A Visualized Sensitized Acupoint Procedure for Knee Osteoarthritis: Behavioral, Thermal, and Molecular Assessments

Summary

This study details a rat fixation method suitable for photographing acupuncture and infrared thermography. Furthermore, it demonstrates that electroacupuncture at sensitized acupoints inhibits local inflammatory pain.

Abstract

Knee osteoarthritis (KOA), a leading cause of musculoskeletal pain and functional disability worldwide, remains challenging to manage due to the limited efficacy of current pharmacological and surgical interventions in achieving sustained pain relief. Although acupuncture has demonstrated clinical promise in alleviating KOA-related pain, its underlying mechanisms require further elucidation. Application of acupuncture to sensitized acupoints can markedly enhance the effectiveness of therapy. This protocol establishes a rat model of KOA through intra-articular injection of sodium monoiodoacetate (MIA) and systematically describes an acupuncture protocol targeting sensitized acupoints, including restraint methods, acupoint localization, and acupuncture parameters. Therapeutic efficacy was comprehensively evaluated through pain behavioral assessments, infrared thermographic analysis of the knee joint, inflammatory cytokine detection in serum, and histopathological examination of cartilage tissue. Results indicate that acupuncture at sensitized acupoints significantly alleviates pain phenotypes in MIA-induced model rats by inhibiting the release of pro-inflammatory mediators, blocking nociceptive signal transmission, improving blood microcirculation, regulating local joint temperature, and delaying joint degeneration.

Introduction

Knee osteoarthritis (KOA), a prevalent degenerative joint disorder, is characterized by chronic inflammation and whole-joint pathology, including cartilage erosion, subchondral bone remodeling, osteophyte development, and synovial inflammation^1,2. Clinically, patients typically present with pain, muscle weakness, joint instability, transient morning stiffness, and progressive functional limitations. KOA imposes a substantial global disease burden, affecting approximately 365 million individuals with functional disability, with epidemiological models predicting a 74.9% escalation in case numbers from 2020 baselines by mid-century 2050³. Notably, while pain serves as the primary treatment motivator⁴, it simultaneously exacerbates functional disability, profoundly compromising multiple quality-of-life domains through impaired mobility and reduced daily activity capacity. Consequently, therapeutic strategies aim to alleviate pain, restore function, and delay disease progression⁵. Current first-line pharmacotherapy includes analgesics, non-steroidal anti-inflammatory drugs, chondroitin and glucosamine sulfate, and intra-articular corticosteroids to reduce pain and inflammation⁶. However, prolonged use of these agents is constrained by significant iatrogenic risks, particularly gastrointestinal complications and cardiovascular adverse events⁷, thereby underscoring the critical need for safer, more sustainable therapeutic alternatives.

Acupuncture and electroacupuncture have been extensively utilized in osteoarthritis management, demonstrating efficacy in alleviating knee pain and improving joint functionality^8,9, with the World Health Organization (WHO) recognizing arthritis as a priority condition for acupuncture therapy. As both stimulation targets and effectors, acupoints remain central to acupuncture research, where their morphological appearance, spatial dimensions, and functional responsiveness undergo dynamic changes during disease states, a phenomenon termed "acupoint sensitization"^10,11. Critically, this sensitization process modulates the receptive field area and therapeutic responsiveness of acupoints, thereby determining treatment efficacy. Previous preclinical studies^12,13demonstrated sensitization at Yanglingquan (GB34, located in the depression anterior and inferior to the head of the fibula, near the lateral side of the hindlimb) and Zusanli (ST36, located on the anterior side of the leg, the lateral border of the patellar ligament is depressed to the anterior midpoint, 3/16 of the ankle joint) in KOA animal models. Laser speckle imaging revealed elevated local blood perfusion at these acupoints, while photoacoustic imaging paradoxically showed no significant structural alterations in microcirculation, suggesting that microcirculatory changes during acupoint sensitization in KOA are primarily functional rather than structural. Investigations into underlying mechanisms require the use of rat models; however, poor procedural compliance in animals results in considerable time investment. To address these constraints, a rat immobilization apparatus was developed, specifically designed to facilitate access to lower limb acupoints and enhance the efficiency of electroacupuncture procedures.

