Animal Models Of Human Disease–Original Article

Veterinary Pathology
2015, Vol. 52(6) 1263-1271
Vitamin C Depletion in Prenatal Guinea ª The Author(s) 2014
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Pigs as a Model of Lissencephaly Type II DOI: 10.1177/0300985814561270
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I. Čapo1, N. Hinić1, D. Lalošević1, N. Vučković2, N. Stilinović3,

J. Marković4, and S. Sekulić5

Abstract
Humans and guinea pigs are unable to produce vitamin C, with deficiency resulting in a well-known disorder of collagen synth-
esis. Pial basement membrane structure preservation is essential in the proper migration of neurons. In our study, intrauterine
deprivation of vitamin C in guinea pig fetuses led to a collagen synthesis disorder, weakness, and finally a breach of pial basement
membrane. We found excessive migration of the external germinal layer cells into the subarachnoid space of the cerebellum
through defects in the pial basement membrane. The changes ranged from focal rupture of pial basement membranes to their
complete disintegration. The loss of proper folia formation resulted in macroscopically visible flattening of the cerebellar sur-
face. Different grades of dysplastic changes in the folia of the cerebellar cortex were observed in 2 experimental groups
assigned different limits to mark the time of commencement and duration of vitamin C deprivation. The most severe form
of dysplastic changes was characterized by marked irregularity of the cerebellar cortex similar to that in lissencephaly type
II. Thus, prenatal vitamin C deficiency represents a novel animal model to study the effects of collagen synthesis on develop-
ment of breaches in the pial basement membrane, disordered migration of neurons, dysplasia of cerebellar cortex, and the
pathogenesis of lissencephaly.

Keywords
scurvy, cerebellum, guinea pigs, vitamin c deficiency, lissencephaly, animal model

Lissencephaly type II is a disease caused by alteration to the
pial basement membrane (PBM) proteins, which causes it to
weaken and become porous. This causes a deficiency of the
glia limitans superficialis and radial glial cell connectivity.
As a result, migrating neural cells pass through the pial gaps
to form cell ectopia near the brain surface. This leads to loss
of gyri and sulci, forming irregularities on the brain surface and
a so-called cobbled formation, which is why lissencephaly type
II is also sometimes called cobblestone lissencephaly.9,34
Based on the fact that the structural integrity of the PBM is
necessary for proper migration of the neurons, 2 main
approaches have been established for experimental modeling
of lissencephaly type II. First, animal models with targeted
inactivation of genes involved in membrane protein synthesis
have been studied. The lack of these proteins, such as collagen,
laminin, and others, causes the PBM to weaken and finally
rupture.13,16,17,25,43 The second approach uses chemical
destruction of meningeal cells over the developing cerebellum
through exposure to 6-hydroxydopamine (6-OHDA). The loss
of glio-pial membrane allows for overmigration of neurons
through the pial breaches into the subarachnoid space.1,29,30
Collagen is one of the key structural units of the basement
membrane, and its synthesis is dependent on vitamin C.
Humans, monkeys, and guinea pigs are unable to synthesize
vitamin C because of a lack of L-gulono-g-lactone oxidase.27
It is well known that a deficiency of vitamin C in guinea pigs
impairs the expression of type IV collagen, elastin, and lami-
nin in blood vessels.10,23
Guinea pigs are precocial rodents in that their brain devel-
opment occurs predominantly during the intrauterine phase.21
Our hypothesis was that the fetal vitamin C deprivation during
cerebellar foliation would cause reduced collagen synthesis,
consequentially weakening and rupturing the PBM to produce
the dysplastic changes consistent with lissencephaly type II.
1
Department of Histology and Embryology, Medical Faculty, Novi Sad, Voj-
vodina, Serbia
2
Department of Pathology, Medical Faculty, Novi Sad, Vojvodina, Serbia
3
Department of Pharmacology and Toxicology, Medical Faculty, Novi Sad,
Vojvodina, Serbia
4
Department of Psychiatry, Medical Faculty, Clinical Center of Vojvodina, Novi
Sad, Serbia
5
Department of Neurology, Medical Faculty, Clinical Center of Vojvodina, Novi
Sad, Serbia
Corresponding Author:
I. Čapo, University of Novi Sad, Medical Faculty, Department of Histology and
Embryology, Hajduk Veljkova 3, Novi Sad, Vojvodina, Serbia.
