JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2005, 56, Supp 3, 71–87
www.jpp.krakow.pl
Original articles
1
1
2
3
T. SKRZYPEK , J.L. VALVERDE PIEDRA , H. SKRZYPEK , J. WOLIÑSKI ,
2
1
1
W. KAZIMIERCZAK , S. SZYMAÑCZYK , M. PAW£OWSKA , R. ZABIELSKI
3,4
LIGHT AND SCANNING ELECTRON MICROSCOPY EVALUATION
OF THE POSTNATAL SMALL INTESTINAL MUCOSA
DEVELOPMENT IN PIGS
1
Department of Animal Biochemistry and Physiology, Faculty of Veterinary Medicine,
Agriculture University in Lublin, Lublin, Poland
2
Department of Zoology and Ecology, Catholic University of Lublin, Lublin, Poland
3
The Kielanowski Institute of Animal Physiology and Nutrition,
Polish Academy of Sciences, Jab³onna, Poland
4
Department of Physiological Sciences, Faculty of Veterinary Medicine,
Warsaw Agricultural University, Warsaw, Poland
Modifications in the structure of gastrointestinal mucosa is often used to evaluate gut
function
for
instance
during
the
development
or
in
response
to
particular
food
components. Scanning electron microscopy (SEM) gives a chance to observe the
surface of the gut epithelium in three dimensions. However, this technique is seldom
used due to technical difficulties. The present study attempted to investigate the
intestinal mucosa structure changes in the postnatal pig using light and scanning
electron microscopy technique. Experiments were carried out on sow reared piglets
from birth until 38 days of age. Piglets were sacrificed at birth and at the 3
and 38
th
, 7 , 21
rd
th
st
day of life. The entire gastrointestinal tract was immediately harvested and
the whole thickness tissue samples were taken from the duodenum, jejunum and
ileum for optical and scanning electron microscopy. SEM analyses corroborated with
histometry made by optical microscopy. Moreover, a number of shape modifications
of the villi and its surface have been observed. The development changes in small
intestine mucosa during the first 3 weeks were manifested in shape, size and density
of villi. In conclusion, the structure of small intestinal mucosa undergoes profound
structural changes. SEM gives a new dimension in the investigation of gut mucosa.
Key
w o r d s : development, intestinal villi, enterocytes, goblet cells, apoptosis
72
INTRODUCTION
The pig gut is exposed to a variety of stress factors particularly in the early
postnatal period and just after weaning. In response to that, the gut undergoes
profound
changes
resulting
in
accelerated
tissue
growth
and
functional
maturation (1-4). After birth, changes are induced by ingested nutrients and a vast
number
of
bioactive
substances
present
in
the
colostrum
and
milk,
and
are
mediated by hormones and nerves. Among the bioactive substances, the role of
colostrum and milk epidermal growth factor, insulin-like growth factor-I and -II
β
(IGF-I and -II), insulin, transforming growth factor-
β),
(TGF-
glucagon-like
peptide-2 (GLP-2), and leptin was demonstrated as stimulating gastrointestinal
(GI) tissue growth and function maturation (2, 5-7). It has been reported that the
small intestine in piglets increases up to 70% in total tissue weight, 115% in
mucosal tissue weight, 24% in length, 15% in diameter, 24% in crypt depth and
33% in villus height during the first postnatal week, reaching the maximum
length in the second week after birth (8, 9). After weaning, the environment in the
GI lumen changes drastically due to the replacement of highly digestible sow
milk by solid food, mainly of plant origin. The control provided by milk bioactive
substances is over, although the gastrointestinal tract has to adapt the digestive
processes (e.g. motility and enzyme secretion) to the new type of food, and
control the microbial ecosystem (10-13). In response to weaning the GI tract in
healthy animals undergoes major changes in structure and function, particularly
in
the
small
intestinal
mucosa
(14,
15).
Animals
failing
to
adapt
to
these
challenges, display poor growth, diarrhea or death associated with gut bacteria
overgrowth (16). The most obvious changes in the structure of the small intestine
following weaning are a reduction in villus height and an increase in crypt depth.
Hampson (17) reported that, following weaning at 21 days after birth, villus
height was reduced to 75% of the pre-weaning value one day after weaning, and
then continued to decline to approximately 50% of the pre-weaning value 5 days
after weaning. Villus height reduction is suspected to be the result of an increased
rate of cell loss, which subsequently leads to increased crypt cell production and
increased crypt depth (15). Along with the reduction in villus height and the
increase in crypt depth, the morphology of villi also change from long finger-like
projections before weaning to leaf- or tongue-like structures after weaning (18).
Changes in the structure of gastrointestinal mucosa are often used to evaluate
gut
function
during
the
development
or
in
response
to
particular
food
components. For this purpose villi and crypt size and enterocyte vacuolization
are analyzed (19, 9). Up to date, most studies on the intestinal development have
been done using classical light microscopy or transmission electron microscopy
which give two-dimensional images. In contrast, scanning electron microscopy
(SEM) gives a chance to observe the surface of the gut in three dimensions in a
wide
range
of
(magnification
scale,
10x)
enabling
till
single
observation
microvilli
range
from
the
(magnification
groups
50
of
000x).
villi
This
73
technique,
however,
has
been
seldom
used,
mostly
due
to
difficulties
with
preparation of fragile mucosa tissue (20).
