Histologic Effects of a New Device for High-Intensity Focused Ultrasound Cyclocoagulation

Transcription

1 Glaucoma Histologic Effects of a New Device for High-Intensity Focused Ultrasound Cyclocoagulation Florent Aptel, 1,2 Thomas Charrel, 1 Xavier Palazzi, 3 Jean-Yves Chapelon, 1 Philippe Denis, 2 and Cyril Lafon 1 PURPOSE. To evaluate the histologic effects and clinical outcomes of high-intensity focused ultrasound (HIFU) delivered by miniaturized annular transducers for ciliary body coagulation in an animal study. METHODS. Eighteen eyes of 18 rabbits were insonified with a ring comprising a six-sector transducer, to produce a 12.8-mmdiameter circular lesion. Six sectors were activated in six rabbits (group 1), five sectors in six rabbits (group 2), and four sectors in six rabbits (group 3) at 2 W acoustic power. The rabbits were examined before treatment (day 0) and after treatment on days 1, 7, 15, 21, and 28. Detailed qualitative and semiquantitative histopathologic analyses of the enucleated eyes were performed. RESULTS. In the treated eyes, intraocular pressure changes ranged from 16.6 mm Hg ( 55.3%) at day 28 to 8.9 mm Hg ( 29.7%) at day 7 in group 1, from 4.7 mm Hg ( 25.5%) at day 28 to 1.4 mm Hg ( 7.6%) at day 21 in group 2 and from 7.9 mm Hg ( 28.1%) at day 28 to 2.0 mm Hg ( 7.1%) at day 7 in group 3. No macroscopic abnormalities were observed in the anterior segment or fundus. Histologic examination showed segmental-to-annular lesions in the ciliary processes, caused mainly by coagulation necrosis, whereas the sclera and lens appeared undamaged. Inflammation was very limited. CONCLUSIONS. Ultrasonic coagulation of the ciliary body with HIFU delivered via a circular miniaturized transducer seemed to be an effective and well-tolerated method of reducing intraocular pressure in an animal study. (Invest Ophthalmol Vis Sci. 2010;51: ) DOI: /iovs Ciliary body destruction is an established treatment method for refractory glaucoma. 1,2 Although diode laser transscleral cyclophotocoagulation is currently the clinical standard, many other methods and energy sources for destroying the ciliary processes have been investigated, including endoscopic laser cyclophotocoagulation, cyclocryotherapy, microwave heating, and high-intensity focused ultrasound (HIFU). 3 7 Ultrasonic ablation of the ciliary body using HIFU was extensively studied in the 1980s and 1990s. Results in both From the 1 INSERM (National Institute of Health and Medical Research), U556, Lyon, France; Université de Lyon, Lyon, France; 2 Hospices Civils de Lyon, Department of Ophthalmology, Hôpital Edouard Herriot, Lyon, France; and the 3 Department of Histopathology, Biomatech-NAMSA, Chasse-Sur-Rhône, France. Supported in part by Eyetechcare, Rillieux la Pape, France. Submitted for publication December 29, 2009; revised March 31 and April 28, 2010; accepted May 7, Disclosure: F. Aptel, Eyetechcare (F); T. Charrel, Eyetechcare (F); X. Palazzi, None, J.-Y. Chapelon, Eyetechcare (F); P. Denis, Eyetechcare (F); C. Lafon, Eyetechcare (F) Corresponding author: Florent Aptel, Department of Ophthalmology, Edouard Herriot Hospital, 5, Place d Arsonval, Lyon cedex 03, France; aptel_florent@hotmail.com. animal and clinical studies indicated that HIFU cyclodestruction is an effective and well-tolerated method of reducing elevated intraocular pressure (IOP) In particular, several clinical series have been conducted using a commercially available device (Therapeutic Ultrasound System; Sonocare, Inc., Ridgewood, NJ), for insonification. For example, Maskin et al. 13 achieved a 38.4% reduction in IOP 8 months after HIFU treatment in 158 eyes with refractory glaucoma, with success rates varying from 66% to 72% after a single treatment. In another study, Sterk et al. 14 reported a 42.2% reduction in IOP 3 to 4 months after a single HIFU cyclodestruction in 44 refractory glaucomatous eyes. Compared with the laser, a specific advantage of ultrasound is that the energy can be focused through non optically transparent media without uncontrolled energy absorption, which reduces the effects on the adjacent tissues. Similarly, energy deposition and tissue heating at the focus site do not depend on tissue pigmentation, which may vary greatly, particularly in the ciliary body. Compared to microwave heating or a nonfocused laser, such as the diode laser that is used for cyclophotocoagulation, focused ultrasound can be used to heat and treat a defined and adjustable tissue volume at any depth or location within the eye. Despite these inherent advantages of HIFU, its use for ciliary body destruction was gradually abandoned in the middle 1990s, 6 14 in part, because of the bulky design of the