Modulation of the secondary injury process after spinal cord injury in Bach1-deficient mice by heme oxygenase–1

Laboratory investigation

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Object

Oxidative stress contributes to secondary injury after spinal cord injury (SCI). The expression of heme oxygenase-1 (HO-1), which protects cells from various insults including oxidative stress, is upregulated in injured spinal cords. Mice deficient in Bach1 (Bach1−/−), a transcriptional repressor of the HO-1 and beta-globin genes, express high levels of HO-1 mRNA and protein in various organs. The authors hypothesized that HO-1 modulates the secondary injury process after SCI in Bach1−/− mice.

Methods

Male C57BL/6 (wild-type) and homozygous Bach1−/− C57BL/6 mice were subjected to moderate SCI, and differences in hindlimb motor function, and electrophysiological, molecular biological, and histopathological changes were assessed for 2 weeks.

Results

Functional recovery was greater, and motor evoked potentials were significantly larger in Bach1−/− mice than in wild-type mice throughout the observation period. The expression of HO-1 mRNA in the spinal cord was significantly increased in both mice until 3 days after injury, and it was significantly higher in Bach1−/− mice than in wild-type mice at every assessment point. Histological examination using Luxol fast blue staining at 1 day after injury showed that the injured areas were smaller in Bach1−/− mice than in wild-type mice. The HO-1 immunoreactivity was not detected in uninjured spinal cord, but 3 days postinjury the number of HO-1–immunoreactive cells was obviously higher in the injured area in both mice, particularly in Bach1−/− mice. The HO-1 was primarily induced in microglia/macrophage in both mice.

Conclusions

These results suggest that HO-1 modulates the secondary injury process, and high HO-1 expression may preserve spinal cord function in the early stages after SCI in Bach1−/− mice. Treatment that induces HO-1 expression at these early stages may preserve the functional outcome after SCI.

Abbreviations used in this paper: Bach1−/− = Bach1-deficient; BBB = Basso-Beattie-Bresnahan; HO-1 = heme oxygenase-1; MEP = motor evoked potential; PCR = polymerase chain reaction; RT = reverse transcription; SCI = spinal cord injury.

Article Information

Address correspondence to: Kiyotaka Yamada, M.D., Department of Orthopaedic Surgery, Graduate School of Biomedical Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, 734-8551, Japan. email: yamada.kiyo@leaf.ocn.ne.jp.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Illustration showing that injury to the spinal cord was made at the T11–12 level by compressing the cord laterally from both sides for 10 seconds using forceps with a 0.5-mm spacer according to the method of Faulkner et al. (A). Photomicrographs showing that the injured area is confirmed from the ventral to dorsal side of the spinal cords in wild-type (B) and Bach1−/− (C) mice. H & E; bar = 2 mm.

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    Illustration showing the positioning of needle electrodes for transcranial electrical stimulation and recording of MEPs. Stimulation was carried out using a pair of needle electrodes placed subcutaneously 3 mm on either side of the vertex of the skull. The MEPs were recorded from needle electrodes placed in the hamstring and triceps muscles as a transaction control. A subcutaneous ground electrode was placed in the tail.

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    Hindlimb functional recovery after SCI. A: Bar graph showing the mean BBB scores of wild-type and Bach1−/− mice measured in an open-field test over the course of the 14-day recovery period. Error bars indicate SD. B: Growth curve analysis of the chronological degree of recovery of the BBB scores over the course of 14 days following SCI. The recovery of the BBB score plateaued at 14 days postinjury in both groups of mice. d = days; n.s. = no significant differences between groups.

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    Typical time courses of MEPs. Waveforms of MEPs from a wild-type mouse (left panels) and a Bach1−/− mouse (right panels) are shown before injury (A and B), 1 day after injury (C and D), and 14 days postinjury (E and F). The waveforms are MEPs of the triceps (asterisks) and hamstrings (arrows).

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    Bar graph showing MEP amplitudes of the triceps as a transaction control. The MEP amplitudes of the triceps, which were the transaction controls, showed little change. No significant difference between the groups was observed.

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    Recovery of MEPs after SCI. A: Bar graph showing the mean values of the peak-to-peak amplitudes of MEPs recorded in the hamstring muscles following transcranial stimulation of the cortex. Values are expressed as percentages of preinjury controls. *p < 0.05 (significant difference). B: Growth curve analysis of the chronological degree of recovery of the amplitudes of the MEPs over the course of 14 days following SCI.

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    Expression of HO-1 mRNA in the spinal cord. Agarose gel showing products of RT-PCR amplification of HO-1 mRNA in the spinal cord of wild-type (W) and Bach1−/− (B) mice before (normal) and 1 and 3 days after injury. Amplified β-actin (ACTB) was used for normalization.

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    Bar graph demonstrating the quantitative analysis of HO-1 mRNA expression in the spinal cord measured using realtime RT-PCR. Heme oxygenase–1 mRNA increased significantly in all mice until 3 days postinjury, and was expressed at significantly higher levels in the spinal cord of Bach1−/− mice than those in wild-type mice at each time of assessment. †p < 0.01 (significant difference).

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    Distribution of HO-1 protein in the spinal cord. Typical examples showing immunofluorescence of neurofilament (green) and HO-1 (red) and nucleus (blue) in sagittal sections of spinal cord from wild-type (A and C) and Bach1−/− (B and D) mice 3 days after injury. Bar = 500 μm (A and B); 200 μm (C and D). (Colors can be seen in the online version of this article.)

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    Immunostaining of serial sections in the spinal cord of a wild-type mouse at 3 days postinjury. In a section double-staining of HO-1 (A) and Iba1 (B), a marked induction of HO-1 in microglia/macrophages in both groups is observed at 3 days after injury. Bar = 200 μm, DAPI (C) and merge (D).

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    A–D: Longitudinal sections in the sagittal plane of the spinal cord at 1 day (A and B), and 14 days (C and D) postinjury. Photomicrographs in wild-type mice (upper panels) and Bach1−/− mice (lower panels) were stained with Luxol fast blue. E: Bar graph comparing the injured area at 1 day and 14 days after injury between wild-type and Bach1−/− mice. At 1 day postinjury, the size of the lesion was significantly smaller in Bach1−/− mice than in wild-type mice (wild-type mice 2.15 mm2, Bach1−/− mice 1.69 mm2). However, at 14 days after injury, there was no significant difference in the size of the lesion between groups (wild-type mice 0.87 mm2, Bach1−/− mice 0.80 mm2). Bar = 2 mm.

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