The aim of the present study was to evaluate the development of the small
intestine during 38 days of postnatal life in pigs using light and scanning electron
microscopy. The combination of the two microscopy approaches would give
more detailed information on structural changes in the mucosa in the gut.
MATERIAL AND METHODS
Studies were carried out on the total number of 10 piglets, born on time and housed in standard
farming conditions. The experimental protocol was approved by the Local Ethical Committee.
Piglets were kept together with their sows from birth up to weaning at 35 days of life. During the
suckling period piglets received small amounts of pre-starter solid food, and after weaning a
commercial starter solid food given ad libitum. Animals were sacrificed just after birth (day 0 unsuckling neonates, n=2), at the day 3 (n=2), 7 (n=2), 21 (n=2) and 38 (n=2) after birth by
pentobarbiturate overdose. The gastrointestinal tract tissues were immediately removed, and the
samples of the small intestine were collected. For optical and scanning electron microscopy, 2.5 cm
long whole tissue gut segments were taken from the duodenum 5 cm distal from the pylorus,
jejunum (proximal - 25%, mid - 50% and distal - 75% of the jejunum length), and ileum 5 cm
proximal to the ileo-cecal valve. For optical microscopy the samples were fixed in Bouin solution,
dehydrated and paraffin embedded. Serial histological sections of 5-µm thickness were cut and
stained with hematoxylin and eosin. The slides were analysed with an optical binocular microscope
coupled via digital camera to a computer (7). In each slide, 30 well-oriented villi and mucosa
thickness were measured using LSM5 PASCAL software v.3.2 SP2 (Zeiss, Germany). For SEM, the
gut samples were rinsed with ice cold saline (0.9% NaCl), and then cut into square-fragments (1.5
x 1.5 cm) which were placed on a metal grid and washed in cold physiological saline for one hour.
Washed intestine samples were fixed in 10% buffered formaldehyde. The fixation time depended
on the thickness of the samples and varied between 24 and 48 hours. After fixation the samples were
washed four times in saline solution (15 minutes each change) and dehydrated in a series of alcohol:
10, 30, 50, 70 and 90% and 2 times in absolute ethanol (15 minutes in each change). After drying
in HMDS (1,1,1,3,3,3-hexametyldisilazane), samples were sputter coated (Polaron Range S.C. 7620
Sputter Coater) with 30 nm layer of gold-palladium (Au/Pd) and examined using LEO 1430 VP
scanning electron microscope at an accelerating voltage of 15 kV.
The numerical data are expressed as their means and standard errors of mean (SEM). One wayANOVA followed by the Tukey post-ANOVA test and the unpaired Student's t-test were used to
indicate the statistical differences between the groups (Graph Pad Software version 3.03, San
Diego, CA, USA). In all statistical analysis P<0.05 was taken as the level of significance.
RESULTS
Duodenum
Figure 1 shows the duodenal villi in unsuckling neonatal piglets under light
microscope and SEM. The length of villi and mucosa thickness in the duodenum
measured by optical microscope is given in Table 1. At birth, the surface of the
74
Fig. 1. SEM (left) and optical microscopy (right) micrographs of the duodenal mucosa at birth. Most
of villi are long and finger-like in shape. Mucous was washed out during the preparation of tissues (left).
Note numerous deep incisions (transverse furrows) on the villi (right). Horizontal bars depict scale.
Table 1. The length of villi and thickness of mucosa in the small intestine piglets in the early postnatal and
Table 1. The length of villi and thickness of mucosa in the small intestine piglets in the early
postweaning periods. Values are given as means ± SEM. Different letters in rows indicate statistical significance
postnatal and postweaning periods. Values are given as means ± SEM. Different letters in rows
(one-way ANOVA followed by Tukey post ANOVA test).
indicate statistical significance (one-way ANOVA followed by Tukey post ANOVA test).
Day 0
Mucosa
thickness
Day 7
Mid jejunum
746 ± 81
1174 ± 67
Ileum
537 ± 82a
766 ± 50b
798 ± 107b
282 ± 43c
<0.0001
a
b
c
bc
<0.0001
972 ± 224a
569 ± 75c
<0.0001
ab
c
<0.0001
Duodenum
404 ± 41
Mid jejunum
865 ± 237a
Ileum
703 ± 60
a
851 ± 41
b
1321 ± 72b
904 ± 68
b
535 ± 105
946 ± 42
c
P
a
645 ± 63
b
Day 38
289 ± 36
Duodenum
Villi length
Day 3
a
a
712 ± 107
926 ± 113
350 ± 36
a
<0.0001
314 ± 51
c
<0.0001
665 ± 65
479 ± 63
mucosa is folded and covered by villi of regular shape and size, most of the villi
are finger-like. There are only a few villi which are branched or incompletely
divided (Fig. 2). The gaps between the villi on Krecking Valves are scanty. The
villi surface is not smooth, and many transversal furrows can be seen both under
small and larger magnification (Figs. 2 and 3). The transversal furrows were
defined as the transverse incisions on the villi surface with a length of minimum
one quarter of villi circumference. The transversal furrows are also observed by
optical microscopy as up to 20 µm deep incisions into the villi interior (Fig. 1).