commercially available system and the relative complexity of the procedure, which required a fluid coupling bath of saline heated at 37 C that was made by sticking a plastic sheet to the patient s skin. The transducer, which was bulky (80-mm diameter) and heavy, was attached to an articulated arm. The distance of the placement of the transducer from the eye had to be measured with a diagnostic transducer. Once the correct distance was determined 170 mm between the transducer and the focus and the focal zone of the transducer was positioned, a single application of energy was performed. The transducer was then moved for each of the approximately six applications. The procedure was therefore rather long and complex. Miniaturized transducers can now be used to produce HIFU. Ultrasound focusing is better controlled and miniaturized transducers create small focal zones that better target the treatment areas, particularly for small organs such as the ciliary body. Complex transducers allow for lesions of variable geometries adapted to organs with a complex anatomy, such as the ciliary body. A higher operating frequency allows for a steeper transition between the focal zone and the untreated area, thus reducing the risk of heating the neighboring healthy tissue. Three-dimensional modeling of the shape and size of the resulting necrotic volume allow for a reduced energy dose to be delivered Such transducers are now being used for an increasing number of medical applications, including treatment of prostate cancer, hepatic tumors, or hemostasis during abdominal surgery We investigated the use of miniaturized circular transducers to produce cyclocoagulation. The primary goal of the present study was to evaluate the histologic Investigative Ophthalmology & Visual Science, October 2010, Vol. 51, No Copyright Association for Research in Vision and Ophthalmology

2 IOVS, October 2010, Vol. 51, No. 10 Ultrasonic Circular Cyclocoagulation 5093 characteristics of ciliary body lesions induced by transscleral HIFU in animals. In particular, we evaluated the form and reproducibility of the ciliary body lesions and the absence of injury to the adjacent ocular tissues. We also report the IOP reduction and clinical tolerability of the procedure. MATERIALS AND METHODS HIFU Device The treatment device placed on the eye comprised two parts. First, a coupling cone made of polymer was placed in direct contact with the eye, which allowed good placement of the transducers in terms of centering and distance (Fig. 1). A ring containing six active piezoelectric elements was then inserted in the upper coupling cone. The ring was 30 mm in diameter and 15-mm high. Each of the six transducers was a segment of a 10.2-mm radius cylinder with a 4.5-mm width and a 7-mm length (active surface area of approximately 35 mm 2 ). The focal volume of each transducer has approximately an elliptic cylinder shape (Fig. 2). The axial length of the focal zone is 1 mm (major section of the ellipse), the transverse focal width is 0.1 mm (minor section of the ellipse), and the lateral focal width is 3.5 mm (height of the elliptic cylinder). The six transducers were placed at regular intervals on the circumference of the ring and oriented to create a focal zone consisting of 4, 5, or 6 regularly distributed elliptical cylinder-shaped spots centered on a 12.8-mm-diameter circle. The devices used in the present study were specifically designed for the rabbit eye s anatomy. The resonant frequency of the transducers was 7 MHz, and we operated it at its third harmonic, 21 MHz. The ring was connected to a control module by a cable. The control module was composed of a signal generator producing an electrical voltage that varied according to a sinusoidal curve at a frequency of 21 MHz, an amplifier to enhance the electric voltage produced by the signal generator to a level that ensured that the piezoelectric components were excited and generated an ultrasonic beam, a watt meter that measured the incident and reflected electric power during treatment, an electronic switch controller with a power supply to enable the electric voltage to be sent to the piezoelectric elements to be activated, and a computer that controlled the electronic switch and the signal generator and allowed us to set up the treatment parameters, which included frequency, power, duration of each shot, and number of sectors to be activated. The computer sequentially activated each sector according to a program defined by the operator. FIGURE 1. Placement of the coupling cone (A). Centering of the coupling cone (B). Insertion of the ring in the upper coupling cone (C). Position of the six transducers (D). FIGURE 2. (A, B) Geometry of the six transducers. R c, radius of curvature (10.2 mm). (C, D) focal zone obtained in a thermosensitive gel made of polyacrylamide hydrogel. 