Microplasmin: Ex Vivo Characterization of Its Activity in Porcine Vitreous METHODS

Transcription

1 Microplasmin: Ex Vivo Characterization of Its Activity in Porcine Vitreous Marc D. de Smet, 1,2 Christophe Valmaggia, 1,3 Javier Zarranz-Ventura, 4 and Ben Willekens 5 PURPOSE. Microplasmin is a recombinant protein limited to the enzymatic moiety of plasmin without any of its cringle domains. Its enzymatic activity is similar to that of plasmin enzyme. The present study characterizes in a porcine eye model the vitreolytic ability of microplasmin. METHOD. Freshly harvested porcine eyes were used in these trials. Eyes were injected with escalating doses of microplasmin (62.5, 125, 250, 400 g) for 1 hour or with 125 g microplasmin with increasing time exposures (15, 30, 60, 120 minutes). Eyes were fixed by a very slow dehydration process to preserve the integrity of the vitreous retinal interface. They were examined by light microscopy to determine the degree of posterior vitreous detachment and by scanning electron microscopy (SEM) to study structural changes. RESULTS. Effective separation of the posterior hyaloid appeared to be dose dependent. After 1 hour, the posterior pole was detached in 100% of porcine eyes exposed to 125 g microplasmin and in the midperiphery to 250 g microplasmin. Vitreous at the ora did not detach. At 120 minutes of exposure, midperipheral detachment was observed with 125 g microplasmin. A smooth retinal surface was seen where the enzyme caused posterior vitreous detachment. There was also significant change to the integrity of the vitreous without any obvious structural alterations to the retina by histology or scanning electron microscopy. CONCLUSIONS. Microplasmin caused vitreolysis and posterior vitreous separation in an ex vivo porcine eye model in an apparent dose- and time-dependent fashion. In this model system, the minimal effective dose appeared to be 125 g. (Invest Ophthalmol Vis Sci. 2009;50: ) DOI: /iovs Inducing posterior vitreous detachment by pharmacologic means has been a longstanding goal of vitreoretinal surgeons. 1 3 A few decades ago, reports appeared suggesting that spontaneous posterior vitreous detachment (PVD) would decrease the risk for or lead to the resolution of proliferative disease. 4 6 Surgically producing an atraumatic separation of From the 1 Department of Ophthalmology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 2 Division of Vitreo-retinal Surgery and Uveitis, Department of Ophthalmology, Middelheim Hospital, Antwerp, Belgium; 3 Department of Ophthalmology, Kantonsspital St. Gallen, St. Gallen, Switzerland; 4 Faculty of Medicine, University of Navarra, Pamplona, Spain; and 5 Department of Morphology, Netherlands Institute for Neurosciences, Amsterdam, The Netherlands. Submitted for publication April 19, 2008; revised June 21 and September 3, 2008; accepted November 3, Disclosure: M.D. de Smet, ThromboGenics Ltd. (F, C), P; C. Valmaggia, ThromboGenics Ltd. (F); J. Zarranz-Ventura, Thrombo- Genics Ltd. (F); B. Willekens, ThromboGenics Ltd. (F) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked advertisement in accordance with 18 U.S.C solely to indicate this fact. Corresponding author: Marc D. de Smet, Department of Ophthalmology, Middelheim Hospital, Lindendreef 1, Antwerp 2000, Belgium; mddesmet1@mac.com. the posterior vitreous face achieves the same goals but may be difficult to induce. Incomplete separation can lead to disease progression, whereas firm adhesions between the hyaloidal face and the underlying retina can lead to damage of the underlying structures. 7 Several compounds, both enzymatic and nonenzymatic, have been tried as vitreolytic agents One of the most promising and extensively tested in humans has been the serine protease plasmin. Its efficacy in producing PVD has been documented in a number of studies However, producing autologous plasmin is a labor- and time-intensive process that most clinical laboratories are unable or unwilling to carry out. The final product is difficult to characterize and is inherently unstable. A more suitable alternative should ideally retain the biological activity but be pharmacologically fully characterized. Microplasmin is a recombinant protein, a truncated form of human plasmin that lacks all five kringle domains but retains the protease activity. 