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动物模型,脑缺血动物模型,脑缺血
z-bo
2020-07-17
873
Simulating the pathogenesis of human ischemic cerebrovascular disease, and establishing a reproducible and physiologically controlled animal model of standardized cerebral ischemia are always the goals pursued by researchers. With the deepening of the research on the mechanism of ischemic neuron injury and repair, higher requirements have been put forward for the establishment of animal models of cerebral ischemia. In today's rapid development of neuroscience, although it is not a new topic to discuss the establishment of standardized cerebral ischemic animal models, it has important practical significance and necessity.
In the study of animal models of cerebral ischemia, especially in the control of various physiological indicators and influencing factors in the process of making animal models, we have a big gap with foreign countries.
1. In-depth study on the pathophysiological mechanism affecting cerebral ischemia in China
2. Evaluation of the definite curative effect of brain protective agents in my country
3. The research results cannot be generally recognized by foreign counterparts, and research papers are difficult to publish and communicate in world-class magazines.
How to establish a standardized and standardized animal model of cerebral ischemia under the existing conditions, and use it reasonably and selectively for basic research on cerebral ischemic damage is a question worth exploring. Analysis of the current status of research and application of domestic cerebral ischemic animal models, still exists:
1. Blindly choose the type of animal model
2. Improper use of anesthetic
3. Inadequate control of physiological indicators and influencing factors
4. The criterion for the success of the model is not equal.
the reason
1. Some research units have limited research funding and are unable to match the corresponding laboratory animal monitoring equipment
2. A small number of researchers do not have enough knowledge about the impact of various physiological indicators and influencing factors on cerebral ischemia research, and they do not understand the formation principles and characteristics of various cerebral ischemia models, resulting in irregular model selection and production processes, leading to research The conclusion is quite contradictory and poorly repeatable.
At present, this phenomenon has attracted widespread attention from domestic scientific research management institutions and experts. In 1997, the bidding guidelines for the National Natural Science Foundation's key project "Research on the Mechanism of Ischemic Neuronal Death and Intervention Measures" clearly stated the requirements for establishing a "standardized cerebral ischemic animal model".
The correct selection of cerebral ischemia model is the prerequisite and basic condition of standardization. Appropriate selection of animal models can not only promote the advantages and avoid the shortcomings according to its characteristics, but also achieve the function of highlighting the research purpose. In the specific selection process of cerebral ischemia model, we must first clarify the formation principle and characteristics of the selected model, and then select it according to its principle combined with the purpose of the experiment.
Usually in mild ischemia/hypoxia, the brain's compensation mechanism protects the central nervous system from damage, but when the degree of ischemia increases, irreversible nerve damage will occur, leading to a series of clinical symptoms and even death. Clinically, cerebral vascular accidents, myocardial infarction, shock, neonatal asphyxia and brain trauma can cause ischemic damage to neurons. Therefore, it is of great scientific significance to actively explore the damage mechanism and preventive measures of cerebral ischemia. A physiologically controllable and reproducible animal model is necessary to systematically and comprehensively study the pathophysiological process of cerebral ischemia and test new treatment methods.
First of all, although the incidence of cerebral ischemia is high in clinic, more cases can be provided. However, human cerebral ischemia is very diverse, and its manifestations, causes, and anatomical location of ischemic areas are very different. This diversity prevents the possibility of performing statistical analysis and setting controls.
Second, accurate histopathological analysis, biochemical and physiological studies, often require invasive surgical procedures and direct brain tissue sampling analysis.
Thirdly, the observation of the early events of ischemic brain injury (minutes or even seconds) can only be done in experimental animals.
Finally, because ischemia is a blood supply abnormality, vascular factors play an important role in it, and changes in vascular factors cannot be simulated by tissue cell culture or brain slice incubation.
Therefore, making animal models of cerebral ischemia occupies a very important position in the study of cerebral ischemic injury mechanism and drug treatment.
Through the comparative study of cerebral ischemia and cerebral ischemia-reperfusion, it was found that even early reperfusion has the problem of secondary damage during reperfusion, but compared with the damage of nerve tissue caused by continuous cerebral ischemia, Reperfusion injury is relatively mild.
1. Reasonable selection of experimental animals and experimental models
Due to the advantages of low cost, good homozygosity within the germline, and similar vascular anatomical characteristics to humans, rats are still common experimental animals for cerebral ischemia research so far.
In addition, rats have extremely strong anti-infective ability and vitality. Routine operation generally does not cause secondary wound infection, and the animal survival time is relatively long. These characteristics also bring convenience to the study.
Note that when analyzing the differences in experimental data, it is necessary to consider not only the animal germline differences, but also the differences caused by different laboratory sources and operators in the same line.
Wistar rats and SD rats are the most commonly used rat species in my country. Each experimenter should choose the rat species according to the actual situation and existing experience.
1. Fischer-344 rat's middle cerebral artery (MCA) variation is small, and the volume of cerebral infarction formed after middle cerebral artery occlusion (MCAO) is consistent. The commonly reported model coefficient of variation is 20%
2. The variation of Wistar-Kyoto rats is relatively large. The coefficient of variation of the model is 33%
3. Sprague-Dawley rats live in between. The coefficient of variation of the model is 49%
4. Mongolian gerbils lack the posterior communicating artery connecting the carotid artery and the vertebral-basal artery system. It is ideal to use it to make a unilateral or bilateral carotid artery (CCA) ligation model of cerebral ischemia.
5. According to the purpose of the study, large animals such as rabbits, cats, dogs and primates were selected as the research objects.
6. It has recently been proposed by foreign countries that the anatomical characteristics of pigs' cerebrovascular vessels are similar to humans, and there are fewer collateral circulations, which is more suitable for basic research on cerebrovascular diseases.
2. Types of cerebral ischemia models
According to the scope of ischemia and the form of injury, cerebral ischemia models can be divided into many types.
