Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

to conventional mechanical vitrectomy (Harooni et al., 1998). However, at higher concentrations, clinical and histological changes were seen in proportion to the concentration and included focal whitening, edema, vitreous haze, vascular abnormalities, and retinal necrosis at the highest doses. Histological evaluation of the retina revealed marked destruction in all layers at higher concentrations (Gottlieb et al., 1990).

The evidence for posterior vitreous detachment induction with hyaluronidase is somewhat conflicting. Hikichi et al. (2000) demonstrated that hyaluronidase was not able to induce a posterior vitreous detachment. However, intravitreal injection of 1IU hyaluronidase and 0.2ml of perfluoropropane gas in the rabbit eye demonstrated induction of posterior vitreous detachment in eyes that had received the enzyme plus the gas, but no posterior vitreous detachment in eyes that had received the enzyme alone (Kang et al., 1995). Similarly intravitreal injection of hyaluronidase, combined with perfluoroethane, can induce posterior vitreous detachment without mechanical vitrectomy (Shen et al., 2004). Intraocular irrigation of hyaluronidase has no effect on the ERG (Winkler and Cohn, 1985).

Hyaluronic acid in human vitreous is likely produced by Muller cells of the retina (Azuma et al., 1990). Hyaluranon is a prominent constituent of the interphotoreceptor matrix, where it may serve to organize the matrix by functioning as a basic scaffold to which other macromolecules in the insoluble interphotoreceptor matrix are attached (Hollyfield et al., 1998). Interphotoreceptor matrix glycoconjugates participate in maintaining retinal adhesion, and thus it is important to determine the toxicity of these compounds on the outer retina after intravitreal injection (Yao et al., 1990). Retinal adhesiveness is weakened by enzymatic modification of the interphotoreceptor matrix with hyaluronidase (Yao et al., 1992). Focal subretinal injections of neuraminidase, chondroitinase, and hyaluronidase in the rabbit lead to a

diffuse loss of retinal adhesiveness beyond the site of injection, suggesting that these molecules are present within the normal interphotoreceptor matrix. Hyaluronidase is used as a spreading agent during the administration of retrobulbar or peribulbar anesthesia (Demediuk et al., 1995).

Hyaluronidase appears to alter the diffusion and movement of substances through the vitreous and, simultaneously, is important in the development of posterior vitreous detachment. The rate of transfer of intravit- really-injected tritiated water from the mid vitreous to the choroid is increased significantly after depolymerization of vitreous hyaluronic acid by injected hyaluronidase (Foulds et al., 1985). Sub-Tenon’s injection of human recombinant hyaluronidase increases the intravitreal movement of dexamethasone in the human eye in vivo after sub-Tenon’s injection (Kozak et al., 2006). In a randomized clinical trial intravitreal ovine hyaluronidase accelerated the clearing of vitreous hemorrhage (Kuppermann et al., 2005a). No serious safety issues were reported after a single intravitreal injection of ovine hyaluronidase. The retinal detachment incidence was not statistically different between groups (Kuppermann et al., 2005b). Hyaluronidase, chondroitinase, and plasmin all increase the amount of vitreous removed with a one port vitrectomy without damage to the inner retina in enucleated pig eyes (Staubach et al., 2004).

Hyaluronidase is present in a biochemical analysis of 66 samples of subretinal fluid from patients with primary rhegmatogenous retinal detachment (Hayasaka et al., 1982). Hyaluronidase activity in the subretinal fluid increased with the duration of the detachment, but there was no correlation between enzyme activity and patient age or the extent of the retinal detachment (Hayasaka et al., 1982).

