KR20180136702A - Use of the autofluorescence, as a marker for diagnosing brain damages - Google Patents

Abstract

The present invention relates to a use of autofluorescence as a phagocytosis marker in microglia / macrophage activated by brain damages. Autofluorescent granules of specific wavelengths are observed in microglia / macrophage activated in lesions of corpus striatum after 3-NP-induced brain damages, and by directly indicating that autofluorescent granules are lipofuscin granule clusters in brain macrophages, the use of autofluorescence as a marker for diagnosing brain damage and for identifying areas of brain damages, and as a marker for phagocytosis after brain damages is confirmed.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an auto-fluorescence (AF)

The present invention relates to the use of autofluorescence as a predatory marker in microglia / macrophages activated by brain injury.

Since brain injury, which causes destruction or degeneration of brain cells, occurs relatively frequently and widely, a wide variety of diagnostic methods are required medically. Brain damage is caused by a wide range of internal and external factors, a common type involving the greatest number of injuries is traumatic brain injury (TBI) due to physical trauma or head injury from external causes, Is also called acquired brain injury (ABI) to distinguish it from damage caused by congenital disorders.

The cause of brain damage has not yet been elucidated, but is known to be caused by long-term hypoxia, poisoning by anomalous substances including alcohol, infection and neurological diseases. Other common causes of brain damage include traumatic brain injury, stroke, aneurysms, surgery, other neurological disorders, and physiological trauma such as heavy metal poisoning. When ischemic injury occurs, the brain responds to acute inflammation and chronic inflammatory processes, which are mainly caused by activation of macrophages, production of inflammatory mediators, infiltration of inflammatory cells such as neutrophils, T cells, monocytes / macrophages do. In the first few hours, leukocytes from the peripheral blood invade the brain parenchyma, where the primary infiltrating cells are neutrophils (Jin et al., J Leukoc Biol. 87 (5): 779-789, 2010). These peripheral neutrophils have been reported to infiltrate into brain tissue within a few hours and play an important role in the initiation of BBB (blood-brain barrier) destruction due to induction of vascular injury, secondary infiltration of other inflammatory cells, and inflammatory response of brain tissue J et al., J. Endotoxin Res. 12 (6): 367-374, 2006; Scholz M et al. Med Res Rev. 27 (3): 401-416, 2007; (5): 779-789, 2010).

It is known that biological tissues emit fluorescence without an exogenous fluorophore, which is called autofluorescence, and it is known that such nonspecific signals can interfere with the intended fluorescence signal resulting in spurious-positive results (Aubin 1979; Croce et al., 1999, Monici 2005). The amount and spatial distribution of endogenous fluorophore may be influenced by pathological or experimental conditions and may lead to changes in autofluorescence emission.

Lipofuscin, a complex mixture of oxidized proteins and lipolytic residues, accumulates gradually over time in lysosomes of cells after mitosis such as myocardial and neuronal cells (Terman and Brunk 1998; Boellaard et al. 2004; Gray and Woulfe 2005; Jung et al. 2007).

3-nitropropionic acid (3-NP) is a neurotoxin that is widely found in plants and fungi. It induces basal ganglia, hippocampus, spinal tracts and peripheral nerve damage in animals. (Amilton and Gould 1987; AC Ludolph et al. 2015).

The inventors of the present invention conducted experiments using 3-NP, a natural mitochondrial toxin known to selectively impair mesothelial striatum neurons, to emit strong autofluorescence at the damaged site 2 weeks after inducing brain injury , It was confirmed that the emission wavelength of the fluorescent dye overlaps with the emission wavelength of the conventional fluorescent dye and the interpretation of the fluorescent label is interrupted. Thus, the present invention has been completed by clarifying the characteristics of autofluorescence occurring in the nerve injury region after brain injury .

The present invention aims to utilize the detailed information of the structure of an autofluorescence signal of a specific wavelength induced by brain damage as a brain damage marker.

To achieve the above object, the present invention provides the use of lipofuscin as self-fluorescence marker as a brain damage marker.