According to the scope of ischemia, it can be divided into chronic global cerebral ischemia model and focal cerebral ischemia model; according to the degree of ischemia, it can be divided into complete cerebral ischemia and incomplete cerebral ischemia models;
According to the time of ischemia, it can be divided into transient ischemia and permanent cerebral ischemia models.
Transient cerebral ischemia can be further divided into one-time transient cerebral ischemia
Multiple transient ischemic model.
In addition, according to the situation of blood flow recovery, it is divided into ischemia and ischemia reperfusion models.
3. Instruments and equipment
The first category is for animal surgery services, including a set of surgical instruments, surgical microscopes, skull drills, brain stereotaxic instruments, temperature control devices (temperature controllers, infrared lamps and heating pads), ventilators;
is a kind of instrument used for monitoring and analysis, including EEG recording device, blood pressure recording device, Doppler blood flow recorder, blood gas and blood glucose analyzer.
Fourth, the animal model of vascular ligation
This model is often used to observe the hippocampus
(1) Animal model of double vessel clamping
Experimental animal: gerbil
The Mongolian gerbil lacks the posterior communicating artery connecting the carotid artery and the vertebral-basal artery system. It is ideal to use it to make a unilateral or bilateral carotid artery (CCA) ligation model of cerebral ischemia.
This animal model is generally used for the study of forebrain ischemia, which is the most common for hippocampal ischemia. The hippocampal CA1 area is the most sensitive to ischemia, and the hippocampal nerve cells are concentrated for easy observation.
Disadvantages: experimental animals are difficult to buy
(2) Creation of a four-vessel ligation cerebral ischemia model
The model used Wistar rats, SD rats, Wistar rats commonly used this method. Severe cerebral ischemia and constant pathological changes can be made in awake and free movement states.
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The model is produced in two stages
The first stage:
Anesthetize the animal, place it on the brain stereotaxic instrument, adjust the height of the ear stem and incisors to tilt the head of the mouse about 30 degrees, and use a rubber bolt to hold the mouse tail to give a slight pulling force to straighten the cervical spine and wings The hole is horizontal for easy observation.
A horizontal midline incision of the first cervical spine under the occipital bone, carefully separating the transverse processes of the first cervical spine on both sides, exposing the wing hole on the transverse process of the first cervical spine, and two vertebral arteries passing under the wing hole, using small unipolar or bipolar coagulation Insert the device into the wing hole and cauterize the vertebral artery.
The animal is then placed in the dorsal position, with a midline incision in the neck to separate the carotid sheath, taking care not to damage the vagus nerve and the cervical sympathetic trunk behind the carotid sheath. Enclose the common carotid arteries on both sides with surgical sutures (do not ligature), hide the thread under the skin, and simply close the incision.
second stage:
Wait for the animal to drink water freely after anesthesia, but after fasting for 12 to 24 hours, take the animal in the awake state, take the back position, and fix the limbs and incisors with a rope to control the animal's struggle. Carefully separate the incision in the front of the neck, identify the suture that is placed on the common carotid artery, gently pull up, tighten the sleeve or clamp the bilateral common carotid artery with microvascular clamps. At this time, within 2 to 3 seconds, the rat will be comatose and lose light reflection. Abnormal electroencephalogram can appear 2 to 3 minutes after ischemia and continue until the end of ischemia.
The closing time of the common carotid artery depends on the experimental design, usually 10 to 20 minutes. The loss of coma and reflex is a sign of ischemia success.
This method is complicated, traumatized, and has a high animal mortality rate.
The presence of spontaneous breathing and corneal reflex during ischemia suggests the presence of blood supply to the brainstem, which is due to the blood supply to the anterior spinal artery.
After the end of ischemia, the common carotid artery is released, and the animal is usually awake quickly. However, some animals will have ischemic convulsions within 24 to 72 hours. If the ischemia lasts for 30 minutes, 20% and 40% of the rats will have ischemic convulsions within 24 hours. Rats should be excluded.
Common causes of unsuccessful ischemia in rats: incomplete coma, death from respiratory failure, infection, etc.
The key to making this model is to burn the vertebral artery. Since the vertebral artery cannot be seen directly, this requires the operator to have certain experience, properly master the time of the burning and the angle of the electrode and the wing hole, and avoid damage to the brain stem.
V. Animal model of focal cerebral ischemia
Focal cerebral ischemia is limited to a certain part of the brain, and there is a pathological central area of infarction and an area around it that has not been completely necrotic. The latter is also known as the semi-dark area. This part of brain tissue can develop into infarct necrosis. It can be saved by timely reperfusion and intervention by various means. Compared with the acute necrosis of neurons in the focal ischemia, the blood supply to the collateral circulation in the ischemic penumbra is not completely interrupted, and the neurons have a relatively long rescue time window. Neuronal necrosis in the penumbra is a recent cerebral ischemia One of the hotspots in the study of injury mechanisms.
A good animal model of focal cerebral ischemia should be: the boundary between the central necrotic foci, penumbra and normal brain tissue is obvious, reliable, stable and reproducible, and the changes in the rat's own life state and neural tissue can be shown The various indicators are relatively comprehensive, which is convenient for studying the effects of various interventions on cerebral ischemia-reperfusion from multiple angles and levels.
There are many animal models of focal cerebral ischemia:
(1) Craniotomy:
Direct electrocoagulation or ligation of the middle cerebral artery via the subtemporal or transorbital approach simulates a permanent embolic human stroke.
Advantage: The surgical effect is intuitive and reliable, and it is not affected by vascular variation. At present, the MCAO model with the most balanced and reliable infarction and the best ischemic effect is also the most widely used classic model of focal cerebral ischemia in the world. It is suitable for neurological impairment after ischemia, drug and rehabilitation treatment efficacy evaluation, etc. Research.
Disadvantages: Animal surgery is complicated and traumatized, cerebrospinal fluid leakage, brain microenvironment changes, no reperfusion and infection are more likely, intracranial operation causes brain damage, intracranial pressure balance, and animal chewing function and other adverse factors.
