November 1999
Volume 40, Issue 12
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Retina  |   November 1999
Nitric Oxide–Producing Cells Project from Retinal Grafts to the Inner Plexiform Layer of the Host Retina
Author Affiliations
  • Yiqin Zhang
    From the Department of Ophthalmology, Lund University Hospital, Lund, Sweden.
  • Rajesh K. Sharma
    From the Department of Ophthalmology, Lund University Hospital, Lund, Sweden.
  • Berndt Ehinger
    From the Department of Ophthalmology, Lund University Hospital, Lund, Sweden.
  • Maria–Thereza R. Perez
    From the Department of Ophthalmology, Lund University Hospital, Lund, Sweden.
Investigative Ophthalmology & Visual Science November 1999, Vol.40, 3062-3066. doi:
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      Yiqin Zhang, Rajesh K. Sharma, Berndt Ehinger, Maria–Thereza R. Perez; Nitric Oxide–Producing Cells Project from Retinal Grafts to the Inner Plexiform Layer of the Host Retina. Invest. Ophthalmol. Vis. Sci. 1999;40(12):3062-3066.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Amacrine cells expressing nitric oxide synthase (NOS) are seen in normal retinas and retinal grafts to extend long processes, which can be followed for long distances. Taking advantage of the morphologic features of these cells, the present study examined whether graft–host connections involve cells capable of producing nitric oxide, a recognized retinal neuromodulatory compound.

methods. Embryonic day 15 rabbit retinas were transplanted to the subretinal space of adult rabbits. The localization of the neuronal form of NOS was assessed by immunocytochemistry in grafts that had reached the equivalent ages of postnatal days 5, 12, 20, 45, 90, and 102.

results. NOS-containing cells and processes were seen in all the transplants. Processes were found to project mainly toward areas within the graft. Yet, at all survival times examined, single immunolabeled fibers could be seen to cross the graft–host border. In fortuitous cases, it was possible to establish that the bridging fiber originated in the graft. Further, bridging fibers were seen to reach the NOS-immunolabeled host inner plexiform layer.

conclusions. Graft NOS-containing cells are not only capable of projecting into the host but also of reaching the appropriate target for NOS-containing fibers within the host retina. This indicates that at least some graft–host connections are established by graft cells that retain their ability to synthesize a modulatory compound and which potentially could contact their partner cells in the host retina.