Email: capo.ivan@gmail.com

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Materials and Methods
Animals and Environment
We studied albino guinea pigs (Cavia porcellus), aged between
4 and 6 months, obtained from the Department of Biochemis-
try, Faculty of Medicine, Novi Sad. The experiments with these
animals were approved by the Ethics Committee of the
University of Novi Sad (No: 01-160/6-2; EK: III-2013-02).
Animals were kept in 400 (wide) 1000 (long)  300 (high)–
mm plastic containers in a harem system: 3 or 4 females per
male. Artificial cycles with 12 hours of light (08:00–20:00) and
12 hours of dark were provided. The ambient temperature was
23 C, and air was fully circulated 6 to 10 times per hour.
Inspection of the female vaginal introitus was performed on a
daily basis. The presence of spermatozoids in a vaginal smear
was designated the first day of gestation and at that time the
dams were housed separately from males. Gestational length
in guinea pigs (C. porcellus) can vary between 62 and 68 days.26
The experimental study included 21 female animals that
maintained pregnancy until they were euthanized. These were
randomly divided in 3 groups: a control group, K (n ¼ 7), and
2 experimental groups, E1 (n ¼ 7) and E2 (n ¼ 7). Experimen-
tal animals that were unable to achieve or maintain pregnancy
during the experiment (0 in K, 1 in E1, and 9 in E2 group)
were excluded from the study and replaced with new dams.
Experimental Protocol
All animals were sustained with a standard vitamin C–free com-
mercial pellet diet. Control animals were supplemented with ad
libitum water enriched with vitamin C (50 mg/100 ml water).
Until the day of vitamin C restriction (see below), females in the
experimental groups had the same diet as the females from
the control group. Vitamin C deprivation began on the 20th day
of gestation for group E1 and on the 10th day for group E2. Vita-
min C deprivation continued for both E1 and E2 groups until the
50th day of pregnancy, when the animals were euthanized.
The experimental design was based on 2 facts. First, the
justification for euthanasia on the 50th day of gestation was
based on our pilot study in which the first signs of cerebellum
foliation could be spotted on the 35th day of gestation and that
the formation of all 10 cerebellum lobules were developed by
the 45th day of gestation. Second, the duration of vitamin C
deprivation was based on the fact that the first symptoms of
deficiency in guinea pigs appeared after 3 to 4 weeks of vita-
min C deprivation.23,28
Perfusion Fixation
On the 50th day of gestation, fetuses were removed by caesarean
section under urethane anesthesia. Perfusion fixation was per-
formed through cannulation of the ascending aorta while simul-
taneously incising the right atrium to allow the egress of blood
and perfusate. Then, 30 to 60 ml of buffer (0.27 mmol/l NaCl,
44 mmol/l dextrose, 47 mmol sucrose, 4.2 mmol/l CaCl2,
3.1 mmol/l Na-cacodylate, pH 7.4) was infused, followed by
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Veterinary Pathology 52(6)
a *150-ml infusion of Zamboni’s fixative. Fetuses were
weighed after perfusion. After fixation, the central nervous
structure (cerebrum, cerebellum, medulla oblongata, and spinal
cord), the eye, and the slice of muscle tissue from the upper
limb were removed from the bodies and immersed in a fixative
for 24 hours. For the purposes of this study, we are only pre-
senting the data derived from the cerebellum.
