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. 2014 Dec;75(6):696-705; discussion 706.
doi: 10.1227/NEU.0000000000000524.

Role of hemoglobin and iron in hydrocephalus after neonatal intraventricular hemorrhage

Jennifer M Strahle  1 , Thomas Garton, Ahmad A Bazzi, Harish Kilaru, Hugh J L Garton, Cormac O Maher, Karin M Muraszko, Richard F Keep, Guohua Xi
Affiliations

Role of hemoglobin and iron in hydrocephalus after neonatal intraventricular hemorrhage

Jennifer M Strahle et al. Neurosurgery. 2014 Dec.

Abstract

Background: Neonatal germinal matrix hemorrhage/intraventricular hemorrhage is common and often results in hydrocephalus. The pathogenesis of posthemorrhagic hydrocephalus is not fully understood.

Objective: To explore the potential role of hemoglobin and iron released after hemorrhage.

Methods: Artificial cerebrospinal fluid (aCSF), hemoglobin, or iron was injected into the right lateral ventricle of postnatal day-7 Sprague Dawley rats. Ventricle size, heme oxygenase-1 (HO-1) expression, and the presence of iron were evaluated 24 and 72 hours after injection. A subset of animals was treated with an iron chelator (deferoxamine) or vehicle for 24 hours after hemoglobin injection, and ventricle size and cell death were evaluated.

Results: Intraventricular injection of hemoglobin and iron resulted in ventricular enlargement at 24 hours compared with the injection of aCSF. Protoporphyrin IX, the iron-deficient immediate heme precursor, did not result in ventricular enlargement after injection into the ventricle. HO-1, the enzyme that releases iron from heme, was increased in the hippocampus and cortex of hemoglobin-injected animals at 24 hours compared with aCSF-injected controls. Treatment with an iron chelator, deferoxamine, decreased hemoglobin-induced ventricular enlargement and cell death.

Conclusion: Intraventricular injection of hemoglobin and iron can induce hydrocephalus. Treatment with an iron chelator reduced hemoglobin-induced ventricular enlargement. This has implications for the pathogenesis and treatment of posthemorrhagic hydrocephalus.

Abbreviations: aCSF, artificial cerebrospinal fluidDAB, 3,3'-diaminobenzidine-4HClGMH-IVH, germinal matrix hemorrhage/intraventricular hemorrhageHO-1, heme oxygenase-1ICH, intracerebral hemorrhagePBS, phosphate-buffered salineSVZ, subventricular zoneTBST, tris-buffered saline with Tween 20.