Transplantation of retinal cell suspensions, of retinal pieces, or of particular cell types to the subretinal space are some of the strategies being tested to improve vision in certain forms of outer retina degeneration. 1 Most cell types and synapses are seen to develop in the transplants, and structural proteins and neurotransmitters found in normal retinas also are expressed by the transplanted cells. However, for in oculo retinal transplants to be functional, it is required that they integrate and form relevant connections with the host retina. Subretinally transplanted photoreceptors cells expressing the lacZ reporter gene product, β-galactosidase, have been found to exhibit well-developed synaptic terminals and to contact host bipolar cells. 2 3 4 Processes projecting from the graft into the host retina also have been observed after epiretinal and subretinal transplantation of the entire neuroretina. Using electron microscopy, prelabeling of the donor tissue, or cell markers to identify distinct retinal cell types, it has been shown that graft–host contacts also can involve cells of the inner retina, for example, contacts between amacrine and bipolar cells. 5 6 7 8 9  
However, the anatomic demonstration of graft–host connections based on synaptic contacts or by using structural markers does not provide information about the functional potential of the connecting cells. Therefore, in the present study we looked at the ability of a chemically and functionally defined retinal cell type to project into the host retina. Nitric oxide is a recognized retinal neuromodulatory compound. 10 It is synthesized by subpopulations of wide-field amacrine cells in the rabbit retina 10 11 12 and has been shown to be expressed by transplanted retinal cells. 12 The processes of cells expressing the neuronal form of its synthesizing enzyme, nitric oxide synthase (NOS), can be followed for long distances, thus providing an effective tool for studying connectivity. We show in the present report that graft cells potentially capable of producing nitric oxide can project to and reach target areas in the host retina. 
Materials and Methods
Animals were handled according to the guidelines set by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, the Declaration of Helsinki, and the local animal experimentation and ethics committee (Djurförsöksetiska nämnden i Lund). 
The preparation of donor tissue and the transplantation procedure have been described previously in detail. 13 Briefly, outbred pigmented rabbits of a mixed strain (embryonic stage [E] 15, normal gestation 31 days) were used as donors for the transplantation. Embryos were placed in Ames solution at 4°C, and the eyes were enucleated. Retinas were dissected free from the retinal pigment epithelium and were stored at 4°C in fresh Ames solution until used for transplantation (4 hours at most). Eleven adult rabbits of the same breed as the donors were used as recipients. Thirty minutes before surgery, the right pupil of the recipients was dilated with 1% cyclopentolate-HCl (Cyclogyl; Alcon-Couvreur, Puurs, Belgium,) and one drop of 10% phenylephrine-HCl (Sanofi Winthrop Pharmaceuticals, New York, NY). The recipient rabbits were anesthetized with 1 ml/kg Hypnorm (10 mg/ml fluanison and 0.2 mg/ml fentanyl; Janssen Pharmaceutica, Beerse, Belgium) and topical tetracaine-HCl (0.5%; Alcon-Couvreur) was applied. Two to four embryonic retinas (in up to 10 μl total volume) were drawn up into a thin polyethylene capillary mounted on a special instrument that was connected to a precision microsyringe. 13 A small scleral incision was made in the recipient eye 2 to 4 mm behind the limbus. The capillary was advanced through the vitreous to the posterior pole of the eye where the retina was penetrated. The embryonic retinas were deposited slowly in the subretinal space in the central retina, below the myelinated streak. The animals were kept in light–dark cycles (12 hours each) and were allowed to survive for 21 to 118 days after the surgery. The transplants thus reached ages equivalent to postnatal days (PN) 5 (n = 2), 12 (n = 2), 20 (n = 3), 45 (n = 1), 90 (n = 1), and 102 (n = 2). No immunosuppressive drugs were used. 
Eyes carrying transplants were quickly enucleated and immersed in a solution of 4% formaldehyde in Sörensen’s buffer (0.1 mM; pH 7.2). After 30 minutes, the eyes were hemisected, and the anterior segment, the lens, and the vitreous were discarded. The remaining eyecups were kept in the same fixative for an additional 90 minutes, after which they were rinsed and cryoprotected in Sörensen’s buffer containing sucrose. The area containing the transplant was cut out, embedded, and frozen. Cryostat sections were incubated with sheep antineuronal NOS serum, followed by incubation with Texas red sulfonyl chloride–conjugated donkey anti-sheep IgG (Jackson ImmunoResearch, West Grove, PA). The NOS antiserum used (gift from Ian G. Charles and Piers C. Emson) was raised against purified rat recombinant neuronal NOS protein and was found to be specific for neuronal NOS in control experiments. 12  
