Name: ウサギの膝関節軟骨のサフラニンO/ファストグリーン染色
Uploaded: 2023-10-20T14:35:00
Description: Watch this Scientific Journal Video about Safranin O/Fast Green Staining of Knee Joint Cartilage in Rabbits at JoVE.com

safranin o/fast green staining of knee joint cartilage in rabbits

修正Videman法を用いたウサギのKOAモデルの確立

Knee osteoarthritis (KOA) is the most frequent form of osteoarthritis, often recognized as a whole-joint disease characterized by articular cartilage degeneration, which manifests clinically as pain, swelling, and limited movement of the affected joints1. According to recent epidemiological statistics, KOA is reported to have affected 654.1 million individuals globally who were 40 years of age or older by 2020. The prevalence and incidence of KOA rise with age, are the highest in middle-aged and older adults, and affect more women than men2. The prevalence of KOA is likely to increase due to the aging population and obesity epidemic worldwide, posing a growing threat to global public health. Age, sex, obesity, trauma, and other complicated risk factors associated with KOA all directly impact knee instability, making a biomechanical imbalance in knee joints one of the primary causes of KOA3.
Under normal physiological conditions, the knee joint is in a state of mechanical balance, ensuring that the mechanical loads in the joint are evenly distributed on the cartilage. Any mechanical imbalance in the knee joint can lead to abnormal stress in cartilage, resulting in cartilage degeneration and the onset of KOA4. The muscle-tendon system is the main dynamic system that maintains the mechanical balance of the knee joint. The coordinated movement of the extensor and flexor muscle-tendon system can evenly distribute the load generated by the movement on the cartilage surface, avoiding the metabolic imbalance of local cartilage stresses beyond its physiological load that results in cartilage loss5. Decreased muscle strength is the main cause of intramuscular movement disorder and cartilage damage, which may occur before symptomatic KOA. 
KOA can also induce arthrogenous muscle inhibition (AMI), manifesting as muscle weakness and decreased muscle strength around the knee6. Among these muscles, the quadriceps femoris group functions as the only knee extensor, an important structure in maintaining knee joint stability. Studies have shown that a decrease in quadriceps cross-sectional area and muscle strength is significantly and positively correlated with KOA progression7. The decline in quadriceps strength affects the gait pattern, knee stability, movement patterns, and many other functions. Moreover, the decline in muscle strength impairs tendon function, manifested as a decrease in tendon stiffness, elastic modulus, and other biomechanical properties8. In long-term strain repair, changes such as adhesion and contracture may occur in the muscles and tendons of the knee joint, damaging their mechanical properties, causing joint instability, and ultimately forming a vicious cycle of pathological changes of KOA. It is, therefore, crucial for KOA treatment to improve the mechanical properties of the muscle-tendon system and restore the joint mechanical balance.
Among the causes of KOA, biomechanical imbalance is the main inducing factor for knee pain, dysfunction, inflammatory lesions, and cartilage degeneration9. Therefore, the key to treating KOA is to restore the biomechanical balance of the knee joint. Acupotomology believes that the etiology and pathogenesis of KOA are "mechanical imbalance." When the mechanical characteristics of the soft tissues around the knee change abnormally, the knee joint loses its mechanical balance, and the abnormal mechanical stress environment of the joint accelerates degeneration, causing inflammatory stimulation to further aggravate the soft tissue adhesions, contractures, and further decline in joint stability. This vicious cycle eventually develops into KOA. By loosening soft tissue adhesions and contractures, as well as reducing stress concentration in the muscles and tendons, acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" improves the soft tissue mechanics and "modulates muscles and tendons," which balances the mechanical stress of the joint, effectively alleviating cartilage degeneration and "treating bone disorders"10. In terms of animal model selection, based on the purpose of this study, we prepared the KOA model by the modified Videman method of left hindlimb extension immobilization.
This paper details the establishment of the KOA model using the modified Videman method of left hind limb extension immobilization and the method of operation and precautions of acupotomy. We demonstrate the effectiveness of acupotomy by testing the mechanical properties of quadriceps femoris and tendon and detecting changes in articular cartilage stress and morphology.

著者スポットライト:変形性膝関節症の治療における鍼切術の作用機序の調査

ウサギの変形性膝関節症モデルにおける鍼切開術の適用

Our research scope is the study of the mechanism of action of acupotomy in the treatment of new osteoarthritis. The question we want to answer, the how acupotomy regulates the mechanical properties of the soft tissues around the knee to protect the cartilage. At present, micro CT, nuclear magnetic resonance, phenetole elemental analysis, and bio mechanical detection techniques can be used to promote research in this field.We found that acupotomy improved the mechanical properties of soft tissue by removing the excisions and pathological contractors around the knee, thereby playing a role in protecting cartilage. To a certain extent, this protocol explains the mechanism and reveals the scientific connotation of acupotomy in the treatment of KOA and fills the gap in the basic research of acupotomy tendon and bone treatment method. In the future, our laboratory will focus on the specific mechanism or action of the acupotomy tendon and bone control method on cartilage protection.

Watch this Scientific Journal Video about Safranin O/Fast Green Staining of Knee Joint Cartilage in Rabbits at JoVE.com

ウサギの膝関節軟骨のサフラニンO/ファストグリーン染色

鍼切開介入後、軟骨軟骨下骨複合組織をパラフィンに埋め込みます。次に、ティッシュワックスブロックをスライスし、スライドの上に置きます。スライドを連続した環境脱ろう溶液でそれぞれ15分間脱パラフィンします。次に、スライドをキシレンと無水エタノールにそれぞれ2〜5分間連続して浸漬し、続いてエタノール濃度を下げて脱水します。スライドを高速の緑色の溶液で1分間染色します。余分な高速緑色溶液を超純水で洗い流した後、1%酢酸溶液でスライドをすばやく数回すすぎ、色を分離します。再度、サフラニンO溶液で10〜15分間染色する前に、スライドを水ですすいでください。スライドをエタノール濃度の上昇にそれぞれ3〜5秒間浸し、次にスライドを連続したキシレン溶液にそれぞれ10分間置きます。スライドの前面に中性共鳴媒体を追加し、組織を避けます。カバーガラスの端をスライドに置き、ゆっくりと下げて中性バルサムを覆います。余分なキシレンと中性バルサムを拭き取ります。一晩乾燥させた後、軟骨の組織学スコアリングのために顕微鏡下でスライドを観察します。対照群では、軟骨表面は平滑で、すべての層で組織化された軟骨細胞配列を示しました。一方、KOA群では軟骨表面が粗く、軟骨細胞の配列が乱れていた。鍼切開術により、軟骨表面の平滑性が改善され、正常な軟骨細胞構造が維持されました。軟骨の完全性スコアは、対照群および鍼切開群と比較して、KOA群で有意に増加しました。

Research

JoVE Journal

Medicine

Safranin O/Fast Green Staining of Knee Joint Cartilage in Rabbits

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...