Morphology and hemodynamics during vascular regeneration in critically ischemic murine skin studied by intravital microscopy techniques.

BACKGROUND: With the understanding of angiogenesis and arteriogenesis, new theories about the orchestration of these processes have emerged. The aim of this study was to develop an in vivo model that enables visualization of vascular regenerating mechanisms by intravital microscopy techniques in collateral arteriolar flap vascularity. METHODS: A dorsal skin flap (15 × 30 mm) was created in mice and fixed into a skinfold chamber to allow for assessment of morphology and microhemodynamics by intravital fluorescence microscopy (IVFM). Laser scanning confocal microscopy (LSCM) was utilized for three-dimensional reconstruction of the microvascular architecture. RESULTS: Flap tpO(2) was 5.3 ± 0.9 versus 30.5 ± 1.2 mm Hg in controls (p < 0.01). The collateral arterioles in the flap tissue were dilated (29.4 ± 5.3 µm; p < 0.01 vs. controls) and lengthened in a tortuous manner (tortuosity index 1.00 on day 1 vs. 1.35± 0.05 on day 12; p < 0.01). Functional capillary density was increased from 121.00 ± 25 to 170 ± 30 cm/cm(2) (day 12; p < 0.01) as a result of angiogenesis. Morphological evidence of angiogenesis on capillary level and vascular remodeling on arteriolar level could be demonstrated by IVFM and LSCM. CONCLUSIONS: Present intravital microscopy techniques offer unique opportunities to study structural changes and hemodynamic effects of vascular regeneration in this extended axial pattern flap model.

What Can Pharmacological Models of Retinal Degeneration Tell Us?

Animal models with pharmacologically induced retinal degeneration including sodium iodate (NaIO3) and N-methyl-N-nitrosourea (MNU) have been extensively used in ophthalmic research to investigate retinal degeneration. NaIO3 induces degeneration of the retinal pigment epithelium (RPE) followed by photoreceptor (PRC) cell death, mimicking features of age-related macular degeneration. In contrast, MNU leads to rapid destruction of the PRCs only, enabling the use of the MNU model to investigate degeneration induced in retinitis pigmentosa. It has been shown that multiple cell death pathways are involved in the cell-specific effects of the toxins. Necrosis has been identified as the cause of the NaIO3-induced RPE loss. PRC degeneration in the described models is mainly induced by programmed cell death, indicated by the upregulation of conventional apoptosis initiator and effector caspases. However, recent research points to the additional involvement of caspase-independent processes as endoplasmic reticulum stress and calpain activation. Since there is still a substantial amount of contradictory hypotheses concerning triggers of cell death, the use of pharmacological models is controversial. Thereby, the advantages of such models like the application reaching across species and strains as well as modulation of onset and severity of damage are not exploited to a full extent. Thus, the present review aims to give more insight into the involved cell death pathways and discusses recent findings in the most widely used retinal degeneration models. It might facilitate further studies aiming to develop putative therapeutic approaches for retinal degenerative diseases including combinatory treatment with cell death inhibitors and cell transplantation therapy.

An improved MTT assay using the electron-coupling agent menadione.

A modification of the MTT based tetrazolium colorimetric assay is described. Using the electron-coupling agent menadione formazan formation by murine splenocytes and P-815 cells was significantly increased whereas dye reduction by macrophages was hardly influenced. These observations suggest that it should be possible to improve the tetrazolium based cytotoxicity assays of murine macrophages against cells of the syngeneic tumour cell line P-815.

Age- and disease-related changes of calcium channel-mediated currents in human Müller glial cells.

PURPOSE: To determine whether the expression of voltage-gated Ca2+ channels in human Müller glial cells changes during normal aging and in cells from patients with proliferative vitreoretinopathy (PVR). METHODS: Müller cells were enzymatically isolated from retinas of healthy donors and from excised retinal pieces of patients with PVR, and the whole-cell, voltage-clamp technique was used to characterize the current densities of transient, low-voltage-activated calcium channels and of sustained. high-voltage-activated calcium channels, respectively. To obtain maximal currents through both channel types, Na+ ions were used as the charge carrier. RESULTS: During normal aging, Müller cells developed a hypertrophy, as indicated by an increase of the cell membrane capacitance. The mean membrane capacitance of cells from aged donors (> or = 60 years old) was elevated by 25% compared with cells from younger donors. The hypertrophy was not accompanied by a changed density of low-voltage-activated currents, whereas the density of the high-voltage-activated currents was enhanced by 76%. The density of the high-voltage-activated currents increased in correlation with the increase of the cell membrane capacitance and with the age of the donors. In the case of PVR, Müller cells displayed a strong hypertrophy accompanied by a downregulation of both current types by approximately 65%. CONCLUSIONS: Both normal aging and PVR cause a gliotic reactivity of human Müller cells, as indicated by their hypertrophy. The type of reactivity, however, differs between the two conditions. Normal aging is accompanied by an increased expression of voltage-gated Ca2+ channels, whereas in PVR Ca2+ channel expression is decreased.

