Ординатура / Офтальмология / Английские материалы / Aging and Age Related Ocular Diseases_Lutjen-Drecoll_2000

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Key Words

Immunocytochemistry W Major histocompatibility complex W Neural development W Rabbit W Retina W Retinal degeneration W Transplant W Ultrastructure W Vitrectomy

Abstract

Embryonic full-thickness rabbit neuroretinal sheets were transplanted to the subretinal space of adult hosts. This was accomplished by using a new transplantation technique involving vitrectomy and retinotomy. The grafts were followed from 10 to 306 days after surgery and were then examined by different histological techniques. In the light microscope, the transplants were seen to develop the normal retinal lamination and fusion with the host retina, especially after long survival times. Ultrastructurally, normal photoreceptor outer segments, well integrated with the host retinal pigment epithelium, were found. Growth cones were present in the zone of fusion between graft and host retina. Immunohistochemical labeling revealed many of the normal retinal components not previously found in retinal transplants, and graft-host connections between neurons in the rod pathway were seen. The morphology of vibratome-sectioned neuroretinal sheets as well as adult full-thickness grafts was also examined. These transplantation types showed

less of the normal morphology compared with embryonic full-thickness grafts. The immunogenicity of embryonic full-thickness and fragmented grafts was compared using major histocompatibility complex immunolabeling. Fragmented grafts elicited a response from the host immune system similar to a chronic transplant rejection. This reaction was absent in the full-thickness grafts which is in accordance with their good long-term survival.

Copyright © 2000 S. Karger AG, Basel

Introduction

Retinal Transplants

The main aim of retinal transplantation is to find a cure for degenerative retinal disease, primarily the group of diseases collectively called retinitis pigmentosa. The diseases within this group are very heterogeneous with varying onsets, severities and other general clinical manifestations, but they all have two things in common:

(1)they manifest degeneration of photoreceptor cells, and

(2)there is no effective treatment available, except for very rare cases where special diets may be beneficial [1, 2]. In recent years, much knowledge on the pathogenesis of

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these disorders has been gained. Many different mutations in genes which control the retinal biochemical machinery have been found, and many of them have also been correlated with defective protein synthesis [3]. The possibility of correcting defective genes is appealing, and experimental studies are in progress [4]. There are, however, both practical and theoretical problems to this approach, such as how to transfer genes specifically to diseased cells and when to perform the procedure. The rationale of retinal transplantation is to replace instead of repair the degenerating cells and thus provide a definite cure.

During the second half of the 1980s, the field of experimental retinal transplantation expanded greatly, following in the footsteps of other successful neuronal transplantation experiments [5, 6]. Three different forms of retina- to-retina transplantation techniques were soon developed. The first, described by Turner and Blair in 1986 [7], involved donor tissue in the form of embryonic retinas which were drawn into a syringe and thereby fragmented into small pieces. When placed in the subretinal space of the host, these grafts do not display the normal retinal appearance but develop into so-called rosettes, earlier seen in tumor-transformed retina, e.g. retinoblastoma. The rosette is a sphere of retinal tissue with layering similar to the adult retina, but with photoreceptor outer segments in the center and inner layers more peripherally. The fragment grafts contain many of the normal retinal cells [8±10] and have also been reported to sprout fibers towards the host retina [11] and to possess light-transduc- ing properties [12]. Small pieces of a fragmented graft can survive for extended periods [13], but the major part loses its organization and degenerates after 4±5 months [14, 15].

A second transplantation technique was developed by del Cerro et al. [16], who used enzymatically dissolved retinal cell suspensions. These grafts show less organization when compared to fragmented counterparts [17] but have been reported to restore vision in light-blinded rats [18]. Cell suspension transplants have recently been performed in humans, but the results have not been encouraging so far [19].

The third method, which has also reached human trials, was developed by Silverman and Hughes [20]. Their concept was to replace only the cells most affected by degenerative disease, namely the photoreceptors. They developed a technique where the retinal graft was embedded in gelatin and then shaved with a vibratome so that only the photoreceptor cells remained. Initially, good results were reported, but they have not been reproduced.

In human experiments, the technique has been reported to be safe, but no improvement of vision has been established in operated patients [21].

