Antioxidant role of melatonin against nicotine’s teratogenic effects on embryonic bone development

Abstract

Objective(s)
This study investigated the possible effects of low (3 mg/kg) and high (6 mg/kg) doses of nicotine on the skeletal development of rat fetuses by the double staining method and the protective role of melatonin (10 mg/kg) against these effects.

Materials and Methods
Eighteen adult female Wistar-Albino rats were divided into six groups (n=3, each) as control, low-dose nicotine, high-dose nicotine, low-dose nicotine+melatonin, high-dose nicotine + melatonin and melatonin. While nicotine was given to the experimental groups on gestation days 1–20, nicotine and melatonin were administered together to the treatment groups. The fetuses were delivered by cesarean section on the 20th day of pregnancy. The skeletal systems of the fetuses were stained using the double staining method. The forelimbs and hindlimbs of the fetuses were firstly investigated under a stereomicroscope, and then their photos were taken. The total bone length, the length of the ossified part and the ossification rate were calculated using the ImageJ program.

Results
The degree of ossification in the bones of the feet and the hands was determined. When the total bone length and the length of the ossified part were evaluated, they were significantly decreased in the nicotine groups (P<0.05), but were close to each other in the treatment and the control groups (P<0.05).

Conclusion
It has been found that the use of nicotine during pregnancy delays skeletal ossification and that melatonin, a powerful antioxidant, eliminates the teratogenic effects of nicotine.

Full text

1465
Int. J. Morphol.,
35(3):1465-1472, 2017.
The Intracranial and Posterior Cranial Fossa Volumes and
Volume Fractions in Children: A Stereological Study
Los Volúmenes de la Fosa Craneal Intracraneal y Posterior y las Fracciones
de Volumen en los Niños: Un Estudio Estereológico
Tolga Ertekin1; Muhammet Degermenci2; Ilyas Ucar3; Ayse Sagıroglu2; Emre Atay4 & Hatice Susar2
ERTEKIN, T.; DEGERMENCI, M.; UCAR, I.; SAGIROGLU, A.; ATAY, E. & SUSAR, H. The intracranial and posterior cranial
fossa volumes and volume fractions in children: A stereological study. Int. J. Morphol., 35(4):1465-1472, 2017.
SUMMARY: The size of intracranial cavity (IC) and posterior cranial fossa (PCF) plays an important role in the pathophysiology
of various disorders. In this study, we aimed at establishing normal volume data of the IC and PCF in Turkish population according to age
and sex by using stereological method. This study was carried out retrospectively on 339 individuals (168 females and 171 males)
between 0 and 18 years old with no medical or neurological disorders that affected the skeletal morphology of the cranial cavity.
Volumetric estimations were determined on computed tomography (CT) images using point-counting approach of stereological methods.
Intracranial volume (ICV) and posterior cranial fossa volume (PCFV) were increased with age in both sexes. They reached adult dimensions
at 5 years of age during the teenage years. According to sex; the mean ICV and PCFV were 1594.51±245.57cm3 and 244.89±53.86 cm3
in males, 1456.34±241.85 cm3 and 228.24±41.38 cm3 in females, respectively. Generally, significant differences were determined in ICV
and PCFV according to sex after they reached maximum growth period. According to age the volume ratios of PCF to IC was ranged
from 13.03 to 17.48 in males and 12.06 to 18.54 in females. This study demonstrated that these volume ratios could help the physician for
both patient selections for surgery, and for the assessment of any surgical technique used to treatment of PCF malformations. However
current study revealed that point counting method can produce accurate volume estimations and is effective in determining volume
estimation of IC and PCF.
KEY WORDS: Computed tomography; Intracranial cavity; Posterior Cranial Fossa; Stereology; Volume.
INTRODUCTION
Intracranial volume (ICV), sometimes referred to as
total intracranial volume, attributes to the estimated volume
of the cranial cavity as outlined by the supratentorial dura
matter or cerebral contour when dura is not clearly detectable
(Eritaia et al., 2000). Recent studies measured ICV to
investigate progressive neurodegenerative brain disorders,
such as Alzheimer’s disease (Dukart et al., 2013), age-related
changes in the structure of premorbid brain (Szentkuti et
al., 2004). ICV consistency during aging makes it a reliable
tool for correction of head size variation across subjects in
studies that rely on morphological features of the brain
(Ikram & DeCarli, 2012). In addition, craniosynostosis, the
premature fusion of one or more cranial sutures, changes
the normal morphology of the growing cranial vault. The
two profound functional concerns with premature fusion are
the probable reduction in craniofacial skeletal growth and
the possible elevation in intracranial pressure. The
relationship between them is still not clear. The earliest
methods for measuring the craniofacial skeleton were based
on anthropometric measurements of head (Bambha, 1961).
