- Email: [email protected]
Pilot Study Evaluating a Rat Model of Radiation-induced Erectile Dysfunction Using an Image-guided Microirradiator
Basic and Translational Science Pilot Study Evaluating a Rat Model of Radiation-induced Erectile Dysfunction Using an Image-guided Microirradiator Masaki Kimura, Andrew R. Zodda, Javed Mahmood, Shiva K. Das, Giao B. Nguyen, Isabel L. Jackson, and Zeljko Vujaskovic OBJECTIVE METHODS
RESULTS
CONCLUSION
To establish a feasible rat model of radiation-induced erectile dysfunction after targeted prostate irradiation using an image-guided irradiation unit specially designed for small-animal radiation research. The X-RAD 225Cx research platform was used in the present study. We first performed quality assurance testing using a rat cadaver. After confirming dosimetry, 24 age-matched, young, adult, male rats were assigned to sham radiation or radiation to the prostate with doses of 15, 20, or 25 Gy. To confirm appropriate prostate irradiation, physiological erectile function was evaluated using intracavernous pressure (ICP) measurements with cavernous nerve electrical stimulation at 9 weeks after radiotherapy. Each animal was weighed at the time of ICP measurement. In addition, we investigated the cyclic guanosine monophosphate level in the penile cavernosa using a commercial enzyme-linked immunosorbent assay kit. Quality assurance results confirmed the accuracy of the irradiation technique. Dose-dependent decreases in ICP in irradiated rats were observed without major toxicity. No difference in body weight was noted among the experimental groups. Cyclic guanosine monophosphate levels were significantly decreased in the group that received 25 Gy compared with the age-matched shamirradiated group. High-precision imaging and targeting capabilities provided by the micro-IGRT platform enable us to develop a reproducible animal model of radiation-induced erectile dysfunction in prostate cancer research. UROLOGY 85: 1214.e1–1214.e6, 2015. 2015 Elsevier Inc.
I
mpairment of erectile function after prostate radiotherapy (RT) is a major concern for patients and clinicians. Development of erectile dysfunction (ED) affects sexual health-related quality of life in prostate cancer patients after routine treatments, including radical prostatectomy, RT, cryotherapy, and hormonal therapy.1-3 In previous study, the 5-year actuarial risk of potency loss was reported to be 60% with 3-dimensional conformal radiotherapy (3DCRT).4 However, a recent study reported that 36% of men receiving RT for prostate cancer have developed ED within 2 years after treatment, if secondary side effects of adjuvant-combined androgen deprivation therapy were excluded.5 Thus, the author mentioned that there was an overestimation of the rate of
Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Urology, Teikyo University, Kaga, Itabashi, Japan; the Division of Translational Radiation Sciences, Department of Radiation Oncology, University of Maryland, Baltimore, MD; the Department of Radiation Oncology, Duke University, Durham, NC; and the Division of Radiation Safety, Department of Radiology, Duke University, Durham, NC Address correspondence to: Zeljko Vujaskovic, M.D., Ph.D., Division of Translational Radiation Sciences, Department of Radiation Oncology, University of Maryland, Medical Sciences Teaching Facility 700A, 685 W Baltimore Street, Baltimore, MD 21201. E-mail: [email protected] Submitted: August 4, 2014, accepted (with revisions): December 12, 2014
1214.e1 ª 2015 Elsevier Inc. All Rights Reserved
ED after RT because hormonal therapy and aging had negative effects for sexual function.5 Because the technology for delivering RT is rapidly improving, recent studies have shown that intensity-modulated RT can deliver higher doses of radiation to the prostate, as well as increase the possibility of sparing a greater amount of erectile structures such as the penile crura and the bulb, which enable to reduce ED after RT.5-7 However, the etiology of radiation-induced ED has not been fully elucidated in large part because of challenges in developing an appropriate animal model. In this context, we previously developed a rat model of radiation-induced ED after targeted prostate irradiation using a clinical 3DCRT device.8,9 However, multiple complex steps were required to establish the model. In addition, because a clinical irradiator was used, our investigations needed to avoid animal contact with patients. New technology has recently been introduced for small-animal research in radiation biology using a conebeam computed tomography (CBCT)ebased smallanimal image-guided irradiation unit (micro-IGRT).10 Acceptable and validated animal models mimicking current clinical RT settings are crucial to advance translational research in radiation biology. These http://dx.doi.org/10.1016/j.urology.2014.12.020 0090-4295/15
Figure 1. Images of quality assurance testing. (A) Interior of the microimage-guided irradiation unit (X-Rad 225Cx), showing the C-arm setup with rat on the 3-dimensional linear translation stage. Detector probe of metal oxideesemiconductor fieldeffect transistor was inserted transrectally. Four different gantry angles (213 , 185 , þ5 , and þ32 ) were illustrated with red arrow. (B) X-ray image for QA test. Prostate and treatment volume were outlined with green and blue lines, respectively. Red circle indicates isocenter. (C) Cone-beam computed tomography image for QA test. White arrow indicates detecting probe, which was located at the level of prostate. Prostate and rectum were outlined with green and orange lines, respectively.
