CN111920541A

CN111920541A - Method for constructing sepsis compound animal model

Info

Publication number: CN111920541A
Application number: CN202010628481.6A
Authority: CN
Inventors: 李川; 程晨; 杜飞飞; 徐方; 王凤清; 余玄; 董凯; 张桂萍; 姚小青
Original assignee: Shanghai Institute of Materia Medica of CAS; Tianjin Chase Sun Pharmaceutical Co Ltd
Current assignee: Shanghai Institute of Materia Medica of CAS; Tianjin Chase Sun Pharmaceutical Co Ltd
Priority date: 2020-07-01
Filing date: 2020-07-01
Publication date: 2020-11-13

Abstract

The invention relates to the field of animal models for basic scientific research, in particular to a construction method of a sepsis composite animal model. The invention combines femoral artery intubation and cecal ligation and puncture to construct a sepsis compound animal model for the first time, and is successfully applied to the pharmacokinetic research of an anti-sepsis drug on a model rat, so that the continuous blood sample collection on the same rat can be realized, the death rate of the whole animal is not influenced, and the number of experimental animals is greatly saved. Meanwhile, reliable pharmacokinetic data of the model animal can be obtained, and the method has important reference significance for developing drug research in related fields.

Description

Method for constructing sepsis compound animal model

Technical Field

The invention relates to the field of animal models for basic scientific research, in particular to a construction method of a sepsis compound animal model.

Background

Sepsis is organ dysfunction syndrome caused by systemic host reaction caused by infection, has fierce illness state and high fatality rate (25-50%), has poor prognosis, and becomes a difficult point faced by modern critical disease medicine. Conventional treatment of sepsis (including septic shock) relies mainly on timely and rational antibiotic anti-infective treatment and regulation of patient abnormal responses, but new drug development is still in the way against the latter, which is still lacking in effective treatment chemicals. The Chinese medicine Xuebijing is the only two kinds of new medicine approved for treating pyemia in China at present (2004), and about 60 million pyemia patients use Xuebijing every year in China, and clinical experiments show that Xuebijing can effectively reduce 28-day mortality of pyemia patients. Various animal models of sepsis are widely used in research and development experiments for different purposes. Among them, the Cecum Ligation and Puncture (CLP) model has a high similarity to clinical sepsis, can better simulate the pathological process of sepsis, and is considered as the gold standard for studying sepsis. Although the development of the CLP model is mature, the difficulty of model construction is high, and the death rate is not stable easily.

The clinical pre-drug pharmacokinetics research is quite important in the research and development process of new drugs, is used for revealing the dynamic change process of the drugs in animal bodies, clarifying the dynamics characteristics of the processes of drug absorption, distribution, metabolism, excretion and the like, provides important dynamics parameters according to a mathematical model, participates in the primary screening of lead compounds, and preliminarily judges whether the drugs have the value of further development and research. For the re-evaluation of Chinese patent medicines after the market, the pharmacokinetics also plays an important role, is beneficial to disclosing the substance basis of the efficacy of the Chinese patent medicines and provides scientific data for the secondary development of large varieties of the Chinese patent medicines.

Rodent rats are commonly used as animals for laboratory drug studies, and there are several methods for blood sample collection from rats: the orbital blood sampling speed is high, but the stimulation to rats is large, and the blood sampling amount is not easy to control. The indwelling needle head for tail vein blood sampling is easy to fall off. Although the common carotid artery and jugular vein are frequently used for blood sampling, the cannula is close to the main organs, so that the death is easy to occur accidentally, and the blood sampling of the rat in a free moving state is difficult to realize. At present, the collection of animal plasma samples in the research of sepsis pharmacokinetics is mainly based on the blood collection of rat orbit, and in order to ensure the data quality, one rat can only collect blood samples at one time point, so that the loss of experimental animals is large.

