Hypothermia and valproic acid activate prosurvival pathways after hemorrhage

Abstract

Background: Therapeutic hypothermia (hypo) and valproic acid (VPA, a histone deacetylase inhibitor) have independently been shown to be protective in models of trauma and hemorrhagic shock but require logistically challenging doses to be effective. Theoretically, combined treatment may further enhance effectiveness, allowing us to use lower doses of each modality. The aim of this study was to determine whether a combination of mild hypo and VPA treatments would offer better cytoprotection compared with that of individual treatments in a hemorrhage model.

Materials and methods: Male Sprague-Dawley rats were subjected to 40% volume-controlled hemorrhage, kept in shock for 30 min, and assigned to one of the following treatment groups: normothermia (36°C-37°C), hypo (30 ± 2°C), normothermia + VPA (300 mg/kg), and hypo + VPA (n = 5 per group). After 3 h of observation, the animals were sacrificed, liver tissue was harvested and subjected to whole cell lysis, and levels of key proteins in the prosurvival Akt pathway were measured using Western blot.

Results: Activation of the proapoptotic protein cleaved caspase-3 was significantly lower in the combined treatment group relative to normothermia (P < 0.05). Levels of the prosurvival Bcl-2 was significantly higher in the combined treatment group relative to sham, normothermia, and normothermia + VPA groups (P < 0.005). The downstream prosurvival protein phospho-GSK-3β was significantly higher in the sham, hypo, and combined treatment groups compared with that in normothermia groups with or without VPA (P < 0.05). Levels of the prosurvival β-catenin were significantly higher in the combined treatment group relative to normothermia (P < 0.01).

Conclusions: This is the first in vivo study to demonstrate that combined treatment with VPA and hypo offers better cytoprotection than these treatments given independently.

Keywords: Apoptosis; Hemorrhagic shock; Hypothermia; Resuscitation.

Figure 1

Schematic diagram of the phosphoinositol 3-kinase (PI3K)/Akt survival pathway. Adapted from Shuja et al. (6).

Figure 2

Mean Arterial Pressure at selected time points. Data are presented as mean ± standard error of the mean. *P < 0.05 between NH, HH, and HH+VPA at the post-hemorrhage (PH) time point. BL, baseline; PS, post-shock; PS1, one hour post-shock; PS2, two hours post-shock; End, time of sacrifice at three hours post-shock.

Figure 3

Western blot of acetylated histone 3 at lysine 9 (ACH3K9) in liver tissue following hemorrhagic shock and resuscitation treatments. Protein band density was quantified as mean ± standard error of the mean. *P < 0.05 compared to Sham, NH, and HH. Representative protein bands from western blots are shown below the densiometric data.

Figure 4

Western blots of proteins in liver tissue following hemorrhagic shock and resuscitation treatments. (A) Cleaved-caspase-3 and pro-caspase-3, with *P < 0.05 compared to NH. (B) Bcl-2, with *P < 0.005 compared to Sham, NH, and NH+VPA. Protein band density was quantified as mean ± standard error of the mean. Representative protein bands from western blots are shown below the densiometric data.

Figure 5

Western blot of phospho-GSK-3β in liver tissue following hemorrhagic shock and resuscitation treatments. Protein band density was quantified as mean ± standard error of the mean. *P < 0.05 compared to NH and NH+VPA. Representative protein bands from western blots are shown below the densiometric data.

Figure 6

Western blot of pro-survival β-catenin in liver tissue following hemorrhagic shock and resuscitation treatments. Protein band density was quantified as mean ± standard error of the mean. *P < 0.01 compared to NH. Representative protein bands from western blots are shown below the densiometric data.