Advances in Bioscience and Biotechnology, 2012, 3, 695-704 ABB
http://dx.doi.org/10.4236/abb.2012.326090 Published Online October 2012 (http://www.SciRP.org/journal/abb/)
Nitric oxide production is associated to increased
lipoperoxidation and active caspase-3 in demyelinated
brain regions of the taiep rat
Guadalupe Soto-Rodríguez1, Daniel Martínez-Fong2, Rosa Arroyo3, Patricia Aguilar-Alonso1,
Hector Rubio4, José Ramón Eguibar3, Araceli Ugarte3, Maricela Torres-Soto1,
Juan Antonio González-Barrios5, Jorge Cebada6*, Eduardo Brambila1, Bertha Alicia Leon-Chavez1
1Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, San Claudio, México
2Departamento de Fisiología, Biofísica y Neurociencias, Cinvestav, México D.F., México
3Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, San Claudio, México
4Facultad de Medicina, Universidad Autónoma de Yucatán, Mérida, Mexico
5División de Medicina Genómica, Hospital Regional 1˚ de Octubre, ISSSTE, Avenida Instituto Politécnico Nacional, México D. F.,
México
6Escuela de Biología, Benemérita Universidad Autónoma de Puebla, San Claudio, Mexico.
Received 15 August 2012; revised 20 September 2012; accepted 30 September 2012
ABSTRACT
We previously showed that the increase in nitric ox-
ide (NO) levels and NO synthase (NOS) expression
correlate with the progression of reactive astrocytosis
and demyelination in the brains of 6-month-old taiep
rats. Increased levels of NO can result in high con-
centration of peroxynitrite and thus cause tissue
damage, which consists of lipoperoxidation of the cy-
toplasmic membrane, such as the myelin, and of
apoptotic and necrotic cell-death. On this basis, we
studied whether the increased NO production is asso-
ciated with lipoperoxidation and cell death in the
cerebellum and brainstem over the age (1, 3, 6, and 8
months) of taiep rats. The results were compared
with those obtained in matched Sprague-Dawley (SD)
rats. We measured the levels of nitrites (NO produc-
tion), malonyldialdehyde, and 4-hydroxya lkena l (lipo-
peroxidation) in brain tissue homogenates. The three
NOS isoforms and cleaved caspase-3 were evaluated
by using ELISA and immunostaining techniques. Our
results showed that NO production and lipoperoxida-
tion increased in the cerebellum and brainstem as the
age of the taiep rats increased compared to SD rats.
The overexpression of nNOS and iNOS were in the
Purkinje cells, magnocellular neurons, and in oli-
godendrocytes, whereas the glial cells showed strong
cleaved-caspase-3 immunoreactivity. In summary our
results suggest that NO plays a role in the demyelina-
tion and cell death in the taiep rat.
Keywords: Hypomyelination; Nitrite;
Malonyldialdehyde; Apoptosis, Necrosis
1. INTRODUCTION
The taiep rat is a myelin mutant with a long survival time.
The taiep rat develops a progressive neurological syn-
drome characterized by tremor, ataxia, immobility epi-
sodes, audiogenic seizures, and hindlimb paralysis [1]. In
addition, taiep rats show hypomyelination at birth and
progressive demyelination that leads to a highly hypo-
myelinated central nervous system (CNS) in adult-hood
[2,3]. The deficit in myelin has been directly associated
with a cytoskeleton alteration of oligodendrocytes, which
show an accumulation of microtubules in their soma
[4,5], and with changes in expression and intracellular
location of myelin-gene products [6,7]. To date, the ge-
netic mutation causing the myelin defect in the taiep rat
remains unknown. It is believed to be of a multifactorial
type. In the taiep rat, contrary to the expected oligoden-
drocyte death, it has been found an apparent increase in
the number of oligodendrocytes at least in some regions
of the central nervous system such as funiculi of the spi-
nal cord, the optic nerve and the cerebellum [2]. To date,
there is no evidence of oligodendrocyte death in the taiep
rat.
Previous studies in the taiep rat have shown an in-
crease of nitric oxide (NO) levels and upregulation of the
mRNA of the nitric oxide synthases (NOS) in cultured
glial cells when stimulated by lipopolysaccharides or the
tumor growth factor (TNF) [8], and in homogenates from
*Corresponding author.

