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Male Sprague Dawley rats weighing about 180 g were injected intraperitoneally with phenylhydrazine hydrochloride at a dose of 40 mg/kg for 5 days. Five days after the injection of phenylhydrazine hydrochloride, the rats were routinely anesthetized. Blood was drawn from the rat’s femoral vein to determine the absence of hemoglobin, haptoglobin, denatured globulin bodies, peripheral red blood cell count, hemoglobin, white blood cells, reticulocytes, and platelets in the plasma. Take a bone marrow smear from the rat femur to observe the proliferation of bone marrow, calculate the ratio of granulocyte red, and measure the weight of liver and spleen. Fix bone marrow, liver and spleen with 10% formaldehyde solution. Then proceed to regular tissue sectioning. Perform HE staining and optical microscope observation.
After 5 days of injection, the model rats reacted slowly, with reduced activity, pale skin on the tail, lips and limbs, and dark yellow urine; red blood cell count and hemoglobin concentration decreased significantly, reticulocytes increased significantly; denatured globulin bodies and free Hb also increased, and the volume and weight of the liver and spleen increased, which was significantly different from the rats in the normal control group. The histopathological observation of the model animals under the microscope showed that a small amount of blood oozing in the bone marrow blood vessels and the proliferation of megakaryocytes and erythroid cells were active. Liver cells showed mild and moderate granular degeneration, liver parenchyma congestion; the red pulp of the spleen was enlarged and scattered, the red blood cells were dense, the splenic sinus was full of red blood cells, squeezing the white pulp, and old bleeding was seen. Five days after intraperitoneal injection of phenylhydrazine hydrochloride, the animals showed obvious signs of anemia, hemolytic anemia of blood and bone marrow, and pathological damage of certain tissues and organs. The model preparation method is simple, reliable, low mortality, and practical.
Hemolytic anemia is a common disease of the hematopoietic system clinically, and there is no better treatment method. Establishing an ideal animal model of hemolytic anemia is of great significance for studying the pathogenesis and mechanism of human hemolytic anemia and observing the efficacy of drugs. In this model, phenylhydrazine hydrochloride was used to induce hemolytic anemia in rats. The main mechanism is that phenylhydrazine can quickly react with oxyhemoglobin to produce methemoglobin (MHB), nitrogen and benzene after being injected into the blood. MHB is an important step in the denaturation of Hb molecules, because the heme of MHB in the body is more likely to be lost than oxidized Hb, and globulin without heme is easy to precipitate. Under normal circumstances, even though red blood cells will keep in contact with oxidants such as peroxide anions (O2--), hydrogen peroxide (H2O2) and lipid peroxides, they can still maintain their integrity. There are three enzymes in red blood cells: catalase, reduced glutathione peroxidase (GSHPx) and superoxide dismutase (SOD). These enzymes can protect red blood cells from oxidative damage. However, when the production of O2- and H2O2 exceeds the ability of SOD and catalase to protect red blood cells from oxidative damage, Hb sulfhydryl groups, enzyme proteins and membrane proteins can be oxidized, and Hb can be oxidized to form MHB and Heinz's body. The deposition of Heinz body on the red blood cell membrane will reduce its fluidity and increase its permeability, leading to oxidative hemolysis. At the same time, phenylhydrazine can cleave some important membrane functional areas, and inhibit CA-dependent ATPase on the red blood cell membrane in vivo or in vitro, resulting in a rapid increase in the concentration of Ca in red blood cells. Therefore, it is speculated that the disorder of the Ca environment in red blood cells is an important factor for hemolysis caused by oxidants, and the lipid peroxidation of the red blood cell membrane can also accelerate the production of hemolysis, because the polyunsaturated fatty acids and the diene chain in the red blood cells directly reduce the membrane The fluidity and the formation of membrane amino propylene chains disrupt the normal function of red blood cell membranes.
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