阅读说明：本技术 一种脓毒血症伴多器官功能障碍综合征大鼠模型构建方法 (Method for constructing sepsis rat model with multiple organ dysfunction syndrome ) 是由 修光辉 李秀玲 凌斌 孙洁 刘萍 陈献忠 邓琼芳 于 2020-11-27 设计创作，主要内容包括：本发明公开一种脓毒血症伴多器官功能障碍综合征大鼠模型构建方法,本发明所述方法为：将脂多糖配制成相应浓度,对大鼠进行脂多糖静脉注射,按住大鼠,保持仰卧位,后肢内侧暴露向上,轻轻拔掉左后肢毛发,酒精棉球擦拭,按压腹股沟,使股静脉充盈；酒精棉球消毒后,1ml注射器平行缓慢进针,确定针尖在血管内后可推药,推药结束后,用干棉球按压注射点止血；止血完毕后常规饲养,定时观察并记录大鼠状态。本发明所述方法构建的动物模型脂多糖剂量低,死亡率仅为50％-70％,是研究脓毒症伴MODS模型的理想方法。(The invention discloses a method for constructing a rat model with sepsis and multiple organ dysfunction syndrome, which comprises the following steps: preparing lipopolysaccharide into corresponding concentration, performing intravenous injection on lipopolysaccharide on a rat, pressing the rat, keeping the rat in a supine position, exposing the inner side of hind limbs upwards, slightly pulling out hair of the left hind limbs, wiping the hair with an alcohol cotton ball, and pressing groin to fill femoral veins; after the alcohol cotton ball is sterilized, a 1ml syringe is inserted slowly in parallel, the medicine can be pushed after the needle point is determined to be in the blood vessel, and after the medicine pushing is finished, the injection point is pressed by a dry cotton ball to stop bleeding; after hemostasis is finished, the rat is raised regularly, and the state of the rat is observed and recorded regularly. The animal model constructed by the method has low lipopolysaccharide dosage and mortality rate of only 50-70 percent, and is an ideal method for researching the sepsis accompanied MODS model.)

1. A method for constructing a rat model with sepsis and multiple organ dysfunction syndrome is characterized by comprising the following steps:

(1) preparing lipopolysaccharide into corresponding concentration, performing intravenous injection on lipopolysaccharide on a rat, pressing the rat, keeping the rat in a supine position, exposing the inner side of hind limbs upwards, slightly pulling out hair of the left hind limbs, wiping the hair with an alcohol cotton ball, and pressing groin to fill femoral veins;

(2) after the alcohol cotton ball is sterilized, a 1ml syringe is inserted slowly in parallel, the medicine can be pushed after the needle point is determined to be in the blood vessel, and after the medicine pushing is finished, the injection point is pressed by a dry cotton ball to stop bleeding; after hemostasis is finished, the rat is bred conventionally, the state of the rat is observed and recorded regularly, and a rat model of sepsis with multiple organ dysfunction syndrome is successfully constructed.

2. The method of claim 1, wherein the rat model of sepsis with multiple organ dysfunction syndrome is constructed by: the injection amount of the lipopolysaccharide femoral vein is 2 mg/kg-3 mg/kg.

Technical Field

The invention relates to a method for constructing a rat model with sepsis and multiple organ dysfunction syndrome, and belongs to the technical field of animal model construction.

Background

Sepsis is one of the important causes of death in clinically critical patients, and infections from various sites throughout the body can induce sepsis; sepsis can cause various pathophysiological changes, such as cell dysfunction, imbalance between inflammatory and anti-inflammatory responses, alteration of body metabolism, and microcirculation disturbance. If the treatment measures are not taken timely and effectively, the important internal organs of the body can be damaged due to excessive inflammatory reaction of the whole body or organ ischemia, and finally the Multiple Organ Dysfunction Syndrome (MODS) is developed, so that the life of a patient is endangered.

