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Experimental transplantation of retinal and iris pigment epithelial cells into the subretinal space
Linköping University, Department of Neuroscience and Locomotion, Ophthalmology. Linköping University, Faculty of Health Sciences.
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A dysfunction of the retinal pigment epithelium (RPE) is the main cause for the development of age-related macular degeneration (ARMD) and visual loss in elderly people. For about 10 years experimental and clinical attempts to transplant RPE cells have been performed. The aims of this study were to elucidate the long term results of RPE allografts, to develop an atraumatic transplantation technique, and to explore the cellular response to RPE allografts, melanin granules, and autologous IPE cells.

Surgery was performed on rabbits with a follow-up period of up to six months. After a pars plan vitrectomy, a subretinal bleb was created into which a suspension of RPE/IPE donor cells or melanin granules was injected. Pigmented RPE donor cells or preparations of melanin granules were implanted subretinally in albino rabbits. Pigmented rabbits were used in IPE transplantation. The eyes were monitored with ophthalmoscopy, fundus photography, light microscopy, electron microscopy and immunohistochemistry.

Transplantation of suspensions of fresh pigmented RPE cells to the subretinal space in rabbits is feasible and induces virtually no complications when an atraumatic surgical procedure is used. The allograft forms a monolayer in conjunction with the native RPE and persists almost intact up to three months. At six months after transplantation, there was a cellular response exhibiting multilayers of cells, such as RPE and macrophages. Damage to adjacent photoreceptors in combination with melanin granules in the subretinal space indicates graft failure. No infiltration of lymphocytes was seen. Whether the cellular response was due to immunological or non-immunological mechanisms could not be determined from this experiment.

Cyclosporine (CsA) could not prevent disintegration of the RPE transplant and graft failure. CsA was not capable of promoting graft survival as compared to the controls. The transplant seems to be disrupted either by immunological mechanisms that are not inhibited by CsA, or by non-immunologic events.

Implantation of melanin granules to the subretinal space of albino rabbits induces a considerable phagocytic cellular response involving the host’s RPE, macrophages and glial cells. The migration of pigment-laden cells into the neural retina was frequently associated with focal photoreceptor damage. The cellular response was identical to that ensuing RPE cell transplantation. These findings support the concept that non-immunological events have a considerable influence on the outcome.

In order to evaluate the impact of non-immunological mechanisms, a technique of transplanting fresh autologous IPE cells to the subretinal space of the same eye was developed. Grafted IPE cells were seen to survive for six months. There was a remodeling of the compound cellular layers in the subretinal space over time where grafted IPE cells joined the native RPE cells. The cellular response that developed exhibited macrophages, but no lymphocytes, and was in this respect similar to that observed following RPE transplantation.

In RPE allografts, the photoreceptors appeared normal on light microscopy at three months, but at six months, the photoreceptors overlying the transplants generally exhibited pathological changes. In autologous IPE grafts, on the other hand, the photoreceptors displayed normal outer segment length and outer nuclear layer on top of grafted IPE cells. Focally, multilayers of both grafted IPE and RPE cells, together with macrophages, induce damage to adjacent photoreceptors as observed at 6 months. Cellular multilayers in the subretinal space, irrespective of genesis, are likely to have adverse effects on photoreceptors. The experiments using autologous IPE grafts show that non-immunogenic mechanisms have a decisive impact on the outcome of the transplant in the subretinal space.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2000. , 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 629
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28029Local ID: 12789ISBN: 91-7219-586-X (print)OAI: oai:DiVA.org:liu-28029DiVA: diva2:248580
Public defence
2000-05-19, Administrationsbyggnadens Aula, Universitetssjukhuset, Linköping, 09:15 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-08-07Bibliographically approved
List of papers
1. Long-term outcome of RPE allografts to the subretinal space of rabbits
Open this publication in new window or tab >>Long-term outcome of RPE allografts to the subretinal space of rabbits
1999 (English)In: Acta Ophthalmologica Scandinavica, ISSN 1395-3907, E-ISSN 1600-0420, Vol. 77, no 3, 247-254 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: To determine the long-term RPE allograft survival in the subretinal space using suspensions of RPE cells and atraurnatic transplantation surgery.

Methods: Nineteen albino rabbits were transplanted with suspensions of pigmented RPE cells from brown rabbits. Following pars plana vitrectomy, the RPE cell suspension was injected through a small retinotomy using a glass micropipette into the subretinal space under microscopic control. No immunosuppression was used. The eyes were monitored by biomicroscopy, color fundus photography, and fluorescein angiography. Rabbits were sacrificed at 1, 3 and 6 months, respectively, and the eyes processed for light and electron microscopy, using monoclonal antibodies for identifying macrophages.

