close

Вход

Забыли?

вход по аккаунту

?

Postnatal-related changes in the size and total number of neurons in the caudal mesenteric ganglion of dogsTotal number of neurons can be predicted from body weight and ganglion volume.

код для вставкиСкачать
THE ANATOMICAL RECORD PART A 286A:917–929 (2005)
Postnatal-Related Changes in the Size
and Total Number of Neurons in the
Caudal Mesenteric Ganglion of Dogs:
Total Number of Neurons Can Be
Predicted From Body Weight and
Ganglion Volume
KARINA MARTINEZ GAGLIARDO,1
JÚLIO CÉSAR DE CARVALHO BALIEIRO,2
ROMEU RODRIGUES DE SOUZA,3 AND
ANTONIO AUGUSTO COPPI MACIEL RIBEIRO1*
1
Department of Surgery, College of Veterinary Medicine, University of São Paulo,
São Paulo, Brazil
2
Department of Basic Science, College of Animal Science and Food Engineering,
University of São Paolo, Pirassununga, Brazil
3
Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo,
São Paulo, Brazil
ABSTRACT
Aging is mostly characterized by a progressive decline of neuronal function that involves
both the central and the peripheral nervous system. The aging process is accompanied by changes
in either the number or the size of neurons. However, these data are controversial and not very
well known in the sympathetic ganglia of large mammals. Hence, the present investigation aimed
to study the dog’s caudal mesenteric ganglion (CMG) in three different periods of postnatal
development, searching for qualitative and quantitative alterations. The CMG is responsible for
the large intestine, internal anal sphincter, and partially the urogenital system innervations.
Nine dead male dogs from the Veterinary Hospital of the College of Veterinary Medicine at
University of São Paulo were divided into three well-defined age groups (1–2 months old, 1–2
years old, and 5–10 years old). The stereological study was pursued using the physical disector
method combined to the Cavalieri principle. The postnatal development was accompanied by an
increase in the nonneuronal tissue amount and in ganglion volume. Additionally, the total
number of neurons also increased during aging (from 70,140 to 1,204,516), although the neuronal
density showed an opposite trend (from 29,911 to 11,500 mm⫺3). Due to the interrelation between
either body weight or ganglion volume and aging in the dogs investigated in this study, it was
possible to predict the total number of neurons in CMG using both body weight and ganglion
volume in an attempt to verify whether or not size and total number of neurons are both
allometrically and aging ruled, i.e., if either the animal’s body weight and ganglion volume or
aging influence these parameters. The prediction of the total number of neurons was very close
to the initially estimated values. © 2005 Wiley-Liss, Inc.
Key words: stereology; disector; caudal mesenteric ganglion; dogs; middle aging
Grant sponsor: Fundação de Amparo à Pesquisa do Estado de
São Paulo; Grant number: 02/04758-0 and 03/03731-4.
*Correspondence to: Antonio Augusto Coppi Maciel Ribeiro,
Departamento de Cirurgia, Faculdade de Medicina Veterinária e
Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando
Marques de Paiva, 87, São Paulo, 05508-000, Brazil. Fax: 55-113091-7805. E-mail: guto@usp.br
©
2005 WILEY-LISS, INC.
Received 17 March 2005; Accepted 21 June 2005
DOI 10.1002/ar.a.20233
Published online 2 September 2005 in Wiley InterScience
(www.interscience.wiley.com).
918
GAGLIARDO ET AL.
INTRODUCTION
The aging process is a normal and inevitable event that
occurs in all animal species. In mammals, this process is
associated with decrements in cellular and physiological
functions and a major incidence of degenerative diseases.
The alterations that happen are the result of an interaction between many factors and aging is therefore a very
complex phenomenon (Szweda et al., 2003). In the nervous
system, the effects of aging are evidenced by a functional
decline that involves the central and peripheral nervous
system. The changes most frequently related are neuron
loss, atrophy, and hypertrophy (Cabello et al., 2002). Nevertheless, these claims are discussed because there are
related differences between various components of the
nervous system and among animal species (Finch, 1993;
Vega et al., 1993).
Our knowledge of the aging process in the sympathetic
ganglia is limited, although in the last 20 years these
ganglia have been studied frequently with the help of
pharmacology, electrophysiology, immunohistochemistry,
and biochemical techniques (Miolan and Niel, 1996).
These ganglia are considered the best models to study and
possibly to solve problems in the gastrointestinal tract
(Gonella et al., 1987). At any time, they control important
functions such as the secretion and absorption of substances from the intestinal wall and blood flux and are
responsible for the arc reflex with the target tissues
(Gonella et al., 1987; Bywater, 1993; Luckensmeyer and
Keast, 1995, 1996; Gabella, 2004).
The degenerative alterations in the neurons of sympathetic ganglia have frequently been correlated with alterations in the dendritic and axonal arbor. However, the
changes are not provoked by the direct effects of aging, but
are secondary to changes in the target tissues (Andrews,
1996). With aging, tissues may decrease or increase their
production of neurotrophic factors (Gavazzi and Cowen,
1996; Bennett et al., 2002; Crutcher, 2002).
The neuronal loss was often a requisite for understanding the effects of the aging process in the nervous system
(West, 1994). Much research in the middle of the 1950s
demonstrated a decrease in neuronal density by area
(packing density) with aging in the brain, but wrongly
described a decrease in the total number of neurons. Furthermore, with the development of accurate and unbiased
procedures to count cells in recent years, it was possible to
verify that the total number of neurons may not decrease
in function with the aging process (Morrison and Hof,
1997).
Due to the lack of quantitative data concerning the
cellular elements in the sympathetic ganglia in different
stages of the development in large mammals and the
functional importance of the caudal mesenteric ganglion
(CMG) in the innervation of the gastrointestinal tract
(sympathetic innervation of the colon and internal anal
sphincter) and partially of the urogenital tract, this research aimed to investigate possible qualitative and quantitative alterations in the dog’s CMG in two different
phases of the postnatal development: maturation (pups to
adult) and middle aging (adult to middle-aged).
In addition, due to the interrelation between either body
weight or ganglion volume and aging in the dogs investigated in this study, it was possible to predict the total
number of neurons in CMG using both body weight and
ganglion volume in an attempt to verify whether or not
size and total number of neurons are both allometrically
and aging ruled, i.e., if either the animal’s body weight
and ganglion volume or aging influence these parameters.
The canine species was chosen given its importance in
veterinary medicine and several gastrointestinal disorders in either middle-aged or aged dogs such as lack of
motility, gastritis, and diarrhea. The causes of these diseases remain unclear and their treatment is often ineffective (Slatter, 1998; Fossum, 2002).
MATERIALS AND METHODS
Animals
In this study, we have investigated nine CMG from nine
dead male dogs obtained from the Veterinary Hospital of
College of Veterinary Medicine at the University of São
Paulo (USP) in connection with other experiments. Animals were divided into three different groups according to
their age. The age group distribution was as follows: group
1 (pups), three CMG (1 month old, 2 months old, and 2
months old; body weights: 0.15, 0.18, and 0.18 kg; cases 1,
2, and 3, respectively); group 2 (adults), three CMG (1 year
old, 2 years old, and 2 years old; body weights: 13, 18, and
15 kg; cases 1, 2, and 3, respectively); group 3 (middleaged), three CMG (5 years old, 7 years old, and 10 years
old; body weights: 20, 23, and 26 kg; cases 1, 2, and 3,
respectively).
