Healthy adult male Sprague-Dawley rats (n=136; weight, 230–280 g) were purchased from the Animal Test Center of Xuzhou Medical University [Lianyungang, China; animal production license no. SCXK (su) 2012011-0003 and animal usage license no. SYXK (su) 2010-0011]. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Affiliated Lianyungang Hospital of Xuzhou Medical College (Lianyungang, China). All the animals had access to food and water ad libitum, lived in natural light with a temperature of 24±1°C and all testing personnel were qualified to experiment on animals. Eight rats were selected at random prior to surgery and used as the conjunctive control, the remaining rats were randomly divided into the LSS (n=64) and sham operation (n=64) groups. The animals undertook flat plate sports every day for 3 days prior to surgery. All the rats in the control group were sacrificed prior to surgery and enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were performed on four of these rats to measure BDNF content and expression. If animals died during the test process then additional animals were added to maintain the original test number.

Silica gel slices were made from 18FR two cavity pure silicone catheter for single use (C. R. Bard, Inc., Covington, GA, USA). A suitable section of silica gel slices were selected and cut into thin slices (length, 4 mm; depth, 1.25 mm and thickness, 1 mm). The outer tip showed a wedge shape and the border was rubbed blunt using abrasive paper (Huifeng abrasive paper Ltd., Guangzhou, China). The silica gel slices were then soaked in 75% alcohol for more than 30 min.

For the operation group, rats were anesthetized with 10% chloral hydrate via intraperitoneal injection (0.5 ml/100 g, additional anesthetic was provided as required). At the two sides of the iliac crest, horizontally level to the L5 vertebra, we performed shaving and skin preparation and 75% alcohol was used for skin degerming. A median incision was made behind the Processus spinosus, the skin and subcutaneous fascia were cut in turn, and the paravertebral muscles were located according to joint motion between L6-S1 and separated. In addition, the supra- and interspinal ligaments of L4-L6 were cut, the S1 Processus spinosus was lifted using forceps and epidural tissues under the lamina of vertebra were separated to expose Dura mater spinalis. Furthermore, the silica gel slice was longitudinally embedded under the L5 lamina of vertebra. At this time, we observed shortened feet in the lower extremities and a tail-flick, which confirmed the accurate location and compression of the spinal cord. Therefore, we removed L5 Processus spinosus, lifted L4 Processus spinosus and the silica gel slice was longitudinally embedded using the previous method. The wound was rinsed with physiological saline and 40 units of gentamicin sulfate were intravenously administerd. The fascia and skin were sutured in turn.

For the sham operation group, we performed the same process as that of the operation group; but, the silica gel slice was not embedded.

The walking distance was measured using a flat plate sport instrument (SportsArt Fitness Industry Corporation, Taiwan, China). All rats undertook flat plate sports 3 days prior to and following surgery until they were sacrificed. The walking speed began at 0.3 m/sec and accelerated to 0.5, 0.8 and 0.9 m/sec every 5 min. Measurements were recorded twice daily at the fixation time and the mean value was calculated. The walking distance was not recorded when rats had continuously fallen off the flat plate three times.

Four rats in the operation group were randomly selected for the CT scan (32 row spirals, GE Company, Fairfield, CT, USA; scan condition was 120.00 KV, 200 MA, 512×512 pixel; slice thickness, 0.625 mm and Dicom format). The scan result was imported into Materialise Mimics 13.0 finite element modeling software (Materialise Company, Leuven, Belgium) to construct finite element modeling of the lumbar in rats. Image-Pro Plus 6.0 (Media Cybernetics Company, PA, USA) was used to measure and calculate the sagittal and transection diameters of the spinal canal in L4 and L5 phase.

PMSF crystal was added to PMSF solvents, and following dissolution and antigrading, 10 ml 100 mM PMSF was prepared. PMSF (1 ml 100 mM) was added to 100 ml NP-40 and the NP-40 lysate of 1 mM PMSF was obtained. The sample was defrosted at room temperature, and then cut into fragments and placed in a centrifuge tube. Lysate (200 μl) was added to each centrifuge tube and following 1 h on ice the tissue homogenate was centrifuged (10,000 × g at 4°C for 15 min) and the supernatant was removed.