The ICV measurement may be more directly related to
intracranial pressure than any linear measurement of the
calvaria. In vivo measurement of ICV was not possible until
the advent of nowadays sectional imaging technics. The
accurate measurement of intracranial pressure is valuable
but generally requires invasive techniques. ICV can be
accurately measured with modern computed tomography and
MR imaging. Approaches at creating a standard reference
of ICV’s have been made in the past but have been limited
either by using volume estimation techniques or by small
1 Department of Anatomy, University of Afyon Kocatepe, Afyonkarahisar, Turkey.
2 Department of Anatomy, University of Erciyes, Kayseri, Turkey.
3 Department of Physical Therapy and Rehabilitation, Ahi Evran University, Kırsehir, Turkey.
4 Vocational School of Health Services, University of Kilis 7 Aralık, Turkey.

1466
sample sizes (Sgouros et al., 1999). The volume of organs
or structures can be obtained using the Cavalieri principle
of stereologic approaches (Cruz-Orive, 1997). The
requirement for the application of this method is a complete
set of parallel two-dimensional slices through the object
which are separated by a known distance, and begin
randomly within the object, and criteria were met by stan-
dard MR imaging and computed tomography (CT) scanning
techniques (Roberts et al., 2000; Sahin & Ergur, 2006). In
addition recents radiologic studies demostrated that point
counting and fluid displacement (the gold standard) methods
were agreement each other for volume estimation of region
of intrest (Nisari et al., 2012).
The posterior cranial fossa (PCF) is a compact region
that contains many structures that are vital to life.
Configuration and size of the posterior fossa (PF) plays an
important role in the pathophysiology of various disorders
of the PF and craniovertebral junction (Wang et al., 1987;
Bagley et al., 2006). A wide spectrum of central nervous
system (CNS) diseases in children have been associated with
alterations in the size of the PCF or its contents. Several
studies have compared PCF dimensions in patients with these
conditions with those in control individuals (Kollias et al.,
1993; Trigylidas et al. 2008; Furtado et al., 2009). Although
normative data could indirectly be derived from the control
groups of these studies, few studies establish normative
values for PCF dimensions (Habibi et al., 2011; Chadha et
al., 2015).
The purpose of current study was to investigate
development and to establish normative data of ICV, PCFV
and volume fraction of PCF in a homogeneous Turkish
population according to age and sex by using stereological
method.
MATERIAL AND METHOD
This study was carried out retrospectively on sagittal
scan images taken from 339 Turkish individuals (168 females
and 171 males) aged between 0-18 years who had been
admitted to Erciyes University Medical Faculty. Subjects
were selected from a larger pool of individuals and children
with any medical or neurological disorders that affected the
skeletal morphology of the cranial cavity were excluded.
Inclusion into the study required a negative computed
tomography report for any pathology, reviewed by a pediatric
radiologist. The CT images were examined to exclude the
maxillofacial deformities, intracranial tumours or infarcts
and other related diseases which could impact the
development of cranium and its morphology and
anatomy.The present study was approved by the ethical
committee of Erciyes University, Turkey.
CT procedure. We analyzed the intact cranial CT images
of all the subjects. The CT images were prepared using the
following protocol. The sagittal CT scans of cranial images
were obtained using a CT scanner (Multislice 16 detector
GE) applying the following parameters; kV: 120, mAs: 130,
FOV (field of view): 24-25 cm, section time: 2.7 sec, slice
thickness: 0.7 mm. CT images were taken from sagittal plan
from Picture Archive and Communication System (PACS).
The margins of the posterior cranial fossa. The posterior
cranial fossa was defined as the osseous anatomical area
with a floor formed by the occipital bone (basiocciput portion
of the clivus and supraocciput portion of the occipital bone
up to the insertion of the tentorium cerebelli, which formed
the superior boundary of this fossa) and the basisphenoid.
The petrous ridges of the temporal bones formed the
anterolateral border of this cavity anteriorly to their
connection (posterior petroclinoid ligament) to the poste-
rior clinoids (Tubbs et al., 2008).
