KR100834564B1 - Method for predicting neuronal death in a mouse model of transient global forebrain ischemia - Google Patents

Abstract

A method for predicting neuronal death in a mouse model suffering from transient global forebrain ischemia is provided to measure the neuronal death more accurately by decreasing severity mutation of brain damage between individuals. A method for predicting neuronal death in a mouse model suffering from transient global forebrain ischemia comprises a step of measuring the change of regional cerebral blood flow from the mouse model and a diametric ratio of posterior communicating artery regarding basilar artery, wherein the change of regional cerebral blood flow is calculated as a percentage by dividing a blood flow value of one minute after the forebrain ischemia damage into a reference value(an average value of a blood flow of 5 minutes before the ischemia) and the regional cerebral blood flow is measured by using a laser doppler flowmeter.

Description

Method for predicting neuronal death in a mouse model of transient global forebrain ischemia}

The present invention relates to a method for measuring neuronal cell death in a transient whole cerebral ischemic mouse model. More specifically, the present invention is characterized by measuring the change in regional cerebral blood flow and the ratio of the diameter of the posterior communicating artery to the cerebral artery in the transient whole cerebral ischemic mouse model. The present invention relates to a method for measuring neuronal cell death in a cerebral ischemic mouse model.

Ischemic brain disease is a disease caused by selective delayed neuronal death in the hippocampus CA1 region, which is known to be particularly vulnerable to ischemia due to reduced blood flow in the brain and lack of oxygen and glucose supply (Kirino). , Brain Res , 239: 57-69, 1982; Petito et al., Neurology , 37: 1281-1286, 1987; Pulsinelli et al., Ann. Neurol ., 11: 491-498, 1982). In addition, the ischemic brain disease is known to be caused by the lack of blood supply to the brain in pathological conditions such as stroke or heart attack (Nedergaard, Acta. Neurol. Scand ., 77: 81-101, 1988; White et. al., Neurology , 43: 1656-1665, 1993). The cause of delayed neuronal death in the hippocampal CA1 region caused by transient cerebral ischemia and reperfusion is caused by the influx of calcium into cells (Hara et al., Brain Res. Bull ., 29: 659-665, 1992; Nedergaard , Acta. Neurol. Scand ., 77: 81-101, 1988), known as free radical related neuronal damage and glutamate-receptor mediated neuronal damage (Won et al., Neurosci. Lett ., 301: 139-142, 2001). That is, when cerebral ischemia occurs, depolarization of neurons occurs, and glutamate of synapses is released, thereby increasing the concentration of extracellular glutamate. In addition, the reversal of active transport resulting from a decrease in ATP accelerates the accumulation of extracellular glutamate. Accumulation of the extracellular glutamate may include N-methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole -propionic acid (AMPA), and metabotropic glutamate receptor. ), Resulting in an over-flow of calcium into cells (Olney et al., Science , 244: 1360-1362, 1989). Intracellular influx of calcium triggers the activity of calcium-dependent proteases, lipases and modulators, and eventually results in the generation of cytotoxic molecules including free radicals, which destroy cell structures such as DNA and membranes, resulting in neuronal death. Known.

Research into the pathogenesis and treatment of such ischemic brain disease has been mainly conducted using animal models such as rodents. In particular, the transient whole cerebral ischemic injury model has contributed to the study of pathophysiology of stroke. The rat model is characterized by delayed neuronal cell death in the hippocampal CA1 region (Pulsinelli & Brierley, Stroke , 10: 267-272, 1979), which is the glutamate excitatory toxicity described above ( It is known to be caused by various mechanisms related to glutamate excitotoxicity, oxidative stress, inflammation, and apoptosis (Lipton, Physiol Rev 79: 1431-1568, 1999; Lo et al., Nat Rev Neurosci , 4: 399-415, 2003; Chan Neurochem Res , 29: 1943-1949, 2004). Recently, bilateral common carotid artery occulsion (BCCAO) using C57BL / 6 mice has been attracting attention as a model of transient whole cerebral ischemia injury.

