August 2003
Volume 44, Issue 8
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Retina  |   August 2003
Transplantation of Syngeneic Schwann Cells to the Retina of the Rhodopsin Knockout (Rho−/−) Mouse
Author Affiliations
  • David J. Keegan
    From the Department of Pathology, Institute of Ophthalmology, London, United Kingdom; the
  • Paul Kenna
    Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland; the
  • Marian M. Humphries
    Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland; the
  • Peter Humphries
    Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland; the
  • Daniel I. Flitcroft
    Mater Misericordiae, Dublin, Ireland; the
  • Peter J. Coffey
    Department of Psychology, University of Sheffield, Sheffield, United Kingdom; and the
  • Ray D. Lund
    From the Department of Pathology, Institute of Ophthalmology, London, United Kingdom; the
    Moran Eye Center, University of Utah, Salt Lake City, Utah.
  • Jean M. Lawrence
    From the Department of Pathology, Institute of Ophthalmology, London, United Kingdom; the
Investigative Ophthalmology & Visual Science August 2003, Vol.44, 3526-3532. doi:10.1167/iovs.02-0097
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      David J. Keegan, Paul Kenna, Marian M. Humphries, Peter Humphries, Daniel I. Flitcroft, Peter J. Coffey, Ray D. Lund, Jean M. Lawrence; Transplantation of Syngeneic Schwann Cells to the Retina of the Rhodopsin Knockout (Rho−/−) Mouse. Invest. Ophthalmol. Vis. Sci. 2003;44(8):3526-3532. doi: 10.1167/iovs.02-0097.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To determine whether subretinal Schwann cell transplantation can prolong the survival of photoreceptors in the rhodopsin knockout (rho−/−) mouse.

methods. Schwann cells were prepared from postnatal day (PN) 5 to 7 mouse pups and grafted subretinally into the eyes of PN35 rho−/− mice. RT-PCR was performed on similarly prepared cells to determine growth factor production in vitro. Eyes were retrieved at PN70 for anatomic and statistical analysis. Control animals received grafts of fibroblasts or sham surgery.

results. RT-PCR demonstrated the presence of message for ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic factor (GDNF) in the cultured Schwann cells. Schwann cell grafts produced a statistically significant rescue of photoreceptors in a restricted area of retina at PN70, but the effect was lost by PN140. Preserved inner segments could be identified, but outer segments were never present. Sham surgery also resulted in photoreceptor rescue but at a reduced level. Fibroblast grafts appeared to produce little or no rescue effect. Grafts of Schwann cells or fibroblasts and sham surgery induced a reactive Müller glial response.

conclusions. Schwann cells can prolong photoreceptor survival in the rhodopsin knockout mouse until at least PN70.