Sepsis, a problem in the ICU and even the whole medical community, has been studied for decades, but the mortality rate remains high, and approximately 200000 people die of sepsis in the united states every year. The development of sepsis into MODS undoubtedly increases the difficulty of treatment, and later patients often die of multiple organ dysfunction or even failure. Thus, increasing the cure rate in patients with sepsis alone or already with MODS may also save the patient's life to a greater extent. At present, multiple researches on sepsis are based on animal experiments, and methods for establishing sepsis animal models mainly comprise three methods: injecting lipopolysaccharide endotoxin or zymosan; ② injecting bacteria or viruses; (iii) disruption of the endogenous barrier, resulting in abdominal infections, such as in the caecum ligation-perforation (CLP) model. Some researchers have also used improved "second hit" (blood loss + endotoxin or blood loss + CLP) to make animal models of sepsis, but this approach is complicated and time consuming and therefore less applicable. The infection is the basic etiology of the animal model, accords with the clinical pathogenesis of patients with sepsis, can better replicate the pathogenesis of patients with sepsis, and has important reference value for the research of sepsis. Lipopolysaccharide-induced sepsis is often accompanied by damage to the heart, liver, lung, kidney, small intestine, and even multiple organs. Bruno et al found that sepsis induced by lipopolysaccharide can cause rapid decrease of cardiac output and myocardial dysfunction. Zhang et al showed that intravenous injection of lipopolysaccharide successfully induced the acute liver failure model. Several studies have shown that a rat model of sepsis with MODS induced by endotoxin lipopolysaccharide is feasible.

Disclosure of Invention

The invention aims to provide a method for constructing a rat model with sepsis and multiple organ dysfunction syndrome, which specifically comprises the following steps:

(1) preparing lipopolysaccharide into corresponding concentration, performing intravenous injection on lipopolysaccharide on a rat, pressing the rat, keeping the rat in a supine position, exposing the inner side of hind limbs upwards, slightly pulling out hair of the left hind limbs, wiping the hair with an alcohol cotton ball, and pressing groin to fill femoral veins;

(2) after the alcohol cotton ball is sterilized, a 1ml syringe is inserted slowly in parallel, the medicine can be pushed after the needle point is determined to be in the blood vessel, and after the medicine pushing is finished, the injection point is pressed by a dry cotton ball to stop bleeding; after hemostasis is finished, the rat is raised regularly, and the state of the rat is observed and recorded regularly.

The injection amount of the lipopolysaccharide femoral vein is 2 mg/kg-3 mg/kg.

The invention has the beneficial effects that:

according to the embodiment of the invention, lipopolysaccharide with different administration modes and different doses is adopted to prepare a rat model with sepsis accompanied with MODS; the results of comparison of the mortality, the ethology, the corresponding biochemical indexes and the histopathological changes of all groups of animals show that the survival rate of the animals is relatively high, the animals have a plurality of organ dysfunction and meet the requirements of the required model, the dose of lipopolysaccharide is relatively small, the cost is low, and the optimal modeling method for researching sepsis and MODS is provided.

Drawings

FIG. 1 is a sample macroscopic view of a sham operation group and a model group;

FIG. 2 is a HE staining of lung pathological sections of a sham operation group and a model group;

FIG. 3H staining of liver pathological sections HE of the sham operation group and the model group;

FIG. 4 shows HE staining of kidney pathological sections in the sham operation group and the model group;

FIG. 5 HE staining of small intestine physical sections in the sham and model groups;

FIG. 6 HE staining of cardiac pathology sections of the sham and model groups.

Detailed Description

The present invention is further described in detail with reference to the following specific examples, but the scope of the present invention is not limited to the above description.

Example 1

Experimental materials used in this example: lipopolysaccharide (Sigma-L2880(O55: B5)); chloral hydrate (Solarbio); HITACHI-7171A automatic biochemical analyzer (Hitachi, Japan); organization microscopy videography system (national institute of science and technology, university of Sichuan); BH-Z common light microscope (OLYMPUS); surgical instruments (Shanghai surgical instruments factory).

To further illustrate the advantages of the methods of the present invention, this example designed 9 sets of experiments, where 7 sets (a, B, C, D, E, H, I) were used as comparative experiments.

The specific grouping process is as follows: clean grade healthy SD rats 180, female, body weight (220 + -10) g, randomized number method into 9 groups of 20 rats. Group A: in a sham operation group, PBS is injected into femoral vein at 2.5 mL/kg; group B: mild blood loss plus 30mg/kg of lipopolysaccharide intraperitoneal injection; group C, mild blood loss and lipopolysaccharide femoral intravenous injection of 5 mg/kg; group D, lipopolysaccharide femoral intravenous injection 5 mg/kg; group E, lipopolysaccharide femoral intravenous injection 4 mg/kg; group F, lipopolysaccharide femoral intravenous injection 3 mg/kg; group G, lipopolysaccharide femoral intravenous injection 2 mg/kg; group H, lipopolysaccharide femoral intravenous injection 1 mg/kg; group I lipopolysaccharide femoral intravenous injection 0.5 mg/kg.

