Electroacupuncture may promote the repair of spinal cord injury in rats by regulating the perineuronal net through Chst11

doi:10.21203/rs.3.rs-3404696/v1

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Research Article

Electroacupuncture may promote the repair of spinal cord injury in rats by regulating the perineuronal net through Chst11

https://doi.org/10.21203/rs.3.rs-3404696/v1

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The aim of this study was to assess the expression of perineuronal net(PNN) and parvalbumin positive interneuron(PV⁺IN) in spinal cord injured rats and to characterize the effect of electroacupuncture(EA) on the modulation of PNN and PV⁺IN via Chst11. EA stimulation of jiaji acupoints (EX-B2) was started on day 1 after preparation of the spinal cord injury(SCI) model using the IH 0400 spinal cord impactor for 14 days. And chondroitinase ABC was used to treat spinal cord injured rats and compared with EA. A specially adapted adeno-associated virus (Chst11) was also injected into the T9 spinal cord of rats and combined with Basso-Beattie-Bresnahan scoring, in vivo fibre optic calcium imaging, western blotting and immunofluorescence. The results suggest that EA can effectively ameliorate the destruction of PNN structure and function after SCI, increase the activity of PV⁺IN, promote the regeneration of chondroitin sulfate and reverse the inhibitory effect of Chst11 on injury repair, maintain the balance of neuronal plasticity and stability, and promote spinal cord repair after injury.

spinal cord injury

electroacupuncture

perineuronal net

parvalbumin positive interneuron

Chst11

With the rapid development of the global economy and the increasingly severe aging process of the population, the incidence of spinal cord injury(SCI) remains high^[1], and its disability rate has become the second leading cause of paralysis^[2], placing a heavy burden on the social health care system and causing great economic and psychological pressure on patients and their families. The pathological mechanisms of SCI are complex and diverse, and effective treatments for SCI are still lacking.

Perineuronal net(PNN) is one of the important extracellular environments around parvalbumin positive interneuron(PV⁺IN) in the central nervous system(CNS). It plays an important role in neuroprotection and maintenance of neuronal plasticity and stability. PNN is a network structure within the extracellular matrix(ECM) that is enriched with chondroitin sulfate proteoglycan(CSPG)^[3]. It is worth noting that CSPG has been shown by a large number of studies to be an important factor in the action of PNN on the CNS^[4], which is mainly formed by covalent bonds between chondroitin sulfate glycosaminoglycan(CS-GAG) chains and various core proteins. According to the difference of chondroitin sulfate sulfotransferase have different mode of sulfation, will happen in the process of the development of significant change^[5–7], and chondroitin 4-sulfation(C4S) and chondroitin 6-sulfation(C6S) are the two main types. A large number of studies believe that the negative charge carried by CS-GAG plays a crucial role in maintaining the plasticity and stability of neurons, and the degradation of CS-GAG after nerve injury will cause serious damage to the CNS^[4]. At the same time, studies have also shown that the absence of chondroitin sulfate(CS) will reduce the spike reaction of PV⁺IN wrapped in PNN^[8], and the maturation of PV⁺IN will promote the development of PNN and promote the sulfation mode of CS-GAG. In turn, the sulfated mode mode of CS-GAG also promotes the maturation of PV⁺IN and maintains cell function^[8]. Chondroitin-4-sulfotransferase-1(C4ST-1)/carbohydrate sulfotransferase-11(Chst11), an enzyme widely distributed in the CNS that specifically promotes CS synthesis, regulates C4S expression and plays an important role not only in CS 4-O-sulfation but also in the amount of CS synthesis^[9]. Research has demonstrated that the knockout of C4ST-1 can expedite the regeneration of neuronal axons after SCI in juvenile and adult zebrafish. In contrast, the knockout of D4ST-1 has no effect. These findings suggest that Chst11/C4ST-1 may inhibit axonal regeneration and various cellular parameters that play a role in the restoration of motor function following injury^[10].

Electroacupuncture(EA) administered on the jia-ji acupoints constitutes a reliable and effective clinical approach that safely enhances the peripheral milieu following SCI. It efficiently repairs the nerve function of the spinal cord, fosters axon regeneration, and ultimately accelerates the restoration of motor function^[11]. The team's previous research confirms the impact of spinal cord EA on SCI. EA plays a significant role in promoting nerve recovery and promoting motor and sensory function recovery for SCI patients. Additionally, EA interventions during the acute stage can have a more significant neuroprotective effect^[11]. Previous research extensively studied the role of PNN in memory and the brain, but its changes and effects after SCI require further investigation. The mechanism of EA on local PNN in SCI rats lacks strong research support.

