PEER REVIEWED

Macular Surgery and Enzymatic Manipulation of the Vitreous Cavity: An Introduction to Chemical Vitrectomy

Polly A. Quiram, MD, PhD • David Goldenberg, MD

The vitreoretinal interface plays an important role in the pathogenesis of many retinal disorders. Traction at the vitreoretinal interface contributes to retinal pathology seen in proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy, macular pucker, diabetic macular edema (DME), vitreomacular traction (VMT) syndrome, and other conditions. VMT is thought to be mediated by the vitreous cortex and by fibrocellular proliferation, and therefore complete removal of the cortical hyaloid is often a principal goal of vitreoretinal surgery.¹ Surgical management of these disorders often targets separating the posterior hyaloid from the internal limiting membrane (ILM), thereby creating a posterior vitreous detachment (PVD). Mechanical separation of the vitreoretinal interface with vitrectomy may not remove all of the vitreous or completely separate the vitreoretinal junction, as cortical vitreous fibrils are left behind on the internal limiting membrane.² Incomplete removal of the vitreous may result in surgical failure.³

Vitrectomy with peeling of the ILM necessitates direct mechanical manipulation of the macula. Although it is generally safe, it can potentially result in retinal trauma.⁴ Several authors have proposed substances, enzymatic and nonenzymatic, to cleave the vitreoretinal juncture or liquefy the central vitreous as an adjunct to vitreoretinal surgery or a method to resolve vitreous traction or clear vitreous opacity. Enzymatic induction of a PVD and vitreous liquefaction may facilitate surgical separation of the posterior hyaloid, thus reducing intraoperative time, especially when using smaller 25- and 23-g vitrectors.

HISTORY OF ENZYMATIC MANIPULATION OF THE VITREOUS

With the advent of a better understanding of vitreous anatomy and biochemistry, enzymatic agents have been used to cleave the vitreoretinal junction and manipulate the vitreous.^3,5-10 Dispase was initially thought to be a good candidate for pharmacologic vitreolysis due to its ability to hydrolyze several proteins including type 4 collagen and fibronectin. In fact, dispase can lead to the creation of a PVD; however, there are some reports of dispase-induced anterior chamber and vitreous inflammation, epiretinal membranes, pre- and intraretinal hemorrhages, cataract, electroretinogram (ERG) amplitude reductions, and ultrastructural damage to the retina.^11-13 However, dispase toxicity may be due to endotoxin contamination, as several authors have reported using dispase without toxicity.^8,14

Another agent, chondroitinase, lyses the proteoglycan chrondroitin sulfate, which is associated with the vitreoretinal interface, but it fails to produce a PVD.^6,15 More recently, enzymatic manipulation has been performed with agents with a more favorable profile, such as hyaluronidase, plasmin, and microplasmin.^16-18

Hyaluronidase (Vitrase, ISTA) is a highly purified ovine enzyme that primarily digests the proteoglycan hyaluronan that comprises a large component of the vitreous body.^17,19 Hyaluronidase was originally targeted toward patients with dense vitreous hemorrhages in hope of promoting vitreous liquefaction and clearing of blood to allow for laser photocoagulation in cases of PDR. A recent prospective, double-masked phase 3 trial concluded that hyaluronidase is slightly more effective than placebo injections in clearing vitreous hemorrhages.¹⁹ Although it has been shown to decrease vitreous macromolecule size, suggesting a role for vitreous liquefaction, it cannot inducea PVD.^12,20,21

Figure 1. Scanning electron microscopy of vitreoretinal interface in animal models. A. Control showing surface with dense network of cortical vitreous. B. Microplasmin: Smooth retinal surface free of cortical vitreous following induction of PVD. C. Hyaluronidase: Condensed cortical vitreous is present at the vitreoretinal interface following vitreous liquefaction without posterior vitreous detachment (PVD).

IDEAL PHARMACOLOGIC AGENTS: PLASMIN AND MICROPLASMIN

In order for enzymatic manipulation of the vitreous to be beneficial, 2 successful components are necessary: (1) vitreous liquefaction, and (2) complete vitreoretinal interface separation (PVD). Plasmin and microplasmin (ThromboGenics, New York) have been shown to achieve both of these goals without damage to the retina.^22-25 These substances can eliminate vitreous traction and result in ILM clean of cortical vitreous as shown in animal and human studies (Figure 1).^20,26,27

Plasmin

Plasmin enzyme is a nonspecific protease capable of hydrolyzing glycoproteins such as laminin and fibronectin,²⁸ which bridge vitreous collagen fibers between the posterior vitreous cortex and the ILM.²⁹ Gandorfer and associates reported that a single injection of plasmin enzyme is able to cleave the vitreoretinal junction without causing morphological changes to the retina in postmortem porcine eyes.² The authors concluded that the degree of vitreoretinal separation depends on the concentration and length of exposure to plasmin. Plasmin enzyme reaches its peak activity in 15 to 30 minutes and remains at this peak for approximately 90 minutes, with the activity decreasing over the next several hours to an immeasurable level.