Retinitis pigmentosa (RP) comprises a group of inherited retinal degenerative diseases characterized by photoreceptor loss, vascular changes, invasion of the inner retina by retinal pigment epithelial (RPE) cells, and blindness. 1 2 Most mutations target photoreceptors, but some affect RPE cells directly. 3 4 5 6 7 Mutations in more than 100 genes have been identified that cause photoreceptor loss. 8 Their effects are widely distributed involving such processes as movement of materials in cells, movement of molecules between photoreceptors and RPE cells (by targeting transport proteins) and phototransduction. 9  
Photoreceptor loss can be limited in experimental models of RP by delivery of growth factors, either by direct injection into the eye 10 11 12 13 14 15 16 or by gene therapy approaches. 17 18 19 20 Direct injection of various growth factors (e.g., bFGF, ciliary neurotrophic factor [CNTF], glia-derived neurotrophic factor [GDNF]) has been shown to limit photoreceptor loss in a number of rodent models, 10 11 12 13 14 15 16 although there is evidence of variance of efficacy among models, 15 and, in some situations, combinations of growth factors 21 22 are effective, whereas the same factors delivered individually are not. Our previous work 23 showed that Schwann cells grafted subretinally into the dystrophic RCS rat support photoreceptor survival for up to 9 months after surgery. The rationale for using these cells was that they produce such retinally active growth factors as CNTF, 24 brain-derived neurotrophic factor (BDNF), 25 GDNF, 26 and bFGF 27 ; and, after transplantation to the central nervous system, they can promote neural regeneration. As such, they potentially present an additional trophic factor delivery approach for the eye. To assess whether the rescue potential of Schwann cells may extend beyond the RCS rat to include RP models in which there is an intrinsic photoreceptor defect, we examined whether such grafts are capable of protecting photoreceptors in the rhodopsin knockout mouse. In this animal, the retina has a normal complement of photoreceptors at birth, although rod outer segments never develop. 28 By 8 weeks, an electroretinogram (ERG) is no longer recordable, 29 and by 3 months nearly all photoreceptors are lost except for some residual cones. The degree of degeneration is variable, depending on the background on which the mouse is bred. Photoreceptor degeneration is more rapid in the 129 rho−/− mouse than in the C57 rho−/− mouse. 30 By transplanting Schwann cells into the subretinal space at an age before the loss of significant numbers of photoreceptors, it is possible to explore whether rescue of the remaining cells can be achieved. 
Materials and Methods
Donor Cells
Preparation of Schwann Cells.
Because the rhodopsin knockout mouse used in the study was derived on a 129 mouse background, donor Schwann cells were also taken from 129 mice (Harlan Sprague-Dawley, Crawley Down, UK) to reduce the risk of graft rejection. Sciatic nerves were dissected from postnatal day (PN)5 to 7 pups (n = 17) and transferred to Leibowitz’s L15 medium. After removal of contaminating tissue, the nerves were chopped into 100-μm pieces in a McIlwain tissue chopper and digested in a collagenase-trypsin mixture 31 for 90 minutes at 37°C. The digestion was stopped with Dulbecco’s modified Eagles medium (Life Technologies, Paisley, UK) plus 10% fetal calf serum (DMEMF; Harlan Seralab, Crawley Down, UK). After cells were spun at 1000 rpm for 5 minutes, they were resuspended in medium and triturated through a glass capillary and a 25-gauge needle. Cells were plated onto poly-l-lysine (Sigma-Aldrich Co., Irvine, UK)–coated 35-mm dishes (Nunc, Paisley, UK) in DMEMF, plus glutamine, pyruvate, and penicillin-streptomycin 32 and incubated at 37°C-5% CO2. After 24 hours, the medium was changed to remove unattached cells and debris. One week later, cells were removed from the dish with trypsin-EDTA 32 and suspended in DMEM. This suspension was panned 31 by incubating at 37°C in a 100-mm dish previously coated with rabbit anti-rat IgG (Dako, Glostrup, Denmark) and Thy 1.2 IgM (the latter being a supernatant from a mouse 129 hybridoma cell line, generous gift of Roger Morris, Kings College, London, UK). Most fibroblasts had attached to the dish after 10 minutes, and floating cells were removed, centrifuged and resuspended in DMEMF before replating to poly-l-lysine–coated dishes. Cells were ready for transplantation within 24 to 48 hours. After counting the number of contaminating fibroblasts (5%–7%), cells were removed from the dish as before. Because Schwann cells are far less adherent to the dish than fibroblasts, it is possible to reduce further the number of contaminating cells by selecting only those cells that move into suspension rapidly. 33 Cells were counted and transplanted in suspension to rho−/− animals (n = 15) in DMEM plus DNase (DNase 1, 5 μL/mL; Sigma-Aldrich). DNase reduces the formation of cell aggregates. 
Preparation of Fibroblasts.
To control for any potential rescue effect conferred by contaminating fibroblasts, fibroblasts left attached to the Thy1.2-coated dishes after panning were trypsinized off the dish and transferred to flasks containing DMEMF (10% FCS) as before. Cells were plated at low density, and the fibroblast cultures were left to expand almost to confluence. Before transplantation, cells were trypsinized off the flasks, counted, and suspended in carrier medium (DMEM) with DNase. 
Growth Factor Production by Schwann Cells In Vitro.
To examine growth factor production by the donor Schwann cells, cells were prepared as for transplantation but left in culture for a further 7 days after purification. Cells were trypsinized from the dish, and the enzyme activity quenched with DMEMF. The cells were centrifuged at 1000 rpm for 5 minutes, resuspended in a large volume of DMEM and spun again. The supernatant was removed, and the cells were suspended in a small volume of DMEM and transferred to a tube (Eppendorf, Fremont, CA). The cells were centrifuged once more, and as much DMEM as possible was removed. They were frozen rapidly before analysis by reverse transcription–PCR (RT-PCR) according to the manufacturer’s protocol (Life Technologies). Messenger RNA was extracted with a kit (RNeasy; Qiagen, Crawley, UK). 
The growth factors assayed were BDNF, CNTF, GDNF, nerve growth factor (NGF), bFGF. Details are given in Table 1 . The primers were developed at Sigma-Genosys (Poole, UK). 