Histology
After death using an overdose of anesthetic (acepromazine 0.1 mg/kg body weight injected i.v., followed by Na
pentobarbitone, tiopental 50 mg/kg body weight injected
i.v.), the abdominal cavity was opened by a midline incision and the intestines were slightly turned away to facilitate the identification of the ganglia and their connections to spinal cord, intermesenteric and pelvic plexuses,
and also to facilitate the visualization of the main vessels
of the abdomen (abdominal aorta and caudal cava vein).
About 20 ml of washing-up solution of phosphate-buffered saline (PBS; Sigma; 0.1 M; pH 7.4) containing 2% of
heparin (Roche) and 0.1% sodium nitrite (Sigma) was
perfused through the abdominal aorta close to the emergence of the caudal mesenteric artery. The caudal cava
vein was cut to clear the circulatory system. Next, 10 ml of
a 5% glutaraldehyde solution (Merck) and 1% formaldehyde (Sigma) in sodium cacodylate buffer (EMS; 0.125 M;
pH 7.4) were also perfused via abdominal aorta.
Then, the ganglia were dissected out together with their
connections and the caudal mesenteric artery, which
passed toward the ganglia. Afterward, all ganglia were
measured for their length (long axis) using a digital
pachymeter (Starret) and ganglia from group 1 were fully
immersed in the same fixative. Ganglia from groups 2 and
3 were sliced along their length, giving five slabs used for
the volume estimate. Each slab was separately immersed
in the same fixative solution in which they remained for
up to 72 hs, keeping the original craniocaudal slab’s orientation.
Then, ganglia (group 1) and ganglion slabs (groups 2
and 3) were washed in sodium cacodylate buffer (EMS),
postfixed in 2% osmium tetroxide (EMS), block-stained
with a uranyl acetate-saturated aqueous solution (Reagen), dehydrated in graded ethanols and propylene oxide
(EMS), and embedded in Araldite (502 Polyscience). The
resin was cured for 3 days at 60°C. Later on, 2 ␮m sections
NEURONS IN CAUDAL MESENTERIC GANGLION
were cut using a glass knife, stained with toluidine blue
(Nuclear), dried on a hot plate, and mounted under a
coverslip with a drop of Araldite.
Morphometry
For the morphometric study, 70 – 80 serial sections were
cut at 2 ␮m thickness. For each ganglion, 30 consecutive
sections were collected on glass slides, stained with toluidine blue, and mounted in Araldite. A test system comprised of eight different unbiased counting frames was
landed over each section field’s image projected on a computer screen. A fraction (1/fr) of the counting frames was
randomly, uniformly, and systematically sampled using a
random start between 1 and fr. The sampled field’s images
were observed on a computer screen using a Leica DMR
Microscope coupled with a DFC 300FX Leica digital camera. In each counting frame (with a surrounding guard
area), only the neurons located inside the counting frame
and not landing the forbidden lines were measured (Gundersen, 1977). This approach was also adopted in a 3D
view “brick counting frame” (Howard and Reed, 2004),
where the neurons from the three first sections were not
measured. Each neuron received the same number in all
serial sections, and its largest perikaryon profile as well as
its largest nuclear profile were identified and therefore
measured for the cross-sectional area using the image
analysis system Q-Win Leica. The nucleus-cytoplasm ratio was also calculated by dividing the nuclear area by the
cytoplasmic area. A total of 54 neurons and 33 neuronal
nuclei were measured per ganglion, accounting for 162
neurons and 99 neuronal nuclei in each age group.
Stereology
A combination of the physical disector method and the
Cavalieri principle was pursued to obtain an unbiased
estimate of the total number of neurons (N) in the CMG
(Gundersen et al., 1988, 1999; Pakkenberg and Gundersen, 1988; Mayhew and Gundersen, 1996).
Ganglion Volume (Volume Reference; Vref)
The Cavalieri principle was used to estimate the volume
of the CMG. The volume is obtained using a uniform
random systematic sampling. Given the small ganglion
size (mean ⫽ 6.7 mm), all ganglia from group 1 were
initially processed for histological study and afterwards
exhaustively sectioned with a glass knife (at 2 ␮m thickness). In groups 2 and 3, due to the fact that the ganglia
were larger (mean ⫽ 12 and 12.3 mm in groups 2 and 3,
respectively), for full sectioning, all ganglia were macroscopically and transversally slabbed by means of a tissue
slider before histological processing. The resulting five
consecutive slabs had the same mean thickness, which
was obtained by dividing the length of the ganglion by the
total number of slabs using the same approach adopted by
Mayhew and Olsen (1991) and Ribeiro et al. (2004). The
ganglion volume was estimated by multiplying the sum of
the section areas in group 1 or the sum of the sectional
slab areas in groups 2 and 3 (¥a) by the distance between
sections or slabs (k). The volume was estimated as Vref ⫽
k ⫻ ¥a
In group 1, ganglia were exhaustively sectioned at 2 ␮m,
which yielded an approximate total of 3,000 sections. In
this approach, the interval between sampled sections (k)
was 200 sections. From the first field containing ganglion
919
tissue, the first section was sampled by taking a random
number between 1 and 200. In groups 2 and 3, the mean
slab thickness was 2.5 mm. In all groups, either section
areas or slab areas were measured using the image analysis system Q-Win Leica. Furthermore, ganglion section’s
images were captured and projected on a computer screen
using a DFC 300FX Leica digital camera coupled with a
Leica DMR Microscope and the images of ganglion slabs
were projected on a computer by means of a TK 1280OU
JVC camera coupled with an Axioscopic Microscope Zeiss
(L08). The area of the caudal mesenteric artery was not
considered in the estimate of either section or the ganglion
slab area.
The accuracy of the volume estimate was evaluated
through the coefficient of error of the Cavalieri principle
(Gundersen and Jensen, 1987). The formula used to assess
the error coefficient (CE) was CE ⫽ 1/¥A ⫻ [1/12 (3a ⫹ c ⫺
4b)]1/2, where ¥A is the sum of all section or slab areas; a
is the sum of all products a ⫻ a; b is the sum of all products
a ⫻ (a ⫹ 1); and c is the sum of all products a ⫻ (a ⫹ 2).
The tissue shrinkage effects were calculated using the
following formula: shrinkage ⫽ volume before processing ⫺ volume after processing/volume before processing.
The tissue shrinkage using Araldite embedding was 15%.
Therefore, the volume of the reference space estimated
using the Cavalieri principle was corrected as follows:
Vref (corrected) ⫽ V(ref) ⫻ (1 ⫺ shrinkage) (Braendgaard
et al., 1990; Howard and Reed, 2004).
Neuronal Density (Nv)
From the serial 2 ␮m pairs of sections (disectors), each
one consisted of a reference and a look-up section with a
fixed height (h) of 10 ␮m, were selected throughout the
extent of the ganglia. A test system comprised of eight
different unbiased counting frames was landed over the
image of each section field on a computer screen. A fraction (1/fr) of counting frames was randomly, uniformly,
and systematically sampled using a random start between
1 and fr. The sampled field’s images were observed on a
computer screen. The total sampled area (a) in the test
systems was 10,000 ␮m2 for group 1 and 40,000 ␮m2 for
groups 2 and 3. Neuronal profiles that appeared in a
specific area of the reference section but did not appear in
the same area in the look-up section were counted, being
referred to as Q⫺. Then, the neuronal density was calculated by the sum of Q⫺ in all disectors divided by the sum
of all disector volumes, i.e., the product between the area
test (a) and the height (h) of the disector (V(dis) ⫽ a ⫻ h).
៮ ¥V(dis).