Results
In all grafts, cells of the outer nuclear layer were organized as rosettes, and in between the rosettes, cells of the inner retina were observed (Fig. 1 A). The outer host retina degenerated with time in most areas adjacent to the graft (see below), whereas the innermost layers appeared to have retained their normal structure (Figs. 1A 1C 2 3) . With the antibody used, NOS immunoreactivity was found in normal adult rabbit retinas restricted to a few cell bodies in the innermost cell row of the inner nuclear layer, to the inner plexiform layer (Fig. 1B) , and to some cells in the ganglion cell layer (not shown). This distribution pattern was preserved in the host retinas even in the areas adjacent to the graft (Figs. 1C 2 3) . The number of labeled cells found in the host retina also was not different in areas adjacent to the graft or when compared to normal retinas. Further, there were no indications that after transplantation, cell types other than those seen in a normal rabbit retina expressed NOS immunoreactivity in the host retina. The only difference was noted in the host inner plexiform layer, where stronger and denser labeling often was seen compared with nonoperated animals. Only sections in which no morphologic disturbance of the host inner retina could be observed in the areas adjacent to the graft were analyzed. The presence of labeled amacrine cells in the proximal inner nuclear layer and of immunoreactivity in the inner plexiform layer as a continuous plexus, running parallel to the vitreal border of the host retina and the graft–host border, were used as an indication of the relative integrity of the inner host retina. NOS-immunoreactive cells were judged to belong to the host or to the transplant, depending on their location. 
NOS-immunoreactive cells and processes were seen in all grafts examined. Immunolabeled processes occasionally could be found in the grafts, close to the graft–host border. However, in regions where one or more photoreceptor cell rows of the host outer nuclear layer remained, such processes were seen to run parallel to the border, without entering the host retina (Fig. 1C) . Rows of photoreceptor cells could be observed in the host retina at the shorter survival times and at the edges of the bleb created in the host retina by the graft (Fig. 1C)
Nevertheless, labeled fibers originating in the graft could at times be seen crossing the graft–host border. Such bridging was observed at all survival times, but not in all specimens examined (in 8 of 11), and only in regions where the host photoreceptor layer was absent. In fortuitous cases, it was possible to follow a labeled process from the graft all the way into the host inner plexiform layer. Examples of this are given in Figures 2 and 3 . A long thin bridging process is seen originating in a graft corresponding to PN 12 (28 days after transplantation; Fig. 2A ) and a shorter one is seen in Figure 2B in a transplant corresponding to PN 20 (36 days after transplantation). As a result of the degeneration of the host outer layers, NOS-containing cells and fibers occasionally were found in the grafts relatively near the host inner plexiform layer. In Figure 2B 2a bridging process is seen to run between the host inner plexiform layer and a more distal area where several immunolabeled structures are observed. Because no NOS-immunoreactive fibers were seen external to the inner plexiform layer in a normal rabbit retina (Fig. 1B) , the bridging process seen in Figure 2B is also likely to connect structures within the graft with the host retina. In Figure 3A , several stained processes are seen within a graft corresponding to PN 45 (61 days after transplantation). In addition, a process arising from a NOS-immunolabeled cell located in the graft is seen to extend toward the host inner plexiform layer. Serial sections confirmed the location of this cell. Further, a stained cell body also is seen in the amacrine cell layer of the host retina, and strong and continuous labeling is noted in the host inner plexiform layer, reflecting the relative organization of the inner host retina. The immunolabeled fiber, seen in Figure 3A 3B 3C 3D 3E 3F 3G 3H 3I 3J 3K 3L 3M 3N 3O 3P 3Q 3R each almost perpendicularly the host inner plexiform layer, can thus be judged as a bridging process that originates in the graft. In Figure 3B 3a stained cell body is seen in the host retina 106 days after transplantation. One strongly labeled cell body also is seen in the transplant (corresponding to PN 90) to project to a region within the graft where faintly stained fibers are seen. In addition, one large NOS-immunoreactive cell body is seen to project to the same region within the graft and to emit a more weakly labeled process toward the host inner plexiform layer. As mentioned above, a progressive loss of the outer host retina is normally observed, which with time brings the graft closer to the host inner layers. Again, a labeled cell is seen in the host retina in its expected position, next to the inner plexiform layer. The connecting cell could therefore, judging from its position, belong to the transplant. However, it is not possible to determine conclusively in this case whether the bridging cell indeed belongs to the graft. 
