The protocol of all experimental procedures was approved (approval no., KW-2000113-1) on 7th Feb. 2020 by the Ethics Committee of Kangwon National University (Chuncheon, Gangwon, Korea). Animal handling stuck to the “Guide for the Care and Use of Laboratory Animals” [20]. In addition, all efforts were made to reduce pain in the animals and minimize the numbers of the animals used.

Adult male gerbils at six months of age (body weight, 72–78 g) were used for this study. The gerbils were obtained from the Experimental Animals Center of Kangwon National University (Chuncheon, Republic of Korea). The gerbils were housed in pathogen-free environment under standard laboratory conditions (temperature, 23 $\pm$ 0.5 ${}^{\circ }$C; relative humidity, 55 $\pm$ 5%) on 12:12 hour light–dark cycle.

A total of 126 gerbils were blindly and randomly divided into eight groups as follows: (1) sham IR plus (+) vehicle group (Sham+vehicle group; n = 14) was subjected to sham IR operation, treated with vehicle (saline), and sacrificed at zero and 5 days after sham IR operation; (2) IR+vehicle group (n = 21) was given IR operation, treated with vehicle, and sacrificed at two and five days after IR operation; (3), (4) and (5) sham+25, 50, and 100 mg/kg PYC group (n = 14, respectively) was subjected to sham IR operation, treated with 25, 50, and 100 mg/kg of PYC, respectively, and sacrificed at zero and five days after IR; (6) and (7) IR+25 and 50 mg/kg PYC group (n = 14 respectively) was given IR operation, treated with 25 and 50 mg/kg of PYC, respectively, and sacrificed at five days after IR operation and (8) IR+100 mg/kg PYC group (n = 21) was given IR operation, treated with 100 mg/kg PYC, and sacrificed at two and five days after IR operation.

IR injury was induced in the forebrain containing the hippocampus by occlusion of bilateral common carotid arteries in accordance with previously described method [15]. In brief, all gerbils were anesthetized with 2–2.5% isoflurane (657801261, Hana Pharm. Co., Ltd., Gyeonggi-Do, Korea) using an inhaler. A middle incision was made in the ventral surface of the neck, the common carotid arteries were isolated from the carotid sheath and clamped with aneurysm clips for five minutes, and the clips were removed for reperfusion. Complete blockage and reperfusion of arterial blood was observed by observing blood flow in the central retinal arteries using HEINE K180 ophthalmoscope (C-182.27.388, Heine Optotechnik, Herrsching, Germany). For body temperature, normal temperature (37 $\pm$ 0.5 °C) was controlled using thermometric blanket using TR-200 rectal temperature probe (21052-00, Fine Science Tools, Inc., Foster City, CA, USA). The gerbils in all sham groups received the same surgery without occluding the arteries.

PYC was purchased from Horphag Research Ltd. (1049951, Horphag Research Ltd., Geneva, Switzerland), and PYC (25, 50, and 100 mg/kg in saline, respectively) or saline was intraperitoneally administrated immediately, at one day and two days after IR, respectively (Fig. 1).

Fig. 1.

Experimental timeline RAMT was daily conducted for three days before and for five days after IR induction. PAT was carried out at one day before and five days after sham and IR, respectively. Gerbils were given IR injury for five minutes and, thereafter, they were respectively treated with vehicle (saline), 25, 50 and 100 mg/kg PYC immediately, at one day and two days after sham and IR.

RAMT was conducted to examine change in spatial learning memory following IR injury at designated times (Fig. 1) according to published protocols [6, 21]. In short, a central octagonal plate consisting of a non-transparent acryl board (diameter, 20 cm) with eight radial arms (5 cm wide; 9 cm high, and 35 cm long) (60150, Stoelting Co., Wood Dale, IL, USA). The gerbils were trained three times for three days (once a day) prior to IR (Fig. 1). The substantive test was conducted once a day for five days from one day after IR (Fig. 1). In detail, Pellet feed (RodFeed, DBL Co Ltd., Chungbuk, Korea) was put at the end of each arm, and the gerbil was placed onto the central platform. The trial (test) was wrapped up when the gerbil consumed the feed. The numbers of errors were calculated for re-entering the arms that had already been entered.

To evaluate IR-induced change in short-term memory, PAT was performed at designated times (Fig. 1) according to previous methods [22, 23] with some modification. Gemini Avoidance System (GEM 392, San Diego Instruments Inc., San Diego, CA, USA) was used. This apparatus consists of dark and light rooms that were divided by vertical door. The test had two sessions (training and testing sessions). For training session, at one day before IR (Fig. 1), the gerbil was placed in the light room with its back towards the dark room, allowed to freely explore both rooms for three minutes, and the dark room was electrified for five minutes. If the mouse stepped into the dark compartment, it would receive a mild foot shock. Testing session was performed five days after IR (Fig. 1). The gerbil was placed to the light room and allowed to explore for five minutes, and the latency time (seconds) to enter the dark room was recorded within three minutes.