Upregulation of P2X(7) receptor currents in Müller glial cells during proliferative vitreoretinopathy.

PURPOSE: Müller glial cells from the human retina express purinergic P2X(7) receptors. Because extracellular adenosine triphosphate (ATP) is assumed to be a mediator of the induction or maintenance of gliosis, this study was undertaken to determine whether the expression of these receptors is different in human Müller cells obtained from retinas of healthy donors and of patients with choroidal melanoma and proliferative vitreoretinopathy (PVR). METHODS: Human Müller cells were enzymatically isolated from donor retinas, and whole-cell patch-clamp recordings were made to characterize the density of the P2X(7) currents and the activation of currents through Ca2+-activated K+ channels of big conductance (I:(BK)) that reflects the increase of the intracellular Ca2+ concentration. RESULTS: Stimulation by external ATP or by benzoylbenzoyl ATP (BzATP) evoked both release of Ca2+ from thapsigargin-sensitive intracellular stores and opening of Ca2+ -permeable P2X(7) channels. These responses caused transient and sustained increases in I:(BK). In Müller cells from patients with PVR, the mean density of the BzATP-evoked cation currents was significantly greater compared with cells from healthy donors. As a consequence, such cells displayed an enlarged I:(BK) during application of purinergic agonists. ATP and BzATP increased the DNA synthesis rate of cultured cells. This effect could be reversed by blocking the I:(BK). CONCLUSIONS: The increased density of P2X(7) receptor channels may permit a higher level of entry of extracellular Ca2+ into cells from patients with PVR. Enhanced Ca2+ entry and the subsequent stronger activation of I:(BK) may contribute to the induction or maintenance of proliferative activity in gliotic Müller cells during PVR.

Human Müller glial cells: altered potassium channel activity in proliferative vitreoretinopathy.

PURPOSE: To determine differences of K+ channel activity between Müller glial cells obtained from retinas of healthy human donors and of patients with retinal detachment and proliferative vitreoretinopathy. METHODS: Müller cells were enzymatically isolated from retinas of healthy donors and from excised retinal pieces of patients. The whole-cell and the cell-attached configurations of the patch-clamp technique were used to characterize the current densities of different K+ channel types and the activity of single Ca2+ -activated K+ channels of big conductance (BK). RESULTS: Cells from patients displayed a less negative mean membrane potential (-52.8 mV) than cells from healthy donors (-80.6 mV). However, the membrane potentials in cells from patients scattered largely between -6 and -99 mV. The inwardly rectifying K+ permeability in cells from patients was strongly reduced (0.3 pA/pF) when compared with cells from healthy donors (6.0 pA/pF). At the resting membrane potential, single BK channels displayed a higher mean activity (open probability, Po, and channel current amplitude) in cells from patients (Po, 0.30) than in cells from healthy donors (Po: 0.03). The variations of BK current amplitudes were correlated with the variations of the membrane potential. CONCLUSIONS: The dominant expression of inwardly rectifying channels in cells from healthy donors is thought to support important glial cell functions such as the spatial buffering of extracellular K+. The downregulation of these channels and the less negative mean membrane potential in cells from patients should impair spatial buffering currents and neurotransmitter clearance. The increased activity of BK channels may support the proliferative activity of gliotic cells via feedback regulation of Ca2+ entry and membrane potential.

Effective chemokines and cytokines in the rejection of human retinal pigment epithelium (RPE) cell grafts.