Sympathetic nerve fibers attached to MCA can be damaged by ligation or electrocoagulation, impairing the self-regulating function of blood vessels.
Methods: Under animal anesthesia, incise the scalp, separate the temporalis muscle, take the midpoint of the connection between the external canthus and the external auditory canal, open the skull with a dental drill or circular skull drill, expose the middle cerebral artery, and identify the middle cerebral artery under the operating microscope For its branches, under the branches, which is near the middle cerebral artery (MCA), separate the ligated or cauterized MCA, and take the neck incision to separate the common carotid artery on the same side. When separating the ligation or cauterization of the MCA, it should avoid damaging the nearby brain tissue. Usually, a blunt-headed right-angled hook is used to fix the locator. Operate the locator knob, gently lift the MCA, and then cauterize or ligate.
The key to the operation of this model is twofold:
(1) Accurately locate the ligation site: it must be below the MCA branch of the striated artery. If it is ligated above it, there will rarely be cortical and basal ganglia infarcts. However, during the experimental operation, the branch of MCA is often not recognized. At this time, the olfactory groove can be used as a sign and the ligation can be performed below it.
(2) Permanent ligation of the common carotid artery on the same side can aggravate the degree of ischemia in the MCA blood supply area and is a necessary condition for the occurrence of a constant cerebral infarction, sometimes even with a temporary contralateral common carotid artery ligation.
(2) Non-cranial:
(1) A small blood clot made of microspheres or autologous blood injected into the internal carotid artery
The biggest disadvantage of this method is that the embolization site is not constant, the repeatability of animal experiment results is poor, and the reliability of the experiment results is not good.
Microembolism embolization cerebral ischemia model, because it can not predict the location and scope of ischemia, can not be communicated, therefore, limited to thrombolytic therapy and platelet microthrombosis after brain pathological changes and other special studies.
⑵Creation of cerebral infarction artery model by photochemical induction method
Model principle: The photochemical cerebral infarction model is a new animal model of focal cerebral ischemia. Its mechanism is that after certain light-sensitive dyes are injected into the body, a photochemical reaction occurs under the action of light waves of a specific wavelength to produce and release some active substances. Such as oxygen free radicals. These active substances cause damage to vascular endothelial cells, platelet adhesion, and then a release reaction, which stimulates the coagulation process in blood vessels, leading to thrombosis.
Most of the ischemic foci formed by this method are caused by microvascular endothelial damage, resulting in terminal artery occlusion, which is different from the pathological changes of human large blood vessel occlusion, and such models cannot be reperfused, and are not suitable for simulating human cerebral infarction or thrombolytic drug treatment Research on posterior recanalization. There is a big difference from the clinical pathogenesis of the ischemic area caused by embolization of large vessels.
Commonly used photosensitizing dyes: Nine Rose Red B (Tetraiodotetrachlorofluorescein disodium, rose bengal)
Operation process: After the animal is anesthetized, fix the head with a brain stereotaxic instrument, inject the photosensitizing dye through the tail vein, cut the skin after a few hours, use a halogen lamp or a xenon lamp as the light source, filter out the infrared and ultraviolet light parts, directly or optically Fibre conduction is projected into the brain. The light passes through the skull and illuminates the selected cortical area for 5 to 10 minutes, and the local irradiation intensity is 30 to 40 k/x (thousand lux). Extensive platelet aggregation in the blood vessels of the soft membrane and brain parenchyma, destruction of the blood-brain barrier and vasogenic edema, rupture of the vascular intima, irregular vascular caliber, and degeneration of deep cortical neurons can be seen within a few minutes of irradiation. ～24 hours is the most serious. The experimenter can control the size and depth of the infarct by adjusting the irradiation range, intensity, time and dose of photosensitizing dye. The edge of a typical infarct is neatly shaped like a bowl, penetrating the entire cortex and extending to subcortical structures.
Advantages:
1. The positioning is accurate and the surgical operation is relatively simple, avoiding the defects of craniotomy and manipulation of sympathetic nerve fibers in the blood vessel wall.
2. The photochemically induced cortical cerebral ischemia model also has adjustable cortical infarct size and depth.
3. The characteristics of infarcts in different parts can be made selectively, which provides conditions for the study of cortical function localization.
4. So far, in all models of focal cerebral ischemia, the animals with the photochemically induced cortical cerebral ischemia have the longest survival time.
Disadvantages:
1. Microvascular damage is prominent, early blood-brain barrier opening and vasogenic edema are obvious, and reperfusion is not possible.
2. During the process of photochemically induced thrombosis, the ischemic brain injury system after thrombosis and the vascular endothelial damage-coagulation and fibrinolysis system during thrombosis are activated at the same time. Therefore, the pathological damage of brain tissue is higher than that of other cerebral ischemic animal models More serious.
Scope of application:
This model is suitable for the study of antiplatelet drugs, antithrombotic drugs and endothelial cell protective agents.
(3) Local ischemia model of intravascular thrombus technique (insertion method)
In 1986, Japanese scholars first invented the thread embolization method to prepare a rat model of focal cerebral ischemia. Only in 1989 have there been reports in the literature. It was only used in laboratories in my country in 1996. This method does not craniotomy, causes little damage to animals, and does not require special equipment.
This method is to produce focal cerebral infarction without craniotomy, and can achieve blood flow recanalization by pulling out the thrombus.
Scholars at home and abroad have continuously improved it, and have developed and improved their understanding of the model principle, tether preparation, animal selection and operation process and experimental processing factors.
At present, this animal model is recognized as the standard model of experimental focal cerebral ischemia, and it is widely used at home and abroad.
But in practice, it was found that the preparation of the animal model is a relatively complicated system engineering, and there are still some problems that have not been satisfactorily resolved.
Ischemic site:
Striatum and the corresponding cerebral cortex
Model principle:
Rely on the front end of the insertion line to block the root of the anterior cerebral artery and block the blood flow through the anterior branch of the brain, and rely on the side wall of the insertion line to block the blood flow of the internal carotid artery and the posterior communication branch, so that the blood supply area of one side of the middle cerebral artery The blood flow is completely blocked.