Transplantation Procedure
All animals were cared for in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Irish Department of Health and Children. 
Host mice (rhodopsin knockout, n = 30) were anesthetized with a ketamine-xylazine mixture. Rho−/− animals (n = 15) received subretinal transplants of Schwann cells (1 × 104 cells in 2 μL of medium) or sham injections (2 μL of DMEM+DNase: n = 15) at PN35. Grafts were introduced transsclerally into the dorsotemporal quadrant of the right eye with a glass micropipette (outer diameter, ∼150 μm, inner diameter, 75 μm) attached to a 10-μL syringe (Hamilton, Reno, NV). Eyes that showed signs of retinal damage at the time of surgery were eliminated from the study (rho−/− grafted, n = 5; sham surgery, n = 5). The nonsurgical eyes served as controls. A further group of rho−/− animals (n = 5) received subretinal injections of fibroblasts to both eyes. 
Tissue Processing
At PN70, animals (Schwann cell transplanted rho−/−, n = 10; fibroblast transplanted rho−/−, n = 5 [10 eyes]); sham operation, n = 10, nonsurgical control, n = 6) received an overdose of pentobarbital sodium (Euthatal; Rhone Merieux, Cambridge, UK) and perfused with 2.5% paraformaldehyde (TAAB, Aldermaston, UK), 2% glutaraldehyde (TAAB) and 0.01% picric acid in 0.1 M phosphate buffer. A suture was placed in the ventral corneoscleral junction before the eye was excised into fresh fixative to maintain tissue orientation. After overnight immersion, eyes were transferred to 0.1 M cacodylate buffer (TAAB) and the lenses removed through an incision in the cornea. Eyes were postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, followed by dehydration through a graded series of alcohols and epoxypropane before embedding in Agar resin (Agar Scientific, Stanstead, UK). 
Tissue Analysis
Semithin sections were cut through the region of the transplant and stained with toluidine blue (Sigma-Aldrich). A temporal-to-nasal orientation was maintained throughout. Representative sections (adjacent to or within 200 μm of the injection site) from each retina were photographed (see Fig. 2 ) and the photographs montaged. The total number of photoreceptors was counted blind for each sample by the same person, in 100-μm bins (∼50 per eye). Maximum cell counts for each bin location in each experimental group (see Fig. 4A ) and mean cell counts for each bin location in each experimental group (see Fig. 4B ) across the retinas (temporal to nasal) were generated and compared. Analyses were performed on computer (StatView; SAS Corp., Cary, NC) for PC. All group data were subjected to a one-way analysis of variance in the temporal region of the retina (boxed). Post hoc analysis was performed on significant group findings. 
In addition some ultrathin sections were obtained from each group, stained with alcoholic uranyl acetate and lead citrate, and viewed with an electron microscope (model 1010; JEOL, Tokyo, Japan). 
Results
Donor Cells
Schwann cell cultures of high purity (>95%) were obtained according to the described protocol (Fig. 1A) . Schwann cells had the typical spindle-shaped morphology and were readily distinguishable from contaminating fibroblasts that had large nuclei and extensive flattened cytoplasm. Fibroblast cell cultures contained cells with typical flattened morphology and large oval nuclei. 
Reverse Transcription–Polymerase Chain Reaction
Schwann cells expressed mRNA for BDNF, CNTF, and GDNF (Fig. 1B) . There was no evidence of NGF or bFGF message in cells grown in these conditions. 
Rho−/− Retinal Histology
Histologic analysis of Schwann cell graft-recipient eyes (n = 10) at PN70 showed a distinct region of photoreceptor preservation in the temporal retina (Figs. 2J 2K 2L , quantified in Fig. 4 ), six to seven cells deep and approximately 800 μm wide. This was in contrast to fibroblast-grafted (Figs. 2G 2H 2I) , sham-operation (Figs. 2D 2E 2F) , and nonsurgical animals (Figs. 2A 2B 2C) , in which generally only one to two layers of photoreceptors, mostly cones, remained. Although more photoreceptors survived in the retinas with Schwann cell transplants, typical outer segments never formed. However, cone inner segment morphology was distinctly preserved (Figs. 2K 2L)
Schwann cells were identified in the general region of the area of rescue (Figs. 3A 3B) , where, as described previously, 23 they could be identified by their indented nuclei, which were larger than the elongated nuclei of fibroblasts. Some of the fibroblasts may have been transplanted with the donor cells, but because fibrotic scars can be identified after sham surgery or after grafts of other cell types, it is likely that a proportion are host fibroblasts introduced from the sclera at the time of surgery. Because animals that received grafts of pure fibroblasts showed no improved photoreceptor rescue and no cone inner segment preservation, then the rescue seen in the Schwann cell graft recipients (with some fibroblast contamination) is most likely due to the Schwann cells themselves. Although the retina immediately overlying the graft was disrupted, there was marked preservation of photoreceptors. 
Maximal photoreceptor counts for each group, along the length of the retina temporal to nasal, are presented in Figure 4A . The outlined area indicates a region of approximately 2300 μm in the vicinity of the temporal injection site, where there was a marked increase in photoreceptor survival. This region was subjected to statistical analysis. The Schwann cell graft group had significantly more photoreceptors than did the fibroblast graft, sham-surgery, and nonsurgical groups (F = 148.01, df = 3.32, P < 0.001). There was approximately a 55% increase in photoreceptor survival in the Schwann cell transplant group compared with the sham-surgery group and a 225% increase in the Schwann cell transplant group compared with the fibroblast transplant group and nonsurgical rho−/− group. Furthermore, the sham-surgery group had approximately twice as many photoreceptors as the fibroblast and nonsurgical groups, which was significant (sham versus fibroblast-transplant group: t = 13.06, df = 16, P < 0.01; sham versus the nonsurgical control: t = 17.13, df = 16, P < 0.01. A similar area, 2300 μm, (Fig. 4B) was analyzed for the mean data from each group. Once again, a region in the temporal retina showed a significant rescue effect in the Schwann cell transplant group over the fibroblast transplant, sham-surgery, and nonsurgical control groups (F = 33.15, df = 3.38, P < 0.001). There was approximately a 30% increase in photoreceptor survival in the Schwann cell transplant group compared with the sham-surgery group and a 66% increase in the Schwann cell group compared with the fibroblast transplant and nonsurgical control group. As before, a significant increase in photoreceptors was observed in the sham-surgery groups compared with the fibroblast transplant group and nonsurgical control group (sham versus fibroblast-transplant group: t = 5.96, df = 44, P < 0.01; sham versus nonsurgical knockout control: t = 3.43, df = 44, P < 0.01). Thus, there was a focal rescue effect adjacent to the site of injection and in this particular area the effect was significant. 
