Multiphoton Imaging and Laser Ablation of Rodent Spermatic Cord Nerves: Potential Treatment for Patients With Chronic Orchialgia

Multiphoton Imaging and Laser Ablation of Rodent Spermatic Cord Nerves: Potential Treatment for Patients With Chronic Orchialgia Ranjith Ramasamy, Joshua Sterling, Philip S. Li, Brian D. Robinson, Sijo Parekattil, Jie Chen, Diane Felsen, Sushmita Mukherjee, Marc Goldstein and Peter N. Schlegel* From the Departments of Urology (RR, PSL, JC, DF, MG, PNS), Biochemistry (JS, SM) and Pathology and Laboratory Medicine (BDR), Weill Cornell Medical College, New York, New York, and Department of Urology, Winter Haven Hospital, University of Florida (SP), Winter Haven, Florida

Purpose: Microsurgical denervation of the spermatic cord has been done to treat chronic orchialgia. However, identifying the site of spermatic cord nerves is not feasible with an operating microscope or robotic stereoscope. We used multiphoton microscopy, a novel laser imaging technology, to identify and selectively ablate spermatic cord nerves in the rat. Materials and Methods: The spermatic cords of adult male Sprague-Dawley® rats were initially imaged in vivo under a low power multiphoton microscopy laser. After assessing the number, diameter and site (vasal vs perivasal) of the nerves a higher power laser using the same objective was used to ablate the nerves. The precision of nerve ablation and the preservation of surrounding structures were determined by histological analysis. We assessed the heterogeneity of the number of nerves with the Wilcoxon signed rank test. Results: The average number of nerves per spermatic cord was 10, which was similar bilaterally (p ⫽ 0.13). The vas and perivasal structures had a similar number of nerves (p ⫽ 0.4). The median diameter of all nerves was 32 ␮m. Confirmation of nerve ablation, and preservation of the vas deferens and vasculature were anatomically validated by histological analysis. Conclusions: Multiphoton microscopy can identify and ablate nerves selectively in vivo in the rat. It can potentially be used for spermatic cord denervation to treat chronic orchialgia. Such imaging may increase the efficacy of nerve ablation and can avoid the potential risks of testicular atrophy and hydrocele associated with spermatic cord microsurgical denervation.

Abbreviations and Acronyms MPM ⫽ multiphoton microscopy Submitted for publication May 9, 2011. Study received institutional review board approval. Supported by a Weill Cornell Medical College Clinical and Translational Science Center planning award (RR). * Correspondence: Department of Urology, 525 East 68th St., Starr 900, New York, New York 10065 (telephone: 212-746-5491; FAX: 212-7468425; e-mail: [email protected]).

Key Words: testis; spermatic cord; microscopy, fluorescence, multiphoton; pain; denervation CHRONIC orchialgia, defined as intermittent or constant, unilateral or bilateral testicular pain more than 3 months in duration, significantly interferes with daily activity and often creates a challenging management dilemma for urologists.1 Microsurgical denervation of the spermatic cord is

currently the most effective surgical treatment for chronic orchialgia.2 However, this procedure is effective in 70% of men at best.3 It is also associated with about a 1% risk of testicular artery injury due to the extensive dissection of the spermatic cord necessary to ligate the nerves.3

0022-5347/12/1872-0733/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION

Vol. 187, 733-738, February 2012 Printed in U.S.A. DOI:10.1016/j.juro.2011.09.143

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A technical limitation of current microsurgical technology is that the nerves cannot be directly visualized due to their small diameters. Thus, a noninvasive, real-time imaging modality that could specifically identify and selectively ablate nerves while preserving testicular vasculature and spermatic cord lymphatics is highly desirable to improve outcomes and decrease operative time and complications. MPM is a novel optical imaging technique4 that relies on the simultaneous absorption of 2 or 3 low energy, near infrared photons to cause an excitation equivalent to that created by a single photon of blue light. Excitation only occurs where there is sufficient photon density, ie at the laser focus point, allowing highly precise control of the part of the tissue being illuminated. Using 2-photon excitation at low laser power MPM is a noninvasive technique to visualize tissue structures that does not require specimen staining or fixation.5,6 At higher laser power the same laser source can be used as a precise microsurgical tool to ablate cells and subcellular structures.5,7,8 Groups at our institution previously reported visualizing cavernous nerves in ex vivo prostate specimens with MPM.9,10 In medicine MPM was recently applied to diagnose human dermal neoplasms in vivo11 and in animal studies in pulmonology12 and ophthalmology.7,13 We investigated in vivo imaging of nerves in the rat spermatic cord combined with nerve ablation by the same laser at higher output power. Since this

study was designed as a preclinical surgical feasibility assessment, we only used an objective that can image tissue at approximately 1 to 1.25 inches from the tissue surface. Thus, it is easier to translate to the clinical scenario, where surgical field sterility must be stringently maintained.

