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Intl. J. of Molecular Zoology, 2012, Vol.2, No.1, 1
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3.2 Animals
Male Wistar neonatal rats originating from Valproate
-treated females were randomly selected from the two
experimental groups (ten pups from each group)
before any of the above treatment and were subjected
to blood tests to investigate autism (study 1). Thirty
newborn rats (of sixty five) were used as follows: the
1
st
group served as treated control and were assigned
randomly to be mother-fed after injection of interferon
– γ and administration of gliadin (10 pups). Ten pups
for the second group were collected from their
mothers immediately after birth to prevent suckling
of maternal milk. No foster nursing took place
because the major objective was to study pup
viability. The animals were weighed and then placed
in an infant incubator to control body temperature
and assigned to hand-fed of bovine colostrum every
3–4 h using a silicone rubber tube. This method is
time and labor demanding yet essential because
gastrostomy of newborn rats is associated with a
very high surgery related death rate. In addition, ten
normal dams fed normal littermates served as
normal controls in this study.
3.3 Postnatal growth and maturation development
On PND 0, pup weights were determined and
examined for malformations. Measurement of pup
weights was repeated on PND 7, 14, and 21 when the
offspring were weaned from their mothers for all
experimental groups.
3.4 Blood test
3.4.1 Study 1 (to investigate autism)
Blood samples ten pups/each experimental group that
will serve as group 1 and group 2 in study 2 were
pooled for the analyses before administration of
interferon - γ and gliadin. In addition, ten pups from
control females that were kept on normal standard diet
and injected with physiological saline were served as
control. The samples were taken from the eye ball
using capillary tubes and collected in EDTA tubes
containing aprotinine at 0
℃
, centrifuged at 1 600 g
for 15 minutes. Plasma was analyzed for the
following: 1-Epinephrine and norepinephrine Plasma
catecholamine concentrations were determined by
High Performance Liquid Chromatography HPLC as
previously described (Lang et al., 1989).
The blood samples were taken from the eye ball using
capillary tubes and collected in eppendorf tubes,
Serum was analysed after centrifugation for the
following: 2-Serotonin in plasma was measured by High
Performance Liquid Chromatography HPLC (Kluge et
al., 1999). 3-Calcium & 25-OH vitamin D were
measured by using Chemiluminescent Microparticles
Immuno- assay CMIA (Peterlik, 2009).
3.4.2 Interferon γ
Recombinant rat interferon-γ (PRP24; Serotec, Oxford,
United Kingdom) was used. Interferon-γ was
lyophilized (0.1 mg), reconstituted in 0.3 mL distilled
water, and stored until use in portions of 50 μL
(10 000 U) at
-
70
℃
. A dose of 1 000 U was used for
application per one newborn rat. After application of
interferon-γ the pups were fed with mother's milk or
bovine colostrum.
3.4.3 Gliadin administration
Gliadin (from wheat gluten, G-3375; Sigma, St. Louis,
MO, USA) was diluted in 0.02 mol/l acetic acid (pH
2.9) and administered intragastrically by means of a
silicon tube. Young rats were repeatedly given gliadin
in the following doses: day 0, 0.5 mg in one
intragastric dose; day 3, 3 mg in one intragastric dose.
The pups in each group were killed at 21 days of age.
They received a provocative dose of 30 mg gliadin per
animal 24 hours before the killing (on PND 20).
3.4.4 Blood test
Study 2 (to investigate Coeliac disease in autistic
pups). After application of interferon-γ in group 1
(Suckling pups + gliadin) and group 2 (Bovine
colostrum + gliadin). Serum tissue transglutaminase
tTG antibody titres were measured quantitatively by an
enzyme-linked immunosorbent assay (QuantaLite
tTG, Inova Diagnostics, Ca, USA).
All the previous chemical analysis for study 1 and
control group on PND 0 were repeated also on PND
21 in addition to serum tTG assay.
3.5 Gene expression from blood
About 250 µL of blood was collected from different
groups (study 1 on PND0 for
Met
gene expression,
study 2 on PND21 for
Dq2
and
Met
gene expression
as compared to control for each study) in PAXGene
Blood RNA tubes (Qiagen Inc., Valencia, CA, USA)
for downstream RNA isolation, Further RNA was
quantified using NanoDrop2000 and Real Time-PCR
analysis (Enitan et al., 2007) was carried out for
DQ2
and
Met
gene expression using GapDH as a
housekeeping gene.