Discussion
The results show that Schwann cells grafted into the rho−/− mouse can preserve a focal region of photoreceptors up to at least PN70. The results are of interest, because they show that Schwann cells can preserve photoreceptors in an animal model in which the defect lies in the photoreceptors themselves (Figs. 2 4) , in contrast to our previous work showing rescue in RCS rats. In the eyes with evident grafts, the cells appear normal, and they integrate into the subretinal space, although with some contaminating fibroblasts (Fig. 3B) . However, in this animal model, fibroblast grafts appear to produce little or no photoreceptor rescue, and therefore the rescue effect observed is most likely due to the Schwann cells alone. 
None of the cells appeared to provoke inflammatory reactions, although there was a Müller glial response. However, because the glial hypertrophy occurred in the sham-surgery and both transplant groups, this is likely to be a response to surgical intervention. In parallel studies in RCS rats, 23 photoreceptors with outer segments were preserved in the vicinity of the Schwann cell grafts for quite extensive periods. Retinas grafted with fibroblasts or sham surgery showed less extensive preservation of the outer nuclear layer for much shorter times. Outer segments never develop in the rho−/− mouse, nor are they seen after Schwann cell grafting or adeno-associated virus (AAV)-CNTF treatment. 19 However, after Schwann cell grafting, inner segment cone morphology appears to be better preserved. It is presumed that the Schwann cells preserve photoreceptors by delivering growth factors into the subretinal space because, in vitro at least, they are a source of several factors known to preserve photoreceptors from degeneration. The cones could be directly rescued or secondarily through rod rescue in a manner similar to that described in other studies. 34 The primary target of the growth factors is still unclear, but the finding in C57BL/6J mice that intravitreal injections of CNTF, BDNF, and FGF2 (bFGF) activate intracellular signaling mechanisms in Müller glia (and other inner retinal cells), but not in photoreceptors, 35 suggests that the photoreceptor rescue may be through a second-order pathway. 
The degree of rescue was less extensive than that shown by Liang et al., 19 who delivered secretable CNTF mediated by AAV at a much earlier age (PN2–5). Delivery at this earlier time may be important as the peak of apoptosis in the outer nuclear layer of the rho−/− mouse (129Sv background) begins at approximately PN35, 30 proceeding rapidly until approximately PN63, and in the present study grafting did not take place until PN35. We chose the later time to intervene, because it would be more analogous with a potential treatment point in humans. However, it is possible that when transplantation occurs so late, the cell death cycle can only be delayed. In addition, donor cells may be more locally distributed than an AAV-mediated product, thus reducing their overall efficacy. Liang et al. 19 showed that cells engineered with nonsecretable CNTF could not promote photoreceptor survival. Because Schwann cell CNTF may not be in a secretable form and may only be released by cell death, other growth factors produced by them could give rise to the observed rescue effect. 
Although photoreceptors have been preserved in this study, no outer segments were generated, and this was also observed after AAV-CNTF delivery. 19 As indicated in that study, an ERG response could not demonstrated. Nonsurgical control mice are also without outer segments and never show evidence of an ERG. 29 Furthermore, Sieving’s group 36 have determined the electrophysiological relationship between outer segment length and photoreceptor nuclei number and have shown that, without outer segments, it is not possible to generate an a-wave. Thus, the absence of outer segments indicates that an intact ERG response is unlikely. In agreement with these findings, a preliminary ERG investigation of the present group of transplant-recipient animals before fixation (data not shown) failed to demonstrate any significant response. It should also be noted that viral (rAAV or lentivirus) secretable CNTF injected into the eyes of other rodent models of RP 37 38 provided long-term photoreceptor preservation but had no or negative effects on ERG amplitudes. However, patients with RP may also fail to show full-field ERGs and have greatly reduced numbers of photoreceptors with shortened or absent inner and outer segments, yet have some functional vision remaining. 39 Therefore, an absent ERG does not necessarily indicate that there is no visual processing. Rod rescue per se would not be expected to rescue visual function in this animal. However, other investigators 34 have proposed that rescuing rods preserves cones that may otherwise die. The present preparation provides a special situation in which to examine this possibility. 
In summary, Schwann cell grafts limit photoreceptor loss in two very different models of retinal degeneration (RCS rat 23 ; rho−/− mouse). The most parsimonious explanation for this phenomenon is that they function by delivering a range of growth factors. The potential of a steady, continuous release of a mixture of factors from transplanted cells affords some advantage over intravitreal injection. Local release at the site of need avoids the inherent problem of delivering factors at nonphysiological levels to other tissues of the eye or body. The use of syngeneic grafts also obviates the need for immune suppression, which can be a serious problem in transplantation studies. The degree of functional vision that is preserved after such grafts, in comparison with RPE cell grafts, is a matter for continued investigation. 
 
