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Neuroprotective effects of Acer palmatum thumb. leaf extract (KIOM-2015E) against ischemia/reperfusion-induced injury in the rat retina

Purpose The present study aimed to determine whether the administration of Acer palmatum thumb. leaf extract (KIOM-2015E) protects against the degeneration of rat retinal ganglion cells after ischemia/reperfusion (I/R) induced by midbrain cerebral artery occlusion (MCAO). Methods Sprague–Dawley rats were subjected to 90 min of MCAO, which produces transient ischemia in both the retina and brain due to the use of an intraluminal filament that blocks the ophthalmic and middle cerebral arteries. This was followed by reperfusion under anesthesia with isoflurane. The day after surgery, the eyes were treated three times (eye drop) or one time (oral administration) daily with KIOM-2015E for five days. Retinal histology was assessed in flat mounts and vertical sections to determine the effect of KIOM-2015E on I/R injury. Results A significant loss of brain-specific homeobox/POU domain protein 3A (Brn3a) and neuron-specific class III beta-tubulin (Tuj-1) fluorescence and a marked increase in glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) expression were observed after five days in the PBS-treated MCAO group compared to the sham-operated control group. However, KIOM-2015E treatment reduced (1) MCAO-induced upregulation of GFAP and GS, (2) retinal ganglion cell loss, (3) nerve fiber degeneration, and (4) the number of TUNEL-positive cells. KIOM-2015E application also increased staining for parvalbumin (a marker of horizontal cell associated calcium-binding protein and amacrine cells) and recoverin (a marker of photoreceptor expression) in rats subjected to MCAO-induced retinal damage. Conclusions Our findings indicated that KIOM-2015E treatment exerted protective effects against retinal damage following MCAO injury and that this extract may aid in the development of novel therapeutic strategies for retinal diseases, such as glaucoma and age-related macular disease.