Many important facts such as the superiority of embryonic versus adult tissue, the good differentiation of embryonic grafts and the lack of acute graft rejection have been established by work involving the methods mentioned above. They do, however, leave room for improvement, especially in the areas of transplant morphology, long-time survival and graft-host interactions.

Immunological Aspects of Retinal Transplants

The eye is one of the immunologically privileged sites [22] where the immune response to foreign antigen is actively downregulated, thereby minimizing destruction of the surrounding tissue. The presence of the atypical low-keyed immune response in the eye, often named ante- rior-chamber-associated immune deviation [23], is clearly relevant for the survival of retinal grafts, even though recent investigations indicate that the privileged status of the subretinal space is not perfect [24]. Syngeneic embryonic fragmented grafts are well tolerated by the host, but their allogeneic counterparts display an upregulation of major histocompatibility complex (MHC) molecules, announcing an interaction and possibly a rejection by the host immune system [25]. This might explain why fragment transplants diminish considerably and lose their organization after 4±5 months [14]. Conversely, even xenogeneic fragmented transplants can survive for at least 41 weeks in immunosuppressed hosts [26].

The immunological status of the host as well as the immunogenicity of the graft obviously play important roles in the survival of neuronal transplants.

Neural Development

This review describes a new procedure of neuroretinal transplantation [27]. The concept on which it relies is very dependent on basal mechanisms governing neural development, and a brief analysis of this subject is therefore motivated.

In normal embryonic development, the cells of the body go through extensive changes resulting in a multitude of different tissues, all originating from the fertilized egg cell. Embryonic cells possess an immense plasticity, which is evident from the fact that each cell in the very early mammalian embryo can differentiate into any cell

type, depending on its environment [28]. This dependency is very prominent and crucial in neural development, where each cell needs to take part in a highly refined network. To achieve this, the developing neuron goes through two important phases. In the first, the cell after final mitosis migrates to its appropriate location. In this process, glial cells function as a scaffold, guiding the cell. The importance of this scaffold is well exemplified in the developing retina which will form rosettes instead of laminated layers if the prime glial cells, the Müller cells, are disturbed [29]. The second characteristic of neuronal development is axonal and dendritic pathfinding, a process governed by surrounding cells and extracellular matrix. A multitude of adhesion molecules, intercellular signals and growth factors guide the growing neurite towards its target. In the retina, the Müller cells are again important, providing both positional information as well as chemical cues for correct neurite growth [30±32]. The target of the developing neurite also plays an important role in the construction of the neuronal network by releasing neurotrophic factors [33]. This is illustrated by the fact that embryonic retinal cells can be grafted to the brain and form connections with visual centers [34]. Together, these findings suggest that developing neurons are dependent on the integrity of their surroundings and on stimulation from their targets.

Materials and Methods

Donor Tissue

Embryonic Transplants

Time-mated female pigmented rabbits were killed with 5 ml intravenous sodium pentobarbital (60 mg/ml), and the embryos were collected and used as donors in all embryonic transplants. Tissue from stage E15 to E19 (15±19 days after conception) was used [27, 35±39]. The eyes were enucleated, and the neuroretinas carefully dissected out and trimmed at the edges, forming an elliptical sheet of approximately 2 ! 3 mm. This sheet was kept in +4 °C Ames' solution [40] and used either as a full-thickness graft or was further processed by vibratome sectioning or fragmentation (see below).

Adult Transplants

Pigmented rabbits aged 3±5 months were used as donors for adult transplants [39]. The animals were killed with 5 ml sodium pentobarbital (60 mg/ml) intravenously, and both eyes were enucleated. The anterior segments were cut away with dissecting scissors. Using a 2- mm circular biopsy punch (Stiefel), 4±6 disks of full-thickness retina were cut out from each eye. The neuroretinas were gently separated from the retinal pigment epithelium by infusing Ames' solution subretinally. The neuroretinal disks were kept in +4°C Ames' solution and were used either as full-thickness grafts or were further processed by vibratome sectioning (see below).

