3. Results

3.1. TLR4 mRNA Increased in Brain Tissue after ICH
TLR4 mRNA levels were detected by Rt-PCR. The low level of TLR4 mRNA was observed in normal rat brain tissue. In the saline control group, the TLR4 mRNA slightly increased, but was not significantly different from normal group. However, the TLR4 mRNA after ICH significantly increased starting from 6 hours after ICH (P < .05), peaked on the 3rd day after ICH, and then decreased on the 7th day after ICH (P < .05), but still maintained a higher level compared with saline control (P < .05, Figure 1).

3.2. Expression of TLR4 Protein Increased in Brain Tissue after ICH
To investigate the increased TLR4 at the protein level, the expression of TLR4 protein was measured in brain tissue by the method of Western Blots using specific antibodies for TLR4. Our results showed that a low level of TLR4 protein was detectable in the saline control group. The expression of TLR4 significantly increased starting from 6 hours after ICH (P < .05), peaked on the 3rd day after ICH, and then decreased but still maintained at a high level on the 7th day after ICH (P < .05, Figure 2). This observation of TLR4 protein was consistent with the temporal profile of the TLR4 mRNA after ICH.

3.3. Distribution of TLR4 in Brain Subjected to ICH
To explore the distribution of TLR4 protein, the TLR4 was detected by immunohistochemistry (IHC). Results showed that TLR4 immunoreactivity was not detectable in brain tissue from normal and sham control. However, immunoreactivity for TLR4 was consistently demonstrated in peri-hemorrhage areas, the hippocampus, cortex, thalamic nuclei, and some white matter tracts (Figure 3).

3.4. Activation of TLR4-Mediated NF-Kappa-B Signaling
Activation of TLR4 induces the phosphorylation of inhibitor of kappa-B (IκB), which dissociates IκB with NF-kappa-B (NFκB). Then, NFκB translocates into the nucleus and activates and regulates the transcription of genes related to inflammatory responses. To detectd the activation of TLR4-mediated NFκB signaling, the phosphorylation of inhibitor of kappa-B (p-IκB) and activity of NFκB were measured by methods of Western Blots and EMSA. Results showed that the p-IκB increased at 6 hours after ICH (P < .05), peaked on 3 days after ICH, and maintained a high level on 7 days after ICH (P < .05, Figure 5). As expected, the activity of NFκB was also increased in the same manner as that of the p-IκB (Figure 4).