Interaction of aquaporin 4 and N-methyl-D-aspartate NMDA receptor 1 in traumatic brain injury of rats

Abstract

Objectives: methyl-D-aspartate NMDA receptor (NMDAR) and aquaporin 4 (AQP4) are involved in the molecular cascade of edema after traumatic brain injury (TBI) and are potential targets of studies in pharmacology and medicine. However, their association and interactions are still unknown.

Materials and methods: We established a rat TBI model in this study. The cellular distribution patterns of AQP4 after inhibition of NMDAR were determined by Western blotting and immunoreactive staining. Furthermore, the regulation of NMDA receptor 1 by AQP4 was studied by injection of a viral vector targeting AQP4 by RNAi into the rat brain before TBI.

Results: The results suggest that AQP4 protein expression increased significantly (P<0.05) after TBI and was down-regulated by the NMDAR inhibitor MK801. This decrease could be partly reversed using the NMDAR agonist NMDA. This indicated that AQP4 mRNA levels and protein expression are regulated by the NMDA signaling pathway. By injection of AQP4 RNAi viral vector into the brain of TBI rat models, we found that the mRNA and protein levels of NMDAR decreased significantly (P<0.05). This suggested that NMDAR is also regulated by AQP4.

Conclusion: These data suggested that the inhibition of AQP4 down-regulates NMDAR expression, which might be one of the mechanisms involved in edema after TBI.

Keywords: Aquaporin 4; Edema; N-methyl-D-aspartate NMDA receptor; NMDAR1; Traumatic brain injury.

Figure1

The injury animal model. A: The device for injury model; B: The contusion was made by free falling of 20 grams “T” shaped weight at 30 cm height; C: The guiding tube is 30 cm long. Its inner diameter is 15 mm and the outer diameter is 16 mm. A maximum of 2 or 4 mm depression of the brain surface was allowed; D: The demarcation of the brain was made by dividing the brain into ipsilateral or contralateral to the injury. The right side is the ipsilateral side of injury and was divided into three areas, pre-impact (area 1), impact (area 2), and post-impact (area 3). The injury contra-lateral side was divided into three areas also, the contra-pre-impact (area 4), contra- impact (area 5), and contra-post-impact (area 6)

Figure 2

The water contents in different groups. A: Traumatic brain edema by the footplate with 2 mm effective compress length; B: Traumatic brain edema of the brain injured by the footplate with 4 mm effective compress length

Figure 3

The protein expression of aquaporin 4 in different groups. The representative immunoblotting demonstrating AQP4 expression levels of the impact area in the 5 groups, sham, control, MK80, NMDA, and MK80+NMDA, at 24 hr time points after injury; B: Analysis of AQP4 protein expression. The intensity level for each band relative to GAPDH was determined, and the value of the sham group was assigned as 100%. Compared with the sham group, the control group showed a significant increase in AQP4 expression (140±28%, P<0.05). With the treatment of MK801 before and immediately after the injury, the AQP4 protein expression was strongly down-regulated to 68±12%. The effect of MK801 could be reverted to 119±16% by the administration of NMDA after MK801. Furthermore, we didn’t find significant changes in AQP4 expression in the injured area in the NMDA group, which received NMDA before the injury when compared with the sham group

Figure 4

Representative immunofluorescent staining of aquaporin 4 in the hippocampus at the impact area of the brain (scale bars, 250 μm)

Figure 5

Validation of the RNA interference virus of aquaporin 4. A: The decrease of AQP4 in protein expression was observed in vivo after the administration of AQP4 targeting RNAi virus, numbered as LV1, LV2, and LV3. The sequence in LV4 did not significantly lower AQP4 expression; B: Intensity values for each band relative to GAPDH were evaluated by semi-quantify AQP4 protein expression levels. Protein expression level of AQP4 in the control group which was infected by the virus containing the same backbone of RNAi virus but not AQP4 targeting sequence was assigned as “100%”. The four RNAi lentiviruses, LV1, LV2, LV3, and LV4, lowered the expression of AQP4 to 64.42±14.83%, 43.22±8.91%, 58.33±14.21%, and 102.13±27.57% of the control group. Except for LV4, the other 3 viruses all lowered AQP 4 significantly (P<0.05, n=5); C: The mRNA transcription of AQP4 in vivo after RNAi by lentiviruses LV1, LV2 and LV3 and LV4 were also decreased. The transcript level of AQP4 in the control group which was infected by the virus containing the same backbone of RNAi virus but not AQP4 targeting sequence was assigned as “100%”. Compared to the control group, the lentiviruses LV1, LV2, and LV3 significantly decreased the transcription of AQP4 to 43.29±11.10%, 48.02±13.11%, and 44.26±10.84% (P<0.05, n=5). The decreasing trend of LV4 on AQP4 transcription (91.11±17.46%) did not reach significance

Figure 6

The protein expression of N-methyl-D-aspartate NMDA receptor 1 in different groups. A: The protein expression and transcript abundance of NMDAR1 in the impact area of the rat brain after AQP4 RNA interference in vivo. The protein expression of NMDAR1 was reduced significantly by LV1, LV2, and LV3 RNAi virus injection (P<0.05, n=5); B: Relative intensity values for each band were analyzed. Protein expression level of NMDAR1 in the control group, which was infected with the virus containing the same backbone of RNAi virus but not AQP4 targeting sequence was assigned as “100%”. The three RNAi lentiviruses, LV1, LV2, and LV3, lowered the expression of AQP4 significantly in the control group (P<0.05, n=5); C: The mRNA transcript of NMDAR1 also decreased in the tissues treated by AQP4 knockdown induced by LV1, LV2, and LV3 (P<0.05, n=5)