Middle cerebral artery occlusion (MCAO) in rodents is one of the most common experimental paradigms for inducing focal cerebral ischemia [16][17][18]. Retinal ischemia induced by ligation or clamping of the ophthalmic artery is a reproducible model of central nervous system (CNS) stroke that is highly amenable to experimental manipulation [3,9]. Steele et al. reported that MCAO induces retinal ischemic damage because the ophthalmic artery originates from the internal carotid artery and is proximal to the origin of the middle cerebral artery (MCA) [19]. As the ophthalmic artery that provides the main supply to the inner retina originates from the internal carotid artery, occlusion of the MCA will simultaneously interrupt the vascular supply to the retina and the whole eye [20][21][22]. Previous studies have indeed demonstrated that MCAO results in retinal ischemia in rats [22][23][24]. Therefore, MCAO is a useful model for researchers who aim to understand the changes that lead to retinal damage in human patients [19,22] and may also aid in the development of novel therapeutic strategies for retinal ischemia.
KIOM-2015E is an extract of Acer palmatum Thumb. leaves. In this experiment, the hot water extract, KIOM-2015EW, and the ethanol extract, KIOM-2015EE, were used. Plants of the genus Acer (Aceraceae)-commonly known as maple-have been used extensively in the traditional treatment of various diseases in East Asia and North America [25]. Several research groups have investigated the biologic and pharmacological activities of these plants, which contain several phytochemicals and are known to possess antioxidant, anti-tumor, and anti-inflammatory properties [26][27][28]. Clinical studies have revealed that medicinal plants belonging to the Acer genus are highly effective in the treatment of rheumatism, bruises, hepatic disorders, eye disease, and pain and in detoxification [25]. Modern pharmacological studies have confirmed the traditional uses of these important Acer species. Arnason et al. reported the traditional use of several Acer plants by the native population of eastern Canada. For example, A. saccharum Marshall is used to treat shortness of breath, eye pain, and cataracts, while A. rubrum L. and A spicatum Lam. are used to alleviate soreness of the eye [29]. A. tataricum subsp. ginnala (Maxim.) Wesm., which has traditionally been used to reduce redness and swelling of the eyes, has further been observed to exhibit anti-inflammatory activity in vitro [25]. Recently, we demonstrated that KIOM-2015EW has anti-inflammatory and anti-apoptotic effects in a hyperosmolar stress-induced in vitro dry eye model [30].
Although plants of the Acer genus are widely used worldwide for medicinal purposes, until recently little research has been conducted regarding the use of these plants for the treatment of ocular disease. In the present study, we investigated the effect of the novel therapeutic agent KIOM-2015E-a natural extract obtained from Acer palmatum Thumb. leaves-in a rat model of MCAO-induced retinal ischemia injury. Our report is the first to describe the neuroprotective effect of KIOM-2015E in a rat model of retinal ischemic injury induced by MCAO.
Preparation of herbal extracts, KIOM-2015EW and KIOM-2015EE: Acer palmatum: Thumb. leaves were purchased from Korea Medicine Herbs Association (Yeongcheon, Korea), confirmed by Professor K i Hwan Bae of the College of Pharmacy at Chungnam National University (Daejeon, Korea), and then stored in the herbal bank of the Korea Institute of Oriental Medicine (KIOM). To prepare KIOM-2015EW, dried Acer palmatum Thumb. leaves (1700 g) were ground into a fine powder, soaked in 17 L distilled water, and then heatextracted in an extractor (Cosmos-600 Extractor, Gyeonseo Co., Inchon, Korea) for 3 h at 115 °C. To prepare KIOM-2015EE, the fine Acer palmatum Thumb. powder (1700 g) was soaked in 17 L of 25% ethanol and extracted in a shaking incubator at room temperature for 24 h. The KIOM-2015EE extract was filtered using standard testing sieves (150 µm, Retsch, Haan, Germany), following which it was concentrated to dryness in a lyophilizer. KIOM-2015EE powder (50 mg) dissolved in 1 ml of distilled water was filtered through a 0.22-µm disk filter and maintained at −20 °C before use.
Animals: Male Sprague-Dawley (SD) rats weighing 280±10 g (Orient Bio Inc., Gyeonggi-do, South Korea) were used for all experiments. The animals were housed under controlled environmental conditions in an ambient temperature of 23±1 °C and relative humidity of 50±10% under a 12-h light/dark cycle and were given ad libitum access to food and water. All animal experiments were approved by and performed in accordance with the guidelines of the Animal Care and Use Committee of KIOM (Daejeon, Korea; reference number: D-16-004).

MCAO-induced retinal ischemic injury in rats:
MCAO was induced in accordance with standard procedures (Longa et al., 1989). Briefly, rats were anesthetized with 100% oxygen containing 3% isoflurane. Rectal temperature was measured using a rectal probe and maintained at 37 °C with a heating pad (FHC Inc., Bowdoin ME) during the surgical procedure. The left common carotid artery was exposed, carefully separated from the vagus nerve, and ligated at the more proximal side via a right paramedian incision. The external carotid artery (ECA) was also ligated. Ischemia was induced by advancing the tip of a rounded 3-0 nylon suture into the internal carotid artery through the ECA. After placement, the intraluminal suture was secured by tying it around the ECA (90 min). Reperfusion was produced by withdrawing the intraluminal suture. In the sham group, the ECA was surgically prepared for the insertion of the filament, but the filament was not inserted.
Measurement of infarct volume: Following MCAO surgery and 5 days of reperfusion, the rats were sacrificed via decapitation. The brains were cut into coronal slices (2 mm thickness) with the aid of a brain matrix. The coronal brain slices were immediately immersed into 2% 2,3,5,-triphenyltetrazolium chloride (TTC; Sigma) for 10 min at 37 °C, following which they were fixed in 4% paraformaldehyde (PFA) solution (Prolabo, Paris, France) overnight before analysis. Brain slices were scanned individually, and the unstained area was analyzed using an image analysis system (Adobe Systems Inc., San Jose, CA). The areas of MCAO-induced ipsilateral cerebral infarction were determined based on loss of TTC staining. Areas of infarction were traced and analyzed using Image J software (NIH), version 1.32.

