KR101294351B1 - Method of Fluorescent thrombus imaging-based prediction of infarct volume in a stroke model - Google Patents

Abstract

The present invention relates to a method for predicting the size of a cerebral infarct area in a stroke animal model and a method for screening a cerebral infarct therapeutic material to determine its effect by measuring the size of the cerebral infarct area. The present invention relates to a method for accurately predicting the size of cerebral infarction area using near-infrared fluorescent (NIRF) imaging and more accurately evaluating the effect of a cerebral infarction therapeutic substance.
In addition, by combining cerebral blood flow monitoring with the above method, it is possible to more accurately predict the size of cerebral infarction area, and further reduce the false negative result in the screening method of cerebral infarction, thereby more sensitively detecting the neuroprotective effect of the infarct therapeutic material. It's about how to do it.

Description

Method of predicting the size of cerebral infarction in stroke animal model using cerebral thrombosis fluorescence imaging method

The present invention relates to a method for accurately predicting the size of a cerebral infarct area in a stroke animal model and to a method for screening a cerebral infarct therapeutic material based on the measurement of the size of the cerebral infarct area. More specifically, the present invention relates to a method using near-infrared fluorescent (NIRF) imaging of cerebral thrombi, which is combined with blood flow monitoring to more accurately predict the size of cerebral infarct area and thereby It relates to a screening method.

Thrombus is a product of metabolic processes that naturally produce and dissociate during reactions in vivo and is related to blood coagulation. Blood coagulation may occur when blood composition changes, such as damage to the blood vessel wall, increase in the number of platelets in the blood due to changes in blood flow rate after surgery, and increase in fibrinogen.

The cause of thrombus has not yet been accurately identified, but congenital genetic diseases such as disseminated intravascular coagulation (DIC), physiological changes due to aging of the body, stress, etc. Stress caused by overworking in modern people is one of the causes of abnormal thrombus by changing the amount of factors involved in blood clotting.

Thrombosis occurs as part of a hemostatic effect. Hemostasis is caused by shrinking blood vessels and narrowing the internal diameter of the blood vessels to prevent them from leaking out of the blood vessels. The blood coagulation factors are involved and the blood coagulates. The hemostasis process can be roughly classified into four stages: 1) contraction of blood vessels, 2) thrombus formation due to platelet aggregation, 3) blood clotting, and 4) proliferation of fibrous tissue in the clotted blood. In other words, platelets and tissues are collected in collagen exposed at the wound of blood vessels, and blood coagulation is started, and fibrous tissue proliferates at this region, causing blood vessels to be blocked. Thus, the formed blood clot causes a cerebrovascular disease such as stroke. Sometimes. A stroke is a neurological condition in which the blood vessels that supply blood to a part of the brain are blocked or burst, resulting in damage to the brain in that part.

Stroke is one of the leading causes of death in adults worldwide, resulting in cell death due to a lack of blood supply to the brain. Ischemic stroke can be classified into "complete ischemia" and "partial ischemia" according to its blood circulation disorder. When complete ischemia, a part of the brain dies, that is, "brain infarction" because the blood circulation of the localized brain area is completely blocked. This happens. The site of cerebral infarction cannot restore its function, so the disorder caused by cerebral infarction remains permanent and can act as a major disorder.

Since stroke is once irreversible, it leaves irreversible neurological damage and cannot be cured. Therefore, cause analysis and prevention are the most important, and many risk factors are being actively researched.

In general, it is known that about 10% of the cerebral infarction area is determined by whether a patient with cerebral infarction disease can ride a wheelchair or can walk. It is recognized as an important factor in determining the degree.

Many drugs and treatment techniques have been developed for the treatment of stroke, and the effectiveness of such drugs or treatment techniques is evaluated based on the extent to which the infarct size decreases due to the administration or treatment of the drug. Accurately calculating the size is an important part of the treatment and research of stroke.

That is, conventionally, the brain slice image of the stroke animal model is prepared and the expert visually checks it and manually specifies the borderline of the cerebral infarct area.The size of the cerebral infarct area is multiplied by the area of the cerebral infarct border line and the thickness of the cut brain section. The method of calculating the was used. However, in this method, it is difficult to produce uniform results because the determination site may vary according to the expert's skill in determining the cerebral infarction area, and the deviation is very high in accuracy. There was a problem that it takes a lot of time and hassle because the boundary line had to be carried out by hand.

