A total of 273 specific pathogen-free (SPF) rats, including neonatal (20 days after birth; P20), juvenile (P40), and adult (P60) (6–8 rats per group, 36 groups; Fig. 1 and Table 1), were purchased from Unilever (Beijing, China), and all the animal experiments were conducted at the Laboratory A Ⅲ of Center for Animal Experiment of Wuhan University. After one week of early adaptation or feeding acclimatization (newborn rats were fed by their mothers), SNI or sham SNI surgery was performed on the animals [8, 9]. According to the method by Bennett and Xie [10], the surgery involved the blunt separation of the sciatic nerve and its three branches, ligation of the tibial peroneal and tibial nerves and preservation of the small sural nerve, silk ligation and shear of the tibial and common peroneal nerves, and preservation of the small sural nerve to establish the SNI model. Local and intramuscular injections of penicillin were adopted to promote wound healing in rats. Sham operation was also performed in the neonatal, juvenile, and adult rat groups. The skin was incised and the nerve separated but not ligated before suturing. After the operation, the rats were kept in a single cage for 14 days.

Fig. 1.

Experimental design. SNI, spared nerve injury; d, days; h, hour; PWT, 50% paw withdrawal threshold; DAP-5, DL-2-amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7 nitroquinoxaline-2, 3-dione.

The animals were randomized into the SNI, SNI + AEA, SNI + D-AP5, SNI + CNQX, SNI + D-AP5 + AEA, SNI + CNQX + AEA, sham SNI, sham SNI + AEA, sham SNI + D-AP5, sham SNI + CNQX, sham SNI + D-AP5 + AEA, and sham SNI + CNQX + AEA groups. A volume of 0.5 mL of 1 mmol/L [11] AEA (Asc-429, Xiamen Huijia Biotechnology Company, Xiamen, China) or 10 mmol/L [12] D-AP5 (Asc-003, Xiamen Huijia Biotechnology Company, Xiamen, China) or 10 mmol/L [13] CNQX (MCE, HY-15066, NJ, USA) was injected into the caudal vein at 14 days after SNI (sham operation) in SNI/sham + AEA, SNI/sham + D-AP5, and SNI/sham + CNQX groups. After 6 h post-D-AP5 or -CNQX administration, 0.5 mL of 1 mmol/L anandamide (AEA) [11] was injected into the caudal vein in the SNI/sham + DAP-5 + AEA or SNI/sham + CNQX + AEA group. After 4–6 h, mechanical stimulation was performed to shrink the right palm and assess the leg shrinkage threshold (50% paw withdrawal threshold (PWT)). Then, mechanical stimulation and electrophysiological experiments were carried out in the SNI group, following which, the second and third spinal cord segments were collected.

50% PWT was measured before the injury and at 3, 7, and 14 days after the SNI operation. Morphology was observed and 50% PWT was determined in rats as follows. The animals were placed in a von Frey${}^{\text{TM}}$ resin glass cage (10 cm $\times$ 10 cm $\times$ 15 cm) (UGO Basile, Comerio, Italy) at 25 ${}^{\circ }$C $\pm$ 1 ${}^{\circ }$C, and allowed to adapt to the environment for 30 min before the experiment. According to Chaplan et al. [14], a series of stimulations were performed on the dorsal skin of the right foot of rats of different ages using calibrated von Frey silk (Stoelting Company, IL, USA). The initial stimulation intensity started from 2.041 g. In order to avoid agitation or pain to animals caused by frequent stimulation, the interval between two stimulations was set to 5 min, and each pulse lasted for 6–8 s. A sharp twitch of the foot during von Frey silk stimulation was considered a positive response. The formula was 50% PWT = (10lgX + k$\delta$)/10000, where X is the value of the final stimulus using von Frey filaments; k is the tabular value of the positive/negative response pattern, and $\delta$ is the average difference between stimulations.

