Animal model for meningitis / ventricitis caused by klebsiella pneumoniae after craniocerebral trauma and construction method thereof

Abstract

The invention relates to an animal model for meningitis / ventricitis caused by klebsiella pneumoniae after craniocerebral trauma and a construction method of the animal model. The animal model is obtained through immunosuppression, craniocerebral injury and intracranial infection. According to the invention, craniocerebral trauma and a bacterial infection meningitis / ventricular inflammation model are combined for the first time to simulate the pathological state of a patient suffering from meningitis / ventricular inflammation caused by klebsiella pneumoniae after neurosurgery, and the method can be applied to the physiological and pathological research of the diseases; secondly, the model animal is subjected to sampling after administration, tissue penetrability research can be carried out, the pathological characteristics of a clinical patient can be better fitted, and the change of the medicine at a specific part along with time under different infection states is described according to the sampling of a target part and medicine quantification; and thirdly, the method can be applied to preclinical research of meningitis biomarkers, and provides model support for immunological research of cytokine change and the like after meningitis.

Description

Technical Field

[0001] The invention belongs to the field of animal models, and particularly relates to an animal model of meningitis / ventriculitis caused by Klebsiella pneumoniae after craniocerebral trauma and a construction method thereof. Background Art

[0002] In recent years, the attributable mortality rate of central nervous system infection after neurosurgery has remained high, and the cure rate is not high. In particular, anti-infection treatment for infections caused by drug-resistant bacteria is difficult and the prognosis is poor. Even if cured, patients will suffer from permanent neurological sequelae, including brain damage, hearing loss, and learning disabilities. At present, there is still a lack of animal models that are close to the pathological state of such clinical patients for physiological and pathological and drug treatment research.

[0003] At present, the more mature animal models of craniocerebral trauma include the Fluid percussion injury model (FPI), the Weight drop injury model (WDI), the Controlled cortical impact model (CCI), the Penetrating ballistic-like brain injury model, the Blast injury model, and the Closed-head impact model of engineered rotational acceleration model (CHIMERA), etc. Different models correspond to different craniocerebral injuries. For example, the craniocerebral injury corresponding to the CCI model is cortical injury, which requires exposing the skull and directly simulating the cortical injury.

[0004] There have also been relevant case reports of animal models of meningitis caused by Klebsiella pneumoniae. The rat model can be infected through intranasal, oropharyngeal, peritoneal and other routes, and there are cases of direct bacterial infection in rabbit models. The defects of the rat model are that the infection mode of the model is different from the main route of clinical infection after actual neurosurgery; the rabbit model is difficult to operate due to the large size of the animal. Different species have different tolerance to Klebsiella pneumoniae, and the animal's own immune system can generally clear the bacteria strongly, making it difficult to ensure a continuous infection state, resulting in insufficient model stability. In addition, the modeling is prone to "threshold" conditions, that is, when the amount of bacteria inoculated is greater than a certain value, the animal's 24h or 48h mortality rate increases sharply; but below this "threshold", due to the strong immune response, the cerebrospinal fluid bacteria are quickly cleared, making it impossible to evaluate the model stability by colony counts, and it is difficult to conduct pharmacodynamic or pathological evaluations. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an animal model of meningitis / ventriculitis caused by Klebsiella pneumoniae after craniocerebral trauma and a method for constructing the same, so as to provide a model support for the study of the physiology and pathology of such diseases and drug treatment, especially the in vivo pharmacokinetics (PK) / pharmacodynamics (PD) study of antibacterial drugs.

[0006] The invention provides an animal model of meningitis / ventriculitis caused by Klebsiella pneumoniae after craniocerebral trauma. The animal model is obtained through immunosuppression, craniocerebral injury and intracranial infection.

[0007] Preferably, the animal model is a rat, more preferably a male Sprague Dawley (SD) rat, weighing 250-300 g. Rats weighing less than 250 g have a high mortality rate after modeling, and rats weighing more than 300 g will affect the stability of craniocerebral injury modeling due to excessive weight, that is, under the same impact parameters, as the weight increases, the degree of craniocerebral injury may decrease (subsequent neurological evaluation).

