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.

Intracerebral hemorrhage is a subtype of stroke with high morbidity and mortality rates, accounting for ∼ 15% of all deaths from stroke.33 If the patient survives the ictus, the resulting hematoma within brain parenchyma triggers a series of events leading to secondary insults and severe neurological deficits.18,29 A cascade of events triggered by erythrocyte lysis is critical for the delayed development of edema after ICH. Intracranial hemorrhage causes iron overload in the brain. Iron accumulation in brain tissue is toxic and may result in brain damage after ICH. Lysis of RBCs and iron overload contribute to delayed edema formation after ICH.6 Deferoxamine, an iron chelator, reduces ICH-induced and secondary brain damage, which suggests that iron plays an important part in brain injury after ICH.7,15,17,31 Several studies have shown that the degree of brain edema around the hematoma is associated with poor outcome in patients.31,34

Aquaporin 4, a major water channel protein that is expressed in the brain, plays a key role in the maintenance of brain water homeostasis. The role of iron accumulation in the pathogenesis of ICH is controversial. It has been proposed that AQP4 may play an important role in the formation of cerebral edema.1,25 Whether iron overload and AQP4 contribute to perihematomal brain edema formation, and the function of iron chelators after ICH, however, remain unclear. The purpose of our project was to investigate the mechanisms involved in iron overload– induced brain edema following ICH, and to study the temporal relationship among iron accumulation, brain water content, and AQP4 expression levels after ICH in rats. The goal of this study was to investigate the role of AQP4 expression in a rat model of ICH, and to study the correlation between AQP4 expression and iron toxicity and its chelator's possible effectiveness in treatment of brain edema after experimental ICH.

Methods

Animal Preparation

All protocols were approved by the Institutional Animal Care and Use Committee of Central-South University. A total of 144 Sprague-Dawley rats (half of them male and the other half female, provided by the Animal Experimental Center of Central-South University) and weighing between 250 and 300 g were used in this work. They were randomly divided into 4 groups: Group A were normal control rats (12 animals); Group B were surgical control rats that received an infusion of 100 μl saline into the right basal ganglia (48 animals); Group C were ICH rats that received an infusion of 100 μl autologous whole blood into the right basal ganglia (84 animals); and Group D were DFO intervention rats that were randomly chosen from Group C—each rat was administered a dose of 50 mg DFO by intraperitoneal injection twice daily at 24 hours after autologous whole-blood injection (36 animals). At 1, 3, 7, and 14 days, after neurobehavioral evaluations were done, each rat was killed by an overdose of chloral hydrate.

The experiment consisted of 3 parts. In Part 1, we examined brain iron deposition with Perls staining9 and AQP4 expression by using immunohistochemical studies. In Part 2, brain water content was determined by the dryweight/ wet-weight method. In Part 3, the levels of AQP4 mRNA were determined using RT-PCR (6 rats in each group per time point).

Animal Model

The rats were allowed free access to food and water before the procedure. The rats were anesthetized using chloral hydrate (300–350 mg/kg intraperitoneally) and were placed in a stereotactic head frame (model 51600, Stoelting Instruments). A 1-mm cranial bur hole was then drilled on the right coronal suture 2.3 mm lateral to the midline. Then, 100 μl autologous whole blood (derived from the rat's left ventricle) was infused into the right caudate (coordinates: 0.5 mm anterior, 5.8 mm ventral, and 2.3 mm lateral to the bregma) with the aid of a microinfusion pump (Stoelting Instruments). Surgical control rats received 100 μl saline only. The needle was removed and the skin incision was closed using sutures after the infusion. The DFO was dissolved in saline at a concentration of 100 mg/ml, then administered intraperitoneally (0.5 ml DFO solution 24 hours after ICH) every 12 hours for 3, 7, and 14 days.