The present invention also provides a method for providing information for diagnosing brain damage.

The present invention also provides a method of imaging a brain injury site.

The present invention also provides a method for detecting predation after brain injury.

In addition, the present invention provides a screening method of a substance inhibiting the progress of brain injury.

In accordance with the present invention, the presence of autofluorescence granules of specific wavelengths in microglia / macrophages activated by 3-NP-induced brain injury was confirmed, and autofluorescence granules in striate lesions were detected in the lipofuscin granule clusters , It can be used as a marker for diagnosis of brain damage and a marker for brain damage, and it can also be used as a marker for predation after brain damage.

Figure 1 shows the presence pattern of autofluorescence granules found in the central portion of the brain of 3-NP treated rats:
a: striatal sections of saline-treated control (Scale bars: 50 占 퐉);
b: Sclerotic lesion sections (Scale bars: 50 μm) in the experimental group after 3 days of 3-NP treatment;
c: Sclerotic lesion sections (Scale bars: 50 μm) in the experimental group after 7 days of 3-NP treatment;
d: Autofluorescence signal (Scale bars: 100 ㎛) detected with a yellow-green laser (excitation / emission wavelength: 555 nm / 573 nm) in the striatum sections of the experimental group 14 days after treatment with 3-NP;
e: autofluorescence signal (Scale bars: 100 ㎛) detected by blue laser (excitation / emission wavelength: 488 nm / 523 nm) in the striatum sections of the experimental group 14 days after 3-NP treatment;
f: Autofluorescence signal (Scale bars: 100 ㎛) detected with a yellow-green laser in the striatum sections of the experimental group 28 days after 3-NP treatment;
g: Autofluorescence signal (Scale bars: 100 ㎛) detected by blue laser in the striatum sections of the experimental group 28 days after 3-NP treatment;
h: High magnification photographs of autofluorescent granules excited with yellow-green laser at the striatum sections of the experimental group 28 days after 3-NP treatment (Scale bars: 10 ㎛);
i: high-magnification photographs of autofluorescence granules excited by blue laser in scaffold lesion sections of experimental group 28 days after 3-NP treatment (Scale bars: 10 ㎛);
j: High magnification photograph of autofluorescent granules excited with red laser (excitation / emission wavelength: 639 nm / 660 nm) in the striatum sections of the experimental group 28 days after 3-NP treatment (Scale bars: 10 μm);
k: High-magnification photographs of autofluorescent granules excited by all three lasers in scaffold lesion sections of experimental group 28 days after 3-NP treatment (Scale bars: 10 μm); And
(red dashed line) obtained from the blue laser in the region indicated by the red arrow of l: k (green dashed line), the autofluorescence intensity (red solid line) obtained from the yellow- Profile histogram.
Figure 2 shows the cellular identification and characterization of autofluorescent granules in 3-NP treated brain striatum:
a: Autologous fluorescence signals and GFAP-positive astrocytes (Scale bars: 50 ㎛) in striatum lesions in the experimental group 14 days after 3-NP treatment;
b: Autologous fluorescence signals and Iba1-positive microglia (Scale bars: 50 ㎛) in striatum lesions in the experimental group 14 days after 3-NP treatment;
c: Autofluorescence signal and GFAP-positive astrocytes (Scale bars: 50 ㎛) in the striatum lesions of the experimental group 28 days after 3-NP treatment;