preclinical models can play a pivotal role in exploring the nature of radiobiological response, including mechanisms of pathophysiology of recurrence and development of new therapeutic drugs. Previous animal models have been limited by challenges in imaging and in focusing ionizing radiation to small organs using clinical irradiators. We report here on a pilot study establishing a feasible rat model of prostate irradiation and radiation-induced ED using a dedicated micro-IGRT for small-animal radiation research and investigated physiological erectile function. Of note, the nitric oxideecyclic guanosine monophosphate (cGMP) system is the most important pathway for penile erection. Nitric oxide from nonadrenergic noncholinergic fibers activates guanylyl cyclase, which leads to induction of smooth muscle relaxation by catalyzing the conversion of guanosine triphosphate to cGMP, resulting in engorgement of blood in the corpus cavernosum. Therefore, we confirmed cGMP levels in the penile tissue after 9 weeks from prostate irradiation using the micro-IGRT device.
METHODS Animals Fifteen young, adult, male, Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) with an initial age of 12 weeks were randomly divided into 3 groups for prostate irradiation with doses of 15, 20, and 25 Gy. An additional 8 agematched rats were assigned to sham irradiation, and a single rat, UROLOGY 85 (5), 2015
aged 21 weeks, was used for quality assurance (QA) testing, resulting in a total of 24 animals used in this study. Animals were weighed before intracavernous pressure (ICP) measurement at 9 weeks after radiation or sham treatment. All animal handling and procedures were approved by our Institutional Animal Care and Use Committee.
Hardware The X-RAD 225Cx small-animal research platform (Precision X-Ray Inc., North Branford, CT) was used in this study. This micro-IGRT includes a rotating C-arm gantry to facilitate both 360 CBCT scanning and 360 radiation delivery. The 3D stage and gantry are manipulated through a multiaxis motion controller with position encoders on each axis. Both the stages and gantry are housed in a self-shielded cabinet, with x-ray control, detector readout, and motion under computer control.
Quality Assurance Testing A cadaver of a rat aged 21 weeks was used for QA testing. The rat was euthanized using a CO2 chamber. After confirmation of death, the animal was secured in the prone position. A metal oxideesemiconductor field-effect transistor (MOSFET; Best Medical Canada, Ottawa, Canada) was used to perform QA testing. After calibration, the MOSFET was inserted into the rat rectum adjacent to the prostate to confirm the proposed dose used in treatment (Fig. 1A). An x-ray image of QA testing is shown in Figure 1B. The tip of the detector probe was confirmed to be at the appropriate level and location in the rectum. Figure 1C shows the CBCT QA testing image. The CT isocenter was placed at midprostate. 1214.e2
Figure 2. Image of planning and treatment. Cone-beam computed tomography images of case 1 (upper) and case 2 (lower). Prostate, rectum, and treatment volume were outlined with green, orange, and blue lines, respectively. Red circle indicates isocenter.
Irradiation Prostate-targeted irradiation using micro-IGRT was performed by a trained technician in consultation with a radiation physicist. An image of the position of the rat in the micro-IGRT machine is shown in Figure 1A. Rats received continuous isoflurane gas anesthesia via induction in an anesthesia chamber (2 L/min oxygen with 2.0% isoflurane). During imaging and irradiation, rats received continuous isoflurane anesthesia gas via a nose cone. The pelvic cavity was imaged via high-resolution microcomputed tomography (CT) under the following conditions: fluoro (1 1) þ scout (40 kVp, 0.5 mA) þ CBCT (40 kVp, 2.5 mA, 1 1, 7 frames per second, 0.008 voxel, 1 1 downsampling, 0.5 rpm). The prostate was specifically targeted for ionizing irradiation treatment from an x-ray source through micro-CT image guidance. Figure 2 shows representative images of CBCT before irradiation, showing the isocenter located in the middle of the rectum and skin surface at 8 mm above the pelvic joint. Four different gantry angles (213 , 185 , þ5 , and þ32 ) were used for irradiation based on micro-CT imaging. A collimator with a treatment field size of 20 20 mm2 was applied for whole prostate irradiation. The rats were treated at a maximal energy of 225 kVp and 13 mA, with a single dose of 15, 20, or 25 Gy at 5 minutes per treatment.
Physiological Erection Studies To analyze nerve-mediated physiologic erectile function, ICP measurement with cavernous nerve (CN) stimulation was conducted at 9 weeks after irradiation. Animals were 1214.e3
anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital, and the right carotid artery was cannulated with polyethylene-50 tubing containing heparinized saline for continuous monitoring of mean arterial pressure (MAP). The shaft of the penis was then exposed from skin and muscle, and the crus punctured with a 23-gauge needle connected to polyethylene-60 tubing. Both were connected to a pressure transducer (World Precision Instruments, Sarasota, FL). The CN was stimulated using a bipolar electrode connected to a Grass Instruments S48 stimulator (Grass Technologies, West Warwick, RI). Stimulator settings were 2-8 V of 1.5 mA, 16 Hz, 5-ms wide pulses for 1 minute with a minimum interval between stimulation of 5 minutes. Erectile response was determined by maximum ICP, the area under the curve (AUC; mm Hg/s) obtained during ICP elevation, and the ICP-to-MAP ratio. ICP/ MAP was determined using maximum ICP divided by MAP obtained during CN stimulation. All data were recorded using the Biopac MP100 data acquisition system and analyzed using the AcqKnowledge software, version 3.9.1 (Biopac System Inc., Goleta, CA).