Experimental procedure for KIOM-2015EE application:
The rats were randomly divided into eight groups (15 rats in each group)-sham-operated control, MCAO+PBS vehicle, MCAO-KIOM-2015EE 1 mg/ml or 2 mg/ml for eye drops, KIOM-2015EE 100 mg/kg or 200 mg/kg for oral administration or eye drops, KIOM-2015EW 2 mg/ml for eye drop, and positive drug 20 mg/kg (Ginexin-F Tab, 80 mg, SK Chemical, Gyeonggi-do, Korea) for oral administration. Retinal damage was induced by MCAO operation in all rats, with the exception of the sham-operated control group. Beginning the day after MCAO ischemic injury, the right eye of each rat in the experimental groups was treated three times daily (09:00, 15:00, 21:00) with topical drops or once daily with the oral formula. On day 5, all rats were sacrificed. Eye tissues were carefully harvested for histological analysis after the following preparations.
Retinal flat mount: Flat mounting was performed in accordance with previously described methods [31]. All rats were euthanized, following which their eyes were harvested. Harvested eyes were rinsed in PBS and then fixed in 4% PFA for 15 min. Eyes were transferred into a Petri dish, and the lens, sclera, and choroid were removed. The cup-shaped retina was transferred to the plate, and four to five radial incisions were made. Methanol was added dropwise to the surface of the retina until complete coverage was achieved. When the retina turned white, it was placed in methanol for at least 20 min. Fixed retinas were maintained in methanol at −20 °C until staining.

Paraffin embedding and histological examination:
The collected eyeballs were fixed in 4% PFA in PBS for 24 h at 4 °C, following which they were washed twice in PBS. The samples were then dehydrated through an ethanol series, cleared by soaking in xylene, embedded in paraffin, and sectioned into 3-µm slices using a microtome (RM 2125RT; Leica Microsystems, Wetzlar, Germany). Slides containing paraffin sections were deparaffinized in xylene and rehydrated through an ethanol series. Hematoxylin and eosin (H&E) staining was then performed for histopathological examination. The specimens were mounted with Permount (Fisher, Fair Lawn, NJ), and images were captured using a Nikon fluorescence microscope equipped with NIS-Elements BR 4.50 software (Nikon, Tokyo, Japan).
Immunofluorescence staining of flat-mounted retinas: For immunofluorescence staining, the retinal tissues were removed from methanol, washed three times for 30 min each time in PBS, and transferred to glass slides. The specimens were permeabilized and blocked with 0.3% Triton-X/0.2% BSA (BSA)/5% normal goat serum in Tris-buffered saline (TBS; Perm/Block solution) for 1 h at 4 °C. The specimens were then incubated overnight with monoclonal antibodies against Tuj-1 or Brn3a and polyclonal antibodies against GFAP (diluted 1:50-100) in Perm/Block solution. Specimens were then washed three times in TBS, following which they were further incubated with the appropriate Alexa488-or 555-conjugated secondary antibodies at room temperature for 1 h. Specimens were again washed three times in PBS and stained with DAPI (Vectashield mounting medium).
Imaging the whole-mount retina for cell counts: Images were captured using a Nikon fluorescence microscope equipped with NIS-Elements BR 4.50 software (Nikon). Images from each retina were captured from an area located 100 μm from the optic nerve disc. For each condition, we randomly selected five images from among the captured photos and automatically measured the number of ganglion cells (Brn3a-positive cells) using ImageJ software. Total ganglion cell numbers were calculated as the average of three out of five measured results, excluding the minimum and maximum values.

