Neuroprotective effect of minocycline on rat retinal ischemia-reperfusion injury

The retinal ischemia-reperfusion injury model

Adult male Sprague-Dawley rats, weighing 250±10.0 g, were obtained from the Animal Center of Wenzhou Medical University. All animals were housed in an environment with a temperature of 22±1.0 °C, relative humidity of 50±1.0%, and a 12 h:12 h light-dark cycle. All animal studies (including the rat euthanasia procedure) were performed in compliance with the regulations and guidelines of Wenzhou Medical University institutional animal care and conducted according to the American Association for Accreditation of Laboratory Animal Care (AAALAC) and Institutional Animal Care and Use Committee (IACUC) guidelines.

The ischemia-reperfusion injury model was established as follows: Animals were anesthetized with an intramuscular injection of 2% pentobarbital sodium (40 mg/kg bodyweight). Ischemia was induced in the right eyes by increasing the intraocular pressure (IOP). The anterior chamber was infused with sterile normal saline (NS) through a 30-gauge needle connected to a pressure transducer (Geuder®, D-69,126, Heidelberg, Germany) and an NS container via a three-way connector. The anterior chamber pressure was set to 110 mmHg by adjusting the height of the NS container. During the ischemia, the retina paled, and the fundus vasculature was narrowed, as observed with fundoscopy. Increased IOP was sustained for 60 min. Retinal edema was observed with optical coherence tomography (OCT) and indirect ophthalmoscopy.

The sham group was treated by inserting a 30-gauge needle into the anterior chamber through the corneoscleral junction, but not IR. Animals were handled in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Visual Research.

Drug delivery and biomicroscopy observation

The experimental design involved two main groups of rats: the minocycline-treated group (Mino group) and the NT (Control) group that received no treatment. Specifically, the Mino group received intraperitoneal (IP) injections (twice daily for 14 days; initial dosage of 45 mg/kg 1 day before ischemia and then 22.5 mg/kg every 12 h), while the NT group received the same amount of normal saline. In addition, a sham group received normal saline and underwent sham surgery, as described above. All surgical animals were monitored with slit-lamp microscopy and fundoscopy to exclude ones that had complications caused by the operation, such as traumatic cataract, corneal opacity, and end ophthalmitis.

Morphology: Histology and optical coherence tomography

To investigate the effect of minocycline on the IR-induced morphological and retinal thickness changes, we performed OCT and image hematoxylin and eosin (H&E)-stained paraffin sections at 3 and 14 days after reperfusion. At a predetermined time (immediately and 3 and 14 days after reperfusion), six rats were anesthetized using 2% pentobarbital sodium (40 mg/kg bodyweight); both pupils were dilated using a single drop of 0.5% tropicamide and 0.5% phenylephrine eye drops (Santen Pharmaceutical Co., Ltd., Osaka, Japan). Rats were placed and fixed with an examiner to ensure that their eyeball was orthophoric, and the scan beam was perpendicular to the corneal surface. Spectralis HRA OCT (Heidelberg Engineering, Heidelberg, Germany) was used in the examination. Normal saline was administered to eyes every 2–3 min during the examination to keep the corneas lubricated. The retina was directly visualized during centering, and the optic nerve head was used as a landmark and located as the center while OCT images were taken (Software Version 6.3a). For each eye, one coronal image (right to left) and one vertical image (bottom to top) were collected crossing through the center of the optic nerve (ON), with four locations chosen at 0.5 mm intervals from the optic nerve for each image (i.e., the coronal image as shown in Figure 1), resulting in a total of eight locations for each eye. The resultant images were analyzed with ImageJ software to measure the retina’s thickness at the locations described above. Corresponding neural retina thickness at the locations described above was measured by the distance from the ganglion cell layer’s vitreal face to the apical face of the RPE as the whole retinal thickness.

