Rabbit brainstem hemorrhage model and establishment method thereof

Abstract

The invention discloses a rabbit brainstem hemorrhage model and an establishment method thereof. The establishment method comprises the following steps: S1, preoperative preparation: selecting healthy adult New Zealand white rabbits, and fasting experimental rabbits 12 hours before an operation; s2, performing an operation process, namely performing anesthesia by adopting isoflurane gas, dissecting to fully expose the skull, extracting arterial blood of an experimental rabbit, injecting autoblood into cortical spinal bundles of the right brainstem, disinfecting, and suturing an incision. A stereotactic autoblood injection method is adopted to construct the rabbit brainstem hemorrhage model, the pathophysiological process of clinical real brainstem hemorrhage is well simulated, and a reliable foundation is laid for studying the pathophysiological mechanism and scheme of occurrence, treatment and outcome of brainstem hemorrhage. The repeatability and reliability of experimental results are improved by accurately controlling positioning coordinates and injection parameters, and the method is simple in operation step, easy to master and suitable for large-scale experimental application.

Description

Technical Field

[0001] The invention belongs to the technical field of animal experimental model construction, and in particular relates to a rabbit brainstem hemorrhage model and a method for establishing the same. Background Art

[0002] Brainstem hemorrhage is the most severe subtype of hypertensive intracerebral hemorrhage, accounting for approximately 6-10% of all hypertensive intracerebral hemorrhages. Its clinical characteristics are: sudden onset, rapid deterioration of neurological function, poor prognosis, and high mortality. Approximately 60-80% of primary brainstem hemorrhages occur in the pons and are generally believed to be caused by rupture of the pontine branch of the basilar artery. Currently, there is still a lack of effective treatments for brainstem hemorrhage to improve patient prognosis. However, due to the low incidence of primary brainstem hemorrhage, the severity of the disease, and the complex factors affecting prognosis, it is difficult to conduct systematic basic research and large-scale clinical sample studies. At the same time, brainstem and basal ganglia hemorrhages differ greatly in anatomical location, blood supply system, and hematoma injury characteristics. Therefore, research on basal ganglia hemorrhage is not fully applicable to brainstem hemorrhage. Therefore, establishing an effective animal model of brainstem hemorrhage is very important for understanding the pathogenesis and pathological damage mechanism of brainstem hemorrhage and exploring treatment options.

[0003] Currently, the most common primary brainstem hemorrhage models are mainly constructed using rats and rabbits.

[0004] There are two main methods for establishing rat brainstem hemorrhage models: stereotactic injection of autologous blood or type VII collagenase. In 2013, Tim Lekic et al. established the first collagenase-induced rat brainstem hemorrhage model by injecting type VII collagenase into the rat brainstem. In this model, 0.15 U of collagenase (0.2 U / μl) was injected into the right ventral pontine tegmentum at a rate of 0.2 μl / min. After recovery from surgery, the rats developed ataxia, gait, craniocaudal rotation, and cranial nerve and limb dysfunction. Gross pathological examination revealed a well-defined hematoma within the right ventral pons tegmentum, with no subarachnoid (or subdural) hemorrhage. Furthermore, the experimental rats exhibited significant cerebral edema, neurological deficits, and cognitive decline, closely resembling some of the pathological changes and clinical symptoms of human brainstem hemorrhage. However, the 30-day mortality rate of this model was as high as 54%, limiting its application in evaluating the long-term therapeutic efficacy and molecular mechanisms of brainstem hemorrhage. In 2016, Chuanyuan Tao et al. established the first autologous blood injection model of brainstem hemorrhage by injecting autologous blood into the pons of Sprague-Dawley rats. In this experiment, 30 μl of autologous blood was injected into the right pons via the caudal artery at a rate of 5 μL / min. Postoperative MRI showed a stable hematoma with an average volume of 21.6 ± 3.41 mL, with no blood backflow or intraventricular hemorrhage. The rats also developed some cerebral edema, functional deficits, and white matter damage. However, the high mortality rate of this model at 1, 3, and 30 days was 28.1%, 50%, and 60%, respectively, which diminishes its value. Subsequently, Bal Krishna Shrestha et al., building on Tao's work, made improvements in eight aspects, including stereotactic positioning, injection volume, injection method, and anesthesia. They adopted a two-step injection method, injecting 60 μL of autologous blood 1.1 mm outside and 2.5 mm posterior to the fontanelle, at a depth of 9.6 mm, achieving a 90% success rate and establishing a rat model of moderate brainstem hemorrhage. In 2021, Xiangyue Tang et al. used a two-step injection method to inject 100 μL of autologous blood into the pons, establishing an animal model of extensive pontine hemorrhage.

