Effects of γ-oryzanol on motor function in a spinal cord injury model

2.1 Animals

Female C57BL/6 mice (9 weeks old; average weight, 20 g) were purchased from the Comparative Medical Center of Yangzhou University (Yangzhou, China). Mice were housed under a 12 h light/dark cycle, 3–4 per cage, maintained at 20–22°C with 50% relative humidity, and provided water and food ad libitum.

2.2 Grouping and surgical procedures

Mice were randomly divided into three groups. Mice were anesthetized with 2% isoflurane in oxygen. The spinal cord was exposed with T10 as the center, and the left spinal cord was removed with the posterior midline of the spinal cord as the boundary, forming a 2 mm rectangular defect. A medical gelatin sponge was placed bilaterally in the paravertebral muscular space to absorb bleeding and prevent postoperative adhesion between the connective tissue and the dura mater. The muscles were repositioned and sutured, and the skin was stapled. The spinal cord of the sham group was exposed after left laminectomy without further injury.

2.3 Dose calculation

The dose for intraperitoneal injection of γ-oryzanol in mice was calculated as 100 mg/kg. This was based on the dose translation formula from humans to animals, which is based on the body surface area of the daily dosage of γ-oryzanol (MedChemExpress, China) of 500 mg/day in humans [16].

2.4 Postoperative treatment

To prevent postoperative infection, all mice with SCI were intramuscularly injected with ampicillin (150 mg/kg) once daily for 7 days, and the bladder was manually squeezed twice daily until the micturition reflex was restored. Mice in the sham group were only provided antibiotics. The injury group received saline injection intraperitoneally every 2 days, whereas the γ-oryzanol-treated group received 100 mg/kg γ-oryzanol intraperitoneally every 2 days.

2.5 Assessment of functional recovery of the spinal cord

Within 6 weeks of surgery, the recovery of right lower limb function was evaluated weekly using the Basso mouse scale (BMS) score and an oblique plate experiment to analyze the recovery process of motor function.

Gait analysis was performed at week 6 after SCI, the two hind plantar surfaces of each mouse were stained black using non-toxic ink, and the mice were allowed to crawl on a cut white paper track (5 × 15 cm²). The measures included stride length (distance between the centers of adjacent footprints on the same side) and sway distance (vertical distance between the center of the left and right hind limbs) and were statistically compared using the average of three steps for each condition.

2.6 Enzyme-linked immunosorbent assay (ELISA)

The levels of inflammatory factors in the peripheral blood of mice were assessed by ELISA 6 weeks after surgery. After fixation, the tails of the mice were immersed in hot water at 50°C for a few minutes to fill the tail blood vessels. The tail was dried, scissors were used to cut the tail tip to 1–2 mm, a test tube was used to connect the blood outflow, and the tail root to the tail tip was massaged. After blood collection, cotton ball pressure was applied to stop bleeding. In strict accordance with the manufacturer’s instructions, the levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 in the peripheral blood of mice were detected by ELISA using IL-6, TNF-α, and IL-10 ELISA test kits (BYabscience Co., LTD, China).

2.7 Damage area assessment

Magnetic resonance imaging (MRI) was performed on five animals in each group on day 42 after surgery. Mice were anesthetized with halothane (3–4% induction, 1.5–2% maintenance) in oxygen (0.4 L/min) and nitrogen (0.6 L/min). Anesthetized mice were placed on a fixation system in the prone position, and scans were performed using a small-animal MRI system (Bruker Biospec 11.7T animal MR scanner; Bruker AXS GmbH, Germany). The sequence protocol was performed using the following parameters: echo time, 23.000 ms; echo spacing, 7.667 ms; repetition time, 550.000 ms; repetitions, 1; average, 9; rare factor, 8; slice thickness, 1.000 mm; slice orientation, sagittal; read orientation, Ro-Cd.

