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Internal luminal pressure during early chick embryonic brain growthDescriptive and empirical observations.

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Internal Luminal Pressure During
Early Chick Embryonic Brain
Growth: Descriptive and Empirical
Department of Biology, Villanova University, Villanova, Pennsylvania
Department of Mathematical Sciences, Villanova University,
Villanova, Pennsylvania
Division of Pulmonary and Critical Care, Hospital of the University of
Pennsylvania, Philadelphia, Pennsylvania
If the intraluminal pressure of the brain is decreased for 24 hr, the
brain and neuroepithelium volumes are both reduced in half. The current
study measured the intraluminal pressure throughout the period of rapid
brain growth using a servo-null micropressure monitoring system. From
613 measurements made on 55 embryos, we show that the intraluminal
pressure over this time period is appropriately described by a linear model
with correlation coefficient of 0.752. To assess whether sustained hyperpressure would increase mitosis, elevated intraluminal pressure was induced in 10 embryos for 1-hr duration via a gravity-fed drip. The mitotic
density and index of the mesencephalon were measured for the 10 embryos.
Those embryos, in which the colchicine solution was added to the intraluminal cerebrospinal fluid creating a sustained hyperpressure, exhibited at
least a 2.5-fold increase in both the mitotic density and index compared with
control embryos. Based on the small sample size, we cautiously conclude
that sustained hyper-intraluminal pressure does stimulate mitosis.
2005 Wiley-Liss, Inc.
Key words: CNS development; neural tube; neural tube defects;
mitosis; neuroepithelium; cerebrospinal fluid; CSF
cell proliferation; hydrocephaly; neurogenesis
The morphogenesis of the early embryonic central nervous system has fascinated embryologists for over a century. Most likely this fascination has been fueled by the
fact that the central nervous system is the first organ
system to develop and features a large hollow brain at the
head end of the embryo. In fact, the brain grows rapidly as
a result of expansion of its cavity (Desmond and Jacobson,
1977) and coincides with completion of neural tube fusion
along the dorsal midline (neurulation) and coincident occlusion of the neurocoel (Desmond and Jacobson, 1977;
Schoenwolf and Desmond, 1984b, 1986; Pacheco et al.,
1986; Desmond and Field, 1992).
Early Embryo Brain Expansion
Our laboratory, along with others, has focused on both
brain expansion (Desmond and Jacobson, 1977; Desmond,
1985; Pacheco et al., 1986; Li and Desmond, 1991; Des©
mond et al., 1993; Alonzo et al., 1998; Desmond and Levitan, 2002; Gato et al., 2004) and occlusion of the spinal
neurocoel, which is needed to initiate the rapid expansion
Grant sponsor: National Institute of Neurological Diseases and
Stroke; Grant number: NINDS24136; Grant sponsor: National
Institute of Child Health and Human Development; Grant number: NICHHD24710.
*Correspondence to: Mary E. Desmond, Department of Biology,
Mendel Science Center, Villanova University, Villanova, PA
19085. Fax: 610-519-7863. E-mail:
Received 27 September 2004; Accepted 11 April 2005
DOI 10.1002/ar.a.20211
Published online 23 June 2005 in Wiley InterScience
(Desmond, 1982; Schoenwolf and Desmond, 1984a, 1984b,
1986; Desmond and Schoenwolf, 1986; Desmond and
Field, 1992; Desmond et al., 1993; Desmond and Levitan,
2002). Desmond and Jacobson (1977) first demonstrated
that rapid brain expansion occurred coincident with occlusion of the spinal neurocoel. At that time, we intubated the
hindbrains of living chick embryos with a small glass
capillary tube for 24 hr, allowing the release of intraluminal pressure (ILP). These brains had 50% less tissue and
number of cells compared to control embryos of identical
stages (Hamburger and Hamilton, 1955; Desmond and
Jacobson, 1977; Desmond, 1985). More recently, Inagaki
et al. (1997) demonstrated a decrease in size of the posterior fossa of intubated mice embryos in utero as well.
To prove that such differences were not caused by the
traumatic interventive nature of the experiment, Desmond and Jacobson (1977) intubated the hindbrains of a
significant number of embryos with solid glass rods of the
same outside diameter as the capillaries. The tissue volume and cell number of these sham-control embryos did
not differ significantly from the values for the nonmanipulated controls. These results suggest that the decrease in
tissue volume and cell number was directly related to a
decrease in pressure.
To show further that brain growth depends on occlusion
of the spinal neurocoel, Desmond and Levitan (2002) reported a complementary experiment. Desmond experimentally occluded the neurocoel before natural occlusion
occurs and showed that the brains of these embryos expanded earlier than the brains of nonoccluded test mates.
Moreover, the brains of naturally occluded embryos expanded significantly subsequent to the natural occlusion
of the neurocoel (Desmond and Levitan, 2002). Both of
these experiments illustrated the dependence of brain expansion on sealing the spinal neurocoel from the outside.
Both experiments point to the importance of a closed fluidfilled system to generate a positive pressure to initiate and
maintain the expansion.
Embryonic ILP Measurements
Intraluminal pressure measurements of the chick embryo were first reported by Jelinek and Pexieder (1968,
1970). Using a micropipette attached to a transducer and
chart recorder, and then a Landis micromanometer, these
investigators proved that the pressure in the intraluminal
spaces was greater than the surroundings and that there
was a minimum critical pressure (approximately 2.5–3.0
mm H20) for expanding the brain vesicles. Other indirect
implications of intraluminal pressure driving growth can
be found in the early reports of Coulombre (1956), in
which he showed that intraocular pressure is required for
the normal expansion of the eye and also that intubation
of the hindbrain results in folding of the roof of the hindbrain in 3-day-old chick embryos (Coulombre and Coulombre, 1958). Other investigators have demonstrated the
importance of interstitial pressure in maintaining normal
growth of cells and tissues, particularly in rat brain, dog
lung, and frog oocytes (Lai-Fook, 1982; Wiig and Reed,
1983; Kelly and Macklem, 1991).
