Three-dimensional Construction of Micrometer Level in Rat Stomach by Synchrotron Radiation

doi:10.21203/rs.3.rs-38915/v2

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Three-dimensional Construction of Micrometer Level in Rat Stomach by Synchrotron Radiation

https://doi.org/10.21203/rs.3.rs-38915/v2

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published 20 Mar, 2021

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Background: The structural changes of gastric mucosa are considered as an important window of early gastric lesions. This article shows an imaging method of the stomach that does not use imaging agents. X-ray phase-contrast images of different stages of gastric development were taken using micrometer level X-ray in-line phase-contrast imaging (XILPCI) technique on synchrotron radiation facility. The aim of the study was to demonstrate that the imaging technique is an appropriate method for micron imaging of the gastric structures.

Methods: The stomachs of 4-, 6- and 12-week-old rats were removed and cleaned. XILPCI has 1000 times greater soft tissue contrast than that of X-ray traditional absorption radiography. The projection images of the rats stomachs were recorded by an XILPCI charge coupled device (CCD) at 9 μm image resolution.

Results: The X-ray in-line phase-contrast images of the different stages of rats’ gastric specimens clearly showed the gastric architectures and the details of the gastro-duodenal region. 3-dimensional stomach anatomical structure images were reconstruction.

Conclusion: The reconstructed gastric 3D images can clearly display the internal structure of the stomach. XILPCI may be a useful method for medical research in the future.

Biomedical Engineering

Synchrotron radiation phase-contrast imaging

3-dimensional gastric structure images

different stages

The stomach is an important organ in the alimentary tract. However the imaging observation of its development has not been realized yet. This mainly employs gastroscopy which could obtain the image at a millimeter level. There are high-resolution images of X-ray absorption of the human skeleton, but the images of the human abdominal organs remain poor. X-ray in-line phase-contrast imaging (XILPCI), using the X-ray phase-change after an X-ray passes through objects, has emerged as an imaging method and used to acquire micrometre level images of soft tissues.

XILPCI can be combined with computed tomography (CT) and form the phase-contrast CT. It is also a kind of the diffraction CT and will probably be a imaging method for soft tissues without using imaging agents. The micrometre level image resolution of XILPCI of soft tissues can reach 0.37 μm which make it able to obtain the more accuracy gastric structure. With the advantages mentioned above, this type of XILPCI method could be a good research method in the future of medicine.

A growing number of studies have shown that intestinal development has obvious temporal characteristics and is regulated by many factors[1]. The rapid growth and functional maturation of the stomach and small intestine in newborns may reflect an adaptation process, which include being exposed to an open environment and processing nutrient soon after the birth of newborns. Morphometrical analysis revealed that the growth rate is greater in the gastric corpus region than in the cardiac and pyloric regions, and the same increase is observed in the mucosal layer compared with the one in other layers. It has been shown that gastric mucosal cell proliferation was elevated during early postnatal development in the rat model[2]. Some evidence indicate that growth of gut mucosal tissues is associated with an enhanced DNA synthesis rate[3] and a decreased cell turnover rate in neonatal animals[4]. There are lots of researches on gastrointestinal cells, but few studies focus on the characteristic morphological in stomach and intestine of the rats. Our team focused on the XILPCI images of morphology and structure of normal rat stomach. Hence we choose to use the young and adult rats as the objects of study.

Rats’ gastric specimens characteristics in XILPCI projection images

In order to intuitively understand the changes of gastric morphology and structure in different age groups, the XILPCI projection images of gastric specimens are shown in Fig. 1. Changes were observed at different week in rats. Fig. 1A show the ordered and regular tissues of a 4-week-old young gastric normal specimen, and the gastric walls are smooth without any hyperplasia. The XILPCI image are more detailed than the X-ray traditional image of a normal gastric specimen[5-9], aswe could only see the overlapping walls of the stomach in the absorption images, and the images are very fuzzy with an unclear internal texture (as shown in Fig. 2).

On XILPCI images, a 4-week-old image shows a uniform grey level, which indicates that the wall of the 4-week-old gastric corpus is as thick as the gastric fundus. It is obvious that the wrinkles of the fundus are abundant, while the gastric fundus is much thinner. In the middle of Fig. 1B and C, there is a demarcation line between the gastric corpus and the gastric fundus. Fig. 1B demonstrates the characteristics of a 6-week-old adult gastric normal specimen XILPCI image. The gastric wrinkles are more extensive, but the wrinkles of the 6-week-old gastric fundus are fewer than those of the 4-week-old gastric fundus, as shown in the image that the grey of a 6-week-old gastric fundus is lighter than that of a 4-week-old fundus. The 6-week-old gastric corpus is thicker than the 4-week-old gastric corpusfor the grey of a 6-week-old gastric corpus is deeper in image than that of 4-week-old. Fig. 1C demonstrates the characteristics of mature gastric normal tissues from a 12-week-old normal gastric specimen. The wrinkles in the walls of the gastric corpus and duodenum are the most abundant.

