Pregnant Sprague-Dawley rats at the E17 pregnancy stage (n=8) were obtained from DooYeol Biotech (Seoul, Korea) and housed at 22°C and 55±5% humidity with a 12 h light/dark cycle. Rats were fed a commercial diet and provided with water ad libitum throughout the study. All experiments were approved by the Pusan National University (Yangsan, Korea) Animal Care and Use Committee in accordance with the National Institutes of Health Guidelines (Approval number: PNU-2015-0771). After delivery, 40 pups were selected. Induction of the hypoxic-ischemia model in the pups (n=32) was performed as previously described with modifications (20,21). Briefly, pups were anesthetized under 2% isoflurane (Choongwae Pharma Corp., Seoul, Korea) at post-natal day 7 (P7; weight, 12–15 g) and the left common carotid artery was ligated and subsequently sutured. Following recovery from surgery, rats were placed in a humidified hypoxic chamber with 8% O₂/N2 mixture for 3 h in an incubator maintained at 36°C. Pups were returned to their mother until they were fully weaned, following which they were randomly divided into control, hypoxic-ischemia vehicle (HI), treadmill-treated alone (TM), JSQW-treated alone (JSQW), and co-treated with treadmill and JSQW (TM+JSQW) groups (n=8 in each) at age P21. Following weaning, mother rats sacrificed by CO2 overdose. Hypoxic-ischemia injuries were indirectly confirmed at P22 via a cylinder test. Body weight and behavioral assessments were monitored from 4 weeks of age (P22), and subsequently at weeks 5, 6, 7, and 8. All treatments were administered under isoflurane anesthesia using a model VIP 3000 calibrated vaporizer (Midmark Corp., Orchard Park, OH, USA).

JSQW is comprised of nine herbs as follows: 16 g steamed Rehmannia glutinosa Lib. root, 8 g Dioscorea batatas Decaisne rhizome, 8 g Cornus officinalis Sieb. fructus, 6 g Poria cocos Wolf sclerotium, 6 g Paeoniasuffruticosa Andr. bark, 6 g Alisma Canaliculatum rhizome, 6 g Schizandra chinensis Baill. fructus, 6 g Amomun xanthioides Wallich fructus, and 4 g Cervus elaphus L. pilose antler from a stag. These medicinal herbs were obtained from the Korean Medicine Hospital (Pusan National University) and authenticated by Professor Dr. Young Ju Yun (Department of Integrative Medicine, School of Korean Medicine, Pusan National University). A voucher specimen was deposited at the Cerebrovascular Diseases Laboratory of Pusan National University. A total of 66 g JSQW were immersed in 2 l distilled water and boiled at 120±5°C for 3 h. The resultant extract was centrifuged (2,000 × g at 4°C for 20 min) and filtered through a 0.8-µm filter. The filtrate was then concentrated in vacuo for 3 h at 60±5°C under reduced pressure (2.3 kPa) and converted into a fine dried powder using vacuum drying apparatus with a yield of 12.9% (8.52 g). The resulting powder was subsequently dissolved with distilled water for use in further experiments.

From P22, doses of 568 mg/kg JSQW were dissolved with distilled water to final volume 0.3 ml and administered orally for 4 weeks following hypoxic-ischemia in the JSQW and TM+JSQW groups, whereas rats in the control, HI and TM groups were administered distilled water at the same intervals. Rats were trained on a treadmill from P22. In week 4, rats ran at a speed of 5 m/min for 10 min on the first day and gradually progressed to 15 min on the fifth day. This speed and duration was maintained until the end of the second week. During the next 3 weeks, rats were run with an initial warm-up at 5 m/min for 5 min followed by increasing speed up to 6 and 7 m/min for 15 min and then a recovery period of 5 m/min for 5 min.

BrdU (B5002-5G; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) is a synthetic thymidine analog that becomes incorporated into a cell's DNA when the cell is dividing during the S-phase of the cell cycle. All rats in the present study were injected with 50 mg/kg BrdU once daily for 2 weeks (days P22-P35) to label proliferating cells in the subventricular zone of the lateral ventricle in the brain.

The cylinder test was performed to measure the forelimb and assess functional recovery from a unilateral brain lesion. A rat was placed in a glass cylinder 20 cm in diameter and 30 cm tall, and the number of limb-wall contacts was counted with ipsilateral (to the lesion site) forelimb and contralateral forelimb support with both forelimbs against the wall and was recorded during lateral exploration. Total count score = (both forelimbs movement-ipsilateral forelimb movement) / total of 20 contacts.