20 Produced in Pichia pastoris, the recombinant protein is stabilized in a dilute citrate buffer at ph 3.1 and is lyophilized. Its half-life in blood is several times higher than in plasmin, whereas its biological activity in models of peripheral arterial occlusion and ischemic stroke are similar to that in intact plasmin enzyme. Early studies in cat and rabbit models have shown its safety in animal eyes. 14,21 The present study was designed to characterize the enzymatic activity of recombinant microplasmin on the vitreous interface in an ex vivo porcine model system in which artifactual separation of the vitreous from the retinal surface was minimized. In this system, dose and time exposure were evaluated by light microscopy (LM) and electron microscopy (EM). METHODS Enzyme Preparation and Injection Procedure Microplasmin was provided by ThromboGenics Ltd. (Dublin, Ireland). The solution was stored at 20 C until needed. Just before use, the solution was thawed, diluted to the appropriate concentration under sterile conditions with balanced salt solution plus (BSS ; Alcon Laboratories, Fort Worth, TX), and kept on ice until use. The injections were given with a maximal delay of 15 minutes, after which a new vial of microplasmin was prepared. In all experiments, fresh cadaver pig eyes were obtained from a local slaughterhouse. To ensure freshness, the eyes were obtained directly after the animal was killed and were cleaned and gutted. The procedure never took longer than 10 minutes. One hundred microliters of the appropriate concentration of microplasmin or physiologic saline was injected into the eye through the pars plana 3 mm posterior to the limbus. The site of injection was marked on the surface of the sclera with indelible ink. Eyes were kept at ambient temperature (20 C 25 C) for the duration of the incubation period and were then processed as described. Fixation Procedure and Preparation for Light and Electron Microscopy Preliminary studies were conducted to determine the best fixation, embedding, and staining methodology to minimize interference with Investigative Ophthalmology & Visual Science, February 2009, Vol. 50, No Copyright Association for Research in Vision and Ophthalmology

2 IOVS, February 2009, Vol. 50, No. 2 Microplasmin 815 the vitreoretinal interface on LM and on EM. The resultant protocol is summarized in this article. Additional studies were carried out to ensure that the vitreoretinal interface would not undergo any alteration caused by autolysis at the maximal incubation period used in these studies. With fresh porcine globes, it was determined that autolysis becomes significant at 24 hours of incubation at room temperature. Slow progressive dehydration and the use of whole globes was felt to be critical to avoid artifactual separation of the vitreous from the retina. To stop enzymatic reaction and to facilitate intraocular fixation, a 4- to 6-mm scleral opening was made parallel to the pars plana at least 90 away from the site of injection, and between 0.2 ml and 0.4 ml fixative was injected directly into the eye, toward the midvitreous. The eyes were left in a mixture of 1.25% glutaraldehyde and 1% paraformaldehyde in 0.08 M cacodylate buffer (ph 7.4) at room temperature for 3 days. Then they were slowly dehydrated in progressively higher concentrations (up to 100%) of ethanol over a 4-week period. After complete dehydration, the eyes were cut in two equal parts with a sharp scalpel blade. The incision was placed in the anteroposterior axis of the injection site. Photographs were taken of the eyecups. Half of each eye was used for LM, and the other half was used for scanning EM (SEM). The specimen for LM was placed in embedding resin (Technovit 7100; Heraeus Kulzer GmbH, Wehrheim/ts, Germany) in accordance with the manufacturer s instructions. Serial 4- m thick slices were obtained, placed on a glass slide, and stained with 1% toluidine blue. The specimen for EM was immersion dried in hexamethyldisilazane (Sigma-Aldrich, St. Louis, MO), mounted with carbon glue on special stubs, and coated with 8 nm platinum. Slides were examined and photographed under an SEM (XL20; Philips, Eindhoven, Netherlands). Effect of Concentration on Vitreous Separation Experiments were directed at determining the effect of different concentrations of microplasmin on the vitreous interface. For each concentration, four eyes were used and incubated for 60 and 120 minutes at room temperature. Eyes were injected as described. Concentrations tested were 62.5, 125, 250, and 400 g. Eyes were examined for gross changes in the appearance of the vitreous and retina and for the presence of a PVD. At each concentration, two eyes were examined by EM in representative areas of the retina. Effect of Time Exposure to a Fixed Dose of Microplasmin The effect of time exposure to a fixed dose of microplasmin was assessed, with the minimal effective dose of 125 g injected in the midvitreous of fresh porcine eyes. For each time point, four eyes were used. Eyes were injected as described and were incubated at room temperature for 15, 30, 60, or 120 minutes. After processing, the eyes were submitted to LM and were evaluated