Osteoarthritis (OA) is a degenerative joint disease affecting nearly 250 million people worldwide, including 50 million adults in the US.1 With an aging population, the prevalence of OA is projected to increase to nearly 80 million US adults by 2040.1 Due to the primary symptom of pain, OA is a major cause of physical disability and decreased quality of life throughout the world.2,3 Osteoarthritis does not only affect humans; numerous veterinary species, including cats, dogs, sheep, goats, cattle, and horses, can also develop and be impacted by OA.4,5
Currently, there are no approved medications that prevent, halt, or reverse the progression of OA. With no known cure, current treatment strategies are focused on reducing symptoms of joint pain and returning to normal functional capabilities. Common first-line treatments include exercise, weight loss, and pharmacologic pain control with paracetamol and NSAIDs.6 However, these drugs are not effective for all individuals and have serious safety concerns, particularly in regard to gastrointestinal and cardiovascular side effects.6 Therefore, safe and effective treatments for the management of OA pain are still needed.
In fact, many animal and human patients are seeking alternative treatments with minimal to no side effects, such as acupuncture, for pain relief.4,7 Acupuncture is a traditional Chinese practice of medicine that involves the insertion of thin needles into the skin to stimulate specific points on the body. The needles may be stimulated by hand or small amounts of electrical current, termed manual acupuncture and electroacupuncture, respectively. Numerous studies7–9 have reported acupuncture to be beneficial for treating OA pain, although methodological flaws are often noted. Because of these study limitations, there still remains insufficient evidence to provide a general recommendation for acupuncture.7,10 Therefore, further laboratory investigations using animal models are beneficial to investigate the effectiveness of specific acupuncture techniques and loci for use in clinical human and animal patients with OA.
The Dunkin-Hartley guinea pig develops age-related, spontaneous knee OA with histological changes similar to idiopathic human OA. Although clinical reports and trials evaluating acupuncture for OA in patients exist in the literature, the heterogenicity of the patient population complicates investigating the mechanisms by which acupuncture works. Therefore, laboratory studies evaluating mechanisms of action with standardized animal models is desirable. The majority of laboratory studies11–14 evaluating acupuncture for OA have utilized animal models of acute inflammation or surgically induced OA. These animal models of OA may not adequately represent the natural, idiopathic form of OA most commonly diagnosed in humans. Our previous study15 found that 3 weeks of 3 sessions/week of electroacupuncture in combination with manual acupuncture improved symptoms in the guinea pig model of OA compared to untreated control animals. Research evaluation of both manual acupuncture and electroacupuncture has been performed, and it is recognized that these techniques are not equivalent.16 Therefore, the purpose of this study was to evaluate and investigate the needling technique and needling loci17 of manual acupuncture as a therapy for knee OA using the Dunkin-Hartley guinea pig model of spontaneous OA. We hypothesized that guinea pigs treated with manual acupuncture would have increased mobility and decreased markers of inflammation compared to 2 different comparator acupuncture groups.
Methods
Animals
Eighteen 12-month-old male Dunkin-Hartley guinea pigs were purchased from Charles River Laboratories for use in this study. All animals were evaluated by 2 licensed veterinarians prior to the study initiation and deemed of appropriate health and mentation to be included. All guinea pigs were singly housed in USDA-approved facilities at Colorado State University and were provided a red hut and hay cubes for enrichment. Standard laboratory guinea pig chow and filter-sterilized water were provided ad libitum. Animals were housed with a 12-hour light-dark cycle, 20 to 26 °C temperature, and 30% to 70% humidity. All procedures were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals and approved by the Colorado State University IACUC.
Experimental design
Animals were randomly assigned to 1 of the following acupuncture treatment groups: sheath-tap acupuncture (n = 6), off-point acupuncture (n = 6), or manual acupuncture for knee OA (n = 6). These groups will be referred to as “sheath tap,” “off point,” and “acupuncture” respectively. We utilized a randomized allocation process to assign animals to each group with a free online tool available through GraphPad Prism (https://www.graphpad.com/quickcalcs/randomize1/). All animals were anesthetized with isoflurane, and acupuncture or comparator acupuncture treatments were performed once weekly for 3 weeks. Open-field enclosure monitoring was performed 2 weeks prior to initiating treatment. Subsequent enclosure monitoring was performed 24 hours after each treatment. All animals were euthanized 1 week after their final treatment. At euthanasia, blood was collected for CBC, serum biochemistry, and serum protein analysis. Whole knee joints were collected for histological evaluation. Knee cartilage and muscle from acupoint stomach (ST)-36 were saved for gene expression. All outcome assessments were performed blinded to acupuncture treatment groups.