The enterocyte brush border is well developed; the microvilli are approximately
2 µm long and densely packed (40 to 50 microvilli in 1 µm ). At the edge of
2
enterocyte outlines, the microvilli are shorter than in the central apical area of the
cell, and the apical cell surface is raised. This helps to observe pentagonal and
hexagonal outlines of single enterocytes (Fig. 3), making the whole surface of the
villi appear like the surface of a football. The goblet cells are scanty, and can be
75
Fig.2. The duodenal villi at birth. Most of the villi are regular and finger-like. Many transverse furrows
are present along the villi of various length. There are a few not completely divided or branched villi
visible at the lower part of micrograph. Fig.3. SEM micrograph showing the duodenal villi at birth. The
villi are regular in shape, but the surface of the villi is still not developed. The villi seem to be
"crumpled". Mucus secreted by the goblet cells is visible as numerous irregular white buttons on the
villi surface. Fig.4. SEM micrograph showing the tips of villi in the duodenum in 3 d old piglets. Note:
in comparison with the micrographs from older piglets, the tip of the villi is smooth and lacks the
extrusion zone. Fig.5. SEM micrograph of the lateral part of the villus in the duodenum of 3 d old
piglets. The brush border is well developed. Arrows indicate deep transversal furrows on the villus.
recognized by the crater (often containing a droplet of mucus) located on the top
of the cell devoid of microvilli (Fig. 3). The extrusion zone on the top of the villus
indicates nearly no cell shedding and therefore it is hard to be defined in the
unsuckling neonates (Fig. 2). At the day 3 after birth, the villi are finger-like in
shape and their arrangement on the intestinal mucosa surface is regular. The
extrusion zone shows little if any shedding activity, similar to that on the day of
birth (Fig. 4). The surface of the villi corpus is smoother than at the day of birth,
the transversal furrows are thinner but still present (Fig. 5). This corresponds with
the increase of the length of villi observed in few days after birth (Table 1). The
76
Fig. 6. SEM micrograph of duodenal mucosa on day 7 after birth in neonatal piglets. Most of villi
are finger-like in shape but some are also twin-like . The arrow shows villus during dividing process.
Fig. 7. Micrograph of dividing villi in the duodenal mucosa in 7 d old piglets. Longitudinal indention
is visible (arrows). Note, the surface of villi including the apex is smooth. Fig. 8. SEM micrograph
of the apical region of villi in
21 d old piglets, showing the extrusion zone which seems to be more
active than on the earlier micrographs. Arrows indicate the mucus produced by the goblet cells
remaining after washing procedure. Fig. 9. SEM micrograph showing the extrusion zone of duodenal
villi (arrows) in suckling 21 d old piglets. The extrusion zones are localized on the tips of villi. Note,
the extrusion zone on the 21 d old villi seem to be more active than in younger animals.
architecture of villi observed on the day 7 after birth is similar to that of villi from
the day of birth. However, the transversal furrows are less numerous and much
shallower (Fig. 6) as compared to day 0. Again it corresponds to the postnatal
increase in villi length (Table 1).
Though the majority of villi are finger-shaped, there are a few twin-shaped
villi or villi with longitudinal indention (Figs. 6 and 7). SEM micrographs of
duodenal villi on day 21 after birth show further reduction in the number of
transversal furrows, and loss of their regularity in comparison with the unsuckling
neonates. However, the most prominent changes are observed on the apex of
77
Fig. 10. SEM micrograph of the duodenal villi in weaned, 38 d old piglets. A marked reduction in
villi length can be observed. Note that absence of mucus allows observation of crypts outlets. Fig.
11. SEM micrograph of villi in weaned, 38 day old piglet. The villi are either leaf-like or irregular
in shape. Spaces between villi are smaller in comparison with the earlier stages of development.
Fig. 12. SEM micrograph showing the top of villus from the duodenum in a 38 d old piglet. Single
enterocytes can be easily recognized, as they are either collapsed or shed in the extrusion zone. The
cell continuity seems to be interrupted in many places and cell remnants are present between the
functional cells. Note the well developed brush border.
duodenal villi which are not smooth any more. The changes involve the presence
of deep, knife incision-like, furrows and "cell packets" consisting of several
enterocytes (Figs. 8 and 9). The surface of "cell packets" is either elevated or put
below
the
surface
of
the
remaining
enterocytes.
Under
confocal
microscope
following 7-AAD and caspase-3 staining, the "cell packets" were identified as
groups of enterocytes that underwent apoptosis (4). The goblet cells on the top of
villi
produce
mucus
which
is
more
difficult
to
wash
out
using
standard
preparation (Fig. 8). In weaned piglets (38 day old), the length of villi in the
duodenum is reduced as it can be seen in Figure 10 and Table 1. The villi are leaflike with a wide base (Fig. 10) or irregular (Fig. 11) with no transversal furrows.