G. Soto-Rodríguez et al. / Advances in Bioscience and Biotechnology 3 (2012) 695-704 696
the cerebellum of 6-month-old rats [9]. The increased
levels of NO have been related to apoptosis [10] and
necrosis of neurons [11,12], caused by the production of
peroxynitrite [10,12-14]. Accordingly, the inhibition of
the production of NO by administration of N-nitro-L-
arginine methyl ester (L-NAME) and of superoxide by
the addition of superoxide dismutase decrease lipoper-
oxidation, apoptosis, and the infarct zone size in several
models of disease [11,14,15]. In contrast, the increase of
nitrate/nitrite levels in serum, lipoperoxidation products
(malonyldialdehyde plus 4-hydroxyalkenals), and glu-
tathione peroxidase activity have been associated with
remitting multiple sclerosis as shown in patients of a
Mexican population [16]. Both the neuronal (n)NOS and
the inducible (i)NOS have also been associated with the
loss of oligodendrocytes and demyelination [17-19]. It is
known that the iNOS is not significantly expressed by
resident cells unless cellular activation occurs. In this
case, the iNOS is produced by several types of cells,
such as macrophages, microglia, astrocytes [20,21], oli-
godendrocytes [22], and even cerebral endothelial cells
[23].
The oligodendrocyte cell death is mediated by cas-
pases in the pathogenesis of autoimmune demyelination
diseases such as multiple sclerosis (MS). The inhibition
of caspase-3 activation in oligodendrocytes decreased
apoptosis [24]. In addition, other caspases are involved
in the inflammatory and apoptotic processes in oli-
godendrocytes. Accordingly, the caspase-11 and caspase-
1 mRNA and protein levels are significantly elevated in
oligodendrocytes in both acute and chronic MS [24-26].
In support of this, the number of apoptotic oligodendro-
cytes and severity of experimental allergic encephalo-
myelitis (EAE) are reduced in caspase-1- or caspase-
11-deficient animals [25].
These antecedents clearly demonstrate the participa-
tion of NO through peroxynitrite in lipoperoxidation and
oligodendrocyte death by necrosis or apoptosis mecha-
nisms. On this basis, our work aims to demonstrate that
the increased NO production is associated with lipoper-
oxidation and cell death in the cerebellum and brainstem
of taiep rats. We analyzed the production of NO as as-
sessed by nitrite accumulation, the expression of the
three NOS isoforms by using an enzyme-linked immu-
nosorbent assay (ELISA), and the indirect immunofluo-
rescence in the brainstem and cerebellum at ages of 1, 3,
6, and 8 months. In addition, the levels of malondialde-
hyde-4-hydroxyalkenals and caspase-3 immunoreactivity
(IR) were determined and used as the markers of cell
death. Our results provided evidence of the possible role
of NO in the neuron death of necrotic type and demyeli-
nation in the taiep rat, a model of the hypomyelina-
tion-demyelination process.
2. MATERIALS AND METHODS
2.1. Experimental Animals
Taiep rats, ages from 1 to 8 months, were obtained from
the vivarium of the Institute of Physiology, BUAP and
aged-matched Sprague-Dawley rats were from the
CINVESTAV. Animals were maintained in adequate
animal rooms with controlled conditions of temperature
(22˚C ± 1˚C) and light-dark cycle (12 h:12 h light-dark;
light onset at 0700). Food and water were provided ad
libitum. All procedures were in accordance with the
Mexican current legislation, the NOM-062-ZOO-1999
(SAGARPA), based on the Guide for the Care and Use
of Laboratory Animals, NRC. The Institutional Animal
Care and Use Committee (IACUC) approved our ani-
mal-use procedures with the protocol number 410. All
efforts were made to minimize animal suffering.