Acupuncture protocol
Each animal was anesthetized with 2% isoflurane in oxygen. Local acupuncture points at the level of, as well as proximal and distal to, the stifle were chosen for pain modulation. Points were also chosen for autonomic nervous system support. Specifically, large intestine (LI)-11 and ST36 were chosen for the strongest autonomic and anti-inflammatory support; bladder (BL)-40, BL54, gall bladder (GB)-29, GB30, and ST36 were chosen as local points for the stifle; BL11 was chosen to support bones and joints; and BL23 was chosen to support the lumbar spine in relation to stifle lameness. Animals in the acupuncture treatment group were placed in either left or right lateral recumbency, and acupuncture needles (Seirin J-15 No. 01 [0.14 X 15 mm], SEIRIN CORP.) were placed at chosen points. Manual acupuncture was performed on BL11, -23, -40, and -54; LI11; GB29, -30, -34, and -39; and ST36 for 10 minutes. Needles were manually rotated after 3 and 7 minutes. The animals were then positioned on their opposite side, and manual acupuncture was performed on BL11 and -40, GB34, and ST36 for 5 minutes and manually rotated after 2.5 minutes. A needle sheath was tapped on these same acupuncture points in the sheath-tap group at 0, 3, and 7 minutes. For those in the off-point acupuncture group, 2 needles were placed 1 cm apart along the spine of the scapula, where there are no known acupuncture points and off the meridians, for 10 minutes. They were then placed on their opposite side, and the procedure was repeated for 5 minutes. All treatments were performed by a veterinarian with extensive training in veterinary acupuncture.
Open-field enclosure monitoring
Open-field enclosure monitoring was performed using ANY-maze behavioral tracking software (version 6.18; Stoelting Co). The apparatus consisted of a circular plastic blue bin that measured 1.14 meters in diameter and 15.2 centimeters in height with a red security hut placed along the edge. Guinea pigs were randomly selected for each recording. They were placed in the center of the apparatus and allowed to move freely for 10 minutes. Animals were acclimated to the apparatus for 1 week prior to baseline data collection. Baseline recordings were performed the day prior to the first acupuncture or comparator treatment. Subsequent recordings were performed the day prior to and after each treatment. All recordings were performed within the same time period (10 am to 3 pm) by the same handlers (APS, SBB, and CAS). For optimal video recording, all recordings occurred in the dark (with the exception of light from the laptop and a wall-mounted light-emitting diode light bar).
Tissue collection
Guinea pigs were euthanized 1 week after the final treatment session. Animals were euthanized via vaporized isoflurane in oxygen overdose, and death was ensured by exsanguination. The thoracic cavity was opened to expose the heart, and blood was collected via intracardiac puncture using a 20-gauge butterfly catheter. Both hind limbs were removed at the coxofemoral joint. A 1 X 1-cm section of muscle in the area of acupoint ST36, as well as cartilage and menisci from the knee joint, was removed from the right hind limb. These tissues were placed in RNAlater solution and stored at −80 °C. The left hind limb was placed in 10% neutral-buffered formalin for 48 hours. After fixation, the left femur length was measured using calipers. The limbs were then transferred to a 12.5% solution of EDTA at pH 7 for decalcification, and EDTA was replaced 3 times weekly for 8 weeks.
Complete blood counts, serum biochemistries, and serum protein analyses
Blood collected at the time of euthanasia was stored in 0.5-mL EDTA microtubes and red-top serum collection tubes. Once blood had clotted, red-top tubes were centrifuged at 5,000 X g at 4 °C for 15 minutes for serum collection. Ethylenediaminetetraacetic acid microtubes and serum aliquots were submitted to the Colorado State University Clinical Pathology Laboratory for CBCs and serum biochemistries, respectively. Complete blood counts were performed using the Advia 120 Hematology Analyzer (Siemens), and WBCs differentials were performed manually on blood films. Serum biochemistries were analyzed using the Cobas 6000 (Roche). The remaining serum aliquots were stored at −80 °C for serum protein analyses.