After 28 days of d: 3-NP treatment, autofluorescence and Iba1-positive microglia (Scale bars: 50 ㎛) in the striatum lesions of the experimental group;
e: autofluorescence in the cytoplasm of activated macrophages / macrophages at the center of the lesion (Scale bars: 10 μm);
f: Iba1 (Scale bars: 10 μm) in the cytoplasm of activated macrophages / macrophages in the lesion center;
g: autofluorescence (round amoeboid macrophages, arrowheads filled with autofluorescent granules, arrowheads) and Iba1 (activated small cells with Iba1 strongly marked arrows) in the cytoplasm of activated macrophages / (Scale bars: 10 占 퐉) showing the position pattern of the sample;
h: a complementary pattern of autofluorescence and Iba1 in orthogonal view (Scale bars: 10 mu m);
i: a high magnification picture of the box area of figure g (Scale bars: 10 mu m);
j: intensity histogram of Iba1-positive signal and autofluorescence in the blue arrow region of figure i; And
k: scattering degree of Iba1-positive signal and autofluorescence intensity in the blue arrow region of i) x-axis: autofluorescence intensity, y-axis: Iba-positive signal intensity).
Figure 3 shows the results of quantitative analysis of the autofluorescence characteristics of brain macrophages in 3-NP treated striatum of the brain:
a: Autofluorescence signal (Scale bars: 10 ㎛) in striatum lesions of experimental group 28 days after 3-NP treatment;
b: lysosomal marker ED1 / CD68 signal (Scale bars: 10 mu m) in striatum lesions of experimental group 28 days after 3-NP treatment;
c: Distribution pattern of autofluorescence and ED1 / CD68 in brain macrophages of striatal lesions in experimental group 28 days after 3-NP treatment (Scale bars: 10 ㎛);
d: autofluorescence and ED1 / CD68 complementary positional patterns in brain macrophages expressed by orthogonal view (Scale bars: 10 μm);
e: autofluorescence signal (Scale bars: 10 ㎛) in striatum lesions of experimental group 28 days after 3-NP treatment;
f: lysosomal marker Lamp1 signal (Scale bars: 10 쨉 m) in striatum lesions of experimental group 28 days after 3-NP treatment;
g: Distribution pattern of autofluorescence and Lamp1 in brain macrophages of striatal lesions of experimental group 28 days after 3-NP treatment (Scale bars: 10 ㎛);
h: autofluorescence in brain macrophages in orthogonal view and the complementary position pattern of Lamp1 (Scale bars: 10 μm);
i: Number of ED1 / CD68-positive cells in lesions over time after 3-NP treatment ( P <0.05; ^** p <0.01, ^*** p <0.001, ns: not significant); And
( p <0.05, ^** p <0.01, ^*** p <0.001, ns: not significant) in the lesion according to time after 3-NP treatment.
In the control group treated with saline using the correlation method, it is proved that the micrograph does not show autofluorescence:
a: confocal microscope image of Iba1 immunostained half-cut slices in control striatum (white: Iba1) (M = inactivated micrograph) (Scale bars: 5 쨉 m);
b: an image (Scale bars: 5 탆) overlaid with a confocal microscope image (a) and a TEM image (c); And
c: a TEM image at the same site of the image (M = inactive micrograph) (Scale bars: 2 μm).
Figure 5 shows the intracellular location of autofluorescent granules using correlation analysis:
a: Confocal microscope image of Iba1 immunoreactive cutaneous slices on striatum lesion 14 days after 3-NP treatment (white: Iba1, red: Scale bars: 5 ㎛);
b: an image (Scale bars: 5 탆) overlaid with a confocal microscope image (a) and a TEM image (c);
c: TEM image (N = nucleus) at the same lesion of the image (Scale bars: 5 μm); And
(n = nucleus) (Scale bars: 1 ㎛).