Immunohistochemistry
For immunohistochemical analysis, we cut a 5-mm-thin midsa-
gittal section from the region of the cerebellar vermis. After
appropriate dehydration, the section was embedded in paraffin
(Histowax, the Netherlands) and cut on a rotary microtome
(Leica, Germany). Slides were routinely stained with hematox-
ylin and eosin (HE). Methods for immunohistochemical stain-
ing included primary antibodies: mouse anti–collagen IV Ab-3
in a 1:50 dilution (Lab Vision; Thermo Scientific, Rockford,
IL), mouse anti–Ki-67 Ab-4 in a 1:100 dilution (Lab Vision;
Thermo Scientific), and mouse anti-GFAP in a 1:100 dilution
(Lab Vision; Thermo Scientific) using the appropriate visuali-
zation system: UltraVision LP Detection System HRP Polymer
& AEC Chromogen (Lab Vision; Thermo Scientific) and Ultra-
Vision LP Detection System HRP Polymer & DAB Chromo-
gen (Lab Vision; Thermo Scientific).
Ki-67 and GFAP immunostaining required antigen retrieval
using citrate buffer (pH 6.0) in a microwave oven at 850 W for
20 minutes. Collagen IV immunostaining required antigen
retrieval using protease XXV (Lab Vision; Thermo Scientific)
for 10 minutes at 37 C. All the antibodies were applied for 30
minutes at room temperature. Visualization was performed
using either AEC Chromogen (Lab Vision; Thermo Scientific)
or DAB Chromogen (Lab Vision; Thermo Scientific). Mayer’s
hematoxylin was used as a counterstain for immunohistochem-
istry followed by mounting and coverslipping (Bio-Optica,
Italy) for slides. Prepared slides were analyzed using an Olym-
pus BX-43 microscope (Olympus, Tokyo, Japan) and photo-
graphed on an Olympus DP 73 camera (Olympus).
Statistical Analysis
Body weight comparisons were performed using unpaired
2-tailed Student’s t-test. Data were reported as the mean +
standard deviation (SD). For the dysplastic changes compari-
son between 2 groups Mann-Whitney U test was employed.
A difference was considered statistically significant for a P
value less than .05 (P < 0.05).
Results
Effect of Vitamin C Deprivation on Pregnancy in Dams
and Body Weight of Fetuses
During the experiment, spontaneous abortion was present in
0% of the control group and 14.23% and 52.95%, respectively,
of pregnant dams in the E1 and E2 groups. None of dams
developed clinical signs of scurvy during the study.
Čapo et al
Figure 1. Cerebellum, fetal guinea pig, control group. Dorsal view of cerebellum shows regular arrangement of folia in cerebellar hemispheres
and vermis. Zamboni’s fixation. Figure 2. Cerebellum, fetal guinea pig, E2 group (E2 ¼ vitamin C deprivation after the 10th day of gestation).
Dorsal view of cerebellum shows the absence of folia and sulci on the cerebellar surface. Zamboni’s fixation.
On the 50th day of gestation, fetal weights in group E1 (30.9
+ 3.16g, P < .002) and group E2 (27.21 + 1.16g, P < .001)
were both significantly reduced compared with the control
group (33.80 + 1.77g). In addition, some fetuses from the
E2 group were significantly smaller than fetuses in the E1 and
control groups, and the E2 group also had cases of talipes
equinovarus (varus defomity or internal rotation at the carpo-
metacarpal and tarsus) of the forelegs and hind legs with visible
muscular atrophy.
Gross Lesions in Cerebellum of Vitamin
C–Deprived Fetuses
The total number of fetuses (n ¼ 60) was distributed as 20 in
the control group, 21 in group E1, and 19 in group E2. Grossly,
the cerebellar surface in animals from the control and E1
groups had normal development and anatomical arrangement
of folia (Fig. 1). The absence of with smooth surface of the cer-
ebellum was observed in the E2 group. These changes were
most pronounced in the vermis with fused folia and the loss
of borders between hemispheres and vermis (Fig. 2).
Qualitative Pathologic Changes of the Cerebellar Cortex
in Vitamin C–Deprived Fetuses
Dysplastic changes of the cerebellar cortex were detected in
fetuses in the E1 (25%) and E2 (100%) groups. These changes
were not noticed in the control group of animals.