Table 1.
 
Primers Used for the Target Growth Factor Genes
Table 1.
 
Primers Used for the Target Growth Factor Genes
Gene Sequence Product Size (bp) Number of Cycles Annealing Temperature (°C)
NGF 5′-ATGTGGTTCCTGATCCTG-3′ 796 35 48.2
5′-TGTGATACTCAGGGG-3′
bFGF 5′-GCCACTTCAAGGATCCC-3′ 378 35 57.0
5′-GGAAGAAACAGTATGGCCTTC-3′
CNTF 5′-GTAGCCGTTCTATCTGG-3′ 479 35 48.2
5′-GAAGGTCATGGATAGACC-3′
GDNF 5′-AAGCACAGCTACGGGATG-3′ 707 35 57.0
5′-GGGAGGAGCAGCCATTG-3′
BDNF 5′-ACGTCCACGGACAAGG-3′ 489 35 48.2
5′-CACTTGGTCTCGTAG-3′
Figure 1.
 
Mouse Schwann cells in culture. (A) The spindle-shaped appearance of the mouse (Sv129) Schwann cells is shown. The isolation technique involves purifying the culture to remove most of the contaminating fibroblasts. Primary cultures do not divide rapidly—hence, the relatively sparse number of cells. (B) RT-PCR profile of primary Schwann cells harvested from PN5 to 7 pups for growth factor mRNA. The specific factors screened are noted at the head of the lanes. There was strong expression of BDNF and CNTF and weak expression of GDNF. Messenger RNA was undetectable for NGF and bFGF. β-actin was used as the positive control primer.
Figure 1.
 
Mouse Schwann cells in culture. (A) The spindle-shaped appearance of the mouse (Sv129) Schwann cells is shown. The isolation technique involves purifying the culture to remove most of the contaminating fibroblasts. Primary cultures do not divide rapidly—hence, the relatively sparse number of cells. (B) RT-PCR profile of primary Schwann cells harvested from PN5 to 7 pups for growth factor mRNA. The specific factors screened are noted at the head of the lanes. There was strong expression of BDNF and CNTF and weak expression of GDNF. Messenger RNA was undetectable for NGF and bFGF. β-actin was used as the positive control primer.
Figure 2.
 
Comparative anatomy of control (AC), sham-surgery (DF), fibroblast grafted (GI), and Schwann cell grafted (JL) retinas at PN70. (A) Semithin section of a control retina at PN70. There is near complete loss of the outer nuclear layer (ONL). (B) A higher power view of the boxed area in (A) demonstrates these features more clearly. There is total absence of outer segment structure and a grossly compromised inner segment (IS) layer. (✶) Outer limiting membrane; (C) electron micrograph showing stunted photoreceptor inner segments (IS) and OLM (✶). (D) Semithin section of a sham-surgery retina that shares some features of a control retina (i.e., thinning of the ONL and loss of inner segment morphology). (E) A higher power view of the boxed area in (D). (F) An electron micrograph of the same sham-surgery eye shows that inner segments are rare and there are no outer segments. Hypertrophied Müller cell processes (mp) surround remaining photoreceptors. OLM, asterisk (✶). (G) Semithin section of a retina that received a fibroblast transplant. The ONL is reduced and inner segments are much reduced. (H) A higher power view of the boxed area in (G). (I) An electron micrograph of a fibroblast-grafted retina. There is total absence of outer segment structure and a grossly compromised IS layer. Hypertrophied Müller cell processes (mp) surround remaining photoreceptors. (J) Semithin section of a retina that received a Schwann cell transplant. There is considerable preservation of the ONL compared with control, sham-surgery, and fibroblast-grafted retinas (A, D, and G, respectively). (K) A higher power view of the boxed area in (J). There are no outer segments but there is preservation of cone IS morphology (arrows) at PN70 compared with fibroblast, sham-surgery, and control retinas. (L) An electron micrograph from the area of retina illustrated in (G) and (H) that had received a graft. Note the more distinct cone inner segment morphology (arrows) compared with that in (C), (F), and (I). As in those panels there are no demonstrable rod or cone outer segments. Note also that there is some hypertrophy of Müller glial cell processes (mp). Scale bars: (A, B, D, E, G, H, J, K) 25 μm; (C, F, I, L) 2 μm.
Figure 2.
 