Discussion
The present study demonstrates that a subpopulation of graft cells with the ability to express neuronal NOS extend processes into the host retina in rabbit-to-rabbit transplants. Furthermore, it was seen that the NOS-containing processes are capable not only of projecting into the host retina, but also of reaching the host inner plexiform layer, the appropriate target in the host retina. It was not determined here whether synaptic contacts are established by the projecting NOS-accumulating cells. However, previous studies have shown that synaptic terminals of transplanted cells exhibit normal morphology, even after long survival times. 6 7 14 Further, there is evidence that neurites projecting from retinas transplanted to the brain are capable of forming synaptic contacts on reaching the host target tissue. 15 In normal rabbit retinas, NOS is expressed by subpopulations of wide-field amacrine cells, 10 11 12 and it is likely that the NOS-containing cells found in the grafts also are of this type. It has been suggested that nitric oxide produced by amacrine cells may modulate light-induced inward currents in ON cone bipolar cells. 10 Thus, it is possible that the NOS-containing cells found in the grafts contact cone bipolar cells at the level of the host inner plexiform layer. 
At times, the origin of the bridging fiber could not be established. We also have found examples of NOS-immunoreactive neurons projecting to the graft and to the host retina. In the case illustrated in Figure 3B , the possibility that the contacting cell belonged to the host cannot be ruled out. Whatever the origin of the projecting neuron might be, it appears to connect the host inner plexiform layer to an equivalent region within the graft. If so, information from the graft could be conveyed to the host retina not only by those neurons that directly project to the host, but also indirectly. 
Bridging fibers were more often seen at long survival times. This is as expected, considering that also in normal developing rabbit retinas, outgrowth of NOS-containing fibers is a relatively slow process. 11 12 Further, a better graft–host fusion was observed in older transplants, which conceivably should also favor the formation of connections. Bridging, however, was never observed in areas where one or more cell rows were left of the host outer nuclear layer. Labeled processes located next to the graft–host border were in these cases seen to run parallel to the graft–host border, without crossing over, in spite of the fact that the host outer nuclear layer was thinned and that the host inner plexiform layer was located near the graft. This does not seem to be unique for rabbit-to-rabbit transplants or a limitation of NOS-containing fibers only, because the same has been observed in rat-to-rat transplants using additional cell markers. 16  
Furthermore, it was noted that even when present, the number of NOS- immunoreactive fibers crossing the graft–host border was not very large and varied between specimens and also between sections from the same specimen. It may be noted that even in normal retinas, NOS is expressed in only a small population of cells, and high numbers of bridging fibers are not necessarily expected. The variable and small number of NOS-containing cells and fibers within the grafts may be explained in part also by the random organization of the transplants, which results from transplantation of pieces of embryonic retina. 12 13 However, it has been consistently difficult to demonstrate graft–host connections, irrespective of the transplantation method used. The number of connections found has been low also when transplanting photoreceptors alone or large sheets of properly laminated and oriented neuroretina, 2 3 4 5 6 7 8 9 suggesting that there may well be other factors involved than tissue orientation. After transplantation, a rapid loss of the outer layers of the rabbit host retina is observed in areas adjacent to the graft. Numerous dying cells are also found in the grafts during the first weeks after transplantation, mostly next to the host retina. 17 As a result of neuronal cell death, a gliotic response is normally seen, and factors associated with reactive glia have been identified that inhibit neurite outgrowth. 18 Glial cell activation is observed in the host rabbit retina and with time also in the grafts. 19 It is thus possible that a glia-associated factor(s) may, at least in part, influence negatively the formation of connections between graft and host retinas. 
In summary, the present study demonstrates that at least some graft–host contacts involve graft cells capable of synthesizing a neuronal messenger, disclosing the functional potential of these connections. 
 