Serum protein quantification was performed using complement component 3 (C3) (Abcam) and prostaglandin E2 (PGE2; ABclonal Science) guinea pig–specific ELISA kits. All assays were conducted in technical triplicate and performed as indicated by the manufacturers’ protocols.
Histologic grading of OA
After decalcification, coronal sections of the knee joint at the level of the medial tibial plateau were paraffin embedded, and a 5-μm central section was stained with toluidine blue. A blinded veterinary pathologist (KSS) performed histologic grading of the knee joint section according to the Osteoarthritis Research Society International-Histology for guinea pig (OARSI-HISTOgp) recommendations.18 The medial and lateral tibial plateaus and femoral condyles were individually scored based on articular cartilage structure, proteoglycan content, cellularity, and tidemark integrity. Scores for each of the anatomic locations were summed to obtain a total knee joint OA score for each animal.
Transcript expression in ST36 and cartilage using NanoString technology
Total RNA was extracted from cartilage and acupoint ST36 muscle samples using an RNeasy Lipid Tissue Mini Kit (Qiagen). After spectrophotometric quantification via a NanoDrop 2000 (ThermoFisher Scientific), a total of 250 ng of RNA at a 20-ng/mL concentration was submitted to the University of Arizona Genetics Core for analysis. Custom guinea pig–specific probes were designed and synthesized by NanoString Technologies. Data were analyzed with nSolver software 4.0 (NanoString Technologies). The results were reported as absolute transcript counts and normalized to positive controls and housekeeping genes.
Immunohistochemistry
Immunohistochemistry was performed using a rabbit polyclonal nerve growth factor (NGF) antibody (Abcam; ab6199) at a concentration of 10 μg/mL (1:100 dilution). Prior to staining, slides were incubated in citrate buffer for 5 hours at 55 °C for antigen retrieval. Normal goat serum (2.5%) was used as a blocking reagent. Slides were incubated in primary antibody overnight in a humidified chamber at 4 °C, followed by a 30-minute incubation with a biotinylated goat anti-rabbit secondary antibody (1:200). Negative assay controls—rabbit immunoglobulin at 2.5 μg/ml and secondary antibody (1:100) alone—did not result in background immunostaining. Sections were counterstained with hematoxylin, cover slipped, and imaged by light microscopy. Immunostaining was graded using a previously published semiquantitative scoring scheme.19,20 Briefly, the percentage of cells positively stained for NGF were assigned a corresponding score between 0 and 5, with 0 being less than 5% of cells with immunostaining and a score of 5 representing 75% or more positive cells. The intensity of staining was also assigned a score, ranging from 0 to 3. Thus, the maximum score obtained for articular cartilage was 8.
Statistical analyses
Statistical analyses were performed using Prism (version 9.3.1; GraphPad Software). Normality was determined using the Shapiro-Wilk test. Normally distributed data were evaluated by ordinary 1-way ANOVA followed by Tukey multiple comparisons tests. Non-normally distributed data were evaluated by Kruskal-Wallis tests followed by Dunn multiple comparisons tests. A P value of < .05 was determined to be significant.
Results
Sample size calculation
Group size and power were determined using the statistical software at http://homepage.divms.uiowa.edu/~rlenth/Power/. Based upon previous work,21 stride length collected via treadmill-based gait analysis was selected as the principal outcome. Using a within-group error of 0.5 and a detectable contrast of 0.5 in simple comparison of proportions for equal sample sizes, power associated with an α level of 0.05 was calculated as 0.9 with a sample size of n = 6/experimental group. All 6 animals in each experimental group were included in each outcome assessment.
General description of study animals
All guinea pigs were clinically healthy and survived the duration of the study; there were no losses, exclusions, or unintended effects observed for any group. There was no difference in final weights between the sheath-tap, off-point, and acupuncture groups (P = .83). Mean body weight was 1,059 g (95% CI, 924.5 to 1,194 g) in the anesthesia group, 1,052 g (95% CI, 902.1 to 1,201 g) in the off-point group, and 1,019 g (95% CI, 936.6 to 1,101 g) in the acupuncture group.
