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First published online March 30, 2006
Journal of Experimental Biology 209, 1454-1462 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02141
The postnatal development of neocortical neurons and glial cells in the Göttingen minipig and the domestic pig brain
1 Research Laboratory for Stereology and Neuroscience, Copenhagen University
Hospital, Bispebjerg, Denmark
2 PET Centre, Aarhus University Hospitals, Aarhus, Denmark
3 Ellegaard Göttingen Minipigs ApS, Slagelse, Denmark
4 Department of Psychiatry, Copenhagen University Hospital, Bispebjerg,
Denmark
* Author for correspondence (e-mail: forsklab{at}bbh.hosp.dk)
Accepted 2 February 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: cavalieri volume, fractionator, porcine, total cell number, stereology
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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The pig has previously been considered a potential animal model for
evaluating the effects of developmental insults on human brain growth and
development (Dickerson and Dobbing,
1966
; Book and Bustad,
1974; Dobbing and Sands,
1979; Pond et al.,
2000). In general, the mammalian brain appears to go through a
momentary period of rapid growth, exemplified by a characteristic growth rate
curve when the percent of adult brain mass is plotted against age
(Dobbing and Sands, 1979). One
of the most important interspecies differences seems to be the complexity of
the final product as well as the timing of the brain growth spurt in relation
to birth (Dobbing, 1974). The
timing of the brain growth spurt can be used to roughly categorize mammalian
species as prenatal, perinatal or postnatal developers
(Dobbing and Sands, 1973). In
a comparison of seven mammalian species, it was demonstrated that the pig
brain, like that of humans, develops perinatally, with a brain growth spurt
extending from midgestation to early postnatal life
(Dickerson and Dobbing, 1966;
Dobbing and Sands, 1979;
Pond et al., 2000). This is in
contrast to other mammalian species, e.g. the brain of guinea pig, sheep and
monkey, which has a prenatal growth spurt, or the brain of rat and rabbit,
which develops postnatally (Dobbing and
Sands, 1979). The development of the pig brain is also considered
more similar to the human brain with respect to myelination, compositions and
electrical activity (Dickerson and Dobbing,
1966; Fang et al.,
2005; Flynn, 1984;
Pampliglione, 1971; Thibault and
Margulies, 1998).
The traditional view of the primate neocortex is that it is structurally
stable and that neurogenesis and synapse formation occur during early
development (Bourgeois et al.,
1994; Rakic,
1985b). Quantitative studies based on DNA quantification in the
human brain have indicated that the major phase of neuronal multiplication
occurs during the first half of gestation, prior to the numerically larger but
slower phase of glial multiplication, which continues into the first postnatal
years (Dobbing and Sands,
1973; Dobbing,
1974). A two-phased growth pattern has similarly been observed in
a stereological study on total cell numbers in the developing human fetal
forebrain (Samuelsen et al.,
2003). A clear cell discrimination has, however, not been
performed in early fetal life.
In an attempt to further evaluate the pig as a potential model for human
brain development and to provide a quantitative structural basis for
comparative and experimental studies, a number of quantitative examinations on
the neonate and adult pig brain have been initiated. Quantitative data are
obtained using assumption-free stereological methods. The methods are designed
to describe quantitative parameters without assumptions about shape, size,
orientation and distribution of cells in the reference space and are based on
established procedures for systematic, uniformly random sampling that are
superior in precision compared with results obtained by independent sampling
(Gundersen and Jensen, 1987).
In the present paper, neocortical cell numbers were obtained using the optical
fractionator method (Gundersen,
1986; West et al.,
1991). A detailed description of tissue processing and optimal
sampling procedures for application in the pig brain is presented.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Damm Jorgensen, 1998). The
Göttingen minipig in Denmark originates from caesarean sections performed
on German sows in the early 1990s. The offspring was transferred to a barrier
facility and kept in a non-contaminated environment. Only three populations of
limited population size exist in the world today.
The Göttingen minipig is an outbred animal, with less than 5%
in-breeding (Glodek, 1986).
The newborn Göttingen minipig has a body mass of 
350–450 g.
Boars become sexually mature at an age of 3–4 months, weighing 6–8
kg, while sows are sexually mature at an age of 4–5 months, weighing
7–9 kg. The gestational period is 112–114 days and the average
litter size is 5–6 animals. The adult mass of the Göttingen
minipig, at an age of two years, is 35–40 kg
(Bollen and Ellegaard, 1997;
Damm Jorgensen, 1998).
The Göttingen minipig of today is not gnotobiotic but is kept in barriers to avoid bacterial contamination and thereby minimize the risk of any influence on research caused by microbiological organisms. Health monitoring is carried out twice a year, based on FELASA (Federation of European Laboratory Animal Science Associations) guidelines.