The deep penetrating branch of the middle cerebral artery is an important blood supply artery in the basal ganglia area, and its morphological structure is close to that of human beings, and it is also a "prone stroke artery". Its cortical branches are distributed to the frontal area, apical area, and piriform area, and the temporal area. Its blood supply area surrounds 60-70% of the cortical area of each hemisphere, which is also similar to humans (about 40% of the cortical area).
The area of ischemic necrosis after middle artery occlusion includes: frontal dorsal dorsolateral cortex, caudate nucleus, and globus pallidus. Some test results show that it also affects the temporal area and the piriform area, which is more in line with the pathological range of human middle cerebral artery occlusion;
Experimental cerebral ischemia or infarction requires corresponding cerebral vascular occlusion.
The choice of rat cerebral vascular occlusion has undergone four blood vessels (bilateral common carotid artery + bilateral vertebral artery) occlusion, three blood vessels (bilateral common carotid artery + one vertebral artery) occlusion and bilateral common carotid artery occlusion, and finally developed Occlusion of the middle cerebral artery to the selective side.
The resulting infarction range from diffuse global cerebral ischemia, forebrain ischemia, and further narrowed to focal ischemia in the blood supply area of the middle cerebral artery.
In humans, the middle cerebral artery blood supply area is a high incidence area of stroke.
Improvement of surgical approach for middle cerebral artery occlusion has undergone the development of transorbital approach, transtemporal craniotomy to transcervical incision thread embolization. Realize the change from craniotomy to non-craniotomy. The way of blood flow blocking is a single permanent occlusion, which further achieves ischemia-reperfusion.
Middle cerebral artery occlusion methods include craniotomy mechanical occlusion method, thread embolism method, microembolic embolization method, chemical stimulation leading to thrombosis occlusion method, photochemically induced thrombosis method and endothelin perfusion induced vasoconstriction method.
It is generally believed that an ideal animal model of cerebral ischemia should have the following conditions:
Can repeatedly occlude a single cerebral blood vessel;
Can cause changes in blood flow or infarction in the corresponding blood supply area;
Can achieve reperfusion of ischemic infarction area.
The focal rat model prepared by the thread plug method can meet the above requirements.
Internal carotid artery thrombosis method for the preparation of focal ischemia rat model has almost become the first choice for experimental cerebral ischemia animal model.
The principle of thread embolism for ischemic infarction:
The diameter of the internal carotid artery gradually narrows during running, but the diameter of the tether wire does not change. During the insertion process, the tether wire and the internal carotid artery tend to be tight, so it can gradually reduce and occlude the internal carotid artery blood flow. The tip of the tether is inserted into the beginning of the anterior cerebral artery and stopped here because its diameter is larger than the inner diameter of the anterior cerebral artery.
The blood flow near the anterior cerebral artery is blocked, and it is impossible to return to the middle cerebral artery.
The far segment of the anterior cerebral artery can still obtain the compensated blood flow to the opposite side through the anterior communicating artery without ischemic changes.
The distal cerebral arteries can compensate for blood flow through the basilar artery system from the posterior communicating artery without obvious ischemic changes.
Therefore, the ischemic area prepared by the thread embolization method mainly occurs in the blood supply area of the middle cerebral artery.
And the anterior cerebral artery and posterior cerebral artery blood supply area can basically guarantee a more normal blood supply.
Nylon thread selection:
measured by intravascular resin perfusion technique and found that the internal diameter of the internal carotid artery neck and the proximal end of the anterior cerebral artery were 0.3 and 0.2 mm, respectively.
If the insertion wire is too thick or too thin, it will cause the operation to fail or the result to be unstable. Therefore, the selection of patch cords is very important.
The wire insertion should have a certain hardness and toughness. Use tweezers to clamp the front end of the wire 4mm and pierce the skin of the back of your hand, which can cause a feeling but no pain. The line is straight and not bent. The length of the front end is more than 6mm. A certain bending is the selection criterion.
Processing of plug-in front end:
1. Before use, the front end of the nylon thread is formed into a small spherical bulge by heating, which is convenient for advancement.
2. Dip the nylon thread in the silicone rubber mixed with hardener to increase the hardness of the nylon thread.
3. Immerse the patch cord in nitric acid to soften it.
4. Nail polish method: Cut the cylindrical nylon fishing line into several sections of 30mm, stick one end of it with a small amount of alcohol to dilute the appropriate concentration of nail polish, put it upside down, let it dry naturally, and make black marks at 18mm and 20mm away from the treatment end. .
In order to prevent blood clotting during propulsion, it is necessary to treat the nylon thread with heparin.
The front end of the insertion line is very critical. For example, if the insertion end is not smooth, it is easy to damage the vascular endothelium during the insertion, and thrombosis is formed in the blood vessel.
Steps:
1. A midline incision in the neck exposes, frees, and ligates one common carotid artery.
2. Separate the branched occipital artery, superior thyroid artery, and ascending pharyngeal artery of the external carotid artery.
3. Then separate the internal carotid artery, taking care not to damage the adjacent vagus nerve and hypoglossal nerve.
4. The main part of the free internal carotid artery (ICA) was trunked, threaded and tied up for use, and the ICA was clamped with atraumatic microvascular clips.
5. Separate the occipital artery backward and forward along the ICA. At the beginning, the occipital artery was burned with an electric cautery. Carefully separate the internal carotid artery from the only branch of the cervical pterygopalatine artery and suspend it for use.
6. Cut a small mouth of CCA near the bifurcation of ECA and ICA, insert the thread plug into the pre-soaked heparin saline with a concentration of 2.4×106U/, tighten the pre-prepared live thread knot, and loosen the microvascular clamp , Slowly into the skull through ICA, in order to reduce the damage to the vascular endothelium, make the insertion more accurate and convenient, moderately adjust the angle of the wing jaw process arterial suspension, until the insertion line has a slight sense of resistance, indicating that the embolus has crossed the middle cerebral artery Reaching the beginning of the anterior cerebral artery, completely blocking the blood flow of the middle cerebral artery, resulting in the focal cerebral ischemia state of the blood supply range of the middle cerebral artery.