Therefore, this study utilized adeno-associated virus(AAV) and/or EA intervention, along with behavioral and molecular biological detection and in vivo calcium imaging to investigate the importance of preserving the integrity of PNN in neuronal recovery following SCI and the therapeutic benefits of EA. Research has found that using jiaji EA after SCI can reverse the inhibitory effect of Chst11 on the functional changes of PNN and PV⁺IN, and promote the recovery of nerve function after SCI.

Reagents and experimental instruments

The Mini-protean vertical electrophoresis and membrane transfer system was purchased from Bio-Rad(Hercules, CA, USA). VARIOSKAN LUX was purchased from Thermo Fisher Technologies in the United States. Electronic constant temperature addition table(GT20302) was purchased from Mona Biotechnology Co., LTD.(Suzhou, China). The tricolor optical fiber recorder was purchased from Qianaoxing Konanjing Biotechnology Co., LTD.(Nanjing, China). Sodium pentobarbital and N,N,N',N'-tetramethylenediamine(TEMED) were purchased from Sigma-Aldrich(St. Louis, Missouri, USA). Pierce™ BCA protein test kit purchased from Thermo Fisher Scientific Inc.(Fairlawn, NJ, USA).

Animals

Healthy adult male SD rats(8 weeks of age, 200-220g) were purchased from Shanghai Sipur-Bike Laboratory Animal Co., LTD.(Animal license No. : SCXK(Shanghai) 2018-0006), and were raised in Laboratory Animal Center of Zhejiang Chinese Medicine University and approved by the China Laboratory Animal Management Evaluation and Accreditation Association(AAALAC, Animal license No. : SYXK(Zhejiang) 2018-0012). The rats were kept in controlled conditions and had free access to water and food. All animal experiments were conducted in accordance with all relevant animal testing and research ethics regulations and in accordance with the animal protocol approved by the Animal Ethics Committee of Zhejiang University of Chinese Medicine(ZSLL, 2017183). All experimental protocols strictly follow the National Institutes of Health(NIH) guidelines on the use of laboratory animals(NIH Publication No. 8023).

Preparation of rat SCI model

Healthy adult male SD rats were deeply anesthetized with 3% pentobarbital solution(2ml/kg) and placed on a constant temperature operating table at 37℃. The T10 cone was exposed and the lamina was removed to expose the T10 spinal cord. An acute SCI model was created by impinging the T10 spinal cord with a force of 200 kDynes using the IH Impactor 0400 SCI Impinger(American PSI impactor). Signs of successful modeling: ①body spasmodic tremor; ②spasmodic movement of the tail; ③Intradural congestion or hematoma, the above three signs appear is successful modeling. Basso Beattie Bresnahan(BBB) score was performed on the next day of modeling, and rats with scores ≥3 were excluded and supplemented with the same conditions. Postoperative management: ①Penicillin (100U/d) was injected into the abdominal cavity for anti-infection for 3 consecutive days; ②Keep room temperature at 20-25℃; ③Massage the bladder twice a day until it appears to urinate autonomously.

Electroacupuncture therapy

The T9-T11 jiaji acupoints(EX-B2) on both sides of the spinous process of the back of the rats were treated with electroacupuncture on the first day after surgery. The operation is as follows: Insert a disposable sterile stainless steel acupuncture needle with a diameter of 0.18mm(Beijing Zhongyantahe Medical Instrument Co., LTD., Beijing, China) into the position of 1.5mm apart from the median of spiny process, with a depth of 4-5mm, until the tip of the needle contacts the lamina, and connect it to the HANS200A electroacupuncture instrument(Nanjing Jisheng Medical Technology Co., LTD., Nanjing, China). The parameters are set as follows: the frequency is 2/100 Hz, the current intensity is maintained at 1 mA, once a day for 20 min a day. The electroacupuncture treatment period was fourteen days.

Behavioral test

The Basso–Beattie–Bresnahan(BBB) hind limb movement test was scored on a scale of 0-21(0= no visible hind limb movement, 21= normal movement), and the hind limb movement of rats with SCI was tested in an open field, including hind limb joint movement, weight support, foot step, coordination, paw position, paw fine movement, and trunk and tail control. The BBB test was designed to assess the recovery of hind limb motor function in rats. BBB testing began on the first day after the model was established. Each rat was placed in an open field and evaluated by 2 experimenters who were not aware of the experimental group for 3 min each time, one of whom recorded the score, and all rats were evaluated before the SCI model was established to ensure that there were no baseline differences. Each score is averaged to make the final score.