The domestic pig
The domestic pigs used in the present study are all crossbreeds between
Danish Landrace and Yorkshire. This combination is normally used for
production of mother animals. Danish Landrace is a long and lean breed known
for high fertility and good motherhood. The litter size is around 13.5 piglets
per litter. The Yorkshire pig has a high lean meat percentage, high daily gain
and good meat quality. The mothering characteristics are good, with a litter
size of around 12 piglets per litter. The National Committee for pig
production mainly organizes The Danish Pig breeding program.
The newborn Landrace/Yorkshire domestic pig has a body mass of
1.3–1.9 kg. Boars become sexually mature at an age of 6 months,
while sows are sexually mature at an age of 7 months. The gestational period
is 114 days and the average litter size is 10–14 animals. The adult mass
of the domestic pig, at an age of two years, is 200–300 kg
(Bollen et al., 2000). In
Denmark, most of the domestic pigs are SPF-tested (Specific Pathogen Free) but
are not submitted to the same strict control procedures as the Göttingen
minipigs.
Experimental set-up
A total of 10 Göttingen minipigs and 12 domestic pigs were used in the
study. All Göttingen minipigs [five neonate polts (1–2 days old)
and five adult sows (1.5–3 years old)] were perfusion fixated by a
procedure approved by the Danish Animal Research Inspectorate. The pigs were
anesthetized with an intramuscular injection (1 ml per 10 kg body mass) of a
mixture of 6.5 ml Narcoxyl®Vet (20 mg ml–1; Intervet,
Denmark), 1.5 ml Ketaminol®Vet (100 mg ml–1; Intervet,
Denmark) and 2.5 ml Methadone DAK (19 mg ml–1; Nycomed,
Denmark) added to one bottle of Zoletil®50 Vet (Virbac Laboratories,
France) without additional solvent. The pigs were then placed in a supine
position on the operation table and supplied with a lethal dose of
Pentobarbital (1 ml kg–1 body mass, 200 mg
ml–1) before intervention. The deeply `anesthetized' pigs
received a midsternal incision followed by a sternal split. The left cardiac
ventricle was punctured by an infusion cannula, the right auricle was cut
open, and a transcardial perfusion with 0.5–2.5 l saline followed by
2.5–7.5 l of 4% paraformaldehyde in 0.15 mol l–1
Sørensen phosphate buffer (pH 7.4) was executed. The procedure took on
average 10–15 min. The brain was removed and postfixed for an additional
24 h at 5°C in 1% paraformaldehyde.
The domestic pigs [six neonate sows (1 day old) and six adult sows (3–4 years old)] were euthanized with a lethal injection of Pentobarbital i.v. or with carbon dioxide in a Danish pig slaughterhouse. Following death, the brains were carefully removed from the skull and fixed by immediate immersion in 4% formaldehyde (0.15 mol l–1 phosphate buffer, pH 7.4) for at least two weeks.
Tissue processing
The cerebral hemispheres were divided medially through the corpus callosum,
and the left or right hemisphere was selected randomly before dehydration and
infiltration in paraffin (Fig.
1A,B). The hemispheres were placed in a container with the
midsagittal surface facing down, embedded in blocks of paraffin and sectioned
into coronal series of 40 µm-thick sections on a Leica microtome
(Fig. 1B,C). Satisfactory
adhesion of the thick sections to glass slides was obtained using Superfrost
Plus glass slides primed with a chromalun–gelatin solution
(Feinstein et al., 1996). A
predetermined fraction of the 40 µm-thick sections was sampled using moist
filter paper and carefully rolled onto 40°C preheated primed slides
covered by a thin layer of distilled water. The sections were dried overnight
at 37°C and a final selection of slides was subsequently stained with
Giemsa.
|
; West et al.,
1991). The optical fractionator involves counting particles with
optical disectors in a uniform random systematic sample that constitutes a
known volume fraction of the region being analyzed. This is achieved by
counting cells on a known fraction of sections (ssf;
Fig. 1D), under a known
fraction of the sectional area of the region (asf;
Fig. 1E) in a known fraction of
the thickness of a section (hsf;
Fig. 1G). The total numbers of
neocortical cells are estimated by multiplying the numbers of cells counted
(
Q–) with the inverse sampling fractions. Finally, the
bilateral cell number can be estimated by multiplying the unilateral number by
two.
 | (1) |
The optical disector may be considered as a three-dimensional probe,
generated with the aid of a microscope with a high numerical aperture (NA=1.4)
and an oil immersion objective, in which it is possible to observe thin focal
planes in relatively thick sections (Fig.
