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. 2011 May;31(5):1243-50.
doi: 10.1038/jcbfm.2010.209. Epub 2010 Nov 24.

Iron toxicity in mice with collagenase-induced intracerebral hemorrhage

He Wu  1 , Tao Wu, Xueying Xu, Jessica Wang, Jian Wang
Affiliations

Iron toxicity in mice with collagenase-induced intracerebral hemorrhage

He Wu et al. J Cereb Blood Flow Metab. 2011 May.

Abstract

Intracerebral hemorrhage (ICH) is a devastating form of stroke. In this study, we examined the efficacy of deferoxamine (DFX), an iron chelator, after collagenase-induced ICH in 12-month-old mice. Intracerebral hemorrhage was induced by intrastriatal injection of collagenase. Deferoxamine (200 mg/kg, intraperitoneal) or vehicle was administrated 6 hours after ICH and then every 12 hours for up to 3 days. Neurologic deficits were examined on days 1 and 3 after ICH. Mice were killed after 1 or 3 days of DFX treatment for examination of iron deposition, neuronal death, oxidative stress, microglia/astrocyte activation, neutrophil infiltration, brain injury volume, and brain edema and swelling. Collagenase-induced ICH resulted in iron overload in the perihematomal region on day 3. Systemic administration of DFX decreased iron accumulation and neuronal death, attenuated production of reactive oxygen species, and reduced microglial activation and neutrophil infiltration without affecting astrocytes. Although DFX did not reduce brain injury volume, edema, or swelling, it improved neurologic function. Results of our study indicate that iron toxicity contributes to collagenase-induced hemorrhagic brain injury and that reducing iron accumulation can reduce neuronal death and modestly improve functional outcome after ICH in mice.

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Figures

Figure 1
Figure 1
Deferoxamine (DFX) decreases iron deposition and neuronal death. (A) Perls staining showed that DFX significantly reduced iron deposition. Insets represent higher magnification of Perls-positive amoeboid cells. Scale bar=25 μm. (B) Bar graph shows quantification analysis (**P<0.01 versus vehicle-treated group). (C) Fluoro-Jade B (FJB) histologic staining of degenerating neurons. Scale bar=30 μm. (D) Bar graph shows quantification analysis (*P<0.05 versus vehicle-treated group). Values are means±s.d. ICH, intracerebral hemorrhage.
Figure 2
Figure 2
Deferoxamine (DFX) attenuates ROS production. (A) The ROS signal was evident in the perihematomal region on day 1 after ICH in vehicle-treated mice (left); DFX treatment attenuated ROS production (right). Scale bar=20 μm. (B) Fluorescence intensity was quantified in predefined areas of the hemorrhagic striatum (at the injection site and at 360 μm on each side) after subtraction of the color density on the contralateral striatum. Bar graph shows quantification analysis (**P<0.01 versus vehicle-treated group). Values are means±s.d. ICH, intracerebral hemorrhage; ROS, reactive oxygen species.
Figure 3
Figure 3
Deferoxamine (DFX) decreases microglia/macrophage activation and neutrophil infiltration. (A) Activated microglia/macrophages (Iba1-immunoreactive cells), reactive astrocytes (GFAP-immunoreactive cells), and infiltrating neutrophils (MPO-immunoreactive cells) were evident around or at the injury site on day 3 after ICH. DFX treatment significantly reduced the number of activated microglia/macrophages and infiltrating neutrophils, but not activated astrocytes. Insets represent higher magnification of immunoreactive cells. Scale bar=50 μm. (B) Bar graph shows quantification analysis (*P<0.05 versus vehicle-treated group). Values are means±s.d. GFAP, glial fibrillary acidic protein; ICH, intracerebral hemorrhage; MPO, myeloperoxidase.
Figure 4
Figure 4
Deferoxamine (DFX) ameliorates neurologic deficits. (A) Bar graph shows that DFX treatment improved neurologic function of mice on day 3 after ICH compared with the vehicle-treated group (*P<0.05). (B) Neurologic scores for each of the individual tests did not differ significantly between DFX- and vehicle-treated mice at either the 1-day or 3-day time point (all P>0.05). Values are means±s.d. ICH, intracerebral hemorrhage.
Figure 5
Figure 5
Deferoxamine (DFX) does not affect brain injury volume, edema, or swelling. (A) Representative unstained brain sections from vehicle- and DFX-treated groups. Hematomas were observed in the striatum of each group and were similar in size and appearance. (B) Brain injury volume was measured by Luxol fast blue/Cresyl violet staining. Quantification analysis revealed similar brain injury volume in vehicle- and DFX-treated groups (P>0.05). (C) Percentage brain water content was measured 3 days after ICH. The ipsilateral striatum had more water content than did the contralateral striatum in both groups. DFX treatment failed to affect brain water content (P>0.05). (D) Brain swelling, expressed as the percentage of hemispheric enlargement, was determined by quantitative image analysis. DFX treatment failed to affect brain swelling (P>0.05). Values are means±s.d. ICH, intracerebral hemorrhage.
Figure 6
Figure 6
Deferoxamine (DFX) increases body weight loss (*P<0.05). Values are means±s.d. ICH, intracerebral hemorrhage.
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