Open-field enclosure monitoring
Open-field enclosure monitoring was used to assess changes in voluntary mobility and behavior parameters between manual acupuncture–treated and comparator acupuncture–treated groups. There were no significant differences in parameters (total distance traveled, average speed while mobile, maximum speed, and time in red hut) among groups over time (Figure 1).
Change in enclosure monitoring parameters. Final minus baseline measurements of total distance traveled (A), average speed (B), maximum speed (C), and time in red hut (D) in sheath-tap, off-point, and acupuncture groups. Black lines represent mean values for normally distributed data (time in red hut) and median values for non-normally distributed data (total distance traveled, average speed, and maximum speed).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.11.0341
Complete blood counts, serum biochemistries, and serum protein analyses
Complete blood counts, serum biochemistry, and serum protein concentrations of C3 and PGE2 were performed to evaluate systemic inflammation. There were no significant differences in hematologic or biochemical parameters between groups. Concentrations of C3 were significantly decreased in the acupuncture group compared to the sheath-tap group (Figure 2). Prostaglandin E2 concentrations were significantly decreased in the acupuncture group compared to both comparator acupuncture groups.
Serum protein concentrations of complement component 3 (C3; A) and prostaglandin E2 (PGE2; B) in sheath-tap, off-point, and acupuncture groups. Black lines represent mean values. *P < .05; **P < .01; ***P < .001.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.11.0341
Histologic grading of OA
Histologic assessment of the knee joint structure was performed according to the Osteoarthritis Research Society International recommendations for guinea pigs. Toluidine blue photomicrographs and total joint scores are shown in Figure 3. Animals in all groups showed irregular cartilage surfaces, proteoglycan loss, and cell clustering. There was not a significant difference in total joint scores between any groups.
Histologic images and scoring. Representative photomicrographs of toluidine blue–stained medial compartments of knee joints from guinea pigs in sheath tap (A), off-point (B), and manual acupuncture (C) groups. Scale bars = 50 μm. Knee joints in all groups showed irregular cartilage surfaces, regional-to-diffuse proteoglycan loss, and regions of hypocellularity. Osteoarthritis Research Society International histology scores of the total knee joint (D) from sheath-tap, off-point, and acupuncture groups. Black lines represent median values for non-normally distributed total joint data.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.11.0341
Transcript expression in ST36 muscle and knee cartilage using NanoString technology
From muscle at ST36, the transcripts of 5 genes were upregulated, and 1 gene was downregulated in the manual acupuncture group compared to either comparator acupuncture groups (Figure 4).
Transcript quantification from tissue at ST36 acupoint. B-cell lymphoma 2 (BCL-2) in the acupuncture group was greater than both the off-point and sheath-tap groups (A). Matrix metalloproteinase-3 (MMP-3) in the acupuncture group was lower than the off-point group, but no difference was seen with the sheath-tap group (B). Nuclear factor erythroid 2-like 2 (NFE2L2), nuclear receptor-related factor 1 (NURR1), phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1), and suppressor of cytokine signaling 3 (SOCS3) in the acupuncture group were greater than the off-point group, but no difference was seen with the sheath-tap group (C–F). Black lines represent mean values. *P < .05; **P < .01.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.11.0341
B-cell lymphoma 2 (BCL-2): The acupuncture group was greater than both the off-point and sheath-tap groups.
Matrix metalloproteinase-3 (MMP-3): The acupuncture group was lower than the off-point group, but no difference was seen with the sheath-tap group.
Nuclear factor erythroid 2-like 2 (NFE2L2), nuclear receptor-related factor 1 (NURR1), phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1), and suppressor of cytokine signaling 3 (SOCS3): The acupuncture group was greater than the off-point group, but no difference was seen with the anesthesia group.
For the cartilage and menisci gene expression, manual acupuncture resulted in the downregulation of 13 transcripts in the cartilage/menisci (Figure 5).
