論文の公開元へAn analysis of intestinal morphology and incretin-producing cells using tissue optical clearing and 3-D imaging
概要
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An analysis of intestinal
morphology and incretin‑producing
cells using tissue optical clearing
and 3‑D imaging
Tomonobu Hatoko1, Norio Harada1*, Shinsuke Tokumoto1, Shunsuke Yamane1,
Eri Ikeguchi‑Ogura1, Tomoko Kato1, Takuma Yasuda1, Hisato Tatsuoka1,
Satoko Shimazu‑Kuwahara1,2, Daisuke Yabe3, Yoshitaka Hayashi4 & Nobuya Inagaki1*
Tissue optical clearing permits detailed evaluation of organ three-dimensional (3-D) structure as well
as that of individual cells by tissue staining and autofluorescence. In this study, we evaluated intestinal
morphology, intestinal epithelial cells (IECs), and enteroendocrine cells, such as incretin-producing
cells, in reporter mice by intestinal 3-D imaging. 3-D intestinal imaging of reporter mice using optical
tissue clearing enabled us to evaluate both detailed intestinal morphologies and cell numbers, villus
length and crypt depth in the same samples. In disease mouse model of lipopolysaccharide (LPS)injected mice, the results of 3-D imaging using tissue optical clearing in this study was consistent with
those of 2-D imaging in previous reports and could added the new data of intestinal morphology. In
analysis of incretin-producing cells of reporter mice, we could elucidate the number, the percentage,
and the localization of incretin-producing cells in intestine and the difference of those between L cells
and K cells. Thus, we established a novel method of intestinal analysis using tissue optical clearing and
3-D imaging. 3-D evaluation of intestine enabled us to clarify not only detailed intestinal morphology
but also the precise number and localization of IECs and incretin-producing cells in the same samples.
Abbreviations
IEC Intestinal epithelial cell
EC Enteroendocrine cell
GLP-1 Glucagon-like peptide-1
GIP Glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide
CUBIC Clear, unobstructed brain/body imaging cocktails and computational analysis
3-D Three-dimensional
GFP Green fluorescent protein
LPS Lipopolysaccharide
The intestine is an important organ involved in digestion, absorption, and energy metabolic regulation such as
appetite regulation and nutrient accumulation through various intestinal hormones1. Intestinal epithelial cells
(IECs) are located at the boundary between the intestine and intestinal lumen and are composed of several
types of cells such as absorptive cells, goblet cells, Paneth cells, and enteroendocrine cells (ECs)2. Two major
incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide/gastric inhibitory
polypeptide (GIP), are secreted from enteroendocrine L cells and K cells, respectively, in response to nutrient
ingestion3–5, and have various biological effects such as enhancing glucose-dependent insulin secretion from
pancreatic β-cells6–8 and regulating body w
eight9–13. Thus, GLP-1 and GIP play a crucial role in glucose and
body weight control.
1
Department of Diabetes, Endocrinology and Nutrition, Kyoto University Graduate School of Medicine, 54
Kawahara‑cho, Shogoin, Sakyo‑ku, Kyoto 606‑8507, Japan. 2Preemptive Medicine and Lifestyle Related Disease
Research Center, Kyoto University Hospital, Kyoto, Japan. 3Department of Diabetes and Endocrinology, Graduate
School of Medicine, Gifu University, Gifu, Japan. 4Division of Stress Adaptation and Protection, Department
of Endocrinology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan. *email:
nharada@kuhp.kyoto-u.ac.jp; inagaki@kuhp.kyoto-u.ac.jp
Scientific Reports |
(2022) 12:17530
| https://doi.org/10.1038/s41598-022-22511-7
1
Vol.:(0123456789)
www.nature.com/scientificreports/
Immunohistological analysis of intestinal tissue sections generally has been used for evaluating the number
and localization of target cells in intestine14. However, it is difficult to envision the three-dimensional (3-D)
structure of villi and crypts and to evaluate the number and localization of IECs and ECs under two-dimensional
(2-D) intestinal tissues.
Tissue optical clearing is an observational method that has shown remarkable progress in recent years. Clear,
unobstructed brain/body imaging cocktails and computational analysis (CUBIC) protocol were established in
2014, making the whole body transparent in mice and enabling detailed evaluation of each organ’s 3-D structure
and its cells by tissue staining and autofluorescence15–17. Thus, CUBIC protocol may facilitate not only visualization of 3-D intestinal imaging but also 3-D intestinal structure e valuation18,19.
In the present study, we succeeded in obtaining detailed 3-D intestinal imaging of reporter mice using the
CUBIC protocol and analyzing intestinal morphology, IECs, and ECs such as incretin-producing cells.
Materials and methods
Animals. Villin, encoded by Villin1, is an actin binding protein expressed throughout the crypt-villus axis in
small intestine and colon of mice20,21. Villin1-Cre transgenic mice and Ai14 mice were previously generated (JAX
stock #004586, #007908) (Jackson Laboratory, Bar Harbor, ME)21,22. Villin1-Cre and Ai14 heterozygous (Villin1Tomato) mice, which enable visualization of IECs by tdTomato fluorescence, were generated by crossbreeding
Villin1-Cre transgenic mice and Ai14 homozygous mice. Analysis of intestinal morphology and IECs was performed using 13-week-old male Villin1-Tomato mice.