Descriptive scoring of dysplastic changes in the cerebellum
was not evident in current literature, so we applied an ordinal
method of classification regarding these changes following
principles of histopathologic scoring.7 Scoring (1–4) was based
on the intensity of dysplastic changes in the cerebellar cortex
and the number of affected folia:
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1265
First stage: preserved structure of the cerebellar cortex with-
out detectable lesions
Second stage: focal ectopic clusters of external granular
layer (EGL) cells in subarachnoid space of the fissure
from one folium. The pial membrane has minor damage
(breaches), but the structure of the adjacent cerebellar
cortex was normal (Figs. 3, 4).
Third stage: focal to complete obliteration of 1 or 2 fissures
due to the fusion of opposing folia. In the affected areas,
the cerebellar cortex had a loss of normal linear
arrangement of Purkinje cells with a clear protrusion
of the EGL and molecular layer into the subarachnoid
space (Figs. 5, 6).
Fourth stage: complete fusion of 3 or more fissures. This
level of damage represents a serious disturbance of cer-
ebellar cortex structure and Purkinje cell arrangement.
The intensity of dysplastic changes in the cerebellum
was the most intense, and it conformed to pathologic
findings in lissencephaly type II (Figs. 7, 8).
Anti–collagen IV immunohistochemical staining in the
control group determined the baseline for PBM integrity
(Fig. 9). PBM in animals from group E1 was mostly intact
with rare signs of rupture, while the PBM structure in the
E2 group was completely disrupted in the form of multiple
ruptures and fragmentations. In most cases, individual frag-
ments of pial cells and basal membrane were covered with
ectopic content made up of the EGL cells. Due to the exces-
sive migration of granular cells, pial membrane fragments
were usually pushed deep into the molecular layer.
Despite dysplastic changes of the cerebellar cortex, ectopic
cells of EGL retained anti–Ki-67 positivity (Fig. 12). Pro-
nounced atrophy of the internal granular layer was observed
in the areas of the cerebellar cortex with dysplastic changes.
1266 Veterinary Pathology 52(6)

Figure 3. Cerebellum, guinea pig, E1 group (E1 ¼ vitamin C deprivation after the 20th day of gestation). Low magnification of midsagittal
section of vermis with visible second stage of dysplastic changes presented in black frame area. Hematoxylin and eosin (HE). Figure 4.
Cerebellum, guinea pig, E1 group. High magnification of black frame area from Fig. 3. Note small groups of granule cells in
subarachnoid space in the fissures (arrow). HE. Figure 5. Cerebellum, guinea pig, E2 group. Low magnification of midsagittal section of vermis

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Čapo et al 1267

Application of anti-GFAP staining in the control group
showed Bergmann glial extensions spreading parallel through
the molecular layer from the Purkinje cells to the PBM (Fig.
13). Pial membrane disruption in the E2 group resulted in com-
plete alteration of the direction and structure of Bergmann glial
cells. The ectopic mass of EGL cells in the subarachnoid space
was very often permeated with intense gliosis (Fig. 14).
The most common occurrence of PBM rupture was
observed in the depths of fissures with similar changes extend-
ing to proximal parts of the folia.
Quantification and Distribution of Pathologic Changes of
the Cerebellar Cortex in Vitamin C–Deprived Fetuses
Dysplastic changes were not observed in the control group.
In the E1 experimental group, 76% (16 of 21) were scored
as first stage, 19% (4 of 21) were scored as second stage,
and 5% (1 of 21) were scored as third stage dysplasia. In the
E2 group, 11% (2 of 19) were scored as second stage, 42%
(8 of 19) were scored as third stage, and 47% (9 of 19) were
scored as fourth stage dysplasia. Statistical analyses of the
dysplasia scoring are presented in Table 1.
In group E1 animals with dysplastic lesions (n ¼ 5), the
lesion was localized to the seventh folia in 5 animals, while
1 also had lesions extending into the fifth and sixth folia
(Fig. 15). The fetuses from group E2 showed changes in all
folia except the tenth. The highest incidence of dysplastic
changes was observed in the folia belonging to the central
lobe (VI, VII, and VIII folia) (Fig. 16).