METHODS Animals and Surgery Adult male Sprague-Dawley rats were housed at a constant temperature (20C to 23C) and illumination cycle (12-hour light/12-hour dark cycle with light on at 8:00 a.m.) with free access to food and water. The rats were anesthetized using a ketamine/xylazine cocktail. A midline laparotomy was made, followed by exposure of the bilateral spermatic cords (fig. 1, A). The surgical setup was then moved to the microscope stage. A glass slide was placed under the exposed spermatic cord and the spermatic cord was imaged with MPM under low magnification using an XLFluor 4⫻, 0.28 numerical aperture dry objective (Olympus®) (fig. 1, B). Rat body temperature and respiration rate were monitored throughout the procedure. After intravital imaging the rat was sacrificed without reviving from anesthesia. The spermatic cord was excised and sent for histopathological analysis.

Imaging All imaging was done with the state-of-the-art FV1000MPE MPM system (Olympus). The laser source was a SpectraPhysics® Mai Tai® DeepSee™ with automated dispersion compensation and a tuning range of 690 to 1,040 nm that delivers about 80 femtosecond pulses at 780 nm. Images were collected as 2 separate gray scale images using

Figure 1. A, rat spermatic cord exposure. B, MPM imaging setup.

MULTIPHOTON IMAGING AND LASER ABLATION OF RODENT SPERMATIC CORD NERVES

highly sensitive photomultiplier tube detectors. Images were then color coded and merged to generate the final images presented. The emission channels used were 1) second harmonic generation signal (360 to 400 nm, color coded red) and 2) autofluorescence from nerve axons (400 to 490 nm, color coded yellow). After locating the nerves in the spermatic cord on MPM imaging laser power was increased to its maximum for that wavelength (about 0.5 W at 780 nm under the 4⫻ objective). A scanner zoom of 25⫻ to 40⫻ was used to achieve a concentrated beam to ablate the nerve at the laser focus. The fiber number was determined by systematically counting all nerves seen on 1 surface throughout the spermatic cord length. The spermatic cord was then flipped and imaged on the opposite side in similar fashion. The whole rat was moved on the imaging stage to ensure that vasal and perivasal areas were imaged. All image pseudocoloring and color merging were done using MetaMorph® v7.0r4. As required, further image processing, such as adjustment of brightness and contrast, Gaussian filtering to remove single pixel noise, placement of scale bars and cropping, were done with Photoshop®. All image processing algorithms were documented to enable reproducible comparisons among specimens.

Histopathological Correlations Specimens were processed for routine histopathology. The spermatic cord was serially sliced along the vertical plane into 2 mm thick sections and placed into cutting blocks. Specimens were placed in 10% neutral buffered formalin for 24 hours, followed by treatment with 70% to 100% ethanol and xylene. Specimens were embedded in paraffin and sectioned at 5 ␮m by a microtome. Two slides per

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specimen were stained, including 1 with hematoxylin and eosin, and 1 with monoclonal mouse anti-human neurofilament antibody (M0762, Dako, Glostrup, Denmark) to identify nerves. Slides were observed with a BX41 multihead microscope (Olympus) and digitally photographed. A urological pathologist with prior MPM experience reviewed and compared all histopathology slides and multiphoton images.

Statistical Analysis Data statistical analysis was done using SPSS®. Groups were compared with the Wilcoxon rank sum test for nonparametric data with significance considered at p ⬍0.05.

RESULTS Real-Time Imaging MPM imaging of the rat spermatic cord in vivo showed a median of 10 nerve fiber bundles per spermatic cord, measuring 20 to 50 ␮m in diameter, and in some cases their finer branches. The nerves showed a strong yellow autofluorescence signal, as expected (fig. 2, A). The surface of the spermatic cord produced a predominant second harmonic red signal due to the high collagen content of the sheath surrounding the vas. This plexus of nerves along the vas and the perivasal structures was confirmed by hematoxylin and eosin, and immunohistochemical staining (fig. 2, B and C). The blood vessels and lumen of the vas were preserved (fig. 2, B). While most nerves in the cord surround the vas, nerves

Figure 2. A, low magnification MPM of rat spermatic cord reveals nerve plexus surrounding vas. Nerves showed strong autofluorescence signal (yellow areas). Spermatic cord surface produced predominant second harmonic signal (red areas). Reduced from ⫻4, scale bar represents 0.5 mm. B, staining of same specimen reveals similar nerve (circles) distribution. H & E, scale bar represents 0.1 mm. C, anti-neurofilament antibody labeling of nerves (circles). Scale bar represents 0.1 mm.