Results: Transplanted RPE cells were present in the subretinal space in all eyes at 6 months. There was no fluorescein leakage. Generally, the RPE allograft formed a monolayer, but focal fragmentation and disruption with dispersion of melanin pigment occurred. Foci of multilayers of cells in the subretinal space, containing large macrophages, were associated with adjacent photoreceptor damage. There was no infiltration of lymphocytes but macrophages and glial cells were contiguous to the transplant. Cells harboring intracytoplasmatic melanin pigment were observed in the neural retina.

Conclusion: Transplantation of RPE cell suspensions to the subretinal space generally forms a monolayer that persists at 6 months, However, in areas of multilayers of RPE cells and macrophages, graft failure occurs in combination with adjacent photoreceptor damage. Graft failure is not associated with the infiltration of lymphocytes, but other mechanisms seem to occur.

Keyword
retina, RPE transplantation, allograft, subretinal space, graft survival, graft failure, macrophages
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27595 (URN)10.1034/j.1600-0420.1999.770301.x (DOI)12325 (Local ID)12325 (Archive number)12325 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-08-07Bibliographically approved
2. Cyclosporine treatment of RPE allografts in the rabbit subretinal space
Open this publication in new window or tab >>Cyclosporine treatment of RPE allografts in the rabbit subretinal space
2000 (English)In: Acta Ophthalmologica Scandinavica, ISSN 1395-3907, E-ISSN 1600-0420, Vol. 78, no 2, 122-129 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: To determine the effects of systemic cyclosporine A (CsA) on the survival of retinal pigment epithelial (RPE) allografts in the subretinal space in an animal model using atraumatic transplantation surgery.

Methods: Following pars plana vitrectomy, an RPE cell suspension from brown rabbits was injected with a glass micropipette into the subretinal space of 39 albino rabbits. For immunosuppression, 22 rabbits were given an injection of CsA, 20 mg daily intramuscularly, 17 rabbits with RPE grafts were controls. The grafts were monitored by biomicroscopy, color fundus photography, and fluorescein angiography. Rabbits were sacrificed at 1, 3 and 6 months, respectively, and the eyes processed for light and electron microscopy including immunohistochemistry.

Results: After three months, the transplanted RPE cells, in both the CsA group and the controls, formed a monolayer in the subretinal space. Although a few macrophages were encountered, there was no massive cellular infiltration and the photoreceptor layer was well preserved. After six months, however, there was a disruption of grafted RPE cells in both groups, characterized by dispersion of melanin pigment in the subretinal space, and invasion of macrophages with focal photoreceptor damage but no infiltration of lymphocytes in the retina or choroid. No significant differences between the CsA treated and the control eyes were discernible.

Conclusion: Although the subretinal space has been considered an immunologically privileged site, we found that the survival of RPE allografts was limited. CsA did not prevent RPE allograft destruction in the subretinal space. The transplant seems to be disrupted either by immunological mechanisms that are not inhibited by CsA, or by nonimmunologic events.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27598 (URN)10.1034/j.1600-0420.2000.078002122.x (DOI)12328 (Local ID)12328 (Archive number)12328 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-08-07Bibliographically approved
3. Cellular migration into neural retina following implantation of melanin granules in the subretinal space
Open this publication in new window or tab >>Cellular migration into neural retina following implantation of melanin granules in the subretinal space
2000 (English)In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 238, no 8, 682-689 p.Article in journal (Refereed) Published
Abstract [en]

Background: In some retinal diseases and following transplantation of retinal pigment epithelium (RPE), melanin granules are liberated to the subretinal space. Our aim was to investigate the cellular response to implanted extracellular melanin.

Methods: After pars plana vitrectomy, 17 albino rabbits received a suspension of melanin granules in the subretinal space. Postoperative examination included ophthalmoscopy, color fundus photography, histology using monoclonal antibodies identifying RPE cells (AE1/3), macrophages (RAM 11), B-lymphocytes (CD20) and T-lymphocytes (CD45), and electron microscopy. The follow-up time was 2 weeks, 4 weeks and 6 months.