1G). A counting frame with `exclusion' and `inclusion' lines is
superimposed on the magnified image of the tissue on a computer screen, and
the orientation in the z-axis is measured with a digital microcator
with a precision of 0.5 µm. The purpose of `exclusion' and `inclusion'
lines of the counting frame is to exclude edge effects arising from
sub-sampling (Gundersen,
1978). Similarly, upper and lower guard zones protect the counting
frame to prevent bias as a consequence of loss of cells close to the section
surfaces (Fig. 1G). All cells
that come into focus within the frame and are not in focus at the uppermost
position are counted as the focal plane is moved through the section. All cell
counts were obtained using a BH-2 Olympus microscope and CAST-GRID software
(Olympus, Ballerup, Denmark).
Counting criteria
The cells were identified as neurons if they had a combination of dendritic
processes, a Giemsa-positive cytoplasm, a clearly defined nucleus with a pale
and homogeneous nucleoplasm and a dark and condense centrally located
nucleolus. The nucleus was used as the counting item, and around 250 neuronal
nuclei were counted per brain
(
;
Table 1). The glial cells were
usually smaller and identified by the absence of a Giemsa-positive cytoplasm,
the presence of heterochromatin clumps within the ovoid or irregularly shaped
nucleus and the lack of a clearly identifiable nucleolus. Also here, the
nucleus was used as the counting item, and an average of 360 glial nuclei were
counted per brain
(
;
Table 1). No differentiation
was made between astrocytes, oligodendrocytes or microglia. Endothelial cells
were easily recognized by their dark and elongated nucleus and were excluded
from all counts.
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The section sampling fraction
A sampling scheme was designed based on systematic uniform random sampling
(SURS) to ensure that all parts of the neocortex had an equal probability of
being sampled. Based on a pilot study, every 50th or 100th section was sampled
from the neonate and adult Göttingen minipig brain, respectively, whereas
every 100th or 200th section was sampled from the domestic pig
(Table 1). The first section
was selected randomly using a random number within the sampled section period.
In the event of poor technical quality, e.g. due to the presence of folds or
breaks, the subsequent section was sampled instead. Extra sections were also
sampled to circumvent potential damage of sections during staining procedures.
These deviations are of no consequence to the results, provided the overall
sampling scheme is maintained. The section fraction represents the section
sampling fraction (ssf) and provided 11–13 sections for final
quantitative analyses in the Göttingen minipig and 7–9 sections in
the domestic pig.
The area sampling fraction
In each of the sampled sections, counts of neurons or glial cells were made
with optical disectors at regular predetermined x,y positions in the
neocortex. The neocortex was defined as the isocortex and mesocortex. Again,
the first disector was positioned randomly within the first x,y
interval by the CAST-GRID software. The area of the counting frame of the
disector, a(frame), is known relative to the area associated with
each x,y-step. The area of the sampling fraction (asf) is
accordingly:
 | (2) |
The height sampling fraction
The height of the disector (hdis) should be known
relative to the thickness (t) of the section. To prevent bias as a
consequence of loss of cells, the disector is guarded by upper and lower guard
zones. The size of the guard zones depends upon the size of the particles
counted. For neocortical neurons it should usually be at least 5 µm in the
top and bottom of the section. To compensate for potential deformation of the
sections in the z-axis, the height sampling fraction (hsf)
depends on the Q– weighted mean section thickness
(
Q–):
 | (3) |
 | (4) |
(Dorph-Petersen et al.,
2001).
Volume estimates
The systematic uniform random placement of disectors in the fractionator
design was used to estimate volumes in accordance with the unbiased principles
of the Cavalieri-estimator:
 | (5) |
P is the number of frame upper-corner-points hitting the
neocortex, a(p) is the x,y-step area, t is
the block or microtome advance and k is the section sampling
fraction. Note that estimated volumes are not used for the estimation of total
cell number; the fractionator estimates of total cell number are independent
of the containing volume and its shrinkage and deformation.
Statistical analyses and estimation of precision
The differences between means were analyzed using an unpaired two-tailed
Student's t-test. Group variability is shown in parentheses as the
coefficient of variation (CV=s.d./mean)
(Table 2). The precision of the
estimate of the total cell number in each subject was estimated as the
coefficient of error (CE=s.e.m./mean), caused by sampling error related to the
counting noise, the systematic uniform random sampling and variances in
section thickness (Table 3).
