Laboratory for Experimental Urology, Organ Systems, Department of Development and Regeneration, University of Leuven, Leuven, Belgium
Marcus M. Ilg
Faculty of Health, Education, Medicine and Social Care, Medical Technology Research Centre, Anglia Ruskin University, Chelmsford, UK
Onur Omer Cakir
Department of Urology, University College London Hospitals NHS Trust, London, UK; Division of Surgery and Interventional Science, UCL, London, UK
Department of Urology, University College London Hospitals NHS Trust, London, UK; Division of Surgery and Interventional Science, UCL, London, UK; NIHR Biomedical Research Centre University College London Hospitals, London, UK
Delphine Behr Roussel
Pelvipharm, Montigny-le-Bretonneux, France UMR1179, Université Versailles Saint Quentin en Yvelines, Montigny-le-Bretonneux, France
Laboratory for Experimental Urology, Organ Systems, Department of Development and Regeneration, University of Leuven, Leuven, Belgium
Servicio de Histología-Investigación, Unidad de Investigación Traslacional en Cardiología (UFV-IRYCIS), Hospital Universitario Ramón y Cajal, Madrid, Spain
Endocrinology Unit, Medical Department, Azienda USL, Maggiore-Bellaria Hospital, Bologna, Italy
Division of Urology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
Flare-Health, Amstelveen, The Netherlands
Department of Urology, University College London Hospitals NHS Trust, London, UK, Division of Surgery and Interventional Science, UCL, London, UK, Unit of Urology, Division of Experimental Oncology, Urological Research Institute (URI), IRCCS Ospedale San Raffaele, Milan, Italy, Departement of Urology, Vita-Salute San Raffaele University, Milan, Italy
Rodent animal models are currently the most used in vivo model in translational studies looking into the pathophysiology of erectile dysfunction after nerve-sparing radical prostatectomy.
This European Society for Sexual Medicine (ESSM) statement aims to guide scientists toward utilization of the rodent model in an appropriate, timely, and proficient fashion.
MEDLINE and EMBASE databases were searched for basic science studies, using a rodent animal model, looking into the consequence of pelvic nerve injury on erectile function.
Main outcome measures:
The authors present a consensus on how to best perform experiments with this rodent model, the details of the technique, and highlight possible pitfalls.
Owing to the speciﬁc issue—basic science—Oxford 2011 Levels of Evidence criteria cannot be applied. However, ESSM statements on this topic will be provided in which we summarize the ESSM position on various aspects of the model such as the use of the Animal Research Reporting In Vivo Experiments guideline and the of common range parameter for nerve stimulation. We also highlighted the translational limits of the model.
The following statements were formulated as a suggestive guidance for scientists using the cavernous nerve injury model. With this, we hope to standardize and further improve the quality of research in this ﬁeld. It must be noted that this model has its limitations.
Weyne E, Ilg MM, Cakir OO, et al. European Society for Sexual Medicine Consensus Statement on the Use of the Cavernous Nerve Injury Rodent Model to Study Postradical Prostatectomy Erectile Dysfunction. Sex Med 2020;8:327–337.
Copyright © 2020, The Authors. Published by Elsevier Inc. on behalf of the International Society for Sexual Medicine. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Animal Model; Erectile Dysfunction; Radical Prostatectomy; Cavernous Nerve
The advent of prostate specific antigen screening has led to the early detection of prostate cancer (PCa). As a conclusion, more and more patients will have good long-term oncologic outcome after radical prostatectomy (RP).1–3 In contrast, it has been reported that only 23% of men younger than 60 years regain their complete erectile function (EF) after bilateral nerve-sparing RP (NSRP).4
In accordance with the most recent literature,5 patient preoperative and intraoperative factors (including age, preoperative EF, comorbidities), type of surgery (unilateral vs bilateral nervesparing), grade of nerve sparing (ie, intrafascial vs interfascial vs extrafascial surgeries), and surgical skill represent the key significant contributors to EF recovery after NSRP.5
In nerve-sparing surgery, erectile dysfunction (ED) occurs as a consequence of iatrogenic damage to the periprostatic neurovascular bundle, which results in neuropraxia of the cavernous nerves (CNs).5–9 Neurapraxia, as per the Seddon classification, is a temporary failure of nerve conduction because of a damage to the myelin sheath.10,11 The Seddon classification of nerve injury separates injury based on the scale from microscopic to macroscopic along with severity of tissue injury, prognosis, and time for recovery. Seddon described 3 types of nerve injury—neurapraxia, axonotmesis, and neurotmesis12 with each type having a different potential for regeneration. Neurapraxia, the temporary block of the CN conduction, results in a decrease of the rate and quality of both daily and nocturnal erections, and it promotes a persistent cavernous hypoxia.10,11 In vitro and in vivo studies suggested that the neurapraxia and the consequent penile hypoxia might result in collagen accumulation, smooth-muscle apoptosis, and fibrosis.13,14
Finally, these penile changes lead to venous leakage and permanent ED before complete recovery of the nerve integrity can be achieved up to 2 years after surgery.15 It has been shown that ED occurring after RP differs from classical vasculogenic ED.16–19 In human corpus cavernosum tissue, endothelial function was preserved in patients after RP, whereas significant disturbances were seen in neurogenic relaxation with sympathetic hyper innervation. Accordingly, molecular analysis of protein expression showed significant changes of neuronal proteins in post-RP ED which differs from that observed in vasculogenic ED.16 In line to what is observed in humans, it was reported that penile endothelial function was preserved in rats after crushing of the CNs.18,19 In contrast, imbalanced neurogenic responses favoring adrenergic contraction over nitrergic relaxations have been observed in the human tissue and rat model.17,19 This concept is very important because recovery of endothelial function has been used as an end point in many interventional studies in rats investigating therapeutics in post-RP ED.20
Animal models mimicking this CN injury (CNI) have played a role in the advancement of the field.21,22 Rodent models have become the standard in early phases of in vivo research method because of their relatively low cost compared with other animal species.21–23 Both mouse and rat animal models are available, with mice potentially allowing higher throughput in some cases, while also providing options for genetic knockout or modification. 24 However, despite the possibility of genetic engineering providing a valuable tool for investigation, evaluation of EF may be more challenging in the mouse than in the rat model.25Various preclinical studies, using NSRP rodent models, have demonstrated that vacuum erection devices and several medications (ie, alprostadil injections, phosphodiesterase type 5 inhibitors, and so on) are able to promote EF recovery, improve the cavernosal smooth-muscle/collagen ratio, increase penile smoothmuscle replication, reduce penile apoptosis, preserve penile endothelial function, increase antioxidant enzymes, and promote neuroprotection during and after neuropraxia.22
In spite of promising results generated in the rodent models, most of the well-designed clinical trials have failed to confirm any structural or lasting benefit of this type of treatment in improving the recovery of EF after RP.5,7,8,26–28
One of the reasons for the discrepancy between the clinical and animal data may be because of variance in the methodology used when conducting the basic science studies.22 The variance in the methodology and the lack of consensus guidelines for the use of the NSRP rat model (also called bilateral CN crush, transection, excision, dissection, freezing, electrocautery, and irradiation model) have led to the publication of studies whose results are often equivocal and impossible to compare.29
The aim of this review is to review the current state of art, highlight possible pitfalls, and provide statements for experimental technique and reporting in the use of the NSRP rat model; to provide further standardization and improvement in the quality of research in this field on behalf of the European Society of Sexual Medicine (ESSM).