Nv ⫽ ¥Q
The sampling scheme employed was based on the results of a pilot study in which about 34 disectors were
considered in group 1 and 20 disectors in groups 2 and 3 (4
disectors being applied in each ganglion slab). The interval (k) between each disector was 100 sections in group 1
and 250 sections in groups 2 and 3. The first reference
section was randomly chosen between 1 ⫺ k, being a
section between 1 and 100 in group 1 and between 1 and
250 in groups 2 and 3. Each ganglion from group 1 yielded
30 disectors, whereas each ganglion from groups 2 and 3
gave us 24 disectors.
The reference and look-up sections were projected on a
computer screen. The reference section was drawn up on a
transparency and then compared to the image of the
look-up section seen on the computer screen.
920
GAGLIARDO ET AL.
Total Number of Neurons (N)
The total number of neurons was obtained as the product of the ganglion volume (Vref) and the numerical density (Nv) following the equation: N ⫽ Vref (corrected) ⫻ Nv
Volume Density (Vv)
The same reference sections used to calculate the numerical density were considered for the volume density
estimate. Volume density represents the fraction of total
CMG volume occupied by neurons. It is obtained by randomly throwing a point grid system over the reference
sections. Next, the total number of points falling within
the reference space was counted (P(rs)). Also, the total
number of points landing in cell bodies was counted (P(cb)).
Then, the Vv was estimated as: Vv ⫽ ⌺P(cb)/⌺P(rs). The
results were expressed as a percentage (Howard and
Reed, 2004).
Mean Neuronal Volume (Vn)
From estimates of volume density and numerical density, the mean neuronal volume was obtained (Mayhew,
1989). It was calculated as the ratio between Vv and Nv:
Vn ⫽ Vv/Nv.
Statistical Analysis
Morphometric and stereological data were analyzed using the Statistical Analysis System (SAS, 1995). The analysis of variance (ANOVA) was performed between each
age group in all morphometric and stereological parameters to assess the effect of age, especially on the total
number of neurons (N). When the results were considered
significant (P ⬍ 0.05), the Tukey test was pursued.
The correlation analysis between all stereological parameters and body weight was carried out using Pearson’s
product moment. Correlation can be classified as higher
intensity (0.7–1.0), medium intensity (0.5– 0.7), and lower
intensity (0.1– 0.5).
The stereological results were also analyzed through a
(logistic-based) regression model (linear regression) to test
the interrelation between body weight and all stereological parameters, i.e., numerical density, ganglion volume,
total number of neurons, volume density, and mean neuronal volume, and also the interrelation between the ganglion volume and the same former parameters. From linear functions, Y ⫽ a ⫹ by,x 䡠 X (where Y is a dependent
variable, a is the intercept of the regression line with the
x-axis, by,x represents the slope of regression line, and x is
the independent variable), it was possible to predict the
values of the stereological parameters plotted against either body weight or ganglion volume. The straight correlation between both predicted and estimated values was
verified through the determination coefficient (R2), which
ranged from 0 to 1. When R2 is higher (almost 1), the
function is adequate and by all means both predicted and
estimated values are very close to one another. Conversely, when R2 is lower (⬍ 0.5), the function is inadequate, meaning that there is no correlation between estimated and predicted values.
RESULTS
Macrostructural Organization
The CMG was located close to the abdominal aorta
involving the emergence of the caudal mesenteric artery.
Fig. 1. Microstructure of the dog’s CMG in a pup showing the typical
arrangement in three ganglionic lobes or ganglionic masses (1, 2, and 3)
surrounded individually by a capsule that contained connective tissue
and vessels. Toluidine blue. Scale bar ⫽ 30 ␮m.
The dorsal portion of the ganglion was connected to the
sympathetic trunk by lumbar splanchnic nerves and its
cranial portion to the celiac-mesenteric ganglion through
intermesenteric nerves. Three different nerves left the
caudal pole of the ganglion: the main hypogastric and the
left and the right hypogastric nerves. The main hypogastric nerve followed the caudal mesenteric artery to the
colon. The left and the right hypogastric nerves were directed caudally toward the pelvic cavity reaching the pelvic ganglia.
The main hypogastric nerve divided into two separated
branches following the two branches of the caudal mesenteric artery. The former branch running cranially was
called the left colonic nerve, whereas the second, running
caudally, was named the cranial rectal nerve. The cranial
rectal and left colonic nerves gave rise to branches to the
descending colon and rectum.
In all groups analyzed, a single ganglionic mass was
macroscopically seen. The ganglion length ranged from
6.0 to 7.0 mm in pups (group 1; 6.7 ⫾ 0.06), it was constant
in adult dogs (group 2) at 12.0 mm, and ranged from 10.0
to 16.0 mm in middle-aged dogs (group 3; 12.3 ⫾ 0.32).
Microstructural Organization
Under the light microscopy, the structure of the CMG
was remarkably different, especially between pups (group
1) and adult or middle-aged dogs (groups 2 and 3). In pups
(group 1), it was observed that the CMG was not constituted of a single ganglionic mass surrounded by a capsule
as in adults (group 2) and middle-aged dogs (group 3).
However, it was formed by 2– 4 ganglionic masses (or
ganglionic lobes) surrounded individually by a capsule
that contained connective tissue and vessels (Fig. 1).
In adults (group 2) and middle-aged dogs (group 3), the
capsule sent septa of connective tissue inside the CMG,
dividing it into ganglionic units. Each ganglionic unit was
composed of various cell types and the units were separated from each other by nerve fibers, intraganglionic
capillaries, and septa of collagen fibers. Given this cytoarchitectural arrangement, the CMG was described as a
true ganglionic complex. The ganglion capsule thickness
NEURONS IN CAUDAL MESENTERIC GANGLION
921
ranged from 11.8 to 21.3 ␮m (15.2 ⫾ 3.6) in pups, from
17.6 to 21.3 ␮m (19.9 ⫾ 2.0) in adults, and from 16.7 to
28.3 ␮m (23.5 ⫾ 5.7) in middle-aged dogs.
The main cell types observed in the CMG were ganglion
neurons, glial cells, and SIF cells. Ganglion neurons were
generally spindle-shaped and readily distinguishable due
to their large size, clear nucleus, and the evident nucleolus. The nucleus was predominantly eccentric. In pups, it
was observed that the nucleolus was not a single structure, but in fact two nucleoli were frequently observed.
Furthermore, it was found that 3% of neurons were binucleate in pups. In middle-aged dogs, an aging pigment, i.e.,
lipofucsin granules, was distributed toward the cytoplasm.
Ganglion neurons were surrounded by a thin glial capsule containing one to three glial cell nuclei around the
neuron (Figs. 2– 4). Moreover, SIF cells were seen arranged in two different ways, i.e., close to neurons and
encompassing tight clusters comprised of 2–3 cells in the
proximity of blood vessels.
Morphometric Study
Neuronal cross-sectional area. The cross-sectional
area of 162 neurons in each age group was calculated. It
ranged from 125.5 to 1,035.5 ␮m2 (435.0 ⫾ 177.0) in group
1 (pups), from 109.1 to 1,966.1 ␮m2 (980.2 ⫾ 359.5) in
group 2 (adults), and from 100.0 to 3,539.1 ␮m2 (1,185.8 ⫾
569.8) in group 3 (middle-aged dogs; Fig. 5).
In group 1, the majority of neurons had a cross-sectional
area varying from 100 to 400 ␮m2 (45.7%), which was close
to the cross-sectional area frequency observed from 400 to
700 ␮m2 (43.2%). In groups 2 and 3, the majority of neurons, 37% and 27.3%, respectively, presented a cross-sectional area sized from 700 to 1,000 ␮m2 or from 1,000 to
1,300 ␮m2, particularly for group 3. In the middle-aged
dogs group, a small percentage of neurons (9.3%) varied
from 1,900 to 3,600 ␮m2, which are the largest neurons
recorded (Fig. 5).