24 (E, F) Focal zone obtained in a thermosensitive gel made of polyacrylamide hydrogel. Magnification: (E) 3; (F) Procedures Eighteen male adult New Zealand White rabbits (Charles River Laboratories, l Arbresle, France) were treated. This study was approved by Biomatech Institutional Review Board and Ethical Committee (May 15, 2009; Biomatech-NAMSA [North American Science Associates, Inc.], Chasse-Sur-Rhône, France), and was conducted in accordance with the requirements of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rabbits were anesthetized by inhalation of an O 2 -isoflurane (1% 4%) mixture and drops of tetracaine (Faure, Annonay, France) applied to the surface of the eye. Dexmedetomidine hydrochloride (0.06 mg/kg intramuscular Dexdomitor; Pfizer, Paris, France) and ketamine hydrochloride (20 mg/kg intramuscular Ketamine 1000; Virbac, Carros, France) were administered before the HIFU treatment. The coupling cone was applied so that the truncated side was in contact with the peripheral cornea at the limbus. The base of the cone, adapted to the shape of the eyeball, was introduced under the eyelids. Once the eyelids were correctly placed, the cone was centered by using the pupillary centering device (Fig 1). The active ring with the six piezoelectric elements was placed on the cone at the following areas sectors 1 through 3 at the top and sectors 4 through 6 at the bottom to avoid placement of piezoelectric elements against parts of the globe corresponding to the clock hours 3 and 9. Once the ring was placed on the cone, 5 ml of saline solution was used to fill the device until the fluid level reached the upper ring aperture, acting as coupling fluid and to cool the transducers. Before each treatment, the type of shot was configured on the computer. The parameters set were number of sectors to be activated, duration of shots, and acoustic power. Treatment was then started after the programmed sequence. The computer sent instructions to the electronic switch to provide each programmed sector with electric power during the scheduled time of shooting. Thus, all programmed

3 5094 Aptel et al. IOVS, October 2010, Vol. 51, No. 10 TABLE 1. Variations in IOP Values of the Treated and Nontreated Eyes between Each Time Period and Day 0 Day 1 Day 0 Day 7 Day 0 Day 15 Day 0 Day 21 Day 0 Day 28 Day 0 Group 1, n 6 Treated eye ( 50.7) ( 29.7) ( 42.7) ( 32.3) ( 55.3) Nontreated eye ( 25.5) ( 10.8) ( 4.8) ( 0.9) ( 18.6) Group 2, n 6 Treated eye ( 20.7) ( 8.2) ( 12.7) ( 4.7) ( 15.7) Nontreated eye ( 5.0) ( 3.4) ( 11.2) ( 4.5) ( 7.3) Group 3, n 6 Treated eye ( 16.0) ( 7.1) ( 19.9) ( 17.8) ( 28.1) Nontreated eye ( 22.3) ( 3.6) ( 7.2) ( 13.7) ( 26.7) Mean SD (relative change, %). sectors sequentially performed a shot until the end of treatment without pause. In six rabbits (group 1), six eyes (three right and three left eyes) were treated with the six transducers activated for 3 seconds at a 2-W acoustic power. In six rabbits (group 2), six eyes (three right and three left) were treated with five of the six transducers activated with the same parameters, and in six rabbits (group 3), six eyes (three right and three left) were treated with four of the six transducers activated with the same parameters. The thermal increase can be calculated with a bioheat transfer equation, as described in detail elsewhere. 16 With an activation time of 3 seconds, a power of 2 W, and an operating frequency of 21 MHz, the temperature increase at the focal point is approximately 39 C, and the absolute temperature is 78 C to 79 C, assuming a normal rabbit body temperature of 38.5 C to 39.5 C. IOP was measured bilaterally on day 0 before treatment and after treatment at days 1, 7, 15, 21, and 28 using a tonometer (Tono-pen XL; Reichert Inc., Depew, NY). The rabbits were anesthetized by inhalation of an O 2 -isoflurane (1% 4%) mixture and topical drops of tetracaine for IOP measurements. The measurements were taken at least 5 minutes after the induction of general anesthesia and under the same conditions at all time points. Five series of measurements were taken by one operator. When the standard deviation for one series (indicated by the tonometer) was over 5%, the measurements were repeated until a standard deviation of less than or equal to 5% was obtained for each series. On days 1 and 15, the lids and the anterior segment were examined with a portable slit lamp biomicroscope (model SL-14; Kowa, Nagoya, Japan) and the fundus with a lens (Super Field; Volk Optical, Inc., Mentor, OH) after pupil dilation