MEDLINE and EMBASE databases were searched for articles looking with the following search terms: (“erectile dysfunction” OR “erectile function” OR “radical prostatectomy” OR “post-RP ED” OR “cavernous nerve injury”) AND (“animal model” OR “model”). Studies from 1980 up to 2019 were included.
Abstracts were screened for relevance (F.C. and E.W.); if it was not clear from the abstract whether the article may contain relevant data, the full article was assessed.
Only the basic science studies, using a rodent animal model, looking into the consequence of pelvic nerve injury on EF were considered relevant for this study. Thereafter, relevant studies were analyzed and summarized after an interactive peer-review process of the panel (F.C., E.W., D.B.R., M.I., A.M., and C.G.) to obtain a narrative review. The statements were internally discussed. Disagreements were resolved by consensus. The study was internally reviewed by senior authors (M.A., C.B., Y.C., J.A.).
Owing to the specific issue—basic science—Oxford 2011 Levels of Evidence criteria cannot be applied. However, ESSM statements on this topic will be provided in which we summarize the ESSM position on various aspects of the model.
Ethical board approval was not required for this work.
General concepts of translational research–the animal research reporting in vivo experiments guideline
Statement #1: The ARRIVE check list should be completed and submitted with the manuscript. In particular, the strain, sex, weight, and age of animals always needs to be reported.
The initial basis for addressing a clinical problem in an experimental setting is the selection of a valid model that corresponds to the human condition in etiology, pathophysiology, symptomatology, and response to therapeutic interventions.
The key to using any method or model is to understand its limits and what outcomes it measures, represents, and is able to forecast. For animal models, comparable biological processes or behaviors to signs, responses, or symptoms of human functions and disease, that is face validity, are important for translational research.30 In sexual arousal, penile erection is an example of a component of male sexual behavior that in several species share substantial physiological and biochemical events and for which methods exist that can be used to assess similar end points.31e35 Human clinical trials are regulated by authorities to reduce biases by patients and investigators, whereas corresponding controls do not seem to be extensively in effect in medical research with animals, that despite more objective end points, still is at risk for unconscious procedural errors.36 To increase the value of research processes with animals, we emphasize the concept that the ARRIVE (Animal Research Reporting In Vivo Experiments) guidelines and recommendations should be followed.25 In particular, the ARRIVE guidelines were developed to improve standards of reporting and ensure that the data from animal experiments can be fully evaluated and used. They consist of a 20-point checklist (https://www.nc3rs.org.uk/sites/default/files/documents/Guidelines/NC3Rs%20ARRIVE%20Guidelines% 20Checklist%20(fillable).pdf) of the essential information that should be included in publications reporting animal research. No study has so far examined how authors of publications involving CNI in rodent models adhered to the ARRIVE guidelines. However, there is a recent survey of 271 publications between 1999 and 2005, which involved the use of live rats, mice, and non-human primates, carried out in the UK and US publicly funded research establishments. This survey provided evidence that many peer-reviewed, animal research publications fail to report important information regarding animal demographics and experimental protocols.37 To mention some examples from this survey, 24% of studies did neither report the age nor the weight of animals, 12% of studies reported random allocation of animals to groups, 5.9% used investigator blinding to treatment, and in 4% of the studies, the statistical method was unclear.37
Similar potential deficiencies in animal research are noted in several medical areas, including sexual medicine, and correlations have been made between insufficient reporting and translational value.36 Few articles in the sexual medicine field have reported the use of the ARRIVE checklist.38–41 This concept is very important because similar to humans, several mammals display —in aging or because of various diseases—signs of ED and alterations of important regulatory pathways of erection.24 The lack of crucial information can prevent the correct evaluation of the final results obtained. The ARRIVE checklist should be completed and submitted with the manuscript involving animal models for radical retropubic prostatectomy.
Physiological investigations of penile erection–the rat animal model
Extensive research suggests that regulatory mechanisms of penile erection exhibit substantial physiological, biochemical, and pharmacologic homologies among mammalian species, including humans.31–33 For example, human, monkey, rabbit, dog, rat, and mouse corpus cavernosum tissue that is precontracted to simulate penile flaccidity responds similarly via neuronal and endothelial signals with relevant nitric oxide (NO)-dependent relaxant “erection-simulating” responses.42–47 Accordingly, a crucial role for the nitric oxide pathway in penile erection has been characterized in vivo during erections in various animals.38, 48–52 However, peculiar species characteristics are also present. Hence, the selection of the correct animal model and the parameters to be tested is essential in transitional medicine (see the following sections).
Measurement of Intracavernous and Corpus Spongiosum Pressure
Statement #2: The use of intracavernous pressure (ICP) registrations should be preferred to corpus spongiosum pressure (CSP) as an end point during erection.
Statement #3: The Methods section should clearly report if ICP or CSP was recorded.