In conclusion, the number of animals lost in carrying out pharmacokinetic studies on the CLP model by adopting a conventional blood sampling method is large, so that the experimental cost is increased, and the protection and the welfare of experimental animals are not facilitated.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a method for constructing a composite animal model of sepsis, which is used for solving the problems in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a method for constructing a sepsis composite animal model, which comprises femoral artery intubation and cecal ligation puncture.

Preferably, the femoral artery cannula comprises the steps of:

1) a 0.5-1.5 cm incision is made in the groin parallel direction, and femoral artery is exposed;

2) clamping the proximal end of the femoral artery, and opening the distal end of the femoral artery;

3) inserting the head end of the cannula from the opening towards the proximal end of the femoral artery, loosening the proximal end of the femoral artery while inserting, ligating and fixing the cannula after continuously inserting for 3-4cm, and sealing the cannula;

4) the tail end of the cannula is led out of the body through subcutaneous puncture of the tail of the animal, fixed at the tail and sutured with the wound.

Preferably, heparin saline is used to seal the cannula in step 3).

Preferably, the cecal ligation puncture comprises the following steps:

1) making a longitudinal incision along the median ventral line to free cecum;

2) ligating from the top of the cecum to 30% -50% of the part below the ileocecal valve;

3) puncturing the cecum to form an intestinal fistula, and extruding excrement around the perforation;

4) the cecum was placed in the abdominal cavity and the incision was closed.

Preferably, the puncture site in step 3) is selected from the site from the ligation site to the root of the cecum.

Preferably, cecal ligation punctures are performed after successful femoral cannulation.

Preferably, the animal model of sepsis comprises a rat model of sepsis.

The invention provides a sepsis compound animal model, which is obtained by the construction method.

In a third aspect, the invention provides the use of the sepsis composite animal model in the development of sepsis therapeutic drugs.

Specifically, the application is the application of the sepsis compound animal model in the research of the pharmacokinetics of the sepsis therapeutic drug.

As described above, the method for constructing an animal model of sepsis according to the present invention has the following advantageous effects:

1) the compound model can be applied to the plasma pharmacokinetics research of the sepsis treatment medicine on model animals.

2) Compare traditional single point and kill, the compound model of this application can realize on same rat continuous blood sample collection and do not influence the mortality of whole animal, can save experimental animals quantity greatly.

3) Reliable animal model pharmacokinetic data can be obtained, and the method has important reference significance for developing drug research in related fields.

Drawings

Fig. 1-1 shows the femoral artery cannulation and cannulation location in the femoral artery cannulation model.

Figures 1-2 show rats with completed femoral cannulation.

FIG. 2-1 shows a caecum ligation map during FAC-combined CLP sepsis drug-induced rat complex model preparation.

Fig. 2-2 shows the puncture image after ligation when FAC combined CLP sepsis drug was prepared for rat composite model.

Figures 2-3 show a plot of the extruded small stool at the time of FAC-combined CLP sepsis drug-substituted rat composite model preparation.

FIGS. 2-4 show the post-operative suture map of FAC combined CLP sepsis drug in rat composite model preparation.

FIGS. 2-5 show rats after suturing when the FAC is combined with CLP sepsis drug in place of the rat composite model.

FIG. 3 shows a composite model animal mortality statistic in accordance with the present invention.

Figure 4 shows the time profile of the plasma drug concentration of the exposure substance in rats of each group after the administration of Xuebijing.

Detailed Description

The invention provides a construction method of a sepsis compound animal model, which comprises femoral artery intubation and cecal ligation and puncture.

Further, the femoral artery cannula comprises the following steps:

In a preferred embodiment, after exposing the femoral artery, a section of surgical silk thread is placed under the proximal end of the femoral artery in advance for standby, so that the situation that the surgical silk thread is inconvenient to take after the femoral artery is opened, the operation time is prolonged, and even the animal dies can be avoided.

In a preferred embodiment, step 2) is performed by forming a bevel in the distal end of the femoral artery, which facilitates insertion of the cannula.