Results: On fundus photographs, the layer of melanin showed focal attenuation with lighter areas at 6 months. Melanin granules were phagocytosed by RPE cells and macrophages at 2 weeks, as identified by monoclonal antibodies. In areas where an abundance of melanin was present, multilayers of macrophages were seen associated with considerable photoreceptor damage. Pigment-laden cells invaded the neural retina. The cellular infiltration of the retina was focal, and when it involved the outer nuclear layer the photoreceptor damage was severe. Electron microscopy demonstrated the presence of melanosomes intracellularly in Müller glia. The process of phagocytosis and removal of melanin granules from the subretinal space was slow and not completed at 6 months.

Conclusion: Our experiments show that implantation of melanin granules in the subretinal space of albino rabbits may induce a considerable phagocytic cellular response featuring the host’s RPE, macrophages and glial cells. The migration of pigment-laden cells into the neural retina was associated with focal photoreceptor damage.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27593 (URN)10.1007/s004170000131 (DOI)12323 (Local ID)12323 (Archive number)12323 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-08-07Bibliographically approved
4. Transplantation of Autologous Iris Pigment Epithelial Cells to the Subretinal Space: I. Morphological Features
Open this publication in new window or tab >>Transplantation of Autologous Iris Pigment Epithelial Cells to the Subretinal Space: I. Morphological Features
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Purpose: To investigate the cellular morphology in the subretinal space following transplantation of iris pigment epithelial (IPE) cells from the same eye.

Methods: Following an iridectomy, fresh IPE cells were prepared. After pars plana vitrectomy, a suspension of autologous IPE cells was injected into the subretinal space in 37 rabbits. The grafts were monitored by ophthalmoscopy and calor fundus photography. Rabbits were sacrificed at 1, 2, 3 and 6 months, respectively, and the eyes examined with light and electron microscopy.

Results: The grafted area retained the same configuration over 6 months but then appeared less pigmented. At 1-3 months, the IPE formed one or more contiguous layers on top of native RPE. At 6 months, cells compatible with grafted IPE were present in the subretinal space forming mono-layer like chains integrating with the native RPE. Depigmented cells of presumed IPE origin were seen. lt was commonly observed that the apical portion of RPE cells disclosed abundant melanin granules. With time the grafted cells appeared to decrease in number but focal clusters of IPE cells and large macrophages were present.

Conclusion: Transplanted IPE cells survived for up to 6 months in the subretinal space. Our observations suggest a scenario of remodeling of the cellular layers in the subretinal space over time where grafted IPE cells formed a compound layer with the native RPE. Transplantation of autologous IPE cells may have a potential as a treatment modality in selected cases of age-related macular degeneration with impending degeneration of RPE and choriocapillaris.

Keyword
Retina, IPE cells transplantation, Autologous transplantation, Subretinal space, Blood-retinal barrier (BRB), Macrophages, Melanin pigment, Photoreceptor damage
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79525 (URN)
Available from: 2012-08-07 Created: 2012-08-07 Last updated: 2012-08-07Bibliographically approved
5. Transplantation of Autologous Iris Pigment Epithelial Cells To the Subretinal Space: II. Photoreceptor Survival and Consequences of Cellular Multilayers
Open this publication in new window or tab >>Transplantation of Autologous Iris Pigment Epithelial Cells To the Subretinal Space: II. Photoreceptor Survival and Consequences of Cellular Multilayers
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Purpose: To study the effects of transplanted iris pigment epithelial (IPE) cells in the subretinal space on the survival of adjacent photoreceptors.

Methods: An upper iridectomy was made on the right eye of 37 pigmented rabbits. Suspensions of autologous IPE cells were prepared and injected into the subretinal space of the same eye. Follow- up examinations was performed using ophthalmoscopy and calor fundus photography. At 1, 2, 3 and 6 months, respectively, the rabbits were sacrificed and the eyes examined with light and electron microscopy.

Results: On histological examination, the photoreceptor cells were well preserved in grafted areas at 1-3 months. At 6 months, also, the photoreceptors generally disclosed a normal nuclear layer and long outer segments when overlying areas with single cells or clusters of IPE. However, multilayers of transplanted I PE, native RPE cells and macrophages in the subretinal space were frequently associated with photoreceptor damage and nuclear drop out from the outer retinal layer.

Conclusion: In this experimental model, the photoreceptors generally survive adjacent to grafted IPE cells for 6 months. Multilayers of subretinal cells are likely to induce adverse effects on adjacent photoreceptors which is a new finding that requires further investigation.

Keyword
Retina, IPE transplantation, Autologous, Subretinal space, Melanin pigment distribution, Macrophages, Photoreceptor damage
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79528 (URN)
Available from: 2012-08-07 Created: 2012-08-07 Last updated: 2012-08-07Bibliographically approved

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