Brain Water Content Measurement

After neurobehavioral evaluations were done, each rat was killed by an overdose of hydrated chloric aldehyde. At 1, 3, 7, and 14 days after the operation, rats were reanesthetized, killed by overdose, and decapitated. The brain was quickly removed and an ipsilateral basal ganglia sample from tissue surrounding the hematoma was taken. Samples were wrapped in preweighed aluminum foil and immediately weighed on an electronic analytical balance (model AE 240, Mettler) to obtain the wet weight, then dried for 72 hours in an oven at 100°C and weighed again to obtain the dry weight. The percentage of brain tissue water content was then calculated according to the following formula: (wet weight – dry weight)/ wet weight × 100%.

Iron Staining

In this study, Perls staining for ferric iron was performed. Rats were anesthetized and underwent intracardiac perfusion with 4% paraformaldehyde in 0.1 mol/L (pH 7.4) phosphate-buffered saline. The brains were removed and kept in 4% paraformaldehyde for 12 hours and then immersed in 25% sucrose for 3 to 4 days at 4°C. The brains were then placed in optimum cutting temperature embedding compound, and 25-μm-thick coronal sections were obtained on a cryostat (Sectioning Series D860, Technical Products International). After the sections were washed with distilled water and incubated in Perls solution (1:1, 5% potassium ferrocyanide and 5% hydrochloric acid) for 45 minutes, they were washed in distilled water 6 times for 5 minutes each. The sections treated with Perls stain were incubated in 1% DAB for 15 minutes, incubated again in 0.03% H2O2 and 0.5% DAB for 10 minutes, washed in distilled water for 10 minutes, and then restained with eosin for 5 minutes. We used 25-μm-thick coronal sections from the blood injection site. Two high-power images (magnification 200) were obtained using a digital camera; the 2 images were taken from the caudate just next to the cavity. Area measurements were performed in 2 areas in each of 6 rat brain sections. Areas of iron staining were assayed using HMIAS-2000 Image analysis software (Tongji Medical University, Wu Han, China).

Immunohistochemical Studies of AQP4

The coronal sections obtained around the hematoma as described above were used for histological examination. The sections were incubated overnight at 4°C with primary antibody (polyclonal rabbit anti–rat AQP4 IgG; diluted 1:300, Boshide Bioengineering Co., Ltd.). Normal rabbit IgG was used for a negative control. The immunohistochemical staining step was performed using an SABC kit (Boshide Bioengineering Co., Ltd.) according to the instructions of the manufacturer. The DAB staining method was used and all samples were restained with hematoxylin. Images were captured with a Sony digital camera; the images were taken from the caudate just next to the cavity. An observer counted the number of AQP4-positive cells in blinded fashion on a light-field photomicrograph obtained at a magnification of 200. Two areas were selected, the number of positively stained cells was determined in each section, and counts were determined from 3 sections per animal.

Determination of AQP4 Expression by Using RT-PCR

From the 100-mg frozen tissue sample obtained near the hematoma or similar site in each rat, total RNA was extracted with Trizol Reagent (TB126–100, Invitrogen) according to the manufacturer's protocol. The cDNAs were generated from 2 μg of total RNA by using an MuLV reverse transcriptase kit (K1622, Fermentas) primed with oligo(dT)18. The cDNAs were amplified with primers based on the sequences of rat AQP4 cDNA: rat AQP4 forward primer, 5′-GGTGGGAGGATTGGGAGTC-3′ and thereverse primer, 5′-CAGCGCCTATGATTGGTC-3′ (Gen-Bank accession no. NM_012825); the fragment size of AQP4 was 302 bp. For β-actin as an internal standard, the cDNAs were amplified with primers based on the sequences of rat β-actin cDNA. Forward primer for rat β-actin was 5′-AACCCTAAGGCCAACCGTGAAAAG-3,′ and reverse primer was 5′-TCATGAGGTAGTCTGTCAGGT-3′ (GenBank accession no. NM_031144); the fragment size of β-actin was 241 bp. The amplification mixture was subjected to 29 thermal cycles as follows: denaturation at 94°C for 30 seconds, annealing at 54.9°C for 30 seconds, and primer extension at 72°C for 45 seconds on a programmable thermal controller. Each RT-PCR product was demonstrated on a 1.5% agarose gel stained with ethidium bromide. A semiquantitative multiplex RT-PCR in the same tube was designed to compare the products of AQP4 gene with those of β-actin gene to determine the relative levels of expression of AQP4 in the rats. The bands of the positive film were scanned and the density of each band was measured using an image analysis system (Gis-1000 digital image gel analysis system, Tanon Science & Technology Co.) to calculate the ratio of the product of AQP4 mRNA to β-actin mRNA.