Measurement of Cavernosal Tissue cGMP Levels Cavernosal cGMP levels were measured at 9 weeks after irradiation in the age-matched sham-irradiated rats and the 25-Gy irradiated rats. Frozen corpus cavernosum tissue was homogenized in 10 volumes of ice cold 5% trichloroacetic acid using a sonic dismembrator (Fisher Scientific, Pittsburgh, PA). The homogenized samples were centrifuged at 1500g for 10 minutes UROLOGY 85 (5), 2015
Figure 3. Results of intracavernous pressure (ICP) measurement. (A) Representative image for ICP curves with sham, 15, 20, and 25 Gy irradiation, (B) ICP/mean arterial pressure ratio, and (C) Cyclic guanosine monophosphate level measured at 9 weeks after irradiation. GMP, guanosine monophosphate; ICP, intracavernous pressure; MAP, mean arterial pressure.
at 4 C, and the supernatant was transferred to a clean test tube. The trichloroacetic acid was extracted with water-saturated diethyl ether. The top ether layer was carefully removed, and samples were heated to 70 C for 5 minutes to remove residual ether from the aqueous layer. Determination of concentrations was made using a cGMP enzyme-linked immunosorbent assay kit (Cayman Chemical, Ann Arbor, MI). All samples were duplicated in this assay.
Statistical Analysis Data on physiological erection response were analyzed by 1-way analysis of variance to compare results in the 4 groups. For multiple comparisons among each group, the Bonferroni-Dunn test was used. Statistical significance was considered when P <.05. All statistical analyses were conducted using Stata software, version 11.2 (StataCorp, College Station, TX).
RESULTS Total irradiation dose was confirmed at the 4 gantry points (213 , 185 , þ5 , and þ32 ). The combined dose rates at these gantry points allowed each side of the UROLOGY 85 (5), 2015
prostate to be irradiated equally. Based on results from QA testing, the total dose recorded by the MOSFET and the planned 20-Gy dose differed by <2.6%. Mean body weight of sham-irradiated and 15-, 20-, and 25-Gyeirradiated groups were 553.6 27.8, 575.0 46.6, 537.5 49.2, and 527.5 12.6 g, respectively, which showed no significant differences (P ¼ .273). A representative image of ICP is shown in Figure 3A, demonstrating a dose-dependent decrease in ICP. Figure 3B represents the results of ICP/MAP with 8-V electrical stimulation compared among sham-irradiated and 15-, 20-, and 25-Gy prostate irradiation. An obvious dose-dependent decrease in maximum ICP, AUC, and ICP/MAP was seen in all groups, which was statistically significant by analysis of variance (all P <.001). In addition, a statistically significant decrease in AUC and ICP/MAP ratio were found in the 20- and 25-Gy group when compared with the shamirradiated group by the Bonferroni-Dunn test. cGMP levels were significantly decreased in the irradiation group that received 25 Gy compared with the age-matched sham-irradiated group (P ¼ .048; Fig. 3C). 1214.e4
COMMENT We have demonstrated a feasible animal model for radiation-induced ED followed by focused prostate radiation using the micro-IGRT. To our knowledge, this is the first reported description of a procedure using a microIGRT system in a prostate irradiation model. In addition, we confirmed decreased physiological erectile function evaluated by ICP measurements along with evidence of decreased cGMP levels in cavernous tissues after prostate irradiation. Prostate cancer research uses small-animal models for developing and investigating underlying pathophysiology and evaluating the effectiveness of novel treatment strategies.11 In radiation biology research on prostate cancer in small animals, both focusing and targeted prostate irradiation have been limited by the unavailability of radiation technologies for small-organ irradiation. In this context, the introduction of specialized image-guided and robotically controlled irradiation technology for small animals can be key in empowering future irradiation experiments. The micro-IGRT device allows researchers to greatly extend the range and variation of available techniques and offers the ability for precise and accurate targeting of regions that may be difficult to pinpoint with conventional technologies.10,12 In addition, the specially controlled application of radiation dose permits studies of dose heterogeneity, dose escalation, and normal tissue effects with greater reliability, which contributes reproducibility to resulting data.10 The favorable characteristics of this device may solve some of the previous challenges in preclinical radiation biology research in prostate cancer and lead to minimally invasive biologic intervention via ionizing irradiation in small-animal models of prostate cancer. The majority of previous clinical radiation-induced ED animal models used a conventional