Nuclear cross-sectional area. The cross-sectional
area of 99 neuronal nuclei was calculated in each age
group. It varied from 63.5 to 178.5 ␮m2 (115.2 ⫾ 24.7) in
group 1, from 85.3 to 297.3 ␮m2 (179.6 ⫾ 41.3) in group 2,
and from 49.7 to 417.7 ␮m2 (187.3 ⫾ 70.66) in group 3 (Fig.
6).
The majority of the nuclei in group 1 had a cross-ssectional area sized between 85 and 120 ␮m2 (42.4%). In
groups 2 and 3, the majority of nuclei, 37.4% and 33.4%,
respectively, had a cross-sectional area ranging from 155
to 190 ␮m2. In the middle-aged dog group, a small percentage of neurons (11.1%) presented a nuclear area varying from 295 to 435 ␮m2, which were the largest nuclei
measured (Fig. 6).
Nucleus-cytoplasm ratio. The nucleus-cytoplasm
ratio (nuclear cross-sectional area/cytoplasm cross-sectional area) was verified in 99 neurons in each age group.
It ranged from 0.14 to 1.53 (0.41 ⫾ 0.22) in group 1, from
0.11 to 1.90 (0.23 ⫾ 0.18) in group 2, and from 0.04 to 0.66
(0.2 ⫾ 0.08) in group 3.
In group 1, 29.3% of neurons had a nucleus-cytoplasm
ratio, varying from 0.34 to 0.44. In group 2, the majority of
neurons presented a ratio class between 0.14 and 0.24
(60.6%). Finally, in group 3, 58.6% of neurons showed a
Fig. 2. Micrographs of a pair of sections (2 ␮m thick and stained with
toluidine blue) used for estimating nerve cell numbers in pup (group 1).
In each pair, the reference section is at the top (A). B is the look-up
section. The separation between reference section and look-up section
is 10 ␮m. Black arrows show two neuronal transects seen in the reference section, which no longer exist in the look-up section (white arrows).
Scale bar ⫽ 30 ␮m.
nucleus-cytoplasm ratio class between 0.14 and 0.24 (Fig.
7).
Stereological Study
Ganglion volume (volume reference; Vref). The
ganglion volume, estimated through the Cavalieri principle, ranged from 1.9 to 2.5 mm3 (mean ⫽ 2.3; CV ⫽ 0.14)
in group 1 (pup), from 78 to 94.5 mm3 (mean ⫽ 87.2; CV ⫽
0.09) in group 2 (adult), and from 93.5 to 124 mm3
(mean ⫽ 104.5; CV ⫽ 0.16) in group 3 (middle-aged). The
error coefficient for the volume estimate was 1.8%, 2%,
and 3% in group 1; 5.3%, 7.6%, and 7.6% in group 2; and
7%, 10%, and 9% in group 3.
922
GAGLIARDO ET AL.
Fig. 4. Micrographs of a pair of sections (2 ␮m thick and stained with
toluidine blue) used for estimating nerve cell numbers in an aged dog
(group 3). In each pair, the reference section is at the top (A). B is the
look-up section. The separation between reference section and look-up
section is 10 ␮m. The black arrow shows one neuronal transect seen in
the reference section, which no longer exists in the look-up section
(white arrow). Scale bar ⫽ 30 ␮m.
Fig. 3. Micrographs of a pair of sections (2 ␮m thick and stained with
toluidine blue) used for estimating nerve cell numbers in an adult dog
(group 2). In each pair, the reference section is at the top (A). B is the
look-up section. The separation between reference section and look-up
section is 10 ␮m. Black arrows show three neuronal transects seen in
the reference section, which no longer exists in the look-up section
(white arrows). In A and B, large white arrowheads show two glial cell
nuclei. Scale bar ⫽ 30 ␮m.
Numerical density (Nv). The numerical density estimated using the physical disector method ranged from
29,143 to 32,258 mm⫺3 (mean ⫽ 29,911; CV ⫽ 0.07) in
group 1 (pup), from 12,500 to 13,158 (mean ⫽ 12,719;
CV ⫽ 0.03) in group 2 (adult), and from 10,978 to 11,818
(mean ⫽ 11,500; CV ⫽ 0.04) in group 3 (middle-aged; Figs.
2– 4).
Total number of neurons (N). The total number of
neurons, estimated using the physical disector method
associated with the Cavalieri principle, ranged from
55,448 to 81,677 (mean ⫽ 70,140; CV ⫽ 0.19) in group 1
(pup), from 975,000 to 1,243,421 (mean ⫽ 1,110,307; CV ⫽
0.12) in group 2 (adult), and from 1,026,468 to 1,452,534
(mean ⫽ 1,204,516; CV ⫽ 0.18) in group 3 (middle-aged).
Volume density (Vv). The volume density ranged
from 32.5% to 39% (mean ⫽ 35.3%; CV ⫽ 0.09) in group 1
(pup), from 27.1% to 31.8% (mean ⫽ 29.1%; CV ⫽ 0.08) in
group 2 (adult), and from 28.8% to 30.6% (mean ⫽ 29.4%;
CV ⫽ 0.03) in group 3 (middle-aged).
Mean neuronal volume (Vn). The mean neuronal
volume ranged from 11,166 to 12,131 ␮m3 (mean ⫽
11,800; CV ⫽ 0.04) in group 1 (pup), from 21,680 to 24,183
␮m3 (mean ⫽ 22,867; CV ⫽ 0.06) in group 2 (adult), and
from 24,488 to 26,261 ␮m3 (mean ⫽ 25,667; CV ⫽ 0.04) in
group 3 (middle-aged). All the stereological parameters
investigated in this study are summarized in Table 1.
NEURONS IN CAUDAL MESENTERIC GANGLION
923
Fig. 5. Histograms documenting the distribution of neuronal sizes
(cross-sectional area of the largest profile of a neuron). The top histogram shows the percentage distribution of sizes divided into classes of
the same size for the three age groups and ranging from the smallest
(100 – 400 ␮m2) to the largest (3,400 –3,700 ␮m2). The other three histograms illustrate a single age group each and present the distribution of
cell body sizes in 11 classes evenly spread between the maximum and
minimum values for those age groups.
Coefficient of Correlation and Analysis of
Regression
In the CMG, the correlation between animal body
weight and all stereological parameters was tested by
means of Pearson’s product moment correlation coefficient
and analysis of regression. The body weight of animals,
total number of neurons, ganglionic volume, and the mean
particle volume demonstrated a positive correlation to
each other. However, when these parameters were plotted
against the numerical density and volume density, a negative correlation was observed. The correlation among
stereological parameters was classified as high intensity
except for volume density, which presented a medium
intensity correlation.
Given the high intensity correlation described for body
weight and ganglion volume when plotted against further
Fig. 6. Histograms documenting the distribution of nuclear sizes
(cross-sectional area of the largest profile of a neuron nucleus). The top
histogram shows the percentage distribution of sizes divided into
classes of the same size for the three age groups and ranging from the
smallest (50 – 85 ␮m2) to the largest (400 – 435 ␮m2). The other three
histograms illustrate a single age group each and present the distribution
of neuron nuclei sizes in 11 classes evenly spread between the maximum and minimum values for those age groups.
stereological parameters, with the exception of volume
density, a linear regression analysis, expressed by the
function Y ⫽ a ⫹ by,x 䡠 X, was pursued in an attempt to
verify the interrelationship between predicted and estimated stereological parameter values (Figs. 8 and 9).
Hence, body weight was assumed to be a fixed value (X)
924
GAGLIARDO ET AL.