The passive avoidance test was used to assess memory function following brain injury using a passive avoidance task (MED-APA-D1; Med Associates Inc. St. Albans, VT, USA). Following 1 day of acclimation to passive avoidance conditions, the rat was placed in the illuminated compartment for the learning trial. Rats who crossed the door into the dark compartment, which then closed automatically, received a single inescapable scrambled foot shock (0.8 mA, 3 sec) delivered through the grid base. In this test, the procedure was repeated and a latency time to enter the dark compartment was measured until 180 sec. The retention test was performed once in the same manner at 24 h and 7 days following the learning trial. A latency of 180 sec was assigned to animals that did not enter the dark chamber during the retention test.

Rats (n=20) were anesthetized with 8% chloral hydrate (50 mg/kg; C8383-100G; Sigma-Aldrich; Merck KGaA) and sacrificed via intracardial perfusion with saline. Each harvested brain tissue from the subventricular zone and adjacent striatum was washed two times in cold 4-(2-hydroxyethyl)-1-piperazineethansulphonic acid buffer (15–630-080; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and homogenized in lysis buffer consisting of 200 mM Tris (pH 8.0; TRI01; LPS Solution Co., Ltd., Daejeon, Korea), 150 mM NaCl, 2 mM EDTA (ED2SS; Sigma-Aldrich; Merck KGaA), 1 mM NaF (SB0850; Bio Basic, Inc., Amherst, NY, USA), 1% NP40 (NDB0385; Bio Basic, Inc.), 1 mM phenylmethane sulfonyl fluoride (P7626; Sigma-Aldrich; Merck KGaA), 1 mM Na3VO4 (S6508; Sigma-Aldrich; Merck KGaA) and protease inhibitor cocktail (P3100-001; GenDEPOT, Inc., Barker, TX, USA) at 4°C for 5 min. Equal amounts (30 µg) of protein were subsequently separated by 8–12% SDS-PAGE and resolved proteins were transferred onto a nitrocellulose membrane (Whatman; GE Healthcare Life Sciences, Chalfont, UK), which was subsequently blocked at room temperature with 5% non-fat milk in Tris-buffered saline containing 0.4% Tween-20 for 1 h. Membranes were incubated overnight at 4°C with primary antibodies (all 1:1,000) against the following: Neuronal nuclei (NeuN; MAB377; EMD Millipore, Billerica, MA, USA), glial fibrillary acidic protein (GFAP; MAB360; EMD Millipore), Iba-1 (016–20001; Wako Pure Chemicals Industries, Ltd., Osaka, Japan), doublecortin (Dcx; sc-8066; Santa Cruz Biotechnology Inc., Dallas, TX, USA), myelin basic protein (MBP; ab40390; Abcam, Cambridge, MA, USA), mature brain-derived neurotrophic factor (mBDNF; ab75040; Abcam), cAMP response element binding protein (CREB; #9197; Cell Signaling Technology, Inc., Danvers, MA, USA), and phospho-CREB (pCREB; #9196; Ser133; Cell Signaling Technology, Inc.). The blots were subsequently incubated at room temperature for 1 h with the following horseradish peroxidase-conjugated secondary antibodies (all 1:1,000) according to the appropriate species: Goat-anti-mouse (SC-2005; Santa Cruz Biotechnology, Inc.), goat anti-rabbit (SC-2004; Santa Cruz Biotechnology, Inc.), donkey anti-goat (SC-2020; Santa Cruz Biotechnology, Inc.), and donkey anti-sheep (A130-100P; Bethyl Laboratories, Inc., Montgomery, TX, USA). Antibody-specific proteins were visualized using an enhanced chemiluminescence detection system (NCI4080KR; Thermo Fisher Scientific, Inc.). β-actin (1:1,000; a2228; Sigma-Aldrich; Merck KGaA) was used as a loading control for all experiments. Densitometric analysis was performed to quantify immunoreactivity corresponding to the total bands using an Image Quant LAS 4000 system (Fujifilm, Tokyo, Japan). The presented assays are representative of at least three experiments.

Rats (n=20) anesthetized with 2% isoflurane were sacrificed via intracardial perfusion with saline and 4% paraformaldehyde in PBS. Brains were harvested, post-fixed in the same fixative for 4 h at 4°C, and immersed in 30% sucrose for 48 h for cryoprotection. Frozen tissues were cut into sections (thickness, 20 µm) and incubated for post-fixation with formaldehyde and blocking with a blocking buffer [1X PBS/5% normal goat serum (S-1000; Vector Laboratories, Inc., Burlingame, CA, USA)/0.3% triton X-100] for 1 h at room temperature. The sections were incubated with primary antibodies against the following: BrdU (1:500; MCA2483; Bio-Rad Laboratories, Inc., Hercules, CA, USA), NeuN (1:500; ABN78; EMD Millipore), GFAP (1:500; AB5804; EMD Millipore), DCX (1:200; sc-8066; Santa Cruz Biotechnology, Inc.), and MBP (1:500; ab40390; Abcam) overnight in PBS at 4°C. Sections were washed three times with PBS for 10 min and subsequently incubated with the fluorescein-conjugated horse anti-mouse (1:200; FI-2000; Vector Laboratories, Inc.) and Texas Red-conjugated goat anti-rabbit (1:200; TI-1000; Vector Laboratories, Inc.) secondary antibodies for 2 h at room temperature in the dark and washed three times with PBS. Slides were mounted in mounting medium (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and images were captured using a fluorescence microscope (Carl Zeiss Imager M1; Carl Zeiss AG, Oberkochen, Germany). IMT i-Solution version 10.1 (3122–0770; IMT, Inc., Cicero, NY, USA) was used for automatic measurement of integrated optical density (IOD).