In particular, the cannula is selected from polyurethane cannulas.

Preferably, heparin saline is used to seal the cannula in step 3). Heparin has strong in vivo and in vitro anticoagulation effect, and has pharmacological actions of anticoagulation, antithrombotic, blood viscosity reduction and the like.

Preferably, the whole process of the femoral artery intubation is carried out on a constant-temperature heating pad, and the femoral artery intubation is moved into a cage after the operation is completed until the animal is completely awake, so that the body temperature can be kept in the operation after anesthesia, and the death of the animal due to hypothermia can be avoided.

Further, the cecum ligation puncture comprises the following steps:

1) making a longitudinal incision along the median ventral line to free cecum;

3) puncturing the cecum to form an intestinal fistula, and extruding excrement around the puncture hole;

4) the cecum is placed into the abdominal cavity and the incision is sutured;

specifically, in step 2), ligation is performed with 4-0 or 5-0 silk thread.

The ligation part in the step 2) is important, different ligation parts can cause different mortality of model animals, and the ligation parts should be kept as consistent as possible in the same experiment.

The puncture site in the step 3) is selected from a section of cecum from the top of the cecum to the lower part of the ileocecal valve.

The type of the puncture needle and the number of times of puncturing the cecum used in the puncturing in the step 3) can be determined according to specific conditions, and the more the number of the pinholes is, the larger the number of the pinholes is, the higher the death rate of the model rat is possibly caused. In one embodiment, an intestinal fistula may be formed by two full-thickness penetrations with a 16G puncture needle.

The amount of the excrement extruded in the step 3) needs to be proper, and is determined according to the target death rate, and abdominal cavity infection caused by the excrement is the main reason of death of the model rat. If the target mortality is 60-70%, the extruded feces are rice grain size.

Preferably, the animal body is recovered to be normal by injecting normal saline subcutaneously after cecal ligation and puncture.

Preferably, the animals are anesthetized by using a conventional anesthetic before femoral artery intubation and cecal ligation puncture, and the supine position of the animal is fixed on an operating table after anesthesia is successful. The surgical area is then shaved, and the skin may be wiped with a disinfectant before and after shaving.

Feeding the stock after femoral artery intubation and cecal ligation and puncture, and recovering the weight before operation for later use. During recovery, the patency of the cannula needs to be checked daily and the tube sealed with heparin saline to prevent clogging.

Either of the femoral intubation and cecal ligation punctures may be performed first. Preferably, cecal ligation and puncture are performed after femoral artery intubation is successful, and because the femoral artery intubation is a minimally invasive operation, the controllability of the mortality can be better ensured by performing the cecal ligation and puncture operation after the rat returns to a normal state.

In a preferred embodiment, the cecal ligation puncture is performed 24-72 hours after successful femoral intubation. The proper interval time can ensure that the animal body is normal after the femoral artery is cannulated.

The sepsis composite animal model is selected from a sepsis rat model.

In a third aspect the invention provides the use of the sepsis animal model in the development of a sepsis therapeutic drug.

Specifically, the application is the application of the sepsis animal model in the research of the pharmacokinetics of the sepsis therapeutic drug.

In one embodiment, the sepsis therapeutic agent is Xuebijing.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Example 1 Femoral Artery Cannulation (FAC) model preparation