with 1% tropicamide (MSD-Chibret, Paris, France). On day 28, the rabbits were anesthetized with an injection of tiletamine-zolazepam 10 mg/kg intramuscular (Zoletil; Virbac) and then terminated by a lethal injection of 1 ml/kg intravenous barbiturate (Dolethal; Vetoquinol, Lure, France). After termination, all the treated and nontreated eyes were removed, intravitreously injected with Davidson s fixative solution ( 0.2 ml), and preserved in Davidson s fixative solution. The orientation of the eyes was marked with sutures. Histologic Analysis After fixation in a Davidson solution, the eyes were dehydrated in alcohol solutions of increasing concentration, cleared in xylene, and embedded in paraffin. They were sectioned coronally in two parts (one rostral, one caudal). The rostral portion was further sectioned in the coronal plane encompassing the ciliary processes. The lens was detached during sectioning and then removed. At least 20 serial sections (5- m-thick, every 100 m) through the ciliary bodies were cut on a microtome (HM 355 S; Microm, Francheville, France) and then stained with safranin-hematoxylin-eosin. Additional sections were performed when necessary to cut throughout the ciliary body. The caudal portion of the eyes was further cut in a parasagittal plane to delimit a slice encompassing the optic nerve. Then, 15 sections (5 m thick, every 500 m) were performed and stained with safranin-hematoxylin-eosin at the level of the optic papilla. The sections were analyzed by light microscopy (DMR microscope fitted with 2.5, 5, 10, 20, 40, or 63 objectives; Leica, Solms, Germany). Qualitative and semiquantitative histopathologic analyses of the sections were performed with special care taken to avoid damaging the ciliary process or inducing necrosis or degeneration and retinal changes. The following parameters were graded from 0 (absence) to 4 (severe): inflammatory reaction FIGURE 3. Variations in mean IOP (mm Hg) in the treated eyes between each time period and day 0. Group 1 (n 6): six transducers activated. Group 2 (n 6): five transducers activated. Group 3 (n 6): four transducers activated.

4 IOVS, October 2010, Vol. 51, No. 10 Ultrasonic Circular Cyclocoagulation 5095 FIGURE 4. Variations in mean IOP (mm Hg) in the nontreated eyes between each time period and day 0. Group 1 (n 6): six transducers activated. Group 2 (n 6): five transducers activated. Group 3 (n 6): four transducers activated. (macrophages, lymphocytes, plasma cells, polymorphonuclear cells, and giant cells), fibrocytes, fibrin, edema, hemorrhage, cellular and tissue degeneration, and necrosis. Statistical Analysis The risk for the statistical analysis was established at 5%. For each group, at each follow-up time, the IOPs obtained in the treated eyes were compared with those of the nontreated eyes by Mann-Whitney test. Moreover, for each group, IOPs of the treated eyes on posttreatment days 1, 7, 15, 21, and 28 were compared with the pretreatment IOPs obtained for the treated eyes at day 0 with a Wilcoxon test (SPSS, ver. 15.0; SPSS, Chicago, IL). RESULTS Clinical Aspects No significant abnormalities were observed during treatment. No macroscopic abnormalities of the eyes were observed after treatment (see Supplementary Fig. S1, cgi/content/full/51/10/5092/dc1). On posttreatment days 1 and 15, no abnormalities were observed during anterior segment and fundus examination. The mean body weight variation at termination was slightly negative ( 3.8%). During the study, 14 of 18 rabbits showed a decrease in body weight, 3 rabbits gained no weight, and 1 rabbit gained weight. This negative variation can be explained by the likelihood of partial anorexia in several rabbits and the stress induced by the anesthesia procedure. None of the rabbits died while the study was in process. During the study, mean IOP values globally decreased from days 0 to 28 in both treated eyes and nontreated eyes (Table 1, Figs. 3, 4). Compared with the IOP at day 0, the IOP at day 28 was significantly reduced in all three groups of treated eyes (P 0.05). Compared with the nontreated eyes, the IOP reduction at day 28 was significantly higher in the treated eyes of groups 1 and 2 (P 0.05), but not in group 3. At all time points, the IOP reduction was significantly higher in the FIGURE 5. Whole coronal section of the globe. Meridians of clock hours 12 and 6 were not treated (four sectors activated) and appeared undamaged (black arrows). Magnification, 10. FIGURE 6. High-magnification photomicrographs showing ciliary processes with coagulation necrosis, loss of the bilayered epithelium, and vascular depletion of the stroma (A, B). Undamaged ciliary processes (C, D). Magnification, 40.