Alternatively, the brain slice image of the stroke animal model was prepared, and the cerebral infarct region was determined by using the difference in the absorption rate of the green visible light in the normal region and the cerebral infarct region on the brain slice image, thereby determining the area of the cerebral infarct region and the brain. A method was used to calculate the size of the cerebral infarction area by multiplying the thickness of the sections. However, this method does not detect cerebral infarcts in the brain slice image that is lighter or darker than a certain threshold for distinguishing cerebral infarcts according to the absorption rate of green visible light, and additional equipment is needed to improve accuracy. There was a problem.

It is also important to see if the size of cerebral infarction is reduced by treatment in determining the effectiveness of a treatment drug for cerebral infarction. However, in the embolic stroke model, there are many variations in the size of the cerebral infarction. Therefore, the effect of the therapeutic drug turns out to be false negative or false positive. This weakens the statistical power of the study and increases the standard deviation of cerebral infarct size in the treatment and control groups.

Accordingly, the present inventors devised a method for predicting the size of the cerebral infarction area, and based on this, by correcting the two factors that determine the size of the cerebral infarction, it is possible to increase the accuracy of the cerebral infarction treatment effect determination of the drug candidate. To this end, the invention was able to more precisely determine the size of the cerebral infarction area by using a blood clot image by labeling a fluorescent substance in a thrombus.

An object of the present invention is to predict the size of the cerebral infarction area in the animal model of stroke, the problem of generating a lot of false negative or false positive results in the conventional neuroprotective drug experiments by using the brain thrombus image of the thrombus labeled with fluorescent material It is to provide a method for accurately predicting the size of the cerebral infarction area that can solve the problem.

Still another object of the present invention relates to a method for screening a cerebral infarction therapeutic material using the prediction method.

In addition, an object of the present invention to provide a more accurate method for predicting the size of cerebral infarction area and screening method for treating cerebral infarction in the prediction of the size of the cerebral infarction area using the cerebral thrombosis image, by further combining the monitoring of cerebral blood flow. have.

In order to achieve the object of the present invention as described above, the present invention provides a method for predicting the size of the cerebral infarction region using a brain thrombus image in a stroke animal model.

The present invention also provides a method for screening a cerebral infarction therapeutic material by predicting cerebral infarct area size comprising measuring a change in the size of a cerebral infarct area using a cerebral thrombosis image.

According to the present invention, it is possible to solve the problem that a large number of false negative or false positive results occurred when predicting the size of a conventional cerebral infarction area by using a brain thrombus image of a fluorescently labeled thrombus in vitro and more accurately predict the size of the cerebral infarction area. Can be. In addition, it is possible to predict the size of the more precise cerebral infarction area by combining the result of cerebral blood flow measurement with the brain thrombus image.

In addition, the above-described method of predicting cerebral infarction can more effectively screen the cerebral infarction therapeutic material using the cerebral infarct size change.

Figure 1 shows the use of FXIII coagulation factor as an enzyme to covalent linking the fluorescent material in the thrombus.
2 relates to a method of causing cerebral infarction using a thrombus bound fluorescent material.
3 relates to imaging the distribution and amount of thrombi in vitro.
4 shows that cerebral blood flow reduction and the amount of thrombus correlate with the size of cerebral infarction.
FIG. 5 relates to predicting the magnitude of cerebral infarction using information on cerebral blood flow reduction and amount of thrombus.
FIG. 6 relates to an increase in cerebral infarct size with decreased cerebral blood flow and increased thrombus.
Figure 7 shows the cerebral artery ring with a thrombus of 1000 pixels at the left middle cerebral artery and forearm cerebral artery bifurcation.
8 shows optical molecular images of blood clots capable of monitoring the effects of thrombolytics in vitro using fluorescent binding blood clots. (A: NIFR macroscopic, B: Microscopic, C, D, E: Hemoglobin release with tPA or saline added)
9 is an optical image of a thrombus showing the visualization of cerebral thrombi in vitro.
10 shows that the present invention reflects the in vivo therapeutic effect of thrombolytics.
11 is a lesion map showing a close correlation between the size and location of cerebral thrombosis and the size and location of cerebral infarction.
FIG. 12 shows a subgroup lesion map further showing the relationship between cerebral thrombosis and induced infarction. (A: group with thrombus in the left proximal middle cerebral artery, B: group with thrombus in the left proximal middle cerebral artery C: group with thrombus in the left proximal cerebral artery
Figure 13 shows that cerebral infarct position and size predictive map are increased using the present invention.
FIG. 14 shows that false negative results are reduced in stroke or neuroprotective drug drug / therapy development studies when thrombosis and cerebral blood flow are considered as computer simulation results.