Unlike rats, the C57BL / 6 strains do not develop well in both posterior communicating artery (PcomA) and thus do not connect the frontal and posterior vascular distributions of the brain (gerbil) (Mayevsky & Breuer). , Brain Res , 598: 242-250, 1992) Temporal ligation of both carotid arteries allows easy and simple induction of whole cerebral ischemia. On the other hand, in rats, the blood vessels of the brain are connected through the posterior artery, so ligation of both cervical arteries and hypotension must be induced together to cause delayed neuronal death in the area vulnerable to ischemia. In addition, the mouse model of the C57BL / 6 strain is genetically engineered, and thus has an advantage over other animal models in studying the mechanism of ischemic injury.

However, despite these advantages, the ischemic brain disease model using the mouse model of the C57BL / 6 strain has a disadvantage in that it is difficult to obtain accurate experimental results because the variation in the severity of brain damage between individuals is relatively large.

Therefore, the present inventors measured the cerebral ischemic blood flow and the diameter of the posterior artery of the cerebral artery in the transient whole cerebral ischemic mouse model while studying a method for reducing the variation of brain injury severity in the transient whole cerebral ischemic mouse model. By predicting neuronal cell death and thereby classifying experimental animals, the present invention was completed by confirming that variation in brain injury severity could be reduced.

Accordingly, an object of the present invention is to reduce the variation in the severity of brain damage between individuals in a transient whole cerebral ischemic mouse model, and to provide a more accurate method for measuring neuronal cell death.

In order to achieve the object of the present invention as described above, the present invention is to change the regional cerebral blood flow and the ratio of the diameter of the posterior communicating artery to the basal artery in the transient whole cerebral ischemic mouse model. It provides a method for measuring neuronal cell death in a transient whole cerebral ischemic mouse model characterized in that the measurement.

In addition, the present invention, after predicting the degree of neuronal cell death of the transient whole cerebral ischemic mouse model using the above method, characterized in that to classify and select the animal model according to the degree of neuronal cell death, between individual individuals of the transient whole brain ischemic mouse model It provides a method of reducing the degree of variation in the severity of brain injury.

Hereinafter, the present invention will be described in detail.

As used herein, the term "ischemic" refers to a condition in which blood supply to tissue is interrupted or reduced due to thrombus, embolism, cerebral pressure rise, blood pressure drop, or the like, thereby causing cell damage. Even after reperfusion of blood after ischemia, nerve cells are damaged and cause various sequelae.

As used herein, the term "ischemic brain disease" refers to a disease caused by an ischemic state of brain tissue. An example of such a disease is stroke. The stroke is the second most common cause of death in Korea and is classified into ischemic and hemorrhagic diseases. Ischemic strokes can be subdivided into thrombosis, embolism, transient ischemic attack, and infarction, such as by brain computer photography. Pathological abnormalities occur in the blood vessels supplying. As a result, the supply of cellular energy and oxygen to the brain tissue is insufficient, causing abnormalities in neuronal function and necrosis of nerve cells. The most common cause is that blood clots from other lesions move through the bloodstream to form blood clots (embolism) and atherosclerosis destroy vascular endothelial cells and secondary platelets or blood clotting systems activate blood clots. May occur. Such cerebral ischemia triggers a series of pathophysiological and biochemical reactions, and a suitable and prudent treatment for reversing progression to ischemic brain tissue damage is undoubtedly hemovascular reperfusion. However, hemorrhagic reperfusion following ischemia itself is known to involve an element of tissue damage, ie, production of free radicals (ROS).

As used herein, the term "transient whole cerebral ischemia" refers to a temporary decrease in blood flow to the forebrain, which causes nerve cell damage in the forebrain.

