All animal procedures in this study are carried out under the national animal care guidelines and approved by local Ethics committee. Animals were kept in 12-hour light/dark cycles and ad libitum access to food and water. In this study, ten newborn Wistar rats were divided randomly into two groups, including; the control group (n = 5) and the oxygen-induced retinopathy (OIR) group (n = 5) (Fig. 1). Based on previous studies subjecting cellular therapy in ROP animal models, the intervention was done at the initiation of phase 2 (hypoxia) of ROP modeling on day 12 p. in animals in the OIR group, one eye received BMMNC suspension (treatment group), and the contralateral eye received the same volume of saline injection (vehicle injection group). At the age of 17, all animals underwent funduscopy, electroretinography examinations were euthanized by Co2 inhalation, and their eyes were harvested for histopathological assessments (18).
Rat model of Oxygen Induced Retinopathy (OIR)
In the current study, we used the conventional protocol of preparing an animal model of ROP described in detail by Smith et al. (19). Briefly, seven-day-old Wistar rats with their nursing dams were incubated for five days in a hand-made isolate chamber with oxygen concentration adjusted to 70–75%. During the incubation period, animals were provided free access to standard food and water, and The Oxygen concentration was monitored continuously by using an oxygen indicator (CityCell. the UK). We placed a heated pad under the chamber to maintain the chamber's temperature at 25 ℃. On date 12 p, animals were returned to free room air (21% O2) for five days (Fig. 1).
BMMNC extraction and Autologous Transplantation
In order to extract bone marrow mononuclear cells, we dissected long bones of one 8- month-old male rat after euthanasia by placing the anesthetized rat in a CO2 chamber. Bone marrow was extracted from the dissected humerus, femur, and tibia from the donor rat with cold sterile Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEMF-12) (Gibco, Thermofisher Scientific, MA) and then filtered through 70 µm cell strainer (BD Biosciences, CA). Then the suspension was centrifuged at 500 g for 5 minutes (Eppendorf, Germany) at room temperature. The supernatant was removed, and cells were resuspended in 5 ml of 1 X RBC Lysis Buffer (Thermofisher Scientific, MA) and incubated for 5 min at room temperature. The lysis reaction stopped by adding 20 ml of Phosphate-buffered saline (PBS), and then the solution was centrifuged again and removed the supernatant. The final solution was achieved by adding 1 ml sterile PBS to the cell pellet. The availability of bone marrow cells was approved by trypan blue staining, and then one syringe was filled with final suspension, ready for the injection procedure.
After general anesthesia with an intraperitoneal injection of ketamine/Xylazine and saline mixture (85/10 mg/kg), we made a small incision on the fissure between the eyelids with a lancet blade (NO 24) to separate them. After enough exposure to the eyes, appropriate ocular surface analgesia was induced by tetracaine eye drops (Anestocaine 0.5%). 10 µl of the BMMNC suspended in sterilized PBS, containing 1.2 x 105 cells, was slowly injected intravitreally with a 30-gauge needle to one eye of each neonate rat in the OIR group and equivolume of sterile PBS was injected into the contralateral eyes.
We captured fundus photographs from control, ROP and treatment groups and compared retinal vasculature and optic disc properties to evaluate ischemic damage to the eyes. All animals on day 17 underwent fundus examination by a slit lamp 78 diopter lens. Vascular tortuosity, variation in veins and arteries caliber, no polygonal vascular pattern, and the hazy appearance of the fundus were considered retinal vascular damage. Optic disc ischemic damage was defined by; edema of the optical disc (papilledema) and; enlarged, pale, and noneven boundaries of the optic disk (20).To capture fundus images, we used smartphone fundus photography. The pupil was dilated using mydriatic eye drop (atropine 0.5%). Examiner sat in a chair while holding a slit lamp 78 diopter lens in his hand, wearing a headlight. Proper alignment of the light source, lens, and animal's eye resulted in authentic aerial images recorded using a smartphone camera. This method captured fundus images containing well demonstrable optic disc and retina vasculature.
Full-field ERGs were recorded using the RETIanimal system (Roland Consult, Germany) and a Ganzfeld (Q 450 SC, Roland Consult, Germany). B and A waves amplitude were measured in both dark and adapted eyes. Briefly, neonatal rats in P17 were dark-adapted for 2 hours' and anesthetized by injection of ketamine/Xylazine and saline mixture (85/10 mg/kg). The pupils of rats were dilated with 1% atropine sulfate ophthalmic solution, and the mouse body temperature was maintained at 35°C using an electric heat pad. After sufficient anesthesia of the corneal surface with tetracaine 0.5% eye drop, 12mm Golden electrodes were placed on the cornea, skin, and tail.
ERG responses under dark-adapted conditions were evoked by 9 flashes ranging from 0. 01 Log cd.s/m2 to 10 Log cd.s/m2 in distinct low/high pass filters (Low-pass filter was 0.05 Hz and high-pass was 500 Hz). In order to prevent attenuating dark adaptation, flash series were shined from the lowest to the highest intensity. A and B waves were evaluated in scotopic and photopic states, respectively. The waveforms were averaged across each flash series, and the A-wave, and b-wave, were taken at 3 Log cd.s/m2 intensity for A and B waves in photopic conditions. Moreover, A-wave, B-wave recorded in scotopic condition with different intensities (-2, 0, 1 Log cd.s/m2 for B wave and 0, 1 Log cd.s/m2 for A wave). The A-wave values were calculated as the absolute value of the minimum amplitude following the flash stimulus, while the B-waves amplitude was calculated from baseline voltage (0 µV) to the peak of the response. Recorded waves were automatically analyzed, and waves' amplitude was measured by RETIanimal software.
We approved death by the absence of heartbeat in all animals euthanatized in p17 by an overdose of ketamine injection. Both animals' eyes were enucleated and immediately fixed in 10% neutral-buffered formalin with the fixative solution, which was replaced every two days until the tissues hardened. In addition, each eye was embedded in a paraffin block, and five µm sections parallel to the sagittal axis of the optic nerve were prepared and stained with hematoxylin and eosin (H&E). According to Wilkinson-Berka et al. Study (21), to score neovascular nuclei in the internal limiting surface of the retina, Slides were examined qualitatively in 40X magnification under a light microscope (Nikon. Japan). Endothelial cells or blood vessels were counted with full lumen, retina layers' thickness (inner nuclear layer, outer nuclear layer, and ganglion cell layer), apoptosis, and organization of layers were compared Qualitatively (22).
We used GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, California, USA) for statistical analysis and graphing. Kolmogorov–Smirnov test was used to confirm the normal distribution of data, and differences between groups were calculated using Anova and T-test. P values less than 0.05 were considered to indicate statistical significance. All values were expressed as the mean ± SEM.