The reagents were purchased from Promega Corporation (Fitchburg, WI, USA). Protein standard dispensing liquid (0.8 ml) was added to 20 mg protein standard solution (bovine serum albumin) following sufficient dissolution to give 25 mg/ml protein standard solution. Moreover, 20 μl of 25 mg/ml protein standard solution was added to 980 μl phosphate buffered-saline (PBS) and diluted to 0.5 mg/ml of the final content. BCA fluid was formed by mixing 0.8 ml BCA reagent B and 40 ml reagent A at a ratio of 1:50. The protein standard solution was added into the 96-well plates at 0, 1, 2, 4, 8, 12, 16 and 20 μl, 10 μl supernatant and PBS were added to increase the concentration to 20 μl in each well. In addition, 200 μl BCA fluid was added into each well and the absorbance was measured at 37°C for 30 min. The optical density (OD) value at 562 nm absorbance was assayed with ELISA, relative to OD 450, it was equal to OD 450 (sample) - OD 450 (control). Protein concentration of the samples was calculated using a standard curve, the practical BDNF concentration was the measurement result multiplied by two.

All reagents were purchased from Promega Corporation. The rats were sacrificed by administrating an overdosed level of anaesthetic, the L4-L5 bilateral nerve root was quickly separated and DRG were identified (four DRG were removed from each animal and considered as the same samples). Nerve fibers (1 cm), including DRG were cut and removed. The blood was washed with PBS and stored in the freezer at −80°C until the ELISA test (Fig. 1).

BDNF protein standard liquid was prepared in the first 24 h after the animals were sacrificed. Sample dilution (1 ml) was added to 10 ng BDNF standard preparation tube and 10 ng/ml BDNF protein standard fluid was obtained. Moreover, 0.2 ml 10 ng/ml was added to 0.8 ml sample dilution and 2000 pg/ml BDNF protein standard fluid was obtained. Six centrifuge tubes were marked and 0.3 ml sample dilution was added to each centrifuge tube; 0.3 ml 2000 pg/ml fluid was added into the first tube; and 0.3 ml from the first tube was added into the second tube, with this procedure continuing in this manner. From the six tubes, 0.1 ml protein standard fluid was added to a 96-well plate, 0.1 ml sample dilution was added to the first well as the blank, and 50 μl sample supernatant and 50 μl sample dilution were added to each empty well. The lid was added and the well plate was incubated for 90 min at 37°C. Furthermore, 100 μl BDNF antibody was added to 9.9 ml antibody dilution (1:100) and 10 ml BDNF antibody fluid was obtained. Following incubation, liquid in the well plate was removed and the plate was dried with absorbent paper as the drying process was slow. Antibody fluid (0.1 ml) was added to each well and incubated for 60 min at 37°C. The plate was washed three times with PBS (1 min per well) and liquid was removed and the plate was dried with absorbent paper. ABC and TMB fluids (Promega Corporation) were also incubated for 30 min at 37°C. Furthermore, 100 μl ABC was added to 9.9 ml ABC dilution and 10 ml ABC fluid was obtained. Following the dilution, 0.1 ml ABC fluid was added into each well and incubated for 30 min at 37°C. The plate was washed five times with PBS (Promega Corporation; 2 min per well), liquid was removed and the well plate was dried with absorbent paper. TMB coloration liquid (90 μl) was added to the plate and incubated for 30 min at 37°C. Additionally, 100 μl TMB stopping buffer was added into each well and the OD value of A450 was assessed using ELISA for 30 min. Relative to OD 45O, it was equal to OD 450 (sample) - OD 450 (control). Protein content of the sample was calculated using a standard curve, practical BDNF concentration was the measured result multiplied by two.

BDNF content equaled the BDNF concentration divided by the BCA concentration (pg/mg total protein).

After the animals were sacrificed, 4% paraformaldehyde was quickly administered into the rat heart. DRG were removed using the previous method, soaked in 4% paraformaldehyde liquid and fixed. Following dehydration, clearing, routine paraffin embedding and 3-μm serial sections, we performed immunohistochemical staining with streptavidin-peroxidase. DRG slices were heated at 60°C overnight, dewaxed in xylol and hydrated with gradient alcohol. To remove endogenous peroxidase, slices were incubated at 37°C for 10 min using 3% H₂O₂. Then slices were washed three times for 5 min in PBS (pH 7.4). Slices were placed into sodium citrate liquid (pH 6.0) and heated to boiling by microwave for 2 min. Slices were allowed to cool for 15 min, then washed three times for 5 min with PBS. In addition, 10% normal goat serum confining liquid was added for 20 min at room temperature and any additional liquid was removed. Rabbit anti-rat BDNF polyclonal antibody (50 μl) (1:200; Abnova Corporation, CA, USA) was added overnight at 4°C and PBS replaced the antibody in the blank. Furthermore, slices were washed three times for 5 min with PBS, biotin-labeled goat anti-rabbit IgG (1:50) was added and slices were incubated for 30 min at 37°C. Slices were then washed three times for 5 min with PBS. Horseradish peroxidase labeling strepto-albumin stock solution (1:300) was added, incubated for 30 min at 37°C and slices were washed three time for 5 min with PBS. In addition, DRG slices were then stained with 3,3′-diaminobenzidine for 30 min and washed three times for 5 min with PBS before being stained again with hematoxylin and eosin. Slices were then dehydrated with alcohol gradient and cleared with xylol. DRG slices were mounted with neutral gum (Promega Corporation) and observed under a light microscope (magnification, ×200). Photographs recorded the staining of the DRG slices. We used Image-Pro Plus software, version 6.0 to carry out the gradation analysis for DRG slices and calculate the mean optical density (MOD) of BDNF-positive action in DRG.

