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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)