After anesthetizing (8ml/kg) the rats with chloral hydrate solution (50mg/ml), the thigh (ventral side), groin and caudal end of the rats were shaved with an electric razor, and the shaved portions were wiped with sterilized alcohol. The rat is placed on a constant-temperature (37 ℃) heating pad with the head facing forwards in the supine position, the limbs are stuck and fixed by medical adhesive tapes, and the operation area is exposed in the visual field of an operation microscope. As shown in fig. 1-1, an incision of about 1.0cm was made parallel to the groin, the femoral artery was exposed after blunt dissection, and a length of surgical thread (about 5cm) for ligation was previously placed under the proximal end of the arterial vessel. Clamping the proximal end of a blood vessel by using an artery clamp, opening an oblique opening on the distal end of the blood vessel by using an ophthalmic scissors, inserting the front end of a polyurethane cannula from the incision along the proximal direction, loosening the artery clamp while inserting, immediately flushing arterial blood into the cannula, continuously inserting for 3-4cm, ligating and fixing by using an operation silk thread, filling the cannula with heparin normal saline (25IU), and sealing. The tail end of the cannula is led out of the body by the hypodermic puncture of the tail part of the rat by a puncture needle, is fixed at the tail part (shown in figure 1-2) by a cannula, is sutured with the wound and is disinfected by alcohol. After the surgery was completed, the rats were kept awake by subcutaneous injection of sterile saline (s.c., 20ml/kg) and continued on a heating pad. After waking up, the rats are fed in 2 cages together and recovered to the preoperative body weight for later use. During recovery, the cannula was checked daily for patency and sealed with heparin saline to prevent clogging.

Example 2 femoral artery intubation (FAC) with Cecal Ligation and Puncture (CLP) sepsis drug preparation for rat composite model

48 hours after the femoral artery intubation rat is successfully prepared, a sepsis model is prepared by adopting a Cecal Ligation and Puncture (CLP) method, and an operation method adopts improved CLP operation. Fixing the supine position of the rat on an operating table after the anesthesia is successful, smearing the skin of the abdominal operation area for 2 times by using the iodine, and scraping the abdominal hairs by using a blade; making a longitudinal incision of about 2cm along the midline of the abdomen, separating skin and muscle layer by layer, dissociating cecum, and ligating with 4.0 silk thread from the top of cecum to 30-50% of the part below ileocecal valve (fig. 2-1); and puncture the cecum 2 times with a 16G puncture needle to form an intestinal fistula (fig. 2-2), and squeeze out a small amount of stool around the puncture (fig. 2-3). The cecum was placed back into the abdominal cavity and the incision was sutured layer by layer (fig. 2-4 and 2-5). The Sham operation (Sham) only takes out the rat cecum out of the abdominal cavity and then returns the rat cecum to the abdominal cavity, and then the abdominal cavity is sutured. Saline (20mL/kg) was injected subcutaneously after surgery. After operation, freely drinking water, keeping the temperature at 20-24 ℃ and conventionally feeding.

Example 3 FAC-Combined CLP Complex model animal mortality assessment

The 28-day mortality rate is the main clinical outcome index of sepsis, and the animal observation period is 7 days. 150 rats were randomly assigned to Sham group (n-30), CLP group (n-30), FAC + CLP group (n-30), CLP + XBJ group (n-30), FAC + CLP + XBJ group (n-30), and XBJ group (n-30), where XBJ represents hyacinone. Each group was treated as follows: (1) sham group: after the operation, 4mL/kg of physiological saline is injected intravenously for 2, 12, 24, 36, 48 and 60 hours. (2) CLP group: 4mL/kg of physiological saline is injected intravenously 2, 12, 24, 36, 48 and 60 hours after CLP operation. (3) CLP + FAC group: FAC rats were injected with 4mL/kg of saline intravenously 2, 12, 24, 36, 48, 60 hours after CLP surgery. (4) CLP + XBJ group: after CLP operation, 4mL/kg of Xuebijing is injected intravenously at 2, 12, 24, 36, 48 and 60 hours. (5) CLP + FAC + XBJ group: FAC rats were injected intravenously with 4mL/kg Xuebijing 2, 12, 24, 36, 48, 60h after CLP surgery. The mortality rates of the groups 1d, 2d, 3d, 4d, 5d, 6d and 7d were observed and recorded. The effect of FAC on mortality in CLP sepsis rat model was examined by comparison of mortality between groups and the therapeutic effect of bifenac was compared on both models.