[0008] Preferably, the immunosuppression is the administration of cyclophosphamide aqueous solution. The working principle or function of the immunosuppression is to significantly reduce the number of neutrophils in rats. Neutrophils play a major role in the bacterial infection process. Reducing the number of neutrophils can reduce the immune response of rats to bacterial infection, allowing the infected strain to grow and maintain the pathological state of intracranial infection. The advantage is that it enhances the stability of the model, allows the strain to grow better, and maintains a high value of the final colony count without increasing the inoculation amount, which is conducive to the analysis of pharmacodynamics and pathological states.

[0009] Preferably, the craniocerebral injury adopts Feeney free fall injury method or controlled cortical injury model method. The working principle or function of the craniocerebral injury: the selected cortical point of impact is the motor cortex control area of ​​the rat brain, and the degree of injury is controlled by selecting different impact parameters. The degree of injury can be evaluated by neurological score (such as mNSS scale, Table 1) within three days after modeling. The advantage is that the degree of craniocerebral injury in the motor cortex control area of ​​the rat brain can be qualitatively / semi-quantitatively evaluated by neurological score to ensure the stability and effectiveness of craniocerebral injury.

[0010] Table 1mNSS rating scale

[0011]

[0012] Preferably, the intracranial infection is carried out by directly injecting bacterial solution into the ventricle. The working principle or function of the intracranial infection is to directly inject the bacterial solution into the ventricle to cause intracranial infection in rats. The advantage is that the operation is convenient and accurate, but it is necessary to master the method of inserting the needle into the ventricle through the foramen magnum; the injection infection position is accurate when the cerebrospinal fluid flows out after the syringe is withdrawn, and it can ensure that the injected bacterial solution enters the ventricle completely, which can avoid the error caused by the loss of bacterial solution compared with other inoculation methods; the cerebrospinal fluid can be collected and cultured at a specific time point to quantify the change in colony count.

[0013] The present invention also provides a method for constructing an animal model of meningitis / ventriculitis caused by Klebsiella pneumoniae after craniocerebral trauma, comprising the following steps:

[0014] (1) Rats were immunosuppressed twice, i.e., cyclophosphamide aqueous solution was administered 4 days and 1 day before intracranial infection;

[0015] (2) craniocerebral trauma 2 days before intracranial infection, using the Feeney free-fall injury method or the controlled cortical injury model;

[0016] (3) The Klebsiella pneumoniae liquid was directly injected into the cerebral ventricle to cause intracranial infection in rats, thus obtaining an animal model of meningitis / ventriculitis caused by Klebsiella pneumoniae after craniocerebral trauma.

[0017] Preferably, in step (2), the skull is exposed and a bone window is opened before craniocerebral trauma to locate the cortical impact point, so as to ensure the stability of neurological function damage at the injury site, which is conducive to the evaluation of the brain injury model through neurobehavioral scoring.

[0018] Preferably, in step (3), cerebrospinal fluid is extracted before intracranial infection and then injected to maintain stable intracranial pressure in rats and prevent the model from being affected by excessively high or low intracranial pressure.

[0019] Compared with the existing models, the main advantage of the present invention is that the two pathological processes of brain injury and meningitis caused by bacterial infection are simulated on the same animal, and the bacterial liquid is directly injected into the rat's cerebral ventricle through the cisterna magna for infection. The infection site is accurate, which is consistent with the pathological state and infection route of patients with postoperative bacterial infection in neurosurgery in clinical scenarios. In addition, the use of rats as model animals avoids the problems of large size and difficulty in operation in the use of rabbit models. The "threshold" problem is solved by giving rats stable and reliable immunosuppressive treatment, so that even when the amount of bacteria inoculated is low, the increase or maintenance of cerebrospinal fluid colony counts can be observed, and the evaluation of model stability and reproducibility as well as subsequent pharmacodynamic or pathological evaluation can be better achieved.

[0020] Beneficial Effects

[0021] The present invention combines craniocerebral trauma with a bacterial infection meningitis / ventriculitis model for the first time to simulate the pathological state of patients with meningitis / ventriculitis caused by Klebsiella pneumoniae after neurosurgery surgery, and can be applied to the physiological and pathological research of such diseases; secondly, sampling after administration of the model animal can be used for tissue penetration research, which can better fit the pathological characteristics of clinical patients, and describe the changes of drugs in specific sites over time under different infection states based on sampling and drug quantification at the target site; thirdly, it can be applied to preclinical research on meningitis biomarkers, and provide model support for immunological research such as cytokine changes after meningitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the process of constructing the animal model of the present invention.