The specific molding process of this embodiment is: the method in the literature was modified based on the preparation of a sepsis model to prepare a rat model of sepsis with MODS.

(1) Compound lipopolysaccharide intraperitoneal injection for mild blood loss

Grasping the skin of the head and back of the rat to expose and engorge the eyeball of the rat, and inserting the eyeball into the vein of the rat by using a broken capillary pipette; after the blood flow, the number of blood drops was counted (25 drops of blood are expected to equal 1ml) and a mild (around 15%) blood loss was caused in the rats in an amount of 10ml/kg of blood loss per rat. About 50 drops of blood are discharged from a 200g rat, after the blood discharge is finished, the eyeball is pressed by a cotton swab for 10-20s to stop bleeding, and the state of the rat is observed. Preparing lipopolysaccharide into corresponding concentration, and performing intraperitoneal lipopolysaccharide injection after bleeding for 6 h. The skin of the back of the rat is grasped, the abdominal wall is exposed, and the position of the head is kept low so as to prevent the important organs from being damaged during puncture. Find a puncture point in the right lower abdomen, take the puncture point as the center, and the iodophor cotton swab sterilizes the peripheral area with the diameter of 2 cm. Taking 1ml of injector, breaking the skin at an angle of 30-45 degrees with the skin, picking up the needle head after falling empty, slightly flattening the needle point, and injecting after no blood or bubble or urine is pumped back; after injection, the rats were kept routinely, observed regularly and the status of the rats was recorded.

(2) Lipopolysaccharide intravenous injection

The rat was held in supine position with the inner hind limb exposed upward. Gently pulling off hair of left hind limb, wiping with alcohol cotton ball, and pressing groin to make femoral vein full; after the alcohol cotton ball is sterilized, a 1ml syringe is inserted slowly in parallel, the medicine can be pushed after the needle point is determined to be in the blood vessel, and after the medicine pushing is finished, the injection point is pressed by a dry cotton ball to stop bleeding; after hemostasis is finished, the rat is raised regularly, and the state of the rat is observed and recorded regularly.

And (4) analyzing results:

the observation and record of 24h A-I group death after each group molding are shown in Table 1

TABLE 1 post-mold 24h A-I mortality

Note: in comparison with the group A,^*P<0.05, comparison with group B^#P<0.05, comparison with group I^△P<0.05。

As can be seen from table 1: rats in group A were active normally with no mortality, and the mortality was higher in all the other groups compared to group A, with p <0.05, and lower in G, H, I groups compared to group B, with p <0.05, with statistical differences, and higher in groups B-F, with p <0.05, with statistical differences compared to group I.

② the behavioristic comparison of 9 groups of rats:

group a (sham surgery): the rat is normal in spirit, diet and hair color, and the anus area is dry and clean.

B. Group C: about 15% of blood loss occurs, the activity of the rat is weakened, the rat is manifested by struggling and weakness, muscle relaxation, slow movement, increased respiratory frequency and the like, after 30min of model building, the rat suffers from listlessness and difficulty in opening eyes, the rat dies continuously after 1h, the death rate is 80% after 24h, the hair color is dark and thin, and the stool is thin.

D. Group E: rats begin to appear listlessness, slow movement, contracture and pain 30min after the model building, and die after 2h, and have dark hair, shapeless stool, no blood, no pus and yellow brown.

F. G, H, I group: after 1h of molding, rats are listened, slowed in movement, compressed and painful, and die after 4h, and are dark in hair color, shapeless in stool, bloodless, purulent and yellowish-brown.

③ carrying out blood detection on 9 groups of rats

After the model is successfully made for 24 hours, 3.6 percent chloral hydrate anesthetizes the rat, the abdominal wall is cut at the abdominal midline after the rat pain reaction disappears and the supine position is fixed, but the diaphragm muscle is not broken through; blunt separation, exposing abdominal aorta, taking rat abdominal aorta blood, and detecting indexes such as ALT, AST, BUN, Cr, CK-MB, LDH, WBC, PLT and the like.

The blood routine test results are shown in table 2:

TABLE 2 results of routine examination of blood in each group 24h after molding: (n＝5)

Note: in comparison with the group A,^*P<0.05, the difference was statistically significant.