Statistical Analysis

All data are presented as the mean ± SD. Data obtained using immunohistochemical or RT-PCR analysis as well as water and iron content were analyzed using the Student t-test, and the significance of differences among groups was evaluated using the Dunnett T3 test. The connection among these variables was analyzed using the Pearson correlation coefficients. Significance levels were measured at a probability value < 0.05. Quantitative data analysis was performed with SPSS version 13 (SPSS, Inc.).

Results

Histochemical Assessment for Iron

As shown in Fig. 1, based on enhanced Perls reaction, iron deposition was found in the perihematomal zone as early as the 1st day post-ICH (2798.55 ± 664.72 in the ICH group vs 1342.69 ± 281.63 in surgical controls, p < 0.05) and reached a plateau after 7 days (34,174.48 ± 6623.51 in the ICH group vs 1673.41 ± 374.13 in surgical controls, p < 0.05). Perihematomal iron deposition remained at high levels for at least 14 days (7022.18 ± 2316.12 in the ICH group vs 1605.21 ± 371.32 in surgical controls, p < 0.05). No positive staining for iron could be seen in the normal control rats. Intraperitoneal administration of DFO starting 24 hours after ICH reduced iron levels by ∼ 75% in the vicinity of the hematoma on Days 7 and 14 post-ICH (8903.15 for DFO intervention compared with 34,174.48 for the ICH group on Day 7, p < 0.01; 1783.39 for DFO intervention compared with 7022.18 for the ICH group on Day 14, p < 0.05). There were no differences between the surgical controls and DFO intervention rats at Day 14 (Fig. 2).

Fig. 1
Fig. 1

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.

Fig. 2
Fig. 2

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.

Brain Water Content

As shown in Fig. 3, brain water content in the ICH group and surgical controls showed no significant difference at 1 day. At 3 and 7 days, the brain water content of samples obtained in the ICH group was significantly higher than that of surgical controls (p < 0.05, p < 0.01 at 3 and 7 days, respectively), with maximum water content observed at 3 days. There were no differences between the surgical controls and ICH rats at Day 14. When DFO treatment was delayed for 24 hours after ICH, this also attenuated brain edema around the hematoma 7 and 14 days after ICH (75.43 ± 2.21% in the DFO intervention group compared with 84.40 ± 6.97% in the ICH group at Day 7, p < 0.05; 74.42 ± 3.59% in the DFO intervention group compared with 76.62 ± 0.76% in the ICH group at Day 14, p < 0.01). Deferoxamine treatment starting 24 hours after ICH, however, failed to reduce brain edema at 3 days (p = 0.056).

Fig. 3
Fig. 3

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.