Immunofluorescence staining of vertical retinal sections:
For immunofluorescence staining, sectioned retinal tissue slides were deparaffinized in xylene in a 65 °C incubator for 1 h, following which they were rehydrated through an ethanol series. The specimens were permeabilized with 0.3% Triton™ X-100 for 10 min and blocked overnight with 5% normal goat serum and BSA in TBS at 4 °C. The specimens were then incubated overnight with monoclonal or polyclonal antibodies against glutamine synthetase, PKCα, parvalbumin, and recoverin (diluted 1:50-100) in 5% BSA. They were then washed three times in TBS, further incubated with the appropriate Alexa Fluor® 555-conjugated secondary antibodies at room temperature for 1 h, washed again three times with PBS, and stained with DAPI (Vectashield mounting medium). Images were captured using a Nikon fluorescence microscope equipped with NIS-Elements BR 4.50 software (Nikon). Red-positive cells were viewed at excitation and emission wavelengths of 543 nm and 633 nm, respectively. The density of red fluorescence was measured using the Color Histogram function of the ImageJ software program.
TUNEL assay: The specimens were subjected to a terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay using an in situ cell death detection kit and fluorescein (Roche Diagnostics, Cat. No. 11 684 795 910), in accordance with the manufacturer's instructions. The positive-control sample was incubated with DNase I (3000 U/mL in 50 mM Tris/HCl), and the negative-control sample was incubated with label solution only. TUNEL-positive cells were viewed using a Nikon fluorescence microscope equipped with NIS-Elements BR 4.50 software (Nikon). The total number of cells and number of TUNEL-positive cells were counted in five individual high-power fields in each of the five samples. All slides were read by an experienced scientist who was blinded to the evaluation, and the scores and average ratio of the number of TUNEL-positive cells to the total number of cells were calculated.
The analytical HPLC data were obtained using a Dionex Ultimate 3000 system equipped with a binary pump, autosampler, column oven, and diode array ultraviolet-visible spectroscopy (UV/VIS) detector (DAD). The output signal of the detector was recorded using the Chromeleon software of the HPLC system. Chromatographic separation was achieved on a Xbridge ® C 18 column (5 μm, 4.6 × 250 mm, Waters Co., Milford, MA) using trifluoroacetic acid (TFA) water (0.1%, v/v); solvent A, and acetonitrile. Solvent B was used as a mobile phase at a flow rate of 1 mL/min. The injection volume of each sample was 5 µL, and the column temperature was maintained at 40 °C. Briefly, the mobile phase consisted of water containing (A) 0.1% TFA and (B) acetonitrile with gradient elution at a flow rate of 1 mL/min. The HPLC elution condition was optimized as follows: 0-3 min, 5% B; Statistical analysis: Data were evaluated using one-way analyses of variance, followed by a Tukey's test. The analyses were performed using GraphPad PRISM software® (GraphPad PRISM software Inc., Version 5.02, San Diego, CA). The results are expressed as the mean ± standard error of the mean (SEM), and the level of statistical significance was set at p<0.05.

Animal models of MCAO-induced retinal ischemia injury:
Five days after MCAO, we examined tissue infarction in the ipsilateral hemisphere of the experimental rats. As shown in Figure 1A, infarct volume in the MCAO model group was significantly higher than that in the sham-operated group. Infarct volumes in the MCAO model group significantly differed from those in the sham-operated control group (0.86±0.13% and 31.55±1.162%; p<0.001, respectively; Figure  1A).

KIOM-2015E promotes RGC survival following MCAOinduced retinal ischemia injury:
Five days after MCAOinduced retinal ischemia injury, flat-mounted retinal tissues were stained with the RGC marker anti-Brn3a, and the captured photos were analyzed to determine the number of cells ( Figure 1B).  Figure 2B, topical eye drops containing KIOM-2015E were associated with a slight increase in cell number after MCAO injury, although this difference was not significant. In contrast, oral administration of KIOM-2015E markedly increased RGC protection in a dose-dependent manner, suggesting that oral administration of KIOM-2015EE was more effective than topical administration.