Discussion
Characteristics of Lissencephaly Type II
Lissencephalies are usually classified into type I and type II, both
being characterized by the perturbation of neuronal migration
but with different mechanisms of pathogenesis.6 Lissencephaly
type I is characterized by abnormal nucleokinesis causing aber-
rant cortical layer formation.18 In contrast, lissencephaly type II
is caused by disruption of basement membranes followed by
overmigration of neurons into the subarachnoid space.6 Lissen-
cephaly type II is usually found in syndromes such as Fukuyama
congenital muscular dystrophy (FCMD), muscle-eye-brain
disease (MEB), and Walker-Warburg syndrome (WWS).
These syndromes are characterized by the presence of muscular
dystrophy and sometimes ocular abnormalities in addition to the
pathologic dysplasia of cerebrum and cerebellum. Aberrant
glycosylation of membrane glycoproteins such as dystrogly-
can appears to underlie the defect in all of these syndromes.9
Dystroglycan represents a heavily glycosylated glycoprotein
composed of 2 connected a and b subunits. b-Dystroglycan
is a transmembrane protein interacting with both the cytoplasmic
the cytoskeleton and extracellular a-dystroglycan. This is a per-
ipheral membrane glycoprotein linking to extracellular mole-
cules such are laminin, perlecan, agrin, and neurexin.14 The
deletion of brain dystroglycan in mice is characterized by disrup-
tions of the glia limitans, which can lead to multiple abnormal-
ities of cerebral and cerebellar development. It includes defects
of cerebral lamination and the fusion of cerebellar folia with
aberrant migration of granule cells,24 which can also be seen
in our study. Recent study of animal models with congenital
muscular dystrophies (CMDs) such as WWS, FCMD, and MEB
disease includes mutation of genes for encoding fukutin
(FKTN),35 POMGnT1 (O-mannose N-acetylglucosaminyl
transferase 1),44 POMT 1 (O-mannosyltransferase 1),4 POMT
2 (O-mannosyltransferase 2),39 and LARGE (like-glycosyltrans-
ferase).15 All of those CMD animal models include disruption of
the integrity of the PBM confirmed by electron microscopy
and immunohistochemical analysis. Cerebellar defects include
the fusion of opposing folia with disorganization of the ext-
ernal granular layer and Purkinje cells malposition. We have
presented similar histologic findings in vitamin C–deprived
guinea pig fetuses with a severe grade of cerebellar dysplasia.
Mouse embryos with a deletion of the Col4a1/2 locus and
nidogen binding site for the laminin g1 subunit have neuronal
ectopia caused by local rupture of PBM with cells protruding
into the surrounding mesenchyme.25,42 Due to the lack of these
essential components, the basement membrane changes in lungs
and kidneys lead to intrauterine or postpartum death. It is impor-
tant to note that postnatal development is possible in mice with a
lack of b1-integrins11 or the a1 subunit of laminin;17 however,
cerebellar fissuration and foliation disorder are also present. The
loss of Purkinje cell polarity, ectopic EGL cells along the fusion
line of 2 opposing folia, and atrophied internal granular layer are
identical to those observed in our study.11
Specificity of Chemically Induced Destruction of PBM as a
Model of Lissencephaly Type II
The most comprehensive analysis of dysplastic changes in
the development of the cerebellum was performed in studies
on an animal model with an intracisternal injection of
6-OHDA. The first signs of damage in the cerebellar pial
fibroblasts and meningeal cells occurred by 24 hours follow-
ing application, and macrophage phagocytosis at the site of
denuded of pia was present by 2 to 5 days.29
The chemically induced destruction of meningeal cells
destabilizes the basal lamina and glia limitans superficialis, fur-
ther disorganizing the glial scaffold.40 Sievers et al30 described

Figure 5. (continued) with visible third stage of dysplastic changes presented in black frame area. HE. Figure 6. Cerebellum, guinea pig, E2
group. High magnification of black frame area from Fig. 5. Note that the deep aspect of the fissure is obliterated with clear fusion of the external
granular layer cells of the opposite folia (arrows). HE. Figure 7. Cerebellum, guinea pig, E2 group. Low magnification of midsagittal section of
vermis with visible fourth stage of dysplastic changes presented in black frame area. HE. Figure 8. Cerebellum, guinea pig, E2 group. High
magnification of black frame area from Fig. 5. Note high disorganization of cerebellar cytoarchitecture. Dysplastic area is composed of mature
neurons and immature granular cell collections with fragments of pial basement membrane. HE.

