JP6106883B2 - A novel collagen fibrosis evaluation model using second harmonic light - Google Patents

Description

  The present invention relates to a fibrosis evaluation method for proliferative eye diseases using second harmonic light (SHG light). More specifically, the present invention relates to an in vivo and in vitro evaluation method using a SHG microscope for collagen proliferative eye diseases such as traction retinal detachment.

  Traction retinal detachment is a disease that causes fissure formation or refractory retinal detachment by forming a fibrotic membrane mainly composed of type I collagen fibers on the front and back surfaces of the retina and pulling the retina. There is still no effective drug therapy for traction retinal detachment, and detailed clarification of the pathological condition is urgent. Known factors that cause tractional retinal detachment include transforming growth factor (TGF) β, connective tissue growth factor (CTGF), and platelet-derived growth factor (PDGF).

  As described above, collagen fibers are greatly involved in the pathological condition of traction retinal detachment, but there are multiple eye diseases involving collagen, such as proliferative diabetic retinopathy and premacular membrane, in addition to traction retinal detachment. Therefore, the establishment of an in vitro and in vivo evaluation method that can appropriately evaluate collagen growth in ocular tissues is expected to greatly contribute to elucidation of pathological conditions and drug development of eye diseases involving collagen.

  Currently, as a method for evaluating collagen in eye tissues, a tissue staining method and an observation method using a scanning electron microscope or a reflection confocal microscope are known. However, the observation method using the tissue staining method and the scanning electron microscope requires fixing the observation target and has a drawback of being invasive. In addition, the reflection confocal microscope can perform in vivo measurement and three-dimensional measurement, but has a drawback that only the collagen fibers cannot be observed. In addition, it is currently impossible to obtain collagen orientation information using a tissue staining method or a reflection confocal microscope.

  On the other hand, Second Harmonic Generation (SHG) is a wavelength that is exactly half the wavelength of incident light after the interaction of two photons with a material that lacks central symmetry (non-centrosymmetric structure). This is a phenomenon that is converted into a single photon. The light obtained by this SHG is called second harmonic light (hereinafter abbreviated as “SHG light”). SHG light has been researched for many years in the electrical and mechanical fields, but in recent years, extensive research has also been performed in the life science field (Non-patent Documents 1 and 2), and technologies relating to collagen detection are also disclosed ( Patent Document 1).

JP2007-049990A

Chang Y et al. J Biol Phys. 2010 Sep; 36 (4): 365-83.Epub 2010 Jun 9. Latour G et al. Biomed OptExpress. 2012 Jan 1; 3 (1): 1-15. Epub 2011 Dec 1.

  In patent document 1, the technique regarding the evaluation method of the collagen culture tissue sample using SHG light is disclosed. However, this technique does not disclose any evaluation method for collagen existing in the eye tissue. In addition, there is no disclosure or suggestion of a specific evaluation method when collagen is present in the ocular tissue.

  In the present invention against the background of the above circumstances, an object of the present invention is to develop an evaluation method for evaluating an eye disease by evaluating collagen proliferation using SHG light.

  The inventors have found that even in the same collagen proliferative eye disease, there is a difference in the observable range and intensity of the SHG light depending on the collagen type characteristic to the pathological condition and the stage progression. The inventors have found that by evaluating this difference, it is possible to objectively evaluate the pathophysiology and severity of collagen proliferative eye disease, and have completed the present invention.

  The present invention comprises the following.

  The first configuration of the present invention includes an observation step of observing an eye tissue of an eye disease animal model using an SHG microscope, and evaluating the severity or stage of the disease state of the eye disease animal model. In vivo evaluation method.

  According to a second configuration of the present invention, the in vivo evaluation method further includes a SHG luminance measurement step of measuring SHG luminance of a normal site or a lesion site with an SHG microscope, and an area measurement step of measuring the area of the lesion site. The in vivo evaluation method according to the first configuration characterized by the above.

  In a third configuration of the present invention, the in vivo evaluation method further includes a calculation step of calculating an average SHG luminance at a lesion site using the SHG luminance in the SHG luminance measurement step and the area value in the area measurement step. The in vivo evaluation method according to the second configuration characterized by the above.

  According to a fourth configuration of the present invention, the in vivo evaluation method further includes a therapeutic effect evaluation step of administering a drug to an animal model of an eye disease and evaluating a therapeutic effect. The described in vivo evaluation method.