Techniques employed for measuring interstitial fluid
pressure (IFP) have improved considerably over the past
60 years. One of the first measurements of micropressure
inside a living organism was made by Picken (1935) when
he impaled the large protozoan, Spiratomum, with a microneedle attached to a closed cavity. This technique was
followed by implanting perforated capsules subcutaneously and measuring the fluid collected in the capsule via
osmotic pressure (Prather et al., 1971). To overcome the
limitations of the capsule technique, a wick system connected to a Statham pressure transducer was devised
(Prather et al., 1971).
Finally, in the 1970s, these highly traumatic and unreliable techniques were replaced by the Sanborn pressure
transducer, which could measure IFP in cats and dogs
that varied from ⫺6.5 to 6.0 mm Hg. The procedure was
used extensively to determine the IFP in isolated dog lung
(Lai-Fook, 1982; Bhattacharya et al., 1984), rat kidney
(Quinn and Marsh, 1979), and rat skin and muscle (Wiig
et al., 1981). Particular to the study we are reporting, Wiig
and Reed (1983) used the micropressure technique to determine the interstitial fluid pressure in rat brain. They
demonstrated a positive correlation between IFP and the
amount of cerebrospinal fluid in rat brain.
Goal of Current Study
Using a sensitive micropressure system available from
World Precision Instruments, we decided to make a critical examination of the ILP of the brain during rapid brain
growth in the chick, based on the finding of Desmond and
Jacobson (1977), Jelinek and Pexieder (1968, 1970), and
Coulombre and Coulombre (1958), that this pressure is
essential for normal brain growth. Most importantly, we
were able to measure the ILP in embryos just after the
time of the formation of the closed neural tube, i.e., at
stage 12. Our goal was first to establish the most appropriate mathematical model to relate ILP with the developmental stage of the brain and then to test whether
application of increased pressure for a defined time interval would stimulate mitosis. This hypothesis is a logical
extension of our earlier findings that a decrease in ILP
resulted in a significant loss of cells comprising the neuroepithelium. Moreover, the hypothesis is consistent with
earlier findings of Abercrombie (1972) that tension across
cells in culture results in cell proliferation.
We measured the ILP throughout the period of rapid
brain growth, HH stages 12–26, with the servo-null micropressure monitoring system. Subsequently, we found
that a linear model (correlation coefficient of 0.752) gave
an appropriate representation of the relationship between
HH stages and ILP. For those embryos in which the ILP
was increased for 1 hr by a constantly added colchicine
infusate into the cavity, the mitotic density and index
were at least 2.5 times greater when compared with the
control embryos.
Servo-null Controlled Micropipette System
For our pressure measurements, we used the World
Precision Instruments, Model 900, micropressure monitoring system (MPMS). This system is based on the fact
that a change in pressure between the inside and outside
of a glass micropipette will cause a change in its electrical
The use of low-compliance tubing and the elimination of
microscopic air bubbles make the frequency response of
the hydraulic system higher than 300 Hz (Fox and Wiederhielm, 1973). A gradient of resistance vs. pressure that
is sharp, reproducible, and relatively independent of the
filler or external salt concentration can be established if
Fig. 1. The ILP is shown to drop precipitously (0.5 mm Hg) when a slit
is made in the roof of the midbrain. Once the initial drop occurs, the
pressure somewhat stabilizes at 1.4 mm Hg.
there is a relatively large difference between the electrode
and the external solution (Fein, 1972). This system provides a fast, accurate, and reliable means of obtaining
micropressure in biological organisms that require an interface of 5 ␮m or less.
Microelectrodes for the MPMS were made from WPI
glass capillaries (o.d./i.d. ⫽ 1.20/0.68 mm with a filament)
utilizing a David Kopf Instruments vertical pipette puller.
The microelectrodes were backfilled with 3 M KCl, and the
tip was beveled to a diameter of 2–5 ␮m. Next, the microelectrode with beveled tip was placed in a microelectrode
holder (WPI-MEH23SW) and electrically and hydraulically attached to the servo-pressure system. A ground wire
was placed in the Petri dish holding the embryo, and ILP
recordings were sent to a Macintosh MacLab chart recorder.
Standardization of Micropressure Monitor
To standardize the monitor system to water pressure,
we measured the water pressure in mm Hg of a waterfilled manometer at different heights. The manometer
consisted of an L-shaped glass tube (o.d./i.d. ⫽ 7/5 mm)
filled with water. Once the electrodes were in position, the
manometer was filled with distilled water; the entire manometer was positioned at 0° (flat on the table), then
raised in 10° increments from 0° to 90° against a ring
stand to which a metric rule was attached. There was a
linear relationship between the pressure in mm H20 and
the height of the water meniscus in the column. This
pressure in mm H20 was converted to the more conventionally used mm Hg.
A systems check on the sensitivity of the MPMS was
performed by monitoring the drop in the ILP of the brain
once a slit was made in the roof of the midbrain. The
pressure dropped precipitously in the first 5-min interval,
then stabilized at approximately 1.4 mm Hg (Fig. 1).
Next, initial experiments were done to test whether the
system would detect an increase in ILP. We noted the rise
in pressure on the MPMS monitor when we blew air into
the brain through a micropipette attached to plastic tubing that had been inserted into the midbrain of HH 20, 22,
and 27 chick embryos (Hamilton and Hamburger, 1951).
Fig. 2. The bottom chart is a typical chart of the ILP taken from the
MacLab recorder. Note that there is a surge of pressure as the microelectrode is inserted into the lumen through the neuroepithelium; then
the ILP stabilizes at a given pressure and returns to baseline once the
electrode is removed. The top chart illustrates a typical recording of the
heart beat in a stage 18 chick embryo. Note how much greater the
pressure in the heart is compared to that of the embryonic brain.
