This study describes the method of intra-articular injection of mono-iodoacetate in rats and discusses the resulting pain-related behaviors and histopathological changes, which provide references for future applications.
The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients' daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 β and TNF α) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.
Osteoarthritis (OA) is the most common joint disease in the world, affecting an estimated 10-12% populations in adults1. The most generally involved joint is the knee, and OA has a higher incidence in older adults, especially women2. As a chronic disease, OA develops progressively over decades into joint failure with symptoms such as cartilage loss, synovial inflammation, osteophytosis, decreased function, and chronic pain3. According to the World Health Organization (WHO), OA is the fourth most prevalent disease in females and the eighth most prevalent disease in males. By 2020, OA may become the fourth most disabling disease in humans4. However, currently available therapies of OA address only symptoms and extend the time until joint replacement surgery5.
The spontaneous OA in human patients often takes a long time to produce clinical symptoms such as joint related pain6. In the early stages of OA, pain is usually intermittent and becomes more frequent and severe as the disease progresses, making it the predominant complaint of patients7. Therefore, extensive animal models for OA pain have been developed over the past half century to promote pain relief therapy. OA models have classically been divided into spontaneous and induced models. Spontaneous models include naturally occurring models and genetically modified models, which can more closely simulate the course of primary OA in humans8. Induced models can generally be divided into two categories: 1) post-traumatic OA induced by surgery or other trauma; or 2) intra-articular injection of chondrotoxic or pro-inflammatory substances3. These models lay a foundation for the pathophysiological study of OA and contribute greatly to the development of drugs to reduce pain and increase function.
Recently, the most widely used inducer for OA modeling is mono-iodoacetate (MIA). MIA, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, can cause changes in cartilage matrix, degradation, loss of cartilage, synovitis and other changes, which are similar to the pathological changes of human osteoarthritis9. It has been noted that intra-articular injection of MIA induced ongoing pain at 28 days after MIA administration, indicating that the MIA model may be useful for investigating chronic nociceptive pain10,11,12. In this study, male Sprague-Dawley rats received intra-articular injections with 0.5, 1.5, or 3 mg of MIA in the knee joints. The severity of MIA-induced joint pain was measured by assessment of mechanical and thermal sensitivity at 1, 7, 14, 21, 28 and 35 days after injections. On this basis, 1.5 mg of MIA was selected as the final concentration to evaluate gait patterns and histological changes at 28 days after injections.
Procedures involving animal subjects have been approved by the Medical Norms and Ethics Committee of Zhejiang Chinese Medical University and are in accordance with the China legislation on the use and care of laboratory animals.
1. Intra-articular injection of mono-iodoacetate in the knee
2. Behavioral assessments
3. Histopathological and immunohistochemical analyses
With this methodology, we established an OA pain model in the rat and detected the resulting changes. MWT and TWL reflected mechanical allodynia and thermal hyperalgesia, respectively. As shown in Figure 1, MIA induced mechanical allodynia and thermal hyperalgesia present in a dose-dependent manner. Remarkably, the decrease of MWT reached a peak from 21 days to 28 days, and then rebounded, suggesting that joint repair may occur at this stage, but MWT of 3 mg MIA group was still at a low level. The change of TWL was roughly consistent with MWT (Figure 2).
On this basis, we selected 1.5 mg of MIA as the final dose and assessed gait patterns and histological changes at 28 days after injection. Gait parameters (total paw area and unit stride length) reflected pain related behaviors. Levels of gait parameters including the total paw area (Figure 3A) and unit stride length (Figure 3B) were significantly reduced in the MIA group after 28 days, suggesting that MIA induced osteoarthritis-related joint pain in rats. With increased Mankin's score on the histopathological slides, degeneration of cartilage, disruption of collagen, and disorganization of matrix were obviously seen in the MIA group (Figure 4). As illustrated in Figure 5, 1.5 mg of MIA caused a significant upregulation of MMP13, and Col10, and significant downregulation of Col2.
Figure 1: Development of MTW after MIA injection. Mechanical withdrawal thresholds of hind paws were assessed after injection of MIA (0.5, 1.5, or 3 mg/rat) and saline (0.9% NaCl), n = 10 rats/group. Values are presented as mean ± SD. **P < 0.01 vs. saline-treated group; One-way ANOVA followed by Fisher’s least significant difference (LSD) comparison. Please click here to view a larger version of this figure.
Figure 2: Development of TWL after MIA injection. Thermal withdrawal latency of hind paws was assessed after injection of MIA (0.5, 1.5, or 3 mg/rat) and saline (0.9% NaCl), n = 10 rats/group. Values are presented as mean ± SD. **P < 0.01 vs. saline-treated group (NC); One-way ANOVA followed by Fisher’s LSD comparison. Please click here to view a larger version of this figure.
Figure 3: Gait analysis at 28 days after MIA injection. (A) Total paw area (cm2). Total paw area: the mean of total area of four paws of each group of rats. (B) Unit stride length. Unit stride length = Average stride length (cm)/body length (cm). n = 10 rats/group. Values are presented as mean ± SD. ##P < 0.01 vs. saline-treated group (NC) on day 28. This figure has been modified from Yan et al.15. Please click here to view a larger version of this figure.
