Schwann cells transplantation improves nerve injury and alleviates neuropathic pain in rats

Abstract

The mechanism of neuropathic pain induced by nerve injury is complex and there are no effective treatment methods. P2X4 receptor expression is closely related to the occurrence of pain. Schwann cells (SCs) play a key protective role in the repair of peripheral nerve injury and myelin sheath regeneration. However, whether SCs can affect the expression of P2X4 receptor and play a role in pathological pain is still unclear. Therefore, this study investigated the effect of SCs on whether they can down regulate the expression of P2X4 receptor to affect pain. The results showed that in the neuropathic pain induced by sciatic nerve injury model, the expression of P2X4 receptor in spinal cord tissue was significantly increased and the pain sensation of rats was increased. While SCs transplantation could down regulate the expression of P2X4 receptors in spinal cord and increase the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats. These data indicate that SCs can reduce the expression of P2X4 receptors to alleviate neuropathic pain, indicating that SCs can mediate P2X4 receptor signalling as a new target for pain treatment.

Introduction

Nerve injury leads to paraesthesia and dysfunction and induces neuropathic pain. The mechanism of neuropathic pain is complex, and its treatment is difficult, which brings serious psychosomatic health effects to patients. Analgesics are usually used to relieve pain (such as opioids) in clinical practice, but long-term use has great side effect and dependence. The important thing is that painkillers can only treat symptoms, but have no effect on repairing injured nerves, promoting axonal regeneration and reconstructing neural function. Therefore, it is very important to explore the treatment of repairing injured nerves and relieving pain. The P2X4 receptor belongs to the ATP-gated ion channel P2X receptor family, which is mainly expressed in microglia. As pain-related molecules, P2X4 receptors play an important role in different types of pain signaling, such as neuropathic pain, inflammatory pain and cancer pain [1, 2]. Generally speaking, up-regulation of P2X4 receptor expression in spinal cord tissue can induce pain after nociceptive stimulation or injury of nerve. The P2X4 receptor plays a major role in inducing this kind of pain [3]. Down-regulation of the expression of this receptor can reduce hyperalgesia [4]. Studies have shown that Dexmedetomidine reduces the expression of the P2X4 receptor, NLRP3 and IL-1β in rats, and relieves the neuropathic pain of diabetes mellitus [5]. Mesenchymal stem cells and their conditioned medium decrease the relative gene expression of P2X4 and P2X7 receptors in spinal cord tissue and relieve neuropathic pain [6]. These studies reveal the important role of P2X4 receptors in the progression of pain, suggesting that the P2X4 receptor can be used as a potential molecular target for pain therapy.

In recent years, some researchers have transplanted functional active cells into the host to induce analgesia. For example, neural stem cell transplantation reduces the overexpression of P2X4 and P2X7 receptors, activates the reconstruction of motor and sensory functions and plays an important role in the regulation of neuropathic pain after spinal cord injury [7]. In our previous study, microencapsulated olfactory ensheathing cells were transplanted into the injured sciatic nerve to reduce the expression of P2X4 receptors in the spinal cord and relieve pain [8]. Schwann cells (SCs) are the main glial cells in the peripheral nervous system and play an important role in the repair of nerve injury. SCs can secrete neurotrophic factors, protect the survival of neurons, improve the local inflammatory microenvironment of nerve injury and promote nerve repair [9]. Exogenous nerve growth factor (NGF) can activate autophagy of dedifferentiated SCs at the early stage of peripheral nerve injury, promote the clearance and phagocytosis of myelin fragments, and promote the regeneration of axons and myelin sheaths [10]. Chitosan scaffold loaded with Loc680254 highly expressed SCs can repair the sciatic nerve defects and promote axonal regeneration and functional recovery [11]. Specialized skin SCs have extensive processes, which are connected with the direct excitatory function of sensory neurons, and transmit harmful heat and mechanical sensitivity [12]. These studies have shown that SCs participate in the regulation of nerve injury repair and pain. Therefore, this study investigated the effects of SCs on neuropathic pain induced by sciatic nerve injury.