Figure 1.
 
(A) Bright field micrograph (hematoxylin–eosin) showing a transplant (T) corresponding to PN 20 (36 days after transplantation). Cells belonging to inner retinal layers (IR) are located between photoreceptor cells [ONL(t)], which are organized in rosettes (R). The host outer nuclear layer and part of the host inner nuclear layer[ INL(h)] have degenerated. (B) Fluorescence micrograph showing the distribution of NOS immunoreactivity in normal adult rabbit retina. Labeled cells (small arrows) are seen in the proximal inner nuclear layer (INL) and their processes in the inner plexiform layer (IPL) (small arrowheads). (C) Distribution of NOS immunoreactivity in a retinal transplant corresponding to PN 12 (28 days after transplantation). A few cell rows are present in the host outer nuclear layer at the edges of the bleb created by the graft. Immunoreactive fibers in the transplant run parrallel to the graft-host border (arrowheads). Scale bars, 30 μm.
Figure 1.
 
(A) Bright field micrograph (hematoxylin–eosin) showing a transplant (T) corresponding to PN 20 (36 days after transplantation). Cells belonging to inner retinal layers (IR) are located between photoreceptor cells [ONL(t)], which are organized in rosettes (R). The host outer nuclear layer and part of the host inner nuclear layer[ INL(h)] have degenerated. (B) Fluorescence micrograph showing the distribution of NOS immunoreactivity in normal adult rabbit retina. Labeled cells (small arrows) are seen in the proximal inner nuclear layer (INL) and their processes in the inner plexiform layer (IPL) (small arrowheads). (C) Distribution of NOS immunoreactivity in a retinal transplant corresponding to PN 12 (28 days after transplantation). A few cell rows are present in the host outer nuclear layer at the edges of the bleb created by the graft. Immunoreactive fibers in the transplant run parrallel to the graft-host border (arrowheads). Scale bars, 30 μm.
Figure 2.
 
(A, B) Fluorescence micrographs showing extension of NOS-immunoreactive fibers from the transplant (T) to the host retina (H). The host retina exhibits immunoreactive cells located within the inner plexiform layer [IPL(h)] and ganglion cell layer [GCL(h)] (arrows), and an immunoreactive plexus (arrowheads) in the inner plexiform layer. (A) PN 12 rabbit retinal transplant (28 days after transplantation) with a rosette (R). A long immunoreactive process (open arrowhead) is seen to cross over from the transplant to the host inner plexiform layer. (B) PN 20 retinal transplant (36 days after transplantation) showing a short process (open arrowhead) connecting an immunoreactive plexus in the transplant (large arrowheads) to the immunoreactive plexus in the host inner plexiform layer (small arrowheads). Scale bars, 30 μm.
Figure 2.
 
(A, B) Fluorescence micrographs showing extension of NOS-immunoreactive fibers from the transplant (T) to the host retina (H). The host retina exhibits immunoreactive cells located within the inner plexiform layer [IPL(h)] and ganglion cell layer [GCL(h)] (arrows), and an immunoreactive plexus (arrowheads) in the inner plexiform layer. (A) PN 12 rabbit retinal transplant (28 days after transplantation) with a rosette (R). A long immunoreactive process (open arrowhead) is seen to cross over from the transplant to the host inner plexiform layer. (B) PN 20 retinal transplant (36 days after transplantation) showing a short process (open arrowhead) connecting an immunoreactive plexus in the transplant (large arrowheads) to the immunoreactive plexus in the host inner plexiform layer (small arrowheads). Scale bars, 30 μm.
Figure 3.
 
(A, B) Fluorescence micrographs showing NOS-immunoreactive cells connecting the transplant (T) to the host retina (H). (A) Specimen taken 61 days after transplantation showing several stained processes (large arrowheads) in a transplant, corresponding to PN 45. One of the processes (open arrowhead) belonging to a NOS-immunolabeled cell located in the transplant (arrow) is seen to project toward the immunoreactive plexus in the host inner plexiform layer [IPL(h)] (small arrowheads). A stained cell body is also seen in the host inner nuclear layer [INL(h)] (small arrow), next to the host inner plexiform layer. (B) PN 90 retinal transplant (106 days after transplantation) showing a strongly labeled cell body (large arrow) connecting the inner plexiform layer of the host (small arrowheads) to an immunoreactive plexus in the transplant (large arrowheads). An immunoreactive cell is seen in the inner nuclear layer of the host (small arrow). Scale bars, 30μ m.
Figure 3.
 
