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Gout is a crystal-related arthritis caused by hyperuricemia due to purine metabolism disorder and (or) reduced uric acid excretion, which leads to the deposition of uric acid crystals. The causes of gout may include hyperuricemia, purine metabolism disorder, kidney disease, drug side effects, alcohol abuse, etc. The most common site of gout symptoms is the thumb joint, and the typical symptom is a sudden onset of severe joint pain.
MSU crystals can accumulate in the joint capsule, cartilage, bone or other periarticular tissues, irritating the synovial membrane and producing pathological reactions such as synovial vasodilation, increased permeability, and leukocyte exudation. Injecting MSU crystal suspension into the rat joint cavity can establish a gout model in a short time, helping to study the occurrence and development of the disease and to discover new treatment methods.
MSU Sodium Hydrochloride Crystal Suspension: Add sodium urate to sterile water containing 3.2% 1 mol/L NaOH to prepare a suspension with a concentration of 5mg/mL. After boiling and dissolving, let it cool naturally, adjust the pH to 7.0, and centrifuge immediately when the solution turns milky white. Centrifuge the supernatant repeatedly until no more crystals precipitate and collect the crystals after cooling at 4℃ for 1 hour. Dilute the MSU crystals with PBS before use to obtain the desired concentration of MSU suspension.
Note: The size of the crystals has a significant impact on the success rate of modeling. After the crystals are prepared, their size can be measured under a microscope, which is convenient for subsequent repetition and evaluation of the impact of crystals on the model.
Use the RWD small animal anesthesia machine to anesthetize the animal, then choose a 1mL syringe to inject into the ankle joint cavity or the foot pad of the hind limb of the rat. When the needle enters the joint cavity, there is a sense of breakthrough, which can be verified by back suction to check whether the needle is accurately inserted.
Use the RWD paw swelling tester to measure the paw volume and the degree of ankle joint swelling and observe the state of the modeled side limbs when the animal is walking and stationary for scoring and recording. Multiple measurements and observations should be conducted within 24 hours of modeling, and then measured once a day for 5-10 consecutive days.
| Performance | Scoring (3 points) |
| Both hind limbs make contact with the ground evenly, resulting in a normal gait | 0 |
| When at rest, there is no significant difference in the way both hind limbs touch the ground. However, during movement, there is a noticeable reduction in the weight-bearing capacity of the modeled limb, leading to mild limping | 1 |
| Both at rest and in motion, there is a marked decrease in the modeled limb’s weight-bearing capacity, resulting in pronounced limping | 2 |
| The modeled limb is unable to bear weight, with complete loss of ground contact, resulting in a tripod gait | 3 |
Use ELISA kits to detect the levels of routine inflammatory factors such as IL-1β, TNF-α, and IL-6 in the serum.
After the experiment, euthanize the animal, take the modeled side ankle joint or foot, remove the surface soft tissue, and expose the joint area. After adequate fixation of the tissue, decalcify it, which can be done using ready-made decalcification fluid, until the bones becomes soft (easy to puncture with a toothpick or needle). The decalcified tissue can be made into paraffin sections, and the RWD rotary microtome can be used for sectioning, and staining can be observed according to the experimental needs.
| Staining Methods | Staining Effects |
| Hematoxylin-Eosin Staining | After Hematoxylin-Eosin staining, the articular cartilage specimen shows a clear four-layer structure, with the smooth integrity of the cartilage surface and the orderly arrangement of chondrocytes being accurately assessed. In addition, the matrix part shows a typical basophilic reaction, while the chondrocytes exhibit a strong basophilic staining characteristic. |
| Safranin O Staining | After Safranin O staining, the articular cartilage specimen has distinct four-layer structures, with the superficial layer staining relatively lighter, and the staining gradually deepening towards the tide mark until the matrix deep layer shows a deep red. |
| Alcian Blue Staining | When using Alcian Blue staining, under the condition of pH 1.0, the cartilage matrix presents a light blue, while the periphery of chondrocytes is strongly stained by Alcian Blue, forming a sharp contrast. As the pH value rises to 2.5, the staining depth increases, and the cartilage matrix shows a deeper blue. |
| Toluidine Blue Staining | In Toluidine Blue staining, due to the unclear tissue structure layers, the cell nuclei are stained blue, showing a clear nuclear staining effect, while the cytoplasm is almost unstained, and the matrix part presents a light blue-purple. |
| Safranin- Alcian Blue Staining | Combined with Safranin O and Alcian Blue for double staining, the cartilage surface and matrix show uneven red, with deeper color in the deep layer, contrasting with the blue staining around the chondrocytes. |
| Safranin-Fast Green Staining | When using the Safranin-Fast Green double staining method, the cartilage matrix presents a uniform red, deeper than when using Safranin O staining alone, and the cartilage subchondral bone shows green, making the boundary between cartilage tissue and bone tissue more distinct. |
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