As used herein, the term "nerve cell death" includes delayed neuronal death, which is a type of ischemic neuronal death. The delayed neuronal cell death does not cause cell damage immediately after ischemia, but refers to cell damage occurring after a certain period of time. The pattern of cell damage is similar to apoptosis (Du C, Hu R, J cerebr). Blood Flow Metab . 16: 195-201, 1996; Kirino T., Brain Res . 239: 57-69, 1982).

The present invention is a method for overcoming the drawbacks of the severity of neuronal damage between individuals of the animal model in experiments with a transient whole cerebral ischemic mouse model (regional cerebral blood flow) in the transient whole cerebral ischemic mouse model The method of predicting neuronal cell death in the transient whole cerebral ischemic mouse model characterized by measuring the change in the diameter of the posterior communicating artery to the cerebral artery and the animal model according to the severity of nerve cell damage. It provides a method for reducing the degree of variation in the severity of brain damage between individuals in a transient whole cerebral ischemic mouse model characterized in that the classification and selection.

The transient forebrain ischemic mouse model can be prepared according to methods known in the art. For example, but not limited to this, both warmth artery ligation, one warmth artery ligation, and both warmth artery ligation under hypotension can be used. In one embodiment of the present invention, transient whole cerebral ischemia was induced by bilateral carotid artery ligation using C57BL / 6 mice (see Example 1).

The local cerebral blood flow can be measured according to methods known in the art after whole cerebral ischemic injury in experimental animals. For example, the present invention may be measured using a laser Doppler flow meter, Doppler ultrasound, autoradiography using ¹⁴ C-iodoantipyrine, continuous arterial spin labeling using MRI, or the like. In one embodiment of the present invention, the local cerebral blood flow of the mice induced transient transient cerebral ischemia from the start of anesthesia to 5 minutes after reperfusion was measured using a laser Doppler flow meter (LDF; Perimed, PF5010, Jarfalla, Sweden) (Example <2-1>). In addition, the change in the local cerebral blood flow can be calculated as a percentage of the value obtained by dividing the blood flow value for 1 minute after whole cerebral ischemia by the reference value (average value of blood flow for 5 minutes before ischemia). Preferably, when the change in the local cerebral blood flow is less than 15% of the reference value, it can be determined as apoptosis.

The diameter of the back traffic artery may be measured according to a method known in the art after sacrificing the experimental animal. For example, it is not limited to this, It can measure using Indian ink dyeing method, carbon black dyeing method, etc. In one embodiment of the present invention, after ligating both warm arteries of mice, three days after reperfusion, euthanized and warmed India ink solution was perfused with the heart, and the brain was taken out to take a photograph of the posterior artery by using a digital camera. Image-Pro Plus software was then used to compare the diameter of the posterior artery to the basallar artery (see Example <2-2>). Preferably, when the diameter of the posterior artery to the cerebrospinal artery is less than 1/3, it may be determined as apoptosis.

In an embodiment of the present invention, the degree of death of cerebral neurons according to the change in focal cerebral blood flow and the ratio of the diameter of the posterior traffic artery to the basal artery was confirmed by tissue staining (see Example 3). As a result, it was confirmed that neurons were damaged in the hippocampus after ischemia in mice with focal cerebral blood flow of less than 15% and the posterior arterial artery of less than one third of the diameter of the posterior cerebral artery. . On the other hand, mice with focal cerebral blood flow of more than 15% and one-third the diameter of the posterior coronary artery to the basal artery showed no neuronal cell damage in the CA1 hippocampus, a region of ischemia vulnerable for 3 days after transient ischemic injury . In addition, CA1 hippocampal neuron damage caused by ischemia was not observed in mice with a local cerebral blood flow less than 15% during ischemic injury (30.8%). 1). Therefore, CA1 hippocampal neurons, which are sites for determining drug effects, are satisfied only when two conditions are satisfied from the above experimental results: local cerebral blood flow is less than 15% and the diameter of the posterior traffic artery to the basal artery is less than 1/3. The damage could be confirmed.