Hereinafter, the present invention will be described in detail with reference to embodiments of the present invention. It should be understood, however, that the invention is not limited thereto and that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents. .

As used herein, the term "detection" or "measurement" means identifying or quantifying the presence of a detected or measured subject.

The term "diagnosis" used in the present invention means confirming a pathological condition. For the purpose of the present invention, the diagnosis is made by confirming the presence or absence of a marker for diagnosing brain damage disease, , And includes checking the presence of the marker to determine the brain injury site (lesion) to determine the generation and alleviation.

As used herein, the term "diagnosis marker" refers to a substance capable of distinguishing cells from brain damage cells from normal cells and includes lipofuscin occurring in cells of the brain injury region do. Lipofuscin, which is a marker for diagnosing brain injuries, provided by the present invention, may exhibit autofluorescence.

The term " brain damage "or" brain damage disorder "as used herein includes all kinds of diseases that occur in the short term (acute) or long term (chronic) by all kinds of brain damage causing destruction or degradation of brain cells And although it is not limited to a particular type of disease, preferably the brain injury disease may be an acute brain injury disease caused by 3-NP.

In one aspect, the invention relates to the use of lipofuscin as an brain damage marker, which exhibits autofluorescence at excitation wavelength 488 nm and emission wavelength 523 nm or excitation wavelength 555 nm and emission wavelength 573 nm after brain injury.

In one aspect, the present invention provides a method for detecting fluorescence emission, comprising: detecting the presence of autofluorescence at a peak emission wavelength of 500 to 600 nm; And judging that a site in which autofluorescence exists is a brain injury site, to a method of providing information for brain injury diagnosis.

In one embodiment, autofluorescence can be detected through a variety of methods known in the art, and in one embodiment autofluorescence is confirmed using confocal microscopy or fluorescence microscopy.

In one embodiment, the autofluorescence may be lipofucin, and the autofluorescence may be excited at 440 to 500 nm and emitted at 500 to 550 nm, or excited at 530 to 570 nm and at 550 to 600 nm Can be released. In one embodiment, the autofluorescence was excited with a blue 488 nm laser and emitted at 523 nm, or excited by a 555 nm yellow-green laser to 573 nm, while a violet laser (excitation / emission wavelength 405 nm / 460 nm ) Or a red laser (excitation / emission wavelength: 639 nm / 660 nm).

In one embodiment, microglia-specific markers Iba1 (ionized calcium-binding adapter molecule 1) and lysosome-specific markers ED1 and Lamp1 (lysosomal-associated membrane protein 1) can additionally be identified .

Methods for identifying the small cell or lysosome-specific marker can be performed by a variety of methods known in the art including, but not limited to, radioimmunoassay (RIA), radioimmunoassay radioimmunodiffusion, and immunoprecipitation assay.

In one aspect, the invention provides a method for determining the presence of lipofuscin by measuring an autofluorescence signal at an excitation wavelength of 440 to 500 nm and an emission wavelength of 500 to 550 nm, or an excitation wavelength of 530 to 570 nm and an emission wavelength of 550 to 600 nm step; And determining that the site in which lipofuscin is present is a damaged site.

In one embodiment, the imaging method of the present invention may employ an optical imaging system and may include a fluorescence microscope, a mercury and xenon arc lamp, a CCD camera, an AOTF (Acoustic Optical Modulation Filter) -based spectral imaging acquisition mechanism, The use of software can be integrated and included. The above tool for providing autofluorescence imaging in brain tissue is integrated in an optical imaging system to provide a visible mimic color representation of the spectral signature extracted from the raw image, indicating the presence or absence of lipofuscin, And location.

In one implementation, the use of stereoscopic microscopes that are tuned to visualize fluorescence and scatter signals at higher resolutions can be integrated into the optical imaging system. A stereoscopic microscope can be equipped with a polychrome V variable wavelength light source. In a further embodiment, a MicroFire color digital camera and one or more magnifying lenses may be integrated into the optical imaging system to improve magnification and image detail. Post analysis using imaging software Image acquisition can be achieved and completed by image segmentation and classification.

In one embodiment, the use of an electron microscope capable of visualizing intracellular organelles can be integrated into the optical imaging system. In one embodiment, the autofluorescence of lipofuscin in brain tissue was confirmed by confocal microscopy, and the same site of tissue was examined using an electron microscope to identify intracellular organs.

In one aspect, the invention provides a method for detecting brain damage, comprising measuring an autofluorescence signal at an excitation wavelength of 440 to 500 nm and an emission wavelength of 500 to 550 nm, or an excitation wavelength of 530 to 570 nm and an emission wavelength of 550 to 600 nm. And a method for detecting postprandial action.

In one example, the presence of lipofuscin granules in activated macrophages / macrophages following brain injury was confirmed, and it was confirmed that the predatory activity of lipofuscin decreased with increasing autofluorescence.