D. Chondroitinase

Chondroitinase ABC is a proteolytic enzyme with specificity for chondroitin

sulfate proteogylcan. Previous workers have demonstrated the presence of proteoglycans in normal vitreous. Goes et al. (2005) have characterized the vitreous intrinsic proteoglycans in normal rabbit eyes by collecting vitreous after treatment with glycosidases. Proteoglycans were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride. Electron microscopic study revealed a network with hyaluronic acid as thin threads coating and connecting collagen fibrils. The elimination of the proteoglycan coat showed chondroitin sulfate granules (8–25 nm) arranged at regular intervals on the fibril surface. Chondroitinase ABC digestion removed the granules and caused formation of thicker bundles of the collagen fibrils; analysis suggested the presence of 3 renewable proteoglycans in the vitreous, which were 1 heparan-sulfate and 2 chondroitin-sulfate proteoglycans. (Goes et al., 2005)

Despite the presence of chondroitin sulfate proteoglycan in human vitreous, there is conflicting evidence on the ability of chondroitinase to induce a posterior vitreous detachment. Chondroitinase failed to induce a posterior vitreous detachment in the porcine eye, which is known to have a particularly thick and tenacious posterior hyaloid; plasmin was able to induce a posterior vitreous detachment in this animal model (Hermel and Schrage, 2006). Although chondroitinase was not sufficient to induce a posterior vitreous detachment acting on its own, simultaneous intravitreal injection of chondroitinase ABC and matrix metalloproteinase-3 has been used in an experimental study of 24 rabbit eyes to induce posterior vitreous detachment (Meng and Zeng, 2004). The experimental group was treated with chondroitinase (0.2U) and matrix metallopro- tease-3(10 nanograms), and the control group received an equivalent dose of balanced salt solution. Complete liquefaction was found in every eye of the experimental group. Histological section showed poste-

rior vitreous detachment to various extents in the experimental group, and no vitreous liquefaction with a confined partial posterior vitreous detachment in one eye in the control group. Synchisis and weakening of vitreoretinal adherence occurred almost simultaneously (Meng and Zeng, 2004).

E. Dispase

As mentioned above there are two ways in which a posterior vitreous detachment can be achieved. First, this can be done by liquefying the gel structure of the vitreous (synchisis) with secondary collapse of the vitreous gel and detachment of the posterior hyaloids from the internal limiting membrane of the retina; this process mimics spontaneous posterior vitreous detachment that occurs as a function of age, and involves some risk of development of retinal tears and/or retinal detachment. Second, it is theoretically possible to use substrate-specific enzymes to weaken the adherence of the posterior vitreous cortex to retina (syneresis) (Czajka and Pecold, 2002). The ability of various enzymes to induce a posterior vitreous detachment depends upon the composition of the vitreous and its attachments to the internal limiting membrane for substrate-specific enzymes. To our knowledge dispase is the only agent that has been used to specifically attack the surface between the posterior hyaloid and inner limiting membrane with the goal of cleaving this attachment (Figure 17.1).

We have demonstrated previously that dispase can be used to induce a posterior vitreous detachment in porcine and human eyes (Tezel et al., 1998). Dispase is a 35.9 kD protease obtained from Bacillus polymyxa (Irie, 1976) which cleaves the basal membrane in various tissues including skin (Green et al., 1979), testis (Merkel et al., 1990), and retinal pigment epithelium (Pfeffer, 1991). Dispase acts on type IV collagen and fibronectin, whereas other components of the extracellular matrix such as

laminin, type V and VII collagens are resistant to the enzyme (Stenn et al., 1989). We have shown that dispase is able to induce a posterior vitreous detachment in enucleated human eyes in vitro and porcine eyes in vivo (Tezel et al., 1998). Substrate-specific induction of a posterior vitreous detachment has the advantage of being a controllable procedure, the control depending less on the skill of the individual practitioner than on selection of an appropriate dose of an enzyme for effective cleavage of type IV collagen and fibronectin at the vitreoretinal junction (Figure 17.1). Fibronectin and type IV collagen are found at the point of attachment of the internal limiting membrane to the posterior vitreous. Dispase is thus able to cleave specifically the proteins which attach the vitreous to the internal limiting membrane, and hence reduce side effects from non-specific protein cleavage (Tezel et al., 1998).