Immunohistochemical Assessment for AQP4

The AQP4 immunolabeling appeared as a brown deposit over glial cell processes surrounding microvessels in the brain. There was a rich expression of AQP4 around the hematoma in tissue from the ICH group. There was no difference in AQP4 immunolabeling between normal control rats and surgical control rats. There was no significant difference in the subgroups (p > 0.05) of the surgical group; the upregulation of AQP4 expression around the hematoma began at Day 1 (35.33 ± 9.14 in the ICH group vs 11.80 ± 2.65 AQP4-positive cells in surgical controls, p < 0.01) and continued for at least 14 days post-ICH (28.67 ± 2.32 in the ICH group vs 10.93 ± 1.09 AQP4-positive cells in surgical controls, p < 0.01). There was no significant difference at 1 to 3 days (p > 0.05) in the ICH group, and counts of AQP4-positive cells showed a plateau at 7 days (43.07 ± 6.08 in the ICH group vs 10.00 ± 2.98 in surgical controls, p < 0.001), then decreased (28.67 ± 2.32 at 14 days vs 43.07 ± 6.08 at 7 days in the ICH group) after ICH (Fig. 4). Intraperitoneal administration of DFO starting 24 hours after ICH resulted in a decrease in AQP4 expression at 7 and 14 days after ICH (30.88 ± 5.79 in the DFO intervention group compared with 43.07 ± 6.08 in the ICH group at 7 days, p < 0.001; 15.71 ± 3.49 in the DFO intervention group compared with 28.67 ± 2.32 in the ICH group at 14 days, p < 0.01). However, there were significant differences between the rats in the control and DFO intervention groups at Days 3 and 7 (p < 0.01). There were no differences between surgical controls and rats in the DFO intervention group at Day 14.

Fig. 4
Fig. 4

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.

The Presence of AQP4 in Each Group Based on RT-PCR

The levels of AQP4 mRNA were analyzed using semiquantitative RT-PCR. As shown in Fig. 5, AQP4 mRNA was detected in the brains of all animals. Similar to AQP4 immunolabeling results, there was no significant difference in the subgroups (p > 0.05) of the surgical group. However, the upregulation of AQP4 mRNA began at 3 days after ICH and reached a plateau simultaneously (225.52 ± 28.80 AQP4-positive cells in the ICH group vs 59.99 ± 9.98 in surgical controls, p < 0.001). It continued for at least up to 14 days post-ICH (103.23 ± 21.61 AQP4-positive cells in the ICH group vs 56.50 ± 8.48 in surgical controls, p < 0.01). There was no significant difference at 7 and 14 days (p > 0.05) in the ICH group. Application of DFO resulted in a decrease in AQP4 mRNA expression at 3 and 14 days after ICH (137.34 ± 16.73 in the DFO intervention group compared with 225.52 ± 28.80 in the ICH group at 3 days, p < 0.05; 71.11 ± 13.79 in the DFO intervention group compared with 103.23 ± 21.61 in the ICH group at 14 days, p < 0.05). Although AQP4 mRNA was also decreased at 7 days, this was not significant (p > 0.05). Similar to the results of AQP4 immunolabeling, there were significant differences between the control and DFO intervention rats at Days 3 and 7 (p < 0.01), and there were no differences between the surgical control and DFO intervention groups at Day 14.

Fig. 5.
Fig. 5.

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.

Correlation of Pearson Coefficients Among Iron Deposit, Brain Water Content, and AQP4 Findings

Iron accumulation showed strong correlation with brain water content (r = 0.445, p = 0.001) and AQP4 (r = 0.471–0.563, p ≤ 0.001). The AQP4 was correlated significantly with brain water content (r = 0.519–0.59, p < 0.001). The expression of AQP4 on immunohistochemical studies showed a significant correlation with AQP4 mRNA (r = 0.467, p < 0.001).

Discussion

The findings in the present study confirmed that there was iron accumulation and edema formation in the brain after ICH and that DFO treatment reduced ICH-induced iron overload and brain edema. Furthermore, infusion of DFO, a chelator of iron, decreased AQP4 overexpression, suggesting a protective effect against AQP4-mediated brain edema after ICH.