Cavalieri principle applied to CT sections and
stereological analysis. Point-counting method is based on
the Cavalieri principle that is used for an unbiased estimation
of volume of any structure (Gundersen et al., 1999). Using
the Cavalieri method (point counting), an estimate of the
volume of a structure of irregular shape and size may be
obtained influentially and with known precision (Roberts et
al.). According to this method, the CT images of a section
series 0.7 mm thickness were used to estimate the region of
interest (ROI) volume. The films were displayed on computer
and the transparent square grid test system with d = 0.3 cm
between the test points was superimposed, randomly
covering the entire image frame (Fig. 1). The points hitting
the ROI-sectioned surface area were counted for each section
and the volumes of ICV and PCFV were estimated using
the modified Equation 1 (Sahin et al., 2007).
Where t is the section thickness of consecutive
sections, SU is the scale unit of the printed film, d is the
distance between the points of the grid, SL is the measured
length of the scale printed on the film. ∑P is the total number
of points hitting the sectioned cut surface areas of region of
interest.
ERTEKIN, T.; DEGERMENCI, M.; UCAR, I.; SAGIROGLU, A.; ATAY, E. & SUSAR, H. The intracranial and posterior cranial fossa volumes and volume fractions in children: A stereological
study. Int. J. Morphol., 35(4):1465-1472, 2017.

1467
Volume fraction estimation. Volume is a simple and very
widely used parameter in biomedical science (Mattfeldt et
al., 2003). It is used to express the proportion of a phase or
component within the whole structure. The volume fraction
of an X phase within a Y reference volume is simply
expressed as follows (Eq. 2):
We estimated the volume fraction of the PCF within the IC
by means of volume fraction approach, i.e. the PCFV within
the ICV using the following formula (Eq. 4).
Fig. 1. Estimation of intracaranial cavity and posterior cranial fossa
on sagittal computed tomography images by superimposing
randomly the point-counting grid.
Where the Vv (X, Y) indicates volume fraction of X
phase within the Y reference volume. Volume fraction rates
change between 0 and 1 and is often expressed as a
percentage (Howard & Reed, 2005).
The volume fraction of a phase can be estimated by
means of the Cavalieri principle on radiological images using
point-counting approach. The volume fraction formula with
the point-counting grid can be written as following Eq. (3).
Where ‘ΣPx’ indicates the number of points hitting
the X phase and ‘ΣPy’ the number of points hitting the
reference space Y.
Where, SPposterior cranial fossa is the total number of points
hitting the components of posterior cranial fossa and
SPintracranial cavity is the total number of points hitting sectioned
surface of intracranial cavity including all parts.
The volume fraction of the PCF volume within the
IC was estimated as:
The coefficient of error (CE) for point counting. The
coefficient of error (CE) of the point-counting method was
calculated using the formula described in previous study
(Gundersen et al.). A lower CE value than 5 % is an
acceptable range according to the literature. It is important
to note that the CE has no real biological meaning. Rather, it
is most useful for evaluating the precision of stereological
estimates. It is also important to note that an appropriate
grid size and the number of slices required for volume
estimation of an object are crucial at the beginning.
Statistical analysis. All statistical analyses were performed
with the Statistical Package for the Social Sciences software
(Version 16.0; SPSS, Chicago, IL, USA).The comparing of
the volume results between sexes were analyzed using the
Independent t test and comparison of the results of ICV and
PCFVwere analyzed using pair samples t test. Results have
been expressed as the number of observations and mean ±
Standard Deviation (SD). A p value less than 0.05 was
considered as statistically significant.
RESULTS
The mean ICV and PCFV’s volumes were
1527.22±253.89 cm
3
and 236.86±48.71cm
3
respectively. The
ICV and PCFV’s results were shown in Table I. Age-related
changes in ICV and PCFV in general similar and positive
correlation was found between age and them. They reached
adult dimensions at 5 years of age during the teenage years
(Table I, Fig. 2).
ERTEKIN, T.; DEGERMENCI, M.; UCAR, I.; SAGIROGLU, A.; ATAY, E. & SUSAR, H. The intracranial and posterior cranial fossa volumes and volume fractions in children: A stereological
study. Int. J. Morphol., 35(4):1465-1472, 2017.

