Decreased Expression of CIRP Induced by Therapeutic Hypothermia Correlates with Reduced Early Brain Injury after Subarachnoid Hemorrhage

Abstract

Early brain injury is considered to be a primary reason for the poor prognosis of patients suffering from subarachnoid hemorrhage (SAH). Due to its pro-inflammatory activity, cold-inducible RNA-binding protein (CIRP) has been implicated in the ischemic brain insult, but its possible interplay with hypothermia in SAH treatment remains to be evaluated. One-hundred and thirty-eight Sprague-Dawley rats (300-350 g males) were randomly allocated into the following groups: sham-operated (Sham); SAH; and SAH + hypothermia (SAH + H), each comprised of 46 animals. After treatments, the brain tissues of the three groups were randomly collected after 12 h, 1 d, 3 d, and 7 d, and the expression levels of the CIRP and mitochondrial apoptosis pathway-related proteins Bax, Bcl-2, caspase-9, caspase-3, and cytochrome c measured using Western blotting and real-time PCR. Brain damage was assessed by TUNEL and Nissl staining, the electron microscopy of brain tissue slices as well as functional rotarod tests. Expression of CIRP, Bax, caspase-9, caspase-3, and cytochrome c as well as reduced motor function incidence were higher in the SAH group, particularly during the first 3 d after SAH induction. Hypothermia blunted these SAH responses and apoptosis, thereby indicating reduced inflammatory signaling and less brain cell injury in the early period after SAH. Hypothermia treatment was found to effectively protect the brain tissue from early SAH injury in a rat model and its further evaluation as a therapeutic modality in SAH patients requires further study.

Keywords: apoptosis; cold-inducible RNA-binding protein (CIRP); early brain injury; hypothermia; subarachnoid hemorrhage (SAH).

Figure 1

The schematic illustration of the experimental workflow.

Figure 2

The rotarod and TUNEL staining. Rotarod test results (A) and TUNEL staining images (B) of the temporal cortexes for the indicated treatment groups (×400) 1 d after SAH induction. Panel (C) represents the apoptosis rates derived from the TUNEL assays. * p < 0.05 SAH vs. Sham, # p < 0.05 SAH vs. SAH + H.

Figure 3

The Nissl staining of the temporal cortex paraffin sections 3 d after SAH. (A) Nissl staining images; (B) The numbers of surviving neurons as determined by microscopy (×400) and cell counting. * p < 0.05 SAH vs. Sham, # p < 0.05 SAH vs. SAH + H.

Figure 4

The distribution of CIRP in the rat brain tissue 1 d after SAH induction as determined by immunofluorescence staining. (A) Co-staining with NeuN; (B) Co-staining with GFAP. Micrographs in the top panels at a magnification of ×400. Bottom panels show the whole cerebral coronal sections.

Figure 5

The analysis of CIRP mRNA and protein expression and mitochondrial apoptosis pathway-related proteins from 12 h to 7 d after SAH. (A,B) Immunohistochemical results of rat brain tissue (×400) 1 d after SAH. (C–E) Expression levels of CIRP mRNA and protein at four time points. (C) Analysis of CIRP mRNA at four time points by RT-PCR. (D,E) Western blot analysis of the CIRP protein expression at four time points. (F–K) Expression of the mitochondrial apoptosis pathway-related proteins including Bax, Bcl-2, caspase-9, caspase-3, and cytochrome c. * p < 0.05 SAH vs. Sham; # p < 0.05 SAH+H vs. SAH.

Figure 6

The transmission electron micrographs of neuronal mitochondria 1 d after SAH. Yellow arrows show normal mitochondrial morphology; red arrows point to abnormal mitochondrial morphology.