AAV stereotactic injection

The rats were anesthetized with 3% pentobarbitone(2ml/kg, intraperitoneally injected), the hair on the back at T9 position was shaved, the rats were fixed to a stereolocator(RWD, 68025, Shenzhen, China), and a 10μl microsyringe was connected to a microinjection apparatus(KDS, LEGATO130, Holliston, MA, USA) and its controller(KDS, LEGATO130, Holliston, MA, USA). The rats were then injected with 0.5ml rAAV-CMV-Chst11-Flag-WPREs, AAV2/9(BrainVTA Co., Ltd., Wuhan, China) and a negative control rAAV-CMV-Flag-WPREs, AAV2/9(BrainVTA Co., Ltd, Wuhan, China) or rAAV-CAG-FLEX-jGCaMP7f-WPRE-SV40pa, AAV2/9(BrainVTA Co., Ltd, Wuhan, China) and rAAV-PV-CRE-bGHpa, AAV2/9(BrainVTA Co., Ltd, Wuhan, China) virus to bilateral T9 spinal cord. Procedure: After the T9 lamina was removed with tweezers, the spinal cord was exposed with reference to the median line, the lateral opening was 1mm, the median blood vessel was 0.3mm, the tilt Angle was 10°, and the virus was injected into a depth of 1.5mm under microscope. The tip of the needle is left in the spinal cord for 5 min after the injection is completed to prevent the virus relux. Blood vessels and nerves are avoided during injection, and the final titer is 5.00E+12vg/ml, 100nl/min, about 500 nl for each.

Immunofluorescence staining

Each mouse was deeply anesthetized with pentobarbital sodium(2ml/kg) and infused with 0.9% physiological saline(4℃) 200ml and 4% paraformaldehyde solution 200ml through the heart. The T9-T11 spinal cord was collected and fixed in 4% paraformaldehyde for 4 h(4°C), then dehydrated with 15% and 30% gradient sucrose solutions for seventy-two hours. The spinal cord was successively cut into 12μm thick transverse sections using a Thermo Scientific CryoStar™ NX70(USA), each group containing five or more consecutive sections. All slides were closed with TBST containing 10% normal goat serum at 37°C for 1 h and then incubated with the corresponding primary antibody at 4°C overnight. The primary antibodies used were rabbit monoclonal antibody Anti-Parvalbumin(1:100, ab181086, Abcam) and wisteria lectin WFA(1:100, B-1355-2, Vector Laboratories, USA). The next day, the slices were rinsed with TBST(6 times, 10 minutes each) and reacted with the corresponding secondary antibody mixture(1:200, Alexa Fluor® 555 and 1:300, Alexa Fluor® 488 Goat Anti-Rabbit IgG). Fluorescence images were obtained using a Zeiss structured illuminated optical slice microscope(Axio Imager M2, Zeiss AG, Germany). For quantitative fluorescence intensity analysis, a uniform microscope shot setting is maintained throughout all image acquisition processes. All stained sections were examined and analyzed by blind method. Five images were randomly selected from each rat tissue, and the positive cells in the same area were counted and averaged.

Western blotting

The T10 segment injured spinal cord was collected on SCI day 14. The rats were deeply anesthetized with 3% sodium pentobarbital solution(2ml/kg) and intravenously injected with 200ml normal saline(4℃), and the spinal cord was immediately removed and stored in a refrigerator at -80℃. The tissue was placed in a RIPA lysis buffer containing a protease inhibitor. After the tissue lysis, the supernatant was collected by centrifugation at 12000rpm at 4°C for fifteen minutes. According to the manufacturer's instructions(Thermo Fisher, USA), use dicinchonic acid(BCA, Pierce™ BCA, Thermo Fisher Scientific Inc, Fairlawn, NJ, USA) method for protein concentration determination, 20 mg protein per lane sample. In order to detect CS protein content, 5U/ml of Chondroitinase ABC(ChABC) was added into the protein supernatant and incubated in a shaking bed at 37℃ for 8h. Protein samples were separated on a 4%-15% SDS-polyacrylamide gel electrophoresis(SDS-PAGE) gel and electrophoretically transferred to a polyvinylidene fluoride(PVDF) membrane(Merck Millipore, Inc., Billerica, MA, USA). The membrane was closed with 5% skim milk(Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at room temperature for one hour, and then incubated at 4℃ overnight in a diluted primary antibody in a closed buffer: β-Actin(1: Five thousand, Ms mAb to beta Actin, abcam, USA), C4S(1:2000, mouse Anti-Chondroitin-4-Sulfate, Merck, USA), C6S(1:2000, mouse Anti-Chondroitin-6-Sulfate, Merck, USA), GAD65, and GAD67(1:2000, Rb mAb to GAD65+GAD67, abcam, USA). The next day, the membrane was incubated with secondary antibodies(1:2000, Goat Anti Mouse and 1:2000, Goat Anti Rabbit, 7074s) at room temperature for two hours. Immunoreactivity was detected using enhanced chemiluminescence and visualized using Image Quant LAS 4000. The absorbance of each strip was measured using the ImageJ analysis software. The relative expression of target protein is(target protein absorbance value)/(actin absorbance value), and the result is expressed as the mean ± standard error.