Since preliminary tests showed that the ILP of the embryonic brain was quite small, we assessed the functioning
of the electrode by routinely monitoring the heart ILP for
each embryo stage group studied. A typical QRS peak
from a stage 18 embryo heart is shown in Figure 2a. Note
that the ILP is approximately 100 times greater in the
heart compared to what is found in the brain of the samestage embryo (Fig. 2b).
Culture, Isolation, and Immobilization of Living
Embryos for Monitoring
Fertile White Leghorn chick eggs were incubated at
38.5°C in a forced-air humidified incubator until the embryos reached the stages of HH 12, 13, 16, 18, 20, 22, 24,
and 26 (Hamburger and Hamilton, 1951). Each egg was
then cracked into a finger bowl containing warm physiological saline (37°C), the vitelline membrane was removed, and the blastodisc was excised from the yolk. Next,
the embryo was rinsed in physiological saline and placed
dorsal side up into a preformed depression made in wax to
immobilize the embryo. The embryo was covered with
prewarmed physiological saline to keep it alive and to
ensure proper functioning of the microelectrode used to
monitor the ILP.
Measurement of ILP
The ILP was obtained by impaling the midbrain an
average of 10 times for at least 10 –20 sec per reading. The
ILP reported is an average of these numerous impalements. No detectable loss of cerebrospinal fluid (CSF) was
viewed on microscopic inspection after each impalement,
and the standard error (within the 95% range) of the
multiple pressure measurements further supports that
any loss of fluid was negligible. Previous to recording the
ILP for the midbrain of different stage embryos, an auxiliary experiment was done in which multiple impalements in each of the three brain vesicles were made in 12
stage 21 embryos to ensure that the pressure was constant
throughout the closed neural tube based on basic principles of hydrodynamics of fluids in a closed system (Gardner, 1973). We found this to be the case.
For both the preliminary and actual experiment measurements, the microelectrode was connected electrically
and hydraulically to the servo-pressure system, and a
ground wire was placed in the physiological saline surrounding the embryo. The ILP recordings were saved on a
Macintosh MacLab chart recorder. Fifty-five embryos of
various stages (HH 12–26) were monitored, with the number of individual embryos per each stage varying from one
to nine.
Application of Sustained Hyperpressure
Two dozen stage 21 chick embryos were placed in shellless culture and observed until stage 21 was attained
(Hamburger and Hamilton, 1951). Elevated ILPs (10 mv)
were induced into these embryos via a gravity-fed drip
from a 10 ml syringe positioned 65 cm above the embryo
and held by a clamp from a ring stand. Both the microelectrode and micropipette from the gravity drip tubing
was placed into the midbrain. The ILP was recorded, then
the solution was released into the midbrain by opening a
clamp on the tubing connected to the micropipette used to
impale the brain. The solution was added to the midbrain
via the drip until the pressure was increased by 1 mm Hg
from the initial ILP, and then the flow of the drip was
clamped off. A solution of physiological saline (0.9% NaCl),
neutral red dye (to detect the entry of the drip solution
into the brain), and colchicine (3.5 ␮g/ml) was used as the
drip solution. The purpose of the colchicine was to arrest
cells of the neuroepithelium in metaphase for counting
mitoses. It has long been established (Sauer, 1935) that
the nuclei of the neuroepithelial cells migrate to the luminal surface when they are in the mitotic phase of the cell
cycle and to the basal surface when they are in DNA
synthesis. By inserting the colchicine solution into the
brain cavity, the neuroepithelial cells would have been the
first to receive the mitotic arrest chemical before it had a
chance to enter the systemic circulation. Elevated pressure was induced in two groups of stage 21 embryos, with
five embryos per group. One group had colchicine in the
infusate while another had physiological saline. The hyperpressure was created by gently releasing the drip solution once the micropipette was positioned inside the
midbrain. The pressure was monitored via a microelectrode also positioned in the midbrain. The embryos were
incubated throughout the duration of the experiment by
submersing them in a finger bowl containing physiological
saline that was maintained at 38°C in a warm room. This
finger bowl was placed in a second glass bowl containing
water maintained at 38°C over a hot plate.
The third group of five embryos was placed into physiological saline to which 3.5 ␮g/ml of colchicine was added.
After allowing 15 min for the colchicine to enter the systemic circulation, the embryos were intubated with both
the microelectrode and a glass rod having the same o.d. as
the micropipette used in the chronic pressure experiments.
Following the 1-hr duration of the experiments, the
embryos were fixed in Carnoy’s solution for 24 hr, followed
by dehydration, clearing, and embedding in TissuePrep
(Fisher Scientific). Serial sagittal sections were cut at 10
␮m, mounted onto glass slides, stained with Harris’s hematoxylin, and coverslipped in DPX.
Volumes, Cell Counts, and Metaphase
The areas of the embryonic brains were measured using
the JAVA morphometrics software from Jandel as described in Pacheco et al. (1986). Only the midbrain was
measured. Volumes were determined by multiplying the
sum of the areas by the section factor and thickness of the
section. The cell number was calculated by counting only
nonrandomized quadrats of the midbrain using an ocular
reticle. The quadrats were located in the caudal, middle,
and rostral third of the midbrain. It was important to
count cells in these three areas because it is well known
that rates of cell proliferation differ for the regions
(Kallen, 1961; Cowan et al., 1968; Wilson, 1973; Desmond
and Haas, 2000).