Vibratome-Sectioned Transplants

To remove inner retinal elements [39], some adult and embryonic full-thickness grafts were embedded in gelatin and sectioned on a vibratome using the method described by Silverman and Hughes [20]. These embedded partial transplants were trimmed to a rectangular shape measuring approximately 1.5 ! 3 mm.

Fragment Transplants

The fragment transplants [36] were obtained by drawing an E19 embryonic neuroretina into a syringe.

Hosts

Pigmented mixed-strain rabbits, aged 3±5 months, were used as hosts in all experiments. In order to determine whether the animals might be syngeneic, 3 rabbits (litter-mates) from our local breeder received skin transplants from each other. All skin transplants were rejected after 7 days, suggesting a low degree of inbreeding among the animals.

Surgical Procedures

Preoperative Preparations

The right eye of the recipient rabbit was instilled with cyclopentolate (1%) and phenylephrine (10%) 30 min prior to surgery. General anesthesia was provided with ketamine (40 mg/kg) and xylazine (5 mg/kg) intramuscularly. Topical tetracaine (0.5%) was applied just before surgery.

Operation

The conjunctiva was incised limbally 180° from 10 to 4 o'clock with a vertical incision at 12 o'clock, creating two flaps. A 20-gauge infusion cannula was sutured to the sclera in the 4-o'clock position 1 mm posterior to the limbus and a balanced salt solution (Endosol®; Allergan Medical Optics) was infused, containing adrenaline (5.5 ! 10±9 M) for pupil dilatation and heparin (5 IU/ml) to minimize postoperative inflammation. A metal ring for support of a contact lens was sutured in place with limbal sutures at 3 and 9 o'clock and sclerotomies were made in the 10and 2-o'clock positions. A vitrectomy contact lens was placed on the cornea with 2% methyl cellulose (Methocel®) as contact medium. As much vitreous as possible was removed, using a vitreous cutter (Ocutome®) and an intraocular illuminator. The large rabbit lens made it impossible to remove more than 50±60% of the vitreous, and due to firm vitreoretinal adhesion the posterior hyaloid could not be removed with certainty.

To detach a small area of neuroretina, a thin flexible polyethylene capillary (outer diameter: 0.6 mm, inner diameter: 0.4 mm) attached to a 1.0-ml syringe filled with Ames' solution and supported by a blunt and slightly bent 20-gauge metal cannula was introduced through the sclerotomy at 10 o'clock. Penetrating the retina 3±4 mm inferior to the optic nerve, it was positioned in the subretinal space. The fluid from the syringe was carefully infused, creating a limited circular retinal detachment with a diameter of approximately 3 mm. A second, smaller retinotomy was then made inferior to the first.

The donor tissue was drawn into a glass cannula (outer diameter: 1.2 mm, inner diameter: 1.0 mm; fig. 1). In some experiments, the cannula had a slightly conical shape with the narrow end measuring 0.8 mm in outer and 0.6 mm in inner diameter. The cannula was

Fig. 1. Important steps in the transplantation procedure. A The full-thickness embryonic grafts are stored in Ames' solution. Prior to actual transplantation, the neuroretina and its accompanying fluid is drawn into a glass cannula. B A thin flexible polyethylene capillary is introduced through the vitreous. Penetrating the retina 3±4 mm inferior to the optic nerve, it is positioned in the subretinal space. Fluid from an attached syringe is infused, creating a limited circular retinal detachment (by permission from the journal Retina). C A second, smaller retinotomy is created (by permission from the journal Retina). D The transplant is now introduced subretinally by means of the glass cannula. The fluid flows out of the second retinotomy, but the transplant stays in place due to its relatively larger diameter (by permission from the journal Retina).

connected to a 1.0-ml syringe via a polyethylene tube, allowing the graft together with a small amount of the surrounding solution to be drawn in. As it entered the cannula, it adopted a curved shape. The graft was then carefully moved towards the end of the cannula by gently pushing the plunger of the syringe. The cannula was now introduced into the eye through the 10-o'clock sclerotomy and its end was positioned against the superior retinotomy. The graft was pushed out of the cannula into the subretinal bleb, and while the accompanying fluid passed out into the vitreous space through the inferior retinotomy, the transplant stayed securely in place subretinally. On ejection, varying degrees of flattening of the transplant occurred.