Fig. 7. Distribution of nucleus-cytoplasm ratio classes and the relative frequencies in all age groups (pup,
adult, and aged). The classes were evenly spread in the same size (0.10).
TABLE 1. Overall view of all stereological parameters investigated in the CMG from dogs at three distinct
ages
GROUP
AGE
BW1
Nv2
Vref3
N4
Vv5
Vn6
I (pup)
1 month old
2 months old
2 months old
0.15
0.18
0.18
0.17
0.1
13
18
15
15.33
0.16
20
23
26
23.00
0.13
29,143
28,333
32,258
29,911
0.07
12,500
13,158
12,500
12,719
0.03
10,978
11,704
11,818
11,500
0.04
2.5
1.9
2.5
2.3
0.14
89
94.5
78
87.2
0.09
93.5
124
96
104.5
0.16
73,294
55,448
81,677
70,140
0.19
1,112,500
1,243,421
975,000
1,110,307
0.12
1,026,468
1,452,534
1,134,545
1,204,516
0.18
32.5
34.4
39
35.3
0.09
28.4
31.8
27.1
29.1
0.08
28.8
30.6
28.9
29.4
0.03
11,166
12,131
12,093
11,800
0.04
22,720
24,183
21,680
22,867
0.06
26,261
26,152
24,488
25,667
0.04
Mean
CV
II (adult)
Mean
CV
III (middle-aged)
1 year old
2 years old
2 years old
5 years old
7 years old
10 years old
Mean
CV
1
Body weight (Kg).
Numerical density (mm⫺3).
3
Ganglion volume (mm3).
4
Total number of neurons.
5
Volume density (%).
6
Mean neuronal volume (␮m3).
2
and all other parameters (Y) were therefore plotted. By
the same token, the ganglion volume was also pointed out
as a fixed value. By all means, the predicted values were
fairly close to the estimated ones, as evidenced by the high
determination coefficient (R2), except for volume density,
which showed a low determination coefficient when body
weight was assumed to be a fixed value (R2 ⫽ 0.55), and
when ganglion volume was considered to be a fixed value
(R2 ⫽ 0.53).
DISCUSSION
Technical Approach
For the quantitative work, it is absolutely necessary to
see and define clearly the counting unit and therefore
avoid shrinkage. Thus, the choice of the adequate fixative,
i.e., a modified Karnovsky solution delivered by vascular
perfusion, and a plastic resin embedding were crucial for
the development of this experiment. Due to shrinkage
problems caused by paraffin embedding, a plastic resin
such as Araldite is preferred instead (Guillery and Herrup, 1997; Von Bartheld, 2002; Gardella et al., 2003) and
also the Araldite-embedded block sectioning is a requirement for both measurements and quantification, which
provides an outstanding resolution and sharpness.
In this study, the cell body rather than the nuclei or
nucleolus was considered the counting unit. The prevertebral ganglia of mammalian comprise not only mononucleate neurons as reported by Miolan and Niel (1996)
but also binucleate neurons, which have been found in the
guinea pig (Szurszewski and Miller, 1994; Ermilov et al.,
2000), capybaras (Ribeiro et al., 2004), and rabbit (Sasahara et al., 2003). Given the reasons above, the choice of
the nucleus as a counting unit was not feasible. Furthermore, the literature shows that binucleate neurons can be
observed in the initial phases of development (Szurszewski and King, 1989; Appenzeller, 1990) and at this stage it
is unknown if in the dogs from group 1 (1–2 months old)
the cell division has been completed. By the same token,
the use of the nucleolus for quantification was not possible
either because of the presence of one or two nucleoli as
reported for the celiac-mesenteric ganglion (Miller et al.,
1996; Ribeiro et al., 2002).
Morphological Aspects: Macro- and
Microstructure
The location of the dog’s CMG was the same in the
different age groups as reported by Goshal (1986), Evans
(1993), and Gagliardo et al. (2003). Macroscopically, the
NEURONS IN CAUDAL MESENTERIC GANGLION
925
Fig. 8. Graphs representing the linear functions between the body weight (kg) and the numerical density (Nv), ganglion volume (Vref), total number
of neurons (N), and mean neuronal volume (Vn). The values were represented by triangles for estimated values and diamonds for predicted values.
Notice that the plots do not contain values for pups.
CMG was seen as a unique ganglion mass, as also reported by Gagliardo et al. (2003). However, Goshal (1986)
has reported that the CMG can be divided into two ganglion masses in the dogs and four ganglion masses in cats.
Although more than one ganglion mass was not macroscopically observed, it was microscopically verified that
the pups’ CMG comprised two to four ganglion masses (or
ganglionic lobes), which is in agreement with the observations made by Goshal (1986).
The microstructural arrangement of the dog’s CMG was
similar between adult and middle-aged dogs, e.g., the
CMG was divided into distinct compartments by capsular
septa of connective tissue as reported for mammalian prevertebral ganglia by Gabella et al. (1988), Szurszewski
and King (1989), Banks (1992), Szurszewski and Miller
(1994), Miolan and Niel (1996), Schmidt (1996), and Gagliardo et al. (2003). In pups, however, two or four ganglion
masses were noticed and they were structurally linked by
connective tissue and each one was ensheathed by a ganglion capsule. The ganglion capsule thickness had a 30%
increase from pup to adult, a 18% increase from adult to
middle-aged, and a 54.6% increase from pup to middleaged animals.
Morphometric Study
The size of the CMG neurons, expressed as their crosssectional area, showed a 2.25-fold increase from pup to
adult and 1.2-fold from adult to aged, which was significant in both cases (P ⬍ 0.01). The results of the present
study are in agreement with those reported for the celiac-
mesenteric and superior cervical ganglion of rats (Baker
and Santer, 1988) and of humans (Schmidt, 1996), for the
rat’s distal vagal ganglion (Soltanpour et al., 1996), and
for the rat’s hypogastric ganglia (Warburton and Santer,
1997).
The increase in size of neurons between pups and adults
can be explained by the incomplete maturation of those at
the early stages of development as reported by Vega et al.
(1993) and Masliukov (2001). However, the significant
neuronal increase between adult and middle-aged is susceptible to some speculation such as the possibility of the
perikaryon enlargement given the accumulation of lipofucsin granules (Finch, 1993; Warburton and Santer,
1997), due to the reduction of the pressure exercised by
the surrounded cells as a result of cell death (Baker and
Santer, 1988), or as a compensation mechanism for the
neuronal loss with development (Finch, 1993; Warburton
and Santer, 1997).
Not only an increase of the cell body but also in the
nuclear size was observed during the development. However, the nuclear increase was significant (P ⬍ 0.01) only
when pups were compared to other groups, a 1.56-fold
increase being verified from pups to adults and a 1.04-fold
increase from adults to middle-aged animals. The nuclear
increase during maturation (from pups to adults) was
already expected since this phase is characterized by an
increase of nuclear volume (McMahon et al., 2003).
The nucleus-cytoplasm ratio of the CMG neurons decreases with aging, as stated by Ledda et al. (2000). This
ratio decreases when the area occupied by the cytoplasm
926
GAGLIARDO ET AL.
Fig. 9. Graphs representing the linear functions between the ganglion volume (Vref) and the numerical density (Nv), total number of neurons (N),
and mean neuronal volume (Vn). The values were represented by triangles for estimated values and diamonds for predicted values. Notice that the
plots do not contain values for pups.
in either adults or aged is larger than the area occupied in
pups, or when the area occupied by the nucleus in either
adults or aged is smaller than those occupied in pups, or
by means of the association of these two factors. In this
study, it has been verified that although both nucleus and
cytoplasm increased in size, the cytoplasm increase was
proportionally larger than that of the nucleus during aging.