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Figures

Figure 1
Figure 1
Intraventricular injection of hemoglobin (Hb), but not aCSF, results in ventricular enlargement. (A) Representative T2-weighted MRIs of lateral (frontal and temporal horns) and 4th ventricles in anesthesia control animals and aCSF and hemoglobin-injected (150 mg/mL) animals 24 hours after injection. (B) Quantification of ventricle volume 24 hours after no injection, aCSF injection, or injection of different concentrations of hemoglobin (50, 100, or 150 mg/mL) (n = 4-30 per group; *P < 0.0001; ANOVA with Dunnett post-hoc test for multiple comparisons with CSF used as reference). Mean ± SEM.
Figure 1
Figure 1
Intraventricular injection of hemoglobin (Hb), but not aCSF, results in ventricular enlargement. (A) Representative T2-weighted MRIs of lateral (frontal and temporal horns) and 4th ventricles in anesthesia control animals and aCSF and hemoglobin-injected (150 mg/mL) animals 24 hours after injection. (B) Quantification of ventricle volume 24 hours after no injection, aCSF injection, or injection of different concentrations of hemoglobin (50, 100, or 150 mg/mL) (n = 4-30 per group; *P < 0.0001; ANOVA with Dunnett post-hoc test for multiple comparisons with CSF used as reference). Mean ± SEM.
Figure 2
Figure 2
Iron, a principle degradation product of hemoglobin, results in ventricular enlargement. (A) Representative T2-weighted and T2* MRIs showing that increasing concentrations of Fe(III)Cl result in corresponding increases in ventricle size 24 hours after intraventricular injection. (B) Quantification of ventricle volume in mm for different concentrations of Fe(III)Cl (Pearson correlation coefficient, r = 0.80; P < 0.0001). (C) Iron-deficient protoporphyrin IX (immediate precursor to heme) does not increase ventricular size 24 hours after injection (n = 4-30 per group; *P < 0.0001; ANOVA with Dunnett post-hoc test for multiple comparisons with CSF used as reference). Mean ± SEM.
Figure 2
Figure 2
Iron, a principle degradation product of hemoglobin, results in ventricular enlargement. (A) Representative T2-weighted and T2* MRIs showing that increasing concentrations of Fe(III)Cl result in corresponding increases in ventricle size 24 hours after intraventricular injection. (B) Quantification of ventricle volume in mm for different concentrations of Fe(III)Cl (Pearson correlation coefficient, r = 0.80; P < 0.0001). (C) Iron-deficient protoporphyrin IX (immediate precursor to heme) does not increase ventricular size 24 hours after injection (n = 4-30 per group; *P < 0.0001; ANOVA with Dunnett post-hoc test for multiple comparisons with CSF used as reference). Mean ± SEM.
Figure 2
Figure 2
Iron, a principle degradation product of hemoglobin, results in ventricular enlargement. (A) Representative T2-weighted and T2* MRIs showing that increasing concentrations of Fe(III)Cl result in corresponding increases in ventricle size 24 hours after intraventricular injection. (B) Quantification of ventricle volume in mm for different concentrations of Fe(III)Cl (Pearson correlation coefficient, r = 0.80; P < 0.0001). (C) Iron-deficient protoporphyrin IX (immediate precursor to heme) does not increase ventricular size 24 hours after injection (n = 4-30 per group; *P < 0.0001; ANOVA with Dunnett post-hoc test for multiple comparisons with CSF used as reference). Mean ± SEM.
Figure 3
Figure 3
Increased expression of key iron handling protein, HO-1, after intraventricular injection of hemoglobin. (A) Western blot demonstrating increased levels of HO-1 in hippocampus and cortex 24 hours after hemoglobin injection (n = 3 per group). (B) Quantification of HO-1 levels (fraction of actin control) (n = 3 per group; *P < 0.01; ANOVA with Dunnett post-hoc test for multiple comparisons with CSF used as reference). Mean ± SEM.
Figure 4
Figure 4
Periventricular HO-1 and iron after intraventricular injection of hemoglobin. (A) Ipsilateral frontal horn of lateral ventricle showing HO-1 immunoreactivity (scale bar = 500 μm) and (B) DAB-enhanced Perls' iron staining of ipsilateral ventricle and periventricular zone (scale bar = 200 μm) 24 and 72 hours after intraventricular injection of hemoglobin.
Figure 4
Figure 4
Periventricular HO-1 and iron after intraventricular injection of hemoglobin. (A) Ipsilateral frontal horn of lateral ventricle showing HO-1 immunoreactivity (scale bar = 500 μm) and (B) DAB-enhanced Perls' iron staining of ipsilateral ventricle and periventricular zone (scale bar = 200 μm) 24 and 72 hours after intraventricular injection of hemoglobin.
Figure 5
Figure 5
Ventricle enlargement at 24 and 72 hours after injection of aCSF, hemoglobin, or Fe(III)Cl. (A) Representative T2-weighted MRIs of lateral ventricles 24 and 72 hours after injection. (B) Quantification of ventricle volume (*P < 0.05; **P < 0.01; 1-tailed unpaired t test). Mean ± SEM.
Figure 5
Figure 5
Ventricle enlargement at 24 and 72 hours after injection of aCSF, hemoglobin, or Fe(III)Cl. (A) Representative T2-weighted MRIs of lateral ventricles 24 and 72 hours after injection. (B) Quantification of ventricle volume (*P < 0.05; **P < 0.01; 1-tailed unpaired t test). Mean ± SEM.
Figure 6
Figure 6
Peripheral treatment with the iron chelator, deferoxamine (DFX), reduces hemoglobin (Hb)-induced ventricular enlargement and cell death. (A) Representative T2-weighted MRIs of lateral ventricles, cerebral aqueduct, and 4th ventricle in vehicle-treated or deferoxamine-treated animals 24 hours after intraventricular hemorrhage. (B) Quantification of ventricle size in mm (n = 13 per group; *P < 0.05). (C) Cell death measured by K+ levels in ipsi- and contralateral hemispheres in vehicle- and deferoxamine-treated animals (n = 4-5 per group; *P < 0.05; 1-tailed unpaired t test). Mean ± SEM.
Figure 6
Figure 6
Peripheral treatment with the iron chelator, deferoxamine (DFX), reduces hemoglobin (Hb)-induced ventricular enlargement and cell death. (A) Representative T2-weighted MRIs of lateral ventricles, cerebral aqueduct, and 4th ventricle in vehicle-treated or deferoxamine-treated animals 24 hours after intraventricular hemorrhage. (B) Quantification of ventricle size in mm (n = 13 per group; *P < 0.05). (C) Cell death measured by K+ levels in ipsi- and contralateral hemispheres in vehicle- and deferoxamine-treated animals (n = 4-5 per group; *P < 0.05; 1-tailed unpaired t test). Mean ± SEM.
Figure 6
Figure 6
Peripheral treatment with the iron chelator, deferoxamine (DFX), reduces hemoglobin (Hb)-induced ventricular enlargement and cell death. (A) Representative T2-weighted MRIs of lateral ventricles, cerebral aqueduct, and 4th ventricle in vehicle-treated or deferoxamine-treated animals 24 hours after intraventricular hemorrhage. (B) Quantification of ventricle size in mm (n = 13 per group; *P < 0.05). (C) Cell death measured by K+ levels in ipsi- and contralateral hemispheres in vehicle- and deferoxamine-treated animals (n = 4-5 per group; *P < 0.05; 1-tailed unpaired t test). Mean ± SEM.
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