OBJECTIVE: In the rejection of transplanted retinal pigment epithelium (RPE) cells, an activation of allografts is probably the pivotal point for long-term success. The detailed immunological interactions involved in the rejection after RPE transplantation are still unknown. The aim of this study is to evaluate the interactions of pro-inflammatory cytokines and chemokines in this activation process in vitro. METHODS: Human RPE cells (2 x 10(5)/ml) were therefore activated through a pre-treatment with different concentrations of interferon (IFN)-gamma (100 or 1000 U/ml), tumour necrosis factor (TNF)-alpha (1 or 10 ng/ml) or combinations of both, or employed in a nonactivated form. Afterwards, the RPE cells were tested by enzyme-linked immunosorbant assay (ELISA) and ribonuclease protection assays (RPA) for the secretion and mRNA content of the different chemokines (RANTES, MCP-1 and IL-8) and cytokines (IL-6) at various time points up to 48 h. MAIN FINDINGS: HRPE cells secrete the investigated cytokines in response to pro-inflammatory activation. This could be demonstrated at both the mRNA (RPA) and the protein levels (ELISA). The secretion was time and dose dependent, and significantly upregulated in comparison to that observed with nonactivated cells. CONCLUSIONS: This study demonstrates that RPE cells efficiently secrete such cytokines as RANTES, MCP-1, IL-6, and IL-8, and have an accountable neutrophil and monocyte chemotactic activity. Thus, it could be indicated that the investigated cytokines play a central role in the activation cascade of RPE and in RPE rejection as well.

Modification of glutamine synthetase expression by mammalian Müller (glial) cells in retinal organ cultures.

One of the key enzymes in glial-neuronal transmitter recycling is glutamine synthetase (GS). In the retina, GS is exclusively expressed by glial (Müller) cells where it serves to convert neuron-released active transmitter substances (glutamate and GABA) into glutamine. Experiments on avian retinae have shown that GS expression is developmentally regulated by glucocorticoid hormones and, to a lesser extent, by a non-hormonal control mechanism(s). Much less is known about GS regulation in mammalian retinae, although either increases or decreases of GS immunoreactivity have been observed in Müller cells in different forms of retinal pathologies. We studied GS expression in postnatal rabbit retinae both in vivo and explanted as wholemounts in vitro, using immunocytochemistry and Western immunoblotting. GS expression was detectable in vivo from the fourth postnatal day, and increased rapidly within the first weeks of life. Levels were lower in vitro than in vivo by an order of magnitude, and could be significantly stimulated (> 60-110%) in vitro by application of hydrocortisone, conditioned medium from cultured retinal pigment epithelium and glutamate or ammonia, but not GABA. It is concluded that GS expression in mammalian Müller cells is dependent on systemic control by glucocorticoid hormones, as observed in birds, but environmental (activity-dependent) factors may play a more important role in mammals.

The local and systemic secretion of the pro-inflammatory cytokine interleukin-6 after transplantation of retinal pigment epithelium cells in a rabbit model.

The rejection of retinal pigment epithelium (RPE) allografts is one of the major problems for long-term success after retinal transplantation. However, the details of the immunological interactions in the subretinal space after transplantation are still unknown. The aim of our study was to investigate the role of IL-6 in the rejection process in the subretinal space and to use IL-6 monitoring for a possible early sign of rejection after transplantation of allogeneic RPE cells. For this we used a model of transplanting pigmented RPE cells, either activated in vitro with 1000 U/ml interferon-gamma (IFN-gamma) for 8 days or non-activated, into 30 albino rabbits. The IL-6 was investigated 3, 5, 7, 9 and 14 days after transplantation. Additionally, sham operated animals and the untreated eyes served as controls. At these time-points the animals were killed, the liquid in the vitreous cavity and serum was collected and the IL-6 present in these samples was quantified with an enzyme-linked immunosorbent assay. Under these conditions, IL-6 was detected in the liquid of the vitreous cavity and in the serum of all RPE-transplanted rabbits. In the group receiving activated RPE two cytokine peaks were measured, 3 and 7 days after transplantation in the vitreous cavity. In non-activated grafts, a maximum was detected on the 5th day after transplantation. Generally, the detected quantity of IL-6 depended on the host status and on the phase of rejection. No significant changes were seen in the sera from either group. Possibly, the host RPE cells are the main source of this interleukin in the transplantation area. The measuring of IL-6 in the rejection model suggests that it plays a role in the immune cascade in the subretinal space.

