Shi-Meng Li, Jian Zhou, Qi-Ping Wei, Jun Wang, Wu Sun, Yan Wang, Jie Wang, Yan-Ting Xia?

1. Beijing University of Chinese Medicine, Beijing 100029, China

2. Department of Ophthalmology, Dongfang Hospital Affiliated to Beijing University of Chinese Medicine, Beijing 100078, China

3. Department of Acupuncture, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing 100700, China

Keywords:Wei’s triple nine needling therapy Traumatic optic neuropathy Transverse quantitative traction method Flash visual evoked potential Rat

ABSTRACT Objective: To investigate the effect of Wei’s triple nine needling therapy on the N2?P2 wave of the flash visual evoked potential (FVEP) in rats of the of the transverse directional pulling model. Methods: Thirty?six Wistar rats were randomly grouped, nine were in normal control group without any treatment, and the remaining 27 were surgically modelled in the right eyes.Eighteen of these rats were randomly divided into a Wei’s triple nine needling therapy group and a model group of nine rats each after the TON model was made using the transverse quantitative retraction method. The other 9 rats were sham?operated, and only the optic nerve was exposed without retraction. On the next day of modelling, the Wei’s triple nine needling therapy group was treated with Wei’s triple nine needling therapy for 20 minutes/1 time/1 day for 14 days. The model group, sham?operated group and normal control group were not intervened. Wei's triple nine acupoints were represented as follows: 1st link: "Jingming" (BL1)and " Chengqi" (ST1); 2nd link: "Sizhukong" (SJ23) penetrating "Taiyang" (EX?HN5); and the third: "Fengchi" (GB 20) and "Taichong" (LV3). The FVEP of each group was observed on 1d,7d and 14d.The FVEP of each group was observed on 1d, 7d and 14d. Results: Compared with the model group, the N2 wave latency and P2 wave latency were shortened in the Wei’s triple nine needling therapy on 1d (P < 0.05, P < 0.01); on 7d, their N2 wave latency was shortened(P < 0.01) and the N2?P2 wave amplitude was increased compared with the model group (P <0.05); on 14d, their P2 wave latency was shortened (P < 0.05) and the N2?P2 wave amplitude was increased. There was no statistical difference in N2 wave latency, P2 wave latency and N2?P2 wave amplitude in the sham?operated group on 1d, 7d and 14d (P > 0.05). The delayed N2 and P2 wave latencies in the model group did not improve from 1d to 14d (P > 0.05) and the amplitude decreased throughout, showing a significant difference on 14d compared to 1d (P < 0.05). In contrast, the N2 wave latency in Wei’s triple nine needling therapy group was not significantly different until 7d to 14d (P < 0.05); its P2 wave latency, although significantly delayed from 1d to 7d (P < 0.05), recovered on 14d and reached a level that was not statistically different from 1d and 7d (P > 0.05). There was also no significant decrease in N2?P2 wave amplitude between 1d and 14d in Wei’s triple nine needling therapy group (P> 0.05). Conclusion: In this experiment, the TON rat model was successfully established by the transverse quantitative retraction method, and the treatment of TON rats with Wei’s triple nine needling therapy reduced the P2 wave delay of the FVEP electrophysiological signal and increased the N2?P2 amplitude, which had a certain positive effect on the repair of optic nerve injury, probably related to its effect of improving the conduction function of the optic nerve and protecting the retinal ganglion cells that had not been degenerated and necrosed.

1. Introduction

Traumatic optic neuropathy (TON) is a blinding eye disease that poses a serious threat to human health and is difficult to treat clinically. Professor Wei Qi Ping of our department summarized and developed Wei's ophthalmic acupuncture therapy and used Wei’s triple nine needling therapy to treat optic nerve injury with definite clinical efficacy, which can improve patients' visual acuity and visual field [1-4], but its mechanism of action is still unclear. In this study, the flash visual evoked potential (FVEP) was applied to observe the optic nerve protection effect of Wei’s triple nine needling therapy in rats with TON model, and the scientific connotation of Wei’s triple nine needling therapy in treating optic nerve injury was initially revealed.