Infrared thermography, as a functional imaging modality, utilizes non-contact infrared sensors to detect thermal radiation from predefined regions of interest, with computer-processed data generating pseudocolor thermograms for quantitative analysis^14,15. This technique enables non-invasive monitoring of cutaneous hemodynamic changes by measuring temperature variations associated with blood flow fluctuations in cutaneous, vascular, and muscular tissues¹⁶, thereby facilitating visual assessment of functional microcirculatory alterations at acupoints. In this study, pain sensitization at GB34 and ST36 acupoints was first validated using an electronic algometer, followed by the combined application of safranin O fast green staining, IL-1β and TNF-α analysis, infrared thermographic imaging, and pain-related behavioral assessments to investigate the analgesic effects of electroacupuncture stimulation at GB34 and ST36 acupoints in monosodium iodoacetate-induced (MIA) knee osteoarthritic rats.

Protocol

All experimental protocols were approved and rigorously supervised by the Animal Ethics Committee of Beijing University of Chinese Medicine (ID: bucm-2024091102-3197), and all procedures strictly adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Thirty male Sprague-Dawley rats (weight range: 210-250 g) were used in this study. The reagents and equipment used are listed in the Table of Materials.

1. Experimental animals and groups

1. House the animals in the dedicated animal facility.
2. Maintain ambient temperature at 22 °C ± 2 °C, relative humidity at 50%-60%, and a 12-h light/dark cycle. Provide ad libitum access to food and water throughout the study.
3. After a 7-day acclimatization period, randomly divide rats into five experimental groups (n = 6 per group): normal control (Con) group, Sham operation (Sham) group, Model (MIA) group, non-fixed (NF) group, and electroacupuncture (EA) group.

2. Establishment of a monoiodoacetate-induced KOA model

1. Place rats in an induction chamber. Anesthetize using 4% isoflurane in oxygen for induction, then maintain under 1.5%-2.0% isoflurane delivered via a facemask (following institutionally approved protocols).
2. Fix rats in left lateral recumbency. Remove hair using a gentle depilatory cream on the right knee joint and surrounding areas.
3. Disinfect the right knee joint three times with povidone iodine from inside to outside, followed by repetition of the same steps with 75% ethanol.
4. Maintain the right knee at 90° flexion. Identify the inferior patellar border and tibial tuberosity. Advance a 22-G needle (0.7 mm × 30 mm) 3-5 mm into the joint space via the lateral or medial border of the patellar ligament at a 30°-45° cutaneous approach angle. Confirm intra-articular placement and slowly infuse 50 µL of MIA solution (1 mg/50 µL). For sham controls, inject an equivalent volume of sterile saline.
5. Perform five passive range-of-motion manipulations to ensure uniform intra-articular drug dispersion. Return the animal to its home cage for postoperative recovery.

3. Procedure for fixing rats

1. Secure the rat in a black restraint sleeve. Create a 1.5-cm-diameter circular aperture in the lower right quadrant through which the right hindlimb is exteriorized, as shown in Figure 1A.
2. Secure paired nylon cable ties (5 mm × 300 mm), spaced 6.8 cm apart, through a plastic mesh frame. Position the superior restraint at the axillary region and anchor the inferior restraint at the pelvic girdle, as illustrated in Figure 1B,C,E.
   NOTE: The NF group does not undergo the procedures described in step 3.2.

4. Electroacupuncture treatment

1. Begin electroacupuncture intervention on the fourth day following a consecutive three-day fixation process.
2. Perform aseptic preparation at the GB34 and ST36 acupoint regions. Refer to Figure 1D for standardized surface anatomical mapping of these loci.
3. Insert disposable sterile acupuncture needles (diameter: 0.25 mm; length: 13 mm) into the acupoints. Connect GB34 to the anode and ST36 to the cathode according to the electrostimulation protocol (Figure 1G).
4. After a 20-min electroacupuncture session, deactivate the device. Detach the electrodes from the needle handles and carefully remove the acupuncture needles to complete the therapeutic procedure. Administer interventions daily over two consecutive weeks.
   NOTE: Except for the NF group, the remaining three groups received no treatment but were immobilized for 20 min daily in the same manner as the EA group.

5. Mechanical Withdrawal Threshold (MWT) test

1. In a quiet environment at a room temperature of 24 °C ±  °C, place rats on a foot test platform with a stainless steel mesh bottom and an organic plastic cage body.
2. Stimulate the center area of the right hind paw or designated acupuncture points vertically using the 0. mm diameter probe of the Electric Von Frey electronic pain detector. Gradually and uniformly increase the intensity of stimulation until a paw withdrawal avoidance response is observed. Record the value displayed at this moment as the mechanical withdrawal threshold.
   NOTE: Each rat is measured once at 10-min intervals, with the mean of three consecutive trials designated as the final result.

