Exploring Barriers to Mass Transfer at the Intestinal Mucosa:
The Mucus Layer
y
Yuen Feung (Ian) Lim, Prof. Roger Lentle,
Dr. Patrick Janssen, A/Prof. Martin Williams
5th July 2011
The Mucus Layer - 1
Secreted by goblet cells and absorptive
enterocytes found around the villi (see
photo).
Largely known for its protective role of the
epithelial surface and a likely significant
contributor to mass transfer barrier known
as the UWL.
Composed of ~ 95% water and ~5% mucins
( glycoprotein
l
t i th
i a product
d t off MUC
(a
thatt is
genes).
A semi-permeable gel-like membrane
membrane.
Microscopic view of the cellular composition of the apex of a villus
p
(Science Photo Library 2011). Goblet cells are stained pink while absorptive enterocytes
are stained green
are stained green The Mucus Layer - 2
Previous macro- (Cone 2010) and micro-rheological (Sellers et al.
1991) characterization of mucus was conducted on post mortem
samples scraped from animal intestinal m
mucosa.
cosa
This procedure disrupts regional variation (Sawaguchi et al
al. 2002)
and may alter mucus physical characteristics as it detaches the basal
layer from the apical membranes of enterocytes.
The current work characterizes the physical and micro-rheological
properties of villus mucus in situ which would provide much needed
information that will enhance our understanding of the role of small
intestinal mucus layer
This is needed before the dynamics of mass transfer of nutrients and
pharmaceuticals across the mucus layer can be realistically modeled
The apparatus used to study living intestinal villi
Design of an experimental apparatus that allowed living ileal intestinal villi to be maintained in the visual field of a laser micro‐rheometer.
D l
Development of a system of micro‐adjustment enabling the focal f
f i
dj
bli
h f l
plane to traverse the length of the villus.
4
Summary of methods
The experimental apparatus allows clear visualization of individual living villus.
Application of mucin stain (Dextran Alexa Fluor 488, Life Technologies) allows mucus coat to be visualized
allows mucus coat to be visualized.
Micro‐rheological analysis is performed in situ and is related to villus
width and distance from the tip.
p
Micro‐rheometry of villus mucus
Fluorescent naked or amine coated polystyrene beads applied to the villi.
Naked beads adhere to mucus and allow its physical limits and intrinsic elasticity to be determined by applying force with
to be determined by applying force with laser tweezers. Amine coated beads float in the mucus layer and allow local apparent viscosity and rheology to be determined. The Brownian motion of beads at various
The Brownian motion of beads at various points along the villi (and its mucous environment) are recorded as videos. Relative movements of the beads are tracked and their movements analyzed.
6
General micro-rheology: results from regional ensembles - 1
r 2 (microns^^2)
Mean Squared Displacement (MSD) plots of ensembles surrounding the apices of villi.
m = 1
m = 1/2
Time (s)
MSD obtained from our experimental work (for a similar sized bead) done in situ of the of the
(for a similar sized bead) done in situ
mucus in its villus environment from small intestinal tissue
MSD obtained by Dawson et al (2003) for i it
in vitro micro‐rheological analysis of mucus i
h l i l
l i f
in sputum of patients with cystic fibrosis Mean Squared Displacement (MSD) plots were comparable with Mean
Squared Displacement (MSD) plots were comparable with
those reported by other researchers of sputum mucins.
Micro-rheology of ensembles - 2
G’(w), G’’(w) in
n Pascals
Elastic modulus (G’) – red and Loss modulus (G’’) ‐ blue
Frequency (rads/sec)
At lower frequencies of shear, mucus behaves as a viscous fluid (seen with the higher magnitude plot of G’’ over G
the higher magnitude plot of G
over G’)) while at higher frequencies of while at higher frequencies of
shear, the mucus exhibits elastic gel‐like properties (seen with the higher magnitude plot of G’ over G’’ after the crossover point).
Our mucus samples thus demonstrates ‘shear‐hardening’ attributes as postulated by Taylor et al. (2003).
Special mucus for the small intestine ?