Transcript quantification from cartilage/menisci. B-cell lymphoma 2–associated agonist of cell death (BAD) and glutathione peroxidase 1 (GPx) in the acupuncture group were lower than both the off-point and sheath-tap groups (B and G). Protein kinase B (AKT), BCL-2 antagonist/killer 1 (BAK), Beclin-1 (BECN1), caspase-9 (CASP9), G-protein pathway suppressor 2 (GPS2), glycogen synthase kinase-3 β (GSK3b), peroxisome proliferator-activated receptor γ (PPARG), Sestrin-2 (SESN2), transforming growth factor beta-1 (TGF-β1), transient receptor potential ankyrin 1 (TRPA1), and v-rel avian reticuloendotheliosis viral oncogene homolog A, also known as nuclear factor NF-kappa-B p65 subunit (P65 RELA) in the acupuncture group were lower than the off-point group, but no difference was seen with the sheath-tap group (A, C–F, and H–M). Black lines represent mean values. *P < .05; **P < .01.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.11.0341
B-cell lymphoma 2–associated agonist of cell death (BAD) and glutathione peroxidase 1 (GPx): The acupuncture group was lower than both the off-point and sheath-tap groups.
Protein kinase B (AKT), BCL-2 antagonist/killer 1 (BAK), Beclin-1 (BECN1), caspase-9 (CASP9), G-protein pathway suppressor 2 (GPS2), glycogen synthase kinase-3 β (GSK3b), peroxisome proliferator-activated receptor γ (PPARG), Sestrin-2 (SESN2), transforming growth factor beta-1 (TGF-β1), transient receptor potential ankyrin 1 (TRPA1), and v-rel avian reticuloendotheliosis viral oncogene homolog A (P65/RELA): The acupuncture group was lower than the off-point group, but no difference was seen with the sheath-tap group.
Immunohistochemistry
A significant increase in NGF immunostaining was present in all 3 layers of articular cartilage of the medial tibial plateau in the manual acupuncture group relative to either comparator acupuncture groups (Figure 6). In contrast, when positive cells were detected in the comparator acupuncture groups, these cells were mostly found in the superficial and middle layers and not the deep layer.
Immunohistochemistry (IHC) images and scoring. Representative photomicrographs of IHC-stained medial tibial plateaus of knee joints from guinea pigs in sheath-tap (A), off-point (B), and manual acupuncture (C) groups. Nerve growth factor (NGF) IHC scores of all 3 layers of articular cartilage of the medial tibial plateau (D) from sheath-tap, off-point, and manual acupuncture groups. Black lines represent median values for non-normally distributed total cartilage data. *P < .05.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.11.0341
Discussion
As the major symptom of OA is decreased and painful mobility, our primary objective was initially to determine the efficacy of different acupuncture needling techniques and needling loci for symptom modification in the Dunkin-Hartley guinea pig model of primary OA. In open-field assessments, manual acupuncture–treated guinea pigs did not have significant changes in activity-based parameters compared to either comparator acupuncture groups. Despite no improvements in symptoms, knee joint OA scores trended toward a decrease in the acupuncture-treated guinea pigs. In contrast, previous studies11,12,14,22–25 have demonstrated acupuncture to significantly alleviate pain and inflammation, as well as improve joint structures, in various rodent models of OA. For example, electroacupuncture decreased hyperalgesia, paw edema, and inflammatory histologic scores in rodent models of adjuvant-induced arthritis.11,22–25 Electroacupuncture also decreased arthritis scores in rat models of adjuvant-induced arthritis11,23,25 as well as surgically induced arthritis.12,14
However, many important differences should be noted between these studies and the current study. An important distinction between this and other studies is that the comparison in the current study is between several types of acupuncture, not a comparison to untreated controls. Also, prior studies have used induced models of arthritis, via chemicals or surgery, whereas our study used a natural model of OA to most accurately represent the pathology of humans. Additionally, acupuncture was performed at least 3 days a week for as long as 12 weeks in these studies compared to only once weekly for 3 weeks in the current study. Another important difference is that guinea pigs received treatment at approximately 1 year of age, which is a stage at which moderate OA has already developed in this model. Other rodent studies began acupuncture treatment at the onset of OA. In a rat model of OA induced with IA monosodium iodoacetate, early administration of electroacupuncture alleviated pain and preserved the joint structure, whereas delayed acupuncture treatment had no beneficial effects.11 If manual acupuncture was initiated earlier in the disease stage of the guinea pig model, perhaps it would have been more effective in reducing pain.