2.2. Nitric Oxide Determination
The cerebellum and brainstem of control or taiep rats (n
= 5 in each group) were mechanically homogenized in
phosphate-buffered saline solution, pH 7.4 (PBS) and
centrifuged at 12,500 rpm for 30 min at 4˚C by using a
17TR microcentrifuge (Hanil Science Industrial Co, Ltd;
Inchun, Korea). The NO production was assessed by the
accumulation of nitrites (2) in supernatants of ho-
mogenates as described elsewhere [8,27]. Briefly, the
nitrite concentration in 100 μL of supernatant was meas-
ured by using a colorimetric reaction generated by the
addition of 100 μL of Griess reagent, which was com-
posed of equal volumes of 0.1% N-(1-naphthyl) ethyl-
enediamine dihydrochloride and 1.32% sulfanilamide in
60% acetic acid. The absorbance of the samples was de-
termined at 540 nm with a SmartSpec 3000 spectropho-
tometer (Bio-Rad, Hercules, CA, USA) and interpolated
by using a standard curve of NaNO2 (1 to 10 µM) to
calculate the nitrite content.
NO
2.3. Enzyme-Linked Immunosorbent Assay
ELISA was used to determine the three isoforms of NOS
in homogenates of the cerebellum and brainstem of con-
trol or taiep rats (n = 5 in each group) as we described
previously [8,28]. The protein content was determined
using the method described elsewhere [29]. Aliquots
containing 5 µg of total protein were placed into wells of
the ELISA plates for the separate determination of nNOS,
iNOS, and eNOS. Volumes of 100 µL of 0.1 M carbonate
buffer were added into each well and the plate was incu-
bated for 18 h at 4˚C. To block nonspecific binding sites,
200 µL of 0.5% bovine serum albumin, IgG free, was
added to each well at room temperature (RT). After a
30-min incubation, the wells were washed three times
with PBS-Tween 20 (0.1%). Mouse monoclonal anti-

G. Soto-Rodríguez et al. / Advances in Bioscience and Biotechnology 3 (2012) 695-704 697
bodies to nNOS, iNOS, or eNOS (1:200 dilution; Sigma-
Aldrich; St. Louis, MO, USA) were added into each well
and incubated for 2 h at RT. After three washings with
PBS, a horseradish-peroxidase-conjugated goat anti-
mouse IgG (1:1000 dilution; Dako A/S Denmark Dako
North America, Inc. Carpinteria, CA) was added and
incubated for 2 h at RT. The antibody-antigen complex
was revealed by adding 100 µL of ABTS containing
0.3% H2O2 into each well. After 15 min, the optical den-
sity (OD) was determined at 415 nm using a Benchmark
multiplate reader (Bio-Rad, Hercules, CA, USA) as de-
scribed previously [8].
2.4. Immunolabeling of NOS and Cleaved
Caspase-3
The cell location of NOS isoforms was analyzed by dou-
ble immunofluorescence to specific NOS and galacto-
cerebroside (oligodendrocyte) or glial fibrillary acidic
protein (GFAP; astroglial cells) in sagital brain slices of
control and taiep rats (n = 3 in each group). Rats were
deeply anesthetized with chloral hydrate and perfused
through the ascending aorta with 100 mL of PBS, fol-
lowed by 150 mL of 4% paraformaldehyde in PBS. Their
brains were removed and maintained in the fixative for
48 h at 4˚C. After overnight maintenance in PBS con-
taining 10% sucrose at 4˚C, each brain was frozen and
sectioned into 35-µm slices on the sagittal plane using a
Leitz cryostat (LEICA 2000R). Slices were individually
collected in a 24-well plate containing PBS and used for
fluorescent immunolabeling of oligodendrocyte (galac-
tocerebroside), astroglial cells (GFAP), and NOS iso-
forms. Slices were incubated with 0.5% IgG-free bovine
serum albumin in PBS-Tween-20 (0.1%) for 20 min at
room temperature. The primary antibodies were mouse
monoclonal antibodies to nNOS, iNOS, or eNOS (1:200
dilution; Sigma-Aldrich; St. Louis, MO, USA), a mouse
monoclonal antibody to galactocerebroside (Galc), and a
rabbit polyclonal antibody to GFAP. The secondary an-
tibodies were an IgG antimouse, made in goats, labeled
with fluorescein and an IgG antirabbit, made in goats,
labeled with rhodamine.