Data are expressed as the mean ± standard deviation and analyzed with SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA). An independent sample t-test was used to compare BDNF content difference and flat plate sports distance in each group, between groups and before and after sports. Two- and one-way analysis of variance and multiple comparison methods were used to analyze BDNF expression changes between each group at all time-points and before and after sports. Bivariate correlation was used to analyze BDNF expression and sports distance. P<0.05 was considered to indicate a statistically significant difference.

The majority of rats in the operation and sham operation groups were able to walk 1 day post-surgery. Eight rats in the operation group and 1 rat in the sham operation group died during the experiment; therefore, animals were added to maintain the sample number. In the operation group, 7 rats were paralyzed in the lower extremities and showed crossing. Unilateral hind legs of five rats were paralysis and one rat was suspected to have an infection. All the rats in the operation group demonstrated drooping tails and were suspected to have uroclepsia or constipation. In the sham operation group, no obvious differences were observed in their walking abilities compared with those prior to surgery and no animals indicated infection.

In the operation group, the sagittal diameter of the spinal canal in L4 phase was 1.31±0.05 mm and the transection diameter was 2.05±0.02 mm. In the L5 phase, the sagittal diameter was 1.16±0.03 mm and transection diameter was 1.99±0.02 mm. Rats were dissected and the lamina of vertebra was cut. The obvious compression of the coccygeal nerve was exposed and compression was ~50–70%.

BDNF total protein levels in the operation group were 202.06±89.25 pg/mg prior to surgery, which was significantly higher than that of the sham operation (123.00±36.06 pg/mg) and control groups (114.28±10.78 pg/mg) (P<0.05). There were no differences between the sham operation and the control groups prior to surgery (P>0.05).

In the operation group, BDNF total protein concentration before and after sports was 169.22±70.99 pg/mg and 234.90±95.50 pg/mg, respectively (P<0.05). There were significant differences at each time point in the operation group (P<0.05), BDNF total protein levels were highest 3 days following surgery and reached 264.93±105.64 pg/mg. Moreover, there were significant differences prior to surgery compared with that 14 days following surgery (140.54±42.27) (P<0.05). There were no significant differences at the remaining time points (P>0.05).

BDNF content following sports in the operation group at the same time points was higher than that prior to sports, but there were no significant differences (P>0.05). Values before and after sports in the sham operation group at each time point showed no significant differences (P>0.05) (Tables I and II, Fig. 2).

In the operation group, BDNF protein expression was upregulated 1 day following surgery. A positive reaction was distributed in each type of neuron, but it was mainly distributed in the intracytoplasm of neurons in the middle of DRG with a small diameter (10–50 μm). BDNF distribution was also observed in nerve fibers and marginal expression was identified in the nucleus (Fig. 3).

MOD in the operation group (0.293±0.034) was significantly higher than that in the sham operation (0.200±0.206) and control groups prior to surgery (0.199±0.028) (P<0.05). There were no differences between the sham operation and control groups (P>0.05). In the operation group, the MOD values before (0.281±0.031) and after sports (0.305±0.033) were significantly different (P<0.05). Multiple comparison with the SNK method demonstrated significant differences at each time point in the operation group (P<0.05). MOD was the highest 3 days post surgery (0.316±0.025) and lowest 1 day post surgery (0.273±0.026) (P<0.05). There were no differences at the remaining time points.

In the operation group, the MOD following surgery was significantly higher after sports than before sports. However, t-test analysis compared the same time points before and after sports and demonstrated that 3 days following surgery BDNF expression levels after sports were significantly different than those before sports (P<0.05). In the sham operation group, MOD at the same time points before and after sports showed no significant differences (P>0.05) (Tables I and II; Fig. 4).

In the operation group, flat plate sports distance following surgery was significantly less than that before surgery (P<0.05). In the sham operation group before and after surgery, walking distance was not significantly different. Walking distance following surgery in the operation group was significantly less than that in the sham operation group (P<0.05) (Table I, Fig. 5).

ELISA confirmed the results of the immunohistochemical staining in the operation group. BDNF expression levels showed a significant negative correlation with walking distance (correlation coefficient r=−0.05 and −0.65, P<0.05); however, the correlativity was not close (Fig. 6).