Ординатура / Офтальмология / Английские материалы / Ocular Neuroprotection_Levin, Polo _2003

II. CLINICAL RETINAL DETACHMENT

Based on general etiological parameters, retinal detachments are thought of as belonging to one of three major forms: rhegmatogenous, traction, and serous [10,11]. In the exudative form of age-related macular degeneration (AMD), choroidal neovascularization can lead to leakage of fluid and growth of blood vessels in the subretinal space, which can produce localized macular separations. Models of AMD are beyond the scope of this chapter. Photoreceptors receive oxygen and glucose from the nearby choriocapillaris and even a shallow detachment can result in photoreceptor hypoxia and ischemia [12,13]. Because of their high metabolic activity, photoreceptors are very susceptible to prolonged hypoxic conditions [14]. Whatever the cause of the RD, failure to achieve retinal reattachment in a timely manner often leads to irreversible loss of vision in the affected region. For example, prolonged detachment of the macula results in significant cone alteration or degeneration in the fovea and invariably leads to permanent loss of visual acuity, distortion of color vision, and metamorphopsia, even following successful retinal reattachment [9].

Rhegmatogenous retinal detachment is the most common form of retinal detachment and occurs as a result of tears or holes in the retina. In the older eye, the vitreous loses some of its gel-like characteristics and can become liquefied, and these retinal holes or tears permit liquefied vitreal fluid to enter and accumulate in the subretinal space, thus creating and enlarging the detachment. A variety of surgical techniques (scleral buckle, pneumatic retinopexy, and vitrectomy) are used to reattach the retina. Retinal reattachment surgery often affords an initial success rate of 70 to 90% and a final success rate of higher than 95% following repeat surgeries [15,16]. Scleral buckle surgery is the most common technique for treating rhegmatogenous RD and can result in overall reattachment success rates higher than 90% following a single operation. Thus retinal reattachment surgery is thought to be largely successful. However, success in RD surgery is measured primarily, if not exclusively, in terms of achieving retinal reattachment. Unfortunately, this anatomical success does not parallel recovery in visual function. In a retrospective study, Burton found that of patients treated successfully with a single operative procedure, 42% achieved better than 20/50 visual acuity. For cases involving patients with macula-off detachments, only 20% of reattached macula achieved postoperative visual acuities of 20/50 or better [9]. Although decreased postoperative visual acuity can occur as a result of proliferative vitreoretinopathy, cystoid macular edema, or macular pucker, it is generally thought that degenerative loss of macular cone function is the primary underlying cause of permanent visual acuity impairment and color distortion following “successful” reattachment surgery [9,15].

Tractional forces within the vitreous body or along the inner surface of the retina can exert sufficient mechanical force on the retina to pull it away from the

RPE, thereby creating a traction retinal detachment. Traction RD is the second most common form of retinal detachment and is thought to result in part from an inappropriate cellular “wound healing” response along the vitreal-retinal and retinal-RPE interface. Proliferating, hypertrophic, reactive, or otherwise altered glial cells (Mu¨ller cells and astrocytes), RPE, pericytes, and endothelial cells are believed to contribute to the formation of the tractional forces [17]. These cells can proliferate and form a clinically evident membrane structure that contracts and pulls either perpendicularly (via interactions with vitreous strands) or tangentially to the retinal surface. Traction RD occurs in diabetic retinopathy, penetrating trauma (secondary to internal bleeding), retinopathy of prematurity and proliferative vitreoretinopathy [10,11]. It can also lead to permanent visual loss, and is generally treated with vitrectomy, which allows the surgeon to remove the tractional forces. Tractional forces can also contribute to the formation of a retinal tear by pulling a patch of retina from the RPE, usually creating a “horseshoe” tear and increasing the likelihood for the development of rhegmatogenous RD.

Diseases and abnormalities of the choroid or RPE can lead to serous or exudative retinal detachment [10]. Serous RD occurs without retinal holes or tears and without apparent tractional forces on the retina, and it is thought in most cases to originate from choroidal fluid leaking into the subretinal space across a compromised RPE [10,18]. Conditions that can lead to serous detachments include central serous retinopathy, severe hypertension, toxemia of pregnancy, Harada’s disease, and various choroidal inflammatory disorders. Treatments for serous RD are generally nonsurgical.

Many aspects of rhegmatogenous, traction, and serous RD are studied by employing an experimental model of non-rhegmatogenous retinal detachment, which has been used extensively to study photoreceptor degeneration, retinal alterations, and, more recently, to test potential therapeutic approaches for preventing retinopathic changes. This model is described below.