As shown in fig. 3 and table 1, the mortality rates of CLP rats and CLP + FAC rats are similar, which indicates that FAC is a minimally invasive surgery, has little influence on the mortality rate of CLP model rats, and the composite model is successfully constructed. In addition, the therapeutic effects of Xuebijing on the two models are similar.

TABLE 1 mortality in rats of each group

Example 4 FAC-incorporation CLP Complex model Bisph plasma pharmacokinetics study

42 rats were randomly assigned to the normal XBJ group (n ═ 6), CLP XBJ group (n ═ 30), CLP + FAC XBJ group (n ═ 6). Each group was treated as follows: (1) normal rat XBJ group: 2h after CLP operation of FAC rats, injecting 4mL/kg of Xuebijing through tail vein, continuously collecting blood plasma samples 10min, 30min, 1h, 3h and 6h after administration on the same rat through femoral artery, and paralleling 6 rats; (2) CLP rat XBJ group: 4mL/kg of Xuebijing is injected through tail vein 2h after CLP operation, plasma samples are collected at eye sockets 10min, 30min, 1h, 3h and 6h after administration respectively, and 6 rats are killed at each time point; (3) CLP + FAC rat XBJ group: FAC rats were injected with 4mL/kg of Xuebijing via tail vein 2h after CLP operation, and the collection protocol was the same as (1), i.e. plasma samples were continuously collected via femoral artery on the same rat 10min, 30min, 1h, 3h and 6h after administration, and were paralleled to 6 rats. Collected plasma was analyzed for concentration of essential Xuebijing substances by LC-MS/MS after sample pretreatment. Pharmacokinetic parameters (including C10min, AUC0-6h, AUC0- ∞, t1/2, MRT, CLtotal, VSS) were analyzed using a non-compartmental model using Innaphase kinetic 2000TM software (version 5.0; Thermo Scientific, Philadelphia, Pa., USA). The experimental data are expressed as mean ± standard deviation (mean ± SD).

As shown in table 2 and fig. 4, compared with the normal group, the trend of the change of the in vivo exposure substance in the CLP + FAC group and the CLP group was consistent, and the values of the CLP + FAC group and the CLP group were very close. Proved that the pharmacological data obtained by continuous blood sample collection of FAC is accurate and reliable, and the use number of animals is greatly reduced by adopting FAC + CLP to carry out pharmacokinetic research.

TABLE 2 comparison of the pharmacokinetic parameters of the exposed substances in rats of each group after administration of Xuebijing

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims

1. A construction method of a sepsis compound animal model is characterized by comprising femoral artery intubation and cecal ligation puncture.

2. The method of constructing according to claim 1, wherein the femoral artery cannula comprises the steps of:

3) inserting the head end of the cannula from the opening to the proximal end direction of the femoral artery, loosening the proximal end of the femoral artery while inserting, ligating and fixing the cannula after continuously inserting for 3-4cm, and sealing the cannula;

3. The method of claim 2, wherein the step 3) of sealing the cannula with heparin saline.

4. The method of construction according to claim 1, wherein said cecal ligation puncture comprises the steps of:

1) making a longitudinal incision along the median ventral line to free cecum;

2) ligating from the top of the cecum to 30-50% of the part below the ileocecal valve;

4) the cecum was placed in the abdominal cavity and the incision was closed.

5. The method of constructing according to claim 4, wherein the puncture site in step 3) is selected from the group consisting of ligation site to the root of the cecum.

6. The method of claim 4, wherein the cecal ligation puncture is performed after successful femoral cannulation.

7. The method of construction of claim 1, wherein the animal model of sepsis is selected from a rat model of sepsis.

8. A sepsis composite animal model, which is constructed by the construction method according to any one of claims 1 to 7.

9. Use of the sepsis composite animal model of claim 8 in the development of a sepsis therapeutic drug.

10. Use according to claim 9, wherein the use comprises use in the pharmacokinetic study of a sepsis therapeutic.