For histology, six rats of each group were euthanized by intramuscular injection of 2% pentobarbital sodium (60 mg/kg body weight) at 3 and 14 days after reperfusion. Their eyes were enucleated and oriented by perpendicularly incising the wall toward the corneoscleral junction at the 3 o’clock location, and then fixed in an admixture of formalin, dehydrated alcohol, and glacial acetic acid (formalin, alcohol, and acetic acid [FFA] fixation fluid) for 24 h. The globes were embedded in paraffin and dissected vertically through the optic nerve head in 5 μm serial sections, which were stained with H&E. Histopathologic images were also analyzed with ImageJ software and measured for eight locations chosen at 0.5 mm intervals from the optic nerve for each image using the same method as the OCT images. The differences between them were the locations and the retinal layers. There were two more locations for the histology analysis than OCT in the perpendicular, all taken at 0.5 mm intervals. Because the histopathologic images were much clearer than OCT, the thickness of the different retinal layers was measured, including the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL; RNFL + GCL), inner plexiform layer (IPL), and inner nuclear layer (INL).

Full-field ERG

After reperfusion for 1 and 2 weeks, six rats from each group were used for full-field electroretinography (ERG) examination with the International Society for Clinical Electrophysiology of Vision (ISCEV)-ERG program. We adopted the RETIport system with a custom-built Ganzfeld dome (Roland Consult, Wiesbaden, Germany), which was used to record standard full-field ERG. The pupils were dilated with a single drop of 0.5% tropicamide and 0.5% phenylephrine eye drop solution (Santen Pharmaceutical Co., Ltd., Osaka, Japan) and left to adapt in the dark for 2 h. Under dim red light, the animals were anesthetized as described above. A small amount of 2.5% methylcellulose gel was then applied to each eye, and a copper loop electrode designed specifically for rats was placed on the cornea to record the ERGs. Needle reference and ground electrodes were inserted into the cheek and tail, respectively. Dark-adapted ERG responses, including a rod response (dark adapt 0.01 ERG), standard combined ERG (dark adapt 3.0 ERG), and oscillatory potentials (OPs), were then recorded. The animals were allowed to adjust to the light for 5 min under a light intensity of 25 cd/m² and light-adapted responses, including a cone response (light adapt 3.0 ERG) and 30-Hz flicker. For each of the five stimuli programs, the average amplitudes and implicit times for each group were counted and analyzed.

TUNEL assay

Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay was used to detect apoptotic cells. Twelve hours and 24 h after reperfusion were chosen as the time points for the TUNEL assay based on the preliminary data. Six animals were euthanized at each time point. The globes were quickly enucleated and oriented in the same way as for the frozen section. The globes were fixed in 4% paraformaldehyde (PFA) in 0.1 M PBS (1X; 120 mM NaCl, 20 mM KCl, 10 mM NaPO₄, 5 mM KPO₄, pH 7.4) buffer, for 24 h and dehydrated in 30% sucrose solution in 0.01 M PBS buffer for 4 h. The eyeball wall was sectioned serially at a thickness of 10–12 μm vertically crossing 6 and 12 o’clock through the optic nerve head using a cryostat. Six sections of each animal eye were chosen for TUNEL assay and the TUNEL Apoptosis Detection Kit (fluorescein isothiocyanate [FITC]; PH1456, Phygene Life Science, Fuzhou, China). The eyeball wall sections were digested with proteinase K at 37 °C for 30 min and then reacted with equilibration buffer at room temperature for 20 min. The tissue was reacted with a mixture of terminal deoxynucleotidyl transferase and FITC-dUTP buffer at 37 °C for 1 h. After several washings with PBS, the slides were stained with 4',6-diamidino-2-phenylindole (DAPI; PH0524, Phygene Life Science). Images were acquired with a confocal microscope (Zeiss LSM 710, Jena, Germany). The quantities of the apoptotic cells of the whole retinal layer of every section for each tissue were manually counted in a controlled blind manner and then were statistically analyzed.