According to the in vitro electrophysiological technology [15], the sciatic nerve of P20, P40, and P60 rats received ankylosing electrical stimulation at 14 days after SNI to induce LTP in the superficial layer of the spinal dorsal horn, respectively. The field potential amplitude was processed on the RM6240 multi-channel signal acquisition system (RM6240 model: RM6240BD, VBD5246, CZ 01000158) and subjected to single stimulation; P20, P40, and P60 animals were stimulated with 3 V, 5 V, and 8 V, respectively. In the sham operation groups, P20, P40, and P60 animals were stimulated with 5 V, 10 V, and 15 V, respectively. Then, the evoked waves of spinal dorsal horn A or/and C fibers (basic values) were induced. After high-frequency stimulation (HFS) at 6 V, 10 V, and 12 V, respectively, the sham operation group received four levels of electrical stimulation at 10 V, 20 V, and 30 V, frequency 100 Hz, wave width 0.5 ms, string length 1 s, and string interval 10 s. The evoked waves of spinal dorsal horn A and C fibers were induced through the sciatic nerve to produce field potential changes that were recorded for at least 60 min; 14 days after sham SNI, CNQX, D-AP5, and AEA was injected intravenously 6 hours later, and behavioral tests were carried out after 2 h. Subsequently, the electrophysiological experiments were carried out to observe the evoked field potentials of A and C fibers as well as the potential amplitude changes in AMPAR- and/or NMDAR-mediated FPs (expressed in %) (Fig. 2 and Table 2).

Fig. 2.

50% PWT before and after SNI in rats of different ages.

After the electrophysiological experiment, the chests of the animals were opened, the right atrial appendage was cut off, and 100–150 mL of normal saline and 250 m of 4% paraformaldehyde fixation solution were injected successively and rapidly through the ascending aorta (4 ${}^{\circ }$C). Then, the perfusion rate was slowed down for 30 min. Subsequently, the L4 spinal cord segments of the rats were collected and fixed in 4% paraformaldehyde for 4 h and then immersed in 30% sucrose (4 ${}^{\circ }$C, 48 h). POTC was used for embedding the tissue and sectioning.

After the electrophysiological experiment, the tissue sections were analyzed according to the above operation, followed by phosphate-buffered saline (PBS) hydration, and sealing with skimmed milk. Hematoxylin and eosin (H&E) staining was performed for histological analysis. The cytoplasm stained red with strong reflectivity, and the color changed with SNI, electrical stimulation, and medication. At room temperature, anti-CB1 receptor antibodies (Abclonal, A1447) were added and incubated for 2 h, followed by incubation with secondary antibodies (A0208,1:1000, Beyotime, Shanghai, China) for 1 h. After three PBS washes, the film was sealed and observed under a light microscope.

The Mshot High Definition Color Pathological Graphic Report Management System (Mshot MD50) was used to acquire the images of lumbar 2 and 3 segments of the spinal cord of neonatal, juvenile, and adult rats, respectively. Under a 200$\times$ light microscope, the superficial region of the spinal cord’s posterior horn from rat sections was selected for H&E staining. Densitometric analysis was performed using Image ProPlus 6.0 image analysis system (Media Cybernetics). The optical density (OD) reflected the positive staining intensity of the tissue; the higher the OD, the stronger the staining. All OD measurements were conducted under the same optical conditions.

After the above treatments in various groups, spinal cord tissue samples were obtained, snap frozen with liquid nitrogen, homogenized, and tissue protein extract was obtained by centrifugation. An equivalent of 35 $\mu$g/well total protein was analyzed by SDS-PAGE and transferred to PVDF membranes. After probing with anti-CB1 receptor primary antibodies, the membranes were incubated with secondary antibodies. Enhanced chemiluminescence (ECL) solution was used for color development, and a direct gel imaging CCD system was used for image analysis of CB1 receptor signal expression at 14 days after nociceptive stimulation.

The ImageJ software (version 1.8.0, LOCI, University of Wisconsin, Madison, WI, USA) was used to analyze the gray value of strips. GAPDH (66009-1-Ig, Proteintech, Company) was used as an internal reference for normalization. The secondary antibodies were goat anti-mouse (SA00001-1, Proteintech Inc., Chicago, IL, USA) and goat anti-rabbit (SA00001-2, Proteintech Inc, Chicago, IL, USA).