Marked by the bifurcation of ECA and ICA, the general insertion depth is (18±0.5) mm. Since the length from the bifurcation to the beginning of the middle cerebral artery is 14mm, the front end of the nylon thread enters the anterior cerebral artery, thereby blocking the blood supply to the middle cerebral artery. The anterior cerebral artery can obtain blood flow through the anterior communicating artery.
The operation of simple middle cerebral artery ischemia is to cut off the part of the embolus that is left outside the blood vessel, and suture the incision layer by layer. Animals were killed at different times during ischemia to obtain materials.
The operation of cerebral ischemia reperfusion is to leave the end of the tied knot and the other end of the embolus outside the skin, and suture the incision layer by layer. The ischemia continues to pull out the emboli slowly at different times, tighten the live knot, and the blood supply area of the middle cerebral artery can get the blood supply of the anterior communication artery and the posterior communication artery, forming the reperfusion state of the blood supply area of the middle cerebral artery. Rats were sacrificed at different times to obtain materials.
Whether the pterygopalatine artery is ligated can affect or affect reperfusion.
Because of the ligation of the pterygopalatine artery, thrombus is likely to form around the internal carotid artery, resulting in lumen occlusion and affecting the supply of compensated blood flow during the reperfusion period.
Therefore, the rat model of ischemia-reperfusion should not be permanently ligated.
Therefore, the improvement of the operation of the thread embolism method must be carried out on the basis of mastering the cerebral vascular anatomy and modeling principles of rats. Not only should we consider the practicality, but also whether we can completely block the blood flow into the middle cerebral artery and achieve reperfusion after ischemia.
Ischemic degeneration occurs in the core blood supply area of the middle cerebral artery, that is, outside the parietal cortex and the caudate putamen. If the right common carotid artery is occluded at the same time, as a result, ischemic degeneration also occurs in the anterior cerebral artery on the side of the thrombus and in the frontal lobe of the collateral supply area of the deep perforating branch and the medial part of the caudate nucleus.
The reason is that after the contralateral common carotid artery occlusion, the above-mentioned anterior cerebral artery-middle cerebral artery collateral anastomosis provides reduced blood flow. After 1.5 hours of ischemia, the anterior cerebral artery collateral blood supply area also has ischemic changes.
It can be seen that compared with humans, rats as rodents have less degenerative anastomosis of the anterior cerebral artery, middle artery and distal branch of the posterior artery in the dorsal side of the brain. Strong effect, can provide effective protection against cerebral ischemia.
This can explain why the neurological deficits of rats with thread embolism usually recover around 24h? Why can't young rats simulate obvious and constant ischemic foci in focal cerebral ischemia modeling?
Therefore, the preparation of a cerebral ischemia model with high replication rate, obvious and limited lesions, and constant lesions with small variation needs to safely and effectively reduce the collateral circulation of rat cerebral blood vessels.
Note:
1. Whether the pterygopalatine artery (PPA) is ligated:
In the history of the development of online thrombosis, there is a fierce debate on whether the pterygopalatine artery is ligated, forming a ligated and non-ligated school.
Early advocates of thread embolism believed that the branch of the pterygopalatine artery through the skull base provided collateral blood flow to the basal ganglia. Therefore, to achieve infarction in the frontal parietal cortex and basal ganglia in rats, the artery must be occluded. Permanent pterygopalatine artery ligation can increase the success rate of the non-ligation model by 26% to 40%.
Later scholars also temporarily or permanently occluded the artery during model making. But its purpose is no longer to occlude collateral blood flow, but to improve the success rate of modeling.
Our practical experience is to temporarily clamp the pterygopalatine artery with micro-arterial clips during the operation. The purpose is to prevent the embolism thread from entering the ptalatine artery and not entering the internal carotid artery.
Or wear a suture at the beginning of the pterygopalatine artery to play the role of pulling. When the tether is approaching the frontal artery, pull the tether to temporarily occlude it to ensure that the tether enters the internal carotid artery.
Because the branch of the pterygopalatine artery is diverged from the internal carotid artery to the front and the outside, and the pterygopalatine artery is thicker, the angle of intersection with the internal carotid artery is small, and the plug line easily enters the ptalatine artery and cannot enter the cranial, resulting in the failure of modeling.
With the deepening of thread embolization, more scholars believe that it is not necessary to occlude the pterygopalatine artery. Because the position of the pterygopalatine artery is deep, if the artery is ligated, neuromuscular damage is severe, causing difficulty in eating, causing great damage to rats and increasing mortality.
Especially in ischemia-reperfusion studies, the artery should not be ligated. It can be seen that the attitude to whether the pterygopalatine artery is occluded has gone through three stages of permanent occlusion, temporary occlusion and non-occlusion. Its purpose has also changed the pursuit of simple collateral blood flow occlusion. Considering the feasibility of surgery, reperfusion studies and Comprehensive factors such as animal trauma.
At present, although there are few anatomical data that can confirm the collateral circulation effect of the pterygopalatine artery, we have concluded from practical experience that it is not necessary to forcibly permanently occlude the pterygopalatine artery, only temporary occlusion, which can take into account cerebral ischemic rats Modeling and reperfusion studies also significantly reduce the difficulty of surgery, which is generally adaptable to units that lack experience and conditions in microsurgery.
2. The key to the success of the operation: the diameter of the insertion wire should match the size of the rat's body weight.
As the body weight of rats increases, the inner diameter of blood vessels will increase accordingly. For rats of different ages, different diameters of plugs should be selected to adapt to them, so as to ensure the maximum blocking of blood flow.
Our recommended optimization plan is:
The body weight of the thread plug is fixed at 250-300g, which matches the size 3 cylindrical nylon fishing line made in Korea with a diameter of 0.2864mm. The insertion depth of the thread plug is 17mm or 18mm.