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1268 Veterinary Pathology 52(6)

Figure 9-14. Cerebellum, fetal guinea pig, midsagittal section of vermis. Figure 9. Control animal group (normal). Presence of intact pial base-
ment membrane (PBM) immunopositive for collagen IV (arrow). Immunohistochemistry (IHC) for collagen IV. Figure 10. E2 animal group ver-
mis. Presence of fragmented PBM immunopositive for collagen IV (arrow). Figure 11. Control animal group. Note Ki-67 positivity of the
proliferative zone in the external granular layer (arrow). IHC for Ki-67. Figure 12. E2 animal group. Note anti–Ki-67 positivity of the granular
cells affected by severe grade of dysplasia (arrows). Figure 13. Control group. Note GFAP-positive Bergmann glial extensions with parallel
spreading through the molecular layer (arrow). Subarachnoid space (asterisk). IHC for GFAP. Figure 14. E2 animal group. Note GFAP-positive
fragments of damaged Bergmann glial extensions (arrow) and gliosis of an ectopic mass of external granular layer cells located in the subarach-
noid space (asterisk).
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Čapo et al
Table 1. Intensity Scores of Dysplastic Changes in Cerebellar Folia in
Control, E1 and E2 Groups.
Score
Group No. of Fetuses 1 2 3 4
Control 20 20 0 0
E1a,b 21 16 4 1
E2b,c 19 0 2 8 9
a
Group control vs E1, P ¼ .022, Mann-Whitney U test.
b
Group E1 vs E2, P ¼ .000, Mann-Whitney U test.
c
Group control vs E2, P ¼ .000, Mann-Whitney U test.
that the first changes can be observed when the Bergmann-glia
endfeet lose contact with the PBM. Subsequently, the loss of
integrity through PBM ruptures leads to the excessive growth
of EGL cells, spreading from the Bergmann-glia endfeet into
the subarachnoid spaces of cerebellar fissures.29 As a conse-
quence, this change influences neuronal migration as well as
regular fissuration and foliation of the cerebellum.31,32 Simi-
larly, the weakening and rupturing of PBM in our investigation
(Fig. 10) also caused disorganization in the glial scaffold of the
cerebellar cortex (Fig. 14).
The use of light microscopy and transmission and scanning
electron microscopy shows that the ectopic EGL cells develop
into granule, stellate, and basket cells possessing a morphology
identical to relative cells placed in situ.22,30 We have also
observed that ectopic cells of EGL continue to proliferate (Fig.
12) and form large colonies of granule and stellate cells in these
regions. In addition, we have observed intense gliosis in ectopic
areas (Fig. 14), suggesting that these ectopic cells usually dis-
appear within 1 year.
Specifics of the Intensity and Distribution of Dysplastic
Changes in Vitamin C–Deprived Guinea Pig Fetuses
Our pilot studies (see Materials and Methods) indicated that
the first signs of cerebellar foliation can be observed in guinea
pigs on the 35th day of intrauterine development. Formation
of all 10 folia is completed by the 40th to 45th day of intrau-
terine development, and the specificity of differences in the
distribution of dysplastic changes between the experimental
groups can be explained using Larsell classification.19
Using radioactive isotopes to mark mitotic active structures
during the folia development in the cerebellum of rats, Joseph
Altman2 has identified 3 neurogenetic divisions. The first one
is marked as early generated division and includes the formation
of I, X, and a part of II folia. The second one, intermediate gen-
erated division, includes the formation of a part of II, III, IV, V,
and IX folia. The third one, late-generated division, includes
the formation of VI, VII, and VIII folia. On this basis, we can
assume that the depletion of vitamin C in group E2, as well as
the consequent collagen synthesis disorder and final weakening
and rupture of PBM, took place in the early stage of cerebellar
development. Folia that were developing at the time and all those
that would develop in the future were affected by dysplastic
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1269
changes. Because the early stage defect affected several folia,
the presence of third and fourth stage dysplasia scores were com-
mon in the E2 group. The preservation of folia X can be attrib-
uted to the fact of its early formation.