Spinal cord hematoxylin and eosin (H&E) staining and Luxol fast blue (LFB) staining were performed 6 weeks after surgery. Mice were anesthetized with an intraperitoneal injection of 2% pentobarbital (50 mg/kg). T9–T11 spinal cord samples were rapidly dissected after cardiac perfusion with 4% paraformaldehyde and fixed with 4% (W/V) paraformaldehyde overnight. Tissues samples were dehydrated in a gradient of ethanol and embedded in paraffin. Paraffin-embedded tissues were cut into a thickness of 4 μm (Leica CM 1900, Germany) and subjected to H&E and LFB staining.

2.8 Immunofluorescence analysis

Tissues were processed as described in the aforementioned section, and 20 μm horizontal sections were cut on a cryostat. Spinal cord sections were washed thrice with 0.01 M phosphate-buffered saline (PBS) and incubated with primary antibodies overnight at 4°C. Subsequently, the spinal cord sections were washed thrice with PBS. The primary antibodies used in this study include mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200 dilution; Sigma-Aldrich, USA), to label the astrocytes; rabbit anti-mouse laminin antibody (1:200 dilution; Sigma-Aldrich); and rabbit anti-rat 5-hydroxytryptamine (5-HT) (1:200 dilution; Immunostar, USA), to label the motor neurons. The sections were then incubated with the corresponding secondary antibody for 1 h at room temperature protected from light. Finally, the sections were washed thrice with PBS and gently incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 3 min protected from light. Excess DAPI was removed by washing with PBS, the slides were covered with glycerol and cover slips, and a field was randomly selected using a fluorescence microscope (BX-51; Olympus, Japan).

2.9 Statistical analysis

All experiments were repeated independently at least three times. SPSS software (version 22.0) (IBM, USA) was used for statistical analysis, and GraphPad Prism 8 software (GraphPad Software Inc., USA) was used to draw the graphs. Quantitative data were described as mean ± standard deviation (SD). Independent samples t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. Statistical significance was set at *P < 0.05, **P < 0.01, and ***P < 0.001.

1. Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. All the animal experiments were designed and approved by the Yangzhou University Laboratory Animal Ethics Committee (yzu-lcyxy-n060).

3.1 Intraperitoneal γ-oryzanol injection restores motor function in SCI mice

SCI was modeled in mice as described previously (Figure 1). The restoration of motor function is the most important measure of SCI repair. Six weeks after surgery, mice in the sham group showed a strong and stable normal gait during gait analysis, whereas mice in the injury group showed severe toe dragging, increased swing distance (4.14 ± 0.04), and reduced stride length (2.28 ± 0.09). On the other hand, the stride, swing distance (3.28 ± 0.04), and stride length (3.90 ± 0.12) of mice in the γ-oryzanol-treated group were close to those of the sham group (Figure 2a–c). Additionally, motor behavior was assessed using the BMS score, and the results showed that both the SCI and γ-oryzanol-treated groups showed significant left hindlimb paralysis from days 1 to 7 after injury. However, from week 4 to week 6 post-trauma, the BMS scores improved considerably in the γ-oryzanol-treated group compared to that in the injury group (Figure 2d). In the oblique plate experiment, although the γ-oryzanol-treated group showed a higher response than the injury group, the difference was not statistically significant (Figure 2e).

Figure 1

Protocol for SCI. The spinal cord of mice was exposed with T10 as the center and the left spinal cord was removed with the posterior midline of the spinal cord as the boundary, forming a 2 mm rectangular defect.

Figure 2

Intraperitoneal γ-oryzanol injection restored motor function in SCI mice: (a) Representative footprint patterns of the three groups 6 weeks after SCI. Scale bar = 2.5 cm; (b) and (c) semiquantitative analysis of stride length (b) and sway distance (c) in the gait analysis; (d) BMS score of mice 6 weeks after SCI; and (e) slant plate experiment score of mice 6 weeks after SCI. Data are presented as mean ± SD (n = 5). **P < 0.01 and *P < 0.05.

