Traumatic injury activates MAP kinases in astrocytes: mechanisms of hypothermia and hyperthermia

Abstract

Hyperthermia is common following traumatic brain injury (TBI) and has been associated with poor neurologic outcome, and hypothermia has emerged as a potentially effective therapy for TBI, although its mechanism is still unclear. In this study we investigated the effects of temperature modulations on astrocyte survival following traumatic injury and the involved MAPK pathways. Trauma was produced by scratch injury of a monolayer of confluent astrocytes in culture, followed by incubation at hypothermia (308 degree C), normothermia (378 degree C), or hyperthermia (398 degree C). The activation of MAPK pathways including extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase ( JNK), and p38 MAPK were measured at 0, 15, 30, 60, and 120 min after traumatic injury followed by temperature modulation. Apoptosis of astrocytes was assessed by quantitation of cleaved caspase-3 expression 24 h after injury. Our findings showed that only JNK activation at 15 min after trauma was reduced by hypothermia, and this was associated with a marked reduction in apoptosis. Hyperthermia activated both ERK and JNK and increased apoptosis. The specific JNK inhibitor, SP60025, markedly reduced JNK-induced apoptosis at normothermia and hyperthermia, and showed a dose-dependent effect. In conclusion, the JNK pathway appears to mediate traumatic injury-induced apoptosis in astrocytes. Prolonged hyperthermia as a secondary insult worsens apoptosis by increasing JNK activation. Hypothermia protects against traumatic injury via early suppression on JNK activation and subsequent prevention of apoptosis. Manipulation of the JNK pathway in astrocytes may represent a therapeutic target for ameliorating the devastating progression of tissue injury and cell death after TBI.

FIG. 1.

(A) Representative Western blots show that neither hypothermia nor hyperthermia changed the phosphorylation level of ERK1/2 in uninjured astrocytes. (B) ERK1/2 is rapidly phosphorylated after traumatic injury in astrocytes, while total levels of ERK1/2 remain constant. (C) ERK1/2 phosphorylation was significantly higher post-injury than pre-injury at all temperatures from 15 min to 2 h post-injury. Hyperthermia increased ERK1/2 activation (*p < 0.05 versus normothermia), while hypothermia did not change the phosphorylation level of ERK1/2.

FIG. 2.

(A) Representative Western blots show that neither hypothermia nor hyperthermia changed the phosphorylation level of JNK in uninjured astrocytes. (B) JNK is rapidly phosphorylated after traumatic injury, while total levels of JNK remain constant. (C) JNK phosphorylation was significantly higher post-injury than pre-injury at all temperatures from 15 min to 2 h post-injury. Hypothermia decreased and hyperthermia increased JNK activation (*p < 0.05 versus normothermia).

FIG. 3.

(A) Representative Western blots show that cleaved caspase-3 was activated 24 h after traumatic injury under normothermia, and was increased by hyperthermia. No significant activation of cleaved caspase-3 after trauma was observed under hypothermia. The total levels of caspase-3 remained constant. (B) Western blots of cleaved caspase-3 were quantified from three independent culture experiments using standard densitometry technique (*p < 0.05 versus normothermia).

FIG. 4.

The effect of SP60025 on cleaved caspase-3 activation after trauma. (A) Representative Western blots show that phosphorylation levels of JNK after trauma decreased with increasing doses of SP60025, and that maximal inhibition of JNK activation was achieved by 50 μmol/L SP60025. Cleaved caspase-3 activation 24 h after traumatic injury under either normothermia or hyperthermia was also inhibited with increasing doses of SP60025, and they returned to baseline level at the dose of 50 μmol/L SP60025. (B) Western blots of cleaved caspase-3 were quantified from three independent culture experiments using standard densitometry technique (*p < 0.05 versus normothermia).

FIG. 5.

Micrographs of caspase-3/7 fluorescein stained cells taken 24 h after scratch injury in astrocytes. (A) At 24 h after scratch injury (the dashed line indicates the edge of the scratch injury, marked by the white arrow), the astrocytes began to extend their processes (indicated by the black arrow). (B) The grey arrows indicate the positively-stained cells. Caspase-3/7 expression under hypothermia (30°C) was significantly lower than that seen at 37°C (C) and 39°C (E). Also, 50 μmol/L of JNK inhibitor (SP60025) dramatically reduced caspase-3/7 expression at 37°C (D) and 39°C (F) after trauma (magnification × 200; bar = 50 μm).