III.EXPERIMENTAL NON-RHEGMATOGENOUS RETINAL DETACHMENT

A common method for experimentally creating a retinal detachment is to introduce a small needle or micropipette in the anterior globe (usually through the pars plana), advance the needle or micropipette through the vitreous and into the retina, and then gently inject fluid into the subretinal space (Fig. 1A). The resultant detachment is best described as a non-rhegmatogenous retinal detachment— non-rhegmatogenous because the retinal hole caused by the needle is sufficiently small to prevent shunting of subretinal and vitreal fluid [36]. Experimental nonrhegmatogenous RD is sometimes referred to as a “subretinal bleb” or “serous retinal detachment,” although it is important to recognize that this model does

Figure 1 Diagrammatic representation of techniques used to create experimental non-rhegmatogenous and rhegmatogenous retinal detachments. (A) The micropipette or small needle (usually 32-G) is inserted through the pars plana, slowly advanced into the vitreous, and into the retina. A small outflow is maintained across the needle tip to prevent obstruction by the vitreal gel, but this is usually done with very small diameter micropipette tips. After creation of the subretinal bleb and retinal separation, the micropipette or needle is retracted, leaving a hole that either seals on its own (perhaps through localized blood-clotting) or a hole that is otherwise too small for exchange of subretinal and intravitreal fluid. (B) In preparation for the creation of a retinal hole, the vitreous is collapsed by aspirating and reinjecting hyaluronidase with the needle that is inserted intravitreally through the pars plana (top panel). The retinal hole and surrounding detachment are subsequently created by pressure injection of vitreous against the retina (middle and bottom panels), resulting in a rhegmatogenous retinal detachment [35].

not formally mimic many aspects of a true serous RD. The model is used to study many aspects of retina/RPE interactions, including rates of subretinal fluid reabsorption, forces of retinal adhesion, metabolic and pharmacological effects on the RPE fluid pump, and subcellular and cellular effects on the retina and RPE [1,19–23]. Such retinal detachments are also invariably produced when delivering compounds, proteins, and viral vectors (for gene-based therapy) into the subretinal space [24,25]. More recently, large bullous or complete non-rhegma- togenous retinal detachments are deliberately produced in the clinic as part of an evolving surgical technique called macular translocation surgery for the treatment of age-related macular degeneration [26]. In this approach, the entire macula is detached from the underlying diseased RPE and moved to a presumably healthier region of the RPE. Experimental models of non-rhegmatogenous RD have been produced in rabbit, cat, monkey, and, more recently, rat, ground squirrel, and pig [23,27–33].

A. Preoperative Procedure

Animals undergo general anesthesia in preparation for retinal detachment surgery. A variety of anesthetic combinations have been used for rabbits (singly or in various combinations): thorazin, pentobarbital, urethane, ketamine, and acepromazine maleate. Cats: combinations of ketamine and sodium pentobarbital; or ketamine and acepromazine maleate. Monkeys: combinations of ketamine and xylazine; phencyclidine hydrochloride and sodium pentobarbital; or sodium pentobarbital alone. Rats: a combination of buprenophrine, sodium pentobarbital, and atropine. Topical anesthesia is administered via retrobulbar injection of xylocaine in rabbits and cats. To visualize the injection and fundus, pupils are dilated using (singly or in various combinations): phenylephrine, cyclopentolate, tropicamide, and atropine. Some of the pioneering or prototypical models for experiment RD can be found in these references: [23,30,31,34,35].

B. Surgical Procedure: Retinal Detachment

Modifications of a basic surgical procedure are used for preparing the eye and creating a retinal detachment; these approaches often depend on the species and the investigator. Delineation of the myriad of surgical approaches used will not be attempted here, but a number of general methodologies are discussed instead. In rabbits, the superior sclera is penetrated with a needle (usually 22-G) 3–4 mm behind the limbus to avoid inserting through the retina, and a glass micropipette (15–50 m tip diameter) or small needle (32-G or smaller) is inserted through the scleral opening and advanced into the vitreous under the fine control of a micromanipulator [23,36]. For micropipettes, a small outflow of fluid can be maintained across the tip by applying air pressure at 10–20 lb/in.2 to prevent

vitreous gel from obstructing the tip. The tip is advanced until the retina is gently penetrated and a detachment begins to form. Small and large subretinal blebs can be made this way.