Changes in the mRNA expression of cytokines and chemokines by stimulated RPE cells in vitro.

PURPOSE: The transplantation of retinal pigment epithelial (RPE) cells is a possible therapy for degenerative diseases of the retina. However, the immune response and the subsequent rejection of the allografts are major problems in this field. We investigated the effect of pro-inflammatory factors on the cytokine and chemokine mRNA expression of human RPE cells during long-term observations in vitro. METHODS: Human RPE cells were cultured in the presence of tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml), interferon-gamma (IFN-gamma, 1000 U/ml) or with a combination of both up to 96 hours. Cells were harvested and total RNA was isolated. The changes in expression of mRNA coding for RANTES, the interleukines (IL)-6, 8, 10, 15, IFN-gamma, monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-beta1) during the stimulation were investigated using the ribonuclease protection assay. RESULTS: IL-10 and IFN-gamma mRNA were detected in neither unstimulated nor stimulated cells. Human RPE cells constitutively express the mRNA for IL-6, MCP-1, IL-8, IL-15, TGF-beta1 and, at very low levels, for RANTES. The TGF-beta1 mRNA expression was not influenced by either stimulation. The mRNA of the other factors was up-regulated for 24-48 h dependent on the stimulation. CONCLUSIONS: Human RPE cells are able to increase their mRNA expression for the detected cytokines in response to the pro-inflammatory factors which are detectable in the rejection process. These up-regulated cytokines themselves are known to be involved in several inflammatory and immunological processes, suggesting their role in the rejection of transplanted RPE allografts.

Na(+) currents through Ca(2+) channels in human retinal glial (Müller) cells.

PURPOSE: To detect the presence of voltage-gated Ca(2+) channels in the plasma membranes of freshly isolated Müller glial cells from the human retina and their modulation by GABA(B) receptor agonists. METHODS: Whole cell voltage-clamp recordings were made to study Ca( 2+), Ba(2+), and Na(+) currents through voltage-gated Ca(2+) channels. RESULTS: The vast majority of the investigated cells displayed no resolvable currents through Ca(2+) channels when Ca(2+) ions (2 mM) were present in the extracellular solution. Small-amplitude inwardly directed currents ( approximately 0.6 pA/pF) were detected when Ba(2+) ions (20 mM) were used as charge carrier. However, when Na(+) ions were used as charge carrier in divalent cation-free external solution, currents of large amplitudes ( approximately 7.5 pA/pF) through voltage-gated Ca(2+) channels were detected. Human Müller cells displayed currents through both transient, low voltage-activated Ca(2+) channels and long-lasting, high voltage-activated channels. The Na(+) fluxes through low voltage-activated Ca( 2+) channels were inhibited in a voltage-independent manner in the presence of GABA(B) receptor agonists. CONCLUSIONS: Human Müller glial cells express different kinds of voltage-gated Ca(2+) channels in their plasma membranes that can be activated only under certain physiological or pathophysiological conditions. The record of Na(+) fluxes in divalent cation-free solutions may be a technique to detect the presence of "hidden" voltage-gated Ca(2+) channels in Müller glial cells.

Immunosuppression by IL-10-transfected human retinal pigment epithelial cells in vitro.

PURPOSE: Retinal pigment epithelium (RPE) transplantation seems to be a possible therapy for restoring vision in the case of retinal degeneration. As there is a risk of allergic rejection, a gene-transfer of immunosuppressive cytokines into the graft may diminish this reaction. Therefore, we investigated the transfer of interleukin-10 (IL-10) into an immortalised human RPE cell line (hTERT-RPE1) and its effect on the proliferation of allogeneic immune competent cells. METHODS: The hTERT-RPE1 cells were transiently transfected with the cDNA of human IL-10 using a lipid-based transfection reagent. The expression of IL-10 mRNA was ana-lysed by reverse transcription-polymerase chain reaction and an enzyme-linked immunosorbent assay measured the secretion of the cytokine over 7 days. The effect of the secreted IL-10 on the proliferation of allogeneic T cells with and without homologous macrophages was investigated colorimetrically. To enhance this reaction, RPE cells were pre-activated with interferon-gamma (IFN-gamma). Anti-IL-10 antibodies were used in a neutralising assay. RESULTS: A transfection efficiency of 23.3 +/- 9.03% was achieved. IL-10 mRNA could only be shown in IL-10-transfected hTERT-RPE1 cells. The same was found for the level of cytokine, with a maximum on day 3 (10.34 +/- 0.09 ng/ml). A significant suppressive effect of the secreted IL-10 on T-cell proliferation was detectable on days 5 and 6. This effect could be significantly abolished with anti-IL-10 antibodies. CONCLUSIONS: The IL-10-producing hTERT-RPE1 cells had an immunosuppressive action on T-cell proliferation in vitro. A gene-transfer into RPE allografts before transplantation may be able to promote graft survival.