In one aspect, the present invention relates to a method of screening for an inhibitor of brain damage progression comprising the steps of:

(a) causing brain damage to mammals other than humans;

(b) detecting an autofluorescence signal at an excitation wavelength of 440 to 500 nm and an emission wavelength of 500 to 550 nm, or an excitation wavelength of 530 to 570 nm and an emission wavelength of 550 to 600 nm to measure a brain injury site;

(c) treating the test agent;

(d) measuring an area of brain damage by detecting an autofluorescence signal at an excitation wavelength of 488 nm and an emission wavelength of 523 nm, or an excitation wavelength of 555 nm and an emission wavelength of 573 nm; And

(e) comparing the test agent with the brain injury site of the control group not treated with the test agent to determine whether the test agent inhibits or reduces the extent of the brain injury site.

The term "mammal" as used herein includes human and non-human primates such as chimpanzees, and other apes and monkey species; Farm animals such as cattle, sheep, pigs, goats and horses; Pet mammals such as dogs and cats; And laboratory animals, including rodents such as mice, rats and guinea pigs, and the like. These terms do not indicate a specific age or gender. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of these terms.

In one aspect, the invention relates to the use of lipofuscin as an endocrine marker after brain injury, which exhibits autofluorescence at excitation wavelength 488 nm and emission wavelength 523 nm or excitation wavelength 555 nm and emission wavelength 573 nm after brain injury.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.

Example  One. in vivo  Induce acute brain tissue damage

In order to induce acute brain injury, the present inventors conducted experiments on brain tissues containing adult striatum. In this experiment, male Sprague Dawley rats (250-300 g) at 9-11 weeks of age were used for the experiment and 15 mg / kg of 3-NP (3-nitropropionic acid), a mitochondria toxin, was continuously injected into the peritoneum for 3 days Respectively. The behavioral patterns of animals treated with 3-NP were examined daily and animals with neurological deficits such as hind limb disorders, kyphotic postures, lateral and postural and postural control disorders were selected and used in the experiments. Control rats were injected intraperitoneally with the same volume of saline.

In this model, the lesion core area, which is morphologically identifiable to the striatum of both brains, was confirmed by inducing acute brain injury (data not shown). Therefore, it was confirmed that the above treatment can induce acute brain injury experimentally.

Example  2. Tissue harvest after induction of acute brain injury

The rats were allowed to survive for 3, 7, 14, and 28 days after inducing acute brain injury by the method of 1-1 above. A total of 3 mice and 3 saline-treated rats that caused acute brain injury at each time point (3, 7, 14, and 28 days after the last 3-NP treatment) And anesthetized with 4 mL / kg of chloral hydrate. Anesthetized rats were fixed in 0.1 M phosphate buffer, pH 7.4, using a 4% paraformaldehyde fixative using a transcardial perfusion method. Brain tissues were removed from fixed mice and treated with a solution containing 30% sucrose in 0.1 M phosphate buffer and completely frozen.

All procedures for animal experiments were approved by the Catholic Ethics Committee (CUMC-2016-0014-02) of the Catholic University of Korea.

Example  3. Single and double label immuno-staining chemistry ( immunohistochemistry ) analysis

3-1. In acute brain injury tissue Autofluorescence  Confirm

In order to identify autofluorescence and nuclei in brain tissue induced by acute brain injury, 25 μm thick coronal cryostat sections were incubated with DAPI (4,6-diamidino-2-phenylindole; Roche; dilution ratio 1: 2000) for 10 minutes to label the nuclei. The labeled sections were fixed on a cover glass slide with mounting solution. The slides were observed using a confocal microscope (LSM 700; Carl Zeiss Co. Ltd., Germany) equipped with four lasers (Diode 405, Argon 488, HeNe 555, HeNe 639) and images were converted to TIFF format And contrast levels were adjusted through Adobe Photoshop v.10.0 (Adobe Systems, San Jose, Calif., USA).