(A, B) Fluorescence micrographs showing NOS-immunoreactive cells connecting the transplant (T) to the host retina (H). (A) Specimen taken 61 days after transplantation showing several stained processes (large arrowheads) in a transplant, corresponding to PN 45. One of the processes (open arrowhead) belonging to a NOS-immunolabeled cell located in the transplant (arrow) is seen to project toward the immunoreactive plexus in the host inner plexiform layer [IPL(h)] (small arrowheads). A stained cell body is also seen in the host inner nuclear layer [INL(h)] (small arrow), next to the host inner plexiform layer. (B) PN 90 retinal transplant (106 days after transplantation) showing a strongly labeled cell body (large arrow) connecting the inner plexiform layer of the host (small arrowheads) to an immunoreactive plexus in the transplant (large arrowheads). An immunoreactive cell is seen in the inner nuclear layer of the host (small arrow). Scale bars, 30μ m.
The authors thank Kennerth Wilke, Karin Arnér, and Katarzyna Said for excellent technical assistance. 
Sharma RK, Bergström A, Ehinger B. Retinal cell transplants. Prog Retinal Eye Res. 1995;15:197–230. [CrossRef]
Gouras P, Du J, Kjeldbye H, Kwun R, Lopez R, Zack DJ. Transplanted photoreceptors identified in dystrophic mouse retina by a transgenic reporter gene. Invest Ophthalmol Vis Sci. 1991;32:3167–3174. [PubMed]
Silverman MS, Ogilvie JM, Lett J, et al. Photoreceptor transplantation: potential for recovery of visual function. Christen Y Doly M Droy-Lefaix MT eds. Rétine, vieillissement et transplantation. 1994;43–59. Elsevier Paris.
Gouras P, Du J, Kjeldbye H, Yamamoto S, Zack DJ. Long-term photoreceptor transplants in dystrophic and normal mouse retina. Invest Ophthalmol Vis Sci. 1994;35:3145–3153. [PubMed]
Ehinger B, Bergström A, Seiler M, et al. Ultrastructure of human retinal cell transplants with long survival times in rats. Exp Eye Res. 1991;53:447–460. [CrossRef] [PubMed]
Aramant RB, Seiler MJ. Fiber and synaptic connections between embryonic retinal transplants and host retina. Exp Neurol. 1995;133:244–255. [CrossRef] [PubMed]
Seiler MJ, Aramant RB. Transplantation of embryonic retinal donor cells labelled with BrdU or carrying a genetic marker to adult retina. Exp Brain Res. 1995;105:59–66. [PubMed]
Seiler MJ, Aramant RB. Intact sheets of fetal retina transplanted to restore damaged rat retinas. Invest Ophthalmol Vis Sci. 1998;39:2121–2131. [PubMed]
Ghosh F, Bruun A, Ehinger B. Graft-host connections in long-term full-thickness embryonic rabbit retinal transplants. Invest Ophthalmol Vis Sci. 1999;40:126–132. [PubMed]
Koistinaho J, Sagar SM. NADPH-diaphorase-reactive neurones in the retina. Prog Retinal Eye Res. 1995;15:69–87. [CrossRef]
Perez MTR, Larsson B, Alm P, Andersson K-E, Ehinger B. Localisation of neuronal nitric oxide synthase immunoreactivity in rat and rabbit retinas. Exp Brain Res. 1995;104:207–217. [PubMed]
Sharma RK, Perez MTR, Ehinger B. Immunocytochemical localisation of neuronal nitric oxide synthase in developing and transplanted rabbit retinas. Histochem Cell Biol. 1997;107:449–458. [CrossRef] [PubMed]
Bergström A, Ehinger B, Wilke K, et al. Transplantation of embryonic retina to the subretinal space in rabbits. Exp Eye Res. 1992;55:29–37. [PubMed]
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Figure 1.
 
(A) Bright field micrograph (hematoxylin–eosin) showing a transplant (T) corresponding to PN 20 (36 days after transplantation). Cells belonging to inner retinal layers (IR) are located between photoreceptor cells [ONL(t)], which are organized in rosettes (R). The host outer nuclear layer and part of the host inner nuclear layer[ INL(h)] have degenerated. (B) Fluorescence micrograph showing the distribution of NOS immunoreactivity in normal adult rabbit retina. Labeled cells (small arrows) are seen in the proximal inner nuclear layer (INL) and their processes in the inner plexiform layer (IPL) (small arrowheads). (C) Distribution of NOS immunoreactivity in a retinal transplant corresponding to PN 12 (28 days after transplantation). A few cell rows are present in the host outer nuclear layer at the edges of the bleb created by the graft. Immunoreactive fibers in the transplant run parrallel to the graft-host border (arrowheads). Scale bars, 30 μm.
Figure 1.
 