Stereological Study
Ganglion volume. The increase in both sensory and
parasympathetic ganglion volume during development
has already been reported in the literature, such as in the
rat’s distal vagal ganglion (Soltanpour et al., 1996), rat’s
dorsal root ganglion (Popken and Farel, 1997), rat’s hypogastric ganglion (Warburton and Santer, 1997), and cervical (C5) and lumbar (L4) (Bergman and Ulfhake, 1998).
Similar results were now found in a sympathetic ganglion, i.e., the dog’s CMG, where a 37.9-fold increase was
shown from pups to adults and a 45.4-fold increase from
pups to middle-aged animals. These figures were significant (P ⬍ 0.01), though any increase between adult and a
middle-aged animal was of no significance.
Numerical density. The numerical density in the
CMG of pups was higher and significant (P ⬍ 0.01) when
compared to the additional groups. In adults and in middle-aged dogs, the numerical density was 42% and 38%,
respectively, of that found in pups. Yet, though a decrease
was found between adults and middle-aged animals, these
results were of no significance.
It should also be stressed the lower coefficients of variation (CV) for the numerical density (Nv) within age
groups may express a tight control of the interindividual
and age-related variation in the density of neurons in a
certain volume of the CMG. That may imply a functional
role in the target organ innervation as well as in the
synaptic organization within CMG. Coefficients of variation were of 7% in pups, 3% in adults, and 4% in middleaged dogs.
The biological meaning of CV was stated by Clegg
(1983), who performed morphometric studies of the spleen
of hypoxic mice. In that experiment, coefficients of variation of spleen variables showed a tendency to decrease in
animals exposed to an atmospheric pressure of 72 kPa, but
rose markedly at 52 kPa. This finding indicated that at 72
kPa spleen and red pulp changes are adaptive, but at 52
kPa they indicated an overall relative failure of adaptive
mechanisms, with consequent reduced somatic fitness.
Hence, when things are biologically important for survival
or function, there is often a tight control (Clegg, 1983).
An age-related decrease in neuronal density was also
observed in dorsal root ganglia, demonstrating a reduction
of 50% in neuronal density when 2- to 3-month rats were
compared to 30-month rats (Popken and Farel, 1997) and
also a 40% decrease in hypogastric ganglia’s neuronal
NEURONS IN CAUDAL MESENTERIC GANGLION
density when 4-month rats were compared to 24-month
rats (Warburton and Santer, 1997).
The high numerical density in pups was due to the
small size of neurons and also the volume density was
higher. In addition, with aging, a 17% increase of nonneuronal tissue amount (connective tissue, vessels, and neuropil) was reported in our present investigation and, in
line, a reduction of approximately 2.35-fold and 2.6-fold in
the numerical density was observed when pups were compared to both adults and middle-aged animals, respectively. Yet the aging process was accompanied by a 17%
decrease in the neuronal volume density when pups were
compared to both adults and middle-aged animals. Further, the mean neuronal volume increased significantly
(P ⬍ 0.01) with aging when all groups were compared to
one another.
Total number of neurons. Although it is easy to
think that the phases of development are associated with
a decrease in the number of neurons, despite some controversy about this dogma (Crutcher, 2002), and that apoptosis is a natural process of development, they are no more
than a good deduction (Finch, 1993). The results of this
study demonstrated that in the dog’s CMG there was no
reduction in the total number of neurons from adult to
middle-aged dogs. Moreover, the number of neurons found
in adult and middle-aged dogs is larger and more significant (P ⬍ 0.01) than the total number of neurons estimated in a puppy.
The number of neurons in the CMG of pups represented
only 6.3% of that quantity in adults and 5.8% of that in
middle-aged dogs. The small percentage of neurons obtained in pups in relation to the other groups could be
associated with the small ganglion volume in pups when
compared to the other age groups.
In addition, a nonsignificant 40% increase in the total
number of neurons in the hypogastric ganglion of rats of
24 months compared to rats of 4 months was reported by
Warburton and Santer (1997) and either a 28% significant
increase (using the physical disector method) or a 19%
significant increase (using the point counting method) in
the total number of neurons in the dorsal root ganglia of
rats of 80 days compared to rats of 11 days was reported by
Popken and Farel (1997). It was stated by the latter that
the increase in the number of neurons could be associated
to possible cell division in different developmental periods.
However, Farel (2003) reported that the increase in the
number of neurons in the dorsal root ganglia of rats is not
associated with a possible neurogenesis, but may be the
result of a later maturation or incomplete differentiation,
which makes it difficult to identify the neurons that will
not be quantified due to the fact that they do not show a
typical morphology.
By contrast, a reduction in the number of neurons with
aging was shown in the superior cervical ganglion, where
rats of 4 months presented a not significantly larger number of neurons than rats of 24 months. In that study, the
physical fractionator method was pursued (Santer, 1991).
Similar results were also reported by Bergman and Ulfhake (1998), who observed a reduction in the number of
neurons in the dorsal root ganglia of rats of 3 months
when compared to rats of 30 days using the optical disector.
Hence, the 17-fold increase in the total number of neurons of the dog’s CMG might be related to three main
927
factors: neuronal division, later maturation or incomplete
neuronal differentiation, and binucleate neuron division.
In the literature, there is no robust evidence for cell division, at least not for the rat DRG (Popken and Farel,
1997). Instead, late maturation or incomplete neuronal
differentiation seems to be the case for the rat’s hypogastric ganglion (Farel, 2003).
As for the third possibility, i.e., binucleate neuron division, it seems very unlikely that a 3% population of binucleate neurons as seen in pups (which would account for a
6% increment after the mitotic division) would be responsible for a 17-fold increase in CMG neurons as reported in
this investigation. Nevertheless, cell markers (Activin,
BrdU) should be tested for large mammals’ sympathetic
ganglia in the near future.
Relationship Between All Stereological
Parameters and Body Weight or Ganglion
Volume
Due to the interrelation between either body weight or
ganglion volume and aging in the dogs investigated in this
study, it was possible to predict the total number of neurons in CMG using both body weight and ganglion volume
in an attempt to verify whether or not size and total
number of neurons are both allometrically and aging
ruled, i.e., if either the animal’s body weight and ganglion
volume or aging influence these parameters.
From the linear regression analysis, it was possible to
predict the interrelationship between body weight and all
stereological parameters, i.e., total number of neurons,
neuronal density, ganglion volume, and mean neuronal
volume, as well as the relationship between ganglion volume and the following stereological parameters: total
number of neurons, neuronal density, and mean neuronal
volume. It has been shown that the results estimated in
this study allow a close correlation to the predicted results, verified by the high determination coefficient (R2). A
similar study was pursued by Mayhew (1991) using the
relationship between cerebellar weight and the number of
Purkinje cells of different animal species. However, it was
carried out using a different stereological method than
was used in our study, i.e., the fractionator. The results
obtained reflected a positive correlation between number
and Purkinje layer surface area within a given species;
further, the surface area was related to the body weight of
each species.
Although our study shows a close correlation between
both predicted and estimated results (stereological parameters) and either body weight or ganglion volume, the
results using the ganglion volume allow a better correlation with the estimated results. Although some of the
predicted results slightly corresponded to the estimated
values, the reliability of the function expressed by the
determination coefficient (R2) was high for all parameters,
with the exception of the volume density when plotted
against the body weight and ganglion volume, where only
55% and 53% correlations, respectively, were verified between predicted and estimated results.