Furthermore, the present inventors showed the degree of neuronal damage by scattering the degree of neuronal cell damage by applying the conditions of the present invention to a transient whole cerebral ischemic mouse model (see Example 3). As a result, when the two conditions (local cerebral blood flow is less than 15% and the diameter of the posterior traffic artery to the cerebral artery are less than 1/3) are applied, there is no neuronal damage. It can be seen that the ratio of the 0 grade indicating the decrease (see Fig. 4).

Therefore, in the transient whole cerebral ischemic mouse model, the extent of regional cerebral blood flow and the ratio of the posterior communicating artery to the basal artery are measured to predict the extent of neuronal damage and to determine the severity of neuronal damage. Therefore, the classification and selection of animal models can reduce the degree of variation in the severity of brain damage between individuals in the transient whole cerebral ischemic mouse model.

This invention is very useful in the following aspects.

First, the efficacy evaluation of neuroprotective drugs using the transient whole cerebral ischemia mouse model can be improved.

Second, the number of transient whole cerebral ischemic mouse models used in preclinical procedures for drug efficacy evaluation can be greatly reduced.

Therefore, the present invention can provide scientific excellence, economics and bioethical advantages in the process of developing a drug for the treatment of brain diseases, including stroke.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

<Example 1>

Induction of transient forebrain ischemia

As an animal model, 19 male C57BL / 6 mice (8-9 weeks old), weighing 20-24 g were used, and transient whole cerebral ischemia was induced by bilateral common carotid artery occlusion (BCCAO). The mice were kept in an environment in which the room temperature was maintained at 22 W 1 ° C. throughout the experimental period, and was automatically given day and night for 12 hours.

The mice were anesthetized with 1% halotane mixed with 70% nitrous oxide and 30% oxygen, and then exposed to the common carotid artery through a midline incision and fine hemostatic forceps. For 30 minutes Ligation induced ischemia. The hemostatic forceps were then removed and reperfused.

<Example 2>

Prediction of Nerve Cell Damage in Animal Model of Ischemic Brain Disease by the Method of the Present Invention

Local cerebral blood flow changes of the ischemic brain disease mice prepared in Example 1 were measured and neuronal cell death was measured by measuring the diameter of the posterior arterial artery to the basal artery.

<2-1> Measurement of focal cerebral blood flow

From the start of anesthesia to 5 minutes after reperfusion, regional cerebral blood flow (rCBF) of all mice of Example 1 was measured using a laser Doppler flowmeter (LDF; Perimed, PF5010, Jarfalla, Sweden). That is, the probe was fixed at 3.5 mm right position from the bregma of the mouse and rCBF was measured. The change in rCBF was calculated as a percentage of blood flow for one minute after whole cerebral ischemic injury divided by baseline.

Experimental results showed that rCBF varied from 3.7 to 60.6% in 19 mice during BCCAO. Of these, 13 (68%) had a decrease in rCBF of less than 15% (Table 1). Figure 1 shows the result of measuring the change in rCBF.

<2-2> Reversible Measurement of Posterior Carotid Artery

After 3 days of reperfusion, the mice of Example 1 were euthanized by intraperitoneal injection of 15% chloral hydrate and cardiac perfused with 0.1M phosphate buffer (pH 7.4) in saline and 4% paraformaldehyde. 5 ml of warm Indian ink solution was injected into the heart and the brain was taken out, and the posterior communicating artery (PcomA) was photographed using a digital camera (EOS 300D, Canon, Tokyo, Japan). Then fixed for 4 hours in 4% paraformaldehyde solution. The degree of plasticity of PcomA was measured by comparing the diameter of PcomA to the basallar artery using Image-Pro Plus software (version 5.1; MediaCybernetics, Silver Spring, MD). The reversibility of PcomA was evaluated according to a rating from 0 (under developed) to 1 (well-formed), where 0 was less than 1/3 of the diameter of the posterior artery. In this case, the first grade was classified by more than 1/3 cases.