The slices were mounted on glass slides using Vec-
tashield (Vector Laboratories; Burlington, Ontario, Can-
ada) and analyzed on a confocal imaging system equipped
with a krypton-argon laser beam (Bio-Rad MRC-600,
Watford, UK) as we have described elsewhere [9]. The
fluorescence was detected at Ex-Em wavelengths of 488 -
522 nm (green channel) and 568 - 585 nm (red channel).
Ten to twenty consecutive optical sections at 1 µm in-
tervals were obtained in the z-series. The resulting im-
ages were projected on a bidimensional plane and were
overlapped on the screen monitor using green for FITC
and red for Rho. The brain sections processed under
similar conditions in the absence of the primary antibody
were used as negative controls.
The cleaved caspase-3 immunoreactivity was analyzed
by an immunohistochemical method. The fixed brains
were maintained overnight in PBS containing 30% su-
crose at 4˚C. Then, each brain was frozen and sectioned
into 16 µm slices on the coronal plane using a Leica
SM100 cryostat (Leica Microsystems; Nussloch, Ger-
many). Slices were individually collected in a 24-well
plate containing PBS and used for immunohistochemis-
try for cleaved caspase-3. The slices were incubated with
PBS-Triton (0.1%) and later with 10% horse serum in
PBS-Triton (0.1%) for 60 min at room temperature. The
slices were incubated overnight with rabbit polyclonal
antibody against cleaved caspasa-3 (1:300 dilution, Cell
Signaling Technology, 3 Trask Lane, Danvers, MA
01923) and then with the secondary antibody bioti-
nylated anti-rabbit IgG (H + L), made in goats, (1:600
dilution; Vector Laboratories. Burlingame, CA, USA) for
2 hours at room temperature. After rinsing, the slices
were incubated with streptavadin-horseradish peroxidase
conjugate (BRL Inc., Gaithersburg, MD), diluted 1:400,
again for 30 minutes at room temperature. The peroxi-
dase reaction was developed by immersion in a freshly
prepared solution of 0.02% 3,3’-diaminobenzidine (DAB,
Sigma). The slices were counterstained with cresyl violet.
The caspase-3 immunoreactivity was analyzed with mag-
nification of 5×, 20×, and 40× using a Leica DMIRE2
microscope (Leica Microsystems; Wetzlar, Germany).
Images were digitalized with a Leica DC300F camera
(Leica Microsystems; Nussloch, Germany) and analyzed
with workstation Leica FW4000, version V1.2.1 (Leica
Microsystems Vertrieb GmbH; Bensheim, Germany).
The histophathology study of the cerebellum and
brainstem from the brains of each experimental group
was analyzed by hematoxylin-eosin staining in coronary
brain slices at 24-h postreperfusion (n = 3 in each group).
Paraffin-embedded tissue sections of 5 m were stained
with hematoxylin and eosin and examined at a magnify-
cation objective of 40× (Mod BM 1000, Jenopika Cam-
era, Wetzlar; Leica, Germany). Digital photomicrographs
were made from 5 randomly selected fields of each tissue
section of each experimental group at 24-h postreperfu-
sion (Progress capture pro 2.1, Leica).
2.5. Lipoperoxidation
Malonyldialdehyde (MDA) and 4-hydroxyalkenal (HA)
were measured in supernatants of homogenates of the
cerebellum and brainstem using the method described
elsewhere [11]. The colorimetric reaction in 200 μL of
supernatant was caused by the subsequent addition of
0.650 mL of 10.3 mM N-methyl-2phenyl-indole diluted
in a mixture of acetonitrile:methanol (3:1). The reaction

G. Soto-Rodríguez et al. / Advances in Bioscience and Biotechnology 3 (2012) 695-704 698
was started by the addition of 150 μL of methanesulfonic
acid. The reaction mixture was strongly vortexed and
incubated at 45˚C for 1 h and then centrifuged at 3000
rpm for 10 min. The absorbance in the supernatant was
read at 586 nm with a SmartSpec 3000 spectrophotome-
ter (Bio-Rad; Hercules, CA, USA). The absorbance val-
ues were compared to a standard curve in the concentra-
tion range of 0.5 to 5 μM of 1,1,3,3-tetramethoxypropane
(10 mM stock) to calculate the malondialdehyde and
4-hydroxyalkenal contents in the samples.