2. Materials and methods

2.1 Experimental animals

Thirty?six healthy adult Wistar rats, body mass (259.11±11.77)g, SPF grade, male, provided by Sibeifu (Beijing) Biotechnology Ltd [License No.: SCXK (Beijing) 2019?0010], were housed in the animal room of Beijing Baiaosike Biomedical Technology Co. All rats were fed in a 12h light (06:00?17:59)/12h dark (18:00?05:59)environment with a temperature of 20?25°C, 40%?70% humidity and background noise <60dB. Inclusion criteria: No abnormalities in the retina and blood vessels in the fundus of the rat eyes by direct ophthalmoscopic examination. The disposal of animals during the experiment has been approved by the animal ethics committee.

2.2 Main instruments and reagents

Luolan electrophysiological animal platform and Huatuo brand disposable acupuncture needle, specification: 0.25mm×13mm,manufacturer: Suzhou Medical Supplies Co., Ltd, approval number:Su Food and Drug Administration Machinery (approved) No.2270884, 2012.

2.3 Grouping and model preparation

Previous studies in our department [5-7] showed that the transverse quantitative traction method resulted in a more controllable TON model in rats, so this method was used for modelling. Pre?operative direct ophthalmoscopic examination of the rat's fundus retina and blood vessels showed no abnormalities. After 7 d of acclimatization in the animal house, the rats were weighed, numbered and randomly divided into 4 groups of 9 rats each using the random number table method. The rats of normal control group were not subjected to any intervention. The remaining rats were surgically moulded in the right eye, and 18 of them were randomly divided into 9 rats each in the Wei’s triple nine needling therapy group and model group after making a TON model using the lateral quantitative retraction method. The rats were given general anesthesia with 10% chloral hydrate (10 ml/kg) intraperitoneally and carbomer gel was applied to the eye. The skin was cut along the temporal orbital rim of the right eye, part of the orbital fat was removed, and the optic nerve was exposed by bluntly separating it towards the orbital apex, and a 6?0 polyester suture was looped around the optic nerve 1?2 mm behind the ball and knotted. After completion of the preoperative examination and routine anesthesia, the skin of the nine rats in the sham?operated group was cut along the temporal orbital rim of the right eye, about 1 cm long, and part of the orbital fat above was cut off, and the nerve sheath was bluntly separated towards the orbital apex and cut to expose the optic nerve without traction. The rats in all groups were sutured, coated with erythromycin ophthalmic ointment and returned to their cages.

2.4 Intervention method

On the next day after successful modelling, the Wei’s triple nine needling therapy group started the acupuncture intervention by taking the representative points of Wei’s triple nine needling therapy: 1st link: "Jingming" (BL1) and "Chengqi" (ST1); 2nd link:"Sizhukong" (SJ23) penetrating "Taiyang" (EX?HN5); and the third:"Fengchi" (GB 20) and "Taichong" (LV 3). The acupoints of the first and second links are on the affected side, while the acupoints of the third link are on both sides. The acupoints of the first link were not taken needling manipulation. The remaining acupoints were taken even supplementation and drainage. needled once a day, and left for 20 min for 14 days of treatment. The rats were treated once a day,leaving the needles in place for 20min, for 14 days. The acupoints were positioned according to Experimental Acupuncture [8] and the Color Atlas of Rat Tomographic Dissection [9] for selection.①"Jingming": the inner corner of the eye, at the junction of the upper and lower eyelids. Stabbing technique: push away the eyeball and give an oblique insertion inwards and downwards. ②"Chengqi":Between the infraorbital rim and the eyeball below the pupil in plain view. Stabbing technique: push the eyeball and give a perpendicular insertion along the top of the inferior orbital rim. ③"Sizhukong":lateral part of the head, in the orbital bone depression above the lateral aspect of the eye. ④"Taiyang": in the temporal fossa, on the upper posterior side of the external canthus of the eye. Stabbing technique: Sizhukong towards Taiyang. ⑤"Fengchi": in the neck, at the outer 1/3 of the line between the midpoint of the line connecting the posterior angles of the ears and the posterior angle of the ear.Stabbing method: give an oblique insertion 0.5?0.8cm backwards and downwards and take even method. ⑥"Taichong": the depression between the 1st and 2nd metatarsal bones on the back of the hind limbs. Stabbing technique: give a perpendicular insertion 1mm and take even method.