Retrograde labeling and counting of the RGCs and retinal blood vessel staining

Because retinal IR can induce not only the loss of retinal ganglion cells (RGCs) but also impairment of retrograde axonal transport, retrograde labeling is an excellent way to evaluate the quantity of the remaining RGCs, which have the function of retrograde axonal transport after IR. In this study, the animals were anesthetized and prepared by shaving the skin 7 days after retinal refusion. According to the Rat Brain in Stereotaxic Coordinates [19], the operator incised the skin over the skull and located the superior bilateral colliculi, which were located 6.3 mm posterior to the bregma and 1.5 mm lateral to the midline on both sides. After skull trepanation, 2 μl 10% 1,1’-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, cat. no. 42,364, Sigma, St. Louis, MO) in dimethyl sulfoxide was slowly injected to a depth of 4.3 mm below the bony surface. The needle was left there for 5 min and then slowly withdrawn, after which the skin was sutured.

Seven days after the retrograde labeling operation, the rats were euthanized, and the globes were quickly enucleated and orientated. The anterior segment and vitreous were removed after fixation with 4% PFA and dehydration with a 30% sucrose solution. The retina was divided equally into four quadrants and incubated at 4 °C overnight in lectin from Bandeiraea simplicifolia BS-I isolectin B4 (IB4) FITC conjugate (1:100 dilution; cat. no. L2895, Sigma, St. Louis, MO) to visualize the retinal vasculature. After washing with PBS, the retinas were stained with DAPI. Finally, the retina was mounted flat on a slide for subsequent observation and imaging.

Using the same method as that for obtaining the histopathological sections to find the locations for imaging, we took images for each retinal quadrant at the positions from the ON at 0.5 mm intervals (i.e., from 0.5 mm, 1.0 mm, 1.5 mm, and 2.0 mm). Sixteen images were captured for each retina. The Z-stacks of images collected over all depths, on which all positive cells appeared with 200X magnification with the Zeiss LSM 710 confocal microscope, were projected as one composite image to include all labeled cells in the image. Two investigators counted the number of RGCs in each image in a blind manner, and the final data were averaged.

RNA extraction and real-time PCR

At 12 and 24 h after reperfusion, six rats from each group were euthanized, and their retinas were removed and stored at −80 °C for subsequent analyses. Total RNA was extracted from retinal tissues using TRIzol reagent (Invitrogen, Carlsbad, CA), dichloromethane (1,024,451,000, Sigma) and 2-propanol (I9516, Sigma), and then analyzed for purity with a NanoVue^TM ultraviolet/visible spectrophotometer (28,956,057, GE Healthcare, St. Louis, MO). Consequently, 0.2 mg of total RNA from each sample was reverse transcribed with M-MLV reverse transcriptase into first-strand cDNA according to the manufacturer’s instructions (Promega, Madison, WI). The primers were designed using Oligo 7.0 software; the sequences and product lengths are listed in Table 1. PCR reactions were performed in the Applied Biosystems 7500 Real-Time PCR System with 26SYBRH Green PCR Master Mix (PH1456, Phygene Life Science). The condition is 30 cycles of 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 30 s. The results were normalized using the housekeeping gene GAPDH as a reference with the mRNA levels standardized in each group. For relative comparison, the cycle threshold (Ct) of the real-time PCR data was first converted to 2^-ΔCt before statistical analyses.

Western immunoblotting

The eyes of six rats were enucleated at 24 h after reperfusion. The retinas were peeled from the eyecup and immediately homogenized with 0.5 ml ice-cold lysis buffer. Insoluble material was removed with centrifugation at 120,000 xg at 4 °C for 20 min. According to the manufacturer’s specifications, the final protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL). GFAP (1:500 dilution in blocking solution), NRF2 (1:1,000) IL-6 (1:500), and IBA-1 antibodies (1:500), and HIF-1α (1:500) were used for immunodetection, and an anti-GAPDH polyclonal antibody was used to serve as a control to ensure that equivalent quantities of proteins were used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Protein bands were digitally analyzed with Quantity One 1-D Analysis Software (BioRad, Richmond, CA).