In cats, a number of modifications of a basic surgical technique for inducing large retinal detachments have been reported [13,30,31]. The procedure begins with a pre-detachment preparation phase in which an extracapsular lens extraction is performed through an 180° corneal incision. This is followed by excision of the posterior capsule and a partial vitrectomy or, alternatively, the posterior capsule can be left intact. The vitrectomy affords better control of the height and extent of the induced RD. The cornea is sutured closed and allowed to heal for several weeks before the actual retinal detachment is created. For RD surgery, an infusion cannula is sewn in place in the cornea (for example, in the inferotemporal quadrant). A puncture incision is then made with a 20-G needle in an adjacent quadrant (superotemporal) of the cornea, and the posterior capsule and vitreous are removed. Fluid-gas exchange is performed following the vitrectomy. Alternatively, the lens and vitreous can be left in place [13]. A glass micropipette with a flat 80–100 m tip diameter is mounted on a micromanipulator and then inserted into the 20-G incision. The retinal detachment can then be made in a manner similar to that used in rabbits. Variations of this technique have been adapted for use in rhesus monkeys [28].

A rat model of retinal detachment has recently been developed and used for subretinal fluid reabsorption studies [33]. Retinal detachments were made in Long-Evans rats by first inserting a 26-G guidance needle behind the limbus and into the vitreous. A 33-G flat-tip needle attached to a Hamilton syringe is then inserted into the barrel of the 26-G guidance needle to create the detachment and to inject solutions into the vitreous. A specially designed double convex lens for the rat eye is placed on the cornea to view injections and the fundus. Although the 26-G needle used to penetrate the rat eye may seem rather large relative to the size of the eye, this approach does not appear to cause significant changes in intraocular pressure or other complications and seems amenable for studying retinal detachments over a 24-h period.

C.Surgical Procedure: Retinal Reattachment

In some cases, it is necessary to reproducibly induce retinal reattachment. In cats and monkeys, a gas/air exchange procedure has been developed to induce retinal reattachment and theoretically should be applicable for use in other species with eyes of similar or larger sizes [28,31]. In this approach, animals are first given general anesthesia. The eye is then prepared for a mixture of sulfur hexafluoride (SF6) gas and air exchange via an infusion cannula and a drainage cannula at different quadrants of the globe. In the cat, the infusion cannula is placed in the inferotemporal quadrant of the cornea approximately 1.5 mm from the limbus.

(The infusion cannula can be inserted through a 20-G incision, for example, and secured with 5.0 Dacron mattress suture.) A second 20-G incision is then made in the superotemporal quadrant, in which a 20-G blunt beveled tip on a Charles fluted needle is inserted. This allows for a mixture of 50% or 75% SF6 gas and air to be flushed through the eye until complete exchange is achieved.

D. Solutions

A variety of buffers and Ringer’s solutions have been used to experimentally induce retinal detachments. Table 1 shows the effects of some of these buffers and solutions on the duration of detachments made in a number of species. The results shown in Table 1 were taken from various published reports and may represent laboratory-specific findings. Other factors such as height and extent of the induced RD are also important factors in affecting the duration of the detachment. Reattachment of experimental non-rhegmatogenous RD usually occurs over a period of hours to days, assuming that a physiologically based solution is used to create the detachment. Rabbits and rats appear to spontaneously reattach over a period of hours, and cats and monkeys can take many hours or even days [1,27,30,36]. For studies of prolonged, controllable, and semi-reproducible retinal detachments, a useful technique has been developed that enables the researcher to create detachments of fixed lengths of time and consists of adding sodium hyaluronate in the infusion solution; this prevents the retina from reat-

Table 1 Approximate Duration of Non-Rhegmatogenous Retinal Detachments Following Subretinal Injection of Different Solutions in Various Species

taching [22,30,55]. In cats, for example, retinal detachments of up to 14 months in duration have been made with this approach [30]. Furthermore, retinal reattachment can be achieved by using the gas/fluid exchange technique described above. In the aforementioned rat model, it was noted that the constancy of the apparent subretinal bleb size over time depended on the relative osmolarity of the blood serum, thus suggesting that osmotic differences across the retina and RPE play a significant role in determining the rates of subretinal fluid reabsorption [33]. The investigators exploited this finding to devise a modified phosphate buffered saline solution that allowed subretinal blebs to remain relatively constant, at least for an initial 24-h period. This control over the constancy in bleb size has been useful, for example, in evaluating pharmaceutical approaches for enhancing subretinal fluid reabsorption [33].