In order to achieve the above object, an embodiment of the present invention provides a method for accurately predicting the size of the cerebral infarction area in a stroke animal model.

In the present invention, the term “stroke” is a syndrome accompanied by sudden consciousness disorder and motor paralysis caused by a blood circulation disorder of the brain, and the occurrence site of the stroke is largely irreversible damage area (umbra) and the possibility of recovery surrounding it. It is divided into a penumbra (penumbra) which is a part, and the stroke includes both damage of the umbra and penumbra (unless otherwise specified in the present invention).

In determining the effectiveness of a treatment drug for cerebral infarction, it is important to see if the size of the cerebral infarction is reduced by treatment. However, in the embolic stroke model, there are many variations in the size of cerebral infarction. Therefore, the effect of the therapeutic drug turns out to be false negative or false positive. The statistical power of the study is weakened and the standard deviation of cerebral infarct size in the treatment and control groups is also inevitably increased. Therefore, by predicting the size of the cerebral infarction area, the two factors that determine the size of the cerebral infarction are corrected to increase the accuracy of the determination of the effect of treating the cerebral infarction of the candidate drug.

The thrombus of the present invention is preferably a thrombus attached with a fluorescent substance, and the fluorescent substance includes FITC, Cy3, Cy3.5, Texas-Red, Alexa-680, Cy5, Cy5.5, Cy7, Cy3B, and the like. For example, Cy5.5 is near-infrared fluorescent (NIRF), but is not limited thereto.

The thrombi can be used from the same or different types of individuals to be tested, and is not limited to the blood clot of the subject. As used herein, the term “subject” refers to an animal, including a mammal, especially a human being. The subject may be a patient in need of treatment.

Brain thrombus image in the present invention can be imaged without limitation by measurement methods known in the art using fluorescence of various wavelengths, but preferably can be measured using near-infrared fluorescent imaging (NIRF) have.

In addition, according to the present invention, it is possible to more accurately measure the size of the cerebral infarction area by combining the brain blood flow change measurement to the cerebral thrombosis image.

The method of measuring blood flow or changes in blood flow is not limited thereto, but it is preferable to use the Doppler effect and to use electromagnetic induction.

In addition, the Doppler effect can be divided into using a laser or using an ultrasonic wave. In the case of a laser Doppler blood flow meter, a glass fiber is inserted into a blood vessel and a laser is irradiated by measuring the speed of blood flowing through the blood vessel, and the blood flow is precisely measured by using a wavelength change of reflected light. In the case of an ultrasonic blood flow meter, the blood flow is measured by using an externally applied ultrasonic change. The basic principle is the same.

In one embodiment of the present invention, the brain Doppler blood flow meter was used to measure cerebral blood flow changes.

In one embodiment of the present invention can be dyed with a dye in a stroke animal model to identify the area of cerebral infarction. Hematoxylin-eosin (HE) or 2,3,5-triphenyltetrazolium chloride (2,3,5-Triphenyltetrazolium chloride, TTC) is preferred as the dye, but is not limited thereto. Preferably use TTC.

The TTC is a biochromosome that is oxidized by only binding to the respiratory chain (oxidase enzyme) in the normal cell glomeruli, and thus, tissues can be extracted within 24 hours, thereby reducing the variable according to the breeding state after the experiment, and visually identifying a clear boundary. The advantage is that it can be easily and accurately measured.

The present invention also provides a method for screening a cerebral infarct therapeutic material by predicting cerebral infarct area size comprising measuring a change in the size of a cerebral infarct area using a cerebral thrombosis image.

In addition, the present invention provides a method for screening a cerebral infarction therapeutic material using a cerebral thrombosis image in a method for screening a cerebral infarct therapeutic material for measuring a change in the size of a cerebral infarction associated with the treatment of a cerebral infarct therapeutic material.

In the screening of the brain thrombus therapeutic substance as described above, a more accurate method of screening a neuroprotective drug may be provided by combining measured values of brain blood flow changes.

Hereinafter, the present invention will be described in detail through examples. The following examples are merely examples for describing the present invention, whereby the scope of the present invention is not limited.