Materials and methods

Culture and identification of SCs

RSC96 cells are derived from long-term culture of rat primary SCs and purchased from BANA Culture Bank of Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). RSC96 cells were cultured in DMEM/F12 medium containing 10% FBS (Xavier, Wuhan, China), subcultured twice for cell identification and inoculated on a 24 well plate. After the cells adhered to the wall they were washed with PBS for 3 times. Cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS. Then 0.1% TrionX-100 was added to permeate for 15 min and the cells washed with PBS for 3 times. Goat serum blocking was added to the cells and reacted for 30 min. Mouse primary antibody S-100β (1:200, Abcam, Shanghai, China) was added overnight at 4 ℃ and cells then washed with PBS. Subsequently, FITC goat anti mouse fluorescent secondary antibody (1:100, Boshi Biological Technology Co., Ltd., Wuhan, China) was added for a fluorescence reaction for 1 h and cells then washed with PBS. DAPI was added to dye the nucleus for 2 min, and cells then washed with PBS. Then, the labeled positive cells were observed under fluorescence microscope.

Establishment of animal model of chronic compressive injury of sciatic nerve (CCI)

After intraperitoneal anesthesia, the rats were fixed on the operating platform, the right hind limb hair was cut off and the skin was disinfected. Subsequently, the skin and subcutaneous tissue was opened layer by layer to expose the sciatic nerve. Four courses of sciatic nerve were ligated with No. 4 catgut (with a spacing of 1 mm). The ligature force was appropriate for twitching the right hind limb so as not to affect the blood supply to the sciatic nerve.

SCs transplantation and grouping

Forty-eight healthy SD rats, weighing 150-180 g, were provided by the animal room of Nanchang University. All experimental studies and animal use have been approved by the Ethics Committee of Nanchang University. The rats were divided into four groups: sham group, CCI group, DMEM/F12 medium group (Dulbecco's Modified Eagle Medium / Ham's F12 mixture (50%/50% vol/vol)) and SCs group. SCs transplantation: the sciatic nerve trunk was exposed according to the method of CCI group, 0 days after surgery, absorbed on the gelatin sponge with a concentration of 2.4 × 10⁶/ml cells to the ligation of the sciatic nerve, gradually sutured and disinfected with iodophor. DMEM/F12 medium group was given the same amount of medium.

Detection of MWT and TWL of rats

The rats in each group were placed in the Von Frey pain threshold measuring glass instrument at 0, 3, 5, 7, 11 and 14 days after the operation and acclimated quietly for 15 min. Von Frey filaments were used to stimulate the planta of the right hind limb of rats and the stimulation intensity gradually increased until the right hind limb of rats showed foot retraction and licking reactions. Each stimulation time was not less than 15 s. When the hind limbs of rats were stimulated with a filament needle, the right hind limb of rats raised their foot. The minimum stimulation intensity of the foot contraction was the WMT. Repeated 3 times and the average value was taken.

The transparent plexiglass box was placed on the glass plate. BME-410C automatic thermal pain stimulator (Danmic Global, LLC, San Jose, CA, USA) was used to illuminate the plantar of right hind limb of rats, the irradiation time until the lifting and avoidance of the right hind limb of the rat was the TWL.

Quantitative detection of protein

L4-5 spinal cord tissue of rats in each group was taken and lysed in pre-cooled lysis buffer (Wuhan Boshide Biotechnology Co., Ltd., China) for 30 min. The tissue homogenate was then centrifuged (10,000 r/min, 15 min) and the supernatant was collected. Protein concentrations were measured using the BCA assay kit and heated to 95 °C for 10 min. The protein samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and the separated proteins were transferred to a PVDF membrane (Yuheng Technology Co., Ltd., Suzhou, China). At room temperature, the PVDF membrane was blocked for 1 h in 5% skim milk, then placed the PVDF membrane in rabbit P2X4 receptor primary antibody (1:500, Sanying Biotechnology Co., Ltd., Wuhan, China) or rabbit β-actin primary antibody (1:3000, Sanying Biotechnology Co., Ltd., Wuhan, China) and incubated overnight at 4 °C in a refrigerator. The next day, the membrane was washed 3 times with 1xTBST, and then goat anti rabbit secondary antibody (1:5000, Sanying Biotechnology Co., Ltd., Wuhan, China) was used to detect the primary antibody in the blocking buffer, and then was washed with 1xTBST. Subsequently, the reaction was carried out using chemical hypersensitivity solution (Wuhan Boshide Technology Co., Ltd.) and visualization was carried out using the Bio-Rad system (Bio-Rad, California, USA).