  According to a fifth aspect of the present invention, in the first to fourth aspects, the eye disease animal model is an animal model for collagen proliferative eye diseases such as proliferative vitreoretinopathy and preretinal membrane. This is an in vivo evaluation method.

A sixth configuration of the present invention is an in vitro evaluation method for a collagen three-dimensional cell culture system using an SHG microscope, the method comprising seeding cells that perform collagen fiber growth such as fibroblasts in a medium containing collagen; It is an in vitro evaluation method characterized by including the observation process of observing using an SHG microscope, and evaluating the presence or absence of collagen growth.
The seventh configuration of the present invention is the in vitro evaluation method according to the sixth configuration , wherein the cells that proliferate the collagen fibers are eye-derived cells such as retinal pigment epithelial cells.

According to the present invention, it is possible to provide an evaluation method for evaluating an eye disease using SHG light. That is, according to the present invention, in vivo and in vitro evaluation of ocular diseases can be performed by quantitatively and qualitatively evaluating the presence or absence of collagen proliferation using an SHG microscope.

The figure which showed the in-vitro SHG image observation result of a macular hole test subject surgical specimen The figure which showed the in-vitro SHG image observation result of the test sample of a premacular membrane test subject The figure which showed the in vitro SHG image observation result of the proliferative vitreoretinopathy subject's surgical specimen The figure which showed the in-vivo SHG image observation result of the proliferative diabetic retinopathy subject The figure which showed the in-vivo SHG image observation result of the proliferative diabetic retinopathy subject The figure which showed the in-vivo SHG image observation result of the proliferative diabetic retinopathy subject The figure which showed the in-vivo SHG image observation result of the proliferative diabetic retinopathy subject The figure which showed the in-vivo SHG image observation result of the proliferative diabetic retinopathy subject Figure summarizing in vivo SHG microscopic observation results of proliferative diabetic retinopathy subjects Diagram showing changes in collagen SHG in a 3D collagen gel system Diagram showing changes in collagen fibers in a collagen gel 3D system Figure showing changes in collagen fiber bundle diameter in a collagen gel 3D system Diagram showing the effect of SHG change of TGFβ2 in 3D collagen gel Diagram showing the effect of Periostin's SHG change in a collagen gel 3D system

Hereinafter, the in vivo and in vitro evaluation method of the present invention will be described in detail.
The evaluation method for collagen proliferative eye diseases using SHG light of the present invention comprises two evaluation methods, in vivo and in vitro.

First, the in vivo evaluation method will be described.
A practitioner who performs in vivo evaluation uses an eye disease animal model as an evaluation target in the in vivo evaluation method of the present invention. The eye disease animal model to be used is not particularly limited regardless of the presence or absence of collagen growth at the lesion site, and the practitioner can use various eye disease animal models. The practitioner can preferably use animal models of collagen proliferative eye diseases such as proliferative vitreoretinopathy and preretinal membrane. Moreover, although animal models for collagen proliferative eye diseases such as traction retinal detachment, proliferative diabetic retinopathy, and premacular membrane are not known yet, animal models that reflect these diseases may be used. .
With respect to the eye disease animal model to be used, the practitioner can perform pretreatments necessary for performing SHG microscopic observation such as ophthalmic solution administration and anesthesia on the eye disease animal model.

  The practitioner observes the eye tissue of the eye disease animal model with the SHG microscope (hereinafter referred to as “observation process”). In the observation step, it is preferable to observe not only the lesion site but also the entire eye tissue including the normal site. The determination of these normal sites and lesion sites may be performed by combining observations with a fundus camera, which is normally performed.

  The in vivo evaluation method of the present invention can further include an SHG luminance measurement step and an area measurement step. As a result, an objective index can be obtained for the disease state, severity, and stage of an animal model of eye disease.

  In the SHG luminance measurement step, various measurement methods can be adopted as long as the SHG luminance of the eye tissue can be measured. In the area measurement step, various measurement methods can be employed as long as the area value of a lesion or an abnormal site (hereinafter collectively referred to as “lesion site”) can be measured in the eye tissue.