As a result, of the 19 mice used in the experiment, 10 were grade 1, meaning that the diameter of the posterior traffic artery to the cerebrospinal artery was 1/3 or more. The diameter was found to be less than one third (Table 1, Figures 2 and 3).

<Example 3>

Identification of Neuronal Damage in Transient Whole Cerebral Ischemia Mouse Model Using Tissue Staining

Three days after the operation, the brains of the sacrificed mice were stained with cresyl violet to confirm the degree of nerve cell damage. That is, the brain samples extracted in Example 2 were dehydrated in 0.1 M phosphate buffer containing 30% sucrose and embedded in tissue-tech (Sakura Finetechnical, Tokyo, Japan) and immediately cooled using liquid nitrogen. The brain samples were cut into sections using cryotome and transient nerve injury was assessed. Each tissue of striatum and hippocampus was placed on a slide and treated with 0.1% cresyl violet solution for 20 minutes. The slide was immersed in 95% alcohol solution to remove excess dye and dehydrate. After washing with xylene solution, the sections were encapsulated with Canadian balsam and then observed with an optical microscope (BX51, Olympus, Japan). Structural and hippocampal tissue damage was determined by semiquantitative counting of surviving neurons stained with cresyl violet solution. The degree of ischemic neuronal damage is indicated by X when there is no neuronal damage and O by neuronal damage.

Experimental results showed that in Example 1, nine mice with transient whole cerebral ischemia induced less than 15% focal cerebral blood flow and less than one-third diameter PcomA to the basal artery (47.4% of the total). After the ischemic injury, neurons were damaged in the hippocampus and striatum. In contrast, six mice (31.6% of the total) with focal cerebral blood flow greater than 15% and PcomA diameters greater than one third of the basal artery (31.6% of the total) had CA1 hippocampus, a site of susceptibility to ischemia for 3 days after transient ischemia. No neuronal damage at the site was observed.

In addition, in 13 mice with focal cerebral blood flow less than 15%, 4 mice (30.8%) with a PcomA diameter of 1/3 or more of the cerebrospinal artery, the hippocampus at the CA1 site mainly used for the evaluation of drug effects. There was no neuronal damage.

Neuronal Cell Damage in Transient Cerebral Ischemia Mouse Model According to Focal Cerebral Blood Flow and Posterior Carotid Artery Diameter for Cerebral Artery. case parameter Ischemic Neuron Damage PcomA rCBF mCA1 lCA1 CA2 CA3 CA4 CP One + 60.6% X X X X X X 2 + 25.9% X X X X X O 3 + 25.0% X X X X O O 4 + 24.7% X X O O O O 5 + 23.2% X X O O O O 6 + 23.0% X X X X O O 7 + 13.5% X X X X O O 8 + 9.3% X X X X O O 9 + 8.8% X X O O O O 10 + 5.7% X X O O O O 11 - 14.5% O O O O O O 12 - 12.2% O O O O O O 13 - 10.3% O O O O O O 14 - 9.4% O O O O O O 15 - 9.2% O O O O O O 16 - 8.3% O O O O O O 17 - 7.9% X X O O O O 18 - 6.9% O O O O O O 19 - 3.7% O O O O O O

Based on the experimental results, neuronal damage of the hippocampus and striatum was shown semi-quantitatively using a scatter plot (FIG. 4). The degree of neuronal damage was evaluated based on the following criteria. Grade 0 when ischemic damage cells are not observed, grade 1 when ischemic damage cells are observed below 30%, grade 2 when 31 to 64% ischemic damage occurs, grade 3 when more than 65% ischemic damage cells are observed Divided into. In addition, all experimental results were analyzed by Friedman two-way analysis and post hoc non-parametric multiple comparison test to verify statistical significance. A p value of less than 0.05 was considered statistically significant.