All rats underwent flash visual evoked potential (FVEP)examination using a silver needle electrode (impedance <15kΩ) on the Luolan electrophysiological animal platform on 1d, 7d and 14d after the intervention. The recording electrode was placed 3 mm above the occipital ridge, the reference electrode at the midpoint of the line between the eyes, and the ground electrode at the ipsilateral auricle. Flash stimulation parameters: flicker frequency of 2 Hz,number of flashes of 80, flicker luminance of 10 dB, at least 3 consecutive measurements per eye, each at 10 min intervals. When testing the eye, the contralateral eye was completely covered with an opaque black cloth. The stable waveform was recorded 3 times in each eye. The latency and amplitude of the wave were recorded.

2.5 Statistical treatment

SPSS 20.0 statistical software was used for statistical analysis by normality test and chi?square test, if normal distribution and chi?square were satisfied, ANOVA was used, LSD?t test was used for multiple comparisons, if data were skewed or chi?square, Kruskal Wallis Test was used between groups, and Bonferroni correction for multiple comparisons was used for rank sum test P values.Differences were considered statistically significant at P < 0.05.

3. Results

By observing the FVEP patterns of rats, it was found that the homogeneity of repeated measurements was good in all groups of rats, among which the N2?P2 wave was more recognizable, and the P2 wave was equivalent to the P100 wave clinically. So the statistical analysis of this study was conducted around the N2?P2 wave. The results are shown in Tables 1, 2 and 3. The waveforms of the 1d, 7d and 14d groups are shown in Figures 1, 2 and 3.

Figure1 Representative waveforms of each group on 1d

Table 1 Latency of N2 waves(ms)

Table 2 Latency of P2 waves(ms)

Table 3 Amplitude of N2?P2 waves(μV)

Figure2 Representative waveforms of each group on 7d

Figure3 Representative waveforms of each group on 14d

3.1 Comparison of model group, sham-operated group and normal control group

There was no statistical difference in N2 wave latency, P2 wave latency and N2?P2 wave amplitude on 1d, 7d and 14d in the sham?operated group compared to the normal control group (P > 0.05). On 7d and 14d, the N2 peak latency and P2 peak latency were longer in the model group than in the sham?operated group and the normal control group (P < 0.01), and the N2?P2 wave amplitude was not statistically different (P > 0.05). The N2?P2 wave amplitude was lower than that of the sham?operated group and the normal control group (P < 0.05).

3.2 Comparison between Wei’s triple nine needling therapy group and model group

Compared with the model group, the N2 wave latency and P2 wave latency were shorter in t Wei’s triple nine needling therapy group on 1d (P < 0.05, P < 0.01), and there was no statistical difference in the N2?P2 wave amplitude (P > 0.05). On 7d, the N2 wave latency was shorter in Wei’s triple nine needling therapy group (P < 0.01) and the N2?P2 wave amplitude increased (P < 0.05) compared with model group. On 14 d, the P2 wave latency was shorter (P < 0.05) and the N2?P2 wave amplitude was higher (P < 0.05) in Wei’s triple nine needling therapy group than in model group.

3.3 Intra-group comparison

The N2 wave latency, P2 wave latency and N2?P2 wave amplitude were not statistically different in the sham?operated group among 1d, 7d and 14d (P > 0.05). The N2?P2 amplitude in model group was consistently lower and showed a significant difference on 14d compared to 1d (P < 0.05). The delayed N2 and P2 wave latencies in model group remained unimproved from 1d to 14d (P > 0.05).In contrast, the N2 wave latency delay in Wei’s triple nine needling therapy group did not show a significant difference until 7d to 14d (P< 0.05) and its P2 wave peak, although significantly delayed from 1d to 7d (P < 0.05), recovered on 14d to a level that was not statistically different from 1d and 7d (P > 0.05). There was also no significant decrease in N2?P2 wave amplitude between 1d and 14d in the Wei’s triple nine needling therapy group (P > 0.05).