Weight> 350g, thickened intracranial blood vessels, it is difficult to completely block MCA blood flow.
Weight <240g, intracranial blood vessels are too thin, it is difficult to insert intracranial blood vessels.
3. Effects of rat age
Human stroke occurs mostly in the middle-aged and elderly population with multiple stroke risk factors, and stroke models choose young and healthy animals, which is not related to human stroke factors.
This can also partly explain why animal models have proven effective drugs but have no obvious effect in human clinical trials.
Therefore, old animals should be selected as stroke animal models.
However, there are often many factors that cause this problem to be ignored in the practice of the online tether method.
If we use the thread embolism method, the body weight of rats is 250～280g.
And there is a comparative relationship between the weight and age of rats:
Such as rats weighing 250-290g, the age of rats is 200-320d. Therefore, most of the commonly used thread embolism rats are not old rats.
Many practices have also proved that the use of heavier or older rats reduces the adaptability of thread embolism.
For example, a rat with a body weight of 350-450g replicates the focal cerebral ischemia model by thread embolization. The nylon thread has obvious resistance during insertion, and the embolization thread is kinked and it is difficult to reach the predetermined position.
Rats weighing more than 350g often suffer from thickened intracranial blood vessels, which makes it difficult to completely block MCA blood flow and affect the modeling effect. This also suggests that old rats are not suitable for thread embolization. It can be seen that the practice of thread embolization in old rats still needs more practical tests.
Model success criteria:
After the animal is awake, ipsilateral Horner's sign and contralateral forelimb-based hemiplegia symptoms appear, with ipsilateral eyeball depression, increased secretions, closed eyelids, head tilted to the contralateral side, contralateral forelimbs cannot fully extend, and hemiplegic side when crawling Rotate or dump, bend the body to the opposite side when lifting the tail, and the forelimb on the opposite side hangs weakly.
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Questions about Horner's sign:
Horner sign is a symptom of sympathetic nerve fiber damage. Horner sign occurs in almost all rats with thread embolism. For a long time, it is considered to be one of the neurological deficits caused by focal cerebral ischemia.
However, the Horner sign is not a sign of the success of the MCAO model, it can only indicate ischemic injury of the internal carotid artery.
Since the sympathetic trunk of the neck of the rat is accompanied by the internal carotid artery and the common carotid artery, sympathetic nerve fibers are distributed on the internal carotid artery and enter the skull. The sympathetic nerve is susceptible to injury during neck surgery and thread embolization, so Horner's sign may also appear in the sham-operated group.
Craniotomy method for the selective cerebral occlusion of the middle cerebral artery prepared by the focal cerebral ischemia model showed very few Horner signs, which also shows that it is not a reliable sign of nerve defects caused by middle artery occlusion.
Advantages:
(1) No craniotomy
(2) Sympathetic nerve fibers that do not damage blood vessels
(3) Positive effect
(4) can accurately control the ischemic time
(5) Reperfusion and precise control of reperfusion time
Therefore, it is ideal to use it to study the sensitivity, tolerance, reperfusion injury and treatment time window of neurons to ischemia.
At the same time, the model has little effect on the physiological function of the animal's whole body, and the animal has a long survival time. It is also suitable for the study of chronic brain injury.
TTC staining:
Cerebral ischemic infarcts show pale, non-infarcts show red
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experience:
Not ligating the branches of the internal carotid artery in the neck and ligating the pterygopalatine artery does not affect the embolization effect of the middle cerebral artery. It can also simplify the surgical operation and reduce the thrombosis of the internal carotid artery, which is more conducive to reperfusion research.
This model is actually a mechanical artificial embolism stroke, which is different from the common stroke course in the population. Moreover, its surgical operation process is relatively complicated. Many details such as animal strain and weight, embolic thickness and improper insertion depth treatment will obviously affect the experimental results.
VI. Cerebral ischemia model of spontaneously hypertensive rats
Use spontaneously hypertensive rats (SHR), aged rats to make cerebral ischemia models or directly select stroke-type spontaneously hypertensive rats (SHR-strokeprone strain, SHR-SP) for comparison of cerebral ischemia. In accordance with the incidence of human cerebrovascular disease, this cerebral ischemia model is especially suitable for the study of molecular mechanism, genetic characteristics and risk factors of cerebrovascular disease.
But the cost of such experimental animals is currently higher.
1. Spontaneously Hypertensive Rat (SHR)
SHR rats are hereditary hypertensive rats, there are many varieties.
The most commonly used Japanese SHR is Okamoto's selection of Wistar Kyoto rats with higher blood pressure and breeding through genetically directed breeding. The rat's spontaneous blood pressure begins to rise several weeks after birth, and the peak value can reach 26.7kPa (220mmHg) . Although SHRs are not prone to spontaneous stroke, they are much more sensitive to cerebrovascular occlusion than normal rats. Bilateral CCA ligation can cause a significant drop in cerebral blood flow, and the middle cerebral artery ligated by SHR produces larger cortical infarcts than normal blood pressure rats.
2. Stroke-prone hypertensive rats (SHRSP) developed on the basis of SHR
SHRSP rats have higher blood pressure, the peak value can reach 32-33kPa, and the incidence of spontaneous cerebral hemorrhage or cerebral infarction can reach 60-80%.
The location of the stroke is mainly in the anterior medial cortex, occipital cortex and basal ganglia.
The microvascular pathological features of SHR and SHRSP include multifocal blood-brain barrier opening, plasma protein leakage, endothelial injury, vascular smooth muscle fiber-like decay, thickening of the basement membrane, and fibroblast proliferation. Although ligating the middle cerebral artery of SHR and SHRSP mice can produce a larger and more constant infarction, they only represent a small genetic group, which is very different from the nature of patients with essential hypertension. Limited and difficult to raise, it cannot be carried out in large quantities in domestic research.
