Cardiovascular Journal of Africa: Vol 22 No 6 (November/December 2011) - page 24

CARDIOVASCULAR JOURNAL OF AFRICA • Vol 22, No 6, November/December 2011 314 AFRICA
artery and the bifurcation of the internal and external carotid
arteries were exposed. A V-shaped incision was made on the
external carotid artery followed by insertion of a 2F thrombotic
balloon catheter (Edward Life Sciences, USA) deeply into the
common carotid artery. The balloon was dilated by infusing
~ 0.10–0.15 ml of normal saline. The catheter was subsequently
drawn back to cause damage to the intima. Then normal saline
was withdrawn and the catheter was again pushed into the
common carotid artery. The procedure was performed twice in
order to completely remove the intima. Finally, the incision was
sutured and the rats were given free access to food and water.
The rats in group A were intraperitoneally injected with 200
ng/kg/d AMD3100 (octahydrochloride, Sigma, USA) immedi-
ately before surgery for five consecutive days. The rats in group
C were sacrificed two weeks later and those in the other groups
were killed at the designated time. The left common carotid
arteries were removed and rinsed with normal saline. Part of the
artery was stored at –80°C for detection of mRNA or protein
expression (n = 6), and the remainder was fixed for immuno-
histochemistry (n = 6).
Flow cytometric analysis
The peripheral blood (300 μl) was incubated with FITC-
conjugated anti-mouse CD34 (eBioscience, USA) and phyco-
erythrin-conjugated anti-human CXCR4 (eBioscience, USA)
monoclonal antibodies for 30 min at 4°C (n = 12 per group).
The cells were double-labelled with CD34 and CXCR4. The red
blood cells and platelets were subsequently lysed in erythrocyte
lysis buffer for 15 min, followed by centrifugation and washing.
The cells were then re-suspended in phosphate-buffered saline
(PBS) and analysed on an FACS Caliber flow cytometer (BD
FACSCalibur, America).6 Isotype-matched FITC-conjugated and
phycoerythrin-conjugated antibodies (eBioscience, USA) were
used as controls. The number of CD34+CXCR4+ cells was
presented as the absolute number in a total of 50 000 leukocytes.
Enzyme-linked immunosorbent assay of plasma
SDF-1a
The plasma level of SDF-1 a was determined by the enzyme-
linked immunosorbent assay (ELISA) using an ELISA kit (R&D
system, USA) according to manufacturer’s instructions.
Real-time polymerase chain reaction analysis of
SDF-1a and CXCR4
Total RNA was extracted from the injured arteries. For synthe-
sis of cDNA, 1 μg of total RNA was reverse-transcribed with
Promega RT system. Then the transcribed cDNA was amplified
by polymerase chain reaction (PCR) (T3000 PCR instrument,
Biometra, Germany) with specific primers as follows:
SDF-1a forward: 5′- CCAATCAGAAATGGGAACAAGA-3′ ,
reverse: 5′- GTAGGAGGCTTACAGCACGAA-3′ (381 bp);
CXCR4 forward: 5′ - GTGGGCAATGGGTTGGTAAT-3′ ,
reverse: 5′ - GGTGGCGTGGACAATGGCAAGGTAG-3′ (267 bp).
The primers were synthesised by Shanghai Sangon Biological
Engineering Technology & Services Co, Ltd (Shanghai, China).
Reactions involved 10 min at 95°C, 40 cycles at 95°C for 15 sec,
and then 60°C for one min. The products of PCR were detected
with 1.8% agarose electrophoresis and visualised under a gel
imaging and analysis system (Alpha FluorchemTM8900, USA).
Western blot analysis
The artery tissues were lysed in radio-immunoprecipitation assay
(RIPA) buffer (n = 6 per group). The protein concentration in the
supernatant was measured spectrophotometrically at 595 nm.
Forty mg of protein was loaded onto SDS polyacrylamide gel
for electrophoresis (Invitrogen, China) and transferred to PVDF
membranes (Millipore, USA).
The membrane was incubated with anti-CXCR4 antibody
(1:500; rabbit anti-mouse; eBioscience, USA) and anti-<beta>
actin antibody (1:1000; goat anti-mouse, Santa Cruz, USA) over-
night at 4°C. The membrane was then incubated with secondary
antibodies (donkey anti-rabbit antibody, 800DX 1:5000, eBiosci-
ence, USA; donkey anti-goat antibody, 700DX 1:2000 Sigma,
USA) for one hour, followed by detection with an infrared fluor-
escence imaging and analysing system (Odyssey v1.2) (FIAS,
Odyssay LI-COR USA).
Immuno-histochemistry
Sections were then treated with 1.5% peroxide to inactivate
peroxidase activity, followed by blocking with 3% bovine serum
albumin. These sections were subsequently incubated with anti-
CXCR4 antibody (1:250, eBioscience, USA) overnight at 4°C.
The sections were then incubated with biotin-conjugated second-
ary antibody (1:500; Sigma, USA) or immunoglobulin G (1:500;
Santa Cruz, USA).
The sections were stained with haematoxylin/eosin (H&E),
dehydrated and mounted. H&E staining was performed on other
sections from each group (n = 6) to observe the intimal change
after balloon injury. The thickness of the intima was determined
using the Image 45 pro analysis program.
Statistical analysis
Experiments were performed at least three times and data were
presented as the mean ± standard deviation (SD). Statistical anal-
ysis was performed with SPSS version 11.0 (SPSS Inc, Chicago,
IL, USA). The unpaired t-test was used for comparisons between
two groups and one-way ANOVA between multiple groups. A
value of p < 0.05 was considered statistically significant.
Results
As shown in Table 1, compared with group C, the number of
peripheral CD34+CXCR4+ cells in groups S and A was signifi-
cantly increased immediately after intimal injury (S0/A0 ) (p <
0.01), followed by a gradual decline to baseline seven days after
injury. In group A, the number of CD34+CXCR4 + cells was
increased within 24 hours after intraperitoneal administration
of AMD3100, followed by a rapid decline (p < 0.05), which
may have been related to stimulation of the bone marrow by
AMD3100.
As shown in Table 2, the plasma level of SDF-1a after intimal
injury was markedly increased and reached a maximum one day
after injury (p < 0.01), followed by a rapid decrease to baseline
on day seven. The administration of AMD3100 did not affect the
plasma level of SDF-1a.
Total RNA was extracted from the injured common carotid