Measurement data were analyzed using the SPSS 13.0 software (IBM Corp., Chicago, IL, USA) and expressed as mean $\pm$ standard deviation (SD). One-way analysis of variance (ANOVA) was performed for multiple group comparisons, and the treatment and the corresponding sham groups were compared using t-test (PWT) and ANOVA (LTP, immunohistochemistry assessment and Western blot). p 0.05 indicated statistical significance.

The present study evaluated the right paw. No significant difference was detected in 50% PWT between the sham SNI and SNI groups before and 3 days after SNI. In neonatal rats, 50% PWT in sham SNI rats (including those injected with D-AP5, CNQX, and AEA) and the SNI + D-AP5 + AEA group was significantly different from that in the SNI + AEA and SNI + CNQX + AEA groups at 7 and 14 days postoperatively (p 0.05, Table 1). In juvenile rats, 50% PWT in the sham SNI (including injection groups), the SNI + CNQX + AEA, and SNI + D-AP5 + AEA groups differed significantly compared to the SNI, SNI + AEA, SNI + D-AP5, and SNI + CNQX groups (p 0.05, Table 1). In adult rats, 50% PWT of the sham SNI (including injection groups) and SNI + CNQX + AEA groups showed significant differences compared to the SNI, SNI + AEA, SNI + D-AP5, SNI + D-AP5 + AEA, and SNI + CNQX groups (p 0.05, Table 1). These results indicated that CNQX or D-AP5 inhibits PWT in neonatal, juvenile, and adult rats and enhanced the inhibitory effect of AEA on LTP. Specifically, D-AP5 and CNQX exerted a significant inhibitory effect on PWT in neonatal rats and adult rats, respectively. In addition, D-AP5 and CNQX inhibited PWT in juvenile rats and enhanced the inhibitory effect of AEA (Fig. 2).

An amplitude 20% greater than the baseline value and lasting for $>$30 minutes is considered the plasticity threshold for inducing LTP (C or A nerve fibers) evoked by spinal dorsal horn nerve fibers, resulting in long-term enhancement of evoked potential in the spinal dorsal horn. In sham rats, low-intensity (5 V, 10 V, and 15 V for neonatal, juvenile, and adult groups, respectively) electrical stimulation could not induce LTP in the spinal dorsal horn’s C-fiber evoked (or A-fiber evoked) field potential (Fig. 3), while electrical stimulation with high amplitude (30 V) induced LTP. In the SNI group, low-intensity electrical stimulation induced LTP of C-fiber evoked (or A-fiber evoked) field potential in the spinal dorsal horn (Fig. 3). These findings suggested that SNI decreases the plasticity threshold of the C-fiber evoked (or A-fiber evoked) field potential evoked by the dorsal horn of the spinal cord and increased the amplitude of the C-fiber or/and A-fiber evoked potentials. However, after D-AP5 and CNQX administration, the amplitude of this potential was suppressed compared to that in the SNI group. After treatment with AEA (incomplete inhibitor dose), the inhibitory effects of LTP (FPs) on A and C fibers were distinct on field potential at 14 days. Among them, D-AP5 in the neonatal group inhibited the LTP of A fibers. Since C fibers do not play a major role in the superficial layer of the dorsal horn function, LTP formed by A fibers was inhibited (p 0.01, Table 2), and the wave and change in C fibers was not obvious (p $\mathrm{>}$ 0.05, Table 2). D-AP5 and CNQX inhibited the LTP of A and C nerve fibers in the juvenile group (p 0.05, Table 2), which was enhanced by AEA (p 0.01, Table 2). In the adult group, the A and C nerve fibers were mainly inhibited after CNQX administration, and completely inhibited by AEA. The comparison between the SNI and sham SNI groups revealed that the amplitudes of A fibers after ankylosis differed significantly in the neonatal and juvenile groups (p 0.05 or p 0.01, Table 2), and those of C fibers were significantly different in the juvenile and adult groups (p 0.05 or p 0.01, Table 2). The comparison of the SNI + AEA and sham SNI + AEA groups after ankylosis showed that the amplitudes of A fibers were significantly different (p 0.05, Table 2), and those of C fibers differed significantly in the juvenile and adult groups (p 0.05 or p 0.01, Table 2). The comparison of the SNI + D-AP5 and sham SNI + D-AP5 groups did not reveal any significant difference in the amplitude of A fibers after ankylosis in the neonatal period (p $>$ 0.05, Table 2), while the amplitude of C fibers had significant differences between the juvenile and adult groups (p 0.05 or p 0.01, Table 2). The comparison between the SNI + D-AP5 + AEA and sham SNI + D-AP5 + AEA groups after ankylosis did not show any significant differences in the amplitudes of A and C nerve waveforms (p $>$ 0.05, Table 2). The comparison between the SNI + CNQX and sham SNI + CNQX groups after ankylosis showed that the amplitudes of A fibers in the neonatal period differed significantly (p 0.05, Table 2), and those of C fibers differed in the juvenile and adult groups (p $\mathrm{>}$ 0.05, Table 2). The comparison of the SNI + CNQX + AEA and sham SNI + CNQX + AEA groups did not show any significant differences in the amplitudes of A and C nerve waveforms (p $>$ 0.05, Table 2). In order to present the alteration of LTP, we calculated the area of evoked field potentials ($\mu$v s) evoked by A and C nerve fibers during formation and quantification of LTP (Table 3), which was consistent with the results shown in Table 2.