The donor retina was kept with its inner (vitreal) surface up throughout the procedure to maintain correct polarity. When kept in Ames' solution, it had a tendency to fold, adopting its originally slightly curved state. This was favorable, as it needed to adopt this shape inside the glass cannula and also made the drawing in of the graft more atraumatic. In the cannula, the transplant was visualized and the correct orientation kept by twisting the instrument.

The vibratome-sectioned transplants [39] were placed subretinally using a custom-made injector, consisting of a piece of flattened plastic tubing with a plastic piston. This injector had the same dimensions as the one used by Silverman and Hughes [20], with an inner width of 1.5 mm, an outer width of 2.0 mm and a height of 1.0 mm. The injector, being larger than the glass cannula, required a larger sclerotomy and retinotomy for the transplant procedure.

Finally, the vitrectomy lens and its supporting ring were removed, the sclerotomies were sutured and 0.5±1.0 ml of air was injected into the eye to avoid hypotonia. After suturing the conjunctiva, 25 mg gentamicin and 2 mg betamethasone were injected subconjunctivally. The operation times averaged 35 min. The time from sacrifice of the mother to actual transplantation was noted and ranged from 60 to 460 min.

For fragment transplants, 8±10 Ìl of the retinal solution was placed in the subretinal space of the host using a polyethylene catheter inserted through the sclera and vitreous [36].

Postoperative Management and Follow-Up

No postoperative treatment was given. Ophthalmoscopic examinations were made on postoperative days 1 and 7, weekly for 1 month and thereafter monthly. The animals were killed 10±306 days after transplantation.

Tissue Preparation

Light Microscopy

For light microscopy, the eyes were enucleated and fixed for 30 min in formaldehyde (4%, generated from paraformaldehyde) at pH 7.4 in a 0.1 M Sùrensen's phosphate buffer. The anterior segment was then removed and the posterior eyecup postfixed in the same solution for 4 h. Tissue specimens were obtained as approximately 2.5- to 4-mm-wide pieces, including the area of the transplant together with parts of the myelinated fibers and optic nerve. After fixation, most specimens were washed with Sùrensen's phosphate buffer (0.1 M, pH 7.4) and then either washed again using the same solution with added sucrose of rising concentrations (5, 10, 15, 20%) or sectioned at 12 Ìm on a cryostat. The remaining specimens were dehydrated, embedded in paraffin and sectioned at 7 Ìm. The slides were then stained with hematoxylin and eosin. Cryostat sections were used in order to allow immunohistochemical analysis (see below). The vertical length of the transplants was measured directly in the light microscope, whereas their horizontal length was calculated from consecutive sections.

Electron Microscopy

Two transplants with postoperative times of 92 and 141 days were prepared for electron microscopy [37]. These tissue specimens were fixed for 4 h (4% paraformaldehyde mixed with 1% glutaraldehyde in 0.1 M, pH 7.2, phosphate buffer containing 0.15 mM CaCl2 at 4° C). They were then washed overnight in phosphate-buffered

saline (PBS) and then in 0.15 M sodium cacodylate buffer, postfixed in 1% osmium tetroxide in 0.15 M sodium cacodylate buffer for 1 h at 4°C, washed in the buffer, dehydrated through a graded series of alcohol and embedded in Araldite® (Fluka Chemie AG, Buchs, Switzerland). Ultrathin sections were cut on an LKB Ultrotome® (Bromma, Sweden) and contrasted with uranyl acetate and lead citrate according to standard electron-microscopic procedures. The grids were examined in a Jeol 1200 EX electron microscope. Semithin sections were cut and stained with a mixture of methylene blue and azur II blue for preliminary analysis in the light microscope.

Immunohistochemistry

The sections were thawed and washed in 0.1 M sodium PBS with 0.25% Triton X-100 (PBS/Triton, pH 7.2).