Fiber optic calcium imaging and analysis

28 d before modeling, inject rAAV-CAG-FLEX-jGCaMP7f-WPRE-SV40pa, AAV2/9 and rAAV-PV-CRE-bGHpa, AAV2/9 mixed viruses into the spinal cord at the junction of T9 and T10 with Hamilton microinjector. The dose was 500nl per side, 100nl/min rate, and depth was 1.5mm. After the T10 spinal cord was fully exposed, a special 0.01mm thick perforated titanium plate was placed above the spinal cord and secured to the soft tissue and vertebral body with non-absorbable surgical sutures at the four corners. An optical fiber with a diameter of 200um and a length of 2mm(with a stereolocator and optical fiber fixator) was placed under a microscope near the central duct of the spinal gray matter with a depth of 1.5mm, fixed with dental cement and glue, and covered with toner to avoid light. GCaMP7f is excited at two wavelengths(470nm calcium-related signal and 405nm baseline signal), reflected to the dichroic mirror through the amplitude modulation signal, and coupled to the optical fiber for the carrier fiber calcium imaging photometric recording. Load the obtained data into the Matlab system for analysis. Calcium activity was recorded from the first 2 s of the external stimulus to 10 s after the stimulus to capture changes in calcium signaling in the short time after sensory induction(pinching the foot).

Electromyography

The electromyography(EMG) of the gastrocnemius muscle of the right lower limb was collected by Nicolet EDX:Viking QUEST. The rats were put on a special rat sleeve, and the hair of the gastrocnemius muscle was shaved with a hair shaving machine, and the rats were tested in a state of emotional calm. During detection, recording electrodes were inserted into the gastrocnemius muscle of the right lower limb of the rats, and EMG with a frequency of 20-500 Hz was collected for 20-25 consecutive times. The gastrocnemius potential was detected by VikingSelect experimental system, the amplitude and duration were recorded, and the average value was obtained.

Statistical analysis

All analyses and graphs were captured using GraphPad software or Matlab. We used the Shapiro–Wilk test to assess the normality of the distribution of continuous variables, and Student's t test(two-tailed) and one-way analysis of variance, followed by Tukey's post-hoc test, were used to compare the means of two and multiple groups, respectively, for normally distributed data. Alternatively, the Mann-Whitney U test and the Kruskal-Wallis H test were used to compare two or more sets of data with non-normal distributions, respectively. All results are expressed as mean ± SEM, p<0.05 was considered statistically significant.

1. The expression of PNN and GAD decreased significantly after SCI

Since there are no basic studies related to the changes of PNN and PV⁺IN in SCI, we designed SCI model to evaluate both at day 1, 7, and 14. We performed western blot tests for chondroitin sulfate, which is critical for the composition and function of PNN, and the corresponding sulfated forms(C4S and C6S) to quantify PNN expression(Figure 1A, B). The results showed that the expression of C4S and C6S did not change on the first day after SCI, and the expression levels of both decreased significantly on the seventh day, while the expression levels increased to a certain extent on the 14th day, but were still far lower than those in the normal group(Figure. 1C, D). There was no difference in expression between the 7th and 14th days. The results showed that PNN expression decreased significantly after SCI and remained at a low level on day 14. At the same time, quantitative western blotting was performed for glutamate decarboxylase(GAD65/67), an important enzyme for the synthesis of parvalbumin(Figure. 1F, G). Western blotting showed that the expression of GAD65/67 decreased in different degrees after SCI. Among them, GAD65 showed a significant increase on the first day after SCI, and then gradually decreased on the seventh and 14th days. GAD67 showed no significant change on the first day after SCI, then significantly decreased on the seventh day, and increased on the 14th day compared with the seventh day, but still lower than the normal group(Figure 1E). In summary, the results showed that the expression levels of PNN and PV⁺IN were significantly decreased after SCI, indicating that SCI has a significant impact on both.