As a result of autofluorescence emission from the striatum after 3-NP injection, no autofluorescence was observed in the saline-treated rats of the control group (Fig. 1A) (Fig. 1B) and 7 days (Fig. 1C), self-fluorescence began to appear and reproducible and pronounced autofluorescence appeared at the 3-NP injection site on the 14th day after injection (Fig. 1d and e). This phenomenon became more pronounced in the brain tissue of mice 28 days after 3-NP injection (Fig. 1f-k). The autofluorescence was noticeable in both blue laser (excitation / emission wavelength: 488 nm / 523 nm) and yellow-green laser (excitation / emission wavelength: 555 nm / 573 nm) 405 nm / 460 nm) and a red laser (excitation / emission wavelength: 639 nm / 660 nm), no autofluorescence was detected (FIGS. Also, as shown in Figures 1h and i, autofluorescence, which is represented by small granular or spot-like particles, was localized only in the cytoplasm, except for the DAPI-labeled nucleus. In addition, the intensity and amount of autofluorescence varied from cell to cell.

3-2. In acute brain injury tissue Autofluorescence  And glial cell specific Marker  Confirm

In order to identify autofluorescent-filled cells in the striatum lesion caused by acute brain injury, glia-specific markers, astrocytes-specific markers such as glial fibrillary acidic protein (GFAP) (Iba1), a microglia-specific marker, was used for immunohistochemical analysis and confirmed with autofluorescence. Specifically, some frozen sections were incubated with mouse monoclonal antibody (1: 1,500; Millipore, Temecula, CA, USA) for GFAP or rabbit polyclonal antibody against Iba1 (1: 500; Wako Pure Chemical Industries, Ltd., Osaka, Japan ) At &lt; RTI ID = 0.0 &gt; 4 C &lt; / RTI &gt; overnight. In order to minimize the confusion with the signal of the autofluorescence, Alexa Fluor 647 (wavelength more than 650 nm after excitation at 633 nm) of the wavelength at which almost no autofluorescence was detected in the laser of long wavelength, (1: 300; Molecular Probes, Inc., Eugene, OR, USA) as a secondary antibody was incubated with the incubated sections for 2 hours at room temperature. In the control group, the primary and secondary antibodies were omitted from the solvent to confirm that there was no immune response. The slides were observed using a confocal microscope (LSM 700; Carl Zeiss Co. Ltd., Germany) equipped with four lasers (Diode 405, Argon 488, HeNe 555, HeNe 639) and images were converted to TIFF format And contrast levels were adjusted through Adobe Photoshop v.10.0 (Adobe Systems, San Jose, Calif., USA).

As a result of observation, round cells containing autofluorescent granules were located in the striate lesions adjacent to the astroglial scar tissue, and these autofluorescent cells did not show GFAP immunoreactivity and were not astrocytes (Figs. 2A and 2C). On the other hand, immunoprecipitated cells with Iba1 (Ito et al., 1998), a marker for dormant macrophages and activated macrophages / macrophages, were found to be autologous activated macrophages / macrophages (Figures 2b and d). At higher magnification, two types of macrophages / macrophages, morphologically distinct in lesions, rounded brain macrophages and oval cell bodies with rare prominent protrusions and amoeboid cytoplasm, and short, thick protuberances (Fig. 2e-h).

In addition, the autofluorescence granules and Iba1 were found to be complementary to each other in the cells in activated macrophages / macrophages. Specifically, the brain macrophages were filled with autofluorescent granules instead of the Iba1 signal, whereas the activated macrophages showed a weak signal of the autofluorescent granule instead of the strong Iba1 signal (Fig. 2H ).