An example of the SHG luminance measurement process will be described.
The practitioner performs SHG microscopic observation, selects several normal sites and performs measurement. Usually, the SHG microscope has a function of recording a captured image of an arbitrary part together with SHG luminance information for each pixel. Using this function, the practitioner performs SHG microscopic observation, selects several arbitrary sites determined to be normal sites, and records images of the selected normal sites, thereby providing SHG luminance information for each pixel in the normal sites. Can be obtained. By the same operation, SHG luminance information for each pixel in the lesion site can be obtained. With these image recording operations, the SHG luminance measurement process can be completed.

Next, the area measurement process will be described with an example.
According to the SHG luminance measurement process, SHG luminance measurement information in a normal site or a lesion site can be obtained. By using this SHG luminance measurement information, an accurate area value of the lesion site can be calculated. For example, an average value or maximum value of SHG luminance information in a normal part is set as a threshold value. In the SHG image of the lesion site, a pixel having SHG luminance exceeding the set threshold value is newly defined as the lesion site, and the sum of the pixels is set as the area value of the lesion site.

The in vivo evaluation method of the present invention can further include a calculation step, whereby a more objective index as a numerical value can be obtained for the eye disease animal model, and the disease state, severity, and stage of the eye disease animal model can be obtained. It has the effect of making it easier to objectively evaluate.
As an example of the calculation process, the calculation can be performed by dividing the sum of SHG luminances in this area by the area value in the area calculated by the area measurement process.

  The in vivo evaluation method of the present invention can further include a therapeutic effect evaluation step. Thereby, it has the effect that the therapeutic effect of a medicine etc. can be evaluated by an objective index based on SHG light.

Next, the in vitro evaluation method will be described.
In the in vitro evaluation method according to the present invention, the practitioner seeds cells that grow collagen fibers such as fibroblasts in a medium containing collagen as an in vitro evaluation system. Moreover, it is preferable to use an eye-derived cell such as a retinal pigment epithelial cell as a cell or the like that performs collagen fiber growth.
As the collagen gel, a commonly used collagen gel may be used. For example, it can be prepared by mixing and incubating Cell Matrix IA, DMEM, HEPES buffer, and the like.
The prepared collagen gel is seeded with cells for growing collagen fibers and cultured for a predetermined time. It is possible to objectively evaluate collagen growth by observing and measuring the seeded cells that perform collagen fiber growth with an SHG microscope as in the in vivo evaluation method.

  In the following, the in vivo evaluation method and in vitro evaluation method of the present invention will be described in more detail on the basis of examples. However, as a matter of course, the contents of the present invention are not limited to the following.

<< Example 1 >>
1. Under the consent of the subjects, surgical specimens of each subject were collected and fixed with 20% formalin solution. The fixed surgical specimen was uniformly spread on a slide glass and observed with an optical microscope or an SHG microscope.
2. In addition, the fundus image and the OCT image were observed using a fundus camera for the subject before the operation.

<Result>
1. The results are shown in FIGS. In each figure, the upper row is an OCT image or observation image by a fundus camera, the lower left is an enlarged observation image by an optical microscope, and the lower right is an observation image by an SHG microscope. FIG. 1 shows a macular hole, FIG. 2 shows a premacular membrane, and FIG. 3 shows a subject with proliferative vitreoretinopathy.
2. SHG light was not detected at all in the surgical specimens of the macular hole subject mainly with type IV collagen proliferative pathology (FIG. 1). On the other hand, SHG light was detected in surgical specimens of subjects with premacular membranes and proliferative vitreoretinopathy mainly having type I collagen growth pathology (FIGS. 2 and 3).
3. From these results, it was shown that the detection of SHG light differs depending on the type of collagen.
4). Collagen types may differ depending on the pathological condition, and even in the same pathological condition, collagen maturity usually varies depending on the degree of progression. Based on this, it is strongly suggested that changes in the SHG microscopic observation results are observed depending on the pathological condition and the degree of progression of the pathological condition. By objectively evaluating the SHG observation results, an objective evaluation of the pathological condition and the degree of pathological condition is made. It was strongly suggested that an evaluation could be possible.

<< Example 2 >>
<Method>
1. The eyes of proliferative diabetic retinopathy subjects were observed with an SHG microscope.
2. The SHG brightness of the site without proliferating tissue was measured, and the site with the highest brightness was taken as the threshold value for normal tissue.
3. A part having SHG luminance higher than the threshold value of the normal tissue was determined as a part having a proliferation film. Three arbitrary portions (318.5 × 318.5 μm ² ) of the portion having the proliferation membrane were selected, and SHG luminance was measured.
4). The average value per area of SHG luminance was calculated.
5. At the same time, fundus images and OCT images were observed and analyzed with a fundus camera.