< Example 1 > Cy5 .5 NIRF Of the probe  synthesis

In order to detect the activity of FXIII coagulation enzymes, autologous blood clots were labeled with a Cy5.5 near-infrared fluorescent probe. First, the C15 NIRF (Near-Infrared Fluorescent) imaging probe was synthesized using Cy5.5 infrared phosphor. Cy5.5 N-hydroxysuccinimide (Thermo Fisher Scientific, Rockford, IL) in phosphate buffered saline (PBS, 400 μl, pH 7.0; Sigma-Aldrich, St. Louis, MO) to obtain Cy5.5 maleimide; 1 mg, Ex / Em = 675/695) Phosphate buffered saline was dissolved in dimethyl sulfoxide (DMSO, 100 μl; Sigma-Aldrich) N- [β-maleimidopropionic acid] hydrazide, trifluoroacetic acid salt (Thermo Fisher Scientific , 3mg) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (Sigma-Aldrich, 2mg) were mixed for 4 hours.

C18 semi-preparative reversed-phase HPLC was performed to purify the synthesized material (40% to 70% acetonitrile vs. 0.1 M triethylammonium acetate, more than 20 minutes, flow rate 4.0 ml / min.) 95% The purified material was confirmed by HPLC. Cy5.5-maleimide fractions were combined and freeze dried under reduced pressure.

To obtain a C15 NIRF probe, factor XIII substrate peptide (GNQEQVSPLTLLKWC, 1 mg) synthesized using tandard solid-phase Fmoc peptide chemistry (Peptron, Daejeon, Korea) was prepared using Cy5.5-maleimide (3 mg) and PBS (100 μl, pH 7.0) for 4 hours.

< Example 2 > Blood clot preparation.

Thrombi were obtained by drawing 1000 μl of blood from C57 / BL6 mice. Based on the pilot test results, 70 μl of blood was mixed with C15 probe (20 μM, 30 μl) or the same concentration of control phosphor. Thereafter, a 3 ml syringe was used to transfer a 30 cm long polyethylene tube (PE-10 or PE-50). The tube was stored at room temperature for 2 hours and stored at 4 ° C. for 22 hours. The clots were then separated from the tubes and washed three times with PBS.

< Example 3 > Computer - simulated Virtual Neuroprotection Research

To investigate the benefits of NIRF thrombus imaging data, we conducted a virtual neuroprotective study through computer simulation. Virtual simulations quantitatively looked at whether the degree of neuroprotective effect was properly altered by comparing cerebral blood flow (rCBF) and / or thrombometric images. Animals were selected from the study population conducted in the present invention and then selected as control and treatment groups through computer random number generation. In each experiment, control group selection was newly performed. The cerebral infarct size of the treated group was subtracted from the original measured size by one of 0-100% of values for the 21-step virtual neuroprotective effect.

Each case was repeated 1000 times using a Visual Basic application. False positive and negative responses in hypothetical experiments (60,000 trials; 1000 trials / neuroprotection rate / set) were calculated after performing T-tests in the control and treatment groups.

Data are expressed as mean ± SD. Statistical analyzes include Student's t-test, Mann-Whitney test, Pearson correlation, and multivariate regression analysis. P values <0.05 were determined to be statistically significant.

< Example 4 > In vitro  Thrombosis NIRF  Strength measurement.

<4.1> Experimental method.

Blood clots (0.58 mm in diameter and 30 cm in length) were placed in 6 wells each containing 3 ml normal saline. Tissue plasminogen activator (tPA, 5 μg / ml) or the same amount of saline solution was added to each well. . Wells were prepared for various concentrations of Cy5.5 phosphors as reference controls. Before and after the addition of tPA or saline solution, digital photography and NIRF imaging (excitation / emission, 675 nm / 690 nm; 1 second acquisition) were performed for each well.

The amount of hemoglobin from thrombi was measured using a modified cyanomethemoglobin method according to the manufacturer's protocol (Molecular Devices, Sunnyvale, Calif.).

After mixing 150 μl of cyanomethemoglobin reagent and 5 μ of media, the reaction was stirred at room temperature for 15 minutes. Absorbance was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA).

Changes before and after the addition of tPA or saline, a stroke treatment, were expressed as percentages in terms of relative values with a minimum of 0% / a maximum of 100%.

The intensity of the median NIRF signal including the thrombus was measured. The change in the measured value before and after addition of tPA was expressed as a percentage in terms of a relative value with a minimum of 0% / 100%.