Statistical analysis

To compare the difference among groups at different time points, multivariate analysis of variance (MANOVA) tests were used to analyze the data of the retinal thickness measured with H&E staining, OCT, and ERG. One-way ANOVA tests were used to analyze the number of retinal apoptotic cells and the number of RGCs. For PCR, a two-sample Student t test was used. All data were displayed as the mean ± standard error of the mean (SEM). A p value of less than 0.05 was considered statistically significant.

Minocycline prevents morphologic changes in the rat retina, including the formation of blood vessels and retinal thickness after IR injury

The funduscopic examination (Figure 2) revealed hyperplasia, circuity, and dilation 24 h to 2 weeks after IR. However, reduced circuity and dilation were found in the Mino group compared to the NT group (Figure 2). This result was further confirmed with morphologies analysis of the small vessels (Figure 2D–F). Moreover, the NT group’s fluorescence leakage suggested that the blood–retinal barrier was damaged after modeling (Figure 2E). In comparison, there were no fluorescence leakage and less vessel circuity and dilation in the Mino group (Figure 2D). These data suggested that minocycline prevents morphologic changes of the rat retina, including blood vessels induced by IR injury. There was no difference between the fellow eyes of two groups in the fundus photographs and the retinal vessel photographs, which suggested minocycline itself could not induce the retina abnormality.

Next, H&E staining and OCT were used to evaluate the morphology and changes in retinal thickness between groups (Figure 3 and Figure 4). The OCT photographs showed that retina edema and retinal detachment were seen immediately after IR (as the red arrow points out in Figure 4A,B) in the Mino group and the NT group. Compared to the sham surgery eye (Figure 3A), each retinal layer of the NT group at 3 days after IR was edematous (Figure 3C), while no retinal edema was observed in the Mino group (Figure 3B). At 14 days after IR, the retinas of the NT rats (Figure 3E and Figure 4F) were obviously thinner (especially the NFL, the GCL, and the INL) compared to those of the Mino group (Figure 3D and Figure 4F). These results suggested that minocycline may reduce rat retina edema at the early stage of IR and reduce retina atrophy (to some extent) at the late stage of IR injury.

Figure 5 shows the retinal thickness of different locations by measuring H&E staining and OCT photographs of the sham group, the NT group, and the Mino group at 3 days and 14 days after IR. The average retinal thickness measured with the H&E staining images was as follows: The sham group at 3 days was 97.4±2.00 μm, the Mino group at 3 days was 96.1±5.10 μm, the NT group at 3 days was 124.5±1.400 μm; the Mino group at 14 days was 97.4±2.10 μm, and the NT group at 14 days was 74.0±1.00 μm. The results of the respective corresponding group measured with OCT were 203.6±5.800 μm, 205.1±1.800 μm, 223.1±2.900 μm, 195.5±2.700 μm, and 170.3±1.200 μm. The average retinal thickness of the Mino group at 3 days after IR was much thinner than that of the NT group measured using H&E staining (96.1±5.10 μm versus 124.5±1.400 μm, p<0.001) and OCT photographs (205.1±1.800 μm versus 223.1±2.900 μm, p<0.001), which suggested minocycline reduced the rat retina edema caused by IR. Similarly, the average retinal thickness of the Mino group at 14 days after IR was much thicker than that of the NT group measured in H&E staining (97.4±2.10 μm versus 74.0±1.00 μm, p<0.001) and OCT photographs (195.5±2.700 μm versus 170.3±1.20 μm, p<0.001), which suggested minocycline reduced the retina atrophy caused by IR at 14 days. Figure 5 also shows statistical differences of the retinal thickness corresponding to the same position of different locations between the NT and Mino groups by measuring H&E staining and OCT photographs. The results show statistically significant differences in the retinal thickness corresponding to the same position between the two groups at 3 and 14 days.