Fig. 3.

Changes in C and A fiber-evoked potentials induced by SNI in rats of different ages under various treatments. Neonatal (A), juvenile (B), and adult (C) animals were assessed under various treatments.

H&E and immunohistochemistry staining showed eosinophils in the dorsal horn of the spinal cord after electrical stimulation at 14 days after SNI, with bright red, strong, and dark-brown signals. Immunohistochemistry showed that spinal cord cells had brownish-red and dark brown signals at 14 days after SNI. On day 14, the signals were significantly lower in the SNI + D-AP5 + AEA group of neonatal rats compared to the SNI group (p 0.01) and lower in the SNI + CNQX + AEA and SNI + AEA groups compared to the SNI group (p 0.05). The signals were lower in the SNI + CNQX + AEA, SNI + D-AP5 + AEA, and SNI + AEA groups in juvenile rats (p 0.05 or p 0.01), SNI + CNQX + AEA group in adult rats (p 0.01), and SNI + D-AP5 + AEA and SNI + AEA groups compared to the SNI group (p 0.05) (Fig. 4).

Fig. 4.

Histological analysis of samples collected before or after SNI in rats of different ages under various treatments.

The CB1 signals in the SNI groups of neonatal, juvenile, and adult rats were bright red and dark brown and significantly higher than those of the corresponding sham SNI groups on day 14 (p 0.05). After treatment with CNQX or D-AP5, the red and dark brown signals became lighter, indicating decreased expression levels of CB1, which was further decreased by AEA. Strikingly, after treatment with D-AP5 and AEA, the CB1 levels were significantly lower in neonatal rats than the SNI + AEA, SNI + D-AP5, SNI + CNQX, and SNI + CNQX + AEA groups (p 0.05 or 0.01). After treatment with CNQX or D-AP5 and AEA, the expression levels of CB1 in juvenile rats were significantly lower than those of the SNI + AEA, SNI + D-AP5, SNI + CNQX, SNI + D-AP5 + AEA, and SNI + CNQX + AEA groups (p 0.05 or 0.01). After treatment with CNQX and AEA, the expression levels of CB1 in adult animals were significantly lower than those of the SNI + AEA, SNI + D-AP5, SNI + CNQX, and SNI + CNQX + AEA groups (p 0.05 or 0.01) (Table 4, Fig. 5). After treatment with CNQX or D-AP5 and AEA, the CB1 levels in the sham SNI + DAP5 + AEA and sham SNI + CNQX + AEA groups were significantly lower than the corresponding SNI groups (p 0.05).

Fig. 5.

CB1 protein levels detected by immunohistochemistry in samples collected before or after SNI in rats of different ages under various treatments.

Western blot was performed to detect the level of CB1 protein in rats after treatment with AEA, CNQX, and D-AP5 at 14 days after SNI. The changes in CB1 levels were consistent with those detecte3d by immunohistochemistry analysis (Fig. 6). The CB1 protein levels were normalized against the GAPDH density, an internal control (Table 5).

Fig. 6.

CB1 protein levels detected by Western blot in samples collected before or after SNI in rats of different ages under various treatments.