6. Paw Withdrawal Latency (PWL) test

1. Acclimate rats on the thermal platform for 5 min before formal assessment.
2. Maintain the platform surface temperature at 55.0 °C ± 0. °C during testing. Initiate timing when the right hind paw contacts the heated surface and stop timing upon observation of the first positive reaction (paw licking, jumping, or rapid withdrawal). Define the recorded duration as the PWL.
   NOTE: Perform triplicate measurements per rat with 5-min intertrial intervals. Calculate the mean value as the representative PWL.

7. Infrared thermal imaging

NOTE: Depilate the hind limbs the day before thermography to eliminate the influence of hair on temperature measurement.

1. Maintain experimental conditions at 2 °C ambient temperature with 60% relative humidity under sound-attenuated and diffuse lighting.
2. Restrain the rat in a black restraint sleeve with the right hindlimb extended through a circular hole. Place on the operating table for 10 min.
   NOTE: If the rat is not calm after 10 min, extend the waiting time until it becomes quiet before proceeding to the next step.
3. Acquire lateral and ventral stifle joint thermal images using the thermal imager at a 5 cm distance, configured at 0.97 emissivity, 9 Hz frame rate, and 160 × 120 pixel IR resolution.
4. Process the acquired infrared thermographic data using dedicated analytical software to enable precise quantification of thermal parameters through computer-based spectral analysis.

8. Enzyme-Linked Immunosorbent Assay (ELISA)

1. Anesthetize rats by intraperitoneal injection of 20% urethane solution (1.2 g/kg). Open the abdominal cavity and insert the blood sampling needle into the abdominal aorta. Insert the end of the needle into a vacuum blood collection vessel without an anticoagulant.
2. Allow blood samples to clot for 30 min. Centrifuge at 1006. x g for 15 min at  °C. Quantify serum protein levels using the BCA assay.
3. Measure IL-1β and TNF-α concentrations using commercial ELISA kits according to the manufacturer's protocols.

9. Safranin fast green staining

NOTE: Following abdominal aortic blood collection, excise the right stifle joint using surgical scissors, preserving 1 mm osteoarticular segments of both the proximal femur and distal tibia. Carefully debride periarticular soft tissues.

1. Immerse stifle joints in 4% paraformaldehyde (PFA) for 72 h. Rinse thoroughly with phosphate-buffered saline (PBS) under gentle agitation to ensure complete PFA removal.
2. Immerse samples in ethylenediamine tetraacetic acid (EDTA) decalcification solution at  °C for 4 weeks. Replace the EDTA solution every 2 days. Prick the tissue lightly with a needle; absence of resistance indicates completion of decalcification. Rinse decalcified specimens with PBS for 4-6 h to remove residual EDTA.
3. Dehydrate samples using an automatic dehydrator. Embed in paraffin and allow to solidify into rigid blocks. Section paraffin-embedded blocks using a microtome to obtain 4-µm-thick slices. Transfer slices to a water bath. Once expanded, retrieve sections and place them on glass slides. Dry in a temperature-controlled chamber for 12 h.
4. Place slides in xylene (I) for 10 min and xylene (II) for another 10 min. Soak sequentially in anhydrous ethanol (I), anhydrous ethanol (II), 90% ethanol, and 75% ethanol for 5 min each. Rinse with ultrapure water.
5. Stain slides with Fast Green solution for 2 min. Rinse under tap water until cartilage appears glass-like and transparent with clearly visible lacunae under microscopic observation. Differentiate in 1% acetic acid solution for 10 s, monitoring until the residual blue tint is completely removed. Rinse immediately with ultrapure water.
6. Stain slides with Safranin O solution for 15 min until the cartilage matrix shows uniform, intense ruby-red coloration while bone tissue appears pale pink.
7. Soak slides sequentially in anhydrous ethanol (I), (II), (III), and (IV) for 10 s each. Immerse in freshly prepared xylene (I) and xylene (II) for 10 min each.
8. Remove slides and apply neutral resin to the slide surface, carefully avoiding tissue contact. Place the edge of the coverslip on the slide and lower slowly to prevent bubble formation. Wipe off excess xylene and resin. Air-dry slides overnight at room temperature. Place dried sections under an optical microscope for imaging.

10. Statistical analysis

1. Use SPSS software for statistical analysis. Express data as mean ± standard deviation.
2. Analyze MWT and PWL data using repeated-measures ANOVA. Compare other datasets across groups using one-way ANOVA.
3. Express results as mean ± standard deviation (SD). Define statistical significance as p < 0.05.