For labeling with the ABC kit, the sections were incubated with the appropriate normal serum at room temperature for 30 min and then with the primary antibody diluted in PBS/Triton with 1% bovine serum albumin (BSA) at +4°C [35]. After 16±18 h, the sections were washed in PBS/Triton for 2 ! 15 min, incubated with the secondary antibody diluted in PBS/Triton with 1% BSA for 30 min,

washed in PBS/Triton for 2 ! 15 min, and reacted with an immunohistochemical avidin-biotin-peroxidase system (Elite ABC kit standard; Vector Laboratories). They were then washed in PBS/Triton for 2 ! 15 min, developed in 3,3)-diaminobenzidine solution (DAB kit, Vector Laboratories) for 2±10 min, washed in distilled water, dehydrated and mounted in Permount®. For lectin demonstration, sections were incubated in biotinylated peanut agglutinin (Vector Laboratories) at 75 Ìg/ml for 1 h after rinsing in PBS with 0.5% BSA. After incubation, the sections were rinsed and processed with the ABC kit.

For single-labeling fluorescence immunohistochemistry, the sections were incubated with the primary antibody for 16±18 h [35, 36, 38]. They were then rinsed, incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:100) for 30 min, rinsed again and finally mounted in custom-made antifading mounting media.

For parvalbumin and protein kinase C (PKC) double labeling, the tissue was incubated with the parvalbumin antibodies for 18±20 h, rinsed in PBS/Triton and then incubated with the PKC antibodies for 18±20 h [38]. After rinsing in PBS/Triton, the tissue was incu-

bated for 45 min in darkness with a mixture of the secondary antibodies conjugated with two different fluorophores (antirabbit FITC and antimouse Texas red). The dilution of each secondary antibody was 1:100. For calbindin and AB5 double labeling, a different approach was chosen since these antibodies were made in the same species (mouse) [38]. The sections were first incubated with the calbindin antibody for 18±20 h. They were then rinsed in PBS/Triton and incubated with antimouse Texas red. After another thorough rinse in PBS/Triton, the AB5 immunolabeling was performed in a similar manner but with antimouse FITC as secondary antibody. This secondary antibody now recognized all mouse antigen in the tissue and, consequently, the FITC fluorescence was present at both anticalbindin and AB5-positive sites. To differentiate the AB5 and calbindin labeling, separate digital images of the tissue activity of the fluorophores were superimposed on each other. In this composite image, calbindin-positive sites were yellow (green + red) while AB5 was seen as green. Antibodies and dilutions are listed in table 1.

Control experiments included labeling of normal adult rabbit retinas as well as labeling procedures without the primary antibodies on sections from operated animals.

All proceedings and animal treatment were in accordance with the guidelines and requirements of the Government Committee on Animal Experimentation at Lund University and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Results

Surgical Complications

Peroperatively, areas of missing host retinal pigment epithelium (RPE) were often seen after the bleb formation. Other peroperative complications in full-thickness transplantations included limited choroidal bleeding in approximately 10% of the cases and lens touch in another 10%. In a few cases, these complications led to an inflammation of the vitreous and retina with massive epiretinal membrane formation. In eyes receiving vibratome-sec- tioned grafts, complications were more serious and common [39]. Of these eyes, 30% displayed a significant vitreal hemorrhage which often led to epiretinal membrane formation and retinal tractional detachment on postoperative examinations.

Macroscopic Findings

The embryonic full-thickness transplants were easily located at the operation site, under the host retina. Initially, they had the shape of a gray disk, approximately the same size as the optic disk. In time they became more transparent and often included small spots of hyperpigmentation. Vibratome-sectioned grafts were sometimes

found in the vitreous and shrunk with time. Adult grafts generally also diminished considerably after the first postoperative week.

Morphology of Full-Thickness Embryonic Grafts

Light Microscopy

In all series, approximately 10±25% of the full-thick- ness grafts developed into rosettes without distinct lamination (fig. 2). The remainder were all in the form of laminated retinas with layers parallel to the host RPE. Their laminated length measured from 0.5 to 3.2 mm. In the smaller ones, disorganized cells in the form of rosettes were found at the edges of the transplant.