irradiator without an image-guided system, which significantly limited the ability to focus radiation on the prostate in a manner that simulated clinical treatment standards. Some studies used an irradiator for in vitro experiments. Previous models of rat prostate irradiation have resulted in significant toxicity or used large fields, confounding normal tissue studies.13-15 Carrier et al13 identified a dose-response relationship with a bioassay and ICP approach 5 months after RT using a 16-20 cm2 250-kVp anterior field. Merlin et al,14 using a similar treatment design, found a dose-response relationship with increased cavernosal endothelin-1 levels, a vasoconstrictor associated with ED, and reduction in maximum ICP at 1 month after RT. Both studies used lead shielding over the testes but included proximal penile and vascular structures. A more recent study by Qiu et al16 demonstrated that adipose-derived stem cells ameliorated radiation-induced ED. The researchers delivered 4 Gy in 5 fractions followed by adipose-derived stem cell injection to the tail vein 1 week later. In addition, rats were irradiated with 2 Gy in 5 fractions 6 weeks before euthanasia, for a total
1214.e5
of 30 Gy per prostate. However, a conventional irradiator with a 3-mm thick lead shield, and a 25 30 mm window was used for limiting radiation to the prostate, which can result in irradiation of normal tissues adjacent to the prostate and may not adequately mimic current clinical settings. In our previous studies, we established a rat model of radiation-induced ED using an image-guided prostatetargeted irradiation technique. Our previous model demonstrated the time-dependent development of ED with extremely low rodent toxicity, which confirmed the feasibility of this model.9 Furthermore, we found that radiation-induced ED was significantly associated with generation of reactive oxygen species in the prostate as the targeted organ and the penile shaft as an adjacent organ, which can be associated with decreased physiological erectile function.17 Although previous studies have produced fruitful results, including details on the pathophysiology of radiation-induced ED, tremendous efforts were required to develop this model. To conduct image-guided irradiation to the rat prostate, for example, we used the clinical 3DCRT/ intensity-modulated RT irradiator (Varian Medical Systems, Novalis, TX). All procedures were conducted after normal working hours to avoid patient contact with animals. In addition, delivery planning was required for each individual animal, a timeconsuming method that was inappropriate for routine research practice with an animal model in this setting. In the animal model on which we report here, the quality of prostate irradiation was confirmed through a QA test. Irradiating MOSFET detectors for durations that were calculated to deliver 20 Gy to the prostate resulted in differences of <2.6%. We chose to treat animals with single fractions of 15, 20, and 25 Gy, to examine dosedependent decreases in erectile function, as well as the toxicity of ionizing radiation in normal tissue. As a result, no animals showed major or minor toxicities, such as bleeding from the gastrointestinal tract, weight loss, or appetite loss. In contrast, obviously decreased erectile function evaluated by ICP measurement was identified at 9 weeks after irradiation. The radiation dose selection (15, 20, and 25 Gy) was based on previous experiences in our laboratory.8,9 We considered that a single dose of 20 Gy would be expected to have the same effect on these tissues as a fractionated dose of 86-92 Gy.6 In this study, we did not account for limitations such as significant difference in tumor and normal tissue response or radiation durability in human and animal tissues. Therefore, more elucidation of pathophysiology and response with the present model will be informative.
CONCLUSION Our previous methods using a clinical irradiator required multiple and cumbersome steps in each animal—a process poorly suited for routine experimental animal models in the radiation biology research in prostate cancer.
UROLOGY 85 (5), 2015
High-precision imaging and targeting capabilities provided by the micro-IGRT platform enabled us to emulate current patient therapy for prostate cancer with a single step. Nine weeks after prostate irradiation, neither weight loss nor any of the major radiation toxicities were noted. Preliminary data demonstrated significant dosedependent decreased erectile function along with decreased cGMP levels in cavernous tissue after imageguided prostate irradiation. This irradiation technique and strategy may contribute to future radiation biology research in prostate cancer. Acknowledgment. The authors thank Irene Li for her tech-
6.
7.
8.
9.
10.
nical help. 11.