<4.2> C15  Of thrombus shown NIRF  Extracellular Thrombolysis Using Images monitoring .

NIRF macroscopic (FIG. 8A) / microscopic imaging (FIG. 8B) and Cy5.5 fluorescence staining were used as controls to confirm the specificity of C15 to thrombus.

Each well treated with tPA (5 μg / ml) with a thrombolytic agent dramatically reduced the expansion of the NIRF signal in the thrombus as compared to the well treated with control saline. After 360 minutes, the NIFR signal strength of the surrounding media sharply increased (Figure 8c).

It appears that hemoglobin contained in whole blood clots was secreted into the surrounding medium due to tPA treatment, so that the treated wells stained the medium red more rapidly than the control group. (See Fig. 8C)

Using this to compare the treatment with saline (spontaneous thrombolysis; n = 7) and tPA treated media (n = 7) to significantly increase the release of thrombolysis-associated C15 probes (quantify Cy5.5NIRF signal intensity) or thrombus It was confirmed from the increase in the release of hemoglobin from the medium to the medium. As a result, it was confirmed that all reached the plateau during about 1.5 ~ 2 hours (see Figs. 8D and 8E).

Unlike conventional hemoglobin measurement methods, C15 labeling and NIRF imaging methods are not only hemoglobin-containing whole blood clots, but also hemoglobin-free platelet-rich-plasma or platelets. It was confirmed through the present embodiment that there is an advantage that can be applied to the blood clots of the plasma.

< Example 5 > Animal test results

<5.1> Using the Animal Brain NIRF Imaging  Way.

The Embolic stroke model is a model in which a preformed thrombus is injected into the middle cerebral artery (MCA). To prepare this, 10-week-old C57 / BL6 mice (n = 56) were prepared, and CBF was measured for 30 minutes using a laser Doppler flowmeter (Omegawave, Tokyo, Japan), and the thrombus (diameter 0.15 mm, length 15 mm, size 0.07 mm ³ ) was injected at the MCA-ACA intersection and body temperature was maintained at 36.5 ° C. using an animal experimental heat pad (Panlab, Barcelona, Spain). After 24 hours, animals were euthanized, brains were taken, and in vitro imaging was performed using NIRF imaging apparatuses with charge-coupled device cameras (CoolSnap EZ, Roper Scientific, Tucson, AZ).

 In addition, 2,3,5-triphenyltetrazolium chloride (TTC) staining for measuring cerebral infarction was performed using fresh frozen sites (10 μm) of the brain.

In order to check whether NIRF imaging system reflects the thrombolytic effect in vivo, 16 additional mice were treated with tPA (n = 12) or saline (n = 4) 1 hour after stroke induction and then normalized. The pixel of the Cy5.5 near infrared fluorescence signal representing the degree of thrombus in the middle cerebral artery was calculated.

The brain was imaged using NIRF imaging equipment. White light, FITC-channel (1 second acquisition and Cy5.5 NIRF-channel (1 second acquisition), was obtained. Cy5.5 fluorescence (2 second) in cryosections of the brain using fluorescence microscopy (Olympus BX61, Tokyo, Japan). acquisition).

After microscopy imaging, H / E staining was performed to confirm the NIRF signal.

<5.2> C15  Of thrombus shown NIRF  Visualization of In Vitro Cerebral Thrombosis Using Images

Visual examination using white light is difficult to identify cerebral infarction or cerebral thrombosis injected into the left MCA-ACA bifurcation. However, Cy5.5 NIRF thrombus imaging of the present invention confirmed that sporadic bright signal foci were clearly observed symmetrically in the medial cerebral artery of the cerebral artery ring and mainly in the left MCA-ACA branch region. FITC imaging showed autofluorescence signals reflecting cerebral infarction in the cerebral hemisphere, and more clearly in the left hemisphere.

Cy5.5 fluorescence and FITC imaging of consecutive sections of the brain were visualized by NIRF and autofluorescence signals, respectively. FITC auto fluorescence sites were consistent with infarct sites stained with H & E. FITC auto fluorescence sites were also consistent with TTC-non stained infarction sites. (Fig. 10)

<5.3> lesion distribution analysis.

To confirm the distribution of anatomical autologous thrombi, the Cy5.5 signal in the NIRF image was plotted on the template. (Figure 7) The diameter of the blood vessel of the template was approximately four times wider than the original diameter for easy visualization. .