In addition to encouraging results with animal studies, use of plasmin enzyme in human trials has shown promise. Autologous plasmin enzyme (APE), which can be isolated from a patient's serum, has been studied extensively in humans.^22-24 Multiple reports have confirmed that a concentration of 0.4 to 1.2 U of APE can cause vitreous liquefaction and induce a PVD in humans.^22-24 Plasmin has been show to be safe at concentrations 10 times (4 U) the amount needed for PVD formation (0.4 U) without ERG changes or morphologic changes to the ILM and retina.¹²

Human pilot trials with APE-assisted vitrectomy in traumatic macular holes, idiopathic macular holes, and diabetic eyes have been reported.^22-24,27,30 In these eyes, plasmin enzyme allows the posterior vitreous to release from the retina with ease and/or causes spontaneous release of the posterior vitreous. The use of plasmin enzyme in diabetic patients may facilitate membrane peeling by acting on the extracellular matrix fibrin between the cellular pegs of the epiretinal membrane and neurosensory retina, allowing improved exposure and access to the pegs.

Microplasmin

Microplasmin is a recombinant protein that contains the active protease site of human plasmin but lacks many of the remaining "kringle" domains.³ It functions as a direct-acting thrombolytic agent, and it is a much smaller molecule (29 kDa) than human plasmin (88 kDa). It has been proposed that the smaller size of microplasmin enables the molecule to penetrate epiretinal tissue more effectively than plasmin.²⁷ Microplasmin can effectively manipulate the vitreous cavity by causing vitreous liquefaction and cleavage of the vitreoretinal interface with a single intravitreal injection of 62 μg or 125 μg.²⁴ Because it is a recombinant protein, it can be prepared and manufactured in large quantities in limited time. Thus, the venipuncture and labor-intensive preparation that is needed for plasmin can be avoided.

Sebag has shown a dose-dependent reduction in the size of porcine vitreous macromolecules (hyaluronan and collagen) after injection with microplasmin.³³ Using dynamic light scattering, the highest dose of microplasmin (0.8 mg) demonstrated an overall reduction in size of about 85% after just 30 minutes. These results suggest significant vitreous liquefaction is achieved with microplasmin after a relatively brief period of time. Gandorfer and associates²⁷ injected postmortem human eyes and in vivo feline eyes with microplasmin or saline and confirmed the presence or absence of a PVD with scanning and transmission electron microscopy. The microplasmin-injected eyes demonstrated complete vitreoretinal separation in a dose- and time-dependent fashion in both species with a complete vitreoretinal separation induced within 30 minutes after injection.

Figure 2. Results of MIVI 2T Trial: Vitreomacular traction (VMT). A. Optical coherence tomography (OCT) demonstrating evidence of VMT with visual acuity (VA) of 20/100 (left). B-scan ultrasonography demonstrating no evidence of PVD (right). B. OCT image 1 week after intravitreal injection of microplasmin. Vitreoretinal traction is released and VA improved to 20/63 (left). B-scan demonstrates presence of a PVD (arrow) (right). C. OCT image 3 months after intravitreal injection of microplasmin. No evidence of VMT is present and VA is 20/16.

IMPLICATIONS OF "CHEMICAL VITRECTOMY"

Evidence has shown that enzymatic manipulation of the vitreous (chemical vitrectomy) can potentially relieve vitreoretinal traction, alter the influx of molecules, and change oxygen levels in the vitreous cavity. For example, diabetic eye disease has the potential to be treated with microplasmin. A complete PVD is a strong negative risk factor for the progression of diabetic retinopathy (DR)³⁴ and a complete posterior vitreous separation may allow for resolution of DME.²⁴ Multiple growth factors, including vascular endothelial growth factor (VEGF), have been implicated in the pathogenesis of DME and neovascularization.^35-37 It is possible that a PVD may alter the flux of molecules in the vitreous cavity and influence the concentration of vitreal growth factors.³⁸ The author and associates have recently shown that the creation of a microplasmin-assisted PVD increases vitreal oxygen and increases the rate of oxygen exchange within the vitreous cavity in animal models.²⁰ Preliminary data from cat models have suggested that microplasmin-assisted PVD leads to decreased levels of vitreal VEGF.³⁹ It is possible that a microplasmin-induced PVD may increase vitreous oxygenation, thereby decreasing retinal ischemia and altering growth factor production. If so, this may have profound implications for the management of diabetic retinopathy. Microplasmin may be able to delay the progression of DR and DME by inducing a prophylactic PVD. Prospective clinical trials will be needed to address this theory.