2. ChABC can reshape PNN after SCI and promote functional repair after injury

In order to explore the mechanism of PNN involved in spinal cord repair after injury, we treated the injured site with ChABC and observed the changes of CS expression at the injured site thereafter. BBB motor scores showed that the scores of SCI group and PBS solvent control group were significantly lower than those of Sham group on the 7th day after operation. Compared with the model group and the PBS solvent control group, the motor function score of the rats was significantly increased after intramural injection of ChABC, and the motor function of the rats' lower limbs was significantly improved(Figure 2A). To further evaluate the recovery of the hind limb, we performed EMG tests on the gastrocnemius muscle of the hind limb. The results showed that after SCI, the unit EMG of gastrocnemius in the hind limb of rats was significantly weakened, and the amplitude and duration of unit EMG of gastrocnemius were increased after treatment with ChABCcompared with PBS group(Figure 2B-E). These results indicate that ChABC can effectively promote the recovery of motor function after SCI in rats. After that, Western blotting was performed on the relevant rat tissues(Figure. 2F). It can be seen from the results that C4S and C6S expression levels in the ChABC group were significantly increased compared with the PBS solvent control group on the 14th day after SCI, and were close to those in the normal control group(Figure. 2G-H). Immunofluorescence results showed that PNN specific markers WFA and PV in SCI and SCI+PBS groups were significantly less expressed in the area near injury in the gray matter lamina of the T10 spinal cord than in Sham group. Compared with SCI or SCI+PBS, the amount of WFA and PV in the SCI+ChABC group was significantly increased(Figure 3A, B). These results indicate that ChABC can significantly promote the repair of PNN after SCI, and the repair of PNN is consistent with the recovery progress of SCI

3. ChABC can reverse the decreased activity of PV⁺IN after SCI

After confirming that the changes of PNN are closely related to the repair of SCI, we further explored whether similar changes exist in PNN coated PV⁺IN. Therefore, AAV indicating changes in calcium ion signal and labeling PV⁺IN were injected into the rat T9 spinal cord 28 days before modeling, and calcium imaging fiber was implanted 28 days later while the SCI model was prepared. Calcium signals were detected and recorded on day 14 after injury(Figure 4A, B). We recorded calcium signals from 2 seconds before stimulation to 10 seconds after stimulation while maintaining the emotional stability of the rats, and the results showed that PV⁺IN activity decreased significantly after SCI compared with sham group(Figure 4C, D). Compared with the PBS solvent control group, the PV⁺IN activity in the injured site of rats in the ChABC group was significantly increased(Figure 4C, D). In conjunction with the preceding restoration of motor function in rats, ChABC exhibits the potential to facilitate the restoration of SCI while concurrently augmenting the expression level and activity of PV⁺IN. Further WB results showed(Figure. 4G) that compared with PBS solvent control group, the expression of GAD67 was significantly increased after ChABC enzyme treatment, with a statistical difference, while GAD65 had no significant change(Figure. 4H, I).

4. EA reversed PNN and PV⁺IN destruction and loss of motor function after SCI

Having established the role of ChABC in promoting repair after SCI, we aimed to test whether EA would have the same effect as ChABC. The results showed that after EA treatment, BBB motor function scores of rats in both lower limbs were higher than those in model group and sham EA group(Figure. 5A). The results of EMG detection also showed that the unit EMG amplitude and duration of gastrocnemius after EA treatment were significantly higher than those in the sham EA group(Figure. 5B, C). More importantly, the results of western blotting showed that compared with the model group, the protein expressions of C4S and C6S in the injured spinal cord were significantly increased after EA treatment, indicating that EA promoted the repair of PNN after SCI(Figure 5E, F). Moreover, the protein expressions of GAD67 and 65 also increased significantly after EA intervention(Figure. 5H, I). In summary, EA can play the same role as ChABC in promoting the repair mechanism after SCI, and can effectively reverse the loss of PNN and GAD/PV after injury.