It should also be noted that prior studies evaluated electroacupuncture rather than manual acupuncture. Electroacupuncture is typically favored in basic research as it is able to provide a reproducible stimulus.16 We previously demonstrated electroacupuncture to improve symptoms in the guinea pig model of OA, providing evidence that electroacupuncture may be more effective than manual acupuncture in alleviating pain. Similarly, many studies comparing the 2 modalities have shown the analgesic effect of electroacupuncture to be superior to that of manual acupuncture; this is potentially due to the increased frequency of needle stimulation in electroacupuncture compared to manual acupuncture, where needle stimulation occurs by hand. For example, electroacupuncture, but not manual acupuncture, was shown to effectively reduce capsaicin-induced paw edema in rats.26 Using pressure algometry, electroacupuncture was determined to induce a significantly greater analgesic effect than manual acupuncture during brief needle application in humans.27 Further, a meta-analysis of randomized controlled trials determined electroacupuncture to be more effective than pharmacological treatment and manual acupuncture in reducing pain and improving physical function in patients with knee OA.28 However, a randomized controlled trial found no difference between immediate effects of a single session of manual acupuncture compared to a single session of exclusive electroacupuncture, not adjunctive electroacupuncture, on pain, mobility, and muscle strength in humans with OA.29 Despite conflicting evidence, there may be differences in mechanisms of manual acupuncture and electroacupuncture due to electroacupuncture's higher stimulation intensity. Still, the potential differences between the 2 methods are poorly understood and frequently unaddressed.16
In the current study, we also assessed systemic inflammation using CBCs and serum biochemistries. Mice have shown a decrease in the inflammatory response after manual acupuncture, evidenced by a reduction in neutrophils and eosinophils.30 However, there were no differences in CBC or serum biochemistry parameters between groups in the current study. A lack of differences in WBCs may be due to a relatively short-term effect of manual acupuncture as it was only performed once weekly for 3 weeks in the current study. Some systemic effects were expected from the comparator acupuncture treatments, so the lack of differences between any of the acupuncture groups is not unexpected. Thus, more specific biomarkers of systemic inflammation, C3 and PGE2, were also evaluated. Complement Component 3 is an acute-phase protein in guinea pigs.31 Levels of acute-phase proteins, such as C reactive protein, have been shown to be increased in humans with OA.32,33 Complement Component 3 was shown to be significantly decreased in the acupuncture group compared to the sheath-tap group but only trended toward a decrease in the off-point group. These results were encouraging and support that manual acupuncture induces a more targeted and specific effect than both off-point and sheath-tap acupuncture. As the off-point group had needle stimulation, albeit at nondesignated acupuncture points, this still caused a level of systemic effects. Prostaglandin E2 is considered to be the major contributor to inflammatory pain in OA.34 Carrageenan-induced PGE2 inflammation was previously shown to be decreased by electroacupuncture in rat paws.24 In the current study, PGE2 was significantly decreased in the acupuncture group compared to both comparator acupuncture groups, but this did not appear to significantly alleviate pain.
Acupuncture is also known to cause alterations in transcript expression at both the acupoint site and distant tissues. For example, electroacupuncture at ST36 in female BALB/c mice showed alterations in gene expression in the acupoint site and in internal organs.35 In an antigen-induced arthritis model using BALB/c mice, manual acupuncture, when administered daily for 7 days and then every other day for 14 days, significantly ameliorated inflammation.36 Numerous transcripts involved in cartilage structure and antioxidant activity were upregulated in Hartley guinea pigs that received electroacupuncture 3 times weekly for 3 weeks.15 In the current study, with alterations in systemic inflammation in response to manual acupuncture, the gene expression of the ST36 acupoint and the cartilage and menisci of the knee joint was investigated.
