AIM: To propose a simple and effective method suitable for analyzing the angle and distribution of 2-dimensional collagen fiber in larger sample of small intestine and to investigate the relationship between the angles of collagen fiber and the pressure it undergoes.

METHODS: A kind of 2-dimensional visible quantitative analyzing technique was described. Digital image-processing method was utilized to determine the angle of collagen fiber in parenchyma according to the changes of area analyzed and further to investigate quantitatively the distribution of collagen fiber. A series of intestinal slice’s images preprocessed by polarized light were obtained with electron microscope, and they were processed to unify each pixel. The approximate angles between collagen fibers were obtained via analyzing the images and their corresponding polarized light. The relationship between the angles of collagen fiber and the pressure it undergoes were statistically summarized.

RESULTS: The angle of collagen fiber in intestinal tissue was obtained with the quantitative analyzing method of calculating the ratio of different pixels. For the same slice, with polarized light angle’s variation, the corresponding ratio of different pixels was also changed; for slices under different pressures, the biggest ratio of collagen fiber area was changed either.

CONCLUSION: This study suggests that the application of stress on the intestinal tissue will change the angle and content of collagen fiber. The method of calculating ratios of different pixel values to estimate collagen fiber angle was practical and reliable. The quantitative analysis used in the present study allows a larger area of soft tissue to be analyzed with relatively low cost and simple equipment.

Several G.I. diseases can change the collagen fiber structure in the G.I. tract[1-4]. The collagen fiber content and its distribution outside the cell are closely related to some diseases. For example, systemic sclerosis (scleroderma) will replace smooth muscle and deposit large amounts of collagen instead. Gastrointestinal sclerdema is a common clinical disorder; however, due to its complicated etiology, most of them are not easily diagnosed and all the patients with such disease manifested distinct collagen fiber transformations. Early collagen fiber transformations are swelling and homogenization and then become thickened, sclerosed and arranged in a close order[5-9]. Collagen synthesis is increased especially the ratio of fine collagen fibers; at the same time, the smooth muscle fiber bundles become homogeneous, sclerosed and atrophic. There are changes occurred in the orientation of collagen fibers[10-13]. Obstructive diseases could also change the collagen structure and content[14-16]. In normal tissue the submucosal layer consist almost entirely of collagen, which is called the skeleton of the small intestine, and it is well known that the fibers run in a cross-cross pattern with 30 degrees angle in longitudinal direction[17-20]. The directional distribution of collagen fibers has a very important role in studying the function and self-repair of soft tissue[21].

The application of digital image-processing technology in medicine field has offered an accurate, simple, convenient and rapid method for the measurement and analysis of large amounts of medical images, especially for the quantitative analysis of smaller pictures, such as microscopic images. At the present time, the techniques for the quantitatively analyzing the distribution of collagen fibers consist mainly of several kinds as follows: that is, the method of quantitatively polarized light microscopy, small angle beam dispersion, image analysis and X-ray diffraction. These methods will sometimes be constrained by the image size, time cost or image connecting, and 90^o orientation of template etc[23].

The purpose of this paper is to propose a method suitable for analyzing the angle and distribution of 2-dimensional collagen fiber in larger sample and to investigate the relationship between the angles of collagen fiber and the pressure it undergoes. At the same time, this paper also offers an effective method for research of the large area collagen fiber distribution.

According to the steps mentioned above, the calculation results were shown in Table 1, where the first column listed the pressures the tissue were received and the first row was the polarized light angles being rotated. We found that for the same slice, with polarized light angle’s variation, its corresponding area ratio is not identical; for each tissue under different pressures, its biggest area ratio is not identical either. To obtain the smooth curve showing the relationship between the angle and the corresponding ratio, we made a nonlinear normality on the sample data[36], the abscissa of the curve was δ’s integer multiple and the ordinate the series value in array Y (i) (Figure 3).

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Figure 3 Relationship between angles of polarized light and their corresponding pixel ratio in series of slice images.

The relationship between pressures tissue received and their corresponding fiber angle was shown in Figure 4.

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Figure 4 Relationship between pressures tissue received and their corresponding fiber angle.

The relationship between pressures tissue received and their corresponding biggest ratio was shown in Figure 5.

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Figure 5 Relationship between pressures tissue received and their corresponding biggest ratio.

We tested other intestinal slice’s images (of the same animal) and found that their properties above were similar by and large.

From the data in Table 1, we found that in the same tissue sample there exist obvious alterations in the collagen area ratio as the angles of polarized light changed; In different slice’s images with different pressures, its biggest ratio was also not identical (Figure 4), and the biggest value was fairly obvious. However, it can not be determined whether the magnitude of collagen fiber angle and the fiber content are all in direct proportion with the pressure that the intestines received (Figure 4, Figure 5), because this depends some degree on the way how it receives the pressure. Using other methods to change its stress still needs to be tested in future experiments. The method of calculating ratios of different pixel values to estimate collagen fiber angle has its feasibility and reliability, which allows a larger area of soft tissue being analyzed with relatively low cost and simple equipment. The disadvantage of this method is the difficulty in determining an appropriate threshold value as well as a definite scope suitable for analyzing which have very important influence on the study results. At the same time, the less δ is to be selected, the more accurate the angle will be.

This paper has tried to acquire the collagen fiber angle of soft tissue in intestine slice through introducing a quantitative analysis method for calculating different pixel values whose validity is verified with computer program, and suggested a practical and effective method for basic research on G.I. disease.