The human corpus spongiosum is responsible for engorgement of the glans during the erection. The human bulbospongiosus muscle that surrounded the corpus spongiosum has an important role to propel semen during the expulsion phase of ejaculation.53–56 In rats that have a peculiar anatomy of the penis with an almost 180_ frontal flex of the glans, penile striated muscle activity is also essential to compress the erectile compartment to straighten the glans penis (“flips”) to achieve intromission.57 After transection of the CN, the rat can still achieve erections of the glans.57 Furthermore, in contrast to humans, rats also use the bulbospongiosus muscle together with a well-developed rhabdosphincter to forcefully and rhythmically expel urine during micturition.58–60 Consequently, significant pressure responses are recorded during rat micturition, and therefore, for the use of CSP registration in awake and freely moving rats, the behavioral context may need particular attention.60
Some investigators have studied CSP as an end point during erection.61e63 When compared with the corpora cavernosa, the corpus spongiosum has a less-firm tunica albuginea and arteriovenous shunts in the glans and may be considered to be a flowthrough compartment with lower pressures during erection.64,65 Owing to this potential bias, the use of ICP registrations should be preferred to CSP as an endpoint during erection.
ICP and Mean Arterial Pressure
Statement #4: ICP should be normalized by mean arterial blood pressure (MAP); especially when vasoactive substances are used.
Statement #5: Detailed description of the method used to record ICP and MAP should be reported.
Statement #6: Exemplary and representative images of the ICP and MAP traces should be included in or attached to the manuscript.
Activation of the pelvic nerve or the cavernous (penile) nerve by contact electrodes has been shown to induce analogous penile erections in monkeys, dogs, rabbits, rats, and mice that are recorded as characteristic changes in ICP.66–69 Correspondingly, in patients undergoing NSRP or penile surgery, stimulation of the neurovascular bundle posterolateral to the prostate induced subjectively assessed erections or penile tumescence, and activation of the CNs similarly caused visible erections that were correlated to simultaneous increases in ICP.65,70 Hence, under these investigative conditions, techniques to study nerve-induced increases in ICP in animals seem to comprise appropriate translational models for humans.
ICP measurement in rodent can be achieved by implanting the tip of a recording catheter into the corpus cavernosum and connecting to a pressure transducer.62
To register ICP responses, commonly either the crura or the body of the corpus cavernosum is cannulated using either PE tubes or needles. However, few studies exist that compare the cannulation sites.71 The insertion of the catheter can be performed using a small needle connected to the end of the recording catheter or via an incision of the tunica albuginea of the penis, fixing it with a purse-string suture. Before and after the insertion the catheter and the needle, if used, should be flushed with a solution containing heparin to avoid clots. There are no studies that have investigated differences in the outline of the experimental setup. To register systemic blood pressure, a large artery, commonly the aorta or the carotid artery, is cannulated using either PE tubes or needles filled with heparin.72
Because arterial blood pressure may affect ICP responses, a standard experimental setup should include procedures for simultaneous recording of pressures from the erectile compartment and central arteries.73 Consequently, the amplitude and/or area under curve of the erectile responses after electrical stimulation of the CN should be normalized by the MAP during the erectile response and reported as such.64 As ICP can be affected by hemodynamics, it is highly recommended that the ICP is adjusted by the MAP and reported as ICP/MAP especially when vasoactive substance are used.73 Exemplary traces of ICP and MAP are usually included in the manuscript as to provide the reader and reviewers with the option of assessing quality of registration/stimulation.74 The authors should clearly report and describe the measures recorded during the experiment. These should include how many electrical stimulations were performed for each animal, the length of electrical stimulation, and if ICP mean or peak is used in the calculation.
The authors should also clearly report the methodology and the instruments that were used during the experiment including: the site of (crura or corpora) the catheter insertion, the size and the material of the catheters used, the size of the needle used, the amount and the concentration of heparin that was administrated, and the instrument used for the crush and for how long the crush was performed. The version of recording machine and software used should be accurately described.
Type of Rodent Models
Statement #7: Rodent models should be used to mimic post-RP ED. While mouse models might offer higher throughput and options for genetic modification, rat models might offer more ease of use for CN stimulation.
The rat is the most commonly used species for in vivo studies of erection. Electrical stimulation of the CN can cause reproducible ICP responses after appropriate training.29,33,75 The rat bilaterally has a single CN that is readily identified running from the pelvic ganglion on the lateral side of the prostate (Figure 1), whereas corresponding structures in humans are derived from a more diffuse nervous meshwork of the neurovascular bundle.75–77 Behavioral, neurophysiological, and molecular biological or genetic procedures are conveniently available for multimodal investigations when using rats, and animal purchase, housing, and maintenance costs are relatively low. While mice might offer a higher experimental throughput in some cases, the CN stimulation as well as the ICP recording might be easily achieved in the rat model.33,78 It should be noted that the rat is reported to exhibit ancillary penile innervation from the major pelvic ganglion, which accounts for around 45% of ICP responses to supraspinal stimuli after CN transection.76 These ancillary nerves are not damaged in the rat model of RP.
The lack of damage to these ancillary nerves represents an element to be considered in the design of the experiment representing a possible source of bias. However, there are no studies that have tested the long-term effect of the ancillary nerves on the EF of the rat.
Parameters for CN Electrical Stimulation
Statement #8: Detailed description of the parameters for CN electrical stimulation, including: pulse duration, frequency, duration of stimulation, voltage and rest period, should be reported.
Statement #9: The most commonly agreed parameter ranges for cavernous nerve stimulation in rats are pulse duration, 0.5 ms–1 ms, frequency, 10–20 Hz; duration of stimulation, 30ؘ–60 seconds; voltage, 2.5–8V (L4). Frequency- or voltage response curves should be established to manifest optimal and suboptimal stimulation parameters. Rest periods of at least 5–10 minutes between subsequent stimulations should be applied. We recommend using these parameters to improve comparability between studies.