The extrusion zone of the villi is wide and contains deep furrows and a large
number of shedding cells. On the top of villi there are a few micrometer spaces
observed between the epithelial cells as well as partly broken cells (Fig. 12).
Jejunum
In the mid-jejunum of unsuckling neonatal piglets the villi are thin and of
finger-like shape but not uniform in length, as there are shorter villi in between
the taller ones. The villi surface is rough due to numerous deep transversal
furrows (average depth: 14.1 ± 2.7 µm), and the apical enterocyte membrane is
raised (Figs. 13 and 14). As compared to the duodenum, the relative number of
goblet cells in the jejunum is high. At the tip of the villi there are 5 to 6
enterocytes surrounding 1 goblet cell (Fig. 14). On the third day of life, the
mucosa in the middle jejunum shows abundant villi of various lengths (Fig. 15).
78
Fig. 13. SEM micrograph of the middle jejunum at birth. The surface of the villi shows transversal
furrows (arrows). Enterocyte outlines are clearly visible. Villi are packed very densely at their basal
part. Fig. 14. SEM micrograph of the villus tip in the middle jejunum at birth shows enlarged
enterocyte outlines and microvilli. Goblet cells outlets are shown between enterocytes (arrows).
There is no area resembling extrusion zone at the tip of villus. Fig. 15. SEM micrograph of the
middle jejunum mucosa in a 3 d old piglet. Shorter villi are present between the taller ones. At the
basal part the villi are packed very densely, almost "sticking" to each other. Transversal furrows are
visible Fig. 16. SEM micrograph of villus tip in middle jejunum in 3 d old piglet, showing
numerous goblet cell outlets (arrows). Transversal furrows are visible.
The transversal furrows are 11.1 ± 2.8 µm deep and numerous. On the apex of
jejunal
villi
enterocytes
there
with
are
no
many
goblet
microvilli,
thus
cells
no
and
only
well-defined
a
few
single
extrusion
shedding
zone
can
be
observed (Fig. 16). On day 7 after birth, the shape of jejunal villi change from
round
and
finger-like
to
flat
and
tongue-like,
and
the
population
of
villi
is
variable. The length varies from up to 1000 µm long villi to less than 300 µm
short villi (Fig. 17). The transversal furrows are present along the entire length of
villi (Figs. 17 and 18). The extrusion zone is observed with a number of shedding
cells (Fig. 18). At day 21 after birth, the shape of most of jejunal villi changed to
79
Fig. 17. SEM micrograph of the
middle jejunum mucosa
at d 7
after birth. Most villi are tall and
finger-like in shape. Shorter villi
are present between taller ones.
Fig. 18. SEM micrograph of the
apical part of villi. The outline of
the
extrusion
Many
zone
transversal
present
(arrow).
micrograph
Fig.
of
Note,
visible.
19.
the
jejunum mucosa
birth.
is
furrows
are
SEM
middle
at d 21 after
clear
changes
in
shapes of the villi. Fig. 20. SEM
micrograph
of
middle
jejunum
mucosa at d 21 after birth. Dieing
cells are present in the extrusion
zone. Note, that the location and
shape
of
extrusion
zones
on
villus, seem to be dependent on
the villus shape (compare with
micrograph
18).
Fig.
21.
Micrograph of duodenal mucosa
at d 38
after birth. Most villi are
folded and irregular in shape. It is
hard
to
find
"typical
singular"
villus. Shed enterocytes are seen
as
numerous
irregular
white
buttons on the villi surface. Fig.
22. SEM micrograph of middle
jejunum
mucosa
at
d
38
after
birth. Note, active extrusion zone
and
crevices
between
the
epithelial cells.
wide tongue-like, and the villi surface is rough with numerous cells shedding on
the entire length of the villi (Fig. 19). The total number of transversal furrows is
markedly reduced. A few of them are deeper than 13 µm, and most of the
remaining are shallower than in the day 3 and 7 of life. The villi tips show clear
extrusion
zones
with
numerous
goblet
cells,
deep
incisions
and
distorted
continuity of epithelial cells (Fig. 19 and 20). Following weaning on day 38 after
birth, the mucosa in the middle jejunum is thinner (Table 1), and shows a great
variability
in
projections
are
villi
shape;
predominant
tongue-like,
(Fig.
21).
fold-like
No
and
transversal
incomplete
furrows
are
division
observed.
There are numerous epithelial cells shed along the villi surface. At the villi tips
80
Fig. 23. SEM micrograph of surface of
ileum villi at birth showing transversal furrows. Fig. 24. SEM
micrograph of the surface of villus at birth showing enterocytes with well developed microvilli.
Numerous goblet cells are present and a large variability in the enterocyte size is seen. Fig. 25. SEM
micrograph of cross section of ileum villus at birth shows tall, columnar enterocytes. Note, large empty
spaces in the cell body suggesting the localization of a large lysosomal vacuole in the apical region and
the nucleus in the basal region of the enterocyte. Fig. 26. SEM micrograph showing ileum villi at d 3
after birth. Between long ones irregular in shape, often branching villi are visible. At the tip of
a
branched villus some remaining shed enterocytes are present. Transversal furrows are numerous.
the extrusion zone is observed. The shape of epithelial cells in the extrusion zone
is changed from regular, hexagonal to elongated and irregular (Figs. 21 and 22).