5 5096 Aptel et al. IOVS, October 2010, Vol. 51, No. 10 FIGURE 7. High-magnification photomicrograph showing details of necrotic ciliary processes with loss of ciliary epithelium, vascular congestion, and distension of the stromal collagen fibers. Magnification, 120. treated eyes of group 1 compared with those of groups 2 and 3(P 0.01). Histopathology The histologic changes were circumferentially distributed on the ciliary processes, with the location dependent on the specimens (Fig. 5). The maximum intensity was consistently observed in the deepest regions of the ciliary processes, whereas the rostral and caudal regions were less affected. In the affected regions, the distal and intermediate parts of the ciliary processes showed acute inflammatory and necrotic changes ranging from stromal edema (marked distension of collagen fibers) and vascular congestion (distension of vascular lumens by erythrocytes) to coagulation necrosis with loss of surface epithelium and hemorrhage, whereas the basal part of the ciliary processes and the rest of the ciliary body appeared normal (Fig. 6). The bilayered epithelium was preserved in the basal part of most ciliary processes, but it was degenerated or necrotic and sloughed off in the distal parts of the most affected areas (Fig. 7 and Supplementary Fig. S2, iovs.org/cgi/content/full/51/10/5092/dc1). The overall intensity of the findings of the ciliary processes was considered marked to severe. The inflammatory cellular reaction assessed by the presence of macrophages, lymphocytes, plasma cells, polymorphonuclear cells, or giant cells was very limited (Table 2). The sclera appeared normal in front of all treated areas, without any signs of thinning or necrosis. In one specimen of group 2, focal choroidal hemorrhage was associated with focal retinal folds and minimal retinal necroses. There were no signs of retinal degeneration/necrosis or of vascular necrosis of the underlying choroid vessels in the other rabbits. The optic papilla showed no vascular congestion or signs of degeneration. The sclera appeared normal in the region of the optic papilla. DISCUSSION We evaluated the treatment potential of using HIFU delivered by miniaturized circular transducers to produce cyclocoagulation in rabbit eyes. Emphasis was placed on evaluation of the histologic characteristics of the ciliary body lesions, particularly the form and reproducibility of the lesions, and absence of injury to adjacent tissues. Our results indicated that HIFU effectively produced ciliary body destruction. Histologic examination revealed coagulation necrosis at the most intermediate and distal parts of the ciliary processes with loss of the bilayered epithelium, edema, and vascular congestion. These thermal changes in the ciliary processes TABLE 2. Histologic Parameters Graded from 0 (Absent) to 4 (Severe) Group 1 (n 6) Group 2 (n 6) Group 3 (n 6) iliary body Neutrophils Eosinophils Lymphocytes Plasma cells Macrophages Giant cells Fibrocytes Fibrin, exudates (0 2) Stromal edema 2.05 (0 3) 1.85 (0 3) 2.07 (0 3) Congestion/hemorrhage 1.45 (0 3) 0.73 (0 2) 1.55 (0 3) Necrosis 1.94 (0 4) 1.54 (0 2) 2.00 (0 4) Loss of epithelium 2.08 (0 3) 1.88 (0 3) 1.88 (0 3) Choroid and retina Neutrophils 0.07 (0 1) (0 1) Eosinophils Lymphocytes Plasma cells Macrophages Giant cells Fibrocytes Fibrin, exudates Optic nerve edema (0 1) 0 Congestion/hemorrhage 0.20 (0 1) 0.40 (0 1) 0.46 (0 1) Retinal folds/detachment (0 1) 0 Retinal necrosis (0 1) 0 Choroid vessel necrosis Data are expressed as the mean (minimum maximum).