Results

No animals died during the entire experiment. Results of the operation procedure duration and needle detachment count for each rat in both the NF and EA groups indicated that the EA group, following restriction with nylon cable ties, experienced reduced operation time and fewer needle detachments (P < 0.001; Figure 1H,I). Analysis of acupoint pain thresholds revealed no statistically significant differences in mechanical pain sensitivity between GB34, ST36, and n...

Discussion

MIA, a metabolic inhibitor, selectively disrupts cellular aerobic glycolysis, thereby inducing chondrocyte apoptosis^17,18,19. Intra-articular MIA administration results in chondrocyte depletion and subsequent articular cartilage degeneration, characterized by morphological irregularities and histopathological alterations that closely resemble human OA pathology²⁰. Thus, it is widely utilized in research o...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0-10ul Adjustable-Volume Pipettor	Servicebio	IC012395160823
0-200ul Adjustable-Volume Pipettor	Servicebio	IC021029160323
1.5 ml eppendorf tubes	Servicebio	EP-150-M
10ul micropipette tip	Servicebio	TP-10
1ml syringe	Shanghai Zhiyu Medical Equipment Co., LTD	ZSQ-1
200ul micropipette tip	Servicebio	TP-200
22-gauge needle	Kruuse	112416c
2ml eppendorf tubes	Servicebio	EP-150-M
5ml red blood tube	Shanghai Xinle Biotechnology Co. LTD	CXG-5R
75% alcohol	Shandong Anjie Gaoke Disinfection Technology Co. LTD	75-500ml
Adherent slide	CITOTEST	188105
Automatic Plate Washer	Rayto	RT-3100C
Blood collection needle	Jiangxi Ruibang Industrial Group Co. LTD	CXZ-07
Bone tissue safranin and solid green staining solution	Shanghai Yuan Ye Biotechnology Co., Ltd.	R32730
Cover glass	Citotest Labware Manufacturing Co.,Ltd	10212432C
Dehydrator	DIAPATH	Donatello
Electronic balance	Mettler-Toledo	ME203E/02
Electrothermal blast drying oven	Labotery	GEL-70
Embedding machine	Wuhan Junjie Electronics Co., Ltd	JB-P5
Environmental Friendly Dewaxing Transparent Liquid	Servicebio	G1128-1L
Enzyme label detector	BioTeK	Epoch
Ethanol	SCRC	100092683
Ethylenediamine tetraacetic acid	Macklin	60-00-4
Fat-free cotton ball	Caoxian Hualu Sanitary Material Co. LTD	TZMQ-500
High speed refrigerated centrifuge	Dragon	D3024R
High Speed Tissue Grinder	Servicebio	KZ-III-F
Hot Plate Analgesiometer	Kew basis	KW-CT-1
Imaging system	Nikon	NIKON DS-U3
Infrared thermal imager	Testo AG	Testo 865
Iodophor	Shandong Anjie Gaoke Disinfection Technology Co. LTD	DF-500
IR Soft	Testo AG
Isoflurane	RWD life science	R510-22-10
Latex gloves	Beijing Ruijing Latex Products Co. LTD	RJJC-S
Mechanical Paw Poking Test Device	Kew basis	KW-RB
MIA	Sigma	206-165-7
Neutral gum	SCRC	10004160
Normal butanol	SCRC	100052190
Normal saline solution	SHIMEN	SLYS-500ml
Paraffin liquid	Macklin	8012-95-1
Paraformaldehyde Fixative (Neutral)	Servicebio	G1101
Pathology slicer	Shanghai Leica Instrument Co., Ltd	RM2016
Rat IL-1 beta  ELISA Kit GER0002-96t	Servicebio	AC30253754
Rat TNF-alpha ELISA Kit GER0004-96t	Servicebio	AC40253711
SD rats	Beijing Sibefei Biotechnology Co., Ltd	SCXK (jing) 2024-0001
Small Animal Anesthesia Machine	YuYAN	ABM
Surgical mask	winner	WJKZ-1
Tissue spreader	Zhejiang Kehua Instrument Co., Ltd	KD-P
Ultrapure Water Polishing System	Aiken water El	AK-RO-C2
Upright optical microscope	Nikon	NIKON ECLIPSE E100
Urethane	Macklin	2621-79-6
Vortex Mixer	Servicebio	MV-100
Xylene	SCRC	10023418