In laminated transplants, the inner and outer segments appeared to be of normal length, facing the host RPE in the normal fashion. In transplants up to 3 months postoperatively, the outer nuclear layer was often thick and contained a large number of perikarya. In older ones (4±10 months), this layer was thinner and more comparable to the normal adult outer nuclear layer. The outer plexiform layer appeared normal. All inner retinal layers including the ganglion cell layer (GCL) and nerve fiber layer (NFL) were present in the majority of transplants, but the cells of the GCL were often smaller than normal ganglion cells. In young transplants, the GCL and NFL were often present throughout the major part of the transplant. In 4- to 10month specimens, these layers were found in a small section of the transplant if at all present.

Minimal defects in the host RPE were seen in some cases.

There were no signs of inflammatory cell infiltration in any of the specimens.

Electron Microscopy

Both long-term transplants examined in the electron microscope displayed long photoreceptor outer segments with the normal arrangement of stacked disks [37]. The inner segments appeared normal and contained the usual organelles. Well-developed photoreceptor terminals with horizontal and bipolar cell process invaginations were seen in the outer plexiform layer. The inner plexiform layer (IPL) was thinner than normal in the young transplants (92 days postoperatively) but of a more normal thickness in the older ones (141 days postoperatively). Bipolar and amacrine cell synapses were present in this layer but were not as common as in the normal rabbit retina.

Fig. 2. Transplant morphology. A Embryonic E16 transplant (T) 10 days postoperatively. The cells form a neuroblastic cell mass with 10±15 layers and a thin ganglion cell layer. No outer segments are seen. The transplant is folded with the part not against the host RPE forming rosettes. The host retina (H) has degenerated extensively, leaving only a thin inner retinal layer (by permission from the journal Retina). Paraffin section. HE. Scale bar = 200 Ìm. B E19 transplant

(T) 35 days postoperatively. The major part of the graft faces the host RPE and is laminated with correct polarity. The host retina (H) is seen to straddle the transplant and is degenerating in this area (by permission from the journal Retina). Paraffin section. HE.

Scale bar = 200 Ìm. C E19 transplant (T) 86 days postoperatively. The transplant displays all normal retinal layers including photoreceptor outer segments facing the host RPE. The host (H) outer retina has degenerated. Disorganized cells are present between the laminated parts of the host and transplant. Cryostat section. HE. Scale bar = 50 Ìm. D E19 transplant (T) 309 days postoperatively. The laminated structure of the graft can still be appreciated, as well as the well-developed photoreceptors. Fusion of transplant and host has taken place at the level of the inner plexiform layer of the transplant. Cryostat section. HE. Scale bar = 50 Ìm.

Immunohistochemistry

Interphotoreceptor Matrix. Peanut agglutinin labeling of the laminated transplants showed segmentally arranged labeling at regular intervals in the photoreceptor outer segment layer [35]. When compared with the normal retina, the labeling was more compressed, i.e. with increased numbers of labeled structures in a given area, presumably reflecting a higher density of photoreceptor cells. In rosettes, PNA labeling was associated with most or all photoreceptors, as previously described [41].

In 5 of 6 grafts, specific IRBP labeling of the photoreceptor outer segment region was seen. When compared with the host, labeling was somewhat more intense in 4 of these transplants.

Rod Bipolar Cells. In all 14 long-term transplants, PKC-labeled rod bipolar cell bodies were identified in their normal position in the scleral part of the inner

nuclear layer [37]. In the 3-month grafts, they seemed to be as common as in the normal rabbit retina, although no precise cell count was made. In 4- or 10-month grafts, their number had decreased moderately. The cells extended vertical axons with terminal bulbs in the vitreal part of the transplant IPL. The axons in some cases were longer than in the normal retina.

Large and well-labeled structures consisting of minute fibers were seen in a zone more to the vitreal side of the transplant IPL. They were most common in the 3-month specimens and were often in contact with bipolar cell axons in the graft. The structures were interpreted as collections of growth cones [42].

The PKC-labeled rod bipolar cells extended dendritic branches to the most scleral part of the outer plexiform layer in the normal manner.

In the host retina, PKC-labeled rod bipolar cells were often found but were not as common and not as strictly organized as in the normal retina. The cells extended axons with terminals in the most vitreal part of the host IPL. Dendrites extending towards the transplant were seen in specimens of all ages. These dendrites often displayed growth cones with minute filopodium-like processes at their tips.