As a result, in the case of applying the local blood flow change condition (rCBF value less than 15%), the proportion of cases where no ischemic cells were observed (grade 0) was significantly reduced compared to the case where no condition was applied. When the condition for the diameter of the posterior traffic artery with the condition (the diameter of the posterior traffic artery to the cerebral artery) is applied, the proportion of ischemic injury cells not observed (grade 0) is more significant. Decreases (FIG. 4).

The method according to the present invention has the effect of reducing the severity variation of brain damage between individuals used in the experiment by predicting the degree of neuronal cell death in the transient whole cerebral ischemic mouse model. Therefore, the method of the present invention can improve the efficacy evaluation of neuroprotective drugs using the transient whole cerebral ischemic mouse model, and can greatly reduce the number of transient whole cerebral ischemic mouse models used in the experiment.

1 is a result of measuring the regional cerebral blood of a mouse induced transient transient cerebral ischemia by bilateral common carotid artery occlusion using a laser Doppler flowmeter.

1: baseline

2: time point when ischemic injury

3: 1 minute of ischemic injury

4: when reperfusion occurs

Figure 2 is a photograph showing the posterior communication artery (posterior communication artery) of mice induced transient transient cerebral ischemia by bilateral carotid artery ligation.

A: When both posterior artery are 1/3 or more of diameter of basallar artery

B: Right back traffic artery is 1/3 or more of diameter of cerebral artery

C: When both posterior traffic arteries are less than 1/3 of the diameter of the basal arteries,

Figure 3 shows the degree of neuronal cell death according to the change in focal cerebral blood flow and the ratio of the diameter of the posterior traffic artery to the basal artery in the mice induced transient transient cerebral ischemia by bilateral arterial ligation.

A-F: Neuronal cell death is observed in hippocampus (A-E) and striatum (F) when regional cerebral blood flow changes are less than 15% of baseline and the posterior artery is less than 1/3 of the basal artery.

G-L: Neuronal cell death was not observed in hippocampus (G-K) and striatum (L) when regional cerebral blood flow changes more than 15% of baseline and the diameter of posterior traffic artery is 1/3 or more of basal artery.

B, H: CA1 part of the hippocampus

C, I: CA2 part of the hippocampus

D, J: CA4 part of the hippocampus

E, K: CA3 part of the hippocampus

Figure 4 is a graph showing the scattered degree of neuronal damage in mice induced transient transient cerebral ischemia by bilateral carotid artery ligation.

A: If no conditions apply

B: When local blood flow change condition is applied (less than 15%)

C: When the condition of local blood flow change (less than 15%) and the diameter of the posterior traffic artery to the cerebral artery are applied (less than 1/3)

Claims (8)

Changes in regional cerebral blood flow and the ratio of the diameter of the posterior communicating artery to the basal artery in the transient whole cerebral ischemic mouse model were measured. The regional cerebral blood flow change is calculated as a percentage of the value obtained by dividing the blood flow value for 1 minute after whole cerebral ischemia by a reference value (average value of blood flow for 5 minutes before ischemia). . The method of claim 1, wherein the transient whole cerebral ischemic mouse model is prepared by bilateral arterial ligation. The method of claim 1, wherein the local cerebral blood flow is measured using a laser Doppler flow meter. delete The method of claim 1, wherein said local cerebral blood flow is predicted to be apoptosis when it is less than 15% of the reference value. The method of claim 1, wherein the diameter of the posterior artery to the basal artery is measured by Indian ink staining. 2. The method of claim 1, wherein if the diameter of the posterior artery to the cerebral artery is less than one third, it is predicted to be apoptosis. Method for reducing the degree of variation of brain damage severity in the transient whole cerebral ischemic mouse model characterized in that after measuring the degree of neuronal cell death using the method of claim 1, classifying and selecting the animal model according to the degree of neuronal cell death .