Most researchers have reported the stimulus in volts and values ranged from 1 to 15V.75,79–81,71,82,83 Other groups have reported the intensity of the current (ampere) for characterizing the procedure of nerve activation, and this parameter ranged between 0.5–10 mA.71,84–86 The frequencies (Hertz) of the stimuli used by the aforementioned research groups varied between 1 and 30 Hz. Optimal (maximal) ICP responses are generally reported around 6–7.5 V or 1.5 mA and between 10 and 20 Hz.79,71,84,87 Ideally, a voltage-response or intensity-response or frequencyresponse curve is produced to depict erectile responses elicited at several electrical stimulation parameters as this may yield interesting information on the erectile effect a compound/device may produce (at low-, intermediate-, or high-stimulation parameters). Another parameter of interest for nerve stimulation is the width (or duration) of the single pulse to differentiate the threshold for activation of different types of nerves.88 This parameter is reported to vary from 0.05 to 5 ms.80,81,71,84–87,89 Considering settings for optimal stimulation of the vagus nerve in rats, CN activation in dogs, or these used for intraoperative activation of the CN in humans, a pulse width around 0.2 ms is probably appropriate for rodent ICP models.65,90–93 In the mouse ICP model, 0.5–6V, 5–20 Hz, and pulse durations of 1–5 ms have been used to activate the CN.66,94 Maximal ICP responses were reported at 3V and 15 Hz.66
A quite high variability of electrical stimulation parameters used by different research groups has been reported. This may in part be related to the type of stimulators used whether the voltage or the current can be regulated. For voltage stimulators, the resistance of the electrode may affect the intensity of the current needed to activate the nerve. Conversely, stimulators that adjust for the resistance in the electrode delivers stable currents for nerve activation. 65 We recommend using the most commonly used parameters (outlined previously) to improve comparability between studies.
Site for CN Electrical Stimulation
Statement #10: The author should clearly report if a unilateral or bilateral CN stimulation were performed.
Statement #11: The author should clearly report the location of nerve stimulation that is proximally or distal to the point of the nerve damage.
Recording erections with a video camera, Quinlan et al 75 observed better responses upon bilateral stimulation of the CN as compared with unilateral nerve activation. Even so, most of the following studies have implemented unilateral stimulation of the CN, and it may be discussed if the neurovascular mechanisms responsible for ICP responses are fully activated under such conditions, hence the advantage of performing a response curve to several electrical stimulation parameters to reach maximal response. As reported by registration of ICP in dogs, unilateral stimulation of the CN induced full erection of both the corpora cavernosa even if the ICP responses were achieved faster by bilateral stimulation.91
Unilateral CN stimulation may give false low-filling values for tumescence and possibly display a delayed onset of the veno-occlusive mechanisms. It must be noted, however, that most of the studies currently performed in rats use unilateral nerve stimulation. The unilateral CNI model can serve as its own control; the injured and sham groups are in the same animal at the same time, while having the disadvantage of potential compensatory action of the intact nerve. More importantly, the location of nerve stimulation that is proximally or distal to the point of nerve damage nerve damage could influence the ICP response. An electrical stimulation proximal to the point of nerve damage evaluates both the component of nerve damage and those of damage to erectile tissue. Distal nerve damage stimulation evaluates only the component of erectile tissue by bypassing nerve damage.
Type of CNI
Statement #12: Bilateral and not unilateral CNI should be considered as the standard animal model for human RP.
Statement #13: Detailed description of the parameters for CNI including mode of injury (crush, transection, cold, heat,), type of instrument used, and duration of injury and should be reported for the purpose of reproducibility of the model.
Both unilateral and bilateral CNI models have been used to study ED and are believed to imitate the condition in humans after RP. Various types of CNI have been studied in rodent models, especially rats.78 These models were stratified by the type and extent of injury. Various types of injury techniques such as stretching, crushing, freezing, transecting, dissecting, and excising the CN as well as unilateral vs bilateral CNI have been described22 (Table 1). Crushing of CNs is most commonly used to mimic the nerve injury that occurs when using the NSRP technique.22 On the other hand, transection and excision of the CNs are mostly used to mimic RP without nerve-sparing procedure.29 The crush injury model involves various mechanical compressions of the CN over varying periods of compression time.6,26,72,95 The crushing can be induced by several instruments (forceps, hemostatic, or bulldog clamps, microserrefine serrated or not).It is not clear if the instrument used or the timing of the crush can induce any difference in the magnitude and consistency of provoked ED.71 Transection injury consists of direct division of the CN, whereas excision CNI implicates the removal of a segment of CN.96 CNI can be applied unilaterally or bilaterally.
In unilateral nerve injury models, mimicking unilateral NSRP, the nerve supply is partly preserved and therefore penile elections partly maintained.76 Furthermore, compensatory nerve sprouting from the intact contralateral CN is believed to occur.10 In unilateral injury models, the contralateral side is often used as a control, but as mentioned, contralateral sprouting can confound experimental interpretation.10 Because of these reasons, bilateral and not unilateral CNI is considered as the standard model.22,78
Statement #14: Rodent models for erectile dysfunction after NSRP used in experimental studies present important limitations when compared with the clinical settings. These limitations should be reported. Statement #15: The author should consider a possible source of discrepancy between basic science studies and the clinical practice. The limitations are as follows: the age of the rat involved in the experiment, the absence of rat comorbidities, and the spontaneous recovery of erectile function of the rat after 6 months.