Immunohistochemistry

Sciatic ganglion tissue section (10um) was dewaxed, rehydrated and antigen repaired and washed with PBS 3 times. 0.1% TrionX-100 was added dropwise to the tissue, and incubated at room temperature for 15 min, then washed with PBS 3 times. Normal goat serum was then added for 30 min, without washing. Rabbit P2X4 receptor primary antibody (1:100) was added and the tissue placed in 4 ℃ refrigerator to react overnight. The next day, the tissue slice was washed with PBS 3 times. Goat anti-rabbit secondary antibody (1:200) was added at room temperature for 1 h, followed by washing with PBS three times. DABI color developing solution was added dropwise for 2-5 min. When the tissue turned brown it was washed with PBS. Hematoxylin for was then applied for1-2 min. Then the tissue section was dehydrated until transparent. A drop of neutral gum was added to the tissue and it was sealed with a cover slip. It was then under an inverted microscope, photographs taken and the number of P2X4 receptor labeled positive cells counted.

Immunofluorescence

Sciatic ganglion tissue sections were washed with PBS 3 times and then fixed with 4% paraformaldehyde for 15 min and washed with PBS 3 times. Goat serum was added for 30 min without washing. 300 μl rabbit P2X4 receptor primary antibody (1:100) or rabbit IBA-1 primary antibody (1:200, Seville Biotechnology Co., Ltd. Nanchang, China) was added to the tissue and reacted at 4 ℃ overnight. The next day, tissues were washed with PBS 3 times and then 300 μl rabbit fluorescent secondary antibody (1:200, Biyuntian Biological Technology Co., Ltd. Shanghai, China) was added for 1 h and then washed 3 times with PBS. DAPI staining solution was added for 5 min and then washed with PBS. Observed and photographed under an inverted fluorescence microscope.

Statistical method

Data are presented as mean ± SD using Graphpad.prism.6.x.crack-tsrh. Single factor variance and X² test were used for comparison among groups. Two-way analysis of variance was used to compare the difference at different times and between different treatment groups. P < 0.05 was considered statistically significant.

Results

Culture and identification of SCs

Under the microscope SCs were mainly spindle shaped and oval, evenly distributed and they proliferated rapidly (Fig. 1A). Further immunofluorescence identification showed that S100β, a specific marker of SCs, was highly expressed (Fig. 1B). This paved the way for further experiments.

Fig. 1.

Representative diagram of culture and identification results of SCs. A Under the microscope, SCs were mainly spindle shaped and oval and the cells were evenly distributed (indicated by arrows). B Identification results of the S100β specific marker of SCs. S100β was uniformly and highly expressed in cells (green fluorescence)

SCs transplantation alleviates mechanical and thermal hyperalgesia in rats

After transplantation of SCs, behavioral methods were used to detect the changes of pain sensation in the right hind limb of rats at 0, 3, 5, 7, 11 and 14 days. The results showed that, compared with the sham group, the MWT and TWL of CCI group of rats were significantly reduced (Fig. 2A, B). While compared with CCI group, the MWT and TWL of rats in SCs transplantation group were higher. However, compared with CCI group, there was no significant difference in pain perception in DMEM/F12 medium group. These data indicate that SCs transplantation has the effect of relieving hyperalgesia.

Fig. 2.

The effects of SCs transplantation on pain perception in rats were detected by behavioral methods. At 0, 3, 5, 7, 9, 11 and 14 days after transplantation of SCs, behavioral methods were used to detect the changes in the (A) MWT and (B) TWL in the right hind limb of rats. In the CCI group, the MWT and TWL of rats were reduced, however, the MWT and TWL of rats were increased after SCs transplantation treatment. Data are expressed as the mean ± SD of three independent experiments, n = 16. ^a P<0.05 CCI vs sham, ^b P<0.05 DMEM/F12 vs CCI, ^C P<0.05 SCs vs CCI

Effect of SCs transplantation on sciatic nerve myelin sheath

In order to observe the effect of SCs transplantation on the ultrastructure of sciatic nerve, the injured nerve was removed 14 days after surgery, and the changes of myelin sheath observed by transmission electron microscope (Fig. 3A). The results showed that the structure of myelin sheath in the sham group was uniform, regular and clear and the myelinated axons were intact. However, in the CCI group, shedding and separation of myelin fragments and segmental demyelination were observed. It was interesting to find that after SCs transplantation, the demyelination of sciatic nerve was improved, the myelin sheath was reformed, its thickness was uniform and no obvious myelin fragments were found. But in DMEM/F12 group, the change of sciatic nerve myelin sheath was not obvious. It is suggested that SCs transplantation can repair nerve injury.