The precision of an individual estimate is related to the uniformity of the
distribution of particles being counted and the amount of sampling that has
been performed (Table 3). The
sampling was considered optimal when the observed variance of the individual
estimate, CE2, was less than half the observed interindividual
variance, CV2.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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253 million at birth to 324 million in adulthood
(Table 2,
Fig. 2A). This significant
(P=0.01) 28% difference demonstrates a pronounced postnatal
development of neurons in the Göttingen minipig brain. A significant
postnatal development is also observed for neocortical glial cells
(P<0.01), increasing from 382 million in the neonate to
714 million glial cells in the adult Göttingen minipig, an 87%
difference (Table 2;
Fig. 2B). The glial-to-neuron
ratio changes accordingly from 1.5 to 2.2. The total brain mass increases
almost threefold from a mean of 27.8 g at birth to 79.0 g as adult. Meanwhile,
the proportional increase of neocortex volume is more than 500%
(Table 2).
A corresponding postnatal development of neocortical neurons is not
observed in the domestic pig brain. The domestic pig has 425 million
neocortical neurons at birth and 432 million in adulthood
(Table 2;
Fig. 2C), which is 35%
more than in the adult Göttingen minipig neocortex (P<0.01).
The number of neocortical glial cells was not estimated. The total brain mass
increases from a mean of 29.4 g at birth to 134.0 g as adult. The postnatal
proportional increase in neocortex volume is, by comparison, close to 500%
(Table 2).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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A rapid development of the brain prior to the general body growth seems to
be characteristic for all mammals. Differences between species are related to
the gestational time of major cellular multiplication, the developmental stage
at birth and the complexity of the final product
(Dobbing, 1974). At the time
of birth, the human brain constitutes around 23% of its adult mass, and in the
first two postnatal years it increases rapidly to around 75% of its adult mass
(Dobbing, 1974). In
comparison, the brain of the closely related macaque monkey constitutes almost
65% of its adult mass at birth (Dobbing,
1974). The pig brain constitutes around 25% of its adult mass at
birth and seems in this aspect to be more similar to that of humans
(Dickerson and Dobbing, 1966).
The pig is also considered a perinatal brain developer, like human, and
several studies have shown a good correspondence to the developing human brain
with respect to myelination, compositions and electrical activity
(Dickerson and Dobbing, 1966;
Pampliglione, 1971; Fang et al.,
2005; Flynn, 1984;
Thibault and Margulies, 1998).
Accordingly, the pig has been considered an appropriate model for human brain
development (Dickerson and Dobbing,
1966; Book and Bustad,
1974; Pond et al.,
2000).
The present study demonstrates different developmental patterns in the neocortex of two strains of pigs. A significant postnatal development of neuron and glial cells is observed in the Göttingen minipig, whereas the adult number of neurons is established at birth in the domestic pig. This difference in cell number is not represented by a corresponding difference in relative brain mass of the neonates. The brain constitutes 35% of its adult mass in the Göttingen minipig and 22% in the domestic pig, whereas the relative growth of neocortical volume is close to 500% in both strains. The differences can by explained neither by differences in the stereological sampling or counting procedures (see below) nor the different tissue processing. Both fixation methods provided adequate histological sections with no clear difference in cellular morphology. No clinical data corroborate the observed difference; the gestational period is the same and birth mass and behavior is more or less identical in both strains. However, the results do substantiate that strain differences should be considered in future experimental studies using the pig brain and exemplifies the need for a full designation of the specific strain used.
Based on the results, the domestic pig seems to be a more proper model for
evaluating the effects of developmental insults on the human brain than the
Göttingen minipig. Even though recent developments have made it possible
to unambiguously demonstrate that new neurons are added to the adult primate
neocortex as well as to other mammal brains
(Gould et al., 1999;
Gould et al., 2001;
Gould and Gross, 2002), it is
still generally accepted that most neurogenesis in monkeys
(Rakic and Sidman, 1968;
Sidman and Rakic, 1973;
Rakic, 1974;
Rakic, 1978;
Rakic, 1985a;
Rakic, 1985b;
Rakic, 1988) and humans
(Dobbing and Sands, 1973;
Dobbing, 1974;
Samuelsen et al., 2003) is
completed at midgestation or at least before term. The rate of neurons added
to the neocortex in adulthood has no relative influence on the total
neocortical neuron number when compared with the rate of multiplication during
early development (Gould et al.,
2001). Furthermore, recent stereological results from our
laboratory reveal that the total number of neurons in the cortical plate of
human newborns equals the total number in adults
(Larsen et al., in press).