2.6. Statistical Analysis
All values are the mean ± SE obtained from at least 5
independent experiments. After testing for normality
with the Snedecor F-analysis. The significance of differ-
ences were analyzed by an unpaired Student’s t-test. The
data were analyzed using the Graph pad prism software
(version 5.00). The significance value was considered at
P < 0.05.
3. RESULTS
3.1. NO Levels
The production of NO was estimated by determining the
nitrite content in supernatants of cerebellum and brain-
stem homogenates from 1, 3, 6, and 8-month-old taiep
and control rats. In the cerebellum of taiep rats, the NO
level increased by 95% ± 2% at age 1 month and reached
the maximum increase (155% ± 46%) at 3 months.
Thereafter, the levels decreased but remained signifi-
cantly high as long as age 8 months when compared with
SD rats (Figure 1). In the brainstem, the production of
NO in taiep rats significantly increased 73% ± 19% at
age 3 months, 60% ± 23% at 6 months old, and 317% ±
80% at 8 months old when compared with control (SD)
rats (Figur e 1) (P < 0.05).
3.2. NOS Protein Levels
The enhanced production of NO was related to an in-
crease in protein levels of the three NOS isoforms in the
cerebellum and brainstem of the taiep rat as assessed by
ELISA. The increases were statistically significant when
compared with values of the control SD rats and they
were, in the cerebellum (Figure 2), 70% ± 4% for nNOS
at age 6 months, and 19% ± 2% at 3 months, and 107% ±
17% at 6 months for the iNOS. The values of the eNOS
protein in the cerebellum did not show statistical differ-
ences over age when compared with values of SD rats
(Figure 2). The percentage increases in the NOS protein
levels in the brainstem (Figure 2) were 264% ± 3% for
nNOS at age 6 months, 193% ± 13% at age 6 months for
iNOS, but there was a decrease to 55% ± 5% at 8 months.
Figure 1. Increased nitric oxide production in the cerebellum
and brainstem of the taiep rat. Nitrite levels were determined by
using the Griess method. Each value is the mean  SD of 5
independent experiments made in triplicate. SD, Sprague-
Dawley. *Significantly different from the control group, p <
0.05, Student’s t-test.
The increases in the eNOS levels in taiep rats were 32%
± 8% at age 3 months and 39% ± 4% at 6 months when
compared with SD rat values.
3.3. Cell Location of NOS Isoforms
Confocal microscopy and double immunofluorescence
were used to determine the location of the NOS isoform
immunoreactivity (IR) revealed by a secondary antibody
fluorescein-labeled (green channel) in neuronal cell types
and revealed by a secondary antibody rhodamine-labeled
(red channel). The nNOS-IR was seen in magnocellular
neurons and astrocytes in the brainstem (Figure 3) and in
Purkinje cells in the cerebellum, as previously demon-
strated by our group [9,28]. The iNOS-IR was located
mainly in the magnocellular neurons, in oligodendro-
cytes (Figure 3), and in Purkinje cells, as previously
demonstrated by our group [9], whereas the eNOS-IR
was present in oligodendrocytes, astrocytes, and neurons
(Figure 3). The IR intensity in the taiep rat was qualita-
tively higher than that in SD rats.
3.4. Lipoperoxidation Levels
The lipoperoxidation assayed by MDA + 4-HAD levels

G. Soto-Rodríguez et al. / Advances in Bioscience and Biotechnology 3 (2012) 695-704 699
Figure 2. Levels of NOS isoform proteins in the cerebellum and brainstem of the taiep rat.
ELISA was used to determine the protein levels of nNOS, iNOS, and eNOS in separate as-
says. Each value is the mean  SD of 5 independent experiments made in triplicate. SD,
Sprague-Dawley. *Significantly different from the control group, P < 0.05, Student’s t-test.
area of white matter of the cerebellum appears. The cell
morphology of the three layers of cerebellar cortex looks
normal from ages 1 month to 8 months. The cleaved
caspase-3 IR appears in some cells with morphology
similar to glial cells around the Purkinje cells (arrows). It
is interesting that the caspase-3 IR disappeared in the
cerebellum of SD rats with ages from 3 months to 8
months.
was significantly increased in the cerebellum and brain-
stem of the taiep rat as compared to control rats (Figure
4). In the cerebellum, the lipoperoxidation level in-
creased from 142% ± 20% at age 1 month to 325% ±
41% at 3 months, and remained high up to age 8 months.