Seven. Measurement index of cerebral ischemia model
Behavior
Histopathology
Biochemistry
Physiology
molecular biology
Specifically how to choose the evaluation index should be decided according to the experimenter's experiment design and the hypothesis that the experiment designer wants to experiment.
General indicators include mortality, neurological loss, behavioral damage, histopathological damage, etc.
1. Mortality is a relatively simple indicator, which has a certain role in the study of drug efficacy, but the information provided by the research institute of cerebral ischemia mechanism is very limited;
2. Neurology missing score method: This is to simulate the clinical symptoms of human stroke after scoring, but in actual application, the index interval is too large and the objectivity is not enough, the application is limited.
For example, whether the motor damage occurs after the middle cerebral artery ligation is more determined by whether the ischemia involves the internal capsule and has little to do with the size of the infarct.
6h after the operation, the neurological function scored 0 points and 4 points were unqualified and excluded.
Zea Longa Neurology 5-point scoring standard:
No signs of nerve damage: 0 points
Contralateral forelimbs and front paws cannot be fully extended, showing mild focal neuropathy loss: 1 point
Walking and rotating to the side of hemiplegia while walking, for moderate focal nerve damage: 2 points
Tumbled to the side of hemiplegia while walking, showing severe focal nerve damage: 3 points
can't go on its own, and it is accompanied by varying degrees of consciousness disorder: 4 points.
3. Behavioral indicators
Complex behavioral indicators play a role in evaluating ischemic injury.
Such as the jumping platform test to escape reflection, Y-shaped maze and Morris water maze, etc., but such indicators require high, and it requires a special training for researchers and experimental animals, which takes a long time.
4. Molecular biochemistry, histopathology
Many researchers choose more objective measurement indicators, such as brain glucose metabolism and histopathological observation.
It should be noted that the brain interval of such indicators is quite different, and multi-parameter analysis is required.
The gold standard for evaluating ischemic damage among the above indexes is histopathological observation.
For example, after the middle cerebral artery is ligated, the size of the cerebral infarct focus and the size of the penumbra are used to evaluate the degree of ischemic damage.
Total cerebral ischemia model rat hippocampal CA1 area pyramidal cell loss or the number of neuronal death in the striatum to evaluate the degree of transient ischemic damage in the whole brain and the therapeutic effect of certain experimental interventions.
VIII. Related factors affecting cerebral ischemic damage
Because there are many factors that can affect the process and consequences of ischemic brain injury, these factors should be considered and controlled as much as possible when making animal models of cerebral ischemia to ensure the success and results of animal models of cerebral ischemia Of constant. The main factors that affect cerebral ischemic damage are: the type of animal, blood pressure, blood sugar level, anesthesia conditions, body temperature and brain temperature of ischemic animals.
1. Influence of degree and time of cerebral ischemia on brain injury
The degree and duration of cerebral blood flow reduction are the two most important related variables that determine ischemic damage.
Generally, cerebral ischemia that causes hypoxic depolarization on the electroencephalogram (EEG) can last for a few minutes to cause a certain degree of brain damage in all animal species.
The threshold of CBF (ml/100g tissue/min) that causes EEG hypoxic depolarization potential is different in different animals, usually 35-40% of CBF in rats is lower than the normal value, and 25-25 in large animals %, can cause hypoxia depolarization for a few minutes.
The brain damage generally caused by subthreshold ischemia is reversible, but this type of ischemia lasts too long, and it can also cause selective neuronal necrosis and even cerebral infarction.
Therefore, the degree and duration of decrease in cerebral blood flow determines the development process and results of brain damage during ischemia.
In order to control the variation between animals and improve the reproducibility of ischemic damage, CBF and EEG of ischemic animals will be monitored, EEG can be used electrophysiological recorder, CBF can be used laser Doppler flowmeter or hydrogen clearance rate Method determination.
Table 1 Effect of ischemia degree and time on brain damage
Ischemia time (min) Ischemia degree (% control CBF)
35～45%＜35～40%
0～30 reversible dysfunction
Selective neuronal necrosis
～60 selective neuronal necrosis
Infarction
＞60 infarction
Infarction
2. Effects of body temperature and brain temperature on ischemic brain injury
The level of animal body temperature or brain temperature during ischemia and ischemia-reperfusion is another important factor that affects the degree of ischemic brain damage.
Experiment proves that lowering the brain temperature by 3～4℃ can obviously reduce ischemic brain damage.
On the contrary, artificially increasing the temperature of the brain will aggravate ischemic brain damage.
When making a cerebral ischemia model, the animal's body temperature/brain temperature will be affected by many factors and change.
For example, anesthesia can reduce the metabolic rate and body temperature;
Cerebral ischemia itself can reduce the metabolism of brain tissue;
Cut the skin and open the skull during the operation to accelerate the heat dissipation of the brain, etc., which can lower the temperature of the brain and affect the degree of ischemic injury.
Therefore, when making a cerebral ischemia model, the brain temperature should be monitored and managed to maintain a normal level. Accurate brain temperature monitoring is based on the placement of temperature-sensitive electrodes under the dura mater. Many laboratories commonly use the method of monitoring rectal temperature with heating pads and infrared light bulbs to maintain the animal's body temperature at about 37.5°C. However, it should be noted that during a long period of ischemia (> 10 minutes), the rectal temperature does not directly reflect the brain temperature, and it is usually about 2 to 3 degrees lower.
3. Effect of anesthesia on ischemic brain damage
Including preparation and treatment before anesthesia, choice of anesthesia method, administration before anesthesia, judgment of anesthesia depth, first aid for anesthesia accidents, etc.
The effect of anesthetics on brain damage in ischemic model is achieved by affecting cell activity, metabolic rate, body temperature and blood flow. However, the effects of various anesthetics on the above indicators are different. For example, halothane and barbiturate mainly inhibit the activity of the central nervous system, while Ketamin and Freon will sometimes have a central "excitement" effect. Barbiturate has a vasoconstrictive effect, reducing the basic blood flow of the brain. At low doses, halothane can dilate blood vessels and increase cerebral blood flow. Because it is inevitable to use anesthetics when making animal models, experimenters should be familiar with the effects of various anesthetics on various physiological and biochemical activities, choose anesthetics according to their experimental needs, and minimize and control the anesthesia to the experimental results. Negative effect.
