Therapeutic hypothermia attenuates tissue damage and cytokine expression after traumatic brain injury by inhibiting necroptosis in the rat

Abstract

Necroptosis has been shown as an alternative form of cell death in many diseases, but the detailed mechanisms of the neuron loss after traumatic brain injury (TBI) in rodents remain unclear. To investigate whether necroptosis is induced after TBI and gets involved in the neuroprotecton of therapeutic hypothermia on the TBI, we observed the pathological and biochemical change of the necroptosis in the fluid percussion brain injury (FPI) model of the rats. We found that receptor-interacting protein (RIP) 1 and 3, and mixed lineage kinase domain-like protein (MLKL), the critical downstream mediators of necroptosis recently identified in vivo, as well as HMGB1 and the pro-inflammation cytokines TNF-α, IL-6 and IL-18, were increased at an early phase (6 h) in cortex after TBI. Posttraumatic hypothermia (33 °C) led to the decreases in the necroptosis regulators, inflammatory factors and brain tissue damage in rats compared with normothermia-treated TBI animals. Immunohistochemistry studies showed that posttraumatic hypothermia also decreased the necroptosis-associated proteins staining in injured cortex and hippocampal CA1. Therefore, we conclude that the RIP1/RIP3-MLKL-mediated necroptosis occurs after experimental TBI and therapeutic hypothermia may protect the injured central nervous system from tissue damage and the inflammatory responses by targeting the necroptosis signaling after TBI.

Figure 1. Temporal profile of RIP3 and MLKL mRNA and proteins expression after traumatic brain injury.

Rats were sacrificed at the indicated time points after TBI. Total RNA and proteins were prepared from the ipsilateral cortex and hippocampus. (A) Quantitative real-time PCR analysis for RIP3 and MLKL at 6, 24 and 72 hours in cortex in sham-operated animals (SHAM) and normothermic group (TBI) after TBI (n = 6 each); (B) Western blot analysis for RIP3 and MLKL in cortex and hippocampus at 6, 24 and 72 hours after injury (n = 6 each). Lane-loading differences were normalized by levels of β-actin; Data are expressed as mean ± SD and analyzed using unpaired t test vs. SHAM. *P < 0.05.

Figure 2. Hypothermia reduces necroptosis processing in the injured cortex after TBI.

Rats were sacrificed at 6 h after TBI. Total proteins were prepared from the ipsilateral cortex of TBI-Normothermia (NT) and TBI-Hypothermia (HT). (A) Immunoblot analysis of RIP1, RIP3 and MLKL in lysates of ipsilateral cortex after injury (n = 6 each). (B) Quantitative analysis of RIP1, RIP3 and MLKL after TBI. Lane-loading differences were normalized by levels of β-actin; Data are expressed as mean ± SD; Data of RIP1 are analyzed using ANOVA with Dunnett’s post-hoc test vs. SHAM. *P < 0.05.

Figure 3. Characterization of RIP1 and RIP3 expression in cortical neurons.

Rats were sacrificed at 6 h after TBI. The brains of SHAM, TBI-Normothermia (NT) and TBI-Hypothermia (HT) were obtained. (A) Representative photographs of immunostaining for RIP1 and RIP3 in the cortex are shown (white arrows indicate immunoreactivity positive neurons). Original magnification ×100, magnification ×400 for interest area, the scale bars represented 200 μm and 50 μm respectively. (B) Statistical analysis of RIP1⁺ and RIP3⁺ neurons in the respective group (n = 6); Data are expressed as mean ± SD. *p < 0.05; **P < 0.01.

Figure 4. Characterization of RIP1 and RIP3 expression in hippocampal CA1.

Rats were sacrificed at 6 h after TBI. The brains of SHAM, TBI-Normothermia (NT) and TBI-Hypothermia (HT) were obtained. (A) Representative photographs of immunostaining for RIP1 and RIP3 in the hippocampus CA1 are shown. Original magnification ×200, magnification ×400 for interest area, the scale bars represented 100 μm and 50 μm respectively. (B) Statistical analysis of RIP1⁺ and RIP3⁺ neurons in the respective group (n = 6); Data are expressed as mean ± SD. *p < 0.05; **P < 0.01.

Figure 5. Brain lesion volume in the cortex after TBI.

Rats were sacrificed at 6 h after TBI. The brains of SHAM, TBI-Normothermia (NT) and TBI-Hypothermia (HT) were obtained. (A–C) Representative images of HE staining in ipsilateral hemisphere are shown (n  = 2, 6, 6, respectively). All images were shown at bregma level −4.44 mm. Scale bars (A–C): 1600 μm. (D) Statistical analysis of contusion volume in the NT and HT groups. Data are expressed as mean ± SD. **P < 0.01.

Figure 6. The anti-inflammatory effects targeted necroptosis after TBI.

The treatment of hypothermia, DMSO, Nec-1, GSK′ 872 and NSA were performed at 30 minutes after FPI. Rats were sacrificed at 6 h after surgical operation. (A) Cortical expressions of HMGB1, TNF-α, IL-6 and IL-18 were assessed by Western blot analysis. β-actin was used as the loading. (B–E) Quantitative analysis of HMGB1, TNF-α, IL-6 and IL-18 expressions were performed (n = 6 each). Data are expressed as mean ± SD. *p < 0.05, **p < 0.