[0005] However, systematic studies of rabbit brainstem hemorrhage models are limited. In 2005, Jin et al. injected 0.1 mL of autologous blood into the brainstem of Japanese white rabbits through craniotomy to study the effects of acute brainstem hemorrhage on intestinal mucosal congestion and morphological changes, observing increased lateral ventricular pressure. In 2020, Ding et al. developed a rabbit brainstem hemorrhage model using stereotactic collagenase injection. New Zealand rabbits were used for this model. The head was fixed at a position where the anterior bregma was 1.5 mm above the posterior bregma. At a coordinate point 8.0 mm posterior to the anterior bregma, 2.0 mm to the right, and 20.0 mm deep, 0.5 U of type VII collagenase (0.25 U / μl) was injected into the right corticospinal tract at a rate of 0.25 μl / min. A brainstem hemorrhage model was established 24 hours later. The rabbits developed symptoms such as left-sided neck twisting, left-sided hemiplegia, and decreased neurological function scores after surgery. MRI 24 hours after surgery revealed a low-density shadow in the brainstem, and gross pathological sections revealed a significant hematoma. The natural mortality rates within 24 hours and 3 days were 8.6% and 12% respectively, and the mortality rate did not increase further after 3 days.

[0006] Existing research on animal models of brainstem hemorrhage mainly focuses on rat models. However, the brainstem of rats is complex in structure and small in size. Therefore, brainstem hemorrhage modeling surgery requires more precise positioning and higher operational technical requirements. Slight deviations may lead to failure and poor reproducibility. At the same time, as a small animal, rats have poorer tolerance for surgery than large animal models, which often leads to higher postoperative mortality and increases the cost of modeling. In the application evaluation of model results, rats lack white matter in the brain and have a large gap with human brain structure. Many treatment strategies that are successful in rodent models and in vitro experiments often fail to achieve satisfactory results in clinical trials. Therefore, the application value of the rat brainstem hemorrhage model is limited.

[0007] In terms of modeling methods, stereotactic injection of collagenase is one of the commonly used methods for animal models of cerebral hemorrhage. It has the advantage of being less likely to clog the injection needle channel and can simulate the bleeding process of natural blood vessel rupture by destroying the vascular basement membrane. However, compared with the method of stereotactic injection of autologous blood, it still has the following shortcomings. First, collagenase-induced cerebral hemorrhage is often more diffuse and has poor reproducibility, and cannot well simulate the pathophysiological changes caused by blood accumulation and compression after human cerebral hemorrhage. Secondly, as an exogenous substance, collagenase can cause a more intense immune response and local inflammation, causing greater damage to the animal model and failing to well reflect the pathological damage mechanism after cerebral hemorrhage. Finally, the chronic hematoma caused by collagenase through enzymatic hydrolysis of the vascular basement membrane is quite different from the acute cerebral hemorrhage commonly seen in clinical practice, and the space-occupying effect is not obvious, which limits its value in research on therapeutic applications. Summary of the Invention

[0008] The present invention aims to provide a simple and controllable rabbit brainstem hemorrhage model and its establishment method to simulate the pathological process of real brainstem hemorrhage, improve the accuracy and reproducibility of experimental results, avoid immune reactions triggered by exogenous blood and drug injections, and ensure the safety of experimental animals.

[0009] In order to achieve the above objectives, this application adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for establishing a rabbit brainstem hemorrhage model, comprising the following steps:

[0011] S1: Preoperative preparation: Healthy adult New Zealand white rabbits were selected and fasted for 12 hours before surgery;

[0012] S2: Surgical procedure: Using isoflurane gas anesthesia, the skull was fully exposed after dissection, arterial blood was drawn from the experimental rabbit, and autologous blood was injected into the right brainstem corticospinal tract. After disinfection, the incision was sutured.

[0013] In the above technical solution, the experimental rabbits weigh 3.0-3.6 kg.

[0014] In the above technical solution, the rate of autologous blood injection into the right brainstem corticospinal tract is 20 μL / min.

[0015] In the above technical solution, the volume of autologous blood injected is 100 μL.

[0016] In the above technical solution, the specific process of dissecting and fully exposing the skull is as follows: shave the rabbit's head and prepare the skin to fully expose it, use 75% alcohol to disinfect the skin in the surgical area, cut the skin vertically from the midpoint of the line connecting the two eye sockets, peel off the subcutaneous tissue and muscles, and fully expose the skull, anterior fontanelle, and posterior fontanelle; fix the rabbit on a stereotaxic instrument, adjust the position of the rabbit's head so that the anterior fontanelle is 1.5 mm higher than the posterior fontanelle, use the anterior fontanelle as the origin, and use a cranial drill to drill a 2 mm diameter bone window with a coordinate point 8 mm behind the right anterior fontanelle and 2 mm to the right of the midline.