[0023] Figure 2 This is an example of the construction of the animal model of the present invention and the time points of specific matters.

[0024] Figure 3 This is a rat bone window positioning diagram, where the red circle indicates the bone window location.

[0025] Figure 4 The following are the results of routine blood analysis of the animal model of the present invention.

[0026] Figure 5 This is the change in cerebrospinal fluid colony counts in the animal model of the present invention.

[0027] Figure 6 This is the HE-stained coronal section of the brain tissue of the euthanized animal model rat of the present invention, wherein the red circle is the brain injury site. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0029] Example 1

[0030] The present invention relates to an animal model, and the main operation process is established according to the timeline, such as Figure 2 As shown in the figure, the specific methods and usage environment are introduced by taking the pharmacokinetics / pharmacodynamics study of antimicrobial drugs as an example, starting with the timeline. Since bacterial infection is involved, bacterial infection and subsequent animal care must be carried out in a biological secondary laboratory (P2 animal room). DX is used below to represent the Xth day of bacterial supply, for example, D-4 refers to the 4th day before bacterial supply, D0 refers to the day of bacterial supply, and D2 refers to the second day after bacterial supply. CTX represents cyclophosphamide, and TBI represents craniocerebral injury.

[0031] D-14~D-7: Rats were placed in the SPF animal room, adapted to the environment, and raised normally.

[0032] D-4: Weigh and select rats with a body weight of 250-300g to be included in the model establishment group. For immunosuppression, 40mg cyclophosphamide was administered per kg of rat. The drug preparation was a cyclophosphamide aqueous solution with a concentration of 40mg / ml, which needed to be prepared in advance (preparation method: weigh 40mg cyclophosphamide powder and dissolve it in 1ml ultrapure water, and heat it at 37℃ for 10min to dissolve it by ultrasound). The final administration volume was 1ml / kg, that is, 1ml of CTX aqueous solution was given per kg of rat, and injected intraperitoneally.

[0033] D-2: Perform TBI operation. Rats were weighed and anesthetized by injection (10mg / kg Zota 50 + 5mg / kg Xylazine mixed solution intramuscular injection). The hair in the middle part of the head and neck of the rats was shaved, and the rats were disinfected by iodine and alcohol wipes. Erythromycin eye ointment was applied to the rats' eyes and surrounding areas. The scalp was cut along the middle of the head, and the soft tissue and periosteum were bluntly separated to expose the skull. 3% hydrogen peroxide was applied to the skull to reveal the sagittal suture. A circular bone window with a diameter of 5 mm was opened with a skull drill (the diameter of the impact rod head was 4 mm) 1.5 mm behind the coronal suture and 2.5 mm to the left of the midline, keeping the dura mater intact. Figure 3 shown.

[0034] If it is the Feeney free fall striking method, the striker is placed outside the dura mater, and no weight is placed at this time. The striker is adjusted upward by 2-3mm, so that when a 40g weight is used to hit the striker from a height of 20cm along the catheter in a free fall (the baffle is suddenly pulled out), the compression depth can be 2-3mm, without penetrating the dura mater, causing local brain contusion and laceration, resulting in an impact amount of 800g cm (40g×20cm). After the strike, the striker is immediately released from the injury position to avoid secondary injury. If it is the CCI method, the striker is placed outside the dura mater, the striking depth is set to 1mm, the striking speed is set to 3.5m / s, and after setting the parameters, the instrument can be controlled to strike. After the striking, stop bleeding in time, close the bone window with bone wax, suture the scalp layer by layer, disinfect the wound with iodine, and use an appropriate amount of sulfonamide powder for anti-inflammatory. The rats are placed on a constant temperature pad until they wake up and then raised in a single cage.

[0035] D-1: Immunosuppression is performed, and the method and dosage are the same as D-4. Neurological scores of rats can also be performed to screen out rats with moderate brain damage evaluated by the mNSS scoring scale.