4. Discussion

TON is an optic nerve injury that occurs after various direct or indirect violence to the orbital region of the skull or eye. Clinical symptoms are loss of visual acuity, colour vision impairment,relative afferent pupillary defect and fundus changes. Patients' visual acuity can rapidly decrease to finger count, light perception or even no light perception after the injury. And it has become an important blinding eye disease in China [10]. The currently accepted treatment at home and abroad remains the empirical application of high?dose methylprednisolone shock therapy, or combined with optic canal decompression. As for hormonal shock therapy, several studies in recent years have shown that neither alone nor in combination with surgery can significantly improve the prognosis of TON [11-12]. And most patients with severe disease have a poor prognosis.

This disease belongs to the category of "zhuangjibaomang" in traditional Chinese medicine. Injury to the eyes can result in damage to the true qi in the eyes, resulting in stagnation of the blood vessels and channels in the eye connector; or trauma to the eye connector can result in damage to the blood vessels and channels, resulting in blood stasis. Both of these can lead to a loss of nourishment in the eye system, resulting in optic nerve atrophy in later stages.

The pathological mechanism of this disease is damage to the channels and stagnation of Qi and blood. The Spiritual Pivot says:"The eyes is the gathering place of all the meridians". The eyes are able to see everything and distinguish colours because they are nourished by the qi, blood and internal organs. And this close physiological relationship between the eye and the internal organs is achieved through the passage of the meridians and the continuous transport of Qi and blood, which provides a theoretical basis for the treatment of TON by acupuncture. This experiment follows the latest advances in optic nerve injury protection research, and blocking the apoptosis of RGCs fits well with the concepts of holism and balance of acupuncture. It has been shown that acupuncture intervention in a rabbit eye model of non?arteritic anterior ischemic optic neuropathy has an anti?apoptotic effect, and the effect of intraocular needling combined with extraocular needling group was particularly significant [13].

Wei’s triple nine needling therapy is the crucial acupuncture prescription in treatment of eye diseases in Wei’s academic school of ophthalmology. Wei’s triple nine needling therapy includes the three points near to the eyes, the three groups of points for penetrating acupuncture around the eyes and the acupoint selection based on the general differentiation of syndrome. In our previous clinical studies, we have shown that Wei’s triple nine needling therapy can improve the visual function of patients with optic nerve atrophy,including many patients with traumatic optic nerve atrophy, and that Wei’s triple nine needling therapy can improve their visual acuity and visual field [1-4]. And patients' P100 peak latent time after acupuncture were improved compared with before the acupuncture,the effect of acupuncture on the change of pattern visual evoked potential (PVEP) is facilitation [14-15]. We also hope that further basic research will be conducted to investigate the mechanism of action of the Wei’s triple nine needling therapy treatment for TON.

The transverse quantitative traction method directly exposes the optic nerve for quantitative injury, which has the characteristics of high controllability, uniform force and consistent quantification,better simulates the clinical optic nerve injury process, and is an ideal and easily quantifiable model of optic nerve injury. In previous studies by our team [5], partial loss of RGCs could be caused after a TON model was established by pulling the rat’s optic nerve horizontally with a pulling force of 0.1N perpendicular to the optic nerve for 20s [6-7],

Timely understanding of visual function after optic nerve injury is an important indicator to guide treatment and determine prognosis.But subjective visual function tests are often affected by a variety of factors and patients are often in coma due to cranial trauma.Therefore, the FVEP test is important as an objective and sensitive indicator for clinical management of trauma when visual function tests are not available, and even more so in animal experiments.FVEP is an important method of evaluating optic nerve damage.And the function of the visual pathway between the retina and the visual cortex can be evaluated by observing the decreasing trend of amplitude and the latency of the FVEP’s wave crest. The P2 wave of FVEP is the most stable waveform and is equivalent to the P100 wave of PVEP in clinical practice, reflecting the level of visual dysfunction in TON rats [16], and is widely used in basic and clinical studies of optic nerve protection [17-19]. Therefore, the statistical analysis of this experiment revolves around the N2?P2 wave.