Previous reports have shown that O₂ levels in the vitreous cavity increase following pars plana vitrectomy.⁴⁰ In addition, stabilization of PDR and DME often occurs following vitrectomy, possibly due to increased oxygenation and removal of the vitreous scaffold.^41-45 It is not known whether "chemical vitrectomy" has the same effect, but preliminary data are promising.

Figure 3. Results of MIVI 2T Trial: Macular Hole. A. OCT demonstrating evidence of a full thickness macular hole with VA of 20/63. B. OCT image2 weeks after intravitreal injection of microplasmin. Macular hole is decreased in size with VA of 20/63. At this visit SF6 was injected into the eye and patient was positioned face down. C. OCT image 6 months after intravitreal injection of microplasmin. Macular hole is closed and VA improved to 20/25.

RESULTS OF CLINICAL TRIALS WITH MICROPLASMIN

The European MIVI 2T was a phase 2, randomized, double-masked clinical trial of microplasmin intravitreal injection for nonsurgical treatment of VMT. Inclusion criteria was evidence of VMT by ultrasound and optical coherence tomography with the presence of macular thickening >250 μm. Common conditions included VMT, macular holes, or tractional DME. Visual acuity (VA) had to be 20/40 or worse in the study eye and 20/400 or better in the fellow eye. Two cohorts of 15 patients received 75 μg or 125 μg of microplasmin or sham with a 4:1 randomization of microplasmin vs sham. Follow-up data were collected for 6 months. Of the 30 patients enrolled, 16 (53%) had VMT, 6 (20%) had macular holes, and 8 (27%) had tractional DME. Intravitreal injection of microplasmin was well tolerated with no adverse events including retinal tears, retinal detachments, or endophthalmitis reported.

Results showed complete VMT resolution in 9 eyes (40%) with the best results (50% resolution) seen with the higher dose of microplasmin (125 μg) (Figures 2, 3, and 4). Closure of macular holes without vitrectomy occurred in 50% of eyes (Figure 3). A >3-line improvement of VA occurred in 4 patients (17%). Interestingly, microplasmin has been reported to create a PVD with 100% effectiveness in patients with an apparently normal vitreoretinal interface.²⁴ From the MIVI 2T trial we know that microplasmin relieves VMT in 40% of patients with an abnormal vitreoretinal interface. Certainly a phase 3 clinical trial examining the nonsurgical treatment of macular holes and VMT is warranted. Currently, the phase 2b MIVI III trial is enrolling patients to determine the use of microplasmin as a surgical adjunct in vitrectomy surgery and early results appear encouraging. We are awaiting the final results of this trial.

Figure 4. Preliminary results of MIVI 2T trial. Evidence of vitreomacular traction and early macular hole formation (left). One week following intravitreal injection of microplasmin, vitreoretinal traction is released and hole appears closed (right).

POSSIBLE COMPLICATIONS OF ENZYMATIC MANIPULATION OF THE VITREOUS

Pharmacologic manipulation of the vitreoretinal interface presents the inherent risk of causing a retinal tear or detachment. Although no reports to date have specifically addressed this concern, a retinal tear is an intrinsic risk during PVD creation, especially in patients with abnormal vitreoretinal adhesions, such as lattice degeneration. In addition, increasing the vitreous cavity oxygen in adults can lead to advancing nuclear sclerotic cataract and perhaps late glaucoma.⁴⁶

FUTURE DIRECTIONS

The ability to induce vitreous liquefaction and a complete PVD with a single intravitreal injection of microplasmin has potentially significant implications for management of multiple vitreoretinopathies. If used as a surgical adjunct, it has the potential to facilitate more complete removal of the vitreous gel, decrease surgical time, and reduceintra-operative complications. If used as prophylactic therapy, its uses may include VMT syndrome, PDR, macular holes, and pretreatment before pneumatic retinopexy to decrease the incidence of new retinal breaks. If successful, it may even replace surgical intervention for select cases. RP