At the ST36 acupoint, there were differences in the transcript profiles between the manual acupuncture group and the comparator acupuncture groups. The acupuncture group had an upregulation of antiapoptotic, antioxidant, and chondrocyte regulating genes. First, gene transcripts for BCL-2 and phosphoinositide-3-kinase regulatory subunit 1 were upregulated by manual acupuncture. B-cell lymphoma 2 is 1 of the BCL-2 family proteins that is antiapoptotic and promotes cell survival.37 Its expression in fibroblasts is essential for their survival and homeostasis.38 Phosphoinositide-3-kinase regulatory subunit 1 encodes for a regulator subunit of phosphoinositide 3-kinase, which is involved in regulating chondrocyte apoptosis.39 Next, manual acupuncture also upregulated nuclear factor erythroid 2-like 2 transcript, which is related to NFE2-like BZIP transcription factor 2. NFE2-like BZIP transcription factor 2 plays an important antioxidant and chondroprotective role in OA.40,41 Suppressor of cytokine signaling 3 transcript was increased in the manual acupuncture group and is known to be elevated during OA development.42 While suppressor of cytokine signaling 3 may not be beneficial during early OA due to its potential to decrease chondrocyte growth responses, it also has an anti-inflammatory role in chondrocytes, which may be beneficial during other stages of OA.42 Finally, manual acupuncture increased nuclear receptor-related factor 1 expression and decreased MMP-3 expression. Nuclear receptor-related factor 1 plays a protective role in cartilage homeostasis during inflammation and is a transcriptional repressor of MMPs, of which MMP-3 (stromelysin-1) is involved in degrading key components of the extracellular matrix.43
Like ST36, changes in gene expression were also seen at the actual target tissues—the cartilage and menisci of knee joints. Several proapoptotic transcripts were decreased in the manual acupuncture group, including BCL-2–associated agonist of cell death, BAK, and caspase-9. B-cell lymphoma 2–associated agonist of cell death and BAK are members of the BCL-2 family proteins, but unlike BCL-2 they are proapoptotic.44,45 Caspase-9 also triggers apoptosis and is involved in regulating autophagy, as is Beclin-1.46 Two transcripts related to modulating nuclear factor κ B, peroxisome proliferator-activated receptor γ and v-rel avian reticuloendotheliosis viral oncogene homolog A, were decreased in the manual acupuncture group.47,48 Peroxisome proliferator-activated receptor γ is also involved in regulating autophagy and modulating oxidative stress.47,49 Manual acupuncture also decreased the expression of glutathione peroxidase 1, which regulates oxidative stress and may influence synovial and chondrocyte lesions.50 G-protein pathway suppressor 2 expression was altered in the manual acupuncture group and is involved in mitochondrial functions, including stress response and mitochondrial-nucleus retrograde signaling.51 The expression of AKT, glycogen synthase kinase-3 β, and TGF-β1 were all decreased in the manual acupuncture group and are all involved in cartilage and chondrocyte cellular processes. Protein kinase B is a crucial component of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathway, which is essential for joint tissue metabolism.52 Glycogen synthase kinase-3 β is a protective factor for the degradation of chondrocytes, and when overexpressed in a post-traumatic OA mouse model, it ameliorated cartilage mineralization.53,54 Transforming growth factor β1 favors chondrocyte differentiation and cartilage repair.55 Tyrosinase-related protein 1 was lower in the manual acupuncture group, and there is evidence that decreased expression is beneficial for OA. Chondrocytes from OA patients, then treated with a TRPA1 antagonist, had decreased IL-6 expression, which is known for being essential for driving the pathogenesis of OA.56 Additionally, in a mouse model of OA induced with IA monoiodoacetate, acute inflammation, degenerative cartilage changes, and joint pain were attenuated in TRPA1 knockout mice compared to wild-type controls.57 Sestrin-2 expression was decreased in the manual acupuncture group and has been implicated in models of chronic pain. In a rat model of OA induced with IA monoiodoacetate, the overexpression of Sestrin-2 alleviated pain behaviors and attenuated neuroinflammation.58 Another modulator of pain is the neurotrophin NGF. Nerve growth factor was of particular interest due to the rising concerns about anti-NGF monoclonal antibody treatment effects on cartilage in animals.59,60 To further examine the role of NGF in osteoarthritic joints in response to manual acupuncture, protein localization and rough quantification was performed.