E.Species Considerations

There are significant anatomical differences between human eyes and the eyes of various species used for experimental RD studies, and relevant differences should be taken into account when interpreting the results of animal studies in the context of human retinal detachments. Rabbit retinas are rod-dominant, lack a cone-rich macula, and contain extremely limited intrinsic blood vessels that reside along the nasal-to-temporal myelin wing. The rabbit retina is thought to receive virtually all of its oxygen from the choriocapillaris. Thus a detachment would render the entire retina hypoxic, rather than merely the outer retina. The retinal vascular bed in the cat more closely resembles that of humans, and although the cat retina contains a higher population of cones than the rabbit retina, it is nevertheless rod-dominant and also lacks a cone-dominant macula. Rhesus and cynomolgus monkeys contain true cone-dominant macula and would represent ideal animal models for human RD. The retinas of ground squirrels contains 85 to 90% cones, their color vision is dichromatic, and their cone system has been well characterized electrophysiologically. The vascular bed is extensive, and so squirrels appear to be an attractive alternative to monkeys for RD research. Rats also contain an extensive vascular bed, but the retina is rod-dominant ( 95%) and lacks a macular region. Finally, the porcine eye, which also does not contain a true cone-dominant macula, appears to be an attractive model for studying neurodegeneration and neuroprotection in the context of RD. The size of the eye, the retinal vasculature system, lens/vitreal volume ratio, and the presence of a cone-enriched macula (or visual streak) give strong credence for studying cone alterations in this species.

F. Relevance of Model

There were some early concerns about whether the retinal hole created by the small needle or micropipette was sufficiently large to create a true shunt for

subretinal and vitreal fluid. If so, the model could no longer be thought of as “non-rhegmatogenous” and the ability to use the model to estimate RPE fluid absorption, for example, is questionable. To address this concern, Marmor and colleagues showed that in rabbits the creation of retinal holes with a 15–25 m diameter micropipette had no effect on the rates of subretinal fluid reabsorption [36]. Sealing the hole with cyanoacrylate, mucilage, or an air bubble did not affect the rate of subretinal fluid reabsorption, nor did the creation of multiple holes in the same bleb. Pederson and MacLellan showed that in rhesus monkeys, the creation of a subretinal bleb containing a small retinotomy resulted in spontaneous retinal reattachment, whether or not the eye had a vitrectomy [27,34]. In rats, Mamanishkis and colleagues showed that fluorescein injected into the subretinal space did not diffuse into the vitreous through the hole [33]. These results all provide strong experimental support that small retinal holes do not create a shunt for subretinal and vitreal fluid, hence validating the concept that such retinal detachments are non-rhegmatogenous in nature.

In humans, detachments enlarge over a period of hours to days, whereas in the lab, detachments are usually created in less than a minute. Rhegmatogenous RD in an untreated human eye often progresses to total detachment. In the absence of a significant retinal hole or tear, experimental non-rhegmatogenous RD tends to reattach gradually over a period of hours to a few days, and thus requires additional constituents (such as hyaluronic acid) in the buffer formulations used for subretinal injections to maintain prolonged retinal separation. Because clinical retinal detachments tend to occur in an older eye, the state of the human vitreous is significantly different from that of the experimental models. The vitreal collagen framework collapses in the aging eye, resulting in syneresis, liquefaction, and pooling of fluid within the vitreous gel. Gel liquefaction is highly correlated with posterior vitreous detachments, which in turn sets up tractional forces on areas of vitreoretinal adhesion [37]. Such traction on areas of vitreoretinal adhesion may cause retinal tears and subsequent retinal detachment. Thus, some of the important differences between RD in humans and in animal models are the origin and context of the diseased condition, particularly the role of the vitreous. Despite these limitations, a number of important findings made in the lab have demonstrable correlative findings in humans, and have yielded further insight into the pathology of RD above and beyond what might be expected from clinical or patho-clinical studies alone. Some of these findings are described below.

G.Retinal Changes

For an excellent review of alterations in retina and RPE associated with experimental retinal detachment and reattachment, see Ref. 3. Research in animal models of RD has focused considerably more on rods than cones, and very little of the work on cones is performed on the cone-dominant macula. Of the animal

models employed in experimental studies, only certain primate species (including the rhesus macaques) contain a cone-dominant macula. The retina of the nocturnal owl monkey, which has been used in a series of RD studies, contains a macular region that is primarily made up of rods [38]. Rod and cone outer segments exhibit a number of well-appreciated differences in terms of their structural and biochemical interactions with the interphotoreceptor matrix and the RPE apical membrane [39,40].