REFERENCES

1. Gandorfer A, Rohleder M, Kampik A. Epiretinal pathology of vitreomacular traction syndrome. Br J Ophthalmol. 2002;86:902-909.
2. Gandorfer A, Ulbig M, Kampik A. Plasmin-assisted vitrectomy eliminates cortical vitreous remnants. Eye. 2002; 16:95-97.
3. Trese MT. Enzymatic vitreous surgery. Sem Opthalmol. 2000;15:116-121
4. Russell SR, Hageman GS. Optic disc, foveal, and extrafoveal damage due to surgical separation of the vitreous. Arch Ophthalmol. 2001;119:153-1658.
5. Sebag J. Pharmacologic vitreolysis. Retina. 1998;18:1-3
6. Hermel M, Schrage NF. Efficacy of plasmin enzymes and chondroitinase ABC in creating posterior vitreous separation in the pig: a masked, placebo-controlled in vivo study. Grafes Arch Clin Exp Ophthalmol. 2007;245:399-406
7. Sebag J. Molecular biology of pharmacologic vitreolysis. Trans Am Ophthalmol Soc. 2005;103:473-494.
8. Tezel TH, Del Priore LV, Kaplan HJ. Posterior vitreous detachment with Dispase. Retina. 1998;8:7-15
9. Staubach F, Nober V, Janknecht P. Enzyme-assisted vitrectomy in enucleated pig eyes: a comparison of hyaluronidase, chondroitinase, and plasmin. Curr Eye Res. 2004;29:261-268
10. Wang ZL, Zhang X, Xun X, et al. PVD Following plasmin but not hyaluronidase: implications for combination pharmacologic vitreolysis therapy. Retina. 2005;25:35-43
11. Zhu D, Chen H, Xu X. Effects of intravitreal dispase on vitreoretinal interface in rabbits. Curr Eye Res. 2006:31:935-946.
12. Wang F, Wang Z, Sun X, Wang F. Safety and efficacy of dispase and plasmin in pharmacologic vitreolysis. Invest Ophthalmol Vis Sci. 2004;45:3286-3290.
13. Jorge R, Oyamaguchi EK, Cardillo JA, Gobbi A.. Intravitreal injection of dispase causes retinal hemorrhages in rabbit and human eyes. Curr Eye Res. 2003;26:107-112.
14. Tatebayshi M, Mahmoud TH, Blackmon SM, et al. Dispase facilityates posterior vitreous detachment during vitrectomy in young pigs. Retina. 2002;22:1-3.
15. Hagemann GS, Russell SR. Chondroitinate-mediated disinsertion of the primate vitreous body. Invest Ophthalmol Vis Sci. 1994;35(Suppl):28.
16. Sakuma T, Tanaka M, Mitzota A, Inoue J. Safety of in vivo pharmacologic vitreolysis with recombinant microplasmin in rabbit eyes. Invest Ophthalmol Vis Sci. 2005;46:3295-3299
17. Kuppermann BD, Thomas EL, De Smet MD, Grillone LR. Safety results of two phase III trials of an intravitreous injection of highly purified ovine hyaluronidase (Vitrase^®) for the management of vitreous hemorrhage. Am J Ophthalmol. 2005;140:585-597.
18. Liotta LA, Goldfarb RH, Brundage R, Siegal GP. Effect of plasminogen activator (urokinase), plasmin, and thrombin on glycoprotein and collagenous components of basement membrane. Cancer Res. 1981;41:4629-4636.
19. Kuppermann BD, Thomas EL, De Smet MD, Grillone LR. Pooled efficacy results from two multinational randomized controlled clinical trials of a single intravitreous injection of highly purified ovine hyaluronidase (Vitrase^®) for the management of vitreous hemorrhage. Am J Ophthalmol. 2005;140:573-584.
20. Quiram PA, Leverenz VA, Baker RM, Dang L. Microplasmin-induced posterior vitreous detachment affects vitreous oxygen levels. Retina. 2007;27:1090-1096.
21. Hikichi T, Kado M, Yoshida A. Intravitreal injection of hyaluronidase cannot induce posterior vitreous detachment in the rabbit. Retina. 2000;20:195-198.
22. Trese MT, Williams GA, Hartzer MK. A new approach to stage 3 macular holes. Ophthalmology. 2000;107:1607-1611.
23. Margherio AR, Margherio RR, Hartzer M, Trese MT, Williams GA, Ferrone PJ. Plasmin enzyme-assisted vitrectomy in traumatic pediatric macular holes. Ophthalmology. 1998;105:1617-1620.
24. Williams JG, Trese MT, Williams GA, Hartzer M. Autologous plasmin enzyme in the surgical management of diabetic retinopathy. Ophthalmology. 2001; 108:1902-1905.