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were also present in the perivasal tissue (see table). There was a similar number of nerves in the vas and the perivasal tissue (p ⫽ 0.13). The number of nerves on the left and right spermatic cords was similar (p ⫽ 0.4). Median ⫾ IQR nerve diameter was 32 ⫾ 7.3 ␮m. Multiphoton Laser Ablation The exposed spermatic cord of an anesthetized rat was placed under MPM, as described. A single nerve was identified by MPM imaging (fig. 3, B). With the identical nerve at the laser focus the laser power was increased to about 0.5 W under the objective. A high scanner zoom (25⫻ to 40⫻) was used to focus this intense beam on a portion of the nerve (fig. 3, E). The first impact of this ultrahigh intensity laser light on tissue was the generation of cavitation bubbles with a diameter of 1 to 40 ␮m. During illumination with this intense laser beam the specimen was manually scanned repeatedly in the vertical direction through the thickness of the nerve bundle until no autofluorescence signal from the nerve could be detected. While it took up to 2 minutes under these conditions to ablate a nerve in a living anesthetized animal, this time was 30 seconds or less for excised spermatic cords imaged ex vivo (data not shown). After ablation the gross specimen was similar to an electrocautery burn (fig. 3, D). Selective damage to the targeted area only (the nerve bundle at the laser focus) was confirmed by hematoxylin and eosin staining (fig. 3, F). Preservation of the vas deferens and vasculature was also confirmed by histological analysis.

DISCUSSION We developed and investigated an in vivo model of spermatic cord nerve ablation surgery using a femCharacteristics of rat spermatic cord nerves identified by multiphoton imaging Rat No. (spermatic cord side)

No. Vasal Nerves

No. Perivasal Nerves

Median Diameter (␮m)

Rt Lt

5 9

4 3

31 23

Rt Lt

14 7

3 1

32 24

Rt Lt

5 7

2 4

31 49

Rt Lt

3 5

5 4

34 28

Rt Lt

5 7

3 4

28 35

1:

2:

3:

4:

5:

Figure 3. Before and after MPM laser guided selective ablation of rat spermatic cord nerve. A and D, gross view. Scale bar represents 1 cm. B and E, MPM. Arrow indicates ablation site. Scale bar represents 0.5 mm. C and F, arrow indicates ablation site. H & E, scale bar represents 0.2 mm.

tosecond pulsed laser. Precise ablation of nerves was achieved. Results show that a highly focused laser beam creates distinct nerve lesions with a width of less than 5 ␮m. With the same laser scanning system we identified and selected nerves, and achieved immediate control of successful ablation. Control was attained with low laser energy to safely perform initial imaging. Subsequently increasing laser power and imaging time enabled us to perform ablation at the same site(s). Scanner zoom was used to increase the power per unit area and limit the absolute area being ablated. Collateral tissue damage was restricted, and the vasal lumen and the vessel architecture were preserved after ablation. To our knowledge no comparable studies exist of selective nerve ablation of the spermatic cord. Using MPM to visualize and ablate the nerves has several advantages over the current method of microsurgical denervation. The greatest limitation of the current operation is surgeon inability to pre-

MULTIPHOTON IMAGING AND LASER ABLATION OF RODENT SPERMATIC CORD NERVES

cisely identify and ligate the nerves in the spermatic cord even when using state-of-the-art operating microscopes or robotic stereoscopes. With MPM nerves are easily visualized on the vas and in the perivasal structures comprising the spermatic cord. Thus, MPM offers an immediate advantage over the surgical microscopes and robotic stereoscopes used today. MPM also offers the ability to control laser power and select an area for ablation with an accuracy of 2 to 4 ␮m. Other lasers used in clinical practice to ablate tissue, eg KTP, holmium and Nd:YAG lasers, destroy the entire tissue in the laser path. However, with MPM the use of a microscope objective to focus the beam and the generation of 2-photon excitation exclusively at the laser focus allow a high degree of spatial control. This was evident where undamaged tissue was observed above and below the ablated area (fig. 3, F). Since the depth to which meaningful imaging can be achieved by MPM using a 4⫻ objective at 780 nm is about 0.5 mm, complete imaging throughout the entire spermatic cord is not possible. Thus, manipulation of the spermatic cord was needed from the anterior to the posterior orientation to completely image the cord. Figure 3, E shows that the ablation spot was more extensive than absolutely necessary. This was done on purpose in these preliminary experiments to ensure that the ablated spots could be identified later by hematoxylin and eosin staining. The first sets of studies with smaller ablation spots were harder to identify with confidence by hematoxylin and eosin after all tissue processing steps. In future studies we will perform carefully controlled ablation to ablate only what is clinically necessary and sufficient. Many nerve fibers overlapped each other when observed from 1 direction (fig. 2, B and C). MPM images can be obtained in 3-dimensional fashion. An advantage of MPM imaging is that it allows optical sectioning through the specimen. This means that the same xy area can be imaged at various depths (along the z axis) to identify separate structures that lie on top of each other. The observer does not need to continuously change the microscope focus. Rather, image sets are collected as a stack at user defined z intervals. Based on a combination of autofluorescence and second harmonic generation signals MPM can identify larger nerve bundles with a thick sheath of collagen around them and smaller ones without this collagenous sheath.9,10 The lateral resolution of the microscope with the objective used in this study is about 6 ␮ per pixel, much smaller than the size of the smallest nerve bundles. Thus, MPM will likely visualize all nerves as long they are within 0.5 mm of the tissue surface.