for the presence of complete separation of the posterior hyaloid (in which case a clear posterior hyaloid was seen in the vitreous distant from the retinal surface or the absence of any cellular or fibrillar structure on the retinal surface), partial separation, or complete adherence of the vitreous to the retina. FIGURE 1. Light micrograph showing the retina-vitreous interface. The dose of microplasmin used is shown in the lower right corner of each image. Eyes were treated for 1 hour. Top: no separation of the vitreous face. Middle: partial separation (arrows) adjacent to the retina. Lower: complete separation along the full length of the retina. This was assessed for the posterior pole, midvitreous, and the area proximal to the ora serrata. Assessment of Retinal and Zonular Integrity The integrity of retinal structures was assessed by reviewing the LM slides of eyes injected with ascending concentrations or ascending time exposure for signs of vacuolation within the retina. Transmission electron micrographs were obtained from eyes exposed to 125 g microplasmin for 120 minutes. Zonular integrity was assessed by SEM in the same specimen. RESULTS After the best fixation and staining technique were determining, the effect of varying doses of microplasmin was determined in pig eyes (Table 1). The lowest dose did not cause any hyaloid separation at either 1 or 2 hours of exposure. At 125 g microplasmin, complete separation of the posterior hyaloid along the retinal surface occurred in half the eyes exposed for 1 hour (Figs. 1, 2). Consistent posterior hyaloid separation was achieved at doses of 125 g and higher, injected into midvitreous for 2 hours. As shown in Figure 1, separation was consistently greater in eyes receiving 250 g microplasmin than in eyes receiving 125 g microplasmin, possibly indicating a more TABLE 1. Vitreous Separation Assessed by Light Microscopy after Exposing Eyes at Room Temperature to Various Concentrations of Microplasmin in BSS for 1 Hour Concentration ( g) Near Optic Nerve Mid Periphery Ora Serrata Control N5 N5 N N4 N4 N4 125 C4 C1P3 N4 250 C4 C4 N4 400 C4 C4 N4 C, complete separation; P, partial separation; N, no separation. FIGURE 2. Light micrograph taken of the vitreoretinal interface after 60 minutes of exposure to 125 g microplasmin. Left: complete separation of the posterior hyaloid adjacent to the retina. Right: atthe level of the ora, the vitreous face is still intact, except for one small area overlying the pars plana.

3 816 de Smet et al. IOVS, February 2009, Vol. 50, No. 2 TABLE 2. Vitreous Separation Assessed by Light Microscopy over Time at Room Temperature for Different Locations after Injection of 125 g Microplasmin in Porcine Eyes Time after Injection (min) Near Optic Nerve Mid Periphery Ora Serrata Control (120) N8 N8 N8 MP (15) P3 N1 N4 N4 MP (30) C2 N2 P2 N2 N4 MP (60) C4 C3 P1 N4 MP (120) C4 C4 P1* N3 MP, microplasmin. * Partial separation of the vitreous over the pars plana. effective posterior hyaloid separation. The time course of the vitreous separation was studied with a dose of 125 g (Table 2). Progressively more prominent separation was achieved over time with 1 hour of exposure required for a consistent separation at the posterior pole. No separation was seen over the ora serrata (Fig. 2). The retinal-vitreous interface was studied by EM in eyes injected with 125 g microplasmin for 2 hours and were compared with control eyes (Figs. 3, 4). The retinal surface of the treated eye had a smooth appearance, characterized by total or near total absence of vitreous strands. As with LM, occasional cells were seen on the retinal surface (Figs. 4F, 5). Although their appearance was sporadic at a concentration of 125 g, they were more frequently seen at the highest concentration, though their number per high-power field was limited to three cells or less. Al- FIGURE 3. Composite image taken of a globe injected with physiologic saline. (A) Vitreous remains translucent despite dehydration. (B) Eponembedded fragments and identification of the site studied in (C) located close to the ora serrata. (C) Eponembedded fragment at higher power showing the site studied in (D) and (E) (white rectangle) and in (F) and (G)(black rectangle). (D) Vitreous at higher magnification ( 1500) showing a fibrillar structure in an area overlying a vessel. (E) Higher magnification ( 12000) of the vitreous in (D) showing a meshwork of vitreous fibers. (F) Vitreous overlying the retina proper at 1500 showing a meshwork of vitreous fibers. (G) Higher magnification from (F). (H) Epon-embedded fragment from the posterior pole and identification of the site studied in (I). (I) The vitreous overlying the vessel also has a fibrillar structure. (J) Higher magnification of (I).