In the brainstem of taiep rats, the increases of lipoper-
oxidation levels were 110% ± 20% at 3 months, 33% ±
5% at 6 months, and 196% ± 42% at age 8 months. In the taiep rat, the morphology of cerebellar cortex is
diffusely defined. The Purkinje layer is not morphologi-
cally evident with Nissl staining at the age of one month.
The immunoreactivity against cleaved caspase-3 signifi-
cantly increased with age in the granular layer and Pur-
kinje layer. Some immunoreactive cells are also seen in
the molecular layer, exhibiting morphological character-
istics of glial cells at age 1 month. In older ages, some
neurons were caspase-3 immunoreactive. The Purkinje
cells (arrowheads) were positive to caspase-3 suggesting
3.5. Cleaved Caspase-3 Immunoreactivity
The cleaved caspase-3 IR in the cerebellum and brain
stem significantly increased with age in the taiep rats as
compared to that in matched control regions (Figure 5).
The micrograph of the cerebellum from the one-month-
old SD rat shows from top to bottom the molecular layer,
the Purkinje layer (arrowhead), and the granular layer
with the highest cell density. At the bottom right, a small

G. Soto-Rodríguez et al. / Advances in Bioscience and Biotechnology 3 (2012) 695-704 700
Figure 3. Merged confocal micrographs showing the cell location of the immunoreac-
tivity of nNOS, iNOS, and eNOS in the brainstem of SD and taiep rats at age 3 months.
The immunoreactivity (IR) for nNOS (green; arrowheads) was seen mainly on the
magnocellular neurons and less in astrocytes (red; arrows) of control and taiep rats.
The iNOS-IR (green; arrowheads) was in neurons and oligodendrocytes (red, arrows).
The eNOS-IR (green; arrowheads) was in oligodendrocytes (red; arrows) and neu-
rons (arrowheads). These micrographs are representative of three experiments. GFAP
(glial fibrillary acidic protein); Galc (galactocerebroside); SD (Sprague-Dawley).
Figure 4. Levels of lipoperoxidation in the cerebellum and
brainstem. The malonyldialdehyde (MDA) and 4-hydroxyal-
kenal (4-HAD) concentrations measured by using the Gerard-
Monnier’s method were used as biomarkers of lipoperoxidation.
Each value is the mean  SD of 5 independent experiments
made in triplicate. SD, Sprague-Dawley. *Significantly dif-
ferent from the control group, P < 0.05, Student’s t-test.
that the taiep damage has reached these cells at age 8
months. The density of neuropil in the molecular layer
was also reduced in 6-month-old taiep rats when com-
pared with that of a matched SD rat.
In the brainstem of SD rats, the magnocellular neurons
(arrowhead) showed normal morphological characteris-
tics from one month to eight months (Figure 5). Those
neurons have well-defined neuritic projections, promi-
nent nucleoli, and Nissl bodies. Around some glial cells,
scarce and weak immunoreactivity against caspase-3 is
seen from age one to 8 months. In the brainstem of the
taiep rat, the number of neurons (arrowhead) was less
than that of the matched control at one month, and their
nuclei and nucleoli were well-defined. The number of
neurons, their projections, and their intensity of the Nissl
staining significantly decreased in older ages (6 and 8
months) of the taiep rats. The immunoreactivity against
cleaved caspase-3 significantly increased over age in
comparison with the control rats. There were abundant
glial cells around neurons with high immunoreactivity to
cleaved caspase-3 (arrows).