Grading of posterior vitreous detachment in enucleated eyes in vitro is shown in Figure 17.2. The ability of different concentrations of dispase to induce a posterior vitreous detachment in enucleated porcine eye within 15 minutes is shown in Figure 17.3. A partial or complete posterior vitreous detachment was observed in 3/10 (30%) control eyes (1 complete, and 2 partial), 7/10 (70%) of eyes treated with 1 or 2U/ml of dispase (4 complete, 3/10 partial, p 0.08), 8/10 (80%) of eyes treated with 5U/ml dispase (6 complete, 2 partial, p 0.03), 9/10 (90%) of eyes treated with 10U/ml dispase (8 complete, 1 partial, p 0.01), and 6/10 (60%) of eyes treated with 25U/ml dispase (5 complete, 1 partial, p 0.15). The lower rate of posterior vitreous detachment induction at 25U/ml was associated with a weakening of the attachment of retinal pigment epithelium to Bruch’s membrane by the higher concentration of dispase since the retinal pigment epithelium, sensory retina and vitreous left the eye cup as an intact unit when the eye cup was tilted.

A partial or complete posterior vitreous detachment was present in 2/10 (20%) of

No PVD

Complete PVD

FIGURE 17.2 Grading of posterior vitreous detachment. (a) In control eyes the removed vitreous remains adherent to the retina and the eyes are graded as having no PVD. (b) In dispase-treated eyes the vitreous can be removed without removing the retina, and the eye would be graded as a complete PVD. Some retinal adherence would be graded as partial PVD (not shown)

control eyes (1 complete, 1 partial) after 120 minutes. The rate of complete or partial posterior vitreous detachment increased to 4/10 (40%) (3 complete, 1 partial, p 0.24) with 0.05U/ml dispase and to 14/15 (93%) (12 complete, 2 partial, p 0.0003) with 0.1U/ml dispase (Figure 17.3). Concentrations higher than 0.1U/ml induced a complete or partial posterior vitreous detachment in 90% of eyes. The vitreous did not liquefy at any of these dispase concentrations (Tezel et al., 1998). Nineteen of the 20 (95%) human cadaver eyes injected with 0.5ml of 5U/ml of dispase and incubated for 15 minutes developed a complete posterior vitreous detachment, with 1 eye (5%) developing a partial posterior vitreous detachment. None of the eyes that received phosphate-buff- ered saline had a complete posterior vitreous detachment, and only 1 (5%) eye had a partial posterior vitreous detachment (Tezel et al., 1998).

The retinal architecture of the porcine eye was not affected by dispase treatment at either 0.1U/ml for 120 minutes or 5U/ml for 15 minutes on histological examination (Figure 17.4). Transmission electron microscopy of control eyes with an attached vitreous revealed collagen fibrils

(b)

(a)A

ILM GCL

IPL

INL (c)C

FIGURE 17.4 (a) Control pig retina. Vacuoles are seen in the internal limiting membrane similar to changes noted in dispase-treated eyes (Richardson’s stain). (ILM internal limiting membrane, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer). (b) Dispase treatment at 5U/ml for 15 minutes did not alter the morphology of the pig retina on light microscopy (Richardson’s stain).

(c) Note the retinal vessel wall is intact after dispase treatment at 5U/ml for 15 minutes (Richardson’s stain)

in the posterior hyaloid oriented parallel to the surface of the retina and adjacent to the lamina rara externa of the internal limiting membrane; the lamina densa was observed as a continuous and distinct dark band (Figure 17.5a). In dispase-treated eyes the collagen fibrils of the posterior hyaloid and the lamina rara externa of the internal limiting membrane were not present. The lamina densa lost its distinct borders and became an amorphous granular layer (Figure 17.5b). The internal limiting membrane of control eyes appeared as a homogeneous undulating membrane that obscured the underlying retinal surface structure on scanning electron microscopy (Figure 17.6a). Müller cell footplates could be seen as terminal fan-shaped structures.