6 IOVS, October 2010, Vol. 51, No. 10 Ultrasonic Circular Cyclocoagulation 5097 were circumferentially distributed when all six sectors were activated and segmental when five or four sectors were activated. These findings are consistent with those of previous studies in which transscleral or endoscopic diode or Nd:yag laser cyclophotocoagulation was performed In contrast, the epithelium appeared preserved in the basal part of the ciliary processes. We did not observe regeneration of the treated ciliary processes, especially at the level of the basal membrane and ciliary epithelium. Similarly, examination of the stroma did not reveal any fibroblasts. In some studies, it has been reported that the epithelium regenerates after cyclophotocoagulation, resulting in restoration of aqueous humor secretion. 28 The regeneration process most likely involves the recolonization of an intact basement membrane by epithelial cells coming from unaffected adjacent regions. Using HIFU, the thermal increase is higher, up to 80 C, and is most likely sufficient to produce coagulation and rupture the basement membrane. The demarcations between treated and untreated areas were very sharp and often smaller than 0.1 mm. Similarly, localization of the lesions was relatively constant in all treated eyes, and 75% to 90% of the length of ciliary processes was treated in all rabbits. The very focal damage and constant position of the treated zone are a probable explanation of why no injury to the adjacent ocular tissues was observed. Particularly, the iris base, sclera, and pars plana appeared undamaged. We could not assess the presence or absence of lesions of the lens, because it was removed from the fixed tissues before they were cut for histologic analysis. Nevertheless, we did not observe any opacities of the lens during slit lamp examination after pupil dilation, which we believe is a rather good indicator of the absence of damage. In one rabbit of group 2, we observed focal choroidal hemorrhage associated with minimal retinal necrosis. We had difficulty in anesthetizing this rabbit during HIFU treatment, which resulted in the animal s moving and contracting the eyelids. These movements could have caused a misalignment of the device; in particular, toggling the device off or on could result in damage to the retina and choroid by a focal point. We did not observe any sign of retinal degeneration or necrosis and no signs of vascular necrosis of the underlying choroid vessels in the other rabbits. Our histologic examinations revealed very little inflammation of the ciliary processes and other ocular structures. In particular, we did not observe fibrinous or proteinaceous accumulations around the ciliary processes. This result is consistent with those of the slit lamp examination, which showed no signs of intraocular inflammation during the follow-up. It may explain why none of the treated rabbits presented an increase in IOP in the early follow-up. A limitation of the present study is that we used nonpigmented rabbits. Although energy absorption and deposition in HIFU is independent of tissue pigmentation, the release of melanin that can follow necrosis of the ciliary processes could affect the inflammatory response and tolerability of the procedure. Many mechanisms have been postulated to explain IOP reduction after cyclodestruction or cyclophotocoagulation, including destruction of the pigmented and nonpigmented epithelium, resulting in reduced aqueous production, ciliary body inflammation, enhanced outflow through the thinned sclera, and enhanced uveoscleral outflow due to changes in the ciliary body stroma and damage to the pars plana. 12,29,30 Our histologic and clinical examination did not reveal significant inflammation. Similarly, no injury or thinning of the sclera or the pars plana was observed. Therefore, we assume that the IOP reduction obtained in our experiments was related to the ciliary epithelium damage. The amount of ciliary body tissue that must be destroyed to reduce the IOP is not known. Previous studies reported the likelihood of a threshold beyond which treatment is effective. In our experiments, we obtained a significant IOP reduction that was maintained 4 weeks after treatment by treating the intermediate and distal parts of the ciliary processes. The IOP reduction was significantly higher when the ciliary body was circumferentially treated (all six sectors activated). In summary, HIFU delivered by circular miniaturized transducers in rabbit eyes produced localized, reproducible, and sustainable histologic damage of the ciliary processes and did not damage the adjacent ocular structures. Ultrasonic circular coagulation of the ciliary body using HIFU seems to be an effective and well-tolerated method of reducing IOP. References 1. 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