Ileum
At birth the villi in the ileal mucosa are of various sizes. Numerous flat fingerlike villi are observed which seem to be not completely divided (Fig. 23). The
apical
cell
membrane
of
enterocytes
is
raised
(Fig.
23).
In
contrast
to
the
duodenum and jejunum there is a large variability in the enterocyte size, although
both large and small enterocytes have well developed microvilli and a regular
81
Fig.
SEM
27.
micrograph
of
ileum mucosa at d 7 after birth.
Villi
are
leaf-shaped.
transversal
furrows
are
Many
present.
Note, that villi in ileum are less
regular in shape in comparison to
those
in
middle
the
duodenum
jejunum.
Fig.
and
SEM
28.
micrograph shows ileum villi at d
7 after birth. Transversal furrows
are
visible
SEM
(arrows).
micrograph
mucosa
at
showing
d
Fig.
from
21
after
irregularity
29.
ileum
birth
in
villi
shape. Fig. 30. SEM micrograph
of
the
tips
of
the
villi
(ileum
mucosa at d 21 after birth). Villi
are irregular in shape. Numerous
cells are shedding along the villi
surface
apical
with
emphasis
region.
Fig.
in
31.
the
SEM
micrograph of the ileum mucosa
at d 38 after birth. Villi are leaflike
in
division
shape
is
and
incomplete
seen.
Numerous
shedding cells are visible along
the villi surface. Fig. 32. SEM
micrograph of the ileum mucosa
38 days after birth. Note, irregular
and elongated cells surrounding
the extrusion zone.
hexagonal shape. Goblet cells are numerous (Fig. 24). The cross section of villus
shows the lateral cellular membranes of adjacent enterocytes and microvilli in the
apical
membrane.
Two
large
empty
spaces
in
the
cell
body
suggest
the
localization of a large lysosomal vacuoles in the apical region and the nucleus in
the basal region of the enterocyte (Fig. 25). On the 3
rd
day after birth, the ileal villi
are of flat finger-like shape, many of them are partly-divided. The villi surface is
relatively smooth, but some large enterocytes still remain on the villi top (Fig.
26). On the day 7, the ileal mucosa shows finger-like, leaf-like and tongue-like
villi of various magnitudes (Fig. 27). The villi surface is smooth, but some
transversal furrows can be seen. On some epithelial cells at the top of villi the
microvilli are not visible (Fig. 28). The ileal mucosa on day 21 after birth is
characterized by a great diversity in villi shape and magnitude (Fig. 29). Higher
82
magnification of the villi (Fig. 30) revealed numerous cells shed all along the villi
surface, in particular in the apical region and a large number of goblet cells. In
weaned
piglets,
on
day
38
of
life
the
picture
of
the
villi
in
the
ileum
was
dominated by tongue-like shapes, however single finger-like and leaf-like villi
and incompletely divided villi are present as well (Fig. 31). The length of villi is
reduced as compared to the suckling period (Table 1). The transversal furrows are
not
observed.
numerous
A
cells
clear
shed.
extrusion
Regular
zone
in
hexagonal
the
apical
region
enterocytes
are
is
observed
present
in
with
the
villi
corpus, while longitudinal ones predominate in the extrusion zone (Fig. 32).
DISCUSSION
In the present study, scanning electron microscopy technique was used to
examine the mucosa structure in the duodenum, jejunum and ileum in the
neonatal
and
understand
weaned
piglets.
important
This
details
in
three-dimensional
mucosa
structure,
technique
e.g.
helped
length,
to
shape,
transversal furrows and extrusion zone of villi and membrane structure of the
epithelial cells which are indistinguishable or unclear when analyzed with
other
methods.
Commonly
used
light
microscopy
gives
two-dimensional
images of small gut sections and enables the measurement of the size of certain
structures
such
as
villi
and
crypts,
etc.
SEM,
however,
gives
additional
information like in the figures presented, the surface image of the villi which
can not be evaluated by light microscopy. Furthermore, SEM technique allows
to estimate the location, distribution, density and shape of the villi in lowpower
magnification
micrographs.
High-power
magnifications
allow
recognition of type of epithelial cells lining the mucosal surface, their shape
and number in a given area as well as the microvilli structure. This approach
helps to understand the physiological status of the tissue and cell activity or
self-destruction.
Methodological consideration: light versus scanning electron microscopy
Light
mucosa.
microscopy-based
An
estimation
of
methods
the
allow
histometry
cross-sections
of
the
analysis
mucosa
of
the
structure,
gut
size
(length and area), length of the villi, depth of the crypts, and the area of the cells
could be evaluated. These measurements, however, may not perfectly reflect
the
actual
situation
since
the
tissues
may
change
their
dimensions
during
preparation and cross-sections are usually "blindly" chosen for analysis. The
former may be minimized by selecting solutions for tissue fixation, for instance
Bouin's
solution
affects
the
collagen
fibrils
and
changes
the
actual
tissue
dimensions only to a small extent (20). The latter, however, can be successfully
addressed by histometry analysis under the SEM. Scanning microscopy allows
to observe the shape, surface and density of intestinal villi as well as predict the
83
villi function, e.g. by analyzing the extrusion zone of villi, calculating the
number of epithelial cells per area unit, and evaluation of the cell type and their
location on the villi. Importantly, the resolution capability of SEM (10-20 nm)
is
higher
than
in
light
observation
of
the
observation
to
two
microscopy
epithelial
cell
dimensions
which
allows
structure.
whereas
Light
SEM
much
more
detailed
microscopy
restricts
offers
three-dimensional
observation; thus SEM may help to confirm earlier observations and make new
findings.