Cone Bipolar Cells. Calbindin-labeled cone bipolar and horizontal cells were found in all long-term transplants [37]. The cone bipolar cells were identified by their small size and their thinner, more vertical processes. These processes often ended in a network of fibers on the vitreal side of the transplant IPL. The cells were not as common as in the normal rabbit retina.

In the host, labeled cone bipolar cells with processes terminating in the host IPL were seen, although these cells were not as numerous as in the normal retina. Some remaining horizontal cell processes were also noted in the host retina.

AII Amacrine Cells. Parvalbumin-labeled AII amacrine cells in the 3-month specimens were often organized in 3 layers: (1) in the transplant, with cell bodies in the inner nuclear layer and GCL, and with processes in the IPL, (2) in an intermediate plexiform layer on the vitreal side of the transplant IPL and (3) in the host inner nuclear layer with processes in the host IPL [37]. In the 4- to 10month specimens, the AII amacrine cells around the transplant IPL were fewer, and more labeled cell bodies and processes were seen in the intermediate plexiform layer. The AII amacrine cells in the host retina of these specimens displayed branches in the host IPL. There was no apparent decrease in the total number of labeled cells in the oldest specimens (10 months postoperatively) when compared to younger ones.

Cholinergic Amacrine Cells. Cells displaying choline- acetyltransferase-like immunoreactivity were found along both margins of the IPL of the transplant and host in a similar fashion [35]. When compared with the same section stained with hematoxylin and eosin, the majority of cells in the GCL of the transplant had not been labeled.

Ganglion Cells. AB5-positive cell bodies were found in the GCL of 2 out of 6 short-term transplants, 31 and 49 days postoperatively [35]. The cells were large and showed branching like host retinal ganglion cells. AB5labeled fibers in the NFL were found in one additional graft. NF 160 kD labeling revealed labeled fibers in the NFL of all 6 grafts with horizontal or oblique fibers, either single or in clusters. In long-term specimens, a few AB5-

labeled large cells were seen in the GCL of 2 out of 14 transplants (86 and 126 days postoperatively) [38].

In the host retina, NF-160-kD-labeled fibers were found in the NFL of short-term specimens. AB5-labeled ganglion cells were seen in the GCL of the host in all 18 specimens investigated (1±10 months postoperatively). Their number seemed to be relatively constant in all specimens and they displayed branches in the host IPL as well as axons in the NFL.

Müller Cells. Vimentin-labeled, straight and normally arranged Müller cells extending from the NFL to the outer nuclear layer, were seen in 6 out of 6 short-term transplants [35]. Normal, branched endfeet were seen forming a thin inner limiting membrane. The host Müller cell labeling was moderately stronger.

Müller cells labeled by glial fibrillary acidic protein were found in 3 out of 6 specimens in the host retina [35]. In these specimens glial fibrillary acidic protein could also be detected in the Müller cells of the transplant. Their morphology was normal, but the labeling intensity was less than in the host. In 3 animals, no activity was found in either host or transplant.

Graft-Host Integration

Host Retina. The host retina appeared normal in all eyes, except for the part covering the graft where the outer layers had degenerated completely and the inner layers were affected in varying degrees. The inner nuclear layer, IPL, GCL and NFL of the host were preserved in specimens up to 3 months postoperatively but tended to thin out in older ones (4±10 months postoperatively).

Photoreceptor-RPE Integration. In laminated transplants, photoreceptor outer segments were seen facing the host RPE in the normal manner. Small defects in the host RPE were noted in some of these specimens. In the electron microscope, transplanted photoreceptor outer segments were seen to integrate well with microprocesses extending from the host RPE [37].

Inner Retinal Integration. In specimens up to 3 months postoperatively, an array of disorganized cells, often in the form of rosettes, was evident between the laminated parts of the host and graft. These cells could not with certainty be defined as belonging either to graft or host, and prevented contact between laminated parts of the two. In 4- to 10-month specimens, the disorganized cells were few, and long segments without remaining GCL and NFL in the transplants were evident. This allowed for close contact between host and graft, and fusion was often evident at the level of the IPL of the transplant.