[0017] In the above technical solution, the process of injecting autologous blood into the right brainstem corticospinal tract is as follows: use a microsyringe to draw autologous blood, pierce the brain tissue to a depth of 20.0 mm through the bone window, start the automatic injection pump after reaching the position, inject the autologous blood into the right brainstem corticospinal tract, then leave the needle in for 20 minutes and slowly withdraw it, and seal the bone window with sterile bone wax.

[0018] The above technical solution also includes:

[0019] S3: Modeling result detection: After waking up, the experimental rabbits showed symptoms of twisted neck to the left, hemiplegia of the left limbs, slow reaction, inability to stand, unilateral or bilateral exophthalmos, and congestion. The Purdy score of neurological function was 7-9. On the day of surgery, cranial MRI showed low signal shadows in the right brainstem. Gross pathological anatomy showed reddish-brown hematoma formation in the rabbit brainstem, indicating that the model was successful.

[0020] In a second aspect, the present invention provides a rabbit brainstem hemorrhage model obtained by the above-mentioned establishment method.

[0021] The beneficial effects of the present invention are:

[0022] The present invention provides a novel rabbit brainstem hemorrhage model and its establishment method. This model, constructed using stereotactic injection of autologous blood, effectively simulates the pathophysiological processes of actual clinical brainstem hemorrhage, laying a solid foundation for studying the pathophysiological mechanisms and strategies for the development, treatment, and outcome of brainstem hemorrhage. The present invention improves the repeatability and reliability of experimental results by precisely controlling positioning coordinates and injection parameters. The simple and easy-to-use procedure makes it suitable for large-scale experimental applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The height difference between the anterior and posterior fontanelles of the New Zealand rabbit skull is 1.5 mm.

[0024] Figure 2 It is the location of the New Zealand rabbit pons (PONS) and corticospinal tract (CS) in the rabbit brain atlas.

[0025] Figure 3 This is the MRI imaging of hematoma evolution in the New Zealand rabbit brainstem hemorrhage model from 0 to 30 days (the transverse direction represents the MRI images of the same rabbit brain at different levels at the same time). DETAILED DESCRIPTION

[0026] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the present invention to those skilled in the art. The present invention will be limited only by the claims.

[0027] Example 1

[0028] Preoperative preparation: Healthy adult New Zealand white rabbits of either sex, weighing 3.0-3.6 kg, were selected. The rabbits were fasted for 12 hours before surgery. Neurological function scores (Purdy scores) were performed on the rabbits and they were weighed.

[0029] Surgical procedure: Preheat the heating pad to 37°C. Fix the microsyringe on the stereotaxic apparatus. Use isoflurane gas anesthesia, and the anesthesia is successful when the experimental rabbit breathes steadily and slowly and the pain stimulation reflex disappears. Use an electric shaver to shave the rabbit's head and prepare the skin for full exposure. Use 75% alcohol to disinfect the skin in the surgical area, cut the skin about 4 cm vertically from the midpoint of the line connecting the two eye sockets, peel off the subcutaneous tissue and muscles, and fully expose the skull, anterior fontanelle, and posterior fontanelle. Fix the rabbit on the stereotaxic apparatus and adjust the position of the rabbit's head so that the anterior fontanelle is 1.5 mm higher than the posterior fontanelle. Take the anterior fontanelle as the origin, 8 mm behind the right anterior fontanelle and 2 mm to the right of the midline as the coordinate points, and use a skull drill to drill a bone window with a diameter of 2 mm (without damaging the dura mater). Use a syringe to draw 0.2 ml of the experimental rabbit's arterial blood and quickly transfer it to a sterile tube (to prevent blood coagulation). Use a microsyringe to draw 100 μL of autologous blood and pierce the brain tissue to a depth of 20.0 mm through the bone window. After reaching the position, start the automatic injection pump and inject 100 μL of autologous blood into the right brainstem corticospinal tract at a rate of 20 μL / min. Then leave the needle in for 20 minutes and slowly withdraw it. Seal the bone window with sterile bone wax, disinfect it again, and suture the incision.

[0030] Modeling Results: Upon awakening, the rabbits presented with left-sided neck twisting, left-sided hemiplegia, sluggish reflexes, inability to stand, unilateral or bilateral exophthalmos, and congestion. Neurological function scores (Purdy scores) ranged from 7 to 9. A cranial MRI performed the same day postoperatively revealed a low-intensity shadow in the right brainstem, confirming the presence of an intracranial hematoma. Gross pathological anatomy revealed a reddish-brown hematoma in the brainstem, confirming successful modeling.

[0031] The present invention uses New Zealand white rabbits as model animals and constructs a brainstem hemorrhage model by stereotactic injection of autologous blood.