(A) Bright field micrograph (hematoxylin–eosin) showing a transplant (T) corresponding to PN 20 (36 days after transplantation). Cells belonging to inner retinal layers (IR) are located between photoreceptor cells [ONL(t)], which are organized in rosettes (R). The host outer nuclear layer and part of the host inner nuclear layer[ INL(h)] have degenerated. (B) Fluorescence micrograph showing the distribution of NOS immunoreactivity in normal adult rabbit retina. Labeled cells (small arrows) are seen in the proximal inner nuclear layer (INL) and their processes in the inner plexiform layer (IPL) (small arrowheads). (C) Distribution of NOS immunoreactivity in a retinal transplant corresponding to PN 12 (28 days after transplantation). A few cell rows are present in the host outer nuclear layer at the edges of the bleb created by the graft. Immunoreactive fibers in the transplant run parrallel to the graft-host border (arrowheads). Scale bars, 30 μm.
Figure 2.
 
(A, B) Fluorescence micrographs showing extension of NOS-immunoreactive fibers from the transplant (T) to the host retina (H). The host retina exhibits immunoreactive cells located within the inner plexiform layer [IPL(h)] and ganglion cell layer [GCL(h)] (arrows), and an immunoreactive plexus (arrowheads) in the inner plexiform layer. (A) PN 12 rabbit retinal transplant (28 days after transplantation) with a rosette (R). A long immunoreactive process (open arrowhead) is seen to cross over from the transplant to the host inner plexiform layer. (B) PN 20 retinal transplant (36 days after transplantation) showing a short process (open arrowhead) connecting an immunoreactive plexus in the transplant (large arrowheads) to the immunoreactive plexus in the host inner plexiform layer (small arrowheads). Scale bars, 30 μm.
Figure 2.
 
(A, B) Fluorescence micrographs showing extension of NOS-immunoreactive fibers from the transplant (T) to the host retina (H). The host retina exhibits immunoreactive cells located within the inner plexiform layer [IPL(h)] and ganglion cell layer [GCL(h)] (arrows), and an immunoreactive plexus (arrowheads) in the inner plexiform layer. (A) PN 12 rabbit retinal transplant (28 days after transplantation) with a rosette (R). A long immunoreactive process (open arrowhead) is seen to cross over from the transplant to the host inner plexiform layer. (B) PN 20 retinal transplant (36 days after transplantation) showing a short process (open arrowhead) connecting an immunoreactive plexus in the transplant (large arrowheads) to the immunoreactive plexus in the host inner plexiform layer (small arrowheads). Scale bars, 30 μm.
Figure 3.
 
(A, B) Fluorescence micrographs showing NOS-immunoreactive cells connecting the transplant (T) to the host retina (H). (A) Specimen taken 61 days after transplantation showing several stained processes (large arrowheads) in a transplant, corresponding to PN 45. One of the processes (open arrowhead) belonging to a NOS-immunolabeled cell located in the transplant (arrow) is seen to project toward the immunoreactive plexus in the host inner plexiform layer [IPL(h)] (small arrowheads). A stained cell body is also seen in the host inner nuclear layer [INL(h)] (small arrow), next to the host inner plexiform layer. (B) PN 90 retinal transplant (106 days after transplantation) showing a strongly labeled cell body (large arrow) connecting the inner plexiform layer of the host (small arrowheads) to an immunoreactive plexus in the transplant (large arrowheads). An immunoreactive cell is seen in the inner nuclear layer of the host (small arrow). Scale bars, 30μ m.
Figure 3.
 
(A, B) Fluorescence micrographs showing NOS-immunoreactive cells connecting the transplant (T) to the host retina (H). (A) Specimen taken 61 days after transplantation showing several stained processes (large arrowheads) in a transplant, corresponding to PN 45. One of the processes (open arrowhead) belonging to a NOS-immunolabeled cell located in the transplant (arrow) is seen to project toward the immunoreactive plexus in the host inner plexiform layer [IPL(h)] (small arrowheads). A stained cell body is also seen in the host inner nuclear layer [INL(h)] (small arrow), next to the host inner plexiform layer. (B) PN 90 retinal transplant (106 days after transplantation) showing a strongly labeled cell body (large arrow) connecting the inner plexiform layer of the host (small arrowheads) to an immunoreactive plexus in the transplant (large arrowheads). An immunoreactive cell is seen in the inner nuclear layer of the host (small arrow). Scale bars, 30μ m.
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