G’(w), G’’((w) dynes/cm²
G’(w), G’’(w
w) dynes/cm²
There have been no previous reports on the rheology of mucus in situ or on villus
mucus ‐ our study shows it exhibits ‘shear‐hardening’
This differs from the ‘shear
This differs from the shear‐thinning
thinning’ reported in sputum and mucus harvested reported in sputum and mucus harvested
post mortem (Dawson et al. 2003, Ceilli et al. 2007).
This indicates, either that the rheology of mucus in situ differs from that in bulked samples, or that the behaviour of villus mucus differs radically from that in other regions
Villus mucus is known to be broadly similar in chemical composition to others in the GIT but to differ in the degree of glucosylation and sialation
G’’
G’
Frequency (rads/sec)
Frequency (rads/sec)
11
General resistance to mechanical abrasion?
Gut mucus mayy protect the delicate lining
g of the g
gut from
mechanical injury
Shear rates are normally low as digesta moves slowly through
the gut. Mucus are less viscoelastic at low shear rates and
thus may lubricate and facilitate flow
Shear rates will be higher
g
where a sharp
p edge
g p
projects
j
from a
bolus and is driven against the mucosa. Local stress
hardening will help to to protect the mucosa from damage.
Where do we go from here?
1.
Is mucus evenly deposited around the villus or is there more at the tip?(The latter would hinder mass transfer at the site of active absorption and
latter would hinder mass transfer at the site of active absorption and incapacitate villus vascular countercurrent systems).
Assess the depth of mucus at various points around the villi using a larger bead.
2.
Does mucus only protect the tip of the villus from mechanical abrasion?
A
Assess the micro‐rheological properties of the mucus layer at various distances th i
h l i l
ti
f th
l
t
i
di t
along the length and away from a villus.
Assess the mucus tensile breaking strength of mucus around the villi using strongly adherent polystyrene beads.
3
3.
Are these properties more evident in sites where shear forces are greater e.g. A
th
ti
id t i it
h
h
f
t
the colon?
Compare mucus properties at various locations along the GIT i.e. the small intestine and colon.
13
References
Celli, J. P., Turner, B. S., Afdhal, N. H., Woldt, R. H., McKinley, G. H., Bansil, R., et al.
(2007). Rheology of gastric muscin exhibits a pH-dependent sol-gel transition.
Biomacromolecules 8,
Biomacromolecules,
8 1580
1580-1586.
1586
Cone, R. (2009). Barrier properties of mucus. Advanced Drug Delivery Reviews, 61, 75-85.
Dawson, M., Wirtz, D., & Hanes, J. (2003). Enhanced viscoelasticity of human cystic
fibrotic sputum correlates with increasing microheterogeneity in particle transport. Journal
of Biological Chemistry, 278(50), 50393-50401. doi: DOI 10.1074/jbc.M309026200
Sawaguchi, A., Ishihara, K., Kawano, J., Oinuma, T., Hotta, K., & Suganuma, T. (2002).
Fluid dynamics of the excretory flow of zymogenic and mucin contents in rat gastric
processed
db
by hi
high-pressure
h
f
freezing/freeze
i /f
substitution.
b tit ti
Th JJournall off Histochemistry
The
Hi t h i t and
d
Cytochemistry, 50(2), 223-234.
Science Photo Library - Coloured SEM of a villus of the small intestine. Retrieved 13
June,, 2011,, from
http://www.sciencephoto.com/images/download_lo_res.html?id=805200105
Sellers, L. A., Allen, A., Morris, E. R., & Ross-Murphy, S. B. (1991). The Rheology of pig
small intestinal and colonic mucus: weakening of gel structure by non-mucin components.
Biochimica et Biophysica
Bioph sica Acta,
Acta 1115(2),
1115(2) 174
174-179.
179
Taylor, C., Allen, A., Dettmar, P. W., & Pearson, J. P. (2003). The gel matrix of gastric mucus
is maintained by a complex interplay of transient and non-transient associations.
Biomacromolecules,, 4,, 922-927.
Thank you for your time!!
Any Questions??