From the linear functions obtained, it was possible to
predict that dogs of 180 g, 15 kg, and 23 kg would have in
the CMG, respectively, 106,041, 982,111, and 1,455,023
neurons; 28,945, 15,083, and 7,600 neurons/mm3; a volume of 4.3, 81.1, and 122.5 mm3; a volume density of 34%,
30%, and 28%; and a mean neuronal volume of 12,126,
928
GAGLIARDO ET AL.
22,262, and 27,733 ␮m . In our study, the estimated results for the same dogs were 81,677, 1,243,421, and
1,452,534 neurons; 32,258, 13,158, and 11,704 neurons/
mm3; a volume of 2.5, 94.5, and 124 mm3; a volume density of 39%, 31.8%, and 30.6%; and a mean neuronal
volume of 12,093, 24,183, and 26,152 ␮m3.
By the same token, a similar prediction was possible
from the CMG volume. For instance, a dog with a ganglion
volume of 2.5, 94.5, and 124 mm3 would have, respectively, 77,015, 1,139,524, and 1,480,219 neurons; 29,203,
12,688, and 7,393 neurons/mm3; a volume density of 35%,
30%, and 28%; and a mean neuronal volume of 11,985,
23,989, and 27,838 ␮m3. The estimated results for the
same dogs were 81,677, 1,243,421, and 1,452,534 neurons;
32,258, 13,158, and 11,704 neurons/mm3; a volume density of 39%, 31.8%, and 30.6%; and a mean particle volume
of 12,093, 24,183, and 26,152 ␮m3. The correlation between the stereological parameters and body weight or
between stereological parameters and the ganglion volume can be easily shown in the above example, where an
increase in body weight is accompanied by an increase in
ganglion volume and consequently by an increase in the
total number of neurons.
On the other hand, an increase in both ganglion volume
and body weight was accompanied by a decrease in the
number of neurons per volume unit (mm⫺3; numerical
density) inside the ganglion. Moreover, an increase in the
mean neuronal volume is correlated with an increase in
both body weight and ganglion volume. In this way, it is
possible to show that a dog of 180 g has either a ganglion
volume or a total number of neurons smaller than those in
dogs of 15 and 23 kg. Conversely, both numerical density
and volume density in dogs of 180 g are larger than in dogs
of 15 and 23 kg.
In conclusion, this study sheds light on some aspects of
postnatal development in the dog’s CMG, which was represented here by the period from pups to middle-aged
dogs. The main aspects seen here were the total number of
neurons, CMG’s microstruture, neuronal density, and
neuron size.
Although there is still controversy as to what happens
to the total number of neurons in aging (specially in large
mammals) and in different parts of the nervous system
(enteric nervous system and central nervous system), the
results presented in this investigation, which were obtained by using modern and current unbiased stereological methods (Baddeley, 2001) for number estimation, revealed a nonreduction in this parameter during aging,
which increased instead. Furthermore, both body weight
and ganglion volume are positively correlated to aging in
dogs and either body weight or ganglion volume allows an
accurate prediction of the total number of neurons in the
dog’s caudal mesenteric ganglion during the postnatal
period.
In addition, due to the wide territory of innervation of
CMG in dogs, further studies (using neurotracers) may
focus on specific CMG’s cell populations in order to figure
out what happens, for instance, to the total number of
rectum-innervating neurons or descending colon-innervating neurons during aging in an attempt to elucidate
the causes of several gastrointestinal disorders especially
in middle-aged or aged dogs.
3
ACKNOWLEDGMENTS
A.A.C.M.R. thanks Professor Giorgio Gabella (University College London, U.K.) for his advice with the preparation of the material (fixation, embedding, sectioning)
and cytological interpretation of the results during aging,
Professor Terry M. Mayhew (University of Nottingham,
U.K.) for advice, help with the use of the physical disector,
and the linear regression analysis interpretation performed in this study, as well as Emerson Ticona Fioretto
for his advice with the preparation of the images that
illustrate this study. K.M.G. thanks Naianne Kelly Clebis
for her assistance in computer-assisted image acquisition.
LITERATURE CITED
Andrews TJ. 1996. Autonomic nervous system as a model of neuronal
aging: the role of target tissue and neurotrophic factors. Microsc
Res Tech 35:2–19.
Appenzeller O. 1990. Anatomy and histology. In: Appenzeller O, editor. The autonomic nervous system. Oxford: Elsevier. p 1–10.
Baddeley A. 2001. Is stereology “unbiased”? Trends Neurosci 24:375–
376.
Baker DM, Santer RM. 1988. Morphometric studies on pre- and
paravertebral sympathetic neurons in the rat: changes with the
age. Mech Aging Dev 42:139 –145.
Banks WJ. 1992. Sistema nervoso. In: Banks WJ, editor. Histologia
veterinária aplicada. São Paulo: Editora Manole. p 318 –351.
Bennett MR, Gibson WG, Lemon G. 2002. Neuronal cell death, nerve
growth factor and neurotrophic models: 50 years on. Auton Neurosci 95:1–23.
Bergman E, Ulfhake B. 1998. Loss of primary sensory neurons in the
very old rat: neuron number estimates using the dissector method
and confocal optical sectioning. J Comp Neurol 396:211–222.
Braendgaard H, Evans SM, Howard CV, Gundersen HJG. 1990. The
total number of neurons in the human neocortex unbiasedly estimated using optical disectors. J Microsc 157:285–304.
Bywater RAR. 1993. Activity following colonic distension in enteric
sensory fibres projecting to the inferior mesenteric ganglion in the
guinea-pig. J Auton Nerv Syst 46:19 –26.
Cabello CR, Thune JJ, Pakkenberg H, Pakkenberg B. 2002. Aging of
substantia nigra in humans: cell loss may be compensated by hypertrophy. Neuropathol Appl Neurobiol 28:283–291.
Clegg EJ. 1983. Morphometric studies of the spleen of the hypoxic
mouse. J Microsc 131:155–161.
Crutcher KA. 2002. Aging and neuronal plasticity: lessons from a
model. Auton Neurosci 96:25–32.
Ermilov LG, Miller SM, Schmalz PF, Hanani M, Szurszewski JH.
2000. The three-dimensional structure of neurons in the guinea pig
inferior mesenteric and pelvic hypogastric ganglia. Auton Neurosci
83:116 –126.
Evans HE. 1993. The autonomic nervous system. In: Evans HE,
editor. Miller’s anatomy of the dog. London: W.B. Saunders. p
776 –799.
Farel PB. 2003. Late differentiation contributes to the apparent increase in sensory neuron number in juvenile rat. Dev Brain Res
144:91–98.
Finch CE. 1993. Neuron atrophy during aging: programmed or sporadic? Trends Neurosci 6:104 –110.
Fossum T. 2002. Cirurgia de pequenos animais. São Paulo: Editora
Roca.
Gabella G, Trigg P, Mcphail H. 1988. Quantitative cytology neurons
and satellite glial cells in the superior cervical ganglion of the
sheep: relationship with ganglion neuron size. Neurocytology 17:
753–769.
Gabella G. 2004. Autonomic nervous system. In: Gabella G, editor.
The rat nervous system. London: Academic Press. p 81–103.
Gagliardo KM, Silva RA, Guidi WL, Ribeiro AACM. 2003. Gross and
semi-thin view of the caudal mesenteric complex of the domestic dog
(Canis familiaris—Linnaeus, 1758). Anat Histol Embryol 32:1– 8.
Gardella D, Hatton WJ, Rind HB, Rosen GD, Von Bartheld CS. 2003.
Differential tissue shrinkage and compression in the z-axis: impli-
NEURONS IN CAUDAL MESENTERIC GANGLION
cations for optical dissector counting in vibratome-, plastic- and
cryosections. J Neurosci Methods 124:45–59.