Although iron is essential for normal brain function, iron overload may have devastating effects.2,27,29 After ICH, iron concentrations in the brain can reach very high levels following RBC lysis, possibly contributing to acute brain edema formation (1st week) and delayed brain atrophy (1 month later).6,30 Iron has the potential to mediate a number of deleterious reactions both in vitro and in vivo. Iron accumulation in tissues, particularly if the labile iron pool is increased, is associated with tissue damage.3 Iron overload in the brain can cause free-radical formation and oxidative damage such as lipid peroxidation after ICH.8,15,16,27,33 Brain cells, including neurons, astrocytes, and microglia, show a decreased ability to respond to oxidative stress, particularly with respect to their levels of glutathione and glutathione peroxidase, such that alteration in their iron status may predispose them to iron-induced oxidative stress.28 Packed RBCs do not cause acute edema development after infusion into the basal ganglia or frontal white matter. However, they do cause delayed edema that appears to be related to release of hemoglobin.27,30 Usually, most RBCs start to lyse several days after ICH, but RBC lysis can occur very early. In our study, iron accumulation was observed in the perihematomal zone beginning 1 day after ICH and reached its peak on the 7th day after ICH. Deferoxamine, an iron chelator that works by chelating iron and inhibiting free-radical damage on DNA caused by iron, has been shown to be effective in clinical testing and in clinical application.10,20 We chose to use an intraperitoneal 50-mg injection of DFO every 12 hours starting 24 hours after ICH, because we wanted to know whether a delayed application of DFO was beneficial to delayed brain neuronal damage after ICH. We demonstrated that DFO may offer potentially significant benefits in the treatment of ICH by binding free iron.

The results of our study showed that brain water content reached its peak at 3 days and resolved starting at 7 days after ICH, consistent with peak edema observed after ICH in clinics. Peak edema occurred 3 to 7 days after the hemorrhage and correlated with lysis of RBCs. The edema can be substantial and deleterious, as suggested by a few studies showing that delayed post-ICH brain edema is associated with significant midline shift34 and edema formation after ICH increases intracranial pressure and can result in brain herniation.22,31 In our study we demonstrated that brain peak edema occurred at 3 days and peak accumulation of iron occurred at 7 days post-ICH; these results support the suggestion that there were other toxic factors, including thrombin and hemoglobin, released from a blood clot that may account for acute perihematomal edema formation.9,11,14,17,18,32,36

Because of restricted space within the cranium, salt and water flux in the CNS must be strictly regulated to maintain neuronal functions of the brain. Among the AQP family, a major water-channel in the CNS is AQP4, which is a key molecule for maintaining water balance,23,24 and its dysfunction or structural damage may cause brain edema.5 In the CNS, most of the AQP4 is expressed in perimicrovessel astrocyte foot processes,19,23 and alterations in AQP4 expression are associated with perturbations of brain water homeostasis. After brain injury26 and focal ischemia in rats, there is upregulation of astrocyte AQP4 mRNA in regions where the blood-brain barrier is disrupted. After focal cerebral ischemia, AQP4 knockout mice have less periinfarct edema than controls,13 suggesting that AQP4 aggravates edema formation. The pattern of AQP4 expression was correlated with blood-brain barrier permeability, which was assessed using contrastenhanced CT scanning.23 Our immunohistochemistry results showed that AQP4 was mainly located around blood vessels. The current study provided more direct evidence that AQP4 in perivascular astroglial end feet plays a key role in exchange of water between brain, blood, and cerebrospinal fluid.35 Our results demonstrated that the expression of AQP4 was upregulated at 3, 7, and 14 days post-ICH in the perihematoma. The upregulation of AQP4 mRNA reached a maximum at 3 days after ICH and was earlier than its immunolabeling (at 7 days). This result supports the suggestion that RT-PCR was more sensitive than immunohistochemistry. Nevertheless, gene and protein expression of AQP4 were basically consistent. We found that the peak of AQP4 mRNA was consistent with the maximum of brain water contents, suggesting the potential role of AQP4 in edema formation, and peak accumulation of iron was in accordance with AQP4, suggesting that AQP4 expression was affected by iron status. Upregulation of AQP4 induced by iron overload may cause an increased permeability to water in astrocytic membranes. On the contrary, the decreased AQP4 expression prevents the astrocytes from swelling.