Comparative anatomy of control (AC), sham-surgery (DF), fibroblast grafted (GI), and Schwann cell grafted (JL) retinas at PN70. (A) Semithin section of a control retina at PN70. There is near complete loss of the outer nuclear layer (ONL). (B) A higher power view of the boxed area in (A) demonstrates these features more clearly. There is total absence of outer segment structure and a grossly compromised inner segment (IS) layer. (✶) Outer limiting membrane; (C) electron micrograph showing stunted photoreceptor inner segments (IS) and OLM (✶). (D) Semithin section of a sham-surgery retina that shares some features of a control retina (i.e., thinning of the ONL and loss of inner segment morphology). (E) A higher power view of the boxed area in (D). (F) An electron micrograph of the same sham-surgery eye shows that inner segments are rare and there are no outer segments. Hypertrophied Müller cell processes (mp) surround remaining photoreceptors. OLM, asterisk (✶). (G) Semithin section of a retina that received a fibroblast transplant. The ONL is reduced and inner segments are much reduced. (H) A higher power view of the boxed area in (G). (I) An electron micrograph of a fibroblast-grafted retina. There is total absence of outer segment structure and a grossly compromised IS layer. Hypertrophied Müller cell processes (mp) surround remaining photoreceptors. (J) Semithin section of a retina that received a Schwann cell transplant. There is considerable preservation of the ONL compared with control, sham-surgery, and fibroblast-grafted retinas (A, D, and G, respectively). (K) A higher power view of the boxed area in (J). There are no outer segments but there is preservation of cone IS morphology (arrows) at PN70 compared with fibroblast, sham-surgery, and control retinas. (L) An electron micrograph from the area of retina illustrated in (G) and (H) that had received a graft. Note the more distinct cone inner segment morphology (arrows) compared with that in (C), (F), and (I). As in those panels there are no demonstrable rod or cone outer segments. Note also that there is some hypertrophy of Müller glial cell processes (mp). Scale bars: (A, B, D, E, G, H, J, K) 25 μm; (C, F, I, L) 2 μm.
Figure 3.
 
Schwann cells in the subretinal space of a PN70 mouse that received a graft at PN35. (A) Semithin section showing Schwann cells (SC) grafted into the subretinal space at PN35 and harvested at PN70. The cells are layered over the host retinal pigment epithelium (RPE). The overlying retina is disrupted, but the photoreceptor layer (ONL) is preserved. Inner nuclear layer (INL). (B) A higher power view of the boxed area in (A). The Schwann cells (SC) are interspersed with fibroblasts. The Schwann cells have larger, indented nuclei (arrows) compared with fibroblast nuclei, which are elongated (✶). Fibroblasts may have been introduced as a contaminant at the time of grafting or pushed in from the host sclera at the time of surgery. The mixed cells lie in a group on the host RPE. Scale bar, 50 μm.
Figure 3.
 
Schwann cells in the subretinal space of a PN70 mouse that received a graft at PN35. (A) Semithin section showing Schwann cells (SC) grafted into the subretinal space at PN35 and harvested at PN70. The cells are layered over the host retinal pigment epithelium (RPE). The overlying retina is disrupted, but the photoreceptor layer (ONL) is preserved. Inner nuclear layer (INL). (B) A higher power view of the boxed area in (A). The Schwann cells (SC) are interspersed with fibroblasts. The Schwann cells have larger, indented nuclei (arrows) compared with fibroblast nuclei, which are elongated (✶). Fibroblasts may have been introduced as a contaminant at the time of grafting or pushed in from the host sclera at the time of surgery. The mixed cells lie in a group on the host RPE. Scale bar, 50 μm.
Figure 4.
 
Analyses of photoreceptor numbers in Schwann cell–grafted versus fibroblast-grafted, sham-surgery, and control rho−/− retinas. (A) This graph illustrates photoreceptor counts across the retina from temporal to nasal (left to right). Maximal counts for each group at each bin location are shown. The Schwann cell transplant group tends to have higher cell counts in the temporal (surgical region) than sham-surgery, fibroblast-grafted, or control retinas. This difference is significant (P < 0.001). (B) Mean photoreceptor counts for all groups. Again, the Schwann cell transplant effect is demonstrated in the temporal retinal region. The difference between Schwann cell transplant retinas and sham-surgery, fibroblast-grafted, and control retinas in this temporal region (outlined) is significant (P < 0.001).
Figure 4.
 