1468
According to sex the mean ICV and
PCFV were 1594.51±245.57 cm3 and
244.89±53.86 cm3 in males, 1456.34±241.85
cm3 and 228.24±41.38 cm3 in females,
respectively. In addition, there were significant
differences in the ICV according to sex in 8
age groups (4, 5, 11, 14, 15, 16, 17 and 18 years
of age), for PCFV in 7 age groups (2, 8, 14, 15,
16, 17 and 18 years of age), (Table II). The mean
of CEs for the estimation of ICV and PCFV
were 2, 2.5 %, respectively.
When the correlations between the ICV
and PCFV measurements statistically analyzed,
a positive correlation was determined between
the results. Correlation values (r) for ICV and
PCFV were determined as 0.604 for females,
0.679 for males respectively (p<0.001). By the
Cavalieri principle (point-counting) using
sagittal CT images, the volume ratios of PCF
to IC was given (Fig. 3), (Tables I and II).
According to age the volume ratios of PCF to
IC was ranged from 13.03 to 17.48 in males
and 12.06 to 18.54 in females.
DISCUSSION
ICV is a morphometric measurement of
interest in brain disorders and cognitive aging
and is used as a proxy for head size. The ICV is
directly related to brain growth and is
considered to remain relatively stable after brain
development ceases in youth (Courchesne et
al., 2000; Royle et al., 2013).
Age Param eter N Min Max Mean±SD Volume
fraction
1 ICV 20 450.72 1420. 31 972.77 263.91
PCFV 20 115.34 229.41 157.85 35.16
16.23
2 ICV 16 997.92 1505. 95 1210.26 150.72
PCFV 16 111.78 247.21 181.97 35.22
15.04
3 ICV 19 889.38 1714. 61 1418.62 196.75
PCFV 19 153.90 330.48 252.22 43.89
17.78
4 ICV 20 1166.40 1864.62 1521.61 194.17
PCFV 20 185.33 332.42 271.48 46.57
17.84
5 ICV 18 1339,74 1891,51 1624,43 175,34
PCFV 18 179,82 318,82 260,99 33,63
16,07
6 ICV 16 1392,55 1895,40 1612,96 149,52
PCFV 16 206,39 328,86 258,61 29,45
16,03
7 ICV 21 1251,94 1995,84 1642,29 203,33
PCFV 21 241,38 367,42 279,80 33.56
17,04
8 ICV 18 1184.88 1761.42 1431.63 172.81
PCFV 18 138.07 240.15 182.93 32.08
12.78
9 ICV 20 835.63 2122. 22 1403.61 284.41
PCFV 20 122.50 312.50 209.04 48.11
14.89
10 ICV 18 1059.56 1929.78 1561.41 253.63
PCFV 18 184.53 320.00 236.35 41.43
15.14
11 ICV 20 1296.71 1891.56 1572.20 168.28
PCFV 20 181.33 327.11 236.77 38.86
15.06
12 ICV 19 1417.60 1830.40 1621,96 118,74
PCFV 19 165,33 328,53 244,28 43,27
15,06
13 ICV 17 1310,40 1848,89 1620,99 166,35
PCFV 17 203,38 323,56 257,36 39,84
15,88
14 ICV 20 1306,67 1864,18 1624,55 144,05
PCFV 20 188,44 321,07 240,96 31,71
14.83
15 ICV 18 1445,33 1929,60 1633,64 132,74
PCFV 18 196,44 354,13 245,64 41,65
15,04
16 ICV 20 1442,31 1943,20 1646,35 117,15
PCFV 20 198,22 301,20 244,38 31,22
14,84
17 ICV 19 1333,33 1842,64 1639,67 133,86
PCFV 19 191,64 316,09 251,04 33,78
15,31
18 ICV 20 1448,27 1883,95 1692,25 139,04
PCFV 20 194,84 307,56 242,15 34,36
14,31
Table I. The mean volumes of posterior cranial fossa and intracranial cavity
according to age calculated by using stereological method ICV: Intracranial
volume, PCFV: Posterior cranial fossa volume, *p<0.05.
Fig. 2. The volume values of the ICV and PCFV according to age.
ICV: Intracranial volume, PCFV: posterior cranial fossa volume. Fig. 3. The volume fraction of the PCFV within the intracranial
cavity according to age.
ERTEKIN, T.; DEGERMENCI, M.; UCAR, I.; SAGIROGLU, A.; ATAY, E. & SUSAR, H. The intracranial and posterior cranial fossa volumes and volume fractions in children: A stereological
study. Int. J. Morphol., 35(4):1465-1472, 2017.