Glucagon-green fluorescent protein (GFP) knock-in (Gcg-GFP) heterozygous mice and GIP-GFP knockin (GIP-GFP) heterozygous mice, which enabled visualization of L cells and K cells, respectively, were previously generated23,24. Villin1-Tomato and Gcg-GFP or GIP-GFP heterozygous (Villin1-Tomato+ Gcg-GFP or
Villin1-Tomato+ GIP-GFP) mice, which enabled visualization of both IECs by tdTomato fluorescence and L
cells and K cells by GFP fluorescence, were generated by crossbreeding Villin1-Tomato mice and Gcg-GFP or
GIP-GFP homozygous mice. Analysis of incretin-producing cells was performed using 13-week-old male Villin1Tomato+ Gcg-GFP or Villin1-Tomato+ GIP-GFP mice. All mice had free access to standard rodent chow and
were housed in a temperature-controlled room with a light–dark cycle of 14:10 h. All animal experiments were
performed in compliance with ethical regulations in Kyoto University. Animal care and procedures were approved
by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine (Medkyo18245). All
animals were handled in accordance with the ARRIVE guidelines.
Collection of intestine samples and tissue clearing. Mice were anesthetized with intraperitoneal
injection of pentobarbital sodium (10 ml/kg) and transcardially perfused with ice-cold phosphate-buffered
saline (PBS) followed by ice-cold 4% (w/v) paraformaldehyde (PFA) (Wako Pure Chemical Industries, Osaka,
Japan) after insertion of a 23-gauge needle into the left ventricle. The small intestine and the colon of all mice
were excised. A total of five samples of about 1 cm of small intestine were collected, one each from the oral side
(S1) and the anal side (S5), one from the intermediate area between S1 and S5 (S3) and one each from the intermediate areas between S1 and S3 (S2) and between S3 and S5 (S4) of the small intestine (Supplemental Fig. 1A).
S1, S2–S3, and S4–S5 corresponded to duodenum, jejunum and ileum, respectively. A total of three samples of
colon were collected, one from the oral side (C1), center (C2) and anal side (C3) of the colon. All samples were
immediately immersed in PFA at 4 °C with gentle shaking overnight and washed three times for more than 2 h
each in PBS at room temperature with gentle shaking. All samples were immersed in 50% (v/v) CUBIC-L (Tokyo
Chemical Industry Co., Ltd, Tokyo, Japan) reagent (1: 1 mixture of water: CUBIC-L) at 37 °C with gentle shaking for 24 h followed by CUBIC-L at 37 °C with gentle shaking for 24 h. After washing in PBS, the samples were
immersed in 5 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) (Dojindo Laboratories, Kumamoto, Japan) in PBS
at room temperature with gentle shaking for 30 min followed by washing in PBS. The samples were immersed
in 50% (v/v) CUBIC-R+ (Dojindo Laboratories) reagent (1: 1 mixture of water: CUBIC-R+) for 24 h followed by
immersion in CUBIC-R+ at room temperature with gentle shaking for 24 h.
Lipopolysaccharide (LPS)‑induced intestinal injury and infection model mice. 13-week-old
male Villin1-Tomato mice were injected intraperitoneally with 10 mg/kg LPS from E. coli O111:B4 (SigmaAldrich, St. Louis, MO) in saline or an equivalent volume of saline alone25–27. After 24 h of injection, the mice
were anesthetized and transcardially perfused with the methods described above. Five samples of small intestine and three samples of colon in all mice were collected. In the 3-D imaging group, all samples in each five
LPS-injected mice and saline-injected mice (control mice) were cleared by CUBIC methods. In the 2-D imaging group, all samples in each three LPS-injected mice and control mice were fixed in 4% (w/v) PFA solution,
embedded in paraffin, and stained with hematoxylin and eosin (HE).
Image acquisition. 3-D fluorescence images were acquired by spinning disk confocal microscopy (Dragon-
fly, Andor Technology Ltd., Belfast, UK) on an IX83 (Olympus Corp. Tokyo, Japan) device through a UCPLFLN
20× objective lens (Olympus, numerical aperture [NA], 0.7) using a 405 nm laser for DAPI staining (blue), a
488 nm laser for GFP fluorescence imaging (green), and a 561 nm laser for tdTomato fluorescence imaging (red).
Data were collected in Spinning Disk 40 μm pinhole mode on a scientific complementary metal oxide semiconductor (sCMOS) camera (Zyla4.2Plus USB3) (Andor Technologies), which had a measured pixel size of 0.95 μm
× 0.95 μm. Using the Z scan mode, each sample was scanned every 2.5 μm in small intestine and colon. Acquired
microscopic images were further processed by the deconvolution algorithm for 3D volume reconstruction. ...
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