The obtained thrombus images were averaged using custom-built software. The software provides a semi-automatic view of the movement and part of the lesion from the preclinical or clinical brain image of the test body in the form of a standard brain template. This automatically calculates the left and right hemisphere thrombus position and size.

In order to analyze the distribution of ischemic brain injury, the white infarcts in TTC staining were divided into six brain parts (slice numbers 1 to 6 at 2.96, 0.98, -1.06, -3.08, -4.60, and -6.36 mm, respectively). Distance from the mold). The mean value of the TTC images was obtained using custom-built software and the cerebral infarct size (in pixels) of the left or right brain was obtained. Infarct size was calculated as percentage of left or both hemispheres in the slice of the selected template. The mean data for slices 2-5 showed final left cerebral infarct size vs rCBF (% cerebral blood flow relative to the baseline value) and / or cerebral arterial ring cerebrovascular correlation or multiple relationship analysis at the base of the brain. Was used. Maps of various subgroups based on rCBF and / or thrombus size were obtained.

<5.4> NIRF  Signals and infarcts Mapping  Analysis

 Total fluorescent thrombus signals occurred over a wide range of 0 to 5940 pixels (2279 ± 1270 pixels). In addition, lesion accumulation map images of total thrombus and cerebral infarction (FIG. 11A) confirmed heterogeneous features of the embolic stroke model.

Fluorescent thrombus signals labeled Cy5.5 were mainly observed in the MCA-ACA bifurcation region, but interspersed embolism was frequently observed on the flanks and away areas of the cerebral artery. The results indicated that the location of cerebral infarction was varied as well as the size of the total cerebral infarction (% -infarct-area relative to the total bi-hemispheric area of six brain template slices, 21.0 ± 10.3%, range 4.7-60.3%).

The difference between the experimental group injected with thrombus at the right MCA-ACA bifurcation (see top of FIG. 12B, n = 37) and the untreated control (see bottom of FIG. 11B, n = 10) was examined. As a result, the size and distribution of infarction showed a distinct difference. Infarct size of the right hemisphere in the experimental group (% infarct area is expressed as a relative percentage of total brain slice hemispheres, brain template slices -2, -3: 15.2 ± 10.9%, 21.5 ± 21.6% vs. 6.9 ± 13.4%, 8.2) ± 17.2%; P = 0.04, 0.03), and the infarct size of the left hemisphere was smaller. (template slices 2-5: 32.5 ± 16.2% vs. 45.2 ± 20.8%, P = 0.04)

In addition, the thrombi lysis group showed a smaller thrombus than the non-thrombotic group (p = 0.04) (see Fig. 11C).

Examining the subgroup map of the experimental group, the rats with thrombus in the proximal left major cerebral artery (see Fig. 12A, n = 36) had a greater infarct in the left left brain compared to the control group (Fig. 12B, n = 11) otherwise. It was confirmed. (48.1 ± 20.1% vs 31.0 ± 20.1%, p = 0.02)

Through this, it was confirmed that the location and size of cerebral thrombosis and cerebral infarction can be compared and analyzed using NIRF thrombosis.

< Example 6 > C15 sign NIRF Imaging  Used In vivo  Thrombosis Treatment  evaluation.

At 24 hours treated with saline (n = 4) or tPA (n = 12) after 1 hour of embolic stroke, a more pronounced decrease in thrombus signal was observed in the Cy5.5 treated group compared to the control group (310 ± 135 pixels). vs. 32 ± 43 pixels, P = 0.02). In addition, significant reduction of the final infarct size was confirmed through the reduction of thrombus signal. (25.7 ± 6.6% vs. 15.6 ± 8.2%, P = 0.04)

< Example 7 > Laser Doppler With flow meter (LDF)  Brain blood flow measurement.

Regional cerebral blood flow (rCBF) of the brain was measured with a laser Doppler flowmeter (LDF). When blood clots were generated in the left middle cerebral artery, it was confirmed that a decrease in cerebral blood flow occurred immediately (36.1 ± 16.3%, range 10.1 ~ 72.0%), and then gradually increased to converge to 43.9 ± 17.3% of blood flow. Figure 13A)

As shown in FIG. 13B, there was an inverse linear correlation between the blood flow of the brain measured at 5 minutes and the infarct size of the left hemisphere infarct size (24 hours, template slices 2-5). (P = 0.02, r = -0.34)

 Animals were divided into two groups based on the median of the rCBF values (42%). It was confirmed that infarct size tended to be large in the low rCBF group. (46.8 ± 17.5% vs. 36.2 ± 22.1%, P = 0.08)

In template slice-3, the group with low rCBF had statistically significant cerebral infarction (54.9%) compared with the group with high rCBF. (High rCBF 39.1%, P = 0.02).