(a)

Dispase-treated

(b)

FIGURE 17.5 (a) Transmission electron microscopy of the vitreoretinal junction in a control pig eye. Dense collagen fibrils of the posterior hyaloid (arrowheads) are oriented parallel to the internal limiting membrane. (b) Transmission electron microscopy of the vitreoretinal junction in a dispase-treated porcine eye (5U/ml for 15 minutes). Note that the collagen fibrils of the posterior hyaloid and the lamina rara externa of the internal limiting membrane are not present. A small remnant of the cleaved posterior hyaloid is visible (arrowheads). The lamina densa is amorphous, and its borders (arrow) are less distinct

The internal limiting membrane disappeared after treatment with dispase at either concentration, revealing a mosaic pattern to the inner surface of the porcine retina, which may be due to the Müller cell end footplates (Figure 17.6b). The overall viability of retinal cells did not change significantly after injection of dispase into the vitreous of either porcine or human eyes, and dispase treatment did not alter either the elastic properties or maximal stretching before fracture of the human retina (Tezel et al., 1998).

Thus, dispase, a neutral protease with substrate specificity for fibronectin and type IV collagen (Stenn et al., 1989), can be used to selectively cleave the attachment between the posterior hyaloid and the internal limiting membrane without causing damage to the adjacent retina. Dispase disrupts the

FIGURE 17.6 (a) Freeze-fracture scanning electron microscopy of the normal vitreoretinal junction in a control pig eye. The internal limiting membrane (ILM) is a homogeneous undulating structure that obscures the detail of the cells below. The fan-shaped footplates (MCFP) of the Müller cells (MC) terminate in the internal limiting membrane. (b) Freeze-fracture scanning electron microscopy of the sensory retina after treatment with dispase (at 5U/ml for 15 minutes). Arrows are pointing to the cracked plane. Dispase cleaves the attachment sites between the internal limiting membrane and the posterior hyaloid as well as partially digesting the internal limiting membrane, exposing the mosaic pattern of the inner retinal surface which arises from Müller cell end footplates. Reprinted from Tezel et al. (1998)

collagen fibrils in the lamina rara externa of the internal limiting membrane, with some loss of distinct borders in the lamina densa. However, the footplates of the underlying Müller cells are not altered by dispase treatment. Dispase cleaves the attachment of the posterior hyaloid to the internal limiting membrane without causing liquefaction of the vitreous gel (Kohno et al., 1987b; Russell et al., 1991; Stenn et al., 1989). Presumably this is related to the relative specificity of dispase for fibronectin and type IV collagen, which is present in basal lamina, rather than type II collagen, which is present in the vitreous gel (Nishikawa and Tamai, 1996; Yang et al., 1995). Fibronectin and type IV collagen are also found in the epiretinal membranes of macular pucker and proliferative vitreoretinopathy (Kohno et al., 1987a). Thus, dispase may prove to be a useful adjunct during vitreous surgery for these conditions (Tezel et al., 1998).

V. SUMMARY

Over the first three decades of its history, advances in vitreoretinal surgery were dependent upon improvements in microsurgical instrumentation to facilitate removal of the vitreous gel and epiretinal membranes. Advances in vitreous surgery involved the development of intraocular tamponade such as long-acting gases and silicone oil, the miniaturization of surgical instrumentation, and improvements in visualization and control of fluidics. Currently the instruments are becoming smaller and more refined, but we essentially remove vitreous using the same principles that were described by Robert Machemer more than 30 years ago – combined suction/cutting devices are used to remove the vitreous gel. In the near future there will be development of several therapeutic agents either to induce a posterior vitreous detachment indirectly, or to directly cleave the attachment of the posterior hyaloid to the inner limiting membrane of the retina. Use of the enzymes is likely to lead to significant advances in the ability of the surgeon to remove the vitreous gel completely, while minimizing surgical complications from vitrectomy.

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