Nerve growth factor was assessed and localized via immunohistochemistry. Nerve growth factor is a neurotrophin that has been implicated in both detrimental and regenerative activity and has been implicated in joint pain by sensitizing peripheral nociceptive neurons and stimulating neuronal growth.61,62 As the name implies, it was initially found to be essential for embryonic neuron development.63 It has since been found to be essential for neural regeneration, neurotrophic function, and normalizing peripheral nervous system neuron activity.64 Nerve growth factor has a high-affinity receptor and a low-affinity receptor, tyrosine kinase A receptor (TrkA) and p75, respectively. However, TrkaA is limited to neurons, whereas p75 expression is more diverse.60 Historically, NGF inhibition has been a therapeutic target for treating OA pain. However, clinical trials were temporarily halted due to adverse events, specifically rapidly progressive OA.65 More recently, the regenerative properties of NGF have been investigated as potential therapeutic targets.66 Nerve growth factor, TrkA, and p75 expression has been described in bone and articular cartilage of the femur in mice.67 In nondecalcified femurs from 10-day-old C57BL/6J, there was robust NGF staining at the subchondral bone/articular cartilage interface that was likely mesenchymal progenitor cells or undifferentiated chondrocytes.67 Tyrosine kinase A receptor–positive cells were not found in the articular cartilage, and p75-positive cells were in the deep and middle layers.67 In adult horses, increased NGF-positive staining cells in the middle and deep layers of articular cartilage were seen in horses with severe OA compared to those that were healthy or had mild-to-moderate OA.68 More recently, NGF-p75 signaling specifically has been established as initiating anti-inflammatory and anabolic reactions to repair and stabilize OA in mice.60 In the present study, the manual acupuncture for the knee OA group had a significant increase in NGF immunostaining in all 3 layers of articular cartilage of the medial tibial plateau. Even when NGF-positive cells were present in either comparator acupuncture groups, those cells were mostly in the superficial and middle layer but not the deep layer. Particularly in the deep layer, the increase in articular cartilage cells expressing NGF could represent an increase in the population of undifferentiated chondrocytes, indicating a level of regeneration in the cartilage of the acupuncture guinea pigs but not in either comparator acupuncture group.
In general, there are many potential variables in acupuncture research, including the duration and frequency of treatments, type of needle stimulation, and acupuncture point selection. Manual acupuncture treatments may need to be performed more frequently and for a longer duration to see an improvement in symptoms and pathology. It is possible that more statistical differences could have been appreciated in the acupuncture groups if they were compared to a completely untreated control group or anesthesia-only control group; however, relevant differences were still observed. It is acknowledged that the time of acupuncture treatment was not even on each side of the animals within each treatment session; however, treatment times were alternated side to side over the study period as to not overly bias 1 side. This approach was performed based on clinical experience in pet guinea pigs in part to minimize their time under anesthesia and, more importantly, to minimize any risk of overstimulating their autonomic nervous systems. Another potential limitation of the current study was the use of anesthesia. In order to reduce stress and ensure the accessibility of acupuncture points, all guinea pigs were anesthetized with isoflurane. However, anesthesia is known to reduce the neurophysiological response to acupuncture, although the mechanism is still unclear.69
In summary, there were no difference in the symptoms of OA in this model between 3 weekly sessions of manual acupuncture, off-point acupuncture, and needle sheath taps. However, this study did not compare manual or comparator acupuncture with untreated OA model controls. Interestingly, manual acupuncture decreased markers of systemic inflammation and trended toward an improvement in the articular cartilage structure compared to comparator acupuncture. Additionally, the increase in NGF immunostaining seen with manual acupuncture could indicate the initiation of beneficial regenerative remodeling processes. This study supports manual acupuncture as an effective needling technique compared to sheath taps and that targeted acupuncture loci are more effective than off-point acupuncture at inducing beneficial systemic and cartilage changes. Future studies should investigate mechanisms of long-term structural modification in this model of primary OA.
Acknowledgments
The authors would like to thank the Colorado State University Department of Laboratory Animal Resources for their care of the animals used in this study.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
This work was supported by funding provided by Colorado State University's College of Veterinary Medicine and Biomedical Sciences. Dr. Spittler was supported by a Clinical and Translational Science Award One Health Alliance Translational Fellowship funded by U01 TR002953.
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