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

Laboratory investigation

Restricted access


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.


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.


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.


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:

© AANS, except where prohibited by US copyright law.



  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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).

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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).

  • View in gallery

    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.)

  • View in gallery

    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).

  • View in gallery

    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.


  • 1

    Abe YNakamura HYoshino OOya TKimura T: Decreased neural damage after spinal cord injury in tPA-deficient mice. J Neurotrauma 20:43572003

  • 2

    Agrawal SKFehlings MG: The effect of sodium channel blocker QX-314 on recovery after acute spinal cord injury. J Neurotrauma 14:81881997

  • 3

    Anderson DKHall ED: Pathophysiology of spinal cord trauma. Ann Emerg Med 22:9879921993

  • 4

    Barut SCanbolat ABilge TAydin YCokneseli BKaya U: Lipid peroxidation in experimental spinal cord injury: timelevel relationship. Neurosurg Rev 16:53591993

  • 5

    Basso DMBeattie MSBresnahan JC: A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1211995

  • 6

    Blight A: Mechanical factors in experimental spinal cord injury. J Am Paraplegia Soc 11:26341988

  • 7

    Blight A: Remyelination, revascularization, and recovery of function in experimental spinal cord injury. Adv Neurol 59:911041993

  • 8

    Curt AKeck MEDietz V: Functional outcome following spinal cord injury: significance of motor-evoked potentials and ASIA scores. Arch Phys Med Rehabil 79:81861998

  • 9

    Demediuk PSaunders RDClendenon NRMeans EDAnderson DKHorrocks LA: Changes in lipid metabolism in traumatized spinal cord. Prog Brain Res 63:2112261985

  • 10

    Ducker TBKindt GWKempf LG: Pathological findings in acute experimental spinal cord trauma. J Neurosurg 35:700 7081971

  • 11

    Dwyer BENishimura RNLu SY: Differential localization of heme oxygenase and NADPH-diaphorase in spinal cord neurons. Neuroreport 6:9739761995

  • 12

    Faulkner JRHerrmann JEWoo MJTansey KEDoan NBSofroniew MV: Reactive astrocytes protect tissue and preserve function after spinal cord injury. J Neurosci 24:2143 21552004

  • 13

    Fujikoshi Y: Selection of covariables in the growth curve model. Biometricka 78:7797851991

  • 14

    Ghaly RFStone JLAldrete JALevy WJ: Effects of incremental ketamine hydrochloride dose on motor evoked potentials (MEPs) following transcranial magnetic stimulation: a primate study. J Neurosurg Anesthesiol 2:79851990

  • 15

    Gruner JAWade CKMenna GStokes BT: Myoelectric evoked potentials versus locomotor recovery in chronic spinal cord injured rats. J Neurotrauma 10:3273471993

  • 16

    Hall ED: Lipid peroxidants in acute central nervous system injury. Ann Emerg Med 22:102210271993

  • 17

    Hall ED: The neuroprotective pharmacology of methylpredonisolone. J Neurosurg 76:13221992

  • 18

    Ilhan AYilmaz HRArmutcu FGurel AAkyol O: The protective effect of nebivolol on ischemia/reperfusion injury in rabbit spinal cord. Prog Neuropsychopharmacol Biol Psychiatry 28:115311602004

  • 19

    Joshi MFehlings MG: Development and characterization of a novel, graded model of clip compressive spinal cord injury in the mouse: part 1. Clip design, behavioral outcomes, and histopathology. J Neurotrauma 19:1751902002

  • 20

    Loy DNNagnuson DSZhang YPOnifer SMMills MDCao QL: Functional redundancy of ventral spinal locomotor pathways. J Neurosci 22:3153232002

  • 21

    Maines MD: Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J 2:255725681988

  • 22

    Maines MD: New developments in the regulation of heme metabolism and their implications. Crit Rev Toxicol 12:241 3141984

  • 23

    Mautes AEBergeron MSharp FRPanter SSWeinzierl MGuenther K: Sustained induction of heme oxygenase-1 in the traumatized spinal cord. Exp Neurol 166:2542652000

  • 24

    Mautes AEKim DHSharp FRPanter SSato MMaida N: Induction of heme oxygenase-1 (HO-1) in the contused spinal cord of the rat. Brain Res 795:17241998

  • 25

    Nashmi RImamura HTator CHFehlings MG: Serial recording of somatosensory and myoelectric motor evoked potentials: role in assessing functional recovery after graded spinal cord injury in the rat. J Neurotrauma 14:1511591997

  • 26

    Omura SSuzuki HToyofuku MOzono RKohno NIgarashi K: Effects of genetic ablation of bach1 upon smooth muscle cell proliferation and atherosclerosis after cuff injury. Genes Cells 10:2772852005

  • 27

    Rawe SELee WAPerot PL: Spinal cord glucose utilization after experimental spinal cord injury. Neurosurgery 9:40 471981

  • 28

    Saunders RDDugan LLDemediuk PMeans EDHorrocks LAAnderson DK: Effect of methylpredonisolone and the combination of alpha-tocopherol and selenium on arachidonic acid metabolism and lipid peroxidation in traumatized spinal cord tissue. J Neurochem 49:24311987

  • 29

    Schumacher PASiman RGFehlings MG: Pretreatment with calpain inhibitor CEP-4143 inhibits calpain I activation and cytoskeletal degradation, improves neurological function, and enhances axonal survival after traumatic spinal cord injury. J Neurochem 74:164616552000

  • 30

    Stocker R: Induction of heme oxygenase as a defence against oxidative stress. Free Radic Res Commun 9:1011121990

  • 31

    Stocker RYamamoto YMcDonagh AFGlazer ANAmes BN: Bilirubin is an antioxidant of possible physiological importance. Science 235:104310461987

  • 32

    Sun JBrand MZenke YTashiro SGroudine MIgarashi K: Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network. Proc Natl Acad Sci U S A 101:146114662004

  • 33

    Sun JHoshino HTakaku KNakajima OMuto ASuzuki H: Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene. EMBO J 21:521652242002

  • 34

    Tator CH: Biology of neurological recovery and functional restoration after spinal cord injury. Neurosurgery 42:696 7081998

  • 35

    Xu WChi LXu RKe YLuo CCai J: Increased production of reactive oxygen species contributes to motor neuron death in a compression mouse model of spinal cord injury. Spinal Cord 43:2042132005

  • 36

    Yano YOzono ROishi YLambe MYoshizumi MIshida T: Genetic ablation of the transcription repressor Bach1 leads to myocardial protection against ischemia/reperfusion in mice. Genes Cells 11:7918032006


Cited By



All Time Past Year Past 30 Days
Abstract Views 81 81 12
Full Text Views 56 56 0
PDF Downloads 80 79 0
EPUB Downloads 0 0 0


Google Scholar