The
elaborated
method
of
gut
mucosa
preparation
allowed
us
to
observe the surface of villi devoid of artifacts. Cells retained their shape and
structure,
the
microvilli
and
cell
junctions.
Our
analysis
using
SEM
demonstrated that the measurement of such simple parameters as villi length
and
their
density
per
surface
unit
in
2-D
approach
may
be
charged
with
substantial errors and is sometimes worthless. It is due to the fact that the shape
of the villi and the distribution of their size (long versus short villi) show
dynamic changes during gut development.
Postnatal changes in the intestinal villi in piglets
Observations based on SEM analysis indicate very dynamic growth-related
changes that occur in the first weeks of life. In the unsuckling piglets the villi
were short and dense. Their length and spaces between villi extended with age.
Numerous deep transversal furrows were present on the entire length of the villi
in newborns. Within a few postnatal days the villi length increased, and the
number and depth of transversal furrows was reduced suggesting that elongation
of villi may enhance the absorptive area of the gut to the lover extent than it was
reported
previously.
In
the
unsuckling
neonates
the
villi
are
twitched.
They
stretch following the first feedings due to a reduction in basal vascular resistance
simultaneous with dramatic increase in local intestinal blood flow and lymph
formation (21). Thus the analysis of transversal furrows may be presumably
considered for indexing hemodynamic conditions of the neonatal gut.
These two features (smoothing of the villi and reduction in their density)
suggest
the
presence
of
an
"indifferent
absorptive
area"
at
birth.
Word
"indifferent" implies that this area, despite of its great potential ability is not
entirely employed in nutrient absorption. The SEM micrographs of villi from the
following days: 4, 7 and 21 indicate the dynamic progress in the smoothing of the
villi. Basing on SEM micrographs, it seems quite possible that the piglets are born
with an already developed absorptive area in the gut, and the first feedings trigger
a
shift
from
the
"indifferent
absorptive
area"
to
"active
absorptive
area"
in
accordance with changes in local circulation in the gut mucosa. Rapid postnatal
hemodynamic changes in the gut microcirculation are mediated mostly by the
constitutive and stimulated production of nitric oxide and endothelin (21). The
development of intestinal vasculature may be further enhanced by the colostrum
and milk growth factors and hormones (22-24), leading to overall changes in
84
mucosa architecture, and finally formation of the "active absorptive area". Taking
into account, on the one hand, the tasks facing the neonatal intestine, and the
capability to increase cell number in a short time on the other, the solution of an
intestine "ready for use" immediately after birth seems reasonable.
At birth the villi are finger-like and regular. The density of villi is high in all
segments of the small intestine, and the reduction of their density seems to be
associated with mucosa stretching, matching the dramatic increase in length and
width of the entire intestine in the early postnatal period (8, 9). Simultaneously
the villi shape changes from finger- to leaf- or tongue-like and the number of
dividing villi and villi with indentations increase remarkably in time. The analysis
of SEM micrograph showed that the most noticeable changes took place after
weaning during adaptation to a new kind of food. The changes related to weaning
are widely investigated, like the reduction of villi length and mucosa thickness
(25-27), and many of them were confirmed by SEM studies.
Epithelial cell shedding in the small intestine
SEM technique allows to examine in detail the extrusion zone on the tip of
villi
where
shedding
of
epithelial
cells
take
place.
According
to
SEM
micrographs, at birth and 3 days after birth no or almost no detectable cell
shedding is present on the top of villi. A similar picture is observed in all three
investigated segments of the small intestine. In the following postnatal days, the
number of furrows and shedding cells on the villi top increases indicating marked
alteration of the extrusion zone activity with a maximum observed at 3 weeks of
life. Accordingly, immunofluorescence studies revealed that in newborns the
apoptotic epithelial cells are present on the entire length of villi (28), whereas in
the older animals they are present solely on the upper third part of villi (29, 30).
Moreover, in newborns the apoptotic cells were dying in packets of several cells
as shown with 7-AAD and caspase-8 and -3 staining (4). The present study also
confirms the presence of cell packets lifted or collapsed a few micrometers below
the villi surface. The acceleration of extrusion zone activity is observed within a
week after birth, and it may be linked to the intensive rebuilding of the population
of enterocytes from the fetal-type to adult-type, and the enhanced enterocyte
turnover. It was estimated that the turnover of enterocytes after birth (2 to 3 days)
is 10 times faster than during the fetal life (3). The renewal time of enterocytes
may
be
affected
by
the
length
of
villi.