Figure 4: Histopathological observation (HE, SO, and AHB staining) and Mankinʹ s scoring of rat knee joints on day 28 after MIA treatment. n = 10 rats/group. Scale bar = 40 µm. Values are presented as mean ± SD. ##P < 0.01 vs. saline-treated group (NC). One-way ANOVA followed by Fisher’s LSD comparison. This figure has been modified from Yan et al.16. Please click here to view a larger version of this figure.
Figure 5: Immunohistochemical observation of the expressions of MMP13, Col2, and Col10 in rat cartilage on day 28. Scale bar = 50 µm. N = 10 rats/group. This figure has been modified from Yan et al.16. Please click here to view a larger version of this figure.
The rat model of OA induced by MIA is a well-established, widely used model. Intra-articular injection of MIA initially causes severe and acute inflammation, which gives rise to the longer and degenerative phase of OA17,18. In this research, we measured nociceptive sensitivity by MWT and TWL, and assessed gait alterations with an imaging system. Previous reports found that the injection of MIA could raise the sensitivity of afferent knee joint fibers leading to nociception, which is reflected by thermal hyperalgesia and reduced mechanical threshold19,20. It has been proved that gait alterations were related to enhanced nociception, suggesting that gait patterns could be used to evaluate pain models21. Accordingly, MIA-induced models are mainly used to assess OA-related pain and screen oral drugs as well as joint injections drugs3,6.
Although compared with the surgically induced OA model, it is simpler and faster to inject MIA into the joint cavity, there are still critical points in the modeling. First of all, the articular cavity of rats is tiny, and its location should be confirmed before injection. Secondly, MIA is toxic, thus the dose of MIA should be carefully selected. It has been reported that MIA could induce articular cartilage damage in a dose- and time-dependent manner (assessed by the OARSI histological score and the Mankin score), indicating that the progression and severity of articular lesions can be modulated by regulating the concentration of MIA22,23. MIA was found to induce pain and oxidative stress markers at high doses10,18,24. Previous reports suggested that a 1.5 mg dose of MIA injection in rats produced an inflammatory process that is similar to human knee OA18,25. Besides, it is important to use the same experimenter throughout the behavioral test and to familiarize the rats with the environment in advance, to reduce anxiety and avoid affecting the experimental results.
As mentioned above, OA animal models are usually divided into spontaneous and induced models. Intra-articular injection of MIA is widely used due to several advantages: 1) simple operation; 2) ease of induction and reproducibility; 3) controllable dose and severity; 4) short modeling time; and 5) suitability for small animals as well as large animals. However, like other animal models, MIA-induced OA models also have several drawbacks. Extensive cell death and rapid joint destruction after MIA injection are inconsistent with spontaneous or post-traumatic OA in humans26. Moreover, residual MIA in the articular cavity may affect the effects of subsequent intra-articular therapies, resulting in an uncertain outcome by using the MIA model. Whether or not to wash the articular cavity before therapeutic injection in this model remains an unanswered question. Overall there is no single animal model that perfectly recapitulates all aspects of human OA, but the wide variety of models available makes it possible to apply multiple models to most relevant questions synthetically.
The authors have nothing to disclose.
This study was funded by the Zhejiang Provincial Natural Science Foundation of China (Grant No: LY17H270016), the National Natural Science Foundation of China (Grant No: 81774331, 81873049, and 81673997), and the Zhejiang Provincial Science and Technology Project of Traditional Chinese Medicine of China (Grant No: 2013ZQ007 and 2016ZZ011).
Anti-Collagen II antibody | Abcam(UK) | 34712 | Primary antibody for immunohistochemistry (IHC) |
Anti-Collagen X (Col10) antibody | Abcam(UK) | 49945 | Primary antibody for IHC |
DigiGait Imaging System | Mouse Specifics (Boston, MA, USA) | Equipment for gait patterns analyses | |
Eosin | Sigma-Aldrich | 861006 | The dye for HE staining |
Fast Green FCF | Sigma-Aldrich | F7252 | The dye for SO staining |
Goat anti-mouse antibody | ZSGQ-BIO (Beijing, China) | PV-9002 | Secondary antibody for IHC |
Goat anti-rabbit antibody | ZSGQ-BIO (Beijing, China) | PV-9001 | Secondary antibody for IHC |
Hematoxylin | Sigma-Aldrich | H3163 | The dye for HE staining |
MIA | Sigma-Aldrich | I4386-10G | powder |
MMP13 | Cell Signaling Technology, Inc. (Danvers, MA, USA) | 69926 | Primary antibody for IHC |
Modular tissue embedding center | Thermo Fisher Scientific (USA) | EC 350 | Produce paraffin blocks. |
Plantar Test apparatus | UgoBasile (Italy) | 37370 | Equipment for TWL assay |
PrimeScript RT reagent Kit (Perfect Real Time) | TaKaRa Biotechnology Co. Ltd. (Dalian, China) | RR037A | Extracte total RNA from cultured cells |
Rotary and Sliding Microtomes | Thermo Fisher Scientific (USA) | HM325 | Precise paraffin sections. |
Safranin-O | Sigma-Aldrich | S2255 | The dye for SO staining |
Tissue-Tek VIP 5 Jr | Sakura (Japan) | Vacuum Infiltration Processor |