KIOM-2015EE prevents degradation of RGC nerve fibers in MCAO injury:
We also investigated whether KIOM-2015E inhibits RGC nerve fiber degradation via whole-mount immunohistochemical staining using the β-III tubulin (Tuj-1) antibody-a marker for RGCs and neurons-in rats with MCAO-induced retinal ischemia injury ( Figure 1C). In the sham-operated control retina, Tuj-1-positive nerve fibers exhibited numerous branches with thick lines connecting the optic disc to the outer region of the retina. However, in the MCAO+PBS vehicle group, Tuj-1-positive nerve fibers underwent degenerative changes. The few remaining nerve fibers in the MCAO+PBS vehicle group were very weak and exhibited few connections in the optic disc region. .76, p<0.01, respectively) exerted greater neuroprotective effects against damage due to retinal ischemia than the use of topical eye drops, and these effects were concentration-dependent. Taken together, our results suggest that KIOM-2015EE administration following MCAO injury has a neuroprotective function.

KIOM-2015E decreases GFAP expression in MCAO injury:
Expression of GFAP serves as a marker of developmental processes and as an indicator of gliosis in response to injury [32]. GFAP is found in two glial cell types, astrocytes and Müller cells. We investigated whether KIOM-2015E regulates activation of GFAP in flat-mounted retinal tissue using immunoreactive staining for GFAP (Figure 2A). In the shamoperated control group, GFAP-positive fluorescence appeared uniformly scattered throughout the retina, although GFAP was expressed at low levels (100%±5.23). In contrast, GFAP-posi-

Treatment with KIOM-2015E attenuates the loss of inner retinal neurons and histological changes in photoreceptor cells following MCAO injury:
Changes in other cells of the retina were observed using specific antibodies to each, such as PKCα, parvalbumin, and recoverin as markers for bipolar cells, amacrine cells, and photoreceptors, respectively ( Figure  4). The number of bipolar cells was significantly lower in the MCAO injury group than in the sham-operated control group (sham-operated: 55.0349±5.97%; MCAO+ PBS vehicle: 33.96±5.96, p<0.001, respectively). No obvious difference in the staining pattern for bipolar cells was noted between ischemic and KIOM-2015E-treated retinas ( Figure 4A, upper panels; Figure 4B). Amacrine cells were labeled using antiparvalbumin, and photoreceptor cells were labeled using anti-recoverin ( Figure 4A, middle and lower panels). In the retina of sham-operated control rats, strong binding to the