All data are expressed as the mean ± standard error of the mean. Analysis of variance (ANOVA) was performed using the Sigmastat statistical program version 11.2 (Systat Software, Inc., San Jose, CA, USA). Statistical analysis of data was performed using Student's t-test when comparing two groups, or one-way ANOVA via Tukey's post hoc comparison when comparing more than two groups. P<0.05 was considered to indicate a statistically significant difference.

Behavioral analysis was performed to determine the potential benefits of JSQW and treadmill training in hypoxic-ischemia rats. Body weight increased with age and no significant differences were observed among groups (Fig. 1A). A cylinder test was performed for analysis of sensorimotor recovery, and the numbers of cylinder wall contacts with impaired forelimb were found to be significantly decreased in the HI group compared with the control group (P<0.05); however, significant improvements in the use of the impaired forelimb were observed in the group treated with treadmill training alone at 8 weeks (P<0.05) and co-treated with JSQW and treadmill at 6 and 8 weeks (P<0.05 and P<0.01, respectively; Fig. 1B). Retention latency in the passive avoidance test was significantly decreased by hypoxic-ischemia at 7 and 8 weeks (P<0.001 and P<0.05, respectively), whereas a significantly longer latency was observed in rats treated with JSQW (P<0.05) or co-treated with JSQW and treadmill (P<0.01) at 8 weeks compared with the HI group (Fig. 1C). These results suggest that JSQW and treadmill training have a significant beneficial effect on memory capabilities and sensorimotor recovery resulting from hypoxic-ischemia injury.

Morphological and western blot analyses were performed to determine whether functional recovery occurs via myelin components and expression of proteins. The thickness of MBP in the corpus callosum were found to be markedly decreased and the IOD significantly decreased (P<0.001) in the HI group compared with the control group; however the thickness was significantly recovered in the group treated with treadmill training (P<0.01). The IOD of MBP in the corpus callosum was also significantly recovered in both the JSQW and treadmill groups (P<0.01 and P<0.001, respectively). The increases in thickness and IOD of MBP were significantly greater in rats treated with treadmill training alone compared with the group treated with JSQW alone (P<0.05 for thickness, P<0.001 for IOD; Fig. 2). Protein expression of cell components and protective markers in the brain were also investigated. The expressions of NeuN and Dcx were significantly decreased in the HI group compared with the control group (P<0.001 and P<0.05, respectively; Fig. 2A-C). However, NeuN expression was significantly recovered by treatment with JSQW or treadmill training (P<0.05 and P<0.001, respectively) and Dcx expression was significantly increased by treatment with JSQW (P<0.05). Furthermore, the expression of MBP was significantly higher in the treadmill only group compared with the control and HI groups (both P<0.01; Fig. 3). These results suggested that JSQW treatment results in increased expression of Dcx and NeuN, and treadmill training in increased expression of NeuN and MBP, and enhanced thickness and myelin density of the corpus callosum. These changes in protein expression may be associated with the recovery of functional activities from hypoxic-ischemia injury.

Finally, immunohistochemical analysis was performed to determine whether functional recovery occurs via neurogenesis. The number of cells positive for BrdU was observed to determine levels of neural progenitor cell proliferation. The results demonstrated that there was a significant increase in BrdU positive cells in the treadmill group compared with the HI group (P<0.01 or P<0.001; Figs. 4–6). However, a limited number of cells showed a BrdU/NeuN or Dcx and GFAP double-positive reaction, which is indicative of differentiated cells from neuronal progenitor cells. A significant increase in BrdU/NeuN or Dcx and GFAP double-positive cells compared with the HI group was observed only in the group co-treated with JSQW and treadmill training (P<0.05, P<0.01 and P<0.001 for BrdU/NeuN, BrdU/Dcx and BrdU/GFAP, respectively; Figs. 4–6). These results suggest that activity-based treadmill training increased the proliferation of neural progenitor cells, and co-treatment with treadmill and JSQW enhanced differentiation to mature neurons and astrocytes.