For rat animal studies, male rats between the age of 10 and 12 weeks or weighing between 300 and 400 grams are generally used.29,22 It has been criticized that the age of these rats corresponds with adolescence in humans and not middle-aged men such as typically seen in patients with PCa .97 Days or months after the CN injuries, the EF is evaluated as per the Quinlan et al75 model by the electrical stimulation of the injured nerve.98 In rodent studies, the exact time frame in which maximal nerve regeneration and EF recovery occurs is not uniformly agreed upon. Commonly, EF is evaluated 4 weeks after nerve injury in rats because it is generally believed to represent the 2-year time in humans.99 Indeed, in humans, EF continues to improve up to 24 months after RP.100 As early as 1 day and up to a week after CN crush, only 30–40% of axons survive and maximal ICP response to CN stimulation are 4 times lower than in control animals.101,102 It has been shown that EF is decreased 48 hours after bilateral crush CNI and starts to recover at 60 days after injury.103 Spontaneous complete recovery of EF has been seen 6 months after bilateral CN crush injury after injury.104
Rodent models have been used to study pathophysiologic mechanisms and assorted pharmacologic, surgical, and regenerative treatments.22 Several preclinical and translational studies have shown a benefit of penile rehabilitation therapies such as phosphodiesterase type 5 inhibitor/GC activator treatment,105 immunomodulation, neurotrophic factor administration, and regenerative medicine options such as stem cell therapy in animals. 22 However, most of these approaches have either failed clinical translation or have yet to be studied in human subjects.11
The reason of these clinical translation failures may lie in the limits that characterize the CNI model and in the design bias which can occur in basic science studies:
1) The EF evaluation in rats is performed by an objective method (ICP evaluation, electrical stimulation of CN) and, on the other hand, in the clinical practice the evaluation is performed via questionnaires.11
2) As mentioned, there are reports that the ancillary nerves provide more than 50% of the proerectile innervation to the rat penis.76 These ancillary nerves remain unaffected in the CNI model, and it is unclear if thismight have an impact on the severity of ED in rats compared with humans.More important, theCNImimics a “perfect “bilateral intrafacial nerve-sparing procedure difficult to perform in all the patients in the clinical setting.
3) The CNI studies, commonly use 10- to 12-week-old and thus healthy young rats without any baseline ED.29,22 This does not reflect the current clinical landscape. Patients with PCa are commonly older than 50 years of age and may be suffering from different comorbidities (diabetes, cardiovascular disease) which cause baseline ED and can impair nerve repair mechanisms after the NSRP.15 This discrepancy between basic science studies and the clinical practice is illustrated in several clinical studies which showed that young patients without comorbidities have a better recovery of EF after NSRP compared with other patients.5,8,106 Moreover, preoperative EF and Carlson Comorbidity Index are considered independent predictors for EF recovery.15
4) The spontaneous recovery of EF in the CNI rat model104 has important scientific-translational consequences. Indeed, this model can only be used to assess if a treatment can modify the time to return of EF recovery. Conversely, this model cannot be used to test if there is an absolute variation in terms of EF recovery rate between 2 or several treatments. The spontaneous recovery of EF in the CNI rat model should be considered as one of the major limitations of this model.
5) Several studies in this field lack high quality and correct in vitro experimental methodology. Describing the various in vitro techniques in detail is beyond the intent of this ESSMstatement; however, we provided more information on the different available in vitro methods (such as common stainings and marker proteins) to assess fibrosis, neural, and endothelial in tissues in the supplementary data (supplementary data -Table 1)
Today, the rat is commonly used as an animal model to mimic post-RP ED by bilaterally crushing the CNs. In this document, we provided ESSM statements for the correct and reproducible use of this CNI model. We hope to increase comparability between reports which could advance the field as a whole. It must be noted that this model has its limitations. This may help to narrow the gap between preclinical studies and their translation in clinical practice.
Corresponding Author: Fabio Castiglione, MD, PhD, Departement of Urology, University College London Hospital, 16-18 Westmoreland St, Marylebone, London W1G 8PH. Tel: þ447460050365; E-mail: firstname.lastname@example.org
Conflict of Interest: The authors report no conflicts of interest.
Statement of Authorship
(a) Conception and Design
Fabio Castiglione; Emmanuel Weyne; Marcus M. Ilg
(b) Acquisition of Data
Fabio Castiglione; Emmanuel Weyne; Marcus M. Ilg
(c) Analysis and Interpretation of Data
Delphine Behr Roussel; Fabio Castiglione; Emmanuel Weyne; Marcus M. Ilg
(a) Drafting the Article
Delphine Behr Roussel; Fabio Castiglione; Marcus M. Ilg
(b) Revising It for Intellectual Content
Maarten Albersen; Onur Omer Cakir; Giovanni Corona; Yacov
Reisman; Javier Angulo
(a) Final Approval of the Completed Article
Giovanni Corona; Yacov Reisman; Asif Muneer
- Filson CP, Shelton JB, Tan H-J, et al. Expectant management of veterans with early-stage prostate cancer. Cancer 2016; 122:626-633.
- Villa L, Capitanio U, Briganti A, et al. The number of cores taken in patients diagnosed with a single microfocus at initial biopsy is a major predictor of insignificant prostate cancer. J Urol 2013;189:854-859.
- Capitanio U, Suardi N, Matloob R, et al. Staging lymphadenectomy in renal cell carcinoma must be extended: a sensitivity curve analysis. BJU Int 2013;111:412-418.
- Haglind E, Carlsson S, Stranne J, et al. Urinary Incontinence and erectile dysfunction after Robotic versus open radical prostatectomy: a Prospective, Controlled, Nonrandomised trial. Eur Urol 2015;68:216-225.
- Salonia A, Castagna G, Capogrosso P, et al. Prevention and management of post prostatectomy erectile dysfunction. Transl Androl Urol 2015;4:421-437.
- Matsui H, Sopko NA, Hannan JL, et al. M1 Macrophages are Predominantly Recruited to the major pelvic ganglion of the rat following cavernous nerve injury. J Sex Med 2017;14:187- 195.
- Weyne E, Castiglione F, Van der Aa F, et al. Landmarks in erectile function recovery after radical prostatectomy. Nat Rev Urol 2015;12:289-297.
- Castiglione F, Ralph DJ, Muneer A. Surgical techniques for Managing post-prostatectomy erectile dysfunction. Curr Urol Rep 2017;18:90.
- Vignozzi L, Filippi S, Morelli A, et al. Cavernous neurotomy in the rat is associated with the onset of an overt condition of hypogonadism. J Sex Med 2009;6:1270-1283.
- Weyne E, Mulhall J, Albersen M. Molecular pathophysiology of cavernous nerve injury and identification of strategies for nerve function recovery after radical prostatectomy. Curr Drug Targets 2015;16:459-473.
- Fode M, Ohl DA, Ralph D, et al. Penile rehabilitation after radical prostatectomy: what the evidence really says. BJU Int 2013;112:998-1008.
- Kaya Y, Sarikcioglu L. Sir Herbert Seddon (1903-1977) and his classification scheme for peripheral nerve injury. Vol. 31, Child’s nervous system : ChNS. Official Journal Int Soc Pediatr Neurosurg Germany 2015:177-180.
