Eight 6-week-old Sprague-Dawley (SD) rats (Orient Bio, Seongnam, Korea) were used in this experiment. Four out of eight rats were injected with GFP-expressing MSCs after retinal damage was induced and the other animals were used as controls after laser treatment. All experiments were conducted in accordance with the ARVO statements for the Use of Animals in Ophthalmic and Vision Research.

Bone marrow-derived MSCs were extracted from six SD rats under deep anesthesia with phenobarbital sodium (10 mg/kg body weight, Jeil Pharm., Seoul, Korea). In each rat, the femur and tibia were isolated from the adjacent tissues, which were rinsed in 70% ethanol. Under sterile conditions, the edge of each bone was cut, Dulbecco's modified Eagle's medium (DMEM) was injected into the bone marrow using a 25-guage syringe, the bone marrow cells were pulled out to the opposite side, and this maneuver was repeated until the solution became transparent. The bone marrow cell extracts were centrifuged at 500 ×g for five minutes and then washed using new DMEM. Finally, cell suspensions were prepared using new DMEM containing 0.2% fetal bovine serum (FBS), 1% L-glutamate and 0.1% phosphate buffer solution (PBS).

Prior to cell culture, cells were inoculated with DMEM with a high glucose content in which 10% FBS, 1% penicillin-streptomycin and 2% glutamine were added. Cells were then suspended at a concentration of 2 × 10⁵/cm² on a 75 cm² tissue culture flask and were incubated at 37℃ in a 5% CO₂ incubator. To make sure those cells were well engrafted, cultivating cells were incubated for 72 hours. Three rounds of washings were conducted in which non-adhesive cells were removed with medium replacement. Following this procedure, the culture medium was replaced at 4-day intervals. The cell concentration was maintained at 1 × 10⁶ cells per flask.

A retrovirus expressing green fluorescence protein (GFP) was generated as previously described [20].

Prepared MSCs were seeded in fibronectin-coated 6-well plates (Becton Dickison, San Jose, CA, USA) containing 5 µg/mL polybrene. MSCs were applied at a multiplicity of infection of 10. Cells were disclosed to viral supernatant, making a final volume of 3 mL in which the density was 1 × 10⁶ cells per plate. Centrifugation was conducted at 800 ×g at 3 2℃ for 30 minutes. Then, an additional transduction was done for 48 hours. After washing with PBS to remove unabsorbed viral particles, trypsin was applied for five minutes. Puromycin was then added, by which GFP-transduced cells were selectively cultured.

Pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine. Two laser-induced retinal injuries were made in the right eye between the major vessels around the optic disc by a Nd:YAG laser (EPIC I; Coherent, Palo Alto, CA, USA) with an intensity of 2.0 to 3.0 mJ under anesthesia. Induction of full thickness retinal damage was confirmed with vitreous and subretinal hemorrhages.

The fundus changes were studied one day, three weeks, and five weeks after injury using fundus photography under anesthesia. The change over time in the findings of the vitreous and subretinal hemorrhages and the development of retinal detachment were compared between the two groups.

Twenty-four hours after induction of retinal injury, bone marrow-derived MSCs were transplanted into the four rats of the experimental group. After inhalation anesthesia with diethyl ether, 4 µL of cell suspension containing 1 × 10⁶ cells was systemically administered via tail vein in the experimental group. In the four rats of the control group, 4 µL of 0.1 M PBS was injected.

At five and seven weeks after MSCs injection, two eyes from the experimental group and two eyes from the control group were enucleated for tissue preparation. Xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (10 mg/kg) were intramuscularly injected for anesthesia. The thoracic cavity of each animal was opened, then 500 mL of 4% paraformaldehyde (Merck, Darmstadt, Germany) was perfused through the left ventricle. The eyeballs were enucleated and then placed in 4% paraformaldehyde in 0.1 M PBS overnight. To prevent tissue injury in making a frozen section, the eyes were placed in 10%- and 20%-sucrose solutions for one hour, respectively. Following this, they were placed in a 30% sucrose solution for another 12 hours. The horizontal plane of the eyeball was positioned parallel to the cutting plane. A tissue section 4 µm in thickness was prepared. Changes to the injured retinal lesions were observed using a light microscope (E400; Nikon, Kanagawa, Japan) after Wright stain. To evaluate the homing capacity of transplanted MSCs to the retina, the presence of GFP expressing cells was detected using a confocal microscope (ECLIPSE TE300, Nikon).

The expression of GFP in these cells was confirmed by confocal microscopy. After adhesion to the plastic culture flask, MSCs exhibited the typical spindle-like morphology and were arranged in a foray fashion. Adhesion to the culture flask also served as a criterion to distinguish MSCs from free floating hematopoietic cells.

The injury sites were studied at one day after retinal injury and at three weeks and five weeks after MSCs transplantation using fundus photography (Fig. 1). For the fundoscopic examination at day one after treatment with the Nd:YAG laser, hemorrhagic retinal elevations were observed at the injury site in all eight eyes (Fig. 1A and 1D). Three weeks after transplantation, the extent of subretinal and vitreous hemorrhages decreased in all four eyes of the experimental group and proliferative lesions were not observed in all four fundi of the experimental group (Fig. 1B). During that time, the hemorrhagic components in the eyes of the control group seemed to diminish in size. However, definitive elevations in the neurosensory retina were detected around the thick hemorrhage site (Fig. 1E). In the experimental group, the hemorrhagic components were much decreased and the ruptured retinal sites could not be detected in the color fundus photograph at five weeks after transplantation (Fig. 1C). However, the retinal detachment that developed in the eyes of the control group was more progressed with the proliferative changes and the hemorrhagic components remained at the same time point (Fig. 1F).

Histological analysis with Wright staining and GFP expressing cell detection using confocal microscopy were performed on animals at five and seven weeks after MSCs transplantation. At five weeks after treatment with intravenous injection of 1 × 10⁶ MSCs, the laser-induced retinotomy site was partially closed with uncertain types of cell cluster. Round-shaped, dark-pigmented cells were observed at the basal area of the full thickness damaged site. Cellular components of the injured site were certainly distinguished as compared with surrounding healthy retinal tissues. Only two or three layered unidentified cells were detected in the damaged site and there were no properly arranged neuronal networks between the various types of neuronal cells (Fig. 2A and 2B). To investigate whether bone marrow-derived MSCs were incorporated into the damaged chorioretinal tissues, we searched the serially sectioned slides around the damaged site. GFP expressing bone marrow-derived cells were detected only in the laser-induced injury site (Fig. 3A-3C). The placements of GFP expressing cells corresponded to those of round dark-pigmented cells observed in Fig. 2B. These findings, therefore, suggest that transplanted adult bone marrow-derived MSCs can contribute to the repair of injured chorioretinal tissues in the retinal trauma model. The round pigmented cells, as shown in Fig. 2B, were not detected in the injured retina at seven weeks post transplantation, but the laser-induced retinal break at the damaged site was much recovered with unidentified numerous cellular populations (Fig. 2C and 2D). Compared to the injured site at five weeks, the thickness of the cellular component increased and the injured site was much stabilized at seven weeks. Although normal retinal structure could not be identified in the lesion site, retinal detachment was not developed and the number of GFP expressing cells significantly increased in the laser-induced retinotomy site at seven weeks after transplantation (Fig. 3D-3F).