Research ReportMild hypothermia enhanced the protective effect of protein therapy with transductive anti-death FNK protein using a rat focal transient cerebral ischemia model

We previously reported that the protein transduction domain fused FNK (PTD-FNK) protein, which was derived from anti-apoptotic Bcl-xL protein and thereby gained higher anti-cell death activity, has a strong neuroprotective effect on rat focal brain ischemia models. The aim of this study was to investigate the effect of PTD-FNK protein and hypothermia combined therapy on cerebral infarction. Male SD rats were subjected to 120 min middle cerebral artery occlusion (MCAO) with intraluminal thread. Rats were divided into 4 groups: 1) 37 °C vehicle administration (37V); 2) 37 °C PTD-FNK administration (37F); 3) 35 °C vehicle administration (35V); and 4) 35 °C PTD-FNK administration (35F). PTD-FNK protein was intravenously administered 60 min after the induction of MCAO. Hypothermia (35 °C) was applied during 120 min MCAO. Rats were sacrificed 24 h later; infarct volumes were measured, and Bax, Bcl-2, TUNEL and caspase-12 immunostaining was evaluated. There was significant infarct volume reduction in 37F, 35V, and 35F groups compared to 37V. There was also a significant difference between 37F and 35F. This suggests that hypothermia enhanced the effect of PTD-FNK. Similar results were found in neurological symptoms. Caspase-12 and TUNEL staining showed a significant difference between 37F and 35F; however, Bax and Bcl-2 staining failed to show a difference. In this study we showed an additive protective effect of hypothermia on PTD-FNK treatment, and immunohistological results showed that the protective mechanisms might involve the inhibition of apoptotic pathways through caspase-12, but not through Bcl-2.

► Combination therapy of protein transduction therapy and hypothermia of 35 °C. ► Total infarct volume was additively decreased by combination therapy. ► Bcl-2 immunostaining was not affected by combination therapy. ► Bax, TUNEL, and caspase-12 were significantly decreased by combination therapy. ► Combination therapy may inhibit apoptotic pathway through caspase-12.