This study was conducted to observe the effect of Wei’s triple nine needling therapy on the FVEP in rats with optic nerve damage moulded by transverse quantitative pulling method. The FVEP waveforms in the modeled eyes of the rats in the model group showed significant changes compared to the normal control group on 1d after moulding. At all time points, there were significant differences in wave amplitude and peak latency between the model and normal control groups. Previous studies have confirmed that their delayed latency is due to myelin loss in optic nerve fibres or the absence of thicker diameter fast?conducting nerve fibres and their reduced amplitude is due to a reduction in the number of axons caused by degenerative necrosis of the optic nerve axons.

In this experiment, deep stab of the acupoints of 1st link is used to remove heat to improve vision, dispel wind and unblock the collaterals. The 2nd link penetrating acupoints have a wider stimulation area and gives greater stimulation to the body, which helps to strengthen the effectiveness of the acupoints and reduce the number of needle punctures and the stinging pain of the patient.The acupuncture prescription is Sizhukong penetrating Taiyang. The hand Shaoyang meridian’s acupoint penetrate the extra?meridian’s acpoints, which is intended to draw the qi and blood of the Yang meridian into the extra?meridian to strengthen the healing effect of the acpoints.

The 3rd link acpoints are Fengchi, which can scatter wind to release the exterior and clear the head and eyes, and Taichong, one of the "Four Joints Points", it has the effect of regulating qi and blood and unblocking the meridians, and is the yuan?source point of the liver,which is open to the eyes and can regulate the source qi of the liver up to the eyes [20]. Wei’s triple nine needling therapy dredges the meridians, regulates qi and blood which can inhibit the apoptosis of RGCs in order to protect the optic nerve. In this study, the treatment of TON model rats on the following day with Wei’s triple nine needling therapy significantly delayed optic nerve damage. We found that the amplitude of N2?P2 waves in Wei’s triple nine needling therapy group decreased more slowly than that in model group, and the latency of the N2 and P2 waves was reduced compared with that of model group. At the same time, the P2 wave peak latency improved between 7d and 14d in the Wei’s triple nine needling therapy group, whereas the results in model group were completely opposite, indicating that the optic nerve damage in model group was irreversible.

In this study, after transverse quantitative traction method was used to create a rat TON model, abnormalities in the N2?P2 wave amplitude and N2 and P2 wave latency of the FVEP electrophysiological signal were seen in the model group the next day, suggesting a decrease in optic nerve function. At this time,Wei’s triple nine needling therapy helped to increase the N2?P2 wave amplitude of the FVEP electrophysiological signal and reduce the N2 and P2 wave latency after partial optic nerve injury in TON rats.Therefore, it can be concluded that Wei’s triple nine needling therapy has a positive effect on the repair of optic nerve injury and can restore the function of the optic nerve to some extent. The present study demonstrates the feasibility of establishing a model of optic nerve injury by transverse quantitative traction from a functional point of view, and proves that Wei’s triple nine needling therapy has significant efficacy in the treatment of TON, which may be related to its effect of improving the conduction function of the optic nerve and protecting the RGCs that have not yet degenerated and become necrotic. It provides a new idea for the clinical treatment of TON.

Authors' contribution

Li Shimeng, Wang Jun, Sun Wu, Wang Yan, Wang Jie: participated in animal experiments, index testing and wrote the paper; Zhou Jian and Wei Qi Ping: experimental supervision; Xia Yanting:supervision, project leader.

Authors' conflict of interest statement

All authors declare that they have no conflict of interest.