Increased striatal injury and behavioral deficits after intracerebral hemorrhage in hemopexin knockout mice

Laboratory investigation

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Heme toxicity may contribute to the pathogenesis of intracerebral hemorrhage (ICH). The primary defense against extracellular heme is provided by hemopexin, a serum and neuronal glycoprotein that binds it with very high affinity and mitigates its prooxidant effect. In the present study, the authors tested the hypothesis that hemopexin knockout mice would sustain more injury after experimental ICH than their wild-type counterparts.

Methods:

Striatal ICH was induced by the stereotactic injection of bacterial collagenase or autologous blood. Three days later, striatal protein oxidation was assessed via carbonyl assay. Cell viability was quantified at 8–9 days by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Behavioral deficits were detected with high-resolution digital analysis of 6-hour home cage video recordings and standard testing.

Results:

Perihematomal protein oxidation was increased in wild-type collagenase-injected striata by approximately 2.1-fold, as compared with contralateral striata; protein carbonyls were increased 3-fold in knockout mice. Striatal cell viability was reduced by collagenase injection in wild-type mice to 52.9 ± 6.5% of that in the contralateral striata, and to 31.1 ± 3.7% of that in the contralateral striata in knockout mice; similar results were obtained after blood injection. Digital analysis of 6-hour video recordings demonstrated an activity deficit in both models that was significantly exacerbated at 8 days in knockout mice. Striatal heme content 9 days after blood injection was increased approximately 2.7-fold in knockouts as compared with wild-type mice.

Conclusions:

These results suggest that hemopexin has a protective effect against hemorrhagic CNS injuries. Hemopexin deficiency, which is often associated with sickle cell disease, may worsen outcome after ICH.

Abbreviations used in this paper: ICH = intracerebral hemorrhage; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCR = polymerase chain reaction; TTC = 2,3,5-triphenyltetrazolium chloride.

Article Information

Address correspondence to: Raymond F. Regan, M.D., Department of Emergency Medicine, Thomas Jefferson University, 1025 Walnut Street, College Building Room 813, Philadelphia, Pennsylvania 19107. email: Raymond.Regan@jefferson.edu.

Please include this information when citing this paper: published online December 3, 2010; DOI: 10.3171/2010.10.JNS10861.

© AANS, except where prohibited by US copyright law.

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Figures

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    Upper and Center: Representative 2% agarose gels showing PCR products from hemopexin knockout (KO, −/−), heterozygous (+/−), and wild-type (WT, +/+) mice. Lower: Immunoblot of cortical lysates, stained with anti-hemopexin (HPX).

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    Increased protein oxidation after experimental ICH in hemopexin knockout mice. Immunoblots of lysates from collagenase-injected (inj) and contralateral (con) striata of hemopexin KO and WT mice, collected 72 hours after injection and stained with antibody to derivitized protein carbonyls. Bars represent mean lane densities (± SEM, 6 animals/condition) normalized to the mean value in WT striata contralateral (Contra) to collagenase injection (1.0). ***p < 0.001, compared with the mean signal in KO striata injected with collagenase, Bonferroni multiple comparisons test.

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    Bars represent mean striatal cell viability in hemopexin WT and KO mice 8 days after striatal collagenase injection (mean ± SEM, 8 animals/condition), 9 days after autologous blood injection (14 animals/ condition), or 2 days after striatal hemin injection (5 animals/condition), as assessed by MTT reduction to formazan. Control mice were injected with artificial CSF solution only. The MTT reduction is expressed as a percentage of that in the hemisphere contralateral to the injection site. The CSF group consisted of 4 KO and 4 WT mice, which had similar values. *p < 0.05 and **p < 0.01, versus the ratio in KO mice, Bonferroni multiple comparisons test.

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    Serum haptoglobin is increased in hemopexin knockout mice. Haptoglobin levels were assayed before and 24 or 72 hours after striatal injection of collagenase or artificial CSF. Six animals/condition, *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the corresponding KO condition, Bonferroni multiple comparisons test.

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    Reduced activity in hemopexin knockout mice after collagenase-induced ICH. A: Bar graph represents the mean minutes of activity (sum of minutes spent walking, feeding, vertical hanging, rearing, and jumping, ± SEM, 12–16 animals/condition) during 6-hour evening video recordings conducted 3 and 8 days after striatal injection of collagenase (WT Coll or KO Coll, 12–16 animals) or artificial CSF (31 animals). The WT and KO mice injected with artificial CSF had similar values, so results are combined for ease of display. B–F: Bars represent the minutes spent in each activity. *p < 0.05, compared with the corresponding KO condition, Bonferroni multiple comparisons test.

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    Reduced activity in hemopexin knockout mice after striatal injection of autologous blood. Bar graphs represent the mean minutes of activity (A, sum of minutes spent walking, feeding, vertical hanging, rearing, and jumping; ± SEM; 18 animals/ condition) during 6-hour recordings conducted 8 days after striatal injection of 20 μl autologous blood, or indicated end points of adhesive removal (B), corner (C), or elevated body swing tests (D). *p < 0.05, compared with the corresponding KO condition.

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