Baseline study and mouse-to-mouse transplantation

Baseline Study

A baseline study was performed on normal and dystrophic mouse. Eyes from 9 normal mice and 9 dystrophic rd mice, 7-day-old (n=6), 6-week-old (n=6) and 12- month-old (n=6) were studied for light microscopy (Table 1). Retinal sections were studied and labelled with antibodies. The sections will be labelled for rhodopsin (for rods), protein kinase C (PKC, for rod photoreceptor related bipolar cells), GFAP (for astrocytes and activated Müller cells), RT97 (neurofilaments in ganglion and

horizontal cells and their axons), calbindin (for horizontal cells and some amacrine cells), parvalbumin (for subtypes o f amacrine cells and ganglion cells), F4/80 (for microglia and macrophages), P84 and 6G3 (for species-specific synaptic protein). They were compared with previous published reports and subsequent transplantation results.

Table 1. No. of animals used in baseline histology Normal mice Dystrophic mice

7 day old 3 3

6 month old 3 3

Transplantation studies (Mouse to mouse)

Transplantation studies (Mouse to mouse) used normal mouse retinae as donor material and dystrophic rd mice as recipients (See Flow Diagram below). A total o f 66 six to eight-week-old dystrophic rd mice received bilateral subretinal transplants o f neonatal (PN 7 - 9 ) syngeneic normal mouse retina as described above (a total o f

132 eyes), this included all those that were tested in LDPT (n = 36) and

electrophysiology tests (n =18). The animals were sacrificed at 2 (n = 30), 4 (n = 20), and 6 weeks (n = 16) after transplantation. After harvesting the eyes, 110 eyes (n = 55) were processed for light microscopy and 22 eyes (n = 11) were processed for electron microscopy.

No. o f rd mice that had LDPT

n = 3 6

No. o f rd mice that had LDPT only

n = 22

No. o f rd mice that had retinal threshold measurement

n = 4

No. o f rd mice without any functional test

n = 26

No. o f rd mice that had LDPT and retinal

threshold measurement

n = 14

Total no. o f rd mice with transplants

Table 2. No. of animals sacrificed (mouse to mouse transplant)

Weeks Total no. o f animals sacrificed at each time point (No. o f animals that had LDPT before sacrificed)

2 30 (14)

4 20 (15)

6 16 (7)

Total 66 (36)

Control Experiment

For sham control, the process o f surgery and injection of carrying medium as used in other studies is not used here because in rd mice there is no significant host

photoreceptor cells left for preservation. Therefore the sham control has to include cells for transplantation. Some studies used fibroblasts as sham with partly the reason that they might secret growth factors to preserve the remaining photoreceptors and again it may not be appropriate for the study. Final cerebellar homogenates was chosen as the material o f cell sham control because it could test the possibility that any functional recovery was not specific for the retinal microaggregate graft but instead could be mimicked by other non-visual neural cells, (de Cerro et al., 1995) In my study, a total o f 6 dystrophic rd mice (n = 6) received bilateral subretinal grafts (12 eyes) o f cerebellar homogenates derived fi’om 7-day-old syngeneic normal mice (PN = 7). The homogenates were prepared in the same way as retinal

microaggregates. LDPT tests were carried out on these animals and eyes were harvested two weeks post-transplantation and processed the same way as above.

Light microscopy^ immunohistochemistry and electron microscopy

For light microscopy, animals were perfused under tribromoethanol terminal anaesthesia with phosphate-buffered saline (PBS) and then 4%

periodate/lysine/paraformaldehyde fixative (PLP) (see Section 2.4.4.) (McLean et al.,

1974). A suture was placed at the superior conjunctiva for orientation and the eyes were enucleated. The cornea was perforated and the eye was post-fixed in PLP for 1 hour and rinsed in PBS solution. The eyes were embedded in low-melting point polyester wax. Eight-micron sections were cut serially, mounted on charged glass slides and stored at 4°C until used. One series o f sections from each eye was stained with Cresyl violet in order to locate the area o f transplant, and selected slides from the other series o f the same eyes were labelled using specific antibodies.

Sections were de-waxed briefly in absolute and 95% alcohol and washed with PBS (See Appendix I). The slides were incubated in 5% de-fatted milk (a blocking agent) to reduce non-specific antibody binding for 30 minutes. The following primary antibodies were used as appropriate:- rhodopsin (for rod photoreceptor, rho4D2,

1:3000, generous gift from Dr. R. Molday, University o f British Columbia, Canada), PKC (for rod bipolar cells, MC5, 1:100, Santa Cruz Biotechnology, Inc., USA), RT97 (for horizontal cell and retinal ganglion cell axons; monoclonal anti-200-kd

neurofilament protein, 1:500, generous gift o f Dr. R. Morris, KCL Guy’s Hospital, London, UK), GFAP (for astrocytes and reactive Müller cell processes, 1:1000, Sigma-Aldrich Co. Ltd. UK), Calbindin (mainly for horizontal cells and some subtypes o f amacrine cells, 1:200, Sigma-Aldrich Co. Ltd. UK), Parvalbumin (for subtypes o f amacrine cells, Serotec Ltd., Oxford, UK), F4/80 (for murine microglia and macrophages, Serotec Ltd., Oxford, UK ), P84 (for mouse specific synaptic

protein, 1:1, generous gift from Dr C. Lagenaur, University o f Pittsburgh, USA.) and 6G3 (for rat specific synaptic protein, 1:1, generous gift from Dr C. Lagenaur,

University of Pittsburgh, USA) were applied at the appropriate concentrations diluted in antibody diluent (a mixture o f PBS with 1% Bovine serum albumin). Slides were incubated in a humidified chamber with the primary antibodies overnight at 4°C. After washing in PBS for 3 x 10-minute, they were incubated with the secondary antibodies diluted in antibody diluent and 1% rat or mouse serum for 30 minutes. They were washed in PBS 3 x 10-minute and reacted with an immunohistochemical avidin-biotin-peroxidase complex system (standard Elite ABC kit. Vector

Laboratories). After washing in PBS 3 x 10-minute, they were developed in a mixtured 3-3’ diaminobenzidine solution (DAB; Sigma-Aldrich Co. Ltd. UK) activated with hydrogen peroxide and aqueous nickel ammonium sulphate for 3 minutes, washed in PBS and distilled water, dehydrated through a graded series o f alcohol, cleared in xylene and mounted in DePeX (Merck/BDH, Littleworth, UK). The nickel ammonium sulphate changes the brown DAB product to black, making it easier to detect and to photograph.

For electron microscopy, after animals were deeply anaesthetised with

tribromoethanol terminal anaesthesia, 0.5ml o f fixative was injected into the posterior chamber o f the eye. The fixative consisted o f 2.5% paraformaldehyde, 2.5%

glutaraldehyde and 0.01% picric acid in 0.1 M cacodylate buffer (pH 7.4). The dorsal pole o f each eye was marked with a suture; the eyes were removed and left in the same fixative for 24 hours at 4°C. After washing in cacodylate buffer, the lenses were removed, the retinae were post-fixed in 1% osmium tetroxide for 1 hour. After

embedded in TAAB embedding resin (TAAB laboratories Aldermaston, UK). Semi- thin sections were stained with 1% Toluidine blue in 1% borate buffer, and ultra-thin sections were contrasted with alcoholic uranyl acetate and lead citrate. Grids were viewed on a Jeol 1010 electron microscope.