Histopathological sections were prepared at zero, two, and five days after IR. In brief, as described in a published paper by [15], the gerbils were given deep anesthesia by intraperitoneal injection of pentobarbital sodium (200 mg/kg; 644912121, JW pharm. Co., Ltd., Seoul, Korea). The gerbils were rinsed transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) and fixed immediately with 100 mL of 4% paraformaldehyde (in 0.1 M PB, pH 7.4). The gerbils were decapitated and their brains were removed from the skulls. The brains were then placed in the same fixative overnight and stored overnight in 0.1 M phosphate buffer (PB, pH 7.4) containing 25% sucrose and 0.002% CaCl${}_2$ to prevent the brain from cryosection. Thereafter the brains were cut into 30-$\mu$m thickness of coronal planes using a sliding microtome (SM2020 R, Leica, Nussloch, Germany) equipped with a freezing stage (BFS-40MP, Physitemp Instruments Inc., Clifton, NJ, USA).

CV staining was performed to evaluate change in cellular distribution and morphology in the hippocampus after IR according to a previous study [15] with some modification. In short, the sections intended for CV staining were first mounted on gelatin-coated microscope slides, air dried at room temperature and incubated overnight in 95% ethanol at 56 ${}^{\circ }$C. The sections were then rinsed in distilled water (DW) and immersed in the solution of CV acetate (10510-54-0, Sigma-Aldrich Inc., St-Louis, MO, USA) dissolved at 0.1% in DW for three minutes at room temperature. Thereafter the stained sections were briefly washed followed by decolorized in 50% ethanol for three minutes, dehydrated through 70%, 80%, 90%, 95% and 100% ethanol, cleared in xylene and mounted with cover glasses.

The images of the CV-stained cells were taken using BX53 microscope (BX53, Olympus, Tokyo, Japan), and IR-induced change was examined.

To evaluate the therapeutic neuroprotection by PYC in the hippocampus after IR, NeuN (a marker for neurons) immunofluorescence and FJB (a fluorescent marker for the degeneration of neurons) histofluorescence were performed according to a published method [15] with some modification.

For NeuN immunofluorescence, the sections were incubated in mouse anti-NeuN (diluted 1:1,100; MAB377; Merck Millipore, Burlington, MA, USA) overnight at 4 ${}^{\circ }$C. After washing, the sections were incubated in Alexa Fluor${}^{\mathrm{\circledR }}$ 546-conjugated donkey anti-mouse immunoglobulin G (IgG, diluted 1:500; A10036, Invitrogen, Waltham, MA, USA) for two hours at room temperature. After washing, the sections were dehydrated, cleared and coverslipped.

For FJB histofluorescence was carried out to investigate the therapeutic neuroprotection by PYC in the hippocampus after IR. FJB (AG325-30MG, EMD Millipore, Burlington, MA, USA) solution was prepared by dissolving 0.0003% FJB in acetic acid. According to a published method [15] with some modification, the sections were incubated with 0.06% potassium permanganate for ten minutes at room temperature, washed with DW, and incubated in the FJB solution for 20 minutes at room temperature. After washing, the sections were warmed for the reaction and completely dried. Thereafter, the sections were cleared and coverslipped.

For the analysis of neuronal damage or death (loss), five sections were selected with 140 $\mu$m interval at the corresponding levels of the gerbil brain [24]. According to a published method [15], the digital images of the NeuN- and FJB-stained cells were obtained using a fluorescence microscope using blue (450–490 nm) excitation light (for FJB-stained cells) and green (510–560 nm) excitation light (for NeuN-stained cell). Two blinded experimenters counted the mean numbers of the cells were counted in the same area using an image analyzing system (Optimas 6.5, CyberMetrics, Scottsdale, AZ, USA).

IR-induced BBB leakage and interleukin-1$\beta$ (IL-1$\beta$) change in the hippocampus were analyzed by immunohistochemistry (avidin-biotin complex (ABC) method). According to general method [17] with some modification, the sections were washed and immersed in 0.3% H${}_2$O${}_2$ (in 0.1 M PBS, pH 7.4) for 20 minutes to block endogenous peroxidase activity. Immediately, the sections were washed and incubated in 5% goat or horse serum (in 0.1 M PBS, pH 7.4) for 30 minutes to block non-specific immunoreaction. Thereafter, the sections were immunoreacted with primary antibodies (Table 1) for 24 hours at 4 °C. After washing, the sections were incubated in corresponding secondary antibody (Table 1). Then, the sections were incubated in ABC solution (diluted 1:250; PK-6100, Vector Laboratories, Burlingame, CA, USA). The immunoreaction in the sections was visualized with 3,3${}^{\prime }$-diaminobenzidine tetrahydrochloride (0.06% DAB; 32750-25G-F, Sigma-Aldrich Co, St Louis, MO, USA) in 0.1 M PBS (100 mL) and 30% H${}_2$O${}_2$ (50 $\mu$L) for one minute. Finally, the sections were washed, dehydrated, cleared and coverslipped.