Fig. 3.

Changes of myelin sheath after sciatic nerve injury and SCs transplantation. A The changes of myelin sheath of sciatic nerve on the injured side of rats in each group were observed by transmission electron microscope. In the sham group, the structure of myelin sheath of sciatic nerve was uniform, regular and clear, and the myelinated axons were intact. While compared with the sham group, the shedding and separation of myelin fragments and segmental demyelination were observed in the CCI gorp. After SCs transplantation, the demyelination of sciatic nerve was improved, myelination was reformed, the thickness of myelin sheath of sciatic nerve was uniform, and no obvious myelin fragments were found. B Microscope, immunofluorescence was used to detect the fluorescence changes of MBP in sciatic nerve of rats in each group. In the CCI group, the fluorescence intensity of MBP decreased, while the fluorescence intensity of MBP in the sciatic nerve of rats increased in the SCs group, n = 12. *P<0.05, ** P<0.01

Next, we sought to further verify that SCs transplantation can promote the repair of nerve injury by viewing the fluorescence intensity of myelin basic protein (MBP) in the myelin sheath of sciatic nerve of rats in each group (Fig. 3B). The results showed that this decreased in the CCI group, but increased after SCs transplantation. Similarly, there was no significant difference in MBP fluorescence in DMEM/F12 group. The above results indicate that SCs transplantation promotes the repair of sciatic nerve injury, which is consistent with the results of other studies.

Transplantation of SCs reduces the expression of P2X4 receptor in spinal cord

To further investigate whether SCs affect P2X4 receptors, their expression in spinal cord tissue of rats in each group was measured by immunohistochemistry 14 days after surgery (Fig. 4A). The results showed that the cells expressing the P2X4 receptor were brown/yellow. Compared with the sham group, the number of positive cells in spinal cord of rats in the CCI group increased significantly. After transplantation of SCs, the number of P2X4 receptor-labeled cells was significantly reduced. However, there was no significant difference in the expression of P2X4 receptor between DMEM/F12 group and CCI group.

Fig. 4.

Effect of SCs on P2X4 receptor expression. A Immunohistochemistry was used to detect the expression of P2X4 receptor in the spinal cord (The arrows in the figure show the stained brownish-positive cells). Compared with the sham group, the number of P2X4 receptor labeled positive cells in spinal cord of rats in CCI group increased significantly. After transplantation of SCs, the number of P2X4 receptor labeled positive cells was significantly reduced. However, there was no significant difference in the expression of P2X4 receptor between DMEM/F12 group and CCI group. B and C Immunofluorescence was used to detect the co-expression and fluorescence changes of P2X4 receptor and microglial marker IBA-1 in spinal cord tissue. P2X4 receptor and IBA-1 were co-expressed in microglia. Compared with CCI group, the fluorescence intensity of P2X4 receptor decreased after SCs treatment. Data are expressed as the mean ± SD of three independent experiments. n = 12.** P<0.01

To further verify the above results, we further measured the effect of SCs on P2X4 receptor expression in spinal cord by immunofluorescence (Fig. 4B, C). The results showed that the P2X4 receptor was located in microglia, as indicated by co-expression with the microglia marker, IBA-1, and the fluorescence intensity and the number of P2X4 receptor-labeled cells in CCI group were increased. In contrast, the fluorescence intensity and the expression of P2X4 receptor labeled cells were reduced after SCs treatment. These results suggest that SCs transplantation may play a role in pain relief by down-regulating the expression of P2X4 receptor.

SCs transplantation down regulates P2X4 receptor protein expression

Finally, we determined the effect of SCs transplantation on the level of P2X4 receptor protein expression in spinal cord tissue by using Western-blotting (Fig. 5A, B). The expression of P2X4 receptor protein in the CCI group was higher than that in sham group. It decreased significantly after SCs treatment, but there was no significant difference in the expression in the DMEM/F12 group. These data further confirm the inhibitory effect of SCs therapy on P2X4 receptor expression.

Fig. 5.