References 1. Litwin MS, Flanders SC, Pasta DJ, et al. Sexual function and bother after radical prostatectomy or radiation for prostate cancer: multivariate quality-of-life analysis from CaPSURE. Cancer of the Prostate Strategic Urologic Research Endeavor. Urology. 1999;54: 503-508. 2. Karakiewicz PI, Scardino PT, Kattan MW. The impact of sexual and urinary dysfunction on health-related quality-of-life (HRQOL) following radical prostatectomy (RP). Prostate Cancer Prostatic Dis. 2000;3:S21. 3. Kimura M, Donatucci CF, Tsivian M, et al. On-demand use of erectile aids in men with preoperative erectile dysfunction treated by whole gland prostate cryoablation. Int J Impot Res. 2011;23: 49-55. 4. Potosky AL, Davis WW, Hoffman RM, et al. Five-year outcomes after prostatectomy or radiotherapy for prostate cancer: the prostate cancer outcomes study. J Natl Cancer Inst. 2004;96:1358-1367. 5. van der Wielen GJ, van Putten WL, Incrocci L. Sexual function after three-dimensional conformal radiotherapy for prostate cancer:
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results from a dose-escalation trial. Int J Radiat Oncol Biol Phys. 2007; 68:479-484. DiBiase SJ, Wallner K, Tralins K, et al. Brachytherapy radiation doses to the neurovascular bundles. Int J Radiat Oncol Biol Phys. 2000;46:1301-1307. Roach M 3rd, Nam J, Gagliardi G, et al. Radiation dose-volume effects and the penile bulb. Int J Radiat Oncol Biol Phys. 2010;76: S130-134. Koontz BF, Yan H, Kimura M, et al. Feasibility study of an intensitymodulated radiation model for the study of erectile dysfunction. J Sex Med. 2011;8:411-418. Kimura M, Yan H, Rabbani Z, et al. Radiation-induced erectile dysfunction using prostate-confined modern radiotherapy in a rat model. J Sex Med. 2011;8:2215-2226. Clarkson R, Lindsay PE, Ansell S, et al. Characterization of image quality and image-guidance performance of a preclinical microirradiator. Med Phys. 2011;38:845-856. Pienta KJ, Abate-Shen C, Agus DB, et al. The current state of preclinical prostate cancer animal models. Prostate. 2008;68: 629-639. Wong J, Armour E, Kazanzides P, et al. High-resolution, small animal radiation research platform with x-ray tomographic guidance capabilities. Int J Radiat Oncol Biol Phys. 2008;71:1591-1599. Carrier S, Hricak H, Lee SS, et al. Radiation-induced decrease in nitric oxide synthase—containing nerves in the rat penis. Radiology. 1995;195:95-99. Merlin SL, Brock GB, Begin LR, et al. New insights into the role of endothelin-1 in radiation-associated impotence. Int J Impot Res. 2001;13:104-109. van der Wielen GJ, Vermeij M, de Jong BW, et al. Changes in the penile arteries of the rat after fractionated irradiation of the prostate: a pilot study. J Sex Med. 2009;6:1908-1913. Qiu X, Villalta J, Ferretti L, et al. Effects of intravenous injection of adipose-derived stem cells in a rat model of radiation therapyinduced erectile dysfunction. J Sex Med. 2012;9:1834-1841. Kimura M, Rabbani ZN, Zodda AR, et al. Role of oxidative stress in a rat model of radiation-induced erectile dysfunction. J Sex Med. 2012;9:1535-1549.
1214.e6
RESULTS
CONCLUSION
To establish a feasible rat model of radiation-induced erectile dysfunction after targeted prostate irradiation using an image-guided irradiation unit specially designed for small-animal radiation research. The X-RAD 225Cx research platform was used in the present study. We first performed quality assurance testing using a rat cadaver. After confirming dosimetry, 24 age-matched, young, adult, male rats were assigned to sham radiation or radiation to the prostate with doses of 15, 20, or 25 Gy. To confirm appropriate prostate irradiation, physiological erectile function was evaluated using intracavernous pressure (ICP) measurements with cavernous nerve electrical stimulation at 9 weeks after radiotherapy. Each animal was weighed at the time of ICP measurement. In addition, we investigated the cyclic guanosine monophosphate level in the penile cavernosa using a commercial enzyme-linked immunosorbent assay kit. Quality assurance results confirmed the accuracy of the irradiation technique. Dose-dependent decreases in ICP in irradiated rats were observed without major toxicity. No difference in body weight was noted among the experimental groups. Cyclic guanosine monophosphate levels were significantly decreased in the group that received 25 Gy compared with the age-matched shamirradiated group. High-precision imaging and targeting capabilities provided by the micro-IGRT platform enable us to develop a reproducible animal model of radiation-induced erectile dysfunction in prostate cancer research. UROLOGY 85: 1214.e1–1214.e6, 2015. 2015 Elsevier Inc.
I
mpairment of erectile function after prostate radiotherapy (RT) is a major concern for patients and clinicians. Development of erectile dysfunction (ED) affects sexual health-related quality of life in prostate cancer patients after routine treatments, including radical prostatectomy, RT, cryotherapy, and hormonal therapy.1-3 In previous study, the 5-year actuarial risk of potency loss was reported to be 60% with 3-dimensional conformal radiotherapy (3DCRT).4 However, a recent study reported that 36% of men receiving RT for prostate cancer have developed ED within 2 years after treatment, if secondary side effects of adjuvant-combined androgen deprivation therapy were excluded.5 Thus, the author mentioned that there was an overestimation of the rate of
Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Urology, Teikyo University, Kaga, Itabashi, Japan; the Division of Translational Radiation Sciences, Department of Radiation Oncology, University of Maryland, Baltimore, MD; the Department of Radiation Oncology, Duke University, Durham, NC; and the Division of Radiation Safety, Department of Radiology, Duke University, Durham, NC Address correspondence to: Zeljko Vujaskovic, M.D., Ph.D., Division of Translational Radiation Sciences, Department of Radiation Oncology, University of Maryland, Medical Sciences Teaching Facility 700A, 685 W Baltimore Street, Baltimore, MD 21201. E-mail: [email protected] Submitted: August 4, 2014, accepted (with revisions): December 12, 2014
1214.e1 ª 2015 Elsevier Inc. All Rights Reserved
ED after RT because hormonal therapy and aging had negative effects for sexual function.5 Because the technology for delivering RT is rapidly improving, recent studies have shown that intensity-modulated RT can deliver higher doses of radiation to the prostate, as well as increase the possibility of sparing a greater amount of erectile structures such as the penile crura and the bulb, which enable to reduce ED after RT.5-7 However, the etiology of radiation-induced ED has not been fully elucidated in large part because of challenges in developing an appropriate animal model. In this context, we previously developed a rat model of radiation-induced ED after targeted prostate irradiation using a clinical 3DCRT device.8,9 However, multiple complex steps were required to establish the model. In addition, because a clinical irradiator was used, our investigations needed to avoid animal contact with patients. New technology has recently been introduced for small-animal research in radiation biology using a conebeam computed tomography (CBCT)ebased smallanimal image-guided irradiation unit (micro-IGRT).10 Acceptable and validated animal models mimicking current clinical RT settings are crucial to advance translational research in radiation biology. These http://dx.doi.org/10.1016/j.urology.2014.12.020 0090-4295/15
Figure 1. Images of quality assurance testing. (A) Interior of the microimage-guided irradiation unit (X-Rad 225Cx), showing the C-arm setup with rat on the 3-dimensional linear translation stage. Detector probe of metal oxideesemiconductor fieldeffect transistor was inserted transrectally. Four different gantry angles (213 , 185 , þ5 , and þ32 ) were illustrated with red arrow. (B) X-ray image for QA test. Prostate and treatment volume were outlined with green and blue lines, respectively. Red circle indicates isocenter. (C) Cone-beam computed tomography image for QA test. White arrow indicates detecting probe, which was located at the level of prostate. Prostate and rectum were outlined with green and orange lines, respectively.