These results definitively refute previous results demonstrating a major
postnatal neurogenesis in humans (Shankle
et al., 1998; Shankle et al.,
1999). Regrettably, glial cells were not estimated in the domestic
pig. Results from one neonate and one adult domestic pig (not presented) do
indicate a prenatal development in glial cell number similar to the postnatal
increase observed in the Göttingen minipig. Further studies on several
postnatal ages are considered valuable in order to describe the growth slope
of neuronal increase in the Göttingen minipig.
The Göttingen minipig and the domestic pig have also been considered
as useful non-primate models for a number of human neurological diseases
(McClellan, 1968;
Douglas, 1972). However,
although several neuroanatomical studies have been performed, the
documentation of pig brain anatomy, connectivity and function is still
incomplete. Quantitative information of cell numbers based on systematic
sampling procedures has been limited to subcortical areas, e.g. the
hippocampus (Holm and West,
1994) and the subthalamic nucleus
(Larsen et al., 2004). Here,
we present the total number of neocortical neurons in two strains of pigs. In
the adult Göttingen minipig, the neocortex contains 324 million neurons,
whereas the domestic pig brain contains 432 million neocortical neurons. This
33% strain difference in neocortical neuron number was not an unexpected
finding considering the general relationship between body size and neuron
number (for a review, see Williams and
Herrup, 1988). The total number of neocortical neurons has also
been estimated in a number of other species. There are 3 million
neocortical neurons in the mouse brain
(Bonthius et al., 2004), 21
million in the rat (Korbo et al.,
1990), 12.8 billion in the minke whale (Nina Eriksen and Bente
Pakkenberg, unpublished data) and 19–23 billion in the human neocortex
(Pakkenberg and Gundersen,
1997). When compared to these species, one of the most valuable
findings in the pig brain is the rather low coefficient of variation (CV) from
which the true biological variance can be estimated to be less than 10% (see
the additivity of variances below). The low biological variance seems to be a
reproducible finding for the pig brain
(Holm and West, 1994;
Larsen et al., 2004;
Jelsing et al., 2005a;
Jelsing et al., 2005b) and
supports the continued use of pigs in neurotoxicologic studies, since
perturbations may be detected with great sensitivity.
Stereological design
Estimates of neocortical cell numbers were obtained using the optical
fractionator method, which is efficient and independent of any tissue
shrinkage or expansion that may take place during any stage of tissue
preparation (Gundersen, 1986;
West et al., 1991). It was
considered optimal on paraffin sections in which shrinkage during processing
is significant. Provided that mounted sections maintain a sufficient depth to
accommodate optical disectors, the optical fractionator can also be applied on
other preparations, e.g. cryostat or vibratome sections.
Sampling parameters were optimized to obtain a high efficiency in terms of
precision and effort. A total count of 100–150 cells in
75–100 disectors distributed systematically randomly on 5–10
sections is usually enough to obtain an estimate with a precision appropriate
for most biological structures (Pakkenberg
and Gundersen, 1988; Korbo et
al., 1990; West,
1993). However, because the variance in the experimental groups
appeared to be rather low, it was decided to increase the efficiency of the
fractionator by intensifying the sampling of cells to an average of 250
particles per brain in 140–170 disectors. An additional number of
sections were sampled from the Göttingen minipig. This was done to ensure
enough sections for forthcoming studies of specific glial subpopulations, of
which some have a more heterogeneous distribution in the brain. The efficiency
of the fractionator sampling was evaluated from variance analysis of relative
variances and estimator CE. These two measures are related through the basic
equation (the additivity of variances):
 | (6) |
Even though the efficiency and mathematical unbiasedness of the optical
fractionator method for estimating cells in the pig brain neocortex are
evident, some fundamental requirements have to be fulfilled for a proper
application (West, 1993). A
first requirement is that the whole structure is accessible; secondly, one
must be confident that all cells of interest can be identified unambiguously
and that penetration of staining is complete throughout the thickness of the
section. In the present study, a complete penetration of staining was
optimized beforehand by registering the z-distribution of all cells.
However, difficulty in distinguishing glial cells from neurons appeared when
stained with the modified Giemsa method. This was especially true in the
neonate brains, where the neuronal density was high and morphometric
differences between cells were less pronounced. The lack of clear criteria for
distinguishing neurons and glial cells in cortical regions has previously been
a major problem for stereologists
(Braendgaard et al., 1990;
Davanlou and Smith, 2004) and
may partly explain the somewhat lower hemispheric cell number published in a
screening procedure of young Göttingen minipig
(Jelsing et al., 2005a).
Recently, a number of immunohistochemical markers have been evaluated in the
pig brain (Lyck et al., 2006),
and the combination of immunohistochemistry and stereology may provide a
better approach for quantifying neurons and glial cell populations in future
quantitative studies of the pig brain.
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Acknowledgments |
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