As can be seen from Table 2, WBC and PLT were significantly decreased, B-H was statistically significant (p <0.05), and group I was not statistically significant (p >0.05) compared to the sham group; wherein the proportion of N% is increased, the proportion of L% is decreased in the classification of the white blood cells, B-H have statistical significance (p is less than 0.05), and group I have no statistical significance (p is more than 0.05); the proportion of M% is increased, B-G have statistical significance (p <0.05), and H-I have no statistical significance (p > 0.05).

The blood biochemical test results are shown in table 3:

TABLE 3 Biochemical examination results of each group 24h after molding: (n＝5)

Note: in comparison with the group A,^*P<0.05, the difference was statistically significant.

As can be seen from table 3:

liver function: compared with a sham operation group, ALT is obviously increased, the groups B, C, D, F and G have statistical significance (p is less than 0.05), and the group E, H, I has no statistical significance (p is more than 0.05); AST is obviously increased, and the groups B-F and H have statistical significance (p <0.05) and G, I have no statistical significance (p > 0.05).

Kidney function: compared with a sham operation group, BUN is obviously increased, the groups B-D and F-H have statistical significance (p is less than 0.05), and the group E, I has no statistical significance (p is more than 0.05); cr is obviously increased, groups B-E and G-I have statistical significance (p is less than 0.05), and groups F have no statistical significance (p is more than 0.05).

Myocardium enzymology: CK. CK-MB and LDH were significantly elevated, and the difference between the values was statistically significant (p <0.05) in the groups except group I, in which the CK-MB was elevated (p > 0.05).

And fourthly, morphological observation:

observing the specimen by eyes: in comparison with the sham-operated group (group a), the model group (group H) was visible to the naked eye 24 hours after modeling of each tissue organ: intestinal congestion and edema with different degrees, liver dispersing at the congestion point, liver lobe ischemia, kidney congestion and blood stasis, lung and spleen dispersing at the ischemia focus; as shown in FIG. 1, M is a model group, and N is a sham operation group; in the figure, liver A, kidney B, lung C, spleen D.

Fifthly, HE dyeing

Comparing the artificial hand group (A group), and after the model group (H group) is molded for 24 hours, the myocardial gap is widened and the neutrophil is infiltrated; disorganization of hepatocytes, edema, congestion in the antrum of the liver and infiltration of a large number of inflammatory cells; a large amount of inflammatory cells infiltrate into alveoli and pulmonary interstitium, the nuclei are deeply stained, and the pulmonary interstitium is congested and edematous; the visible cells of the kidney are slightly disordered, the periphery of the glomerulus is obviously congested, and inflammatory cell infiltration can be seen in the kidney tissues; congestion and edema of the small intestine, infiltration of a large number of inflammatory cells, and rupture of the villus portion of the intestine are observed. As shown in fig. 2-6, the lung pathological section HE is stained in the sham operation group (A) and the model group (B) in fig. 2; FIG. 3A and B liver pathological section HE staining of the sham operation group (A) and the model group (B); FIG. 4 shows HE staining of renal pathological sections in the sham operation group (A) and the model group (B); FIG. 5 HE staining of small intestine pathological sections in sham group (A) and model group (B); FIG. 6 is a HE staining of a heart pathology section in the sham operation group (A) and the model group (B).

The following reference indexes exist for evaluating the success of the sepsis animal model: firstly, a high-rank low-resistance hemodynamic state and a high metabolic state; secondly, the natural mortality rate reaches 50 to 70 percent; and the time of organ dysfunction and death lags behind the molding time by 6-12 h. Fourthly, accompanying organ dysfunction. MODS is a clinical syndrome that after serious trauma (such as shock, severe pancreatitis) and serious infection occur for 24 hours, dysfunction of two or more organs occurs simultaneously or sequentially. A rat model of sepsis accompanied MODS is constructed, and the conditions are required to be met simultaneously.

In the embodiment, the rat model with sepsis and MODS is prepared by adopting different administration modes and different dosages of lipopolysaccharide; the results of comparison of the mortality, the ethology, the corresponding biochemical indexes and the histopathological changes of all groups of animals show that the survival rate of the animals is relatively high, the animals have a plurality of organ dysfunction and meet the requirements of the required model, the dose of lipopolysaccharide is relatively small, the cost is low, and the optimal modeling method for researching sepsis and MODS is provided.