How to prepare an animal model of hemolytic anemia?
动物模型,溶血性贫血
z-bo
2020-08-10
539
Hemolytic anemia is a group of anemias that occurs when the destruction of red blood cells increases and the rate exceeds that of hematopoietic compensation. Anemia occurs when the average life span of red blood cells is 15-20 days, and the rate of red blood cell destruction far exceeds the compensatory potential of bone marrow. The basic problem of hemolytic anemia is that the life span of red blood cells is shortened and easily destroyed. Mainly through the following three mechanisms: abnormal changes in the red blood cell membrane; abnormal hemoglobin; mechanical factors.
1 A rat model of hemolytic anemia induced by acetophenazine
(1) Reproduction method Male rats weighing 180-250g are bred routinely and freely drink and eat. On the 1st, 4th, and 7th day of modeling, 2% Acetylphenylhydrazine (APH) physiological saline solution was injected intraperitoneally. The first injection dose was 1ml/100g body weight, and the second and third doses were halved to 0.5ml/ 100g body weight. After the injection of acetophenazine, blood was taken from the tail vein of the rat every morning for hemoglobin measurement, and red blood cell count and white blood cell count were performed. Reticulocytes, Heina body, neutrophils, alkaline phosphatase (AKP), acid phosphatase (ACP), ATPase, succinate dehydrogenase (SKH) and Glucose-6-phosphatase (G-6-P) staining.
(2) Characteristics of the model On the 3rd day after the injection of APH, the model rats began to experience fatigue, sluggishness, lethargy, wheezing; pale face, eyes, ears, tail, and low body temperature. Visual observation shows that the liver and spleen are both enlarged, and the splenomegaly is particularly obvious, which is dark red. Hematological observation indicators: hemoglobin and red blood cells showed a progressive decline, and the total number of reticulocytes, Hey's bodies and white blood cells increased significantly (showing anemic blood picture). One week after injection of APH, the hemoglobin of model animals can be reduced to 40～70g/L; red blood cells are reduced to (200～400)×1000000000/L; white blood cells are reduced to (30～39)×1000000000/L; reticulocytes are increased to From 85% to 95%, the Hai's body rises to 30% to 38% (normally 0). Histochemical staining of blood cells showed that neutrophils AKP, ACP, ATPase, SDH and G-6-P enzymes were abnormal in varying degrees.
(3) Comparative medicine Acetphenazine can cause changes in the growth of bone marrow hematopoietic stem cells and promote their transfer from the bone marrow to the spleen, and bone marrow blood cells increase during the compensatory phase. Red blood cells are accelerated due to poisoning, and bone marrow hematopoietic function is insufficiently compensated, which leads to anemia, and white blood cells and reticulocytes increase sharply. Using APH to make hemolytic anemia is a more classic method. In recent years, the use of gene targeting, genetic modification and other technologies to make this model has appeared, which is not widely used in China, and the procedure is relatively cumbersome and time-consuming. Experimental animals that can be used to make models include mice, rats, and rabbits. Hemolytic anemia refers to anemia caused by rapid destruction of red blood cells and insufficient bone marrow hematopoietic function. The clinical symptoms, blood picture and blood cell biochemical indicators of this model are basically similar to clinically human hemolytic anemia. The animal model of hemolytic anemia induced by acetophenazine is an ideal model for studying human hemolytic anemia.
2 Rat model of hemolytic anemia induced by phenylhydrazine hydrochloride
(1) Reproduction method For male SD rats weighing about 180g, phenylhydrazine hydrochloride was prepared into a 5% solution with sterile water for injection; the phenylhydrazine hydrochloride was injected intraperitoneally at a dose of 40mg/kg body weight for 5 consecutive days. Five days after the injection of phenylhydrazine hydrochloride, the rats were routinely anesthetized, and blood was drawn from their femoral veins to determine plasma free hemoglobin, haptoglobin, denatured globin bodies, peripheral red blood cells, hemoglobin, white blood cells, reticulocytes and platelets ; And take the rat femur bone marrow fluid smear, observe the bone marrow hyperplasia, calculate the proportion of red particles; measure the weight of the liver and spleen, and take the animal bone marrow, liver, and spleen tissues, fix them with 10% formaldehyde solution, and make regular tissue sections. HE staining, observe under light microscope.
(2) Model characteristics After 5 days of injection, the model rats showed unresponsiveness, decreased activity, pale skin on the tail, lips and limbs, and dark yellow urine; the red blood cell count and hemoglobin concentration were significantly reduced, and the reticulocytes increased significantly; degeneration beads Protein bodies and free Hb also increased, liver and spleen volume increased, and weight increased, which was significantly different from normal control rats. The histopathological observation of the model animal microscope showed that there was a small amount of blood oozing in the bone marrow blood vessels, the megakaryocyte lineage and erythroid hyperplasia were active; the liver cells showed mild to moderate granular degeneration, and the liver parenchyma was congested; the spleen red pulp was enlarged and diffused with dense red blood cells. The splenic sinus was filled with red blood cells, the white pulp was squeezed, and old bleeding was seen. After 5 days of intraperitoneal injection of phenylhydrazine hydrochloride in the rat, the animals may have obvious signs of anemia, and their blood and bone marrow pictures show hemolytic anemia changes, and may cause pathological damage to some tissues and organs. The model preparation method is simple, the result is reliable, the animal mortality is low, and the practicability is good.
(3) Comparative medicine Hemolytic anemia is a common clinical disease of the hematopoietic system, and there is currently no better treatment. Establishing an ideal animal model of hemolytic anemia is of great significance to the study of the etiology and mechanism of human hemolytic anemia, and the observation of drug efficacy. This model uses phenylhydrazine hydrochloride to induce hemolytic anemia in rats. The main mechanism is that when phenylhydrazine is absorbed into the blood by intraperitoneal injection, it can quickly react with oxyhemoglobin to produce methemoglobin (MHb), nitrogen and benzene. MHb is an important step in the denaturation of Hb molecules. Because the heme of MHb in the body is more easily lost than the heme of oxygenated Hb, the globin that has lost the heme is easy to precipitate. Under normal circumstances, although red blood cells are constantly 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, namely catalase, reduced glutathione peroxidase (GSHPX) and superoxide dismutase (SOD), which can protect red blood cells from oxidative damage. But when the production of O2- and H2O2 in the body exceeds the ability of SOD and catalase to protect red blood cells from oxidative damage, the sulfhydryl groups of Hb, enzyme proteins and membrane proteins can be oxidized, and Hb oxidation will form MHb and Heinz (Heinz) body. The Heinz body deposits on the red blood cell membrane, reducing its fluidity and increasing permeability, leading to oxidative hemolysis. At the same time, phenylhydrazine can cleave a part of important membrane functional areas. When in vitro or injected into rats, it can inhibit the Ca-dependent ATPase on the red blood cell membrane, resulting in a rapid increase in the Ca concentration in the red blood cell. Therefore, it is speculated that the disorder of Ca environment in red blood cells is an important factor in oxidant-induced hemolysis; lipid peroxidation of red blood cell membranes can also accelerate the production of hemolysis. This is because the loss of polyunsaturated fatty acids and diene chains in red blood cells directly reduces the membrane. The fluidity of the membrane and the formation of cross-chains of the membrane aminoiminopropene, thus destroying the normal function of the red blood cell membrane.