To ensure that the same cell was not counted twice, only
every fourth section was used for measurements (Desmond and Haas, 2000). Once the appropriate sections
were determined for the three different areas of the midbrain, the number of nuclei present in each of the small
squares of the grid was counted. A minimum of 1000 cells
were counted for each location. The total number of cells
for the entire midbrain was extrapolated based on the
relationship: number of cells/1 mm3 ⫽ number of cells/
entire midbrain volume in mm3. To determine the mitotic
index and mitotic density, the number of colchicine-induced metaphases per 1,000 cells was counted using the
ocular reticle. This number multiplied by 100 is the mitotic index. The mitotic density is the mitotic index per
unit volume. All cell counting was done with a 40⫻ objective.
ILP and Embryonic Stages
Each ILP measurement was obtained by subtracting the
baseline pressure from the embryo brain pressure. The
baseline pressure was actually the average of two baseline
pressures: the baseline pressure before inserting the
probe into the brain and the baseline pressure after removing the probe from the brain. Since the pressure measurements were extremely sensitive, this averaging took
into account any variation of outside pressure during the
TABLE 1. Intraluminal pressure (mm Hg) in chick embryo brains
HH stages
Timea (hr)
Standard error
95% CI
Time is the average of the HH approximate interval.
For all embryos.
It was expected that the difference in baseline pressure,
⌬b, would be zero, where ⌬b ⫽ (baseline pressure after) ⫺
(baseline pressure before). However, this was not the case.
It is possible that the viscosity of the intraluminal fluid
had an adverse effect on the sensitivity of the pressure
readings. Nevertheless, as demonstrated in Figure 3, the
boxplots of the data for ⌬b reveal that the average reading
for each stage was very close to zero. In fact, all stages
except one (stage 21) appeared to have a mean value of ⌬b
not significantly different from zero when using the t-test
at the 5% level of significance. The mean value of ⌬b for all
613 observations (across all stages) was ⫺0.032 with a
standard error of 0.0140.
The boxplots of the ILP are presented in Figure 4. Table
1 summarizes the data with the means, standard errors
and 95% confidence limits for the ILP at the various HH
stages that were examined. The scatterplot of the means
from Table 1 is presented in Figure 5. Note that the
amount of time between embryological stages differs. This
is clearly shown in Table 1. It appears that the linear
model1 provides an appropriate description of the data
since the analysis of variance associated with that model,
ILP ⫽ 0.096 (HH stage) ⫹ 0.841, has a P ⫽ 0.005. The
value of the corresponding correlation coefficient was r ⫽
0.752. This model indicates that the ILP appears to increase approximately 10% as the embryo moves from one
designated HH stage to the next.
HH Stage 21 Embryos With Colchicine and
The second aspect of this experiment considered the
impact of increased pressure on the mitotic activity of the
neuroepithilium of HH stage 21 chick embryos. All of the
embryos were treated with colchicine in order to assess
mitosis. Specifically, there were three cases that were
examined: case 1, the embryo was immersed in a colchi-
Various alternative models were considered for the data, such
as the logistic, exponential, and logarithmic models. Using the
size of the P-values associated with these analyses as the criterion for choosing a model produced no better choice than the
aforementioned linear model. Furthermore, incorporating the
principle of parsimony also suggested choosing the linear model.
cine bath with pressure created by infused saline directly
into the midbrain cavity; case 2, the CSF was withdrawn
and immediately replaced by the same volume of colchicine solution with no created increase in ILP; and case 3,
the ILP was increased for 1 hr (1 mm HG) by adding a
colchicine infusate into the fluid within the cavity. It is
important to note that there was minimal collapse of the
neuroepithelium in case 2, most likely because of the very
short time interval (0 –30 sec) between removal of the CSF
and addition of the colchicine solution. The mitotic density
and the mitotic index were the aspects of mitotic activity
that were analyzed in each of these three cases. It is
important that caution be exercised when interpreting the
results of the statistical tests due to the small sample sizes
involved. Because of this, the same data were analyzed by
a variety of nonparametric tests stemming from different
mathematical models.2 In fact, it is only when there was
consistency in the outcomes of all statistical tests applied
that a suggested implication was made. The data are
presented in Table 2 and the associated boxplots in Figures 6 and 7.
The mitotic density is the number of mitotic figures per
unit volume of tissue, while the mitotic index is the number of mitotic figures per number of cells in that tissue.
Having seen no detectable intercellular substance in microscopic sections of stage 21 embryonic brains, we expected that the number of cells per unit volume of tissue
for all chick embryos at stage 21 would be approximately
the same. If this were true, then the mitotic density and
the mitotic index essentially measure the same characteristic of mitotic activity. This is demonstrated by noting
that the correlation coefficients between the mitotic density and the mitotic index for cases 1, 2, and 3 are 0.966,
0.973, and 0.990, respectively.
Since we were examining the potential differences
among three cases for the mitotic density as well as the
mitotic index, we first employed the Kruskal-Wallis one-
There were insufficient data to establish the likelihood of an
underlying Gaussian distribution necessary for a parametric test.
In general, nonparametric tests are less powerful than parametric tests. Therefore, nonparametric tests are less likely to show
differences when they do exist.
Fig. 3. Boxplots of the pressure readings before and after the microelectrode was inserted into the brain. The average reading for each stage
was essentially zero except for the stage 21 data. The horizontal line in
the interior of each box corresponds to the median value associated with
the specific data presented. The horizontal lines at the top and bottom
edges of the boxes correspond to the upper and lower quartiles, respectfully. Therefore, the height of a box is the interquartile range (IQR)
and contains the middle 50% of the data. A circle represents an outlier,
a value 1.5 to 3 times the IQR from the adjacent upper or lower edge of
the box, and an asterisk represents an extreme value, a value more than
3 times the IQR from the adjacent edge. The horizontal lines above and
below the boxes represent the maximum and minimum values for the
specific data set ignoring outliers and extreme values.