5. EA can reverse the inhibition of Chst11

Having verified that EA has the same effect as ChABC, we aim to further investigate whether EA can improve SCI by reversing the inhibition of Chst11. We used adeno-associated virus injection to inject the overexpressed virus rAAV-CMV-Chst11-Flag-WPREs, AAV2/9 and the control virus rAAV-CMV-Flag-WPREs, AAV2/9 into the spinal cord of rat T9 28 days in advance with microinjection needle to allow the virus to fully transfect. The model of SCI was then prepared(Figure 6A, B). BBB motor function evaluation results showed that the recovery of hind limb motor function of SCI+Chst11 group was significantly worse than that of control virus group. After intervention with EA, the hind limb motor function of SCI+Chst11+EA group was significantly improved, which was far better than SCI+Chst11 group and also had differences compared with SCI+Veh group(Figure. 6C). On this basis, we also conducted EMG tests on the hind limbs, and the results showed that the amplitude of unit EMG of the hind limbs in the EA treatment group was significantly increased compared with that in the SCI+Veh and SCI+Chst11 groups, with a statistical difference, while the duration was not significantly different among the three groups(Figure. 6D, E). Western blot results showed that the protein expression levels of C4S and C6S in SCI+Chst11 group were significantly lower than those in SCI+Veh group(Figure. 6F), while the protein expression levels in SCI+Chst11+EA group were much higher than those in the first two groups, and there was a statistical difference(Figure. 6G, H). Glutamate decarboxylase protein expression associated with PV synthesis also showed results consistent with CS(Figure 6I). The protein expression levels of GAD67 and GAD65 in the EA treatment group were significantly higher than those in SCI+Chst11 and SCI+Veh groups, and there was a statistical difference(Figure 6J, K). These results indicated that the overexpressed virus inhibited the expression level of PV⁺IN, and EA could effectively improve this situation and promote the repair of SCI. The immunofluorescence results showed that compared with the SCI+Veh group of the virus control group, the number of WFA⁺PV⁺ cells in the spinal cord of the SCI+Chst11 group was significantly reduced, while the number of WFA⁺PV⁺ cells in the SCI+Chst11+EA group was significantly increased compared with the first two groups, and was more than that in the SCI+Veh group(Figure. 7A, B). In conclusion, EA can reverse the inhibitory effect of overexpressed virus Chst11, and promote the remodeling of PNN and PV⁺IN, as well as the functional repair of SCI.

It has been found that after SCI, the volume and number of individual PNN around motor neurons at the site of injury decreased, and structural and functional remodeling occurs^[12], which is accompanied by atrophy of inhibitory GABAergic interneurons and decreased expression of PV⁺IN ^[13]. The same phenomenon was observed in this study. In addition, we also observed that the calcium ion activity of PV⁺IN in the SCI model was decreased, and the protein level expression of CS and GAD was decreased. The level of GAD also reflected the activity and expression level of PV⁺IN to some extent^{[14, 15]}. More importantly, EA intervention reversed the disruption of PNN integrity due to CS deficiency after SCI, as well as the decreased activity of PV⁺IN, and facilitated motor repair after SCI.

PNN is a densely aggregated form of ECM with a lattice-like network structure and a negative charge that is widely present around many neurons in the CNS^[16–20], predominantly wrapping around the rapidly spiking γ-aminobutyric acidergic PV⁺IN and encircling motor neurons along the spinal cord.^{[21, 22]}. The functions of PNN in the nervous system(e.g., ion buffering, neuroprotection, and neuromaturation) benefits from its structural integrity^[22–25]. When the spinal cord is damaged, the structure of the PNN is disrupted and a reduction in thickness occurs^[26], thus upsetting the balance between plasticity and stability^[4]. In addition, a decrease in PNN leads to increased neuronal excitability^[27], a decrease in negative perineuronal charge, and changes in GABAergic responses in inhibitory neurons, leaving the neurons in a state of characterization similar to that of the developmentally immature period^[4]. As a result of PNN reduction, uncontrolled spinal cord sprouting and increased neuronal excitability occurs^{[28, 29]}, leading to hyperexcitability of spinal cord circuits, which can lead to maladaptive symptoms such as neuropathic pain and spasticity^[30–34]. Thus the integrity of the PNN after SCI is of great importance for the subsequent recovery of spinal cord neurological function, as demonstrated in the previous part of this study and by others in reorganization of the corticospinal tract after injury^[35]. In addition, the importance of maintaining PNN integrity for subsequent recovery has been observed in amyotrophic lateral sclerosis and spinal muscular atrophy^{[36, 37]}. Thus, excessive disruption of PNN disrupts PNN reorganization after injury, leading to increased motor deficits^[38].