1469
Traditionally, CT scans have been extensively used
in craniofacial surgery to confirm the clinical diagnosis of a
craniosynostosis, to obtain preoperative information about
the underlying brain and to objectively evaluate the surgical
results (Posnick et al., 1995). Determination of ICV has been
performed for the purpose of surgical planning. The ICV is
a good parameter to determine for evaluation of the surgical
results, since the operation is a complexre-shaping of the
form of the cranium. There is still a controversial discussion
concerning the ICV of patients with isolated sagittal
synostosis and scaphocephaly as it has been reported by
different authors to be greater, less, or equal to the general
population (Heller et al., 2008; Fischer et al., 2015). A further
problem with ICV measurements has been the absence of
adequate reference material and normative aged and sex-
matched control groups, based on the same examination
skills (Fischer et al.). This raises the question of the validity
in regard to the reference group and/or the comparability of
the measurement techniques used. Many studies investigated
the ICV in healthy and in patients’ groups by using automatic,
semiautomatic or manual methods (Dale et al., 1999;
Jenkinson et al., 2012; Rijken et al., 2015). Although
automated methods to stand out in terms of preventing the
loss of time, the choice of the software should take into
consideration whether the population under study is pediatric
or adult. In one study; researchers used four groups including
adult controls (AC), adults with Alzheimer’s disease (AD),
pediatric controls (PC) and group of pediatric epilepsy
Table II. The mean volumes of posterior cranial fossa and intracranial cavity calculated by using stereological method in both sexes. The
comparison of male-female volume results (p values) for each group. ICV: Intracranial volume, PCFV: Posterior cranial fossa volume, *p<0.05.
ERTEKIN, T.; DEGERMENCI, M.; UCAR, I.; SAGIROGLU, A.; ATAY, E. & SUSAR, H. The intracranial and posterior cranial fossa volumes and volume fractions in children: A stereological
study. Int. J. Morphol., 35(4):1465-1472, 2017.
ICV PCFV
Age Sex N Mean± SD p Mean±SD p
Volume
fraction
1 Male 10 1031.78 228.42 149.68 31.37 14.51
Female 10 913.76 295.18
0.331
166.02 38.45
0.312
18.17
2 Male 8 1248.36 149.48 162.69 33.08 13.03
Female 8 1172.15 151.72
0.329
201.25 26.73
0.022*
17.17
3 Male 9 1500.87 155.34 255.42 43.71 17.02
Female 10 1344.59 207.66
0.083
249.34 46.21
0.773
18.54
4 Male 10 1607.42 163.00 278.54 47.11 17.33
Female 10 1435.81 191.49
0.045*
264.42 47.43
0.512
18.42
5 Male 9 1715.11 115.20 272.21 31.47 15.87
Female 9 1533.76 183.20
0.023*
249.77 33.62
0.163
16.28
6 Male 8 1665.73 130.32 262.76 33.05 15.77
Female 8 1560.18 156.72
0.165
254.46 26.99
0.591
16.31
7 Male 11 1673.11 198.18 292.54 40.72 17.48
Female 10 1608.40 214.01
0.481
265.78 15.62
0.066
16.52
8 Male 10 1484.40 157.43 197.48 30.11 13.30
Female 8 1365.67 178.09
0.153
164.74 25.55
0.026*
12.06
9 Male 10 1418.25 302.71 203.25 51.72 14.33
Female 10 1388.97 280.47
0.825
214.84 46.23
0.604
15.47
10 Male 9 1622.32 251.16 242.60 43.99 14.95
Female 9 1500.50 255.48
0.323
230.09 40.29
0.538
15.33
11 Male 10 1660.21 147.93 243.93 43.70 14.69
Female 10 1484.18 143.81
0.015*
229.61 34.13
0.425
15.47
12 Male 10 1664.42 93.53 231.41 50.95 13.90
Female 9 1574.77 130.86
0.101
258.59 29.26
0.179
16.42
13 Male 9 1692.89 136.00 271.71 33.92 16.05
Female 8 1540.10 167.30
0.055
241.22 41.84
0.118
15.66
14 Male 10 1720.39 118.68 258.70 38.04 15.04
Female 10 1528.71 154.58
0.001*
223.22 30.22
0.008*
14.60
15 Male 8 1731.46 131.21 274.68 49.46 15.86
Female 10 1555.38 67.69
0.002*
222.42 16.79
0.004*
14.30
16 Male 10 1728.53 89.65 268.52 24.87 15.53
Female 10 1564.17 77.01
0.000*
220.24 22.97
0.000*
14.08
17 Male 10 1723.93 83.22 273.00 45.08 15.84
Female 9 1546.06 117.44
0.001*
227.00 32.06
0.001*
14.66
18 Male 10 1791.91 110.60 263.53 34.41 14.71
Female 10 1592.59 80.67
0.000*
220.76 27.71
0.002*
13.86

1470
subjects (PE). Three wellknown software packages
(FreeSurfer Ver. 5.3.0, FSL Ver. 5.0, SPM8 and SPM12) were
examined in their ability to automatically estimate ICV
across the groups. SPM12 with the use of pediatric template
is found to be a more suitable candidate for PE group. SPM12
and FSL subjected to tuning are the more appropriate tools
for the PC group (Sargolzaei et al., 2015). Although there
are many studies to prove the accuracy of the automated
methods, manual methods are still preferred (Pengas et al.,
2009; Nordenskjöld et al., 2013). Many study demostrated
that a stereologic technique (point counting) on CT scans
may provide unbiased organ volume estimations and their
results were similar to gold standard studies (Sahin et al.;
Nisari et al.)