CT images of the same rats' gastric specimens

The XILPCI 3-dimensional slices were rebuilt by means of a filter back projective algorithm. As shown in Fig. 3, the details can be visualized inside the gastric tissue from under the gross anatomy and the gastric inner surfaces clearly presented longitudinal branching wrinkle and gastric pits.

Traditional absorption CT images of a normal gastric specimen were obtained by SIEMENS Inveon Scanners with minimum resolution of 11 μm and Inveon Acquisition Workplace with 1.5 Service Pack. The X-ray energy was 80 keV and 400 μA. The absorption CT image of a gastric specimen was Fig. 4. However, it was not clearer than gastric phase-contrast images and the wall fold of the normal gastric specimen cannot be observed.

The d, e and f parts of the gastric specimens in Fig. 3 were taken out and fixed in formalin solution. Further analysis of these specimens show the same shape as Fig. 5, but XILPCI images could not show the clear internal structure the same as Fig. 5. At present, the gold standard for diagnosis is still biopsy. Hematoxylin-eosin (HE) staining processes are as follow: these gastric specimens were dehydrated and dried, and then the d, e and f part of specimens were embedded in paraffin. Paraffin-embedded sections were made into pathological section with 5 μm thickness and stained with HE to evaluate general morphology. In the body portion of the stomach after HE staining, the micrograph and macrograph showed a cross section of the gastric wall (Fig. 5). Similar to the other parts of the gastrointestinal tract, there are four layers of structure in the gastric wall, which are outer mucosa, inner submucosa, muscular external layer, and serosa. During the growth and development of the rat, the fundus, formed by the upper curvature of the organ from the muscular external layer, is the thickest part at all three ages (Fig. 5d, e, f). There is an obvious line between the gastric fundus and corpus. A mucosa layer is also palpable, but with overt differences seen on HE staining, and we could see that the mucosa layer in the 4-week-old rats show more alkalinity than that in the 6- and 12-week-old rats, especially at 12 weeks (Fig. 5f). The line could also be observed in Fig. 5d, e and f between the gastric fundus and corpus.

Three-dimensional reconstruction images of the same rats' gastric specimens

In order to further reconfirm the advantage of the XILPCI 3-dimensional images, we reconstructed 3-dimensional images of the different stages of the stomach as shown in Fig.6. The XILPCI 3-dimensional images show a clear structure of the interior stomach (showed as video 1), and we could see the clear images of the villus of the fundus in the young rat stomach. There is a clear demarcation line between the gastric fundus and corpus. The wall of the 12-week-old rat stomach are thicker than those of the 4-week-old. The conclusion here shows that XILPCI has a high anatomical accuracy for stomachs images. In addition, the thickness of the gastric wall at various stages were measured in Fig. 6 to compare the thickness of gastric specimens. As shown in Fig. 7, the results indicate that the average value of gastric wall at the same stage are very similar, and the measurement of gastric wall by 3-dimensional images in Fig. 6 has certain reference significance. Just as shown in Fig. 7, the thickness of 4-week-old gastric wall for the right panel is lower than that of the left one, but the thickness of 6-week-old gastric wall is higher for the right panel, probably because of the randomness of specimen selection and the existence of measurement error. However, these results are also in line with the requirements of statistics and within a reasonable range.

Limitations of XILPCI must be overcome if XILPCI is to have wider applications. One limitation is that it takes around one hour to shoot a larger specimen as it requires images shooting in several segments. The experiment is only effective for static specimens, so the device must be improved to shorten the imaging time required for living specimens. In addition, movement artifacts will appear when the specimens being taken are moving. In this experiment, because of the high resolution of the imaging, a merely small movement can produce imaging artifacts. Specimens shrinkage occurs when the specimens are taken on the sample table, which also produces movement artifacts under X-ray irradiation. The current solution is to make the specimen as dry as possible so as to shorten the experiment time. Movement artifacts can also affect the image quality and require further solution in later researches.

The CCD camera of BL13W1 of SSRF can obtain a resolution of 0.37 μm, making the XILPCI helpful for angiography. At present, Applications on humans in the field of phase-contrast mammography have been reported by using both synchrotron radiation and conventional sources[10]. With further development, XILPCI would be a valuable imaging method for medical researches.

In summary, we have applied the XILPCI method to imaging of the rat stomach without the need for imaging agents. The XILPCI projection images showed that the development of the normal gastric structure may cause thickening of the gastric wall and coarseness of the gastric texture.

XILPCI is an imaging method using the X-ray phase variation. This makes it able to achieve micron-scale image resolution of biological tissue and can probably replace the traditional X-ray absorption method with synchrotron radiation sources. Due to the advantages mentioned above, the XILPCI method is likely to be widely used in the future at a low cost.