25. Gandorfer A, Rohlender M, Sethi C, et al. Posterior vitreous detachment induced by microplasmin. Invest Opthalmol Vis Sci. 2004;45:641-647.
26. Verstraeten TC, Chapman C, Hartzer M, Winkler BS, Trese MT, Williams GA. Pharmacologic Induction of posterior vitreous detachment in the rabbit. Arch Ophthalmol. 1993;111:849-854.
27. Asami T, Terasaki H, Kachi S, et al. Ultrastructure of internal limiting membrane removed during plasmin-assisted vitrectomy from eyes with diabetic macular edema. Ophthalmology. 2004;111:231-237.
28. Uemura A, Nakamura M, Kachi S, et al. Effect of plasmin on laminin and fibronectin during plasmin-assisted vitrectomy. Arch Ophthalmol. 2005;123:209-213.
29. Kohno T, Sorgente N, Ishibashi T, Goodnight R. Immunofluorescent studies of fibronectin and laminin in the human eye. Invest Ophthalmol Vis Sci. 1987;28:506-514.
30. Azzolini C, D'Angelo A, Maestranzi G, et al. Intrasurgical plasmin enzyme in diabetic macular edema. Am J Ophthalmol. 2004;138:560-566.
31. Trese MT. Enzymatic-assisted vitrectomy. Eye. 2002;16:365-368.
32. Nagai N, Demarsin E, Van Hoef B, et al. Recombinant human microplasmin: production and potential therapeutic properties. Thromb Haemost. 2003;1:307-313.
33. Sebag J, Ansari RR, Suh KI. Pharmacologic vitreolysis with microplasmin increases vitreous diffusion coefficients. Graefes Arch Clin Exp Ophthalmol. 2007;245:576-580.
34. Ono R, Kakehashi A, Yamagami H, et al. Prospective assessment of proliferative diabetic retinopathy with observations of posterior vitreous detachment. Int Ophthalmol. 2005;26:15-19.
35. Paques M, Massin P, Gaudric A. Growth factors and diabetic retinopathy. Diabetes Metab. 1997;23:125-130.
36. Patel JI, Tombran-Tink J, Hykin PG, Gregor ZJ. Vitreous and aqueous concentrations of proangiogenic, antiangiogenic factors and other cytokines in diabetic retinopathy patients with macular edema: implications for structural differences in macular profiles. Exp Eye Res. 2006;82:798-806.
37. Boulton M, Gregor Z, McLeod D, et al. Intravitreal growth factors in proliferative diabetic retinopathy: correlation with neovascular activity and glycaemic management. Br J Ophthalmol. 1997;81:228-233.
38. Stefánsson E, Loftsson T. The Stokes-Einstein equation and the physiological effects of vitreous surgery. Acta Ophthalmol Scand. 2006;84:718-719.
39. Quiram PA, Leverenz V, Baker R, Loan D. Enzymatic induction of a posterior vitreous detachment alters molecular vitreodynamics in animal models. [ARVO Abstract]. Invest Ophthalmol Vis Sci. 2007;83:E-Abstract 83.
40. Holekamp NM, Shui YB, Beebe DC, Vitrectomy surgery increases oxygen exposure to the lens: a possible mechanism for nuclear cataract formation. Am J Ophthalmol. 2005;139:302-310.
41. Stefansson E, Landers MB How does vitrectomy affect diabetic macular edema? Am J Ophthalmol. 2006;141:984.
42. Grigorian R, Bhagat N, Lanzetta P, et al Pars plana vitrectomy for refractory diabetic macular edema. Sem Ophthalmol. 2003;18:116-120.
43. Recchia FM, Ruby AJ, Carvalho, et al. Pars plana vitrectomy with removal of the internal limiting membrane in the treatment of persistent diabetic macular edema. Am J Ophthalmol. 2005;139:447-454.
44. Gotzaridis IV, Lit IS, D'Amico DJ. Progress in vitreoretinal surgery for proliferative diabetic retinopathy. Sem Ophthalmol. 2001;16:31-40.
45. Rosenblatt BJ, Shah GK, Sharma S et al Pars plana vitrectomy with internal limiting membranectomy for refractory diabetic macular edema without a taut posterior hyaloid. Graefes Arch Clin Exp Ophthalmol. 2005;243:20-25.
46. Chang S. LXII Edward Jackson Lecture: Open angle glaucoma after vitrectomy. Am J Ophthalmol. 2006;141:1033-1043.