One clinical study investigated ICV control (aged 172
± 8 days and 10–486 d
ays.) and sagittal synostosis patients.
The ICV was measured in a semi-automatic MATLAB
program with functions such as region growing, watershed,
and thresholding in axial CT slices. Total mean ICV was
calculated 870±15 ml for the controls. For male and female
controls the mean ICV was 883±18 ml and 834±28 ml,
respectively (Fischer et al.). Kamdar et al. (2009) assayed
ICV of 123 children (ages at presentation ranged from 8
days to 6 years. At presentation, mean age was 24 months).
Intracranial volumes were obtained using a semiautomated
image segmentation technique. They determined that ICV
was between 200-400 cm3 at newborn, 1200-1400 cm3 at 72
months age. In this study of healthy children, growth is most
rapid from birth to 12 months of age, with continued but
slower growth during the first 6 years of age, ICV doubled
by 9 months of age and tripled by 6 years of age. Sgouros et
al. published a series of intracranial volumes obtained from
magnetic resonance imaging data and showed ICV in the
first few months of life is on average 900 cm3 in boys and
600 cm3 in girls, increasing to 1300 to 1500 cm3 by 15 years.
In the first 2 years, 77 percent of growth was achieved.
Freudlsperger et al. (2015) investigated the ICV in 634
healthy boys between the ages of 3 and 13 months by using
three dimensional (3D) photogrammetry. The mean total ICV
plotted against the age in months increased from 1173.3 cm3
to 1593.6 cm3. Our series of 339 individuals provides the
largest series of cranial volume data in children using a
proven, accurate technique that we have found in the
literature to date, and can provide useful reference in the
investigation of any condition influencing cranial vault
growth. We determined that at 1 year of age; the mean ICV
was 972.77±263.91 cm3, according to sex mean ICV values
were 1031.78±228.42 and 913.76±295.18 for males and
females respectively. Similiar to literature ICV showed that
positive correlation to age and growth, is most rapid from
birth to the first 5 years of age. Total mean ICV was
calculated 1031.78±228.42 cm3 and male values were greater
than female and these differences were statistically
significant in some age groups. We thought that numerical
differences between present study and literature are possibly
due to patients series (age and race) and used methods
(radiologic and volume estimation technique).
PCF. The growth of the PCF reflects the way the cerebellum,
ver
mis, brain stem and fourth ventricle develop as a whole.
Abnormal changes in the size of the PCF or its contents
associated with malformations or functional defects are
usually assessed qualitatively. Normative data for the PCFV
could be of value in the study of diseases that cause
alterations in the size of the PCF (Prassopoulos et al., 1995).
A fundamental knowledge of normal anatomy of this region
is important to the clinician for diagnosis and treatment (Jha
et al., 2008). The differences between the radiographic and
the anatomic values and even among the radiographic values
are possibly due to the radiologic and other techniques used
by the different authors (Prassopoulos et al.). Morphometric
analysis of the PCF is required to distinguish clinical cases
occurring with a pathologically small PCF from those in
which the size and volume of the PCF are normal. When
correlated with clinical findings, morphometric assessments
of the PCF provide useful clues about the following
mechanisms of cerebellar tonsil herniation (CTH): 1- cranial
constriction; 2- spinal cord tethering; 3- cranial setting; 4-
intracranial hypertension and 5- intraspinal hypotension. The
differentialdiagnosis of CTH i
s likely to inform management
strategies (Milhorat et al., 2010).