The slices of the cerebellum and brain stem of the SD
and taiep rats age 8 months were stained with hematoxy-
lin-eosin to gain further insight into the brain tissue
damage caused by the taiep pathology. The hematoxylin-
eosin staining revealed some abnormalities. First, there
was a loss of normal morphology in the Purkinje neurons
of cerebellum and magnocellular neurons of brainstem in
8-month-old taiep rats (Figure 6, arrowhead). Second,

G. Soto-Rodríguez et al. / Advances in Bioscience and Biotechnology 3 (2012) 695-704 701
Cerebellum
SD Brainstem taiep SD taie
1M
3M
6M
8M
1M
3M
6M
8M
p
Figure 5. Immunohistochemistry against caspase-3 and Nissl counterstaining in slides of the cerebellum and
brainstem at four different ages. The composite figures comparatively show micrographs of the cerebellum
and brainstem from taiep and SD rats. The labels at the left side of the micrographs are ages of 1, 3, 6, and 8
months. The immunostaining against cleaved caspase-3 is shown by the dark marks (arrows) and the Nissl
stain appears in blue (arrowheads).
Figure 6. Hematoxylin-Eosin staining in the cerebellum and
brainstem slides of taiep and control rats. The micrographs are
representative of three experiments. Thin arrows are neurons
and thick arrows are picnotic glial cells. CE, cerebellum; BS,
brainstem; SD, Sprague-Dawley. P: Purkinje layer; G: granular
layer and M: molecular layer and Mg: magnocellular neuron.
both Purkinje neurons and the magnocellular neurons of
the taiep rat displayed a pale and diffuse staining, sug-
gesting necrosis. Third, there are empty vacuoles or
vacuoles with shrunken cells in the brain regions studied
of the taiep rats. Finally, glial cells in the taiep rat are
more abundant and with a picnotic nucleus, suggesting
condensation of chromatin (Figure 6, arrows).
4. DISCUSSION
Our results strongly suggest that the increase in NO pro-
duction is associated with lipoperoxidation and apoptosis
in the cerebellum and brainstem, the most demyelinated
brain regions [2,3] of the taiep rat from ages 3 to 8
months. The iNOS and nNOS isoforms, which also in-
creased after 3 months, could be involved in the high
production of NO. iNOS and nNOS expressed in the
oligodendrocytes and neurons of old taiep rats might be
participating in the death of those cells as shown by pre-
vious reports [9,25,30,31]. Accordingly, our immunohis-
tochemistry against cleaved caspase-3 and histopathol-
ogy studies clearly showed the presence of apoptosis,
necrosis, and reactive astrogliosis in the taiep rats.
The NO role is dual; first the NO exerts a protective
function on neuronal [31-33] and glial cells and second it
becomes cytotoxic at high and sustained concentrations
[10-14]. Neurons in the Purkinje and granular layers of

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the cerebellum and of the magnocellular nuclei in the
brainstem of the control and taiep rats expressed iNOS
and nNOS. However, the large neurons, where iNOS and
nNOS were overexpressed, died by a necrosis mecha-
nism after age 6 months in the taiep rats, suggesting that
the NO production by iNOS and nNOS might exert a
protective effect on large neurons at ages younger than 6
months. In contrast, small neurons had immunoreactivity
against cleaved caspase-3 from age 3 months, suggesting
that these neurons die by apoptotic activation.
In support of the protective role of NO, other work has
shown that the inhibition of iNOS activity and iNOS
knockout increases the brain inflammation and oli-
godendrocyte death in EAE [33]. In addition, the in-
creased levels of iNOS in the cerebellum have been in-
volved in the protection against oxidative stress caused
by manganese intoxication [34]. Furthermore, the NO
production mediated by nNOS together with BDNF has
been associated with the maintenance of neurogenesis
during the postnatal life [35].
It has been described that the NO-citrulline cycle par-
ticipates in the differentiation of neural stem cells (NSC)
into neurons, astrocytes, and oligodendrocytes, augment-
ing the β-3-tubillin and GFAP [35]. Based on this, we
can suggest that NO participates in the astrogliosis
manifested by an increased GFAP immunoreactivity [28].
On the contrary, other authors have documented the cy-
totoxic role of NO in the cerebellum. Accordingly, the
nNOS and iNOS expressions were detected in the Pur-
kinje layer after portacaval anastomosis or oxygen and
glucose deprivation, thus suggesting that NO is involved
in the pathology of these injury types [36].