In addition, the positions of Iba1 and autofluorescence granules in activated macrophages / macrophages were confirmed (Fig. 2i), and the average signal intensities of the two were quantitatively analyzed and shown in Figs. 2j-k. Specifically, fluorescence intensity values of Iba1-positive and autofluorescent cells were obtained using Zen 2010 (Carl Zeiss Co. Ltd.). Scattering and correlation analysis were performed using Excel 2013 (Microsoft, Bellevue, WA, USA) Respectively. As a result, the intensity of the Iba1-positive signal was relatively weak in the cytoplasm with strong autofluorescence granules, and vice versa, as shown by the scatter of Iba1-positive signal and autofluorescence signal. Therefore, it was found that Iba1 and autofluorescence had a negative correlation coefficient (r = -0.77, R ² = 0.5933, p <0.001) in these cells.

3-3. In acute brain injury tissue Autofluorescence  And lysosome specific Marker  Confirm

To clarify the intracellular location of autofluorescent granules in activated macrophages / macrophages, some frozen sections were stained with lysosomal-associated membrane (ED1 / CD68) or late endosomal / lysosomal marker Lamp1 protein 1) and confirmed with autofluorescence. Specifically, the frozen sections were incubated with rabbit polyclonal antibody (1: 200; Abcam, Cambridge, UK) against mouse monoclonal antibody (1: 100; Bio-Rad, Hercules, &Lt; / RTI &gt; overnight at &lt; RTI ID = 0.0 &gt; 4 C. &lt; / RTI &gt; The sections were then incubated with Alexa Fluor 647 labeled goat anti-rabbit antibody or goat anti-mouse antibody (1: 300; Molecular Probes, Inc., Eugene, OR, USA) at room temperature for 2 hours. In the control group, the primary and secondary antibodies were omitted from the solvent to confirm that there was no immune response. The slides were observed using a confocal microscope (LSM 700; Carl Zeiss Co. Ltd., Germany) equipped with four lasers (Diode 405, Argon 488, HeNe 555, HeNe 639) and images were converted to TIFF format And contrast levels were adjusted through Adobe Photoshop v.10.0 (Adobe Systems, San Jose, Calif., USA).

As a result, the autofluorescent granules and the two lysosomal markers co-localized in the brain macrophages but were present in a complementary pattern in the cells (Figs. 3a to h). The signals of ED1 / CD68 and Lamp1 were very weak in the soma of amoebic brain macrophages, where autofluorescent granules occupied a wide area. On the contrary, almost no autofluorescence granule signal was observed in the areas where two lysosomal markers were strong.

In addition, time-dependent changes of ED1 / CD68-positive cells including autofluorescence were quantified in order to check whether the autofluorescence of brain macrophages increased in time after 3-NP injection. Specifically, after 3-NP injection, the lesion central portion was photographed at 400x magnification using a confocal microscope on the immunorecepted tissue sections of ED1 / CD68 on days 3, 7, 14, and 28, The number of cells with positive ED1 / CD68 and autofluorescence was counted among the visible cells. As a result, in the third day of brain injury, the autofluorescence-emitting brain macrophages were 32.0 ± 5.4% of total brain macrophages and 92.0 ± 2.1% on day 28 (FIGS. 3i and j) The number of positive cells and the number of ED1 / CD68-positive cells expressing autofluorescence gradually increased with time after induction of brain injury in lesions.

Example  4. Optical and electron microscopy (correlative light and electron microscopy)

The optical microscope image alone is not enough to clarify the autofluorescence granule clearly. In order to clarify the microstructure of the autofluorescence granule, optical microscopic signals of specific brain macrophages were compared with electron microscope signals. Specifically, correlation analysis of optical and electron microscope was performed using a control group treated with physiological saline solution and a rat section (n = 3) 14 days after 3-NP injection. The sections cut to a thickness of 100 μm using a vibratome were treated with a solution containing 2.3 M sucrose in 0.1 M phosphate buffer and rapidly frozen using liquid nitrogen. This was cut with a Leica EM UC7 ultramicrotome (Leica, Wetzlar, Germany) equipped with an FC7 cryochamber at -100 ° C into a 2 μm thick semithin section. The half-cut slices were incubated with Iba1 antibody (1: 500) overnight at 4 ° C and then incubated with Alexa Fluor 647 labeled goat anti-rabbit antibody (1: 300) at room temperature for 2 hours. Then, the cells were labeled with DAPI for 10 minutes. The sections were observed using a confocal microscope and the boundary of the cells was clearly distinguished using Differential Interference Contrast. Subsequently, the half-cut slices were postfixed, dehydrated and embedded in Epon 812, and the area of interest was cut and adhered onto the resin block. (70-90 nm thick), followed by staining with uranyl acetate. The same area observed with the confocal microscope was observed with an electron microscope (JEM 1010, JEOL, Tokyo, Japan).