Furthermore, we measured the thickness of different retinal layers, including the RNFL and GCL (RNFL + GCL), IPL, INL, and ONL at different locations with H&E staining (Figure 6). The most obvious difference was the RNFL + GCL and IPL between the NT group and the Mino group at 14 days after IR (23.1±0.40 μm versus 16.3±0.40 μm, p<0.001). The thicknesses of the RNFL+GCL by measuring the H&E staining photographs of the sham group, the Mino group at 3 days, and the NT group at 3 days were 23.8±0.50 μm, 21.9±0.20 μm, and 30.3±1.10 μm, respectively. The thicknesses of INL for the sham group, the Mino group at 3 days, the NT group at 3 days, the Mino group at 14 days, and the Mino group at 14 days were 18.8±0.60 μm, 20.0±0.50 μm, 16.5±0.50 μm, 16.4±0.40 μm, and 12.7±0.90 μm, respectively. These results suggest that IR causes edema at the early stage and atrophy at the late stage in the retina layer, especially the inner layer (RNFL+GCL). At the same time, minocycline could prevent these morphologic changes.

Minocycline partially prevents the rat retina functional damage caused by IR on ERG

We adopted standard full-field ERG to evaluate any functional damage to the rat retina. The evaluating indicators included the b-wave amplitudes of dark-adapted 0.01, the a-wave amplitudes, the b-wave amplitudes of dark-adapted 3.0, the OS₂ wave of the dark-adapted 3.0 oscillatory potential ERG, the a-wave amplitudes and the b-wave amplitudes of light-adapted 3.0, the N1-P1 wave times, and amplitudes of light flare 30 Hz ERG.

Table 2 shows all the ERG measurement parameters and the statistical results between the Mino group and the NT group. In general, all wave amplitudes in the NT group were lower than those in the sham group, and the appearances of some ERG patterns in the NT group were called “extinct,” which means that all wave amplitudes were close to zero (Figure 7B). This appearance suggested that IR caused functional damage of the rat retina. In contrast, the wave amplitudes in the Mino group were higher than those in the NT group, and the two groups showed statistical differences, apart from the light flare 30 Hz ERG (Table 2), which suggested that minocycline may partially prevent the functional retina damage caused by IR in rats.

The dark-adapted ERG programs were used to test the functions of the retinal rod cells. In this study, we adopted the dark-adapted 0.01 ERG and the dark-adapted 3.0 ERG to evaluate damage of the retinal rod cells after IR. The results showed that all wave amplitudes of the dark-adapted ERG in the Mino group were much higher at 1 week after IR than in the NT group, and some were even higher than in the sham group, which suggested that minocycline can prevent damage of the retinal rod cells and excite the retinal rod cells. However, the wave amplitudes of the dark-adapted ERG in the Mino group decreased at 2 weeks after IR compared to 1 week. Nonetheless, they were still higher than in the NT group with statistical significance. The results suggested that minocycline could not completely prevent or perhaps just delayed the damage of rod cells caused by IR.

The dark-adapted 3.0 oscillatory potential ERG is a relatively sensitive indicator that reflects the function of retinal microcirculation [20]. In this study, the OS₂ wave amplitudes had a statistically significant difference between the Mino group and the NT group at both time points, which suggested that minocycline prevents retinal microvascular damage caused by IR.

In the ERG program, the retinal cone cells system’s functions were always tested by the light-adapted ERG programs, including the light-adapted 3.0 ERG and the light flare 30 Hz ERG. The results of light-adapted 3.0 ERG were similar to those of the dark-adapted 3.0 oscillatory potential ERG; the wave amplitudes in the Mino group were higher than those in the NT group (p<0.05) and lower than those in the sham group, yet without statistical significance, which suggested minocycline may partially prevent retinal cone cell damage caused by IR. Furthermore, the b-wave amplitude in the light-adapted 3.0 ERG of the Mino group was lower than that in the sham group at 1 week and turned to normal at 2 weeks, which was different from the dark-adapted ERG programs. However, the wave amplitudes of P1-N1 in the Mino group for the light flare 30 Hz ERG at 1 week and 2 weeks were lower than in the sham group, but without statistical significance, thus suggesting that the IR damage to the retinal cone system is not obvious on the light flare ERG. Overall, minocycline suspended the functional damage of the retinal cell system and microcirculation caused by IR.