CSPGs are the main components of PNN^[39], and all types of CSPGs are composed of core proteins and a variable number of CS-GAG chains^{[40, 41]}, whose high negative charge is one of the most important determinants of PNN function^[42]. This is because the high-intensity negative charge carried by CS-GAG can buffer cations generated after oxidative stress or toxic metal ions, conferring neuroprotection to the PNN^{[43, 44]}. Previous studies have suggested that CS-GAG is sulfated at different positions, including monosulfated position 4(CS-A/C4S), position 6(CS-C/C6S), and disulfated CS-D and CS-E^[45]. C4S and C6S have different properties:C4S inhibits axonal growth and suppresses neuronal plasticity, whereas C6S allows axonal growth and enhances plasticity^{[46, 47]}. Therefore, most studies have been conducted to ensure the presence of C6S after the elimination of C4S or to increase the expression of C6S to promote recovery after CNS injury ^{[46, 48]}. However, since CS-GAG is essential for the stability of the PNN, excessive degradation of the CS-GAG chain leads to excessive plasticity and increases the susceptibility of nerves to neurotoxic stimuli.^[4]. Therefore, it is also necessary to maintain the level of CS expression in the PNN after SCI and the balance of the PNN itself.

In the CNS, the PNN is predominantly wrapped around the fast-spiking PV⁺IN^{[21, 22]}, a type of inhibitory γ-aminobutyric acid interneuron, and there is a close relationship between the timing of its maturation and the development of the PNN. PV⁺IN is characterized by high frequency discharge rates and a lack of spike-frequency adaptation, which requires recruitment of small conductance Ca²⁺-activated K⁺channels channels^[49]. By buffering spike-mediated calcium influx to prevent SK channel activation, PV⁺IN can achieve rapid spike discharge^{[50, 51]}. Thus, reduced expression of PV⁺IN and the concomitant accumulation of intracellular calcium decreases the rapid spike discharge of PV⁺IN and reduces its inhibitory output and effect. ^[52]. After SCI, because the PNN structure is disrupted and the number of PNN wrapped around PV⁺IN is reduced, neuronal excitability is increased, and PV⁺IN is more susceptible to damage from neurodegeneration as well as extracellular oxidative stress and reduced neuronal function^[44]. The present study demonstrated a significant reduction in PV⁺IN after SCI, which is likely the result of the reduction in PNN. As PV⁺IN loses the protective effect of PNN, the resulting increase in inflammatory factors, oxidative stress, and cytotoxicity at the site of injury has a tremendous impact on it, leading to a dramatic decrease in PV⁺IN expression and activity. Increasing the activity of PV⁺IN was also found to reduce mechanically abnormal pain in a mouse model of neuropathic pain after nerve injury in previous studies^[52], suggesting that PV⁺IN plays an important role in repairing nerve injury and inhibiting pain. Interestingly, the expression and activity of PV⁺IN improved significantly after our therapeutic intervention at the SCI site using ChABC, which may be attributed to the increased excitability and activity of PV⁺IN after ChABC promoted the rearrangement of PNN. It also suggests that PV⁺IN is essential for SCI repair.

The negative charge carried by CS-GAG confers PNN neuroprotection, so CS-GAG is also essential for PV⁺IN, and CS deletion reduces the PV⁺IN spike response^[8]. Maturation of PV⁺IN promotes PNN development and similarly promotes the sulfation pattern of CS-GAG. In turn, the sulfation mode pattern of CS-GAG was able to promote the maturation of PV⁺IN and maintain the cellular function^[8], and the stable structure of PNN formed by the participation of CS-GAG also brought neuroprotection to PV⁺IN against extracellular oxidative stress and other factors. Overall, the three are closely and inextricably related and complementary, and together they are involved in the stabilization and development of the CNS, and the absence or imbalance of any one of them can result in incalculable damage to the CNS.

