Brain edema after intracerebral hemorrhage in rats: the role of iron overload and aquaporin 4

Laboratory investigation

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Object

Brain edema formation following intracerebral hemorrhage (ICH) appears to be partly related to erythrocyte lysis and hemoglobin release. An increase of brain water content was associated with an increase of brain iron, which is an erythrocyte degradation product. Expression of AQP4 is highly modified in several brain disorders, and it can play a key role in cerebral edema formation. However, the question whether AQP4 is regulated by drugs lacks reliable evidence, and the interacting roles of iron overload and AQP4 in brain edema after ICH are unknown. The goal of this study was to clarify the relationship between iron overload and AQP4 expression and to characterize the effects of the iron chelator deferoxamine (DFO) on delayed brain edema after experimental ICH.

Methods

A total of 144 Sprague-Dawley rats weighing between 250 and 300 g were used in this work. The animals were randomly divided into 4 groups. The ICH models (Group C) were generated by injecting 100 μl autologous blood stereotactically into the right caudate nucleus; surgical control rats (Group B) were generated in a similar fashion, by injecting 100 μl saline into the right caudate nucleus. Intervention models (Group D) were established by intraperitoneal injection of DFO into rats in the ICH group. Healthy rats (Group A) were used for normal control models. Brain water content, iron deposition, and AQP4 in perihematomal brain tissue were evaluated over the time course of the study (1, 3, 7, and 14 days) in each group.

Results

Iron deposition was found in the perihematomal zone as early as the 1st day after ICH, reaching a peak after 7 days and remaining at a high level thereafter for at least 14 days following ICH. Rat brain water content around the hematoma increased progressively over the time course, reached its peak at Day 3, and still was evident at Day 7 post-ICH. Immunohistochemical analysis showed that AQP4 was richly expressed over glial cell processes surrounding microvessels in the rat brain; there was upregulation of the AQP4 expression in perihematomal brain during the observation period, and it reached maximum at 3 to 7 days after ICH. The changes of brain water content were accompanied by an alteration of AQP4. The application of the iron chelator DFO significantly reduced iron overload, brain water content, and AQP4 level in the perihematomal area compared with the control group.

Conclusions

Iron overload and AQP4 may play a critical role in the formation of brain edema after ICH. In addition, AQP4 expression was affected by iron concentration. Importantly, treatment with DFO significantly reduced brain edema in rats and inhibited the AQP4 upregulation after ICH. Deferoxamine may be a potential therapeutic agent for treating ICH.

Abbreviations used in this paper: DAB = diaminobenzidine; DFO = deferoxamine; ICH = intracerebral hemorrhage; RBC = red blood cell; RT-PCR = reverse transcription polymerase chain reaction.

Article Information

Address correspondence to: Wang Gai Qing, M.D., Department of Neurology, Second Hospital of Shanxi Medical University, 382 WuYi Road, Tai Yuan, Shan Xi, China 030001. email: wanggq08@126.com.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    A: Photomicrograph of perihematomal tissue showing enhanced Perls reaction for an iron deposit in a rat in the surgical control group at Day 7 after operation. Perls staining, original magnification × 200. B: Photomicrograph of perihematomal tissue showing enhanced Perls reaction for an iron deposit in a rat in the ICH group at Day 7 after operation. Perls staining, original magnification × 200. C: Photomicrograph of perihematomal tissue showing enhanced Perls reaction for an iron deposit in a rat in the DFO intervention group at Day 7 after operation. Perls staining, original magnification × 200.

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    Graph showing results of calculations of the iron-positive area in Groups B (surgical controls, black squares), C (ICH models, black triangles), and D (DFO-treated ICH models, Xs). Asterisks indicate significance between values in ICH and DFO intervention rats at the same time (*p < 0.05, **p < 0.01); white triangles indicate significance between values in surgical controls and other groups at the same time (Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001); and black circles indicate significance between values determined at the later time and the earlier time in each group of rats (••p < 0.01), based on the Dunnett T3 test.

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    Graph showing the percentage of brain tissue water content in samples obtained in Groups A (normal controls, black diamonds), B (surgical controls, black squares), C (ICH models, black triangles) and D (DFO-treated ICH models, Xs). Data are presented as unadjusted means ± SD. Asterisks indicate significance between values in ICH and DFO intervention rats at the same time (*p < 0.05, **p < 0.01); white triangles indicate significance between values in surgical controls and other groups at the same time (Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001); and black circles indicate significance between values determined at the later time and the earlier time in each group of rats (p < 0.05) based on the Dunnett T3 test.

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    Graph showing results of immunohistochemical analysis in which AQP4-positive cells were counted in Groups A (normal controls, black diamonds), B (surgical controls, black squares), C (ICH models, black triangles), and D (DFO-treated ICH models, Xs). Data are presented as unadjusted means ± SD. Asterisks indicate significance between values in ICH and DFO intervention rats at the same time (**p < 0.01, ***p < 0.001); white triangles indicate significance between values in surgical controls and other groups at the same time (ΔΔp < 0.01, ΔΔΔp < 0.001); and black circles indicate significance between values determined at the later time and the earlier time in each group of rats (p < 0.05, ••p < 0.01, •••p < 0.001) based on the Dunnett T3 test.

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    Graph showing results of semiquantitative RT-PCR analysis used to assess the expression levels of AQP4 mRNA in Groups A (normal controls, black diamonds), B (surgical controls, black squares), C (ICH models, black triangles), and D (DFO-treated ICH models, Xs). Data are presented as unadjusted means ± SD. Asterisks indicate significance between values in ICH and DFO intervention rats at the same time (*p < 0.05); white triangles indicate significance between values on surgical controls and other groups at the same time (ΔΔp < 0.01, ΔΔΔp < 0.001); and black circles indicate significance between values determined at the later time and the earlier time in each group of rats (p < 0.05, ••p < 0.01) based on the Dunnett T3 test.

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