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A potential confounding factor could be confusion between a small caliber nerve fiber and a capillary since the only way to distinguish them is by the lumen in the capillary. We expect that this will be less of a problem in living tissue, in which capillary lumina should be more obvious since they will be filled with blood. If this appears to be a major problem in an intraoperative context, capillaries can be highlighted by intravenous injection of a Food and Drug Administration approved contrast agent such as fluorescein. Pulsed radio frequency ablation of nerves was proposed as an alternate approach to spermatic cord nerve ablation in case reports.14 Unfortunately with this technology the inability to visualize and selectively ablate nerves remains. Also, this treatment can lead to neuroma and a neuritic reaction that can increase sympathetic discharge and exacerbate pain. Men who do not respond to microsurgical denervation (about 20%) or those who partially respond could have neuroma formation at the cut ends of the nerve. MPM ablation of nerves leaves the nerves in situ, maybe within the sheath, and may lead to improved efficacy due to the lack of neuroma formation. However, to our knowledge this finding remains to be investigated. If the laser beam must be focused further, higher magnification and higher numerical aperture objectives can be used, potentially even at the same surgery, by placing multiple objectives on a rotating turret. The limitation of this study is our inability to determine whether localized nerve ablation eliminated activity along the entire nerve. It is also unclear whether we effectively ablated all nerves compared to spermatic cord microsurgical denervation without dividing the vas deferens. These limitations will be addressed in future studies in which survival surgery will be done in rat models of testicular pain.15 If successful, MPM laser induced nerve ablation may also be extended to other nerve ablative pain surgeries, such as that for pelvic pain due to endometriosis, wrist pain and chronic low back pain. These advances will potentially yield improved functional outcomes and quality of life for patients with chronic pain.

CONCLUSIONS To our knowledge we report the first use of MPM to identify spermatic cord nerves. The inability to identify nerves with microsurgical denervation is a significant current limitation of surgical management of chronic orchialgia. MPM imaging may significantly aid in simultaneous nerve identification. It enables direct nerve ablation and prevents testicular artery injury based on the minimal spermatic

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cord dissection that would be necessary. The unique advantage of this technique is the acquisition of high

resolution images without the application of dyes, stains or fixation agents.

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6. König K, So PT, Mantulin WW et al: Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress. J Microsc 1996; 183: 197. 7. Hild M, Krause M, Riemann I et al: Femtosecond laser-assisted retinal imaging and ablation: experimental pilot study. Curr Eye Res 2008; 33: 351. 8. Toropygin S, Krause M, Riemann I et al: In vitro noncontact intravascular femtosecond laser surgery in models of branch retinal vein occlusion. Curr Eye Res 2008; 33: 277. 9. Yadav R, Mukherjee S, Hermen M et al: Multiphoton microscopy of prostate and periprostatic neural tissue: a promising imaging technique for improving nerve-sparing prostatectomy. J Endourol 2009; 23: 861. 10. Tewari AK, Shevchuk MM, Sterling J et al: Multiphoton microscopy for structure identification in

human prostate and periprostatic tissue: implications in prostate cancer surgery. BJU Int 2011; 108: 1421. 11. Koehler MJ, Zimmermann S, Springer S et al: Keratinocyte morphology of human skin evaluated by in vivo multiphoton laser tomography. Skin Res Technol 2011; 17: 49. 12. Nava RG, Li W, Gelman AE et al: Two-photon microscopy in pulmonary research. Semin Immunopathol 2010; 32: 297. 13. Gibson EA, Masihzadeh O, Lei TC et al: Multiphoton microscopy for ophthalmic imaging. J Ophthalmol 2011; 2011: 870879. 14. Cohen SP and Foster A: Pulsed radiofrequency as a treatment for groin pain and orchialgia. Urology 2003; 61: 645. 15. Yoshioka K, Tanahashi M and Uchida W: Behavioral and urological evaluation of a testicular pain model. Urology 2010; 75: 943.