C4ST-1/Chst11 is one of the enzymes involved in CS sulfation by catalyzing the transfer of the sulfate group from the donor to the carbon 4 position of N-acetylgalactosamine(GalNAc) to complete CS sulfation^[3]. Since sulfation of CS-GAG plays an important role in the PNN, transferase enzymes such as Chst11 have been suggested to be a key factor in fine sulfation homeostasis and indirectly influence neural maturation and stabilization of the CNS^{[9, 53]}. Mice with loss-of-function mutations in Chst11 have been found to have severe embryonic defects that cannot be compensated for by other enzymes, suggesting that Chst11 is indispensable for the development and maturation of organisms^[9]. Chst11 mainly catalyzed the sulfation of C4S/CS-A, which significantly increased the expression level and chain length of CS^{[54, 55]}. In addition, C4S is primarily known for its inhibitory properties^[8], so excessive C4S expression leads to an increase in the inhibitory properties of the PNN, an increase in structural stability, and a decrease in the ability to repair after injury. It was shown that the damaged spinal cord of zebrafish was repaired more rapidly after Chst11 knockdown, while the presence of Chst11 inhibited the progress of spinal cord repair^[10]. Interestingly, we treated rats with spinal cord injections of Chst11 virus, which somehow enhanced the inhibitory ability of PNN, and after modeling SCI on them, Chst11 virus-injected rats recovered significantly worse than uninjected rats. This suggests that too much Chst11 simply leads to a worse neuronal environment and more inhibition at the site of injury.

EA is the stimulation of jiaji acupoints followed by a certain parameter of electrical stimulation, with the double effect of traditional acupuncture and electrical stimulation, is the traditional acupuncture in the modern innovation of the therapy. EA stimulating jiaji acupoint(EX-B2) can dredge the channel qi of the governer vessel and the bladder meridian at the same time, so that Yang Qi can reach the limbs. This stimulation can improve the neuronal microenvironment at the site of SCI, improve local blood supply, alleviate lower body paraplegia caused by SCI, and promote long-term recovery of neurological function in patients. In the current study, we observed that EA significantly promoted the rearrangement and expression of PNN after SCI, enhanced the activity of PV⁺IN, and promoted CS regeneration. In addition, EA effectively reversed the inhibitory effect exerted by Chst11, increased the expression levels of PNN and PV⁺IN, maintained neuronal stability and promoted the restoration of their functions. Based on the above, we believe that EA can effectively ameliorate the disruption of PNN structure and function after SCI by Chst11, increase the activity of PV⁺IN, maintain the balance of neuronal plasticity and stability, and promote spinal cord repair after injury.

Given the complexity of the spinal cord itself and the complex mechanisms of the PNN, it is difficult to categorize the causes of SCI as part of the spinal cord. Therefore, more in-depth research and discovery is needed on the role of PNN remodeling in SCI. Many studies have reported the results of EA for the treatment of neurogenic bladder and neuropathic pain after SCI. And as a safe, low-risk and economical treatment for SCI, EA is expected to become a new treatment modality in the future.

Our study demonstrated that the stabilization of CS expression is very important for the normal function of PNN and PV⁺IN in the nervous system, and is essential for the remodeling of PNN structure and function in rats after SCI. In addition, EA was able to regulate CS and GAD after SCI via Chst11, which effectively promoted the structural recovery and functional improvement of the PNN, increased the activity of PV⁺IN, facilitated the recovery of motor and sensory functions, and maintained the balance of neuronal plasticity and stability.

Ethics approval

The authors of this paper have adhered to the journal's code of ethics and all results and data are true and reliable.

Consent to participate

Not applicable.

Consent for publication

All authors gave their consent for the article and related data to be published in this journal.

Author contributions

Bowen Chen, Rong Hu contributed equally to this work as co-first authors.

Ruijie Ma, Bowen Chen and Rong Hu designed this study. Bowen Chen, Rong Hu, Xingying Wu, Mengting Shi and Jieqi Zhang completed this study. Bowen Chen, Rong Hu and Yi Chen analyzed the data. Bowen Chen, Rong Hu and Xingying Wu wrote the manuscript. Yi Chen, Yi Huang and Xihan Ying participated in the manuscript revision. Ruijie Ma and Dexiong Han validated the manuscript. All authors had read and approved the final version of the paper.

Funding

This study was supported by National Natural Science Foundation of China(No.82174487, No.82205258 and No.82205282). Special project of the Affiliated Hospital of Zhejiang Chinese Medical University(No. 2022FSYYZZ08 and 2022FSYYZY09).

Acknowledgments

We sincerely thank the third clinical college of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China for offering the experimental areas and instruments.

Competing interests

The authors declare that there is no conflict of interest regarding the publication of this paper.

Availability of data and materials

All the data used to support the findings of this study are included within the article.

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Electroacupuncture may promote the repair of spinal cord injury in rats by regulating the perineuronal net through Chst11

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