4 IOVS, February 2009, Vol. 50, No. 2 Microplasmin 817 FIGURE 4. Composite image of a globe injected with 125 g microplasmin and 2-hour incubation. (A) Vitreous is hazy after fixation. (B) Epon-embedded fragment of the peripheral retina. A vitreous strand is visible with surrounding bare retina. Locations of sites studied at higher power in (D) and (E) are indicated. (C) Epon-embedded posterior pole of the same eye showing location magnified in (F). (D) Smooth retinal surface with minimal vitreous strand close to vitreous base. (E) Vitreous fragment showing the lack of fibrillar structure. (F) Smooth retinal structure devoid of vitreous elements. (G) Fragment showing location of higher magnification images (H) and (I). (H) Ciliary processes and zonules appear intact. (I) Vitreous in this location over the peripheral retina retains a normal fibrillar appearance. though their nature was not determined, they had a dendritic appearance and were invariably located on the retinal surface. Vitreous adjacent to areas with a smooth retinal surface had a ground-glass appearance (Fig. 4). Along the vitreous base, the vitreous retained a more fibrillar consistency. Gross examination FIGURE 5. Cell with numerous dendritic extensions on the surface of the retina. These cells were observed primarily with higher concentrations of microplasmin and at higher exposures. (A) SEM image. (B) Transmission EM. FIGURE 6. Image taken of a globe opened after fixation and showing circumscribed elevations of retina and RPE after 120-minute exposure to 400 g microplasmin.

5 818 de Smet et al. IOVS, February 2009, Vol. 50, No. 2 showed that the vitreous of injected eyes in which vitreous degradation had occurred, based on LM or EM, had a hazy appearance after fixation (compares Figs. 4 and. 3). By gross examination and on LM, pigment epithelial separation was seen in 25% of the eyes injected with the highest concentration (400 g) at 120 minutes (Fig. 6). A similar result was observed in a repeat experiment using the same concentration and exposure. The overlying retina appeared normal in appearance, as did the RPE. Examination of the ciliary body and zonules adjacent to the site of injection on SEM did not reveal any significant abnormality (Fig. 4H). DISCUSSION The present study demonstrates the efficacy of microplasmin in separating the posterior hyaloid from the inner limiting membrane. In this porcine eye model, complete separation at the vitreoretinal interface was achieved in a time- and dosedependent fashion without morphologic changes. The separation appeared to first develop in the posterior pole region and to extend to the periphery. As with other vitreolytic enzymes, no separation was observed in the region of the pars plana. 16,22 The observed activity profile is similar to that of plasmin enzyme. The effective microplasmin dose of 125 g is comparable to that of plasmin (2 U), which causes complete vitreoretinal separation in porcine and human eyes. 12,19 In our current series, a consistent effect was noticed at 1 hour rather than 30 minutes, as reported by other investigators 14,21 for the cat and human vitreous. This difference may lie in the tighter vitreoretinal junction in the relatively young animals used and in the effect of a lower incubation temperature. Slow dehydration and processing would also minimize artifactual separation between the retina and the posterior vitreous face, which could occur as a result of incubation with a vitreolytic enzyme. Indeed early attempts in nontreated eyes with a faster dehydration process or the use of a punch biopsy did lead to artifactual vitreoretinal separation. Staining and processing of the tissue for light microscopy was also complicated because the interface is often damaged during the cutting process. This was minimized using the embedding resin (Technovit 7100; Heraeus Kulzer GmbH) fixative. Our experiments suggest that after injection of the enzyme in midvitreous, the process starts close to the optic nerve and moves to the periphery. Higher