In the E1 experimental group, dysplastic changes were identi-
fied only in folia VI, VII, and VIII. This clearly indicates that the
0
0
critical moment of PBM rupture came during the period spanning
E40 to E45 or in the period of late-generated division. Consider-
ing the relatively short period for progression of dysplastic
changes leading to euthanasia, these cumulative observations
clarify why this group had second or third stage dysplasia scores.
Dysplastic changes in the depth of the fissure were fre-
quently identified in our study. This can be attributed to the
mechanisms of folia formation that involve their stretching and
lengthening to the bottom of the fissure.20 It is probable that the
fragile PBM is immediately affected by the pressure, which
leads to its rupture.
Although we have analyzed static sections of immature
cerebellar tissue, based on the statements by Sievers et al,30
we can expect some of this dysplastic tissue to develop into
mature tissue. Functional deficit will probably depend on the
extent of folia involvement and the stage of cerebellar dysplasia.
For the fourth stage of cerebellar dysplasia, we can predict
development of severe functional deficit with mental and
motor retardation, which can be witnessed in some human
syndromes.9 Those with focal and mild dysplastic changes
in the first stage of dysplasia will probably develop slight
functional deficits or present with clinical features such as
epilepsy, autism, or cognitive defects.36,41
Significance of the Prenatal Vitamin C Deprivation Model
in Understanding Brain Developmental Disorders
The pathogenesis of cerebellar damage in human lissence-
phaly is not well defined, but the progression of folial disease
in our study might help explain the focal appearance in corti-
cal dysplasia and the usually well-defined junction between
normal and malformed brain tissue in humans.9
Recent investigation of vitamin C deficiency in the period
of prenatal38 and early postnatal life37 has detected a reduc-
tion of the hippocampal neurons in guinea pigs. However, the
experimental groups of animals that received a diet low in
vitamin C (100 mg/kg) were not as fully restricted as in our
experiment. We speculate that lissencephaly was not diag-
nosed in that study because the collagen damage had insuffi-
cient time to develop in the vitamin C–deficient state.
The observation of talipes equinovarus with gross muscular
atrophy in vitamin C–deprived guinea pig fetuses also gave an
opportunity to use this animal model in the investigation of
congenital muscular dystrophy associated with brain defects.
The disruptions of PBM were clearly present in collagen
IV–deficient mouse embryos with early intrauterine lethality.25
In our animal model, we were able to vary the intensity and dis-
tribution of dysplastic changes by modulating the initial time
and duration of vitamin C deprivation.
1270
Figure 15 (E1 group) and 16 (E2 group). Line drawings of cerebellar vermal midsagittal sections. Gray discoloration indicates the presence of
dysplastic changes in the observed folia. The numbers are the percentage of fetuses from each group (Fig. 15, E1 group, n ¼ 21 total; Fig. 16, E2
group, n ¼ 19 total) that had dysplastic changes in the analyzed folia.
Conclusions
Guinea pigs represent a unique animal model with regard to pre-
natal vitamin C deprivation and the consequential disturbance in
collagen synthesis. The results of this study suggest that a vita-
min C deficiency in the fetal guinea pig influences PBM rupture
and the sequential development of dysplastic changes in the
cerebellar cortex. The fact that neither humans nor guinea pigs
are able to synthesize vitamin C creates an opportunity for
further research of the impact of prenatal deprivation of vita-
min C in the development of lissencephaly type II in humans.
Author Contribution
Conception or design: IC, NH, DL, NV, NS, JM, SS. Data acquisition,
analysis, or interpretation: IC, NH, DL, NV, NS, JM, SS. Drafting the
manuscript: IC, NH, DL, NV, NS, JM, SS. All authors participated in
critically revising the manuscript, gave final approval, and agree to be
accountable for all aspects of work to ensure integrity and accuracy.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: Funding
this study was supported by the Serbian Ministry of Science and
Environmental Protection, Grant Number 175006/2011.
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