- Milenkovic U, Albersen M, Castiglione F. The mechanisms and potential of stem cell therapy for penile fibrosis. Nat Rev Urol 2019;16:79-97.
- Castiglione F, Bergamini A, Bettiga A, et al. Perioperative betamethasone treatment reduces signs of bladder dysfunction in a rat model for neurapraxia in female urogenital surgery. Eur Urol 2012;62:1076-1085.
- Briganti A, Di Trapani E, Abdollah F, et al. Choosing the best candidates for penile rehabilitation after bilateral nervesparing radical prostatectomy. J Sex Med 2012;9:608-617.
- Karakus S, Musicki B, Burnett AL. Phosphodiesterase type 5 in men with vasculogenic and post-radical prostatectomy erectile dysfunction: is there a molecular difference? BJU Int 2018;122:1066-1074.
- Martinez-Salamanca JI, La Fuente JM, Fernandez A, et al. Nitrergic function is lost but endothelial function is preserved in the corpus cavernosum and penile resistance arteries of men after radical prostatectomy. J Sex Med 2015;12:590- 599.
- Martínez-Salamanca JI, Zurita M, Costa C, et al. Dual Strategy with oral phosphodiesterase type 5 inhibition and intracavernosal Implantation of Mesenchymal stem cells is Superior to Individual approaches in the recovery of erectile and cavernosal functions after cavernous nerve injury in rats. J Sex Med 2016;13:1-11.
- Martinez-Salamanca JI, La Fuente JM, Martinez- Salamanca E, et al. alpha1A-Adrenergic Receptor Antagonism improves erectile and cavernosal responses in rats with cavernous nerve injury and Enhances neurogenic responses in human corpus cavernosum from patients with erectile dysfunction Secondary to radical prostatectomy. J Sex Med 2016;13:1844-1857.
- Soebadi MA, Moris L, Castiglione F, et al. Advances in stem cell research for the treatment of male sexual dysfunctions. Curr Opin Urol 2016;26:129-139.
- Di Trapani E, Nini A, Locatelli I, et al. Development of the first model of radical prostatectomy in the mouse: a feasibility study. Eur Urol 2018;73:482-484.
- Haney NM, Nguyen HMT, Honda M, et al. Bilateral cavernous nerve crush injury in the rat model: a Comparative review of pharmacologic interventions. Sex Med Rev 2018;6:234-241.
- Füllhase C, Russo A, Castiglione F, et al. Spinal cord FAAH in normal micturition control and bladder overactivity in awake rats. J Urol 2013;189:2364-2370.
- Jin H-R, Chung YG, Kim WJ, et al. A mouse model of cavernous nerve injury-induced erectile dysfunction: functional and morphological characterization of the corpus cavernosum. J Sex Med 2010;7:3351-3364.
- Ghatak K, Yin GN, Choi M-J, et al. Dickkopf2 rescues erectile function by enhancing penile neurovascular regeneration in a mouse model of cavernous nerve injury. Sci Rep 2017; 7:17819.
- Hannan JL, Kutlu O, Stopak BL, et al. Valproic acid prevents penile fibrosis and erectile dysfunction in cavernous nerveinjured rats. J Sex Med 2014;11:1442-1451.
- Salonia A, Abdollah F, Capitanio U, et al. Preoperative sex steroids are significant predictors of early biochemical recurrence after radical prostatectomy. World J Urol 2013; 31:275-280.
- Gandaglia G, Albersen M, Suardi N, et al. Postoperative phosphodiesterase type 5 inhibitor administration increases the rate of urinary continence recovery after bilateral nervesparing radical prostatectomy. Int J Urol Off J Jpn Urol Assoc 2013;20:413-419.
- Cellek S, Bivalacqua TJ, Burnett AL, et al. Common pitfalls in some of the experimental studies in erectile function and dysfunction: a consensus article. J Sex Med 2012;9:2770- 2784.
- Sams-Dodd F. Strategies to optimize the validity of disease models in the drug discovery process. Drug Discov Today 2006;11:355-363.
- Christ GJ, Lue T. Physiology and biochemistry of erections. Endocrine 2004;23:93-100.
- Andersson K-E. Mechanisms of penile erection and basis for pharmacological treatment of erectile dysfunction. Pharmacol Rev 2011;63:811-859.
- Giuliano F, Pfaus J, Srilatha B, et al. Experimental models for the study of female and male sexual function. J Sex Med 2010;7:2970-2995.
- El-Sakka AI, Hassan MU, Selph C, et al. Effect of cavernous nerve freezing on protein and gene expression of nitric oxide synthase in the rat penis and pelvic ganglia. J Urol 1998; 160(6 Pt 1):2245-2252.
- Yamashita S, Kato R, Kobayashi K, et al. Nerve injury-related erectile dysfunction following nerve-sparing radical prostatectomy: a novel experimental dissection model. Int J Urol Off J Jpn Urol Assoc 2009;16:905-911.
- Landis SC, Amara SG, Asadullah K, et al. A call for transparent reporting to optimize the predictive value of preclinical research. Nature 2012;490:187-191.
- Kilkenny C, Parsons N, Kadyszewski E, et al. Survey of the quality of experimental design, statistical analysis and reporting of research using animals. PLoS One 2009; 4:e7824.
- Castiglione F, Hedlund P, Weyne E, et al. Intratunical injection of human adipose tissue-derived stem cells Restores collagen III/I ratio in a rat model of chronic Peyronie’s disease. Sex Med 2019;7:94-103.
- Castiglione F, Hedlund P, Weyne E, et al. Intratunical injection of stromal vascular fraction prevents fibrosis in a rat model of Peyronie’s disease. BJU Int 2019;124:342-348.
- Hakim L, Fiorenzo S, Hedlund P, et al. Intratunical injection of autologous adipose stromal vascular fraction reduces collagen III expression in a rat model of chronic penile fibrosis. Int J Impot Res 2020;32:281-288.
- Castiglione F, Dewulf K, Hakim L, et al. Adipose-derived stem cells Counteract urethral Stricture formation in rats. Eur Urol 2016;70:1032-1041.