doses lead to a more complete effect with a greater separation between the posterior hyaloid face and the retinal surface (Fig. 1). Such a separation may indicate that the vitreoretinal interface is detached over a larger retinal surface area, allowing more retraction of the vitreous body. Indeed, the vitreolytic effect did not appear to be homogeneous throughout the eye after fixation. The area proximal to the site of injection frequently appeared to be more detached than areas distal to the site of injection. It is likely that the clinical effect will also be polarized because it is more prominent in the area proximal to the injection site. Accurate placement of the enzyme close to the area where its effect is anticipated may be important. Electron micrographs show that incubation with 125 g microplasmin does not affect the structural integrity of the retina. It provides a smooth retinal surface devoid of vitreous strands. Adjacent to these areas, vitreous takes on a granular appearance and loses its fibrillar structure. However, more distal to the injection site (e.g., adjacent to the ora, as in Fig. 4I), the vitreous fibrillar structure is maintained. Although this may in part reflect a difference in chemical structure in vitreous adjacent to the vitreous base, 23 it may reflect a lack of exposure to the enzyme that must diffuse to this particular location from its site of injection. Protein diffusion through a macromolecular matrix is slower than through a liquid 24 and may take many hours to occur. In the course of these experiments, we examined ascending doses of microplasmin to determine which doses would be safe for further in vivo evaluation. Doses up to 250 g appeared to differ only in their ability to induce progressively more pronounced PVD. The highest dose tested, 400 g, was associated with the appearance of dendritic-like cells on the retinal surface (Fig. 5) and the appearance of circumscribed elevations of the retina and RPE in serous-like detachments measuring approximately 2 to 3 disc diameters (Fig. 6). These were present in approximately 25% of the eyes examined, always in association with the dendritic-like cells. They were more frequent with more prolonged incubations (120 minutes). When present, multiple lesions were observed in the same eye. These observations suggest an upper limit on the potential therapeutic use of microplasmin in ocular tissues. In other experiments, porcine eyes were exposed to 125 g microplasmin for up to 24 hours. We did not observe the development of these RPE detachments over this prolonged exposure time. Caution should be taken in extending our results to an in vivo setting. Our experiments were carried out in an ex vivo model. Although we took care to avoid incubations that would lead to visible autolysis (intracellular vacuolation in retinal tissue), autolysis is likely to begin shortly after death. It is likely to affect layers adjacent to the pigment epithelium, where autolysis is most active. In summary, microplasmin is an effective agent to separate the posterior hyaloid. The effect appeared, in this limited study, to be dose and time dependent and to extend in a centrifugal fashion from its initiation point. An effective dose appeared to be approximately 125 g, with a possible upper limit placed at 400 g in this porcine eye model. Two hours seemed to be the time needed for complete separation out to the ora with the 125- g dose. References 1. Bhisitkul RB. Anticipation for enzymatic vitreolysis. Br J Ophthalmol. 2001;85: Trese MT. Enzymatic vitreous surgery. Semin Ophthalmol. 2000; 15: Sebag J. Is pharmacologic vitreolysis brewing? Retina. 2002;22: Davis MD. Vitreous contraction in proliferative diabetic retinopathy. Arch Ophthalmol. 1965;74: Foos RY, Kreiger AE, Forsythe AB, Zakka KA. Posterior vitreous detachment in diabetic subjects. Ophthalmology. 1980;87: Akiba J, Arzabe CW, Trempe CL. Posterior vitreous detachment and neovascularization in diabetic retinopathy. 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