3.2 Intraperitoneal γ-oryzanol injection reduces the damage area in SCI mice

To verify the in vivo neuroprotective effects of intraperitoneal γ-oryzanol injection, we performed MRI of the thoracic spinal cord (T10) in mice. MRI showed that intraperitoneal γ-oryzanol injection (1.58 ± 0.13) reduced the lesion area compared to that in the injury group (2.04 ± 0.20) (Figure 3a and d). H&E staining also showed similar results; considerable spinal cord tissue loss was observed in the injury group 6 weeks after injury. However, the γ-oryzanol-treated group exhibited a considerable reduction in the area of tissue loss 6 weeks after injury (Figure 3b). Similar results were observed with LFB staining with respect to the protection of the myelin sheath (Figure 3c).

Figure 3

Intraperitoneal γ-oryzanol injection reduced the damage area in SCI mice: (a) Representative sagittal and coronal MRI images. Scale bar = 1 mm; (b) and (c) spinal cord tissue with H&E (b) and LFB (c) staining; and (d) quantitative analysis of the lesion area. Data are presented as mean ± SD (n = 5). **P < 0.01.

3.3 γ-Oryzanol treatment suppresses neural scar formation and 5-HT neuron loss in SCI mice

The formation of glial scar and fiber scar after SCI is not conducive to the regeneration and repair of the spinal cord. To understand the effect of intraperitoneal γ-oryzanol injection on the growth of nerve scars, spinal cord tissue samples were double-stained for GFAP and laminin. As shown in Figure 4, GFAP expression was lower in the γ-oryzanol-treated group (3.66 ± 0.21) than that in the injury group (4.48 ± 0.20) (Figure 4a and b). It is noteworthy that although laminin expression in the γ-oryzanol-treated group (3.82 ± 0.13) was lower than that in the injury group (4.60 ± 0.33), the difference was not significant (Figure 4a and c). Additionally, the intensity of 5-HT axons is an important indicator of the degree of retention of response motor function. The intensity of 5-HT axons (3.28 ± 0.20) in the γ-oryzanol-treated group was closer to that in the sham group (5.30 ± 0.28) (Figure 4d and e), indicating the retention of motor function.

Figure 4

γ-Oryzanol treatment suppressed neural scar formation and 5-HT neuron loss in SCI mice: (a) Analysis of the inhibition of glial scar formation by double immunofluorescence labeling of GFAP (green) and laminin (red) after 6 weeks of treatment. Nuclei were stained with DAPI (blue). Scale bar = 50 μm; (b) and (c) quantitative analysis of fluorescence intensity of GFAP- (b) and laminin- (c) positive cells (c); (d) analysis of axon growth by immunofluorescence labeling of 5-HT (green) after 6 weeks of treatment. Nuclei were stained with DAPI (blue). Scale bar = 50 μm; and (e) quantitative analysis of the fluorescence intensity of 5-HT-positive cells. Data are presented as mean ± SD (n = 5). ***P < 0.001.

3.4 Intraperitoneal γ-oryzanol injection reduces the inflammatory response in SCI mice

Furthermore, we investigated the effect of γ-oryzanol on inflammation in SCI mice. To this end, the levels of IL-6, TNF-α, and IL-10 in the peripheral blood serum of SCI mice were measured by ELISA. As shown in Figure 5, compared with the sham group, the levels of IL-6 (160.82 ± 5.82) and TNF-α (142.35 ± 5.77) were considerably increased in the injury group, while the levels of IL-10 (44.77 ± 3.71) were considerably decreased. However, γ-oryzanol treatment decreased the levels of these pro-inflammatory factors (IL-6 and TNF-α), but increased the level of IL-10 (84.79 ± 3.41). These data suggest that γ-oryzanol mitigates the systemic inflammatory response in mice after SCI.

Figure 5

Intraperitoneal γ-oryzanol injection reduced the inflammatory response in SCI mice: (a)–(c) ELISA analysis of IL-6 (a), TNF-α (b), and IL-10 (c) levels in the peripheral blood serum of SCI mice. Data are presented as mean ± SD (n = 5). ***P < 0.001.