[0036] D0: The model rats were transferred to the P2 animal room. 200 μl of blood was collected from the tail vein for routine blood analysis and evaluation of immunosuppression results (such as Figure 4 ).Depend on Figure 4 It can be seen that the white blood cell count (WBC), red blood cell count (RBC), neutrophil (NEUT), and lymphocyte (LYMPH) of the two groups of animals (immunosuppression and non-immunosuppression group) on D3 were significantly different, and the immunosuppression group was reduced, indicating successful immunosuppression. On D11, the WBC in the TBI+immunosuppression group increased, and there was no significant difference from the TBI+non-immunosuppression group. The NEUT of both groups increased abnormally, especially the TBI+immunosuppression group, and the LYMPH in the TBI+immunosuppression group was significantly lower than that in the TBI+non-immunosuppression group, indicating bacterial infection. After the rats were weighed, they were injected with anesthesia (10mg / kg of Zotai 50 + 5mg / kg of Xylazine mixed solution was injected intramuscularly). After the adjustment was completed using the rat brain orientation instrument according to the requirements of the positioning map, the anesthetized rat head was fixed on the fixed frame, the head and neck were trimmed and disinfected, a longitudinal incision (about 2cm) was made along the posterior midline, and the dorsal neck muscles were bluntly separated with scissors. Use a spreader to spread the innermost muscle, insert a needle (1 ml insulin syringe) into the triangular depression (foramen magnum), and after a slight breakthrough (needle insertion 2-4 mm), extract 50 μl of cerebrospinal fluid, and then use a 1 ml insulin syringe to extract 50 μl of a 1×10 6CFU / ml of Klebsiella pneumoniae liquid was injected into the large hole of the needle pillow as described above. Aspirate the syringe before injection. If clear liquid flows back without bubbles, it means that the needle has entered the ventricle. Slowly inject all the liquid into the ventricle. After the injection, slowly rotate the needle and slowly withdraw it, and immediately remove the spreader, suture the muscles and scalp, disinfect the wound with iodine, and place the rat on a constant temperature pad until it wakes up and is kept in a single cage.

[0037] D1: Pharmacokinetic studies can be conducted, i.e. blood and cerebrospinal fluid samples can be collected after administration. For example, after tail vein injection of antibiotics, blood and cerebrospinal fluid can be collected at designated time points for quantitative analysis of antibiotics in rats.

[0038] D1-3: Pharmacodynamic studies can be conducted by collecting rat cerebrospinal fluid at designated time points to observe changes in the bacterial counts in rat cerebrospinal fluid (e.g. Figure 5 ).Depend on Figure 5 It can be seen that without anti-infection treatment, the colony count of the final model group (TBI+immunosuppression group) bottomed out at 24 hours and no longer decreased, and rebounded at 48 hours, indicating that the infection state persisted. The mortality rate and neurological scores of rats can also be observed.

[0039] D4-7: The 7-day survival rate can be observed, 200 μl of blood can be collected from the tail vein for routine blood analysis, or brain tissue samples can be collected after euthanasia for slice analysis (such as Figure 6 ).Depend on Figure 6 It can be seen that brain damage and cell debris were observed in the largest section of the hippocampus / hypothalamus, and deformation of the ventricular structure was observed in other sections.

Claims

1. An animal model of meningitis / ventriculitis caused by Klebsiella pneumoniae after craniocerebral trauma, characterized in that: The animal model is obtained through immunosuppression, craniocerebral injury and intracranial infection.

2. The animal model according to claim 1, characterized in that: The animal model is a rat with a body weight of 250-300 g.

3. The animal model according to claim 1, characterized in that: The immunosuppression is administration of cyclophosphamide aqueous solution.

4. The animal model according to claim 1, characterized in that: The craniocerebral injury is performed using the Feeney free-fall injury method or the controlled cortical injury model method.

5. The animal model according to claim 1, characterized in that: The intracranial infection is carried out by directly injecting bacterial solution into the cerebral ventricle.

6. A method for constructing an animal model of meningitis / ventriculitis caused by Klebsiella pneumoniae after craniocerebral trauma, comprising the following steps: (1) Rats were immunosuppressed twice, i.e., cyclophosphamide aqueous solution was administered 4 days and 1 day before intracranial infection; (2) craniocerebral trauma 2 days before intracranial infection, using the Feeney free-fall injury method or the controlled cortical injury model; (3) The Klebsiella pneumoniae liquid was directly injected into the cerebral ventricle to cause intracranial infection in rats, thus obtaining an animal model of meningitis / ventriculitis caused by Klebsiella pneumoniae after craniocerebral trauma.

7. The construction method according to claim 6, characterized in that: In the step (2), the skull is exposed before craniocerebral trauma and a bone window is opened to locate the cortical impact point.

8. The construction method according to claim 6, characterized in that: In the step (3), cerebrospinal fluid is extracted before intracranial infection and then injected.

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