Analyses of photoreceptor numbers in Schwann cell–grafted versus fibroblast-grafted, sham-surgery, and control rho−/− retinas. (A) This graph illustrates photoreceptor counts across the retina from temporal to nasal (left to right). Maximal counts for each group at each bin location are shown. The Schwann cell transplant group tends to have higher cell counts in the temporal (surgical region) than sham-surgery, fibroblast-grafted, or control retinas. This difference is significant (P < 0.001). (B) Mean photoreceptor counts for all groups. Again, the Schwann cell transplant effect is demonstrated in the temporal retinal region. The difference between Schwann cell transplant retinas and sham-surgery, fibroblast-grafted, and control retinas in this temporal region (outlined) is significant (P < 0.001).
The authors thank Rhona Mirsky for advice at the beginning of the project, Robin Howes for preparing the histologic material, and Tim Pheby for assistance. 
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Figure 1.
 
Mouse Schwann cells in culture. (A) The spindle-shaped appearance of the mouse (Sv129) Schwann cells is shown. The isolation technique involves purifying the culture to remove most of the contaminating fibroblasts. Primary cultures do not divide rapidly—hence, the relatively sparse number of cells. (B) RT-PCR profile of primary Schwann cells harvested from PN5 to 7 pups for growth factor mRNA. The specific factors screened are noted at the head of the lanes. There was strong expression of BDNF and CNTF and weak expression of GDNF. Messenger RNA was undetectable for NGF and bFGF. β-actin was used as the positive control primer.
Figure 1.
 
Mouse Schwann cells in culture. (A) The spindle-shaped appearance of the mouse (Sv129) Schwann cells is shown. The isolation technique involves purifying the culture to remove most of the contaminating fibroblasts. Primary cultures do not divide rapidly—hence, the relatively sparse number of cells. (B) RT-PCR profile of primary Schwann cells harvested from PN5 to 7 pups for growth factor mRNA. The specific factors screened are noted at the head of the lanes. There was strong expression of BDNF and CNTF and weak expression of GDNF. Messenger RNA was undetectable for NGF and bFGF. β-actin was used as the positive control primer.
Figure 2.
 
Comparative anatomy of control (AC), sham-surgery (DF), fibroblast grafted (GI), and Schwann cell grafted (JL) retinas at PN70. (A) Semithin section of a control retina at PN70. There is near complete loss of the outer nuclear layer (ONL). (B) A higher power view of the boxed area in (A) demonstrates these features more clearly. There is total absence of outer segment structure and a grossly compromised inner segment (IS) layer. (✶) Outer limiting membrane; (C) electron micrograph showing stunted photoreceptor inner segments (IS) and OLM (✶). (D) Semithin section of a sham-surgery retina that shares some features of a control retina (i.e., thinning of the ONL and loss of inner segment morphology). (E) A higher power view of the boxed area in (D). (F) An electron micrograph of the same sham-surgery eye shows that inner segments are rare and there are no outer segments. Hypertrophied Müller cell processes (mp) surround remaining photoreceptors. OLM, asterisk (✶). (G) Semithin section of a retina that received a fibroblast transplant. The ONL is reduced and inner segments are much reduced. (H) A higher power view of the boxed area in (G). (I) An electron micrograph of a fibroblast-grafted retina. There is total absence of outer segment structure and a grossly compromised IS layer. Hypertrophied Müller cell processes (mp) surround remaining photoreceptors. (J) Semithin section of a retina that received a Schwann cell transplant. There is considerable preservation of the ONL compared with control, sham-surgery, and fibroblast-grafted retinas (A, D, and G, respectively). (K) A higher power view of the boxed area in (J). There are no outer segments but there is preservation of cone IS morphology (arrows) at PN70 compared with fibroblast, sham-surgery, and control retinas. (L) An electron micrograph from the area of retina illustrated in (G) and (H) that had received a graft. Note the more distinct cone inner segment morphology (arrows) compared with that in (C), (F), and (I). As in those panels there are no demonstrable rod or cone outer segments. Note also that there is some hypertrophy of Müller glial cell processes (mp). Scale bars: (A, B, D, E, G, H, J, K) 25 μm; (C, F, I, L) 2 μm.
Figure 2.
 
Comparative anatomy of control (AC), sham-surgery (DF), fibroblast grafted (GI), and Schwann cell grafted (JL) retinas at PN70. (A) Semithin section of a control retina at PN70. There is near complete loss of the outer nuclear layer (ONL). (B) A higher power view of the boxed area in (A) demonstrates these features more clearly. There is total absence of outer segment structure and a grossly compromised inner segment (IS) layer. (✶) Outer limiting membrane; (C) electron micrograph showing stunted photoreceptor inner segments (IS) and OLM (✶). (D) Semithin section of a sham-surgery retina that shares some features of a control retina (i.e., thinning of the ONL and loss of inner segment morphology). (E) A higher power view of the boxed area in (D). (F) An electron micrograph of the same sham-surgery eye shows that inner segments are rare and there are no outer segments. Hypertrophied Müller cell processes (mp) surround remaining photoreceptors. OLM, asterisk (✶). (G) Semithin section of a retina that received a fibroblast transplant. The ONL is reduced and inner segments are much reduced. (H) A higher power view of the boxed area in (G). (I) An electron micrograph of a fibroblast-grafted retina. There is total absence of outer segment structure and a grossly compromised IS layer. Hypertrophied Müller cell processes (mp) surround remaining photoreceptors. (J) Semithin section of a retina that received a Schwann cell transplant. There is considerable preservation of the ONL compared with control, sham-surgery, and fibroblast-grafted retinas (A, D, and G, respectively). (K) A higher power view of the boxed area in (J). There are no outer segments but there is preservation of cone IS morphology (arrows) at PN70 compared with fibroblast, sham-surgery, and control retinas. (L) An electron micrograph from the area of retina illustrated in (G) and (H) that had received a graft. Note the more distinct cone inner segment morphology (arrows) compared with that in (C), (F), and (I). As in those panels there are no demonstrable rod or cone outer segments. Note also that there is some hypertrophy of Müller glial cell processes (mp). Scale bars: (A, B, D, E, G, H, J, K) 25 μm; (C, F, I, L) 2 μm.
Figure 3.
 