Setup and specimens

Twenty-one Sprague-Dawley healthy male rats were purchased from the Animal Centre of Capital Medical University in China. The rats were randomly divided into three groups with seven rats in each one. The rats were housed in cages under a controlled temperature of 22.0±1.0°C and 12 hours light–dark cycles. They were fed on standard laboratory chow and water and allowed to acclimate for more than 7 days. The rats were starved for 12 hours and sacrificed under anaesthesia with an intraperitoneal injection of pentobarbital sodium at 4, 6 and 12 weeks of age. The stomach of the rat was removed and the stomach cavity is filled with 10% formalin to maintain the morphological structure of the stomach for X-ray image scanning. Stomach specimens were shown Fig. 8. These stomach tissues were cleaned with normal saline, fixed in 10% neutral phosphate-buffered formalin solution for 24 hours and embedded in paraffin. 5 μm thick of the specimens were obtained from the stomach and stained with HE staining. This animal study was in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal welfare Committee on the Ethics of Animal Experiments of Capital Medical University (Protocol No. AEEI-2016-079) approved the experiment protocol. All surgery was performed under pentobarbital sodium anaesthesia, and all efforts were made to minimize suffering.

Imaging principle of XILPCI

XILPCI experiments were done at synchrotron radiation[11] facility. Synchrotron radiation is electromagnetic radiation which is emitted by relativistic charged particles traveling along a turning orbit under the action of electromagnetic field. Synchrotron radiation, as a light source, has an obvious high-brilliance and flux, wide energy spectrum and very short pulses.

The gastric XILPCI experiments were performed using the BL13W1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF). The BL13W1 beamline partial facility of SSRF was depictedas shown in Fig. 9. A. Snigirev[5,9,12-14] obtained phase-contrast images by using a synchrotron radiation light in 1995. The XILPCI method can use multi-colour light sources, therefore eliminate the need for the burdensome complexity of a monochrome system.

The complex refractive index n can be used to depict the XILPCI characteristics. The refractive index n is smaller than the number 1 and n formula is shown as:

After X-rays go through an object, their phase and amplitude change. Real component presents the phase changes, while imaginary part presents the amplitude attenuation. In XILPCI of lighter elements (C, H, O, etc.) of the object, δ is 1000 times greater than β, so the phase change quantity is much larger than the change quantity of X-ray absorption attenuation. There is a phase-contrast imaging of micron scale image resolution, so XILPCI images can show microstructures of objects.

Steps of XILPCI

Specific experimental methods: First, in order to reduce the XILPCI artifacts caused by specimen deformation, the specimens are placed in the air and dried for a period of time. Then the dry specimens were wrapped with insulating materials and placed on the sample table.

After evaluating different levels of X-ray energy, we chose the energy level of 17.5 keV to be used for this experiment. The energy value can be changed and is determined according to the specimen size. Different specimens have different energy values in order to obtain clear X-ray phase-contrast images. The images were whiter if the energy was higher than 17.5 keV, and the imaging exposure time also increased when the energy was lower than 17.5 keV. Images will be dark if the exposure time is not appropriate. It will also take a longer time to shoot those images required for CT if the exposure time increases and gastric specimens will undergo serious deformation. Therefore, 17.5 keV is an optimal parameter of comprehensive factors. The distance from the light source to the specimen was 59.3 m and the detector was 60 cm from the specimen to CCD, with 9 μm image resolution and an exposure time of 8 ms. It took more 20 minutes to obtain XILPCI projection images of a gastric specimen by using 0.1 degree steps from 0 to 180 degrees over the gastric specimen.

Histology and pathology scoring

Stomach tissue specimens were dewaxed and stained with the HE staining. Sections were fixed on microscope slides and observed with an Olympus DP72 MacroView (Japan). Histological scoring was based on a previously adapted scoring system.

Statistical analysis

The data were analyzed by using GraphPad Prism 5.0 software package (GraphPad Software Inc., San Diego, CA, USA) of variance of the gastric wall thickness and all values were expressed as mean and standard error of mean (S.E.M.); n was the number of animal in each experiment. The differences among groups were analyzed using a one-way analysis of variance followed by Dunnett’s multiple comparison. A p value less than 0.05 was considered statistically significant. The power of the results was 86.5% by the software power analysis and specimen size software.

XILPCI: X-ray in-line phase-contrast imaging;

CCD: charge coupled device

BL13W1: Imaging and Biomedical Application Beamline;

SSRF: Shanghai Synchrotron Radiation Facility

3D: three dimensional

Acknowledgement

Our deeply gratitude goes the assistance of all the staff in BL13W1 from Shanghai Synchrotron Radiation Facility.

Authors’ contributions

QT completed the experimental data, worked on the algorithm design, measured the thickness of gastric wall and drafted the manuscript. JDX conceived the study, contributed to specimen preparation and conclusions and manuscript modification. XHT and CCG contributed to the animal handling. SZY reconstructed the 3-dimensional reconstruction images. All authors read and approved the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (Grant No. 81673671 and 81274173); the Natural Science Foundation of Beijing (Grant No. 7144189 and 7122017); and the Science Foundation of Capital Medical University (Grant No. 17ZR24).

Ethics approval and consent to participate

Date of our animal study trial registration: 2015-07-11. Registered with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. (Protocol No. AEEI-2016-079).

Data Availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

Competing Interests

The authors declare no competing interests.

Consent for publication

All authors consent for publication.

Electronic supplementary material

Video 1.3D video of a 4-week-old rat stomach

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stomachvideo.mp4

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Three-dimensional Construction of Micrometer Level in Rat Stomach by Synchrotron Radiation

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Abstract

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Results

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Conclusion

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Abbreviations

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