4. Influence of blood factors on cerebral ischemic damage
Blood factors include blood pressure, blood sugar, blood viscosity, blood CO2 and O2 tension. Hyperglycemia aggravates cerebral ischemic damage has been reported by many laboratories, the mechanism is still unclear, and it may be related to the increase of anaerobic glycolysis and lactate accumulation in the ischemic phase. Therefore, animals are usually fasted before undergoing ischemic surgery, and their blood glucose levels are monitored during the ischemic period and after preparation. The influence of blood CO2 and O2 partial pressure on the consequences of ischemia is direct. In addition, arterial perfusion pressure plays a role in the consequences of cerebral ischemia through its influence on CBF. Usually cerebral perfusion pressure maintains CBF relatively constant in a wider range, but when arterial pressure drops to a certain level (human 50～60mmHg, Rats 60～70mmHg), the ability to automatically regulate cerebral blood flow will gradually be lost. Therefore, monitoring blood CO2, O2 partial pressure and arterial blood pressure are necessary operating procedures for making animal models of cerebral ischemia.
The preparation of experimental cerebral ischemic animal model is to provide a powerful research object for ischemic cerebrovascular disease, so that we can increase our understanding of ischemia from different aspects. In recent years, the development trend of the research and application of cerebral ischemia models is the miniaturization of animals (mainly rats or gerbils), emphasizing the controllability and repeatability of ischemic injury, and at the same time strictly controlling various interference factors and trying to Avoid the influence of these interference factors on the experimental results.
Second, strictly control the experimental conditions and interference factors
The results of a large number of animal experiments have confirmed that the changes in experimental conditions and surrounding environmental factors have a significant impact on the experimental results, such as the choice of anesthetics, ambient temperature, animal blood glucose, blood gas and mean arterial pressure control. How to strictly control the experimental conditions during the animal experimental research? What experimental conditions must be controlled? Do animals need to use a ventilator during the study of cerebral ischemia?
1. Reasonable selection of anesthetics: Animal models of cerebral ischemia are all based on animal anesthesia, which is different from the occurrence of human cerebrovascular disease. Therefore, the effects of anesthetics on the experimental results must be strictly excluded. At present, the domestic use of chloral hydrate, phenobarbital sodium, pentobarbital sodium, or uratan under general animal anesthesia.
And a large number of studies have shown that general anesthetics can directly or indirectly affect cerebral blood flow, intracranial pressure, brain metabolism and neurotransmitter transmission.
Take the rat thread embolization MCAO model as an example:
Conventional anesthesia of phenobarbital sodium and urethane can significantly reduce brain temperature;
Chloraldehyde hydrate and phenobarbital sodium can reduce the respiratory rate of rats by 50%, resulting in obvious acidosis and carbon dioxide retention in animals, of which pentobarbital sodium has the smallest effect on the physiological indexes of rats.
Artificially assisted ventilation can significantly correct the effects of anesthetics on the physiological indexes of rats.
At present, the general anesthesia protocol for the study of cerebral ischemia in the world is to use muscle relaxants and halothane inhalation anesthesia under artificial assisted ventilation, and continuous monitoring of electrocardiogram, blood pressure and blood gas during the operation. The whole process is close to the operation of human anesthesia.
In the process of cerebral ischemic animal experiments in China, air is used as the source of air, and animals are given artificial assisted ventilation. During the operation, the respiratory frequency and tidal volume are well controlled. Anesthesia with pentobarbital sodium can also achieve a more ideal effect.
In laboratories lacking the above conditions, I recommend using 2% pentobarbital sodium and 45 mg/kg body weight for intraperitoneal anesthesia. Rats with severe dyspnea or obvious apnea in the experiment should be eliminated.
2. Control the experimental environment temperature, animal blood sugar, blood oxygen and blood gas levels:
Among the interference factors of the experiment, the environmental temperature, animal blood sugar and blood gas level have the most obvious influence on brain function.
In the process of making animal models of cerebral ischemia, the influence of brain temperature on experimental animals comes from two aspects. First, the conventionally used anesthetics can significantly reduce the temperature of the brain parenchyma, thereby producing a mild hypothermia brain protection; on the other hand, such as the surrounding Excessively high temperature in the environment or the local area of the operation causes the temperature of the brain parenchyma to rise, causing obvious damage to brain cells.
Therefore, in the process of studying the pathological mechanism of cerebral ischemia, especially the protective measures of the brain, attention should be paid to the influence of environmental temperature on the experimental results, and anesthetics with less influence on brain temperature should be selected as much as possible, such as pentobarbital sodium and chloral hydrate, Keep the ambient temperature around 25-30℃.
Note: The anal temperature of the experimental rat can not accurately reflect the brain parenchymal temperature, and the deep brain parenchymal temperature should be directly monitored if possible.
Focal or global cerebral ischemia, animal blood sugar increased slightly, and high blood sugar itself can significantly increase the area of cerebral infarction. In order to control the blood sugar level of the animals, fast for 12-24 hours before the experiment.
Hypoxemia and acidosis have a greater impact on brain tissue metabolism, especially the vasomotor function of cerebral blood vessels. The purpose of the research is to understand the damage mechanism of ischemia and hypoxia on neurons. Therefore, cerebral ischemia The basic research has stricter requirements on blood gas, blood oxygen and blood acidity of laboratory animals.
Artificial assisted ventilation is the best way to ensure that the above indicators are within the normal physiological range.
In the process of making a cerebral ischemia model by craniotomy or online embolic method, artificial assisted ventilation should be considered.
Photochemically induced cortical cerebral ischemia model or simple carotid artery ligation reperfusion model, because the manufacturing process itself has