Gavazzi I, Cowen T. 1996. Can the neurotrophic hypothesis explain
degeneration and loss of plasticity in mature and aging autonomic
nerves? J Auton Nerv Syst 58:1–10.
Gonella J, Bouvier M, Blanquet F. 1987. Extrinsic nervous control of
the motility of small and large intestines and related spincters.
Physiol Rev 67:902–961.
Goshal NG. 1986. Inervação abdominal, pélvica e caudal autônoma.
In: Getty R, editor. Anatomia dos animais domésticos. Rio de
Janeiro: Guanabara Koogan. p 1628 –1634.
Guillery RW, Herrup K. 1997. Quantification without pontification:
choosing a method for counting objects in sectioned tissues. J Comp
Neurol 386:2–7.
Gundersen HJG. 1977. Notes on the estimation of the numerical
density of arbitrary profiles: edge effect. J Microsc 111:219 –223.
Gundersen HJG, Jensen EB. 1987. The efficiency of systematic sampling in stereology and its prediction. J Microsc 147:229 –263.
Gundersen HJG, Bagger P, Bendtsen TF, Evans SM, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B,
Sorensen FB, Vesterby A, West MJ. 1988. The new stereological
tools: disector, fractionator, nucleator and point sampled intercepts
and their use in pathological research and diagnosis. Acta Pathol
Microbiol Immunol Scand 96:857– 881.
Gundersen HJG, Jensen EB, Kiâu K, Nielsen J. 1999. The efficiency
of systematic sampling in stereology: reconsidered. J Microsc 193:
199 –211.
Howard CV, Reed MG. 2004. Three-dimensional measurement in
microscopy. In: Howard CV, Reed MG, editor. Unbiased stereology.
Oxford: Bios Scientif Publishers.
Ledda M, Barni L, Altieri L, Pannese E. 2000. Decrease in the nucleocytoplasmic volume ratio of rabbit spinal ganglion neurons with
age. Neurosci Lett 286:171–174.
Luckensmeyer GB, Keast JR. 1995. Distribution and morphological
characterization of viscerofugal projections from the large intestine
to the inferior mesenteric and pelvic ganglia of the male rat. Neuroscience 66:663– 671.
Luckensmeyer GB, Keast JR. 1996. Immunohistochemical characterisation of viscerofugal neurons projecting to the inferior mesenteric
and major pelvic ganglia in the male rat. J Auton Nerv Syst 61:6 –
16.
Masliukov MP. 2001. Sympathetic neurons of the cat stellate ganglion
in postnatal ontogenesis: morphometric analysis. Auton Neurosci
89:48 –53.
Mayhew TM. 1989. Stereological studies on rat spinal neurons during
postnatal development: estimates of mean perikaryal and nuclear
volumes free from assumptions about shape. J Anat 162:97–109.
Mayhew TM. 1991. Accurate prediction of purking cell number from
cerebellar weight can be achieved with the fracionator. J Comp
Neurol 308:162–168.
Mayhew TM, Olsen DR. 1991. Magnetic resonance imaging (MRI) and
model-free estimates of brain volume determined using the cavalieri principle. J Anat 178:133–144.
Mayhew TM, Gundersen HJG. 1996. “If you assume, you can make an
ass out of u and me”: a decade of the dissector for stereological
counting of particles in 3D space. J Anat 188:1–15.
McMahon SS, Dockery P, Mcdermott KW. 2003. Estimation of nuclear
volume as an indicator of maturation of glial precursor cells in the
developing rat spinal cord: a stereological approach. J Anat 203:
339 –344.
929
Miller SM, Hanani M, Kuntz SM, Schmalz PF, Szurszewski JH. 1996.
Light, electron and confocal microscopic study of the mouse superior
mesenteric ganglion. J Comp Neurol 365:427– 444.
Miolan J, Niel J. 1996. The mammalian sympathetic prevertebral
ganglia: integrative properties and role in the nervous control of
digestive tract motility. J Auton Nerv System 58:125–138.
Morrison JH, Hof PR. 1997. Life and death of neurons in the aging
brain. Science 278:412– 418.
Pakkenberg B, Gundersen HJG. 1988. Total number of neurons and
glial cells in human brain nuclei estimated by disector and fractionator. J Microsc 150:1–20.
Popken GJ, Farel PB. 1997. Sensory neuron number in the neonatal
and adult rats estimated by means of the stereologic and profilebased methods. J Comp Neurol 386:8 –15.
Ribeiro AACM, Elias CF, Liberti EA, Guidi WL, De Souza RR. 2002.
Structure and ultrastructure of the celiac mesenteric ganglion complex in the domestic dog. Anat Histol Embryol 31:344 –349.
Ribeiro AACM, Davis C, Gabella G. 2004. Estimate of size and total
number of neurons in superior cervical ganglion of rat, capybara
and horse. Anat Embryol 208:367–380.
Santer RM. 2001. Sympathetic neuron numbers in ganglia of young
and aged rats. J Auton Nerv Syst 33:221–222.
SAS. 1995. User’s guide: basic and statistic. Cary, NC: SAS Institute.
p 1686.
Sasahara THC, Souza RR, Machado MRF, Silva RA, Guidi WL, Ribeiro AACM. 2003. Macro- and microstructural organization of the
rabbit’s celiac-mesenteric ganglion complex (Oryctolagus cuniculus). Ann Anat 185:441– 448.
Schmidt RE. 1996. Neuropathology of human sympathetic autonomic
ganglia. Microsc Res Tech 35:107–121.
Slatter D. 1998. Sistema gastrointestinal. In: Slatter D, editor. Manual de cirurgia de pequenos animais. São Paulo: Editora Manole. p
591–760.
Soltanpour N, Baker DM, Santer RM. 1996. Neurons and microvessels of the nodose (vagal sensory) ganglion in young adult and aged
rats: morphometric and enzyme histochemical studies. Tissues Cell
28:593– 602.
Szurszewski JH, King BF. 1989. Physiology of prevertebral ganglia in
mammals with special reference to inferior mesenteric ganglion. In:
Schultz SG, Wood JD, Rauner BB, editors. Handbook of gastrointestinal physiology. Bethesda, MD: American Physiological Society.
p 519 –577.
Szurszewski JH, Miller SM. 1994. Physiology of the prevertebral
ganglia. In: Johnson LR, editor. Physiology of the gastrointestinal
tract. New York: Raven Press. p 795– 878.
Szweda PA, Camouse M, Lundberg KC, Oberley TD, Szweda LI. 2003.
Aging, lipofuscin formation, and free radical-mediated inhibition of
cellular proteolytic systems. Aging Res Rev 2:383– 405.
Vega JA, Calzada B, Del Valle ME. 1993. Age-related in the mammalian autonomic and sensory ganglia. In: Amenta F, editor. Aging of
the autonomic nervous system. London: CRC Press. p 31– 61.
Von Bartheld CS. 2002. Counting particles in tissue sections: choices
of methods and importance of calibration to minize biases. Histol
Histopathol 17:639 – 648.
Warburton AL, Santer RM. 1997. The hypogastric ant thirteenth
thoracic ganglia of the rat: effects of age on the neurons and their
extracellular environment. J Anat 190:115–124.
West MJ. 1994. Advances in the study of age-related neuron loss. Sem
Neurosci 6:403– 411.
Документ
Категория
Без категории
Просмотров
0
Размер файла
676 Кб
Теги
change, predicted, dogstotal, caudal, volume, tota, can, ganglion, mesenteric, weight, size, postnatal, body, number, neurons, related
1/--страниц
Пожаловаться на содержимое документа