DISCUSSION
In the present study, we investigated the neuroprotective effects of KIOM-2015E against MCAO-induced ischemic retinal injury in rats. As shown in Figure 1, these neuroprotective effects were more pronounced in animals treated via oral administration than in those treated with topical eye drops. Significant neuroprotective effects were observed on RGCs and their nerve fibers, cells of the inner retina, amacrine cells, and photoreceptor cells following treatment with eye drops containing high concentrations of the ethanol extract ( Figure 1, Figure 3, and Figure 4) [34].
Retinal I/R is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions [35]. Following the induction of retinal ischemia, approximately 50% of RGCs die within the first 2 weeks after stroke [36]. Retinal ischemia is also a common cause of visual impairment and blindness [3]. Steele et al.. reported that MCAO is a sufficient method for inducing ischemic damage in the retina [19]. In our MCAO model, reperfusion was performed following 90 min of ischemic stress, which resulted in a 30% infarction in the brain after 5 days. Similarly, I/R injury produced a 36% loss of RGCs ( Figure 1A, B), and Tuj-1 staining revealed that the level of nerve fiber expression had decreased to 39% of that observed in shamoperated control group ( Figure 1C).
The MCAO procedure exerts similar damage on the brain and retinal tissues. As the retina is an extension of the diencephalon, retinal blood vessels share similar anatomic and physiologic properties with those in the brain and possess a blood-retinal barrier analogous to the blood-brain barrier [34].
There are two types of macroglial cells in the mammalian retina, astrocytes and Müller cells [37]. Astrocytes appear mainly in the optic nerve head and migrate to the nerve fiber layer and GCL [38]. They are restricted to the vitreous surface of the retina, where they are located largely within the nerve fiber layer [39]. In contrast, the Müller glial cells are specialized radial glial cells that extend from the inner limiting membrane to the outer liming membrane, providing functional and structural support to retinal neurons [40]. GFAP is an intermediate filament protein found in astrocytes and in Müller glial cell end-feet and processes. Although Müller glial cells in the normal rat retina express little or no GFAP [41], such cells exhibit increased GFAP expression in retinal injuries, including ischemia [42] and glaucoma [43]. In retinal diseases associated with I/R injury such as glaucoma, Müller cells and astrocytes display a hypertrophic morphology and an upregulation of GFAP [41,44]. Under normal conditions, Müller cells protect RGCs by releasing neurotrophic factors and secreting glutathione, which exerts antioxidant effects [40,44,45]. However, activated Müller cells negatively affect RGC survival in the ischemic retina [44,46,47]. GS is exclusively expressed in the soma of Müller glial cells [48]. These are the best reflections of altered glial reactivity [49,50].
To investigate changes in macroglial cell activity, we examined the expression of GFAP and GS in retinal sections obtained 5 days after MCAO injury (Figure 2A,B). As shown in Figure 2A, GFAP-positive staining was ubiquitously scattered throughout the flat-mounted retina, although the level of staining was low. However, following MCAO injury, GFAP expression significantly increased throughout the flatmounted retina. Increased GFAP expression due to MCAO injury was reduced to almost normal levels following the application of KIOM-2015EE. In rats with MCAO injury, GS expression was also strongly increased throughout the layers of the vertically sectioned retinal tissue ( Figure 2B). Macroglial activation is a key component of retinal neurodegeneration. Activation of two glial cell marker proteins by I/R injury was reduced by the application of KIOM-2015EE, which may have contributed to the preservation of retinal function. However, it is unknown whether MCAO-induced increases in GFAP and GS expression occurred immediately after surgery or gradually. Similarly, it remains unknown whether drug application produced effects on the first day or gradually. Therefore, future studies should compare the results of drug application among all 5 days. Nevertheless, our study demonstrates that the application of KIOM-2015EE reduces the activation of glial cells associated with I/R injury. Moreover, these effects were much more pronounced following oral administration than following the application of topical eye drops.
Recent advances in the field of neuroprotection indicate that healthy neurons can be protected from injury-and that damaged neurons can be rescued from death-by blocking specific steps in the cell death cascade [51]. Herbal products have been used in the treatment of various conditions, such as Parkinson's disease, coronary artery disease, cardiovascular disease, and traumatic injuries [52,53]. Romano [55]. The major component of this extract is isoquercitrin, which holds promise for the treatment of glaucoma but requires further study [52,55]. Matteucci et al. have also reported that curcumin, a phenolic extract obtained from Curcuma longa, exerted protective effects against NMDA excitotoxicity in both retinal and hippocampal neurons in primary retinal cell cultures [52,56]. Among the compounds identified in KIOM-2015EW and KIOM-2015EE, the major flavonoids were C-glycosyl flavones (orientin, isoorientin, and vitexin). Orientin, isoorientin, and vitexin are components of many natural plant extracts, and many other research groups have reported their anti-inflammatory [57][58][59], antioxidant [60,61], and neuroprotective effects [62][63][64][65][66][67][68]. Because these compounds are included in KIOM-2015EW and KIOM-2015EE, our findings support the notion that they exert neuroprotective effects in MCAO-induced retinal ischemia injury. Therefore, further studies will need to identify the neuroprotective effects of each of the above compounds.
In conclusion, the present study is the first to demonstrate the neuroprotective effect of KIOM-2015E on retinal cells following I/R and the regulation of macroglial cells in neurologic diseases. Taken together, our findings may aid in the development of novel therapeutic strategies against MCAO-induced retinal ischemia injury and other diseases associated with the degeneration of retinal cells, such as glaucoma, diabetic retinopathy, and age-related macular degeneration.

ACKNOWLEDGMENTS
This study was supported by Korea Institute of Oriental Medicine grants (K18101 and KSN1812101). We thank Young Jeung Park, director of Central Ophthalmic Clinic (Daegu, Korea) for advice on retinal disease.