Also
the
migration
speed
of
fetal
enterocytes is markedly slower in comparison with that of adults. Poor activity of
the extrusion zone within the first postnatal week may be the effect of cell death
occurring on the entire villi length as well as the overall reduction of apoptosis in
the intestinal mucosa. Biernat and co-workers (28) suggested that rapid mucosa
growth which occurs within the first two postnatal days is in part caused by a twofold
increased
mitosis/apoptosis
ratio
which
agrees
with
earlier
measurements in the epithelial cells by Widdowson and co-workers (31).
DNA
85
Another striking finding in the active extrusion zone is the presence of numerous
crevices between the epithelial cells suggesting the local loss of cell continuity on
the top of the villi. The crevices of a few micrometers length are often located
nearby the cell remnants presumably as a result of incomplete cell-cell zipping after
the shed cell (or cells). The crevices are observed in all three studied segments of
the
small
intestine.
From
the
micrographs
it
is
unclear
whether
the
crevices
penetrate to the enterocyte basal membrane thus entirely discontinuing the gut
barrier or terminate along the enterocyte lateral space. Nevertheless, their presence
may increases a chance of any potentially harmful substance and pathogen to pass
the gut barrier. Interestingly, the concentration of goblet cells is higher in the
extrusion
zone
suggesting
their
role
in
physiological
restitution
of
epithelium
continuity. Previously, Ikeda and co-workers (32) reported that goblet cells may
play an important role in epithelial cell repair following a superficial damage to
gastrointestinal
mucosa
by
ischemia-reperfusion
injury.
In
addition,
in
our
preparations the mucus produced by these goblet cells was more difficult to remove
than that produced by the goblet cells located on the villi corpus suggesting some
modification in the production of mucus along the goblet cell life. Maybe the mucus
simply clogs the crevices left by dieing cells.
In conclusion, scanning electron microscopy allowed better understanding of
the physiology of the intestinal mucosa development in the neonatal piglets. The
intestinal villi develop intensively during the first 3 weeks which is manifested by
major changes in the shape, size and density of villi. The extrusion zone located
on the top of villi is not active during the first few days after birth, its activity
gradually increases until the maximum at 21
st
day of life. The size and activity of
extrusion zone may be a good marker of epithelial cell turnover though it lacks
quantitative approach at present. Weaning further enhance the changes in the villi
structure involving their shape and size.
Acknowledgments:
This
work
was
supported
by
grants
nr
PBZ-KBN-093/P06/2003
and
EUREKA! nr 2675 from the National Committee for Scientific Research, Poland.
REFERENCES
1.
Le Dividich J, Seve B. Effects of underfeeding during the weaning period on growth, metabolism
2.
Xu RJ, Wang F, Zhang SH. Postnatal adaptation of the gastrointestinal tract in neonatal pigs: a
and hormonal adjustments in the piglet. Domestic Anim Endocrinol 2000; 19: 63-74.
possible role of milk-borne growth factors. Livestock Prod Sci 2000; 66: 95-107.
3.
Trahair JF, Sangild PT. Studying the development of the small intestine: philosophical and
anatomical perspectives. In: Biology of the intestine in growing animals R Zabielski, PC
Gregory, B Weström (eds), Elsevier 2004, pp. 1-54.
4.
Godlewski MM, S³upecka M, Woliñski J, Skrzypek T, Skrzypek H, Motyl T, Zabielski R. Into
the unknown - the death pathways in the neonatal gut epithelium. J Physiol Pharmacol 2005;
58 (S3): in press.
86
5.
Guilloteau P, Biernat M, Woliñski J, Zabielski R. Gut regulatory peptides and hormones of the
small intestine. In: Biology of the intestine in growing animals Zabielski R, Gregory PC,
Weström B (Eds.), Elsevier, Amsterdam 2002: 325-362.
6.
Burrin DG, Petersen Y, Stoll B, Sangild P. Glucagon-like peptide 2: a nutrient-responsive gut
growth factor. J Nutr 2001; 131: 709-712.
7.
Woliñski J, Biernat M, Guilloteau P, Weström B, Zabielski R. Exogenous leptin controls the
development of the small intestine in neonatal piglets. J Endocrinol 2003; 177: 215-222.
8.
Xu RJ, Mellor DJ, Tungthanathanich P, Birtles MJ, Reynolds GW, Simpson HV. Growth and
morphological changes in the small intestine in piglets during the first three days after birth. J
Dev Physiol 1992, 18: 161-172.
9.
Marion J, Rome´ V, Savary G, Thomas F, Le Dividich J, Le Huerou-Luron I. Weaning and feed
intake alter pancreatic enzyme activities and corresponding mRNA Levels in 7-d-old piglets. J
Nutr 2003; 133: 362-368.
10. Zabielski R, Barej W, Leœniewska V, Pierzynowski S. Pancreas and upper gut dysfunctions
around weaning in pigs and calves. In: Production diseases in farm animals. Wensing Th (ed.),
Wageningen Pers 1998, pp. 134-144.
11. Burrin DG. Is milk-borne insulin-like growth factor-I essential for neonatal development? J
Nutr 1997; 127(S5): 975-979.