The thrombus (24h) in the left cerebral hemisphere was analyzed to correlate with the final infarct size. ( P = 0.000, r = 0.504 FIG. 13C).

As a result of dividing into two groups based on the median value of 1865 pixels, it was confirmed that the group with large thrombus had larger cerebral infarction than the group with small thrombus. (50.6 ± 18.7% vs. 31.3 ± 17.0%, P = 0.001).

The rCBF values were combined with the thrombus imaging results to derive a relationship with the size of cerebral infarction. (FIG. 13D-F) The magnitude of cerebral infarction was compared between animals with low rCBF and large thrombus and animals with low rCBF and small thrombus, and it was confirmed that the former animal group had larger cerebral infarction. (P = 0.001) was also confirmed in the case of the former bihaeseodo animals having a high small clots rCBF and the size of the cerebral infarction is great. ( P = 0.005). In addition, the rats with high cerebral blood flow and large thrombus were found to have a greater infarct than those with high cerebral blood flow and small thrombus. (p = 0.04)

Finally, animals with large thrombus and low rCBF were found to have minimal infarct size variation. (Fig. 13D)

< Example 8 > Confirmation of application to neuroprotective studies.

Computer simulations were performed using animals selected from Study cohort (FIG. 13) to confirm that false negative results in neuroprotection studies can be reduced through cross-analysis of rCBF and thrombus of the present invention.

In an experiment involving animals with low cerebral blood flow and large thrombus (n = 12), the infarct size was measured and the false negative result was reduced to 0% when the neuroprotective effect of the specific treatment / treatment was 35%. (See FIG. 14) The neuroprotective effect of the experiments performed on the whole animal without using the present invention and the experimental group other than the group with low cerebral blood flow and large thrombus, should have a neuroprotective effect of 60% or more. It was confirmed that the false negative result is 0%.

Through this, it was confirmed that the neuroprotective effect of the drug of the stroke treatment to be evaluated can be more sensitively screened by combining the brain thrombus imaging and the blood flow measurement method. According to the multiple regression analysis, when the degree of decrease in brain blood flow is corrected, it is estimated that the size of the cerebral infarction increases by about 2.5 times when the size of the thrombus increases by 1000 pixels.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (11)

1) measuring fluorescence thrombus images using near-infrared fluorescent (NIRF) in a stroke-injected animal model with fluorescent labeled thrombus; And
2) measuring brain blood flow of the stroke animal model using a Doppler flow meter; Including, cerebral infarction size or location information providing method of a stroke animal model.
According to claim 1, The fluorescently labeled thrombus is fluorescent with at least one fluorescent material selected from fluorescent materials consisting of FITC, Cy3, Cy3.5, Texas-Red, Alexa-680, Cy5, Cy5.5, Cy7 and Cy3B A method for providing cerebral infarct size or location information of a stroke animal model, characterized in that labeled.
1) measuring fluorescent thrombus images using near-infrared fluorescent imaging (NIRF) in a stroke animal model injected with fluorescently labeled thrombi and selecting animals with large thrombus having a thrombus of 1865 pixels or more; And
2) selecting an animal model having a blood flow rate of 42% or less by measuring the brain blood flow of the stroke animal model selected in step 1) using a Doppler flow meter; and selecting a neuroprotective drug screening animal model.
The method of claim 3, wherein Neuroprotective drug screening animal model is characterized in that the false negative results (false negative) results, screening method of animal models for neuroprotective drug screening.
1) selecting an animal having a large thrombus having a thrombus of 1865 pixels or more by measuring fluorescent thrombus images using near-infrared fluorescent imaging (NIRF) in a fluorescent animal model injected with a fluorescently labeled thrombus;
2) selecting an animal model whose blood flow is 42% or less by measuring the brain blood flow of the stroke animal model selected in step 1) using a Doppler flow meter; And
3) measuring the cerebral infarct size after treatment with a neuroprotective drug candidate in the animal model selected in step 2); screening method of a neuroprotective drug.
The method of claim 5, wherein the neuroprotective drug is a stroke treatment. delete delete delete delete delete

Images