preclinical models can play a pivotal role in exploring the nature of radiobiological response, including mechanisms of pathophysiology of recurrence and development of new therapeutic drugs. Previous animal models have been limited by challenges in imaging and in focusing ionizing radiation to small organs using clinical irradiators. We report here on a pilot study establishing a feasible rat model of prostate irradiation and radiation-induced ED using a dedicated micro-IGRT for small-animal radiation research and investigated physiological erectile function. Of note, the nitric oxideecyclic guanosine monophosphate (cGMP) system is the most important pathway for penile erection. Nitric oxide from nonadrenergic noncholinergic fibers activates guanylyl cyclase, which leads to induction of smooth muscle relaxation by catalyzing the conversion of guanosine triphosphate to cGMP, resulting in engorgement of blood in the corpus cavernosum. Therefore, we confirmed cGMP levels in the penile tissue after 9 weeks from prostate irradiation using the micro-IGRT device.
METHODS Animals Fifteen young, adult, male, Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) with an initial age of 12 weeks were randomly divided into 3 groups for prostate irradiation with doses of 15, 20, and 25 Gy. An additional 8 agematched rats were assigned to sham irradiation, and a single rat, UROLOGY 85 (5), 2015
aged 21 weeks, was used for quality assurance (QA) testing, resulting in a total of 24 animals used in this study. Animals were weighed before intracavernous pressure (ICP) measurement at 9 weeks after radiation or sham treatment. All animal handling and procedures were approved by our Institutional Animal Care and Use Committee.
Hardware The X-RAD 225Cx small-animal research platform (Precision X-Ray Inc., North Branford, CT) was used in this study. This micro-IGRT includes a rotating C-arm gantry to facilitate both 360 CBCT scanning and 360 radiation delivery. The 3D stage and gantry are manipulated through a multiaxis motion controller with position encoders on each axis. Both the stages and gantry are housed in a self-shielded cabinet, with x-ray control, detector readout, and motion under computer control.
Quality Assurance Testing A cadaver of a rat aged 21 weeks was used for QA testing. The rat was euthanized using a CO2 chamber. After confirmation of death, the animal was secured in the prone position. A metal oxideesemiconductor field-effect transistor (MOSFET; Best Medical Canada, Ottawa, Canada) was used to perform QA testing. After calibration, the MOSFET was inserted into the rat rectum adjacent to the prostate to confirm the proposed dose used in treatment (Fig. 1A). An x-ray image of QA testing is shown in Figure 1B. The tip of the detector probe was confirmed to be at the appropriate level and location in the rectum. Figure 1C shows the CBCT QA testing image. The CT isocenter was placed at midprostate. 1214.e2
Figure 2. Image of planning and treatment. Cone-beam computed tomography images of case 1 (upper) and case 2 (lower). Prostate, rectum, and treatment volume were outlined with green, orange, and blue lines, respectively. Red circle indicates isocenter.
Irradiation Prostate-targeted irradiation using micro-IGRT was performed by a trained technician in consultation with a radiation physicist. An image of the position of the rat in the micro-IGRT machine is shown in Figure 1A. Rats received continuous isoflurane gas anesthesia via induction in an anesthesia chamber (2 L/min oxygen with 2.0% isoflurane). During imaging and irradiation, rats received continuous isoflurane anesthesia gas via a nose cone. The pelvic cavity was imaged via high-resolution microcomputed tomography (CT) under the following conditions: fluoro (1 1) þ scout (40 kVp, 0.5 mA) þ CBCT (40 kVp, 2.5 mA, 1 1, 7 frames per second, 0.008 voxel, 1 1 downsampling, 0.5 rpm). The prostate was specifically targeted for ionizing irradiation treatment from an x-ray source through micro-CT image guidance. Figure 2 shows representative images of CBCT before irradiation, showing the isocenter located in the middle of the rectum and skin surface at 8 mm above the pelvic joint. Four different gantry angles (213 , 185 , þ5 , and þ32 ) were used for irradiation based on micro-CT imaging. A collimator with a treatment field size of 20 20 mm2 was applied for whole prostate irradiation. The rats were treated at a maximal energy of 225 kVp and 13 mA, with a single dose of 15, 20, or 25 Gy at 5 minutes per treatment.