Schwann cells in the subretinal space of a PN70 mouse that received a graft at PN35. (A) Semithin section showing Schwann cells (SC) grafted into the subretinal space at PN35 and harvested at PN70. The cells are layered over the host retinal pigment epithelium (RPE). The overlying retina is disrupted, but the photoreceptor layer (ONL) is preserved. Inner nuclear layer (INL). (B) A higher power view of the boxed area in (A). The Schwann cells (SC) are interspersed with fibroblasts. The Schwann cells have larger, indented nuclei (arrows) compared with fibroblast nuclei, which are elongated (✶). Fibroblasts may have been introduced as a contaminant at the time of grafting or pushed in from the host sclera at the time of surgery. The mixed cells lie in a group on the host RPE. Scale bar, 50 μm.
Figure 3.
 
Schwann cells in the subretinal space of a PN70 mouse that received a graft at PN35. (A) Semithin section showing Schwann cells (SC) grafted into the subretinal space at PN35 and harvested at PN70. The cells are layered over the host retinal pigment epithelium (RPE). The overlying retina is disrupted, but the photoreceptor layer (ONL) is preserved. Inner nuclear layer (INL). (B) A higher power view of the boxed area in (A). The Schwann cells (SC) are interspersed with fibroblasts. The Schwann cells have larger, indented nuclei (arrows) compared with fibroblast nuclei, which are elongated (✶). Fibroblasts may have been introduced as a contaminant at the time of grafting or pushed in from the host sclera at the time of surgery. The mixed cells lie in a group on the host RPE. Scale bar, 50 μm.
Figure 4.
 
Analyses of photoreceptor numbers in Schwann cell–grafted versus fibroblast-grafted, sham-surgery, and control rho−/− retinas. (A) This graph illustrates photoreceptor counts across the retina from temporal to nasal (left to right). Maximal counts for each group at each bin location are shown. The Schwann cell transplant group tends to have higher cell counts in the temporal (surgical region) than sham-surgery, fibroblast-grafted, or control retinas. This difference is significant (P < 0.001). (B) Mean photoreceptor counts for all groups. Again, the Schwann cell transplant effect is demonstrated in the temporal retinal region. The difference between Schwann cell transplant retinas and sham-surgery, fibroblast-grafted, and control retinas in this temporal region (outlined) is significant (P < 0.001).
Figure 4.
 
Analyses of photoreceptor numbers in Schwann cell–grafted versus fibroblast-grafted, sham-surgery, and control rho−/− retinas. (A) This graph illustrates photoreceptor counts across the retina from temporal to nasal (left to right). Maximal counts for each group at each bin location are shown. The Schwann cell transplant group tends to have higher cell counts in the temporal (surgical region) than sham-surgery, fibroblast-grafted, or control retinas. This difference is significant (P < 0.001). (B) Mean photoreceptor counts for all groups. Again, the Schwann cell transplant effect is demonstrated in the temporal retinal region. The difference between Schwann cell transplant retinas and sham-surgery, fibroblast-grafted, and control retinas in this temporal region (outlined) is significant (P < 0.001).
Table 1.
 
Primers Used for the Target Growth Factor Genes
Table 1.
 
Primers Used for the Target Growth Factor Genes
Gene Sequence Product Size (bp) Number of Cycles Annealing Temperature (°C)
NGF 5′-ATGTGGTTCCTGATCCTG-3′ 796 35 48.2
5′-TGTGATACTCAGGGG-3′
bFGF 5′-GCCACTTCAAGGATCCC-3′ 378 35 57.0
5′-GGAAGAAACAGTATGGCCTTC-3′
CNTF 5′-GTAGCCGTTCTATCTGG-3′ 479 35 48.2
5′-GAAGGTCATGGATAGACC-3′
GDNF 5′-AAGCACAGCTACGGGATG-3′ 707 35 57.0
5′-GGGAGGAGCAGCCATTG-3′
BDNF 5′-ACGTCCACGGACAAGG-3′ 489 35 48.2
5′-CACTTGGTCTCGTAG-3′
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