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Propofol ameliorates calpain-induced collapsin response mediator protein-2 (CRMP2) proteolysis and affords
neuroprotection following traumatic brain injury in rats Yun Yu, M.D.; Ruquan Han, M.D., Ph.D.
Department of Anesthesiology, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China
BACKGROUND
Collapsin response mediator protein-2 (CRMP2), a cytosolic protein highly
expressed in the brain, plays an important role in axonal guidance,
specification, elongation and branching, neurotransmitter release, resistance
to glutamate toxicity and neuronal cell death. The proteolysis of CRMP2
mediated by calpain following traumatic brain injury (TBI) may be a possible
inhibiting factor for post-traumatic neurite regeneration. Propofol, a widely
used intravenous anesthetic, is similar to the natural antioxidant vitamin E
structurally and show potential protective effects against oxidative injury.
OBJECTIVES
We examined the hypothesis that lipid peroxidation (LP) involves in triggering
post-injury calpain-mediated CRMP2 proteolysis and whether propofol would
attenuate calpain-induced CRMP2 degradation and programmed cell death
after moderate TBI.
Figure 1 Immunofluorescence staining confirmed the attenuation of LP–derived 4-HNE by
propofol and U83836E at 24 h following TBI. Horizontal arrows indicate representative
neurofilament (NF; green). Round rings and arrowheads indicate representative 4-HNE
(red) expressed in cytoplasm and nucleus respectively. Vertical arrows and boxes
represent 4-HNE positive neurons and other cells respectively. ** P < 0.01 vs. sham group; ∆∆ P < 0.01 vs. TBI group. Bar=50 μm. (n=3)
CONCLUSIONS
Propofol postconditioning could alleviate calpain-mediated CRMP2 proteolysis
and provide neuroprotective effects following moderate TBI through
counteracting lipid peroxidation and reducing calpain activity.
METHODS A unilateral moderate controlled cortical impact (CCI) injury was induced in
adult male Sprague-Dawley rats.
Animals were equally divided into seven groups: sham group, TBI group,
three propofol-treated groups (propofol 1 h group, propofol 2 h group,
propofol 4 h group), U83836E (a potent LP inhibitor) group and fat
emulsion group. Propofol 12.5 mg/kg was i.v. injected within 5 min at 1 h, 2
h and 4 h after TBI in propofol 1 h group, propofol 2 h group and propofol 4
h group respectively, followed by propofol 40 mg/kg/h i.v. infusion for 2
hours. U83836E was freshly diluted in normal saline to 0.75 mg/ml.
Dilutions were made to deliver initial 2 mg/kg i.v. injection at 15 min after
TBI followed by 7 mg/kg i.p. injection. The dose and infusion rate of fat
emulsion were same as propofol 1 h group.
We examined ipsilateral cortex tissues harvested 24 hours post-TBI when
CRMP2 proteolysis is most significant. CRMP2 proteolysis and calpain
activity were determined by western blot analysis. Immunofluorescent
staining of 4-hydroxynonenal (4-HNE) was measured to evaluate lipid
peroxidation. Programmed cell death was detected by TUNEL staining for
assessment of brain injury.
There were not significant differences in hemodynamics and blood gases in experimental groups compared to sham group both before craniotomy and at the end of the experimental treatment. PaO2 decreased in propofol 1 h group at the end of propofol infusion and PaCO2 decreased in U83836E group at the end of U83836E i.p. injection (P < 0.05), within acceptable range. This observation suggested that propofol, U83836E and fat emulsion used in this study did not influence animals’ circulation and respiration significantly (Table 1).
Group Time points HR (bpm) MAP (mmHg) PaO2 (mmHg) PaCO2 (mmHg)
Sham Before 290 ± 5 80 ± 15 106 ± 27 46 ± 7
TBI Before 277 ± 16 77 ± 9 109 ± 11 42 ± 11
After 277 ± 10 78 ± 7 109 ± 11 41 ± 8
Prop 1h Before 286 ± 6 81 ± 8 113 ± 18 41 ± 9
After 274 ± 16 86 ± 12 102 ± 11** 40 ± 4
Prop 2h Before 273 ± 14 77 ± 8 102 ± 19 43 ± 10
After 265 ± 13 83 ± 7 110 ± 19 38 ± 8
Prop 4h Before 274 ± 26 80 ± 10 99 ± 12 46 ± 5
After 277 ± 16 85 ± 8 112 ± 12 39 ± 9
U83836E Before 276 ± 22 75 ± 4 104 ± 8 43 ± 7
After 277 ± 13 81 ± 8 113 ± 7 38 ± 5
FE Before 267 ± 27 75 ± 4 98 ± 9 42 ± 8
After 284 ± 13 82 ± 7 108 ± 15 38 ± 7**
RESULTS
Table 1. Data are presented as means ± SD. Statistical differences (one-way ANOVA and
paired t-test), **p < 0.05 vs. base values.
Figure 4 TUNEL staining demonstrated the reduction in programmed cell death after the
administration of propofol and U83836E following TBI. White arrows signify TUNEL
positive cells. ** P < 0.01 vs. sham group; ∆∆ P < 0.01 vs. TBI group. Bar=50 μm. (n=3)
Figure 3. Western blot analysis of αII-spectrin
in ipsilateral cortex at 24 h following TBI. (A)
Brain tissue lysates were immunoblotted with
anti-αII-spectrin (top) and anti-β-actin (bottom)
antibodies respectively. (B) The ratio of
calpain-cleaved 145-kDa spectrin breakdown
product to intact αII-spectrin. ** P < 0.01 vs.
sham group; ∆∆ P < 0.01 vs. TBI group. (n=6)
Figure 2. Western blot analysis of CRMP2
proteolysis in ipsilateral cortex at 24 h
following TBI. (A) Brain tissue lysates were
immunoblotted with anti-CRMP2 (top) and
anti-β-actin (bottom) antibodies. (B) The
ratio of breakdown product (55 kDa) to
intact CRMP2 (62 kDa). ** P < 0.01 vs.
sham group; ∆∆ P < 0.01 vs. TBI group. (n=6)