Immunohistochemical detection of substance P and vasoactive intestinal peptide fibres in the auricular lymph nodes of sensitized guinea pigs and mice.

The presence of substance P (SP) and vasoactive intestinal peptide (VIP) in auricular lymph nodes of normal and sensitized guinea pigs and mice has been investigated using polyclonal antibodies directed against these neurotransmitters and the unlabelled peroxidase-antiperoxidase procedure. Positive immunoreactivity could be observed only in draining lymph nodes of sensitized animals. SP-immunoreactivity was found in mice and guinea pigs but a VIP-positive reaction only in mice. The immunopositive sites were always associated with blood vessels. The present study suggests that the neurotransmitters SP and VIP are involved in mechanisms of sensitization taking place in secondary lymphoid organs.

In-vitro methods to decrease MHC class II-positive cells in retinal pigment epithelium cell grafts.

Rejection of RPE transplants may be caused by expression of MHC class II molecules on the graft. We wanted to determine if passage level, hyperoxia, low temperature, or magnetic separation had an influence on this expression or on several other physiological parameters of bovine RPE cells. For this, bovine RPE cells were cultured under normal conditions for several passages (1-5) or were incubated with a high level of oxygen (75%) for 24 h or at low temperature (24 degrees C) for seven days. Magnetic beads coated with monoclonal antibody against MHC class II antigens were used to separate the positive cells from the total cell population. The number of MHC class II-positive RPE cells was not affected by increased passage, oxygen concentration, or low temperature. Using magnetic beads, approximately 7.5% of the cells were separated from the total RPE population as MHC class II-positive cells. Hyperoxia initially increased the number of RPE cells to 178% that of the untreated controls, but the subsequent MTT test showed a decrease in proliferation to 95% of control levels. Similarly, low temperature increased cell number of 110% that of controls, but prolonged proliferation decreased to 76% of the controls. None of the experimental conditions had a significant influence on the viability of the cells. Culture conditions can be modified to increase the yield of RPE cells, and MHC class II-positive RPE cells can be detected and selectively separated from the total cell population, both of which may prove to be useful for RPE cell transplantation.

Minor influence of the immunosuppressive cytokines IL-10 and TGF-beta on the proliferation and apoptosis of human retinal pigment epithelial (RPE) cells in vitro.

Undesirable immune reactions such as uveitis or graft rejection after corneal and subretinal transplantations are serious inflammations in the eye. Minimizing this process by means of physiological suppressors, either through systemic or intraocular administration with or without gene therapy, is a future therapeutic possibility. In our study, we used different concentrations of transforming growth factor-beta (TGF-beta; 5, 10, and 50 ng/ml) and interleukin-10 (IL-10; 100, 200, and 500 U/ml), both known as modulators of the suppression process, to treat human retinal pigment epithelium (RPE) cells in vitro. The influence of both cytokines on the viability and proliferation of the RPE cells was measured. Furthermore, the secretion of typical markers of the apoptosis process, such as Fas, soluble Fas ligand, and bcl-2, was investigated. Our results show that the concentrations of TGF-beta and IL-10 used have only a slight influence on RPE cells. Cell proliferation under the influence of TGF-beta was significantly reduced, whereas more Fas protein could be found in the cell lysate of the IL-10 samples. In general, IL-10 seemed to have less effect on the physiology of RPE cells. The discussion of the therapeutic use of an immunosuppressive factor in the eye should therefore be focused more on this cytokine.

[The placing and the correcting of the position of central venous catheters with Seldinger's technique (author's transl)]. / Plazierung und Lagekorrektur von zentralen Venenkathetern mit der Seldinger-Spirale.