The effect of SCs on the expression of P2X4 receptor protein in spinal cord of rats. A and B Western blotting was used to detect the effect of SCs transplantation on P2X4 receptor protein expression in spinal cord. The expression level of P2X4 receptor protein from low to high was sham, SCs, DMEM/F12 and CCI groups. While compared with the CCI group, the expression level of P2X4 receptor protein decreased after SCs transplantation. Data are expressed as the mean ± SD of three independent experiments. n = 8. * P<0.05

Discussion

In this study, we found that SCs could alleviate P2X4 receptor-mediated neuropathic pain, significantly reduce the expression of P2X4 receptor in the spinal cord and reduce hyperalgesia in rats. This is consistent with previous studies showing that P2X4 receptors contribute to neuropathic pain [8, 13, 14]. Although our experimental data reveal that SCs transplantation plays a role in pain relief, the specific molecular mechanism is not very clear. Possibilities include: (1) SCs reconstitute the myelin cells of the peripheral nerve, which can protect and support the survival of neurons. During the repair of peripheral nerve injury, SCs can improve the microenvironment around nerve injury, promote axonal regeneration and re-myelination by changing their phenotypic changes. This supports the repair of nerve injury and pain relief [15–17]. (2) SCs can also provide a nutritional basis for nerve regeneration and repair by secreting a variety of neurotrophic factors (such as nerve growth factor, brain-derived nerve growth factor, ciliary nerve growth factor and neurotrophic factor 3/4) [15, 18]. (3) SCs provide a good basis for nerve regeneration by secreting chemokines, inducing phagocytosis, including of myelin debris and apoptotic cells, and recruiting macrophages to migrate and gather to the injured site [19]. Studies have shown that exogenous nerve growth factor can activate autophagy of dedifferentiated SCs in the early stage of peripheral nerve injury, promote the clearance and phagocytosis of myelin fragments, and promote axon and myelin regeneration [10]. ANXA1 is an extracellular trigger for the proliferation and migration of SCs. It activates FPR2 receptor and downstream AMPK signal pathway to induce SCs proliferation and migration in vitro, and promotes peripheral nerve regeneration [20]. All these show that SCs transplantation can promote axonal regeneration and myelination, improve neurological function, and lay a good foundation for the treatment of pain.

In this study, we found that the P2X4 receptor was localized in spinal cord microglia. In the CCI group, its expression was significantly up-regulated in spinal cord tissue, and hyperalgesia was also significantly increased in rats. However, SCs transplantation can significantly down-regulate the expression of P2X4 receptor in spinal cord tissue, thus playing a role in pain relief, which is consistent with the results of other previous studies [8, 13, 14]. Therefore, We speculate that the possible mechanism is that SCs can recover the sciatic nerve injury, protect the survival of neurons, reduce the expression of P2X4 receptor in microglia, and alleviate the progress of pain by secreting some nutritional factors.

These data indicate that SCs transplantation reduces the expression of P2X4 receptor and relieve pain. However, how SCs affect the mechanism of P2X4 receptor for pain relief needs to be further studied. We are just preliminary to explore the application of P2X4 receptor mediated by SCs in pain treatment. These data also provide new data support for the treatment of SCs in pain in the future, and bring new hope for the treatment of clinical pain patients in the future.

Wen-jun Zhang

is mainly engaged in research in the fields of cell transplantation and pain. He has been engaged in research in this field for many years and has solid experience.  

Author contribution

Wen-jun Zhang: Completed this research, wrote this article and processed the data. Xi Li: Completed method and data collection. Jun-xiang Liao: Completed data collection and references. Dong-xia Hu: Completed the revision of the article. Song Huang: Completed the revision of the article.

Funding

These studies were supported by grants from the National Natural Science Foundation project (82460233), Youth Science Foundation of Jiangxi Province (20224BAB216030). Project of Education Department of Jiangxi Province (GJJ2200238). Natural Science Foundation of Jiangxi Province (20232BAB206048). Jiangxi Province Traditional Chinese Medicine Science and Technology Plan (2023B1213). Incubation Program of National Natural Science Foundation of the second affiliated Hospital of Nanchang University (2022YNFY12006). Doctor's start-up Fund of the second affiliated Hospital of Nanchang University (B3091).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval and consent to participate

Ethical approval has been exempted by the Ethics Committee of the Second Affiliate Hospital of Nanchang University. All protocols were approved by the Animal Care and Ethics Committee, China.

Consent for publication

Not applicable.

Competing of interest

The authors declare no competing interests.