Physiological Erection Studies To analyze nerve-mediated physiologic erectile function, ICP measurement with cavernous nerve (CN) stimulation was conducted at 9 weeks after irradiation. Animals were 1214.e3
anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital, and the right carotid artery was cannulated with polyethylene-50 tubing containing heparinized saline for continuous monitoring of mean arterial pressure (MAP). The shaft of the penis was then exposed from skin and muscle, and the crus punctured with a 23-gauge needle connected to polyethylene-60 tubing. Both were connected to a pressure transducer (World Precision Instruments, Sarasota, FL). The CN was stimulated using a bipolar electrode connected to a Grass Instruments S48 stimulator (Grass Technologies, West Warwick, RI). Stimulator settings were 2-8 V of 1.5 mA, 16 Hz, 5-ms wide pulses for 1 minute with a minimum interval between stimulation of 5 minutes. Erectile response was determined by maximum ICP, the area under the curve (AUC; mm Hg/s) obtained during ICP elevation, and the ICP-to-MAP ratio. ICP/ MAP was determined using maximum ICP divided by MAP obtained during CN stimulation. All data were recorded using the Biopac MP100 data acquisition system and analyzed using the AcqKnowledge software, version 3.9.1 (Biopac System Inc., Goleta, CA).
Measurement of Cavernosal Tissue cGMP Levels Cavernosal cGMP levels were measured at 9 weeks after irradiation in the age-matched sham-irradiated rats and the 25-Gy irradiated rats. Frozen corpus cavernosum tissue was homogenized in 10 volumes of ice cold 5% trichloroacetic acid using a sonic dismembrator (Fisher Scientific, Pittsburgh, PA). The homogenized samples were centrifuged at 1500g for 10 minutes UROLOGY 85 (5), 2015
Figure 3. Results of intracavernous pressure (ICP) measurement. (A) Representative image for ICP curves with sham, 15, 20, and 25 Gy irradiation, (B) ICP/mean arterial pressure ratio, and (C) Cyclic guanosine monophosphate level measured at 9 weeks after irradiation. GMP, guanosine monophosphate; ICP, intracavernous pressure; MAP, mean arterial pressure.
at 4 C, and the supernatant was transferred to a clean test tube. The trichloroacetic acid was extracted with water-saturated diethyl ether. The top ether layer was carefully removed, and samples were heated to 70 C for 5 minutes to remove residual ether from the aqueous layer. Determination of concentrations was made using a cGMP enzyme-linked immunosorbent assay kit (Cayman Chemical, Ann Arbor, MI). All samples were duplicated in this assay.
Statistical Analysis Data on physiological erection response were analyzed by 1-way analysis of variance to compare results in the 4 groups. For multiple comparisons among each group, the Bonferroni-Dunn test was used. Statistical significance was considered when P <.05. All statistical analyses were conducted using Stata software, version 11.2 (StataCorp, College Station, TX).
RESULTS Total irradiation dose was confirmed at the 4 gantry points (213 , 185 , þ5 , and þ32 ). The combined dose rates at these gantry points allowed each side of the UROLOGY 85 (5), 2015
prostate to be irradiated equally. Based on results from QA testing, the total dose recorded by the MOSFET and the planned 20-Gy dose differed by <2.6%. Mean body weight of sham-irradiated and 15-, 20-, and 25-Gyeirradiated groups were 553.6 27.8, 575.0 46.6, 537.5 49.2, and 527.5 12.6 g, respectively, which showed no significant differences (P ¼ .273). A representative image of ICP is shown in Figure 3A, demonstrating a dose-dependent decrease in ICP. Figure 3B represents the results of ICP/MAP with 8-V electrical stimulation compared among sham-irradiated and 15-, 20-, and 25-Gy prostate irradiation. An obvious dose-dependent decrease in maximum ICP, AUC, and ICP/MAP was seen in all groups, which was statistically significant by analysis of variance (all P <.001). In addition, a statistically significant decrease in AUC and ICP/MAP ratio were found in the 20- and 25-Gy group when compared with the shamirradiated group by the Bonferroni-Dunn test. cGMP levels were significantly decreased in the irradiation group that received 25 Gy compared with the age-matched sham-irradiated group (P ¼ .048; Fig. 3C). 1214.e4
COMMENT We have demonstrated a feasible animal model for radiation-induced ED followed by focused prostate radiation using the micro-IGRT. To our knowledge, this is the first reported description of a procedure using a microIGRT system in a prostate irradiation model. In addition, we confirmed decreased physiological erectile function evaluated by ICP measurements along with evidence of decreased cGMP levels in cavernous tissues after prostate irradiation. Prostate cancer research uses small-animal models for developing and investigating underlying pathophysiology and evaluating the effectiveness of novel treatment strategies.11 In radiation biology research on prostate cancer in small animals, both focusing and targeted prostate irradiation have been limited by the unavailability of radiation technologies for small-organ irradiation. In this context, the introduction of specialized image-guided and robotically controlled irradiation technology for small animals can be key in empowering future irradiation experiments. The micro-IGRT device allows researchers to greatly extend the range and variation of available techniques and offers the ability for precise and accurate targeting of regions that may be difficult to pinpoint with conventional technologies.10,12 In