immunohistochemistry for caspase 3 expression
   1. Preserve the testicular tissue in Bouin's fluid; wash the tissue with toluene, submerge in paraffin wax, and incubate at 60 °C overnight.
   2. Take ~5 µm-thick sections, deparaffinize and rehydrate them, retrieve the antigen using preheated citrate buffer, and leave to cool for 30 min.
   3. Clean the slides with laboratory wipes, mark the section areas with a hydrophobic pen, and arrange the slides in a humidified chamber.
   4. Incubate the slides for 10 min following blockade of endogenous peroxidase activity using hydrogen peroxide. Rinse the slides with PBS once, apply the protein block, and incubate for 10 min.
   5. Rinse the slides with PBS 2x, apply the corresponding primary antibodies (caspase 3), incubate the slides for 45 min, and rinse 2x with PBS.
   6. Apply the secondary antibody, incubate for 25 min, rinse with PBS 2x, and add the HRP polymer. Repeat the cycle of incubation for 25 min and rinsing 2x with PBS.
   7. Incubate sections for 5 min in diaminobenzidine (DAB) substrate, rinse 2x with PBS, counterstain with Haematoxylin, and rinse with distilled water.
   8. Apply blueing solution to the sections, rinse, dehydrate, clear, and mounte for qualitative examination.
   9. Examine the tissues under a light microscope and take photomicrographs at 100x and 400x magnification with a digital camera attached to the microscope.

4. Statistical analysis

1. Perform statistical analysis with the software of choice. Perform Anderson-Darling test, D'Agostino and Pearson test, and Shapiro-Wilk test to test for normality.
2. Perform an unpaired t-test to test for significant difference between both groups: SO and T/D.
3. Present the data as mean ± standard error of the mean. Set the level of significance at p < 0.05, p < 0.01, and p < 0.001 (as reported in the figure legends).

Results

XO activity, MDA level, and activities of SOD, catalase, and GPx in the testis were employed as markers of oxidative stress. Torsion/detorsion (T/D) markedly increased XO activity and MDA levels and reduced testicular activities of SOD, catalase, and GPx in T/D rats compared with the sham-operated rats (Figure 1), depicting the role of oxidative stress in T/D-induced injury.

Moreover, TNF-α, a media...

Discussion

This paper and the accompanying video give a detailed step-by-step description of the surgical induction of TT for the study of testicular IRI using the anchorage approach. The method is particularly useful in that it obviates the prolonged exteriorization of the testis occasioned by clamping techniques⁷ and at the same time, more closely models the clinical presentation of TT in humans^10,12.

When ischaemia has ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 cc syringes	Not Applicable	Not Applicable	for anaethetic
2-0 chromic suture	Not Applicable	Not Applicable	For surgery
Bard Parker knife handle	Not Applicable	Not Applicable	For surgery
Curved Mosquito artery forceps	Not Applicable	Not Applicable	For surgery
Dissecting scissors	Not Applicable	Not Applicable	For surgery
GraphPad Prism (version 8.0.2)
Ketamine injection	Swiss Parenterals LTD Gujarat India	2321154	Anesthetic
Paracetamol injection	Shanxi Zhongbao Pharmaceuticals, China	220637	Analgesic
Povidone	JAWA International limited Nigeria	135S	Antiseptic wash
Procaine penicillin powder	Anhui Chengshi Pharmaceutical Co Ltd China	201906	For surgery
Surgical blade	Not Applicable	Not Applicable	For surgery
Surgical gloves	Zhenjiang huayang latex products China	210325
Stitch scissors	Not Applicable	Not Applicable	For surgery
Toothed dissecting forceps	Not Applicable	Not Applicable	For surgery
Xylazine injection	Bioveta Czech Republic	396228A	Anesthetic
Light Microscope	Novel Microscope, China	Not applicable	For histopathological evaluation
Digital Camera	Hayear, China	Not applicable	For histopathological evaluation
TNF-α kit	Elabscience, USA	E-EL-H0109	For assay
Thermo Fischer kit (anti-mouse caspase 3 monoclonal for caspase 3 expression )	Thermo Fischer Scientific Inc., USA	Not applicable	For Immunohistochemistry (caspase 3)