As a result, Iba1-labeled control half-cut slices observed with a confocal microscope showed no autofluorescence (FIG. 4A), and the same sections of the same slices were observed under an electron microscope (FIG. 4C) As a result, the inactivated macrophages of the control group showed distinct heterochromatin-rich nuclei and thin cytoplasm (Fig. 4b and c). On the other hand, in rat striatum 14 days after 3-NP injection, autofluorescence was remarkable in Iba1-positive macrophages / macrophages (Fig. 5a), and the same region of the same section was observed with an electron microscope (Fig. 5B), the autofluorescence-emitting brain macrophages were filled with numerous lipofuscin inclusions and showed few cytoplasmic organelles (Fig. 5d and e ). As evidenced by the higher magnification in Figures 5e-f, autofluorescence was identified as lipofuscin granules, which are distinguished by a polymorphic high electron density representing a granular, fibrous, or plate-like pattern. On the other hand, cytoplasm expressing Iba1-positive signal in activated microglobulin / macrophage cells contained cell organs including mitochondria and Golgi complex, but did not contain lipofuscin inclusion bodies (Fig. 5e and f).

Example  5. Statistics

All data were expressed as mean ± standard error and the significance of the differences was assessed using Excel 2013 (Microsoft, Bellevue, WA, USA) and GraphPad Prism, Version 5 on both sides of the Student's t-test. A P value of less than 0.05 was considered statistically significant.

Claims (11)

Detecting the presence of autofluorescence which is emitted at 500 to 600 nm; And determining that a site in which autofluorescence is present is a brain injury site. The method according to claim 1, wherein the fluorescence emitted by lipofuscin is autofluorescence. The method of claim 1, wherein the autofluorescence is excited at 440 to 500 nm and emitted at 500 to 550 nm. The method of claim 1, wherein the autofluorescence is excited at 555 nm and emitted at 573 nm. The method of claim 1, further comprising identifying microglia-specific markers. 6. The method according to claim 5, wherein the small-cell-specific marker is Iba1 (ionized calcium-binding adapter molecule 1). The method of claim 1, further comprising identifying a lysosome-specific marker. 8. The method of claim 7, wherein the lysosome-specific marker is ED1 or Lamp1 (lysosomal-associated membrane protein 1). Measuring the autofluorescence signal at an excitation wavelength of 440 to 500 nm and an emission wavelength of 500 to 550 nm or an excitation wavelength of 530 to 570 nm and an emission wavelength of 550 to 600 nm to confirm the presence of lipofuscin; And determining that the site in which lipofuscin is present is a damaged site. A method for detecting postprandial phagocytosis comprising measuring an autofluorescence signal at an excitation wavelength of 440 to 500 nm and an emission wavelength of 500 to 550 nm or an excitation wavelength of 530 to 570 nm and an emission wavelength of 550 to 600 nm . (a) causing brain damage to mammals other than humans;
(b) detecting an autofluorescence signal at an excitation wavelength of 440 to 500 nm and an emission wavelength of 500 to 550 nm, or an excitation wavelength of 530 to 570 nm and an emission wavelength of 550 to 600 nm to measure a brain injury site;
(c) treating the test agent;
(d) measuring an area of brain damage by detecting an autofluorescence signal at an excitation wavelength of 488 nm and an emission wavelength of 523 nm, or an excitation wavelength of 555 nm and an emission wavelength of 573 nm; And
(e) comparing the test agent with the brain injury site of the control group not treated with the test agent to confirm whether the test agent inhibits or reduces the extent of brain injury site.

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