[0032] In terms of animal selection, 3.0-3.6kg New Zealand white rabbits were used as the modeling subjects. Rabbits are a commonly used large animal with highly developed white matter that is similar in structure and function to the human brain. They can better simulate the clinical manifestations and pathophysiological mechanisms of the occurrence, treatment and prognosis of human cerebral hemorrhage. Compared with rats, rabbit brains are larger and easier to operate on, and they have good postoperative tolerance, which reduces the difficulty of modeling and increases the success rate. Compared with other large animals, rabbits have low purchase and breeding costs, simple surgical anesthesia, and convenient behavioral observation. Selecting New Zealand white rabbits with a weight within the specified range helps to ensure the accuracy of stereotactic positioning and improve the repeatability of modeling.

[0033] In terms of modeling methods, 100 μL of autologous blood was injected. Autologous blood injection is simple to perform, and using one's own blood can effectively reduce the immune and inflammatory responses caused by exogenous substances. At the same time, the speed, morphology, and distribution of the hematoma formed by autologous blood injection are more consistent with clinical human cerebral hemorrhage and are more clinically significant. Selecting a blood volume of 100 μL ensures that the hematoma compresses the target brain area, which can not only ensure the expected significant behavioral changes, but also avoid excessive hematoma leading to animal death and modeling failure.

[0034] In terms of surgical operation, the use of isoflurane gas anesthesia can ensure the anesthetic effect and prevent the animal from struggling during surgery, which may lead to positioning failure. It can also avoid excessive anesthesia, difficulty in waking up after surgery, and even death from anesthesia. When fixing the rabbit's head, ensure that the anterior fontanelle is 1.5mm higher than the posterior fontanelle. At the same time, ensure that the fixation is stable and the position of the stereotactic injection is correct. When injecting autologous blood, the stimulation should be reduced slowly, and an injection speed of 20μL / min should be used. This can reduce the risk of coagulation blockage and prevent the death of the animal due to excessive injection speed. After the injection, the needle should be stopped for 20 minutes and then slowly withdrawn to ensure that the injected blood is fully coagulated, so as to reduce the back seepage of blood along the needle tract.

[0035] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for establishing a rabbit brainstem hemorrhage model, characterized by: The following steps are involved: S1: Preoperative preparation: Healthy adult New Zealand white rabbits were selected and fasted for 12 hours before surgery; S2: Surgical procedure: Using isoflurane gas anesthesia, the skull was fully exposed after dissection, arterial blood was drawn from the experimental rabbit, and autologous blood was injected into the right brainstem corticospinal tract. After disinfection, the incision was sutured.

2. The method according to claim 1, wherein: The experimental rabbits weighed 3.0-3.6 kg.

3. The method according to claim 1, wherein: Autologous blood was infused into the right brainstem corticospinal tract at a rate of 20 μL / min.

4. The method according to claim 1, wherein: The volume of autologous blood injected was 100 μL.

5. The establishment method according to claim 1, characterized in that: The specific process of dissection to fully expose the skull was as follows: the rabbit's head was shaved and the skin was prepared for full exposure. The skin in the surgical area was disinfected with 75% alcohol. The skin was incised vertically from the midpoint of the line connecting the two orbits, and the subcutaneous tissue and muscles were peeled off to fully expose the skull, anterior fontanelle, and posterior fontanelle. The rabbit was fixed on a stereotaxic instrument, and the position of the rabbit's head was adjusted so that the anterior fontanelle was 1.5 mm higher than the posterior fontanelle. With the anterior fontanelle as the origin, 8 mm behind the right anterior fontanelle and 2 mm to the right of the midline as the coordinate points, a 2 mm diameter bone window was drilled with a cranial drill.

6. The method according to claim 5, characterized in that: The process of injecting autologous blood into the right brainstem corticospinal tract is as follows: use a microsyringe to draw autologous blood, pierce the brain tissue to a depth of 20.0 mm through the bone window, start the automatic injection pump after reaching the position, inject the autologous blood into the right brainstem corticospinal tract, then leave the needle in for 20 minutes and slowly withdraw it, and seal the bone window with sterile bone wax.

7. The establishment method according to claim 1, characterized in that: Also includes: S3: Modeling result detection: After waking up, the experimental rabbits showed symptoms of twisted neck to the left, hemiplegia of the left limbs, slow reaction, inability to stand, unilateral or bilateral exophthalmos, and congestion. The Purdy score of neurological function was 7-9. On the day of surgery, cranial MRI showed low signal shadows in the right brainstem. Gross pathological anatomy showed reddish-brown hematoma formation in the rabbit brainstem, indicating that the model was successful.

8. A rabbit brainstem hemorrhage model, characterized by: Obtained by the method according to any one of claims 1 to 7.

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