Prassopoulos et al. investigated 181 brain CT
examinations, to determine normative data for the PCF in
children. The PCFV was calculated by summing consecutive
CT cross-sectional areas. PCFV increased rapidly during the
first 3 years of life and thereafter the rate of growth decreased.
The PCFV at the age of 3 was 165 cm
3
in boys and 155 cm
3
in
girls and at 15 years, 220 cm
3
and 207 cm
3
respectively.
Trigylidas et al. estimated the volume differences between
Chiari I malformations patients and control groups (61
individuals). Volumetric measurements were calculated
manually using the Cavalieri method. They were found as
86.76 cm
3
for 0-9 years (n=13) and 145.14 cm
3
for 10-18 years
(n=7) in control groups. Milhorat et al. calculated PCFV in
patients with Chiari malformation and control subjects
(31.7±11.8 age) with using radiographic analysis software
(ImageJ) and the Cavalieri method on 2D-CT images and
PCFV was calculated as 190.1±7.84 cm
3
in control subjects.
Similarly, we used individuals between 0-18 years, in our study,
PCFV rised rapidly up to 5 years and positive correlation was
found between age and PCVF. We thought that numerical
differences among our study and the others may be derived
from the subject’s race, sex, age, the number differences of
the samples and methods that were used in researches.
ERTEKIN, T.; DEGERMENCI, M.; UCAR, I.; SAGIROGLU, A.; ATAY, E. & SUSAR, H. The intracranial and posterior cranial fossa volumes and volume fractions in children: A stereological
study. Int. J. Morphol., 35(4):1465-1472, 2017.

1471
Kanodia et al. (2012) investigated 100 consecutive
normal computerized tomography (CT) scans of posterior fossa
(age ranged from 16 to 89 years with a mean of 51.3 years)
and 100 dry adult skulls without any bony abnormality. The
PCFV was calculated by two methods. In the first method,
volume was calculated by abc/2, where a is the height, b is AP
diameter, and c is transverse diameter of the PF. In second
method, PCFV was calculated by an advanced work station
of 16-row bright speed CT scan. The mean value of PCFV
where 157.88 (±27.94) cm
3
(range 98.75–216.88 cm
3
) and
159.58 (±25.73) cm3 (range 116.03– 252.99 cm
3
) measured
by method 1 and method 2, respectively. However, the
difference was not statistically significant (P > 0.05). The mean
PCFV was122.49 cm
3
(±14.66), respectively, in dry skull. They
determined that all the dimensions of PCF were larger in male
as compared to female. The volume values calculated with
both methods was similar to our results. In our study according
to sex; the mean PCFV was determined as 244.89±53.86 cm
3
and 228.24±41.38 cm
3
in males and females respectively. In
addition, there was a significant difference in the PCVF
according to sex in 7 age groups (2, 8, 14, 15, 16, 17 and 18
years of age) (Table II).
Furtado et al. investigated PCFV in 21 CMI patients,
matched with an equal number of pediatric controls by using
MR images. They evaluated PCFV to ICV ratio in pediatric
CMI patients, adult CMI group and in the pediatric control
group. The volume results were obtained by using simple
mathematical spheroidal volume formulas. The mean PCFV
was 204.1 cm
3
in patients with CMI, and 252.8 cm
3
in the age
and sex matched control group. The number, age and sex
distribution of both groups were well matched. There was no
statistical difference between ICV in the study and control
groups (944.7 cm
3
vs. 941.1 cm
3
, p = 0.951), validating the use
of the PFV to ICV ratio to compare the pediatric groups. The
PCFV to ICV ratio was significantly lower in patients with
CMI than in the control population (0.216 vs. 0.268, p = 0.001.
There was no significant difference between the PCFV to ICV
ratios obtained in the pediatric and adult CMI group (0.216
vs. 0.236, p = 0.1754). Similiarly Ventureyra et al. (2003)
recorded a statistically smaller PCFV and PCFV to ICV ratio
in his series of 61 patients with pediatric CMI.
Researchers demostrated that lower PCFV to ICV ratio
which pointing to an overcrowded posterior fossa was
important and necessary criterion for the diagnosis and
treatment of the disease related to PCF. However, in the
analysis of the volume ratio in patients they suggested that a
larger study group would have been helpful (Ventureyra et
al.; Furtado et al.).