The Seldinger technique for placing and correcting the position of central venous catheters with a J-shaped guide wire has proved to be a valuable improvement on the technique available at present. The technique allows us to negotiate otherwise impassable "blocks" in the venous system and to correct problematic malpositioned catheters. The success rate in 135 cases of malpositioning was 81.5%. If in addition one had the help of X-ray scan, the catheters could be corrected in nearly every case. The Seldinger technique is particularly valuable in cases of tumours of the neck and upper mediastinum, in anticoagulant and thrombolytic therapy, in burns and for small children. No complications occurred.

[A new plastic-bag transfusion set for dilution of erythrocyte concentrates]. / Ein neues Plastikbeutel-Uberleitungssystem zur Verdünnung von Erythrozytenkonzentraten.

A new transfusion set which contains 100 ml of 0.9% sodium chloride solution to resuspend packed red blood cells is described. The advantages of the system are--the technique is simple and hygienic, it contains an optimal quantity of suspension solution, and there is no danger of air embolus occurring. The rheologic properties of the then diluted red cell concentrates are very good, and allow for a mass transfusion. This new system affords the wider use of packed red blood cells, and it will probably contribute to the abolition of the practically obsolete transfusion of whole blood even for surgical patients.

[Complications of fluorescence angiography of the retina. Symptoms, prophylaxis and therapy]. / Zwischenfälle bei der Fluoreszenzangiografie der Retina. Symptomatik, Prophylaxe und Therapie.

The pathomecanism of the fluorescein incident is described. The anaphylactic reaction is, in accordance with the "all-or-nothing law", independent of the dose. Antihistamines and corticosteroides should be given prophylactically. Severe reactions require anesthesiological treatment. Fluorescein angiography of the retina should be performed only if an anesthesiologist is available. Fluorescein incidents - anaphylactic or anaphylactoid reactions - are very rare (0.1-1.1%). It is estimated that in every 20 000 procedures only one incident endangering life occurs. Fluorescein angiography of the retina should be performed only when the indication is utterly justified and then only if it will provide a guide for treatment. If prophylactic and therapeutic measures can be taken immediately the risk of fluorescein angiography is acceptable.

Mechanisms of graft rejection in the transplantation of retinal pigment epithelial cells.

PURPOSE: The role of activated retinal pigment epithelium (RPE) cells was investigated in the rejection after subretinal transplantation. METHODS: RPE cells from 7 pigmented rabbits were separated and evaluated regarding their MHC class II expression as the sign of activation. The activation of the RPE cells was augmented with a treatment of 1,000 U/ml interferon gamma (IFN-gamma) for 8 days. These cells were then transplanted into 7 albino rabbits. As control, RPE transplantations without a pretreatment were performed in 7 albino rabbits. Six weeks after the transplantation, the transplanted eyes were enucleated and histology was performed. RESULTS: In culture, without IFN-gamma addition, 11.38 +/- 0.94% of the RPE cells presented MHC class II. After IFN-gamma treatment, this quantity increased to 78.26 +/- 1.46% of the RPE cells. These cells transplanted into the rabbits caused an obvious rejection in the transplantation area which was verified histologically. The control group presented a transplantation area without signs of rejection or inflammation. CONCLUSION: In culture, some of the adult RPE cells are activated. These cells may accelerate the rejection cascade after transplantation. An elimination of activated RPE cells from the transplant should be recommended before transplantation.

Immunological problems of transplantation into the subretinal space.

The objective of retinal transplantation is to substitute destroyed or degenerated retina to improve visual function. Photoreceptors and retinal pigment epithelium cells of embryonic and nonembryonic origin have been transplanted into the subretinal space in different animal models. Recently, retinal cells have also been used for transplantation in untreated or immunosuppressed patients with retinitis pigmentosa and age-related macular degeneration. Transplants performed in animals such as the RCS rat have maintained retinal function at the transplantation site for long periods of time, although such transplantations in humans have not proved conclusively, to date, whether there is a real effect or not. One reason for this phenomenon seems to be an immune response to transplanted retinal cells at the transplantation site. The detectable rejection process shows that the posterior part of the eye is not absolutely immunologically privileged and that rejection is a serious problem in human retinal transplantation. Many questions concerning transplantation technique, graft treatment and postoperative treatment will be answered through more intensive experiments and clinical trials regarding the immunology. However, rejection of transplanted material is one of the main reasons why retinal transplantation has not yet proved successful.