Biology Reference
In-Depth Information
analysis. It then highlights imaging methods including an emerging imaging
method, coherent anti-Stokes Raman scattering (CARS) microscopy, which allows
direct visualization of CLDs in cells and tissues without fixation or staining
(
Cheng, 2007
). In addition, biochemical and physiological methods for assessing
dietary fat absorption are included as these methods are needed in combination
for a thorough understanding of the process of dietary fat absorption.
9.1
MATERIALS AND METHODS
9.1.1
Collection of intestine tissue from mice for analysis
The small intestine is a complex organ with three different sections and multiple cell
types. The three sections of the small intestine include duodenum, jejunum, and
ileum. The anatomical marker between the duodenum and the jejunum is the liga-
ment of Treiz. In mice, the ligament of Treiz is found where the duodenum passes
beneath the transverse colon. Unfortunately, the jejunal-ileal junction is not clearly
identified. Therefore, the intestine is measured and proportionally divided for collec-
tion to normalize for similar regions of the intestine being studied. The majority of
dietary fat absorption occurs in the jejunum; however, each section has the capacity
for involvement in the process of dietary fat absorption. Several epithelial cell types
are present lining the lumen of the small intestine, including enterocyte, goblet,
paneth, and enteroendocrine cells. Enterocytes are the epithelial cell type within
the small intestine that are responsible for mediating dietary fat absorption. Entero-
cytes are produced by stem cells in the villus crypt. These cells migrate toward the
villus tip, die, and are sloughed off and excreted in a 3-4 day cycle (
Crosnier,
Stamataki, & Lewis, 2006
).
Collecting intestine tissue from mice
. After euthanizing the mouse, make a mid-
line incision in the abdomen of the mouse, and rapidly remove the small intestine.
Gently remove any mesenteric fat from the outside wall of the small intestine without
damaging the tissue. This is more challenging in obese mice than lean mice. Using
sharp dissecting scissors, gently run the blade along the outside of the intestine wall
to remove mesenteric fat. Tweezers may also be helpful to remove mesenteric fat.
Not removing the mesenteric fat will prevent accurate determination of tissue lipid
content.
Measure the length of intestine by laying the tissue out lengthwise on a clean sur-
face without stretching, cut the desired sections, and immediately put the samples
into ice-cold PBS. In an adult mouse, the length of the intestine can vary anywhere
from 25 to 40 cm, and often correlates with the size of the mouse. Measuring the
length of the intestine and dividing it into six equal length parts allows for the com-
parison of similar regions of the intestine from mouse to mouse. Approximate def-
initions of the regions of the intestine moving proximal to distal in relation to the
stomach: duodenum (section 1), jejunum (sections 2 and 3), jejunal-ileal junction
(section 4), and ileum (sections 5 and 6). If more precise intestine length