Fig. 4. Boxplots of ILP values for all HH stage 12–26 embryos. (See
explanation of boxplots in Fig. 3.)
way analysis of variance test. This tested the hypothesis
that the mitotic density data for each of the three cases all
came from populations with the same median, and that
the mitotic index data for each of the three cases all came
from populations with the same median. However, the
results of these tests suggested that neither the mitotic
density data (exact test, P ⫽ 0.002) nor the mitotic index
data (exact test, P ⫽ 0.0002) came from populations with
a common median.
Subsequently, we did a pairwise comparison of the medians for the various situations using the Wilcoxon-MannWhitney (WMW) test. We also examined the HodgesLehman (HL) shift estimator to determine if there appeared
to be a shift between the competing pairs of distributions. We
did this in two ways. First, we found a 95% confidence
interval for the HL shift estimator. If this confidence interval
contained 0, we concluded that there did not appear to be a
shift in distribution; otherwise, we determined that there did
appear to be such a shift. Next, we employed the permutation test based on a bootstrapping procedure of the HL shift
estimator with n ⫽ 10,000. If the P-values were sufficiently
small, we would conclude that the two treatments did appear to affect the outcomes, suggesting that there was a
corresponding shift in distributions.
We compared case 1 with case 2 for the mitotic density.
It appeared that the WMW test (exact test, P ⫽ 0.571)
suggested that the medians for these two cases were not
different from one another. Furthermore, the 95% confidence interval for the HL shift estimator (CI ⫽ ⫺2.66 to
6.95; in units 1 ⫻ E ⫹ 04) also suggested no shift in
distributions when comparing case 1 and case 2 populations. Lastly, the permutation test for the HL shift estimator appeared to validate the same conclusion (P ⫽
When we compared case 1 with case 2 for the mitotic
index, it followed the results for the mitotic density, with
the WMW test (P ⫽ 0.071), the 95% confidence interval for
the HL shift estimator (CI ⫽ ⫺0.76 to 6.87; in units 1 ⫻
E ⫺ 03), and the permutation test for the HL shift estimator (P ⫽ 0.076). Therefore, it appeared that there were
also no differences in medians or shifts between the two
Next, we compared case 1 with case 3 for the mitotic
density. The WMW test (exact test, P ⫽ 0.036) suggested
that the medians for these two cases were different from
one another. Furthermore, the 95% confidence interval for
the HL shift estimator (CI ⫽ 1.40 to 3.23; in units 1 ⫻ E ⫹
05) also suggested a shift in distributions between these
populations. The permutation test for the HL shift estimator appeared to validate the same conclusion (P ⫽
0.037). The observed values associated with the HL tests
all suggest that there is a positive shift in distribution
from case 1 to case 3.
Once again, the results for the mitotic index followed
those of the mitotic density when comparing case 1 with
case 3, as we observed the WMW test (exact test, P ⫽
0.036), the 95% confidence interval for the HL shift estimator (CI ⫽ 1.06 to 2.73; in units 1 ⫻ E ⫺ 02), and the
permutation test for the HL shift estimator (P ⫽ 0.038).
From the HL observed values, it also appears as if there is
a positive shift in distribution from case 1 to case 3.
Lastly, we compared case 2 with case 3 for the mitotic
density. Here, the WMW test (exact test, P ⫽ 0.008) also
suggested that the medians for these two cases were different. The 95% confidence interval for the HL shift estimator (CI ⫽ 1.11 to 3.03; in units 1 ⫻ E ⫹ 05) also
suggested a shift in distributions between these populations, and the permutation test for the HL shift estimator
appeared to validate the same conclusion (P ⫽ 0.008) as
well. The observed values associated with the HL tests all
suggest that there is a positive shift in distribution from
case 2 to case 3.
Fig. 5.
The scatterplot of the average ILP for each stage with its linear regression model.
As before, the results for the mitotic index followed
those of the mitotic density when comparing case 2 with
case 3 with the WMW test (exact test, P ⫽ 0.008), the 95%
confidence interval for the HL shift estimator (CI ⫽ 0.72 to
2.33; in units 1 ⫻ E ⫺ 02), and the permutation test for the
HL shift estimator (P ⫽ 0.008). Similar to the above, it
appears that there is a positive shift in distribution from
case 2 to case 3.
Summarizing these results, we see that stage 21 embryo
brains placed under sustained hyperpressure for 1 hr appear
to have a higher mitotic density and a higher mitotic index
than embryo brains whose CSF had been replaced by colchicine without pressure. When pressure was increased using
saline, and the colchicine solution was only outside the embryo, the mitotic index and the mitotic density were also
significantly lower than with the experimental embryos.
What makes these results significant is the fact that they
were found using less powerful statistical tests.
ILP With Developmental Stage
It is clear from our measurements of the ILP of embryos
at increasing developmental stages that a linear model is
appropriate to describe the increase in ILP. Moreover, a
key finding in our study is that the ILP of the embryonic
brain appears to increase approximately 10% as the embryo develops from stage to stage. One extrapolation from
such a finding is that it is this amount of pressure that
maintains the normal turgor of the neuroepithelium during brain expansion. In fact, if the internal pressure on the
neural tissue is increased by just 1 mm Hg of pressure
from its normal baseline pressure for at least 1 hr, as our
hyperpressure experiments reported here show, the neuroepithelium doubles its mitotic density and index. Finding that the mitotic activity of the neuroepithelium in-
creased when the tension on the neuroepithelium is
increased parallels the much earlier findings of Abercrombie (1970), who showed an increase in mitotic activity of
cells under tension (stretch) in culture. However, finding
no increase in the tissue volume in response to hyperpressure (case 3 vs. cases 1 and 2) agrees with the findings of
Alonso et al. (1998) and may suggest that the cells are
getting smaller after a round of cell division.