Minocycline prevents retinal cell apoptosis caused by IR

In the preliminary experiment, six time points (3 h, 6 h, 12 h, 1 day, 3 days, and 7 days) after IR were selected for TUNEL assay measurements. We observed that retinal apoptotic cells appeared at 12 h and 24 h after IR. Consequently, 12 h and 24 h were further analyzed. As shown in Figure 8, the results showed fewer retinal apoptotic cells in the retina of the Mino group at 12 h (Figure 8A) and 24 h (Figure 8D) after IR compared to the NT group (12 h: Figure 8B and 24 h: Figure 8E). Apoptotic cells were mainly observed in the ganglion cell layer and the inner nuclear layer. Figure 8G shows the quantitative results (the average cell numbers of each visual field) of retinal apoptotic cells for each group. TUNEL-positive cells were counted in the retina 12 h and 24 h after IR. There was a statistically significant difference in the TUNEL-labeled cells between the Mino group and the NT group at 12 h after IR (7.97±1.06 cells/field versus 22.72±1.660 cells/field, p<0.001). At 24 h after IR, the difference between the Mino group and the NT group was decreased (12.33±2.380 cells/field versus 17.94±0.880 cells/field, p<0.01). To sum up, these results suggested that minocycline reduces retinal apoptosis caused by IR.

Minocycline protects RGCs against damage caused by IR

Evaluation of the survival of RGCs was performed with retrograde labeling. The RGCs labeled with DiI were counted at 14 days following retinal IR injury with or without minocycline treatment. Figure 9 shows pictures of retrograde-labeled RGCs in the retina; the red cells are the RGCs labeled with DiI, which had a good retrograde axonal transport activity. As shown in Figure 9, the number of labeled RGCs in the Mino group (Figure 9B,E) were higher than the NT group (Figure 9C,F).

To more accurately evaluate the surviving RGCs, the average cell number of retrograde labeled RGCs for each group was calculated (Figure 9G). There was a statistically significant difference in the number of RGCs between the Mino group and the NT group (179.81±19.090/field versus 128.00±16.720/field, p<0.01). These results suggested that minocycline protects RGCs against damage caused by IR.

Minocycline affects alterations of retinal gene expression after IR

The TUNEL assay results indicated that minocycline prevents retinal cell apoptosis caused by IR. Next, we hypothesized that minocycline might affect the retinal gene expression of inflammatory factors during the acute phase. We compared the gene expression levels of iNOS, TNF-α, IL-1α, IL-6, Bcl-2, Bax, caspase-3, GFAP, Iba-1, Hif-1α, and Nrf2 in the Mino group and the NT group after IR (Figure 10). In addition, we compared the protein expression level of GFAP, NRF2, IL-6, IBA-1, and HIF-1α in the Mino group and the NT group 24 h after IR (Figure 11). Because the fellow eye (the left eye) of each group did not receive IR injury, we assumed the retinal gene expression of the fellow eye was normal. We then quantified each group’s value by obtaining the ratio of the experimental eye to the corresponding fellow eye.

As shown in Figure 10A, iNOS gene expression at 12 h and 24 h after IR of the NT group was 11.12±2.16-fold and 13.39±4.840-fold that of the corresponding fellow eyes (n = 5), while the corresponding expression of the Mino group was 0.81±0.46-fold and 3.28±1.38-fold (n = 5). The expression level in the Mino group decreased compared to that of the NT group, and the differences were statistically significant at 12 h and 24 h after IR (p<0.001 and p<0.01). iNOS is a type of enzyme that catalyzes nitric oxide (NO) from L-arginine. NO is an essential cellular signaling molecule, which helps modulate vascular tone. However, NO may be responsible for the degeneration of axons of retinal ganglion cells in the glaucomatous optic nerve head [21]. Thus far, many studies have confirmed that NO is harmful to retinal cells in ischemia and reperfusion [22]. Thus, minocycline may protect RGCs in IR by regulating iNOS gene expression.