12. Kimble RM, Breier BH, Gluckman PD, Harding JE. Enteral IGF-I enhances fetal growth and
gastrointestinal development in oesophageal ligated fetal sheep. J Endocrinol 1999; 162: 227-235.
13. McCracken
VJ,
Lorenz
RG.
The
gastrointestinal
ecosystem:
a
precarious
alliance
among
epithelium, immunity and microbiota. Cell Microbiol 2001; 3: 1-11.
14. Xu RJ, Mao YL, Tso MYW. Stability of gastrin in the gastrointestinal lumen of suckling,
weanling and adult pigs. Biol Neonate 1996; 70: 60-68.
15. Pluske JR, Hampson DJ, Williams IH. Factors influencing the structure and function of the
small intestine in the weaned pig: a review. Livestock Prod Sci 1997; 51: 215-236.
16. Odle J, Zijlstra RT, Donovan SM. Intestinal effects of milkborne growth factors in neonates of
agricultural importance. J Anim Sci 1996; 74: 2509-2522.
17. Hampson DJ, Kidder DE. Alteration in piglet small intestinal structure at weaning. Res Vet Sci
1986; 40: 32-40.
18. Cera KR, Manhan DC, Cross RF, Reinhart GA, Whitmoyer RE. Effect of age, weaning and
postweaning diet on small intestinal growth and jejunal morphology in young swine. Anim Sci
1988; 66: 574-84.
19. Biernat M, Zabielski R, Sysa P, Sosak-Swiderska B, Le Huerou-Luron I, Guilloteau P. Small
intestinal and pancreatic microstructures are modified by an intraduodenal CCK-A receptor
antagonist administration in neonatal calves. Regul Pept 1999; 85: 77-85
20. Wiese F, Simon O, Weyrauch K D. Morphology of the small intestine of weaned piglets and a
novel method for morphometric evaluation. Anat Histol Embryol 2003; 32: 102-109.
21. Nankervis CA, Reber KM, Nowicki PT. Age-dependent changes in the postnatal intestinal
microcirculation. Microcirculation 2001; 8: 377-87.
22. Cranwell PD, Moughan PJ. Biological limitations imposed by the digestive system to the
growth performance of weaned pigs. In: Manipulating pig production II, Branett JL, Hennessy
DP. Australian Science Association Publication, Australia 1989: 140-159.
23. Guilloteau P, Le Huërou-Luron I, Le Drean G, Gestin M, Philouze-Rome V, Artiaga A, Bernard
C, Chayvialle JA. Gut regulatory peptide levels in bovine fetuses and their dams between the 3
rd and 9 th months of gestation. Biol Neonate 1998; 74: 430-438.
24. Zabielski R. Regulatory peptides in milk, food and in the gastrointestinal lumen of young
animals and children. J Anim Feed Sci 1998; 7: 65-78.
87
25. Van Beers-Schreurs Hetty MG, Nabuurs MJA, Vellenga L, Kalsbeek-van der Valk HJ, Wensing
T, Breukink HJ. Weaning and the weanling diet influence the villous height and crypt depth in
the small intestine of pigs and alter the concentrations of short-chain fatty acids in the large
intestine and blood. J Nutr 1998; 128: 947-953.
26. Hall
GA,
Byrne
TF.
Effect
of
age
and
diet
on
small
intestinal
structure
and
function
in
gnotobiotic piglets. Res Vet Sci 1989; 47: 387-392.
27. Deprez P, Deroose P, Van den Hende C, Muylle E, Oyaert W. Liquid versus dry feeding in
weaned piglets: the influence of the small intestinal morphology. J Vet Med B 1987; 34: 254-259.
28. Biernat M, Woliñski J, Godlewski MM, Motyl T, Morisset J, Zabielski R. Apoptosis in the gut
of neonatal piglets. Proceedings of the 9
th
International Symposium on Digestive Physiology in
Pigs. University of Alberta, Edmonton, Canada 2003: 46-48.
29. Iwanaga T. The involvement of macrophages and lymphocytes in the apoptosis of enterocytes.
Arch Histol Cytol 1995; 58: 151-159.
30. Shibahara T, Sato N, Waguri S, Iwanaga T, Nakahara A, Fukutomi H, Uchiyama Y. The fate
of effete epithelial cells at the villus tips of the human small intestine. Arch Histol Cytol
1995; 58: 205-219.
31. Widdowson EM, Colombo VE, Artavanis CA. Changes in the organs of pigs in response to
feeding for the first 24 h after birth. Biol Neonate 1976; 28: 272-281.
32. Ikeda H, Yang C-L, Tong J, Nishimaki H, Masuda K, Takeo T, Kasai K, Ihoh G. Rat small
intestinal goblet cell kinetics in the process of restitution of surface epithelium subjected to
ischemia-reperfusion injury. Dig Dis Sci 2002; 43: 590-601.
R e c e i v e d : April 8, 2005
A c c e p t e d : April 18, 2005
Author's address: Henryk Skrzypek, Ph.D., Department of Zoology and Ecology, Catholic
University of Lublin, Al. Krasnicka 102, 20-718 Lublin, Poland, tel. +48-81-4454644.
E-mail: [email protected]