Knowing that the relationship between the cavity pressure and embryonic stage is a linear one is important
because it allows us to make comparisons with brain
growth studies done earlier by our group and others (Desmond and Jacobson, 1977; Pacheco et al., 1986; Desmond
and Levitan, 2002). We have tried to identify and understand the different mechanisms that play a role in the
expansion of the cavity and the increase in volume of the
neuroepithelium during early brain growth ever since our
findings that a decrease in pressure via intubation resulted in a 50% reduction in the size of both.
Although these recent findings suggest a role for ILP
pressure, they do not exclude the role of growth factors,
other proteins, or proteoglycans present in the CSF during
this time period. We know from the work of Reid and
Ferretti (2003) that FGF1, -2, -3, -4 proteins have been
identified in the neuroepithelium of the murine choroid
plexus at E12.5 da. Most likely these FGF receptors respond to FGFs in the CSF. It will be challenging to explore
the interplay between growth factors and cavity pressure
directing growth of the neuroepithelium.
Likewise, the presence of high molecular proteins and
proteoglycans in the embryonic CSF most likely plays a
role in the expansion of the neuroepithelium by increasing
the osmolarity of the CSF (Alonso et al., 1998; Gato et al.,
2004). Proteoglycans in the embryonic CSF have been
shown to originate in the neuroepithelial cells (Alonso et
TABLE 2. HH stage 21 chick embryo brains with colchicine and pressure*
Case 1 (n ⫽ 3)
Cavity volume
Tissue volume
Total volume
Cells (1 ⫻ E ⫹ 06)
Mitotic figures (1 ⫻ E ⫹ 04)
Mitotic density (1 ⫻ E ⫹ 05)
Mitotic index (1 ⫻ E ⫺ 02)
Case 2 (n ⫽ 5)
Case 3 (n ⫽ 5)
*Case 1, embryo immersed in colchicine bath with pressure created by infused saline. Case 2, CSF withdrawn and replaced
with same volume of colchicine solution for 1 hr. Case 3, CSF withdrawn and replaced with same volume of colchicine solution
plus increased pressure for 1 hr.
Fig. 6. Boxplots of the mitotic density for neuroepithelium of the
midbrain of stage 21 chick embryos. (See explanation of boxplots in Fig.
3.) Case 3 is the true experimental case in which the intraluminal pressure was increased for 1 hr via a gravity-fed drip that contained physiological saline and colchicine. Both cases 1 and 2 are control groups for
this experimental group. Case 1 had embryos that were immersed in a
colchicine bath and infused with only physiological saline, whereas case
2 had embryos in which the CSF was withdrawn and replaced with the
same volume of colchicine for 1 hr. There was a significant positive shift
in distribution of the mitotic density for the experimental group (case 3)
relative to the control groups (cases 1 and 2).
al., 1998). On the other hand, the neuroepithelial cells
may simply serve as a conduit for fluid from the blood
plasma, as Gato et al. (2004) reported the protein composition of chick embryonal CSF to be very similar to that of
blood serum for identical stages.
Whatever the relationship of these chemical factors, it is
important to remember that an increase in tension on the
neural tissue created by an increase in internal fluid alone
results in an increase in mitosis. We are not excluding the
role of chemical factors on neuroepithelial cell behavior;
both most likely play a role. However, this experiment
only tested the impact of increased tension. It is our view
that both physical and chemical factors influence mitotic
activity of the neuroepithelium. In contemporary developmental biology, with its emphasis on genetics and molec-
Fig. 7. Boxplots of the mitotic index for neuroepithelium of the
midbrain of stage 21 chick embryos. (See explanation of boxplots in
Fig. 3.) The treatment for the three cases is the same as in Figure 6.
There was a significant positive shift in distribution of the mitotic
index for the experimental group (case 3) relative to the control
groups (cases 1 and 2).
ular-cellular mechanisms, epigenetic factors such as fluid
dynamics are often overlooked. Recently, researchers
showed the impact of intracardiac fluid force on normal
heart development. Embryos with severely blocked blood
flow failed to form several major components of the heart
(Hove et al., 2003).
Comparison of ILP Measurements Compared
With Others’
Our ILP measurements agree remarkably with most of
those reported by others who have made such measurements (Jelinek and Pexieder, 1968, 1970). For example,
our ILP pressure for one stage 24, was 3.76, and they
reported an average ILP of 3.8 for 38 embryos. Our measurements also show the same decrease between stages 24
and 26. Because of the close agreement of our findings
with theirs, we chose to include the measurements when
we only had one or two embryos in the sample. More
importantly, we wanted to be able to model the trend in
ILP over developmental time rather than focus intensely
Fig. 8. A scatterplot of the averages for the cavity and the total brain
is plotted along with the averages for the ILP (data from Desmond and
Jacobson, 1977; Pacheco et al., 1986). The linear model based on the
ILP averages is also shown. Smooth curves are drawn through the cavity
averages and through the total brain averages, respectively. These
curves show how the cavity and the total brain grow in an exponential
manner. One can see how, at some critical points, the increase in ILP
drives the dramatic increase in the cavity and the total brain.
on a particular stage of development. The only other report of ILP measured in stage 23 chick embryos differs by
an order of magnitude from both ours and the JelinekPexieder group (Gato et al., 1998).
Unfortunately, we are unable to make direct comparisons
of our pressure findings with all of the work reported by
Jelinek and Pexieder (1968, 1970; Pexieder and Jelinek,
1970) because they examined the relationship between ILP
and time of incubation or embryo weight, not with embryonic
stage. Most notable about our study in comparison with their
findings is the fact that we verified the ILP reported by
Jelinek and Pexieder (1968, 1970) using more sensitive instrumentation, and we reported ILP for earlier stages than
had been measured in their studies.
bolstered by our assessing the data with stringent and
appropriate statistical tests.
We did not determine the minimum increase in pressure
necessary or the minimum amount of time that the neuroepithelium needs to be exposed to this increase in pressure
for mitosis to be stimulated. Interestingly, Alonzo et al.