Conclusions

Although DFO is an iron chelator, it can have other effects. Thus, it can act as a direct free-radical scavenger and it can induce brain tolerance.2,4,12,21 The latter condition has been demonstrated in vivo and in vitro, and it may be related to a DFO induction of hypoxia-inducible transcription factor 1 binding to DNA.4 Thus, in our study it is possible that the protective effect of DFO was mediated through iron chelation and against AQP4-induced brain damage after ICH. In this study we showed that iron chelation with DFO that was delayed for 24 hours attenuated perihematomal edema and AQP4 expression, suggesting that DFO might inhibit water influx into the cells and attenuate the acute cytotoxic brain edema after ICH. We propose that DFO may act by reducing the brain edema caused by the release of iron from the hematoma and that DFO may be a potential therapeutic agent for ICH.

Disclaimer

The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Please include this information when citing this paper: published online November 21, 2008; DOI: 10.3171/2008.4.JNS17512.

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    Welch KDDavis TZVan Eden MEAust SD: Deleterious iron-mediated oxidation of biomolecules. Free Radic Biol Med 32:5775832002. (Abstract)

  • 29

    Wu JHua YKeep RFNakamura THoff JTXi G: Iron and iron-handling proteins in the brain after intracerebral hemorrhage. Stroke 34:296429692003

  • 30

    Xi GKeep RFHoff JT: Erythrocytes and delayed brain edema formation following intracerebral hemorrhage in rats. J Neurosurg 89:9919961998

  • 31

    Xi GKeep RFHoff JT: Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol 5:53632006

  • 32

    Xi GWagner KRKeep RFHua Yde Courten-Myers GMBroderick JP: Role of blood clot formation on early edema development after experimental intracerebral hemorrhage. Stroke 29:258025861998

  • 33

    Yang QDNiu QZhou YHLiu YHXu HWGu WP: Incidence of cerebral hemorrhage in the Changsha community. A prospective study from 1986 to 2000. Cerebrovasc Dis 17:3033132004

  • 34

    Zazulia ARDiringer MNDerdeyn CPPowers WJ: Progression of mass effect after intracerebral hemorrhage. Stroke 30:116711731999

  • 35

    Zelenin SGunnarson EAlikina TBondar AAperia A: Identification of a new form of AQP4 mRNA that is developmentally expressed in mouse brain. Pediatr Res 48:3353392000

  • 36

    Zhang XLi HHu SZhang LLiu CZhu C: Brain edema after intracerebral hemorrhage in rats: the role of inflammation. Neurol India 54:4024072006. (Abstract)

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

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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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Welch KDDavis TZVan Eden MEAust SD: Deleterious iron-mediated oxidation of biomolecules. Free Radic Biol Med 32:5775832002. (Abstract)

29

Wu JHua YKeep RFNakamura THoff JTXi G: Iron and iron-handling proteins in the brain after intracerebral hemorrhage. Stroke 34:296429692003

30

Xi GKeep RFHoff JT: Erythrocytes and delayed brain edema formation following intracerebral hemorrhage in rats. J Neurosurg 89:9919961998

31

Xi GKeep RFHoff JT: Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol 5:53632006

32

Xi GWagner KRKeep RFHua Yde Courten-Myers GMBroderick JP: Role of blood clot formation on early edema development after experimental intracerebral hemorrhage. Stroke 29:258025861998

33

Yang QDNiu QZhou YHLiu YHXu HWGu WP: Incidence of cerebral hemorrhage in the Changsha community. A prospective study from 1986 to 2000. Cerebrovasc Dis 17:3033132004

34

Zazulia ARDiringer MNDerdeyn CPPowers WJ: Progression of mass effect after intracerebral hemorrhage. Stroke 30:116711731999

35

Zelenin SGunnarson EAlikina TBondar AAperia A: Identification of a new form of AQP4 mRNA that is developmentally expressed in mouse brain. Pediatr Res 48:3353392000

36

Zhang XLi HHu SZhang LLiu CZhu C: Brain edema after intracerebral hemorrhage in rats: the role of inflammation. Neurol India 54:4024072006. (Abstract)

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