<Result>
1. The results are shown in FIGS. In each figure, the upper part shows the observation result with the fundus camera, and the lower part shows the observation result with the SHG microscope. In the upper part of the figure, the left and right are fundus images and the center is an OCT image.
2. In the observation of the proliferative diabetic retinopathy subject of FIGS. 4 to 8 with the fundus camera, the proliferation of collagen was confirmed in all cases.
(1) SHG light was observed extensively in subjects with relatively poor disease progression, but the SHG intensity was not so high (Figs. 4 and 5).
(2) In addition, as shown in FIG. 6, although the range in which SHG light was observed was narrow, some subjects had high SHG intensity.
(3) In addition, SHG light was observed extensively and the SHG intensity was high in subjects whose disease progressed (Figs. 7 and 8).
3. FIG. 9 shows a summary of the area where SHG is observed and the intensity of each subject. Thus, by quantifying the SHG microscopic observation area and the SHG intensity, it becomes possible to objectively evaluate proliferative diabetic retinopathy subjects. In addition, from this result, the SHG microscopic observation area and the SHG intensity are not proportional to each other, but if both of these are high, they correlate with the progression of stage of proliferative diabetic retinopathy, that is, the maturity of the proliferation membrane The possibility was suggested.
4). These results are results showing that SHG observation is useful for evaluating the progression of the stage of proliferative diabetic retinopathy.

<< Example 3 >>
<Experiment method>
1. Collagen gels were first admixed Cell Matrix IA (700μL) and 5-fold concentrated DMEM (200μL), buffer (0.05N NaOH / 260 mM NaHCO 3 / 20mM HEPES 100μL). 200 μL of this mixed solution was added to a 3.5 mm glass petri dish and gelled by incubation at 37 ° C. for 30 minutes to prepare a collagen gel.
2. To the prepared collagen gel, 2 cc of human retinal pigment epithelial cells (hRPE) were seeded at a seeding density of 4.0 × 10 4 cells / mL. Immediately after seeding, TGFβ (3 ng / mL) was added to the culture. The culture medium was changed every 2 days, and the volume was 1.8 mL.
3. Using a phase contrast microscope and an SHG microscope, three visual fields were measured per dish at 24 hours, 4 days, and 8 days, and the average was evaluated as the SHG expression area ratio and average luminance.
4). Using a scanning electron microscope (SEM), the gel that had not been cultured and the surface of the gel that had been cultured for 8 days were observed after trypsin treatment.

<Result>
1. The results are shown in FIGS.
2. In the collagen gel three-dimensional culture system, the SHG expression area and the average luminance increased from the 4th day of culture, and statistically significant differences were observed for both indices on the 8th day (FIG. 10).
3. SEM microscopic analysis of the same condition gel revealed that collagen fibers were densely present in the gel after cell culture (FIG. 11).
4). In comparison of collagen fiber bundle diameters using SEM microscopic analysis, collagen fiber bundles that were statistically significantly thicker were recognized after culture than collagen fibers before culture (FIG. 12).
5. Under TGFβ-stimulated hRPE cell culture, a statistically significant increase in SHG expression area and average brightness was observed from the 4th day of culture, and the effect continued until the 8th day (FIG. 13, lower panel).
6). In SEM microscopic analysis of the same condition gel, collagen fibers were densely present in the gel of TGFβ-stimulated hRPE cells (FIG. 13, upper panel).
7). Collagen three-dimensional culture system using SHG microscope showed that collagen fibrosis and inhibitory effect can be observed in real time without modification such as fixation. Therefore, it is useful as a new in vitro evaluation method for collagen fiber remodeling.

Claims (1)

And the SHG intensity measuring step of measuring the SHG intensity of normal site or lesion of the eye tissue collagen proliferative ocular disease animal model using SHG microscope, the area measuring step of measuring the area of the lesion, the SHG intensity measurements An in vivo evaluation method comprising: a calculation step of calculating an average SHG luminance at a lesion site using SHG luminance in the step and an area value in the area measuring step; and a step of identifying a collagen type from detection of SHG light .