(1998) reported that ␤-D-xyloside-treated embryos exhibited
an 11% increase in the neuroepithelial volume, which was
not statistically significant using Student’s t-test. We simply
showed that a sustained increase in ILP stimulated mitotic
activity in the midbrain. We do know from the work of others
that the rates of mitosis vary in the chick neuroepithelium
for the three main vesicles and between the roof and base
(Kallen, 1961; Fujita, 1962, 1963, 1964; Kallen and Valmin,
1963; Wilson, 1973). Although cell proliferation need not
lead to immediate growth in the epithelium, we know that
there is an increase in cell proliferation in the mesencephalon of the chick between stages 19 and 24 (Kallen, 1961). The
nearly twofold increase in ILP found between stages 18 and
24 reported in our current study parallels the mitotic activity
surge reported by Kallen (1961) and our own fivefold increase in tissue volume for the brain (Desmond and Jacobson, 1977).
Effects of Sustained Hyperpressure on Mitosis
This part of the experiment was very difficult to do and
is the reason for the limited number of cases. Some of the
difficulties encountered were keeping the embryo alive
and warm for over 1 hr outside of an incubator and not
being able to put the gravity-fed drip system inside the
incubator. More importantly, it was extremely difficult to
maintain the increased pressure for over 1 hr. Nonetheless, after analyzing the data by a variety of nonparametric tests, we feel confident in concluding that the data
support a conclusion that an increase in pressure for at
least 1 hr stimulated mitosis as determined by a significant increase in metaphases. Our confidence in the data is
CSF and Growth Kinetics of Early Embryonic
It is relevant to understanding the significance of this
work that it still remains unknown at what point the
choroid plexus begins to secrete CSF. In his early in vitro
experiments, Weiss ((1934) 1955) concluded that neuroepithelial fragments placed in culture secreted fluid on its
ependymal surface. He had no way to illustrate that the
fluid came from within the cells or that it simply passed
through the cells from the medium. However, knowing
that growth factor receptors are present on the periventricular cells of E 12.5 mice ependyma cells, it seems
probable that these same cells are able to secrete CSF.
Likewise, proteoglycans in the embryonic CSF have been
shown to originate in the neuroepithelial cells (Alonso et
al., 1998). On the other hand, the neuroepithelial cells
may simply serve as a conduit for fluid from the blood
plasma, as Gato et al. (2004) reported the protein composition of chick embryonal CSF to be very similar to that of
blood serum for identical stages.
We have shown in an earlier study that the cavity
increases linearly (Pacheco et al., 1986). We assume that
the cavity volume is the same as the fluid volume. We
have yet to discern the direct relationship between the
increase in fluid and the increase in pressure. When plotting the pressure measurements reported here with volume measurements reported by us in earlier studies (Desmond and Jacobson, 1977; Pacheco et al., 1986), we see
that the pressure increases 50-fold between stages 18 and
24 during the same time interval that the cavity volume
increases 60-fold (Fig. 8). This dramatic spurt in cavity
expansion appears directly related to the burst in pressure. Prior to stage 18, the pressure seems to maintain the
cavity, but there is no large increase in cavity size. It
appears that stage 18 is a critical time for embryonic brain
expansion and can be compared to what happens when
trying to blow up a new balloon. After several trials in
increasing the pressure, the walls give way and the brain
immediately expands. Physiologists have explained the
relationship between internal pressure and vessel expansion at any given time by stating that the distending
tension in the wall of a cylindrical vessel at any time at
any given pressure is directly proportional to its radius
and the elastic limit of the wall (law of LaPlace after
Gardner, 1973).
This relationship mirrors our earlier findings that the
brain exhibits spurts of growth over defined time intervals. Furthermore, the expansion of the cavity and growth
of the tissue alternate, i.e., first the cavity expands faster
than the tissue grows, then the tissue grows faster than
the cavity expands (Pacheco et al., 1986). We interpret
this alternation in growth rates as a way to ensure there
is adequate tissue available to expand under the increased
cavity expansion driven by the pressure increase.
The authors thank Dr. Philip Stephens, Department of
Biology, for his critical analysis of the pressure data and
Mrs. Patti Sharples Haas for the tedious task of making
the cell and mitotic counts.
Abercrombie M. 1970. Contact inhibition in tissue culture. In Vitro
6:128 –142.
Alonso MI, Gato A, Moro JA, Barbosa E. 1998. Disruption of proteoglycans in neural tube fluid by ␤-D-xyloside alters brain enlargement in chick embryos. Anat Rec 252:499 –508.
Bhattacharya J, Gropper MA, Staub NC. 1984. Interstitial fluid pressure gradient measured by micropuncture in excised dog lung. Am
Physiol Soc 84:271–277.
Coulombre AJ. 1956. The role of intraocular pressure in the development of the chick eye: I, control of eye size. J Exp Zool 133:211–225.
Coulombre AJ, Coulombre JL. 1958. The role of mechanical factors in
the brain morphogenesis. Anat Rec 130:289 –290.
Cowan WM, Martin AH, Wenger E. 1968. Mitotic patterns in the optic
tectum of the chick during normal development and after early
removal of the optic vesicle. J Exp Zool 169:71–92.
Desmond ME. 1982. Description of the occlusion of the lumen of the
spinal cord in early human embryos. Anat Rec 204:89 –93.
Desmond ME. 1985. Reduced number of brain cells in so-called neural
overgrowth. Anat Rec 212:195–198.
Desmond ME, Duzy MJ, Federici BD. 1993. Second messenger regulation of occlusion of the spinal neurocoel in the chick embyro. Dev
Dyn 197:291–306.
Desmond ME, Field MC. 1992. Evaluation of neural groove closure
and subsequent initiation of spinal cord occlusion in the chick
embryo. J Comp Neurol 319:246 –260.