- Okamura T, Ayajiki K, Toda N. Monkey corpus cavernosum relaxation mediated by NO and other relaxing factor derived from nerves. Am J Physiol 1998;274(4 Pt 2):H1075-H1081.
- Hedlund P, Aszodi A, Pfeifer A, et al. Erectile dysfunction in cyclic GMP-dependent kinase I-deficient mice. Proc Natl Acad Sci U S A 2000;97:2349-2354.
- Hedlund P, Alm P, Andersson KE. NO synthase in cholinergic nerves and NO-induced relaxation in the rat isolated corpus cavernosum. Br J Pharmacol 1999;127:349-360.
- Hedlund P, Larsson B, Alm P, et al. Distribution and function of nitric oxide-containing nerves in canine corpus cavernosum and spongiosum. Acta Physiol Scand 1995;155:445-455.
- Kim N, Azadzoi KM, Goldstein I, et al. A nitric oxide-like factor mediates nonadrenergic-noncholinergic neurogenic relaxation of penile corpus cavernosum smooth muscle. J Clin Invest 1991;88:112-118.
- Ignarro LJ, Bush PA, Buga GM, et al. Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle. Biochem Biophys Res Commun 1990;170:843-850.
- Ayajiki K, Hayashida H, Tawa M, et al. Characterization of nitrergic function in monkey penile erection in vivo and in vitro. Hypertens Res 2009;32:685-689.
- Burnett AL, Nelson RJ, Calvin DC, et al. Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase. Mol Med 1996;2:288-296.
- Trigo-Rocha F, Aronson WJ, Hohenfellner M, et al. Nitric oxide and cGMP: mediators of pelvic nerve-stimulated erection in dogs. Am J Physiol 1993;264(2 Pt 2):H419-H422.
- Mills TM, Lewis RW, Stopper VS. Androgenic maintenance of inflow and veno-occlusion during erection in the rat. Biol Reprod 1998;59:1413-1418.
- Holmquist F, Stief CG, Jonas U, et al. Effects of the nitric oxide synthase inhibitor NG-nitro-L-arginine on the erectile response to cavernous nerve stimulation in the rabbit. Acta Physiol Scand 1991;143:299-304.
- Shafik A. Response of the urethral and intracorporeal pressures to cavernosus muscle stimulation: role of the muscles in erection and ejaculation. Urology 1995;46:85-88.
- Holmes GM, Chapple WD, Leipheimer RE, et al. Electromyographic analysis of male rat perineal muscles during copulation and reflexive erections. Physiol Behav 1991;49:1235- 1246.
- Beckett SD,Walker DF, Hudson RS, et al. Corpus spongiosum penis pressure and penile muscle activity in the stallion during coitus. Am J Vet Res 1975;36(4 Pt.1):431-433.
- Beckett SD, Purohit RC, Reynolds TM. The corpus spongiosum penis pressure and external penile muscle activity in the goat during coitus. Biol Reprod 1975;12:289-292.
- Sachs BD. Role of striated penile muscles in penile reflexes, copulation, and induction of pregnancy in the rat. J Reprod Fertil 1982;66:433-443.
- Andersson K-E, Soler R, Fullhase C. Rodent models for urodynamic investigation. Neurourol Urodyn 2011;30:636-646. 59. Streng T, Santti R, Talo A. Possible action of the proximal rhabdosphincter muscle in micturition of the adult male rat. Neurourol Urodyn 2001;20:193-197.
- Nout YS, Bresnahan JC, Culp E, et al. Novel technique for monitoring micturition and sexual function in male rats using telemetry. Am J Phys
- Soukhova-O’Hare GK, Schmidt MH, Nozdrachev AD, et al. A novel mouse model for assessment of male sexual function. Physiol Behav 2007;91:535-543.
- Schmidt MH, Valatx JL, Sakai K, et al. Corpus spongiosum penis pressure and perineal muscle activity during reflexive erections in the rat. Am J Physiol 1995;269(4 Pt 2):R904- R913.
- Purohit RC, Beckett SD. Penile pressures and muscle activity associated with erection and ejaculation in the dog. Am J Physiol 1976;231(5 Pt. 1):1343-1348.
- Vardi Y, Siroky MB. Hemodynamics of pelvic nerve induced erection in a canine model. I. Pressure and flow. J Urol 1990; 144:794-797.
- Lue TF, Gleason CA, Brock GB, et al. Intraoperative electrostimulation of the cavernous nerve: technique, results and limitations. J Urol 1995;154:1426-1428.
- Mizusawa H, Hedlund P, Hakansson A, et al. Morphological and functional in vitro and in vivo characterization of the mouse corpus cavernosum. Br J Pharmacol 2001;132:1333- 1341.
- Carati CJ, Creed KE, Keogh EJ. Autonomic control of penile erection in the dog. J Physiol 1987;384:525-538.
- Stief C, Benard F, Bosch R, et al. Acetylcholine as a possible neurotransmitter in penile erection. J Urol 1989;141:1444- 1448.
- Creed KE, Carati CJ, Keogh EJ. Autonomic control and vascular changes during penile erection in monkeys. Br J Urol 1988;61:510-515.
- Rehman J, Christ GJ, Kaynan A, et al. Intraoperative electrical stimulation of cavernosal nerves with monitoring of intracorporeal pressure in patients undergoing nerve sparing radical prostatectomy. BJU Int 1999;84:305-310.
- Mullerad M, Donohue JF, Li PS, et al. Functional sequelae of cavernous nerve injury in the rat: is there model dependency. J Sex Med 2006;3:77-83.
- Albersen M, Fandel TM, Lin G, et al. Injections of adipose tissue-derived stem cells and stem cell lysate improve recovery of erectile function in a rat model of cavernous nerve injury. J Sex Med 2010;7:3331-3340.
- Mills TM, Stopper VS, Wiedmeier VT. Effects of castration and androgen replacement on the hemodynamics of penile erection in the rat. Biol Reprod 1994;51:234-238.
- Albersen M, Kendirci M, Van der Aa F, et al. Multipotent stromal cell therapy for cavernous nerve injury-induced erectile dysfunction. J Sex Med 2012;9:385-403.
- Quinlan DM, Nelson RJ, Partin AW, et al. The rat as a model for the study of penile erection. J Urol 1989;141:656-661.
