Animals and surgery
This study was approved by the Ethics Committee of the 908th Hospital of Joint Logistics Support Force of the Chinese PLA. Thirty 3-week-old male SD rats (Hunan STA Laboratory Animal Co., Ltd, China) were housed at room temperature of 22 °C with free access to food and water and 12-h light/dark cycle. All animals were randomly divided into experimental group (uPA group, n = 15) and control group (saline group, n = 15), both of which sufferer surgery. Surgical produce refers to the previous literature [12, 13]. Briefly, after general anesthesia with sodium pentobarbital (10 mg/100 g), the shoulder muscles of the rat forelimbs were incised to expose the shoulder joints, the forelimbs and scapula were separated from the joint capsule. A 1.5-cm medial incision was made on the left side of the back of the rat to expose the L5–L6 vertebral bodies, and 5ul uPA (2 mg/L, dissolved in normal saline) was injected into the joint cavity with a 34-gauge needle. The control group was injected with 5ul normal saline. Then these rats were placed on a constant temperature heating pad to maintain their body temperature and returned to the special rat cage after they woke up. The average body length of the rats in each cage was measured once a week, and the height of food and water was adjusted according to the body length to maintain the rats in an upright posture (Fig. 1).
Mechanical hyperalgesia testing
The mechanical hyperalgesia threshold was measured before surgery and on postoperative days 3, 7, 14, 28, and 42 (n = 3). Rats were placed on a wire mesh with a uniform size grid, covered with a transparent plexiglass box. After the rat was acclimated to the environment for 15 min, after the rat was quiet, a standardized series of (1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, 26 g) von Frey test needles were used to vertically stimulate the rat through the wire grid. The mid-plantar of the hind limb on the operated side of the rat was bent slightly into an S shape for 6–8 s, and the paw withdrawal response was observed. The rat removed the von Frey needle within the stimulation time or when the time expired (North Coast Medical, 2007). USA), the rapid paw withdrawal response was recorded as a positive response, while the paw withdrawal response of other activities was not recorded. The test needle is gradually increased from 1.0 g, and the interval between each stimulation is 30 s. When the stimulation of the test needle cannot cause a positive response, change to a test needle with one level of strength. When testing the pressure value (g) of the needle at which 50% of the rat (measured 10 times with at least 5 consecutive paw withdrawals) withdrawal response, the paw withdrawal threshold on the side of the rat was measured.
Thermal hyperalgesia testing
The thermal withdrawal latency of the ipsilateral hind paw was measured using a thermoalgesia instrument (BW-PL-200, Ruanlong, Shanghai) before injection and on postoperative days 3, 7, 14, 28, 42, and 56 (n = 3). Before test, rats were placed on a constant temperature glass plate with free movement for 15 min. Then, a thermoalgesia instrument below the glass plate was used to vertically irradiate the middle and posterior 1/3 of the hind limbs. The paw withdrawal latency refers to the ranging time(s) from irradiation to the appearance of the paw withdrawal reflex. The test procedure was repeatedly applied for 3 times with 10-min interval. To avoid thermal damage to the plantar skin, the light intensity was set to 80%, and each irradiation did not exceed 60 s.
Cartilage toluidine blue staining
On the 7th, 14th, 28th, and 42nd days after surgery, three rats were randomly selected from two groups and sacrificed by injecting overdose of pentobarbital sodium. The L5–L6 spines were excised en bloc, fixed in 4% paraformaldehyde for 48 h, decalcified for 3 weeks, and embedded in paraffin. Then the blocks were cut into 4-µm sections on the coronal plane and stained with toluidine. Blue microscopic examination was conducted to evaluate the degree of articular cartilage degeneration according to e Osteoarthritis Research Society International (OARSI) histopathology grading system [17].
Hematoxylin–eosin (HE) staining of the synovium
HE staining of L5–6 facet joints synovium was performed in order to evaluate synovial inflammatory condition. Synovitis scoring was based on three criteria including synovial cell proliferation, subsynovial inflammation and angiogenesis. The sum of each the terms’ scores (3-point scale) was considered as total pathology score (0–9). Synovial cell proliferation was scored as follows: 0 point for less than 3 layers, 1 point for 3–4 layers, 2 points for 5–6 layers, and 3 points for more than 6 layers. Inflammation degree was scored as follows: 0 point for no lymphocyte infiltration, 1 point for lymphocyte aggregation, 2 points for lymphoid follicles, and 3 point for lymphoid follicles with germinal center formation. Angiogenesis was scored as follows: 0 for none, 1 for mild, 2 for moderate, and 3 for severe.
Immunohistochemial staining
The sections were incubated with primary antibodies (IL-1β (bs-0812R, Bioss, Beijing, China; 1:200); TNFa (AF7014, Affinity, Ohio; 1:200); iNOS (AF0199, Affinity, Ohio; 1:200)) at 4℃overnight. HRP-AffiniPure Goat Anti-Rabbit IgG (H + L) (BM3894, Boster, Wuhan, China; 1:1000) was incubated at 37℃for 30 min. The staining was displayed using Diaminobenzidine. Cells with only blue nuclei are negative, and cells with brownish yellow or tan granules in the cytoplasm are positive cells. The staining intensity was scored as the following: 0 point for no staining; 1 point for weak positive (light yellow staining); 2 point for moderated positive (brownish yellow); 3 point for strong positive. The positive cells in three randomly selected field were scored as the following: 0 point for negative; 1 point for less than 10%; 2 point for 11–50%; 3 point for 51–75%; 4 point for more than 75%.
Statistical analysis
All data were expressed as mean ± standard deviation. SPSS 22.0 software (SPSS, US) was used for statistical analysis, and t-test was used to determine significant differences, and p < 0.05 was regarded as statistically significant.