Previous studies had shown that NO is able to produce
lipoperoxidation [14] and mitochondrial injury in LPS-
stimulated oligodendrocytes [19,32]. In our work, we
found enhanced NO production, iNOS and nNOS ex-
pression in oligodendrocytes, and lipoperoxidation in the
cerebellum and brainstem of taiep rats. These results
suggest that NO causing lipoperoxidation and mitochon-
drial injury might affect the myelin sheath, thus becom-
ing one of the mechanisms responsible for the demyeli-
nation process of taiep animals. In support of this idea,
the appearance of lipoperoxidation (at age 3 months)
found in our work coincides with the appearance of de-
myelination in brain regions such as the spinal cord and
diencephalon of the taiep rat [37].
In our work, we showed active caspase-3 IR in the
cerebellum and brainstem of the SD rats and a higher IR
intensity in those regions of the taiep rats at age 1 month.
These results suggest that the caspase-3 expression at
younger ages is involved, at least in part, in the physio-
logical process during the postnatal ontogeny of the brain.
Accordingly, some reports have found that the content of
caspase-3 mRNA at postnatal day 40 in the cortex and
lower levels in the brainstem did not directly correlate
with variations in the number of brain cells [38]. In the
taiep rat, the caspase-3 immmunoreactivity was located
in cells of the Purkinje layer, but not within the Purkinje
cells. In this layer, the caspase-3 IR was located in small
cells (8-µm diameter) neighboring to the Purkinje cells,
suggesting that the Bergman cell bodies were inmunore-
active to cleaved caspase-3. It is possible that caspase-3
expressed in the Bergman cells might be involved in cell
differentiation during the postnatal development of the
cerebellum, as reported previously [31]. Some results
provide evidence for a nontraditional role of caspases in
cellular function that is independent of cell death and
suggest that caspase activation is important for the astro-
glial cytoskeleton remodeling following cell injury [30].
Recently, a study in vitro has been reported that the
presence of caspase-3 in the neonatal rat astrocytes of
two models of astrogliosis does not correlate with apop-
tosis [39]. Based on these reports, we can suggest that
the presence of caspase-3 in the cerebellum and brain
stem of the taiep might be associated with astrogliosis, a
key sign in taiep rats [28], rather than apoptosis.
In our work, the results with immunohistochemistry,
Nissl counterstaining, and hematoxylin-eosin suggest
that the Purkinje and magnocellular neurons in the old
taiep rat died mainly by necrosis, which might be caused
by lipoperoxidation. The increase in lipoperoxidation
strongly suggests an increase in reactive oxygen species
(ROS) and reactive nitrogen species (RNS), which might
cause the rupture of the external mitochondrial mem-
brane and consequently the activation of necrosis, as
reported by other authors [40-42]. The progressive ax-
onal degeneration in taiep rats, a chronic model with
nonimmune-mediated demyelination [43], might account
for the neuronal death in the cerebellum and brainstem of
old taiep rats.
In summary, our results showed that the NO produc-
tion that increases in an age-dependent manner in taiep
rats might have a dual role; a protective role involved in
the glial response to the hypomyelination-demyelination
process, and a cytotoxic role mainly associated with de-
myelination (lipoperoxidation) and necrosis at least in
the death of the Purkinje and magnocellular neurons.
However, there are other factors that should be studied to
gain insight into the taiep pathology, such as growth fac-
tor and cytokine activation that are important neuro-
chemical responses in the myelin mutant taiep rats.
5. ACKNOWLEDGEMENTS
This work was supported by J-34143 (BAL-C) and 106694 (JRE)
grants from CONACYT and VIEP-BUAP G/Nat/2012 (BAL-C) and
G/SAL/2012 (JRE). Guadalupe Soto-Rodriguez and Rosa Arroyo was
recipient of scholarships from CONACYT. We thank the Institute of

G. Soto-Rodríguez et al. / Advances in Bioscience and Biotechnology 3 (2012) 695-704 703
Physiology of BUAP and CINVESTAV for the production and mainte-
nance of both rats. Thanks to Dr. Ellis Glazier for the editing of the
English-language text.
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