- Sato Y, Rehman J, Santizo C, et al. Significant physiological roles of ancillary penile nerves on increase in intracavernous pressure in rats: experiments using electrical stimulation of the medial preoptic area. Int J Impot Res 2001;13:82-88.
- Walz J, Burnett AL, Costello AJ, et al. A critical analysis of the current knowledge of surgical anatomy related to optimization of cancer control and preservation of continence and erection in candidates for radical prostatectomy. Eur Urol 2010;57:179-192.
- Chung E, De Young L, Brock GB. Investigative models in erectile dysfunction: a state-of-the-art review of current animal models. J Sex Med 2011;8:3291-3305.
- Mills TM, Wiedmeier VT, Stopper VS. Androgen maintenance of erectile function in the rat penis. Biol Reprod 1992; 46:342-348.
- Escrig A, Gonzalez-Mora JL, Mas M. Nitric oxide release in penile corpora cavernosa in a rat model of erection. J Physiol 1999;516(Pt 1):261-269.
- Steers WD, Mallory B, de Groat WC. Electrophysiological study of neural activity in penile nerve of the rat. Am J Physiol 1988;254(6 Pt 2):R989-R1000.
- Bivalacqua TJ, Diner EK, Novak TE, et al. A rat model of Peyronie’s disease associated with a decrease in erectile activity and an increase in inducible nitric oxide synthase protein expression. J Urol 2000;163:1992-1998.
- Giuliano F, Rampin O, Bernabe J, et al. [Experimental approach to reflex erection in rats: modeling and functional neuroanatomy of the involved nerve pathways]. Prog Urol 1996;6:81-86.
- Christ GJ, Rehman J, Day N, et al. Intracorporal injection of hSlo cDNA in rats produces physiologically relevant alterations in penile function. Am J Physiol 1998;275(2 Pt 2):H600-H608.
- Bochinski D, Lin GT, Nunes L, et al. The effect of neural embryonic stem cell therapy in a rat model of cavernosal nerve injury. BJU Int 2004;94:904-909.
- Angulo J, Peiro C, Sanchez-Ferrer CF, et al. Differential effects of serotonin reuptake inhibitors on erectile responses, NOproduction, and neuronal NO synthase expression in rat corpus cavernosum tissue. Br J Pharmacol 2001;134:1190- 1194.
- Giuliano F, Rampin O, Bernabe J, et al. Neural control of penile erection in the rat. J Auton Nerv Syst 1995 Oct;55:36-44.
- Tsui B. Electrical nerve stimulation. In: Atlas of Ultrasound and nerve stimulation-Guided Regional Anesthesia. Springer; 2008. p. 9-17.
- Ueno N, Iwamoto Y, Segawa N, et al. The effect of sildenafil on electrostimulation-induced erection in the rat model. Int J Impot Res 2002;14:251-255.
- Berntson GG, Quigley KS, Fabro VJ, et al. Vagal stimulation and cardiac chronotropy in rats. J Auton Nerv Syst 1992; 41:221-226.
- Lue TF, Takamura T, Umraiya M, et al. Hemodynamics of canine corpora cavernosa during erection. Urology 1984; 24:347-352.
- Terada N, Arai Y, Kurokawa K, et al. Intraoperative electrical stimulation of cavernous nerves with monitoring of intracorporeal pressure to confirm nerve sparing during radical prostatectomy: early clinical results. Int J Urol 2003;10:251- 256.
- Burnett AL, Teloken PE, Briganti A, et al. Intraoperative assessment of an implantable electrode array for cavernous nerve stimulation. J Sex Med 2008;5:1949-1954.
- Sezen SF, Burnett AL. Intracavernosal pressure monitoring in mice: responses to electrical stimulation of the cavernous nerve and to intracavernosal drug administration. J Androl 2000;21:311-315.
- Qiu X, Villalta J, Ferretti L, et al. Effects of intravenous injection of adipose-derived stem cells in a rat model of radiation therapy-induced erectile dysfunction. J Sex Med 2012; 9:1834-1841.
- Burnett AL, Becker RE. Immunophilin ligands promote penile neurogenesis and erection recovery after cavernous nerve injury. J Urol 2004;171:495-500.
- Sengupta P. The Laboratory rat: Relating its age with Human’s. Int J Prev Med 2013;4:624-630.
- Canguven O, Burnett A. Cavernous nerve injury using rodent animal models. J Sex Med 2008;5:1776-1785.
- Mulhall JP, Muller A, Donohue JF, et al. The functional and structural consequences of cavernous nerve injury are ameliorated by sildenafil citrate. J Sex Med 2008;5:1126- 1136.
- Rabbani F, Stapleton AM, Kattan MW, et al. Factors predicting recovery of erections after radical prostatectomy. J Urol 2000;164:1929-1934.
- Sezen SF, Lagoda G, Burnett AL. Neuronal nitric oxide signaling regulates erection recovery after cavernous nerve injury. J Urol 2012;187:757-763.
- Sezen SF, Lagoda G, Burnett AL. Role of immunophilins in recovery of erectile function after cavernous nerve injury. J Sex Med 2009;6(Suppl 3):340-346.
- Weyne E, Albersen M, Hannan JL, et al. Increased expression of the neuroregenerative peptide galanin in the major pelvic ganglion following cavernous nerve injury. J Sex Med 2014; 11:1685-1693.
- Kim HJ, Kim HY, Kim SY, et al. Spontaneous recovery of cavernous nerve crush injury. Korean J Urol 2011;52:560- 565.
- Oudot A, Behr-Roussel D, Poirier S, et al. Combination of BAY 60-4552 and vardenafil exerts proerectile facilitator effects in rats with cavernous nerve injury: a proof of concept study for the treatment of phosphodiesterase type 5 inhibitor failure. Eur Urol 2011;60:1020-1026.
- Castiglione F, Nini A, Briganti A. Penile rehabilitation with phosphodiesterase type 5 inhibitors after nerve-sparing radical prostatectomy: are we targeting the right patients? Eur Urol 2014;65:673-674.
Supplementary data related to this article can be found at https://doi.org/10.1016/j.esxm.2020.06.007.