addition, the specially controlled application of radiation dose permits studies of dose heterogeneity, dose escalation, and normal tissue effects with greater reliability, which contributes reproducibility to resulting data.10 The favorable characteristics of this device may solve some of the previous challenges in preclinical radiation biology research in prostate cancer and lead to minimally invasive biologic intervention via ionizing irradiation in small-animal models of prostate cancer. The majority of previous clinical radiation-induced ED animal models used a conventional irradiator without an image-guided system, which significantly limited the ability to focus radiation on the prostate in a manner that simulated clinical treatment standards. Some studies used an irradiator for in vitro experiments. Previous models of rat prostate irradiation have resulted in significant toxicity or used large fields, confounding normal tissue studies.13-15 Carrier et al13 identified a dose-response relationship with a bioassay and ICP approach 5 months after RT using a 16-20 cm2 250-kVp anterior field. Merlin et al,14 using a similar treatment design, found a dose-response relationship with increased cavernosal endothelin-1 levels, a vasoconstrictor associated with ED, and reduction in maximum ICP at 1 month after RT. Both studies used lead shielding over the testes but included proximal penile and vascular structures. A more recent study by Qiu et al16 demonstrated that adipose-derived stem cells ameliorated radiation-induced ED. The researchers delivered 4 Gy in 5 fractions followed by adipose-derived stem cell injection to the tail vein 1 week later. In addition, rats were irradiated with 2 Gy in 5 fractions 6 weeks before euthanasia, for a total
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of 30 Gy per prostate. However, a conventional irradiator with a 3-mm thick lead shield, and a 25 30 mm window was used for limiting radiation to the prostate, which can result in irradiation of normal tissues adjacent to the prostate and may not adequately mimic current clinical settings. In our previous studies, we established a rat model of radiation-induced ED using an image-guided prostatetargeted irradiation technique. Our previous model demonstrated the time-dependent development of ED with extremely low rodent toxicity, which confirmed the feasibility of this model.9 Furthermore, we found that radiation-induced ED was significantly associated with generation of reactive oxygen species in the prostate as the targeted organ and the penile shaft as an adjacent organ, which can be associated with decreased physiological erectile function.17 Although previous studies have produced fruitful results, including details on the pathophysiology of radiation-induced ED, tremendous efforts were required to develop this model. To conduct image-guided irradiation to the rat prostate, for example, we used the clinical 3DCRT/ intensity-modulated RT irradiator (Varian Medical Systems, Novalis, TX). All procedures were conducted after normal working hours to avoid patient contact with animals. In addition, delivery planning was required for each individual animal, a timeconsuming method that was inappropriate for routine research practice with an animal model in this setting. In the animal model on which we report here, the quality of prostate irradiation was confirmed through a QA test. Irradiating MOSFET detectors for durations that were calculated to deliver 20 Gy to the prostate resulted in differences of <2.6%. We chose to treat animals with single fractions of 15, 20, and 25 Gy, to examine dosedependent decreases in erectile function, as well as the toxicity of ionizing radiation in normal tissue. As a result, no animals showed major or minor toxicities, such as bleeding from the gastrointestinal tract, weight loss, or appetite loss. In contrast, obviously decreased erectile function evaluated by ICP measurement was identified at 9 weeks after irradiation. The radiation dose selection (15, 20, and 25 Gy) was based on previous experiences in our laboratory.8,9 We considered that a single dose of 20 Gy would be expected to have the same effect on these tissues as a fractionated dose of 86-92 Gy.6 In this study, we did not account for limitations such as significant difference in tumor and normal tissue response or radiation durability in human and animal tissues. Therefore, more elucidation of pathophysiology and response with the present model will be informative.
CONCLUSION Our previous methods using a clinical irradiator required multiple and cumbersome steps in each animal—a process poorly suited for routine experimental animal models in the radiation biology research in prostate cancer.
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High-precision imaging and targeting capabilities provided by the micro-IGRT platform enabled us to emulate current patient therapy for prostate cancer with a single step. Nine weeks after prostate irradiation, neither weight loss nor any of the major radiation toxicities were noted. Preliminary data demonstrated significant dosedependent decreased erectile function along with decreased cGMP levels in cavernous tissue after imageguided prostate irradiation. This irradiation technique and strategy may contribute to future radiation biology research in prostate cancer. Acknowledgment. The authors thank Irene Li for her tech-
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nical help. 11.
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