Desmond ME, Haas PA. 2000. Experimental manipulation and morphometric analysis of neural tube development. In: Tuan RS, Lo
CW, editors. Methods in molecular biology, developmental biology
protocols. Totowa, NJ: Humana Press.
Desmond ME, Jacobson AG. 1977. Embryonic brain enlargement
requires cerebrospinal fluid pressure. Dev Biol 57:188 –198.
Desmond ME, Levitan ML. 2002. Brain expansion in the chick embryo
initiated by experimentally produced occlusion of the spinal neurocoel. Anat Rec 268:147–159.
Desmond ME, Schoenwolf GC. 1986. Evaluation of the roles of intrinsic and extrinsic factors in occlusion of the spinal neurocoel during
rapid brain enlargement in the chick embryo. J Embryol Exp Morph
97:25– 46.
Fein H. 1972. Microdimensional pressure measurements in electrolytes. J Appl Physiol 32:560 –564.
Fox JR, Wiederhielm CA. 1973. Charactistics of the servo-controlled
micropipet pressure system. Microvasc Res 5:324 –335.
Fujita S. 1962. Kinetics of cellular proliferation. Exp Cell Res 28:52–
Fujita S. 1963. The matrix cell and cytogenesis in the developing
central nervous system. J Comp Neurol 120:37– 42.
Fujita S. 1964. Analysis of neuron differentiation in the central nervous system by triated thymidine autoradiography. J Comp Neurol
Gardner WJ. 1973. The dysraphic states from syringomyelia to anacephaly. Amsterdam: Excerpta Medica.
Gato A, Martin P, Alonso MI, Martin C, Pulgar MA, Moro JA. 2004.
Analysis of cerebro-spinal fluid protein composition in early developmental stages in chick embryos. J Exp Zool 301:280 –289.
Hamburger V, Hamilton HL. 1951. A series of normal stages of the
development of the chick embryo. J Morphol 88:49 –92.
Hove JR, Koster RW, Forouhar AS, Acevedo-Bolton G, Fraser SE,
Gharib M. 2003. Intracardiac fluid forces are an essential epigenetic
factor for embryonic cardiogenesis. Nature 421:172–177.
Inagaki T, Schoenwolf GC, Walker ML. 1997. Change in the posterior
fossa with surgically induced spina-bifida aperta in mouse. Pediatr
Neurosurg 26:185–189.
Jelinek R, Pexieder T. 1968. The pressure of encephalic fluid in chick
embryos between the 2nd and 6th day of incubation. Physiol Bohemoslov 17:297–305.
Jelinek R, Pexieder T. 1970. Pressure of the CSF and the morphogenesis of the CNS: I, chick embryo. Folia Morphol 18:102–110.
Kallen B. 1961. Studies on cell proliferation in the brain of chick
embryos with special reference to the mesencephalon. Zeit Anat
Entwick 122:388 – 401.
Kallen B, Valmin K. 1963. DNA synthesis in the embryonic chick
central nervous system. Z Zellforsch 60:491– 496.
Kelly S, Macklem P. 1991. Direct measurement of intracellular pressure. Am J Physiol 260:C652–C657.
Lai-Fook SJ. 1982. Perivascular interstitial fluid pressure measured
by micropipettes in isolated dog lung. Am Physiol Soc 82:9 –15.
Li XY, Desmond ME. 1991. Modulation of Na⫹/K⫹ ATPase pumps in
the heart of the chick embryo influences brain expansion. Soc Neurosci 17:2116.
Pacheco MA, Marks RW, Schoenwolf GC, Desmond ME. 1986. Quantification of the initial phases of rapid brain enlargement in the
chick embryo. Am J Anat 175:403– 411.
Pexieder T, Jelinek R. 1970. Pressure of the CSF and the morphogenesis of the CNS: II, pressure necessary for normal development of
the brain vesicles. Folia Morphol 18:187–192.
Picken LE. 1935. A note on the mechanism of salt and water balance
in the Heterotrichous cilate, Spirostomum ambiguum. J Exp Biol
Prather JW, Bowes DN, Warrell DA, Zweifach BW. 1971. Comparison
of capsule and wick techniques for measurement of interstitial fluid
pressure. J Appl Physiol 6:942–945.
Quinn MD, Marsh DJ. 1979. Peritubular capillary control of proximal
tubule reabsorption in the rat. Am J Physiol 236:F478 –F487.
Reid S, Ferretti P. 2003. Differential expression of fibroblast growth
factor receptors in the developing murine choroid plexus. Brain Res
Dev Brain Res 141:15–24.
Sauer FC. 1935. Mitosis in the neural tube. J Comp Neurol 62:377–
Schoenwolf GC, Desmond ME. 1984a. Neural tube occlusion precedes
rapid brain enlargement. J Exp Zool 30:405– 407.
Schoenwolf GC, Desmond ME. 1984b. Descriptive studies of occlusion
and reopening of the spinal canal of the early chick embryo. Anat
Rec 209:251–263.
Schoenwolf GC, Desmond ME. 1986. Timing and positioning of reopening of the occluded spinal neurocoel in the chick embryo.
J Comp Neurol 246:459 – 466.
Weiss P. (1934) 1955. Special vertebrate organogenesis, neurogenesis.
In: Willier BH, Weiss PA, Hamburger V, editors. Analysis of development. Philadelphia: Saunders. p 372.
Wiig H, Reed RK, Aukland K. 1981. Micropuncture measurement of
interstitial fluid pressure in rat subcutis and skeletal muscle: comparison to wick-in-needle technique. Microvasc Res 21:308 –319.
Wiig H, Reed RK. 1983. Rat brain interstitial fluid pressure measured
with micropipettes. Am Physiol Soc 83:H239 –H246.
Wilson DB. 1973. Chronological changes in the cell cycle of chick
neuroepithelial cells. J Embryol 29:745–751.
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