0262
EFFECTS OF UNILATERAL
TRANSPLANTATION OF GENETICALLY MODIFIED MYOBLASTS PRODUCING BRAIN-DERIVED
NEUROTROPHIC FACTOR INTO CORTICES OF NEONATAL RATS SUBJECTED TO
HYPOXIC-ISCHEMIC ENCEPHALOPATHY ON LOCOMOTION AND NEUROBEHAVIOR Hong XR1, Qu S2, Shi J3,
Ye LY1 and Chen XM1 1 Department of Pediatrics,
Fuzhou General Hospital, Fuzhou, China 2 Department of
Neurobiology, Second Military Medical University, Shanghai, China 3 Department of
Neurobiology, Shanghai Second Medical University, Shanghai, China Objective: Hypoxic-ischemic encephalopathy (HIE) in survivors of perinatal asphyxia is a
severe brain condition frequently encountered in clinic for which there is
currently no effective therapy except to the symptomatic treatment.
Brain-derived neurotrophic factor (BDNF), owing to its dramatic effect on
protection of responsive neurons against cell death in HIE in the
developing brain, has been regard as a putative candidate of agent in the
treatment for asphyxia in the perinatal period, although its mechanism
still remains to be exhaustively described. Because of difficulties hindering
the application of exogenous BDNF, e.g., short period of half-life in the
brain, inability to penetrate through the blood brain barrier and the deficiency
of source. We have developed a genetically engineered rat myoblast cell
line that is confirmed to express biologically active BDNF in vitro for the brain
transplantation. We also investigate locomotive and neurobehavioral changes
of the injured rats weeks after the procedure. Methods: Fifty-eight 7-day-old
pups from 6 litters weighing 13.3～17.8 g were randomized into sham-operated
group (C, 11 pups), HIE+BDNF transplantation group (B, 21 pups) and
HIE+mock-transplantation group (A, 26 pups). A rat myoblast cell line
expressing BDNF (BDNF（+）/L-6TG) was constructed
by the infection of its original myoblast cell (L-6TG) with a retroviral
vector PN2A carrying rat BDNFcDNA. Identification of BDNFmRNA
expression and BDNF bioactivity was performed by Northern blotting and
bioassay with PC12 cells. A unilateral stereotaxical
intracerebroparenchymal transplantation of either BDNF（+）/L-6TG (group B) or BDNF（-）/L-6TG (absence of BDNF,
group A) at 0.8μl of cell suspension (4×104/μl) into the left cortex
of the brain was carried out shortly after HIE undergone by ligation of
left common carotid artery followed by a 2.5 h inhalation of humidified 8%
O2+92% N2 at 37℃. The location of
microinjection in relation to lambda was 2.1 mm rostral, 1.5 mm lateral to
the left, and 1.5 mm deep to the skull surface. Locomotive and
neurobehavioral changes were
observed once every even- or odd-numbered days during 29 d～42 d after the graft by
both declinational critical angle test and open-field test including
crossing and rearing movements, respectively. A sham-operated group served
as basal control. Results: (1) It was demonstrated
that genetically modified myoblasts expressed and released BDNF in vitro for the fact that the
expression of BDNF was confirmed by Northern blotting and that culture
supernates were verified to promote the survival of PC12 cells and
outgrowth of their dendrites. (2) Mortality of the injured pups in 42 d
after HIE was significantly reduced in group B (6/21) vs group A (15/26, P＜0.05＝. (3) Declinational
critical angles were markedly increased in group B compared to group A at
all the time points observed, although they were still smaller than those
in group C. (4) Similar change patterns were seen in open field test of
both crossing and rearing movements.
Conclusion: Present data
suggest that myoblasts genetically modified express and secrete bioactive
BDNF in vitro. Being transplanted
into the cortex of HIE brain, they exert a protective function to reduce
locomotive and neurobehavioral abnormalities as well as the mortality rate
of HIE rats.