To the best of our knowledge, there was no study in
the literature that investigated the development of ICV, PCFV
and volume ratios of PCF to IC in children by the stereological
technique. In conclusion, we demonstrated increasing ICV and
PCFV’s in children related to age. In this study, growth is most
rapid from from birth to first 5 years of age, ICV and PCFV
nearly doubled by 5 years of age. The volume ratios of ICV to
PCFV ranged from 13 % to 18 % according to age. We thought
that these volume ratios could help the physician for both
patient selections for surgery, and for the assessment of any
surgical technique used to treatment of PCF malformations.
However current study revealed that point counting method
can produce accurate volume estimations and is effective in
determining volume estimation of intracranial and posterior
cranial fossa. The point-counting method is a reliable, simple,
inexpensive, and efficient method for estimating volumes in
CT images.
ERTEKIN, T.; DEGERMENCI, M.; UCAR, I.;
SAGIROGLU, A.; ATAY, E. & SUSAR, H. Los volúmenes de
la fosa craneal intracraneal y posterior y las fracciones de volu-
men en los niños: un estudio estereológico. Int. J. Morphol.,
35(4):1465-1472, 2017.
RESUMEN: El tamaño de la cavidad intracraneal (CI) y
la fosa craneal posterior (FCP) desempeñan un papel importante
en la fisiopatología de diversos trastornos. En este estudio, se
pretende establecer los datos de volumen normal de la CI y FCP
en la población turca, de acuerdo a la edad y el sexo, mediante el
uso de métodos estereológicos. Este estudio se realizó retrospec-
tivamente en 339 individuos (168 mujeres y 171 hombres) entre
0 y 18 años sin trastornos médicos o neurológicos que afectaron
la morfología esquelética de la cavidad craneal. Las estimacio-
nes volumétricas se determinaron en imágenes de tomografía
computarizada (TC) utilizando el conteo de puntos de los méto-
dos estereológicos. El volumen intracraneal (VIC) y el volumen
posterior de la fosa craneal (VFCP) aumentaron con la edad en
ambos sexos. Alcanzaron dimensiones adultas a los 5 años de
edad durante la adolescencia. Según el sexo, el promedio de VIC
y VFCP fue de 1594,51 ± 245,57 cm
3
y de 244,89 ± 53,86 cm
3
en
los hombres, 1456,34 ± 241,85 cm
3
y 228,24 ± 41,38 cm
3
en las
mujeres, respectivamente. En general, se determinaron diferen-
cias signifi
ca
tivas en VIC y VFCP de acuerdo con el sexo des-
pués de alcanzar el período de crecimiento máximo. Según la
edad, las proporciones de volumen de FCP a CI oscilaban entre
13,03 a 17,48 en los hombres y 12,06 a 18,54 en las mujeres. Este
estudio demostró que estas proporciones de volumen podrían
ayudar al médico tanto en la selección de pacientes para la ciru-
gía, como para la evaluación de cualquier técnica quirúrgica uti-
lizada en el tratamiento de malformaciones de FCP. Además, el
estudio actual reveló que el método de conteo de puntos puede
producir estimaciones precisas de volumen siendo eficaz para de-
terminar la estimación de volumen de IC y FCP.
PALABRAS CLAVE: Tomografía computarizada; Ca-
vidad intracraneal; Fossa craneal posterior; Estereología; Vo-
lumen.
ERTEKIN, T.; DEGERMENCI, M.; UCAR, I.; SAGIROGLU, A.; ATAY, E. & SUSAR, H. The intracranial and posterior cranial fossa volumes and volume fractions in children: A stereological
study. Int. J. Morphol., 35(4):1465-1472, 2017.

1472
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Corresponding author:
Tolga Ertekin
Associated Professor - Department of Anatomy
University of Afyon Kocatepe - School of Medicine
Afyonkarahisar - TURKEY
E-mail: tolga.ertekin@yahoo.com.tr Received: 21-03-2017
Accepted: 11-08-2017
ERTEKIN, T.; DEGERMENCI, M.; UCAR, I.; SAGIROGLU, A.; ATAY, E. & SUSAR, H. The intracranial and posterior cranial fossa volumes and volume fractions in children: A stereological
study. Int. J. Morphol., 35(4):1465-1472, 2017.