The density of IgG and IL-1$\beta$ immunoreactivity was evaluated as follows. Five sections per gerbil were chosen, and the images of the immunoreacted structures were captured in the same way as described above using the microscope equipped with cellSens Standard software (version 1.4.1, Olympus, Tokyo, Japan). The density was compared as a ratio of the relative optical density (ROD) using Image J software (version 1.46; National Institutes of Health, Bethesda, Rockville, MD, USA).

SPSS software (version 15.0, SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. In addition, Kolmogorov and Smirnov test was applied to evaluate normal distributions, and Bartlett test was used to calculate identical standard error of the mean (SEM). All presented data were taken for the normality test. The statistical significances of the mean between all experimental groups were determined by two-way analysis of variance followed by post hoc Tukey’s test for all pairwise multiple comparisons. All presented data were displayed as the mean $\pm$ SEM. Differences were regarded as statistically significant at p 0.05.

In this study, we examined IgG immunoreactivity for BBB leakage in CA1 after IR operation. In all sham groups, IgG immunoreactivity was mainly detected in blood vessels in CA1; it was hardly found in CA1 parenchyma (Fig. 5Aa,Ad). In the IR+vehicle group, IgG immunoreactivity at two days after IR was in blood vessels (ROD: about 980% versus sham+vehicle group) in CA1, and, at five days after IR, a significantly increased IgG immunoreactivity was dominantly found in CA1 parenchyma (ROD: about 3800% versus sham+vehicle group) (Fig. 5Ab,B). On the other hand, in the IR+100 mg/kg PYC group, IgG immunoreactivity at two days after IR was significantly attenuated (ROD: about 21% versus IR+vehicle group) as compared with that at the corresponding time point of the IR+vehicle group (Fig. 5Ae,B). At five days after IR, IgG immunoreactivity was also significantly lower (ROD: about 25% versus IR+vehicle group) than that at the corresponding time point of the IR+vehicle group (Fig. 5Af,B).

Fig. 5.

IgG immunohistochemistry. (A) IgG Immunohistochemistry in CA1 of the sham+vehicle (a), IR+vehicle (b,c), sham+100 mg/kg PYC (d), IR+100 mg/kg PYC (e,f) at two and five days after sham or IR operation. In the IR+vehicle group, IgG immunoreactivity is significantly increased in parenchyma at five days after IR, however, in the IR+100 mg/kg PYC group, IgG immunoreactivity is significantly low when compared with the IR+vehicle group. (B) ROD of IgG immunoreactive structure. The bars indicate the means $\pm$ SEM (n = 7, respectively; ${}^{*}$p 0.05 vs. sham+vehicle group, ${}^{\mathrm{\#}}$p 0.05 vs. corresponding time point IR+vehicle group and ${}^{\mathrm{†}}$p 0.05 vs. pre-time point group).

In this study, we examined IL-1$\beta$ immunoreactivity for neuroinflammation in CA1 after IR operation. IL-1$\beta$ immunoreactivity in all sham groups was fundamentally shown in the SP of CA1 (Fig. 6Aa,Ad). In the IR+vehicle group, IL-1$\beta$ immunoreactivity in the SP was significantly enhanced (ROD: 165% versus sham+vehicle group) at two days after IR (Fig. 6Ab,B). In this group, at five days after IR, IL-1$\beta$ immunoreactivity in the SP was remarkably reduced (ROD: about 30% versus sham+vehicle group) (Fig. 6Ac,B). On the other hand, in the IR+100 mg/kg PYC group, IL-1$\beta$ immunoreactivity in the SP was significantly low (ROD: about 110% versus sham+vehicle group) at two days after IR as compared with the IR+vehicle group (Fig. 6Ae,B), and, at five days after IR, IL-1$\beta$ immunoreactivity in the SP was similar (ROD: 105% versus sham+vehicle group) to that found at two days after IR (Fig. 6Af,B).

Fig. 6.

IL-1$\beta$ immunohistochemistry. (A) IL-1$\beta$ Immunohistochemistry in CA1 of the sham+vehicle (a), IR+vehicle (b,c), sham+100 mg/kg PYC (d), IR+100 mg/kg PYC (e,f) groups at two and five days after IR. In the IR+vehicle group, IL-1$\beta$ immunoreactivity is significantly increased in SP (asterisk) at two days after IR and significantly reduced (arrows) at five days after IR. However, in the IR+100 mg/kg PYC group, IL-1$\beta$ immunoreactivity was not significantly changed when compared with the sham+vehicle group (B) ROD of IL-1$\beta$ immunoreactive structure. The bars indicate the means $\pm$ SEM (n = 7, respectively; *p 0.05 vs. sham+vehicle group, #p 0.05 vs. corresponding time point IR+vehicle group and ${}^{\mathrm{†}}$p 0.05 vs. pre-time point group).