Several inflammatory genes increased statistically significantly at 12 h and 24 h after IR compared to the corresponding fellow eye. These genes included TNF-α (Figure 10B), IL-1α (Figure 10C), and IL-6 (Figure 10D), and their ratios of the experimental eye to the fellow eye were much larger than 1 at 12 h and 24 h after IR. However, compared to the NT group, the expression of TNF-α of the Mino group at 12 h and 24 h was much lower (p<0.05), which suggests that minocycline downregulates the expression of TNF-α. Moreover, the expression of IL-1α at 24 h and IL-6 at 12 h between the Mino group and the NT group differed (p<0.05). The protein expression of IL-6 of the Mino group was lower than that of the NT group (p<0.05; Figure 11). These results suggested minocycline decreased the damage by downregulating the expression of inflammatory genes at the early stage after IR.

To test whether minocycline can inhibit the activation of glial cells, we detected the expression of GFAP, a marker of microglia cells, and Iba-1, a marker of a microglial cell. As shown in Figure 10E,F, minocycline can inhibit the activation of macroglia cells and microglia cells at 12 h and 24 h after IR. For GFAP gene expression, the inhibitory effect of minocycline was more statistically significant at 12 h after IR (Mino group versus NT group: 3.48±1.77 versus 22.59±8.140, p<0.001), whereas for Iba-1, the inhibitory effect of minocycline was much more statistically significant at 24 h after IR (Mino group versus NT group: 2.98±1.79 versus 226.06±13.73, p<0.001). The western blotting results further verified the changes from protein expression, as shown in Figure 11.

Hif-1α is overexpressed when cells trigger an adaptive response to hypoxia conditions. Interestingly, as shown in Figure 10G, the Hif-1α gene expression of the model eyes at 12 h and 24 h after IR was much lower than in the corresponding fellow eyes (12 h: 0.2601±0.1319-fold; 24 h: 0.021±0.008-fold). Minocycline statistically significantly abated the low expression of the Hif-1α gene at 12 h and 24 h after IR (12 h: 0.5259±0.2035-fold, p<0.05; 24 h: 0.2868±0.1078-fold, p<0.001). Moreover, the corresponding protein expression of Hif-1α in the Mino group at 24 h was lower than that in the NT group, as shown in Figure 11.

Nrf2 is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes [23]. As shown in Figure 10H, Nrf2 genes in the Mino group and the NT group were similarly highly expressed 12 h after IR (Mino group versus NT group: 2.55±0.5825-fold versus 3.53±0.71-fold). Twenty-four hours after IR, minocycline statistically significantly increased the expression of the Nrf2 gene (Mino group versus NT group: 57.61±19.46-fold versus 26.65±9.91-fold, p<0.05).

According to the TUNEL assay results, IR can cause retinal cell apoptosis; therefore, we detected the expression of genes, including Bcl-2 (Figure 10I), Bax (Figure 10J), and caspase-3 (Figure 10L), associated with cell apoptosis. The Bax and caspase-3 genes were overexpressed at 12 h (14.40±1.94-fold and 3.67±1.45-fold) and 24 h (1.99±0.44-fold and 2.86±1.18-fold) after IR in the NT group. Bcl-2, an antiapoptosis gene, remained at low levels after 12 h (0.21±0.11-fold) and 24 h (0.17±0.07-fold) after IR, which was consistent with the TUNEL assay. In the Mino group, caspase-3 gene expression was lower than in the NT group at the corresponding time points (12 h: 1.18±0.51-fold, p<0.01; 24 h: 0.99±0.43-fold, p<0.05), which decreased statistically significantly compared with the NT group. The ratio of the Bax to Bcl-2 expression is more meaningful for evaluating cell apoptosis; thus, the Bax/Bcl-2 ratio was used to evaluate the expression of the two genes. Twelve hours after IR, the expression of Bax/Bcl-2 (Figure 10K) in the Mino group was much higher than in the NT group (68.73±8.780-fold versus 10.39±2.570-fold, p<0.01).