Recent studies provide some evidence that rods and cones respond differently to retinal detachment and reattachment. Surviving rods continue to express high levels of rhodopsin and other cell-specific markers in a detached retina, whereas cones respond with a rapid reduction in a variety of protein and cellular markers, such as short wavelength (S)-cone opsin, calbindin D, carbonic anhydrase, peanut agglutinin, and possibly medium to long (M,L)-cone opsin [3,41– 43]. Labeling of apoptotic cell death with the TUNEL (TdT-mediated duTPbiotin nick end labeling) technique demonstrated that both rods and cones die during retinal detachments [42,44,45]. However the significance of detachmentmediated reductions in measurable expression of cone-specific markers remains elusive in the context of cone cell death. That is, reductions in the expression of these markers do not necessarily indicate inexorable death of cones. Further investigation into the time-course and extent of recovery of these markers needs to be conducted to appreciate the correlation, if any, between the decrease in cone-specific cell markers and cone death [42].

There is some experimental and clinical evidence that S-cones are more susceptible to prolonged or irreversible damage than M,L-cones in both human and experimental retinal detachment. Distortion in blue-yellow color vision has been described as a relatively common visual defect following successful retinal reattachment. Using enzyme histochemistry for carbonic anhydase and immunocytochemical localization of S antigen to differentially identify blue cones and red/green cones, Nork and colleagues provided evidence for increased loss of blue cones in human rhegmatogenous RD [43]. Clinical electroretinography (ERG) has also provided some insight into alterations in cone-mediated function in patients. Cone ERG results from 19 patients taken prior to and at multiple time points following successful retinal reattachment surgery provide evidence that the capacity for S-cone b-wave amplitudes to recover is significantly reduced when compared with those of L- and M-cone b-wave amplitudes [46,47]. In another study, multifocal ERG recordings taken from detached and attached areas of the retina before retinal reattachment surgery were compared with similar recordings taken from the same areas after successful reattachment surgery [48]. These ERG recordings were then compared with corresponding visual field measures. The authors found that improvement in ERG responses from both areas were relatively modest and surprisingly did not correlate with the more significant improvements in visual field results. Taken together, these ERG recordings in human RD point to the utility of conventional full-field as well as multifocal

ERG in extracting information about retinal function, but also suggest that ERG parameters may not correlate with visual function.

There have been some published reports on experimental RD that have focused on scotopic and photopic ERG responses during detachment and following reattachment [48–52]. Kim and colleagues recorded photopic focal ERG from small retinal detachments in adult rabbits and showed that b-wave amplitudes decreased significantly 30 min following retinal detachment and recovered back to baseline 3 days later [52]. However, immediately following the detachment, b-wave implicit times increased and remained significantly higher than control eyes even up to 28 days following reattachment. Because reattachment in rabbits occurs within a few hours following the induction of a small, bullous detachment, these results indicate that changes in retinal cone function persisted for weeks following retina/RPE reapposition and that photopic b-wave implicit time may be a useful parameter to assess loss and recovery of cone function. ERG recordings made from eyes of ground squirrels with RD of varying sizes showed a clear depression in cone-mediated ERG function in the detachment zone [77]. However, unlike observations made in human RD, no apparent differences in S- cone and M-cone ERG amplitudes were observed in squirrel. The reasons for this discrepancy remain unresolved, but may be a result of species differences or differences in experimental versus surgical conditions [77].

Although most of the basic and clinical research on retinal detachments have focused on changes in photoreceptors, significant alterations in the inner retina and RPE have also been observed and are believed to play a significant role in the aptly named “retinopathy of detachment” [3]. One of the leading causes of failure in retinal detachment surgery is the development of proliferative vitreoretinopathy, which is thought to be mediated by de-differentiated, proliferating, and hypertrophic non-neuronal cells, including retinal astrocytes, Mu¨ller, and RPE cells. The proliferative response can lead to eventual contraction of cells on the vitreal surface of the retina and result in traction retinal detachment. Understanding the causes of proliferative vitreoretinopathy may provide some insight into ways of preventing its development after successful reattachment surgery [15,17].

IV. POTENTIAL THERAPEUTIC OPPORTUNITIES

A variety of experimental parameters have been employed to quantitatively or semiquantitatively define the retinopathic effects of experimental retinal detachment and to measure the efficacy of a potential therapeutic approach. A number of these parameters are listed here:

Immunohistochemical labeling of rhodopsin and cone-opsin Quantification of cell proliferation with MIB-1 antibody