Hydroxyl radicals generated in the rat spinal cord at the level produced by impact injury induce cell death by necrosis and apoptosis: Protection by a metalloporphyrin

F. Bao, D. Liu

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61 Citations (Scopus)

Abstract

We previously measured the time courses of hydrogen peroxide (H 2O2), hydroxyl radical (OH), and catalytic iron increases following traumatic spinal cord injury (SCI). This study determines whether the SCI-elevated level of OH causes cell death. OH was generated by administering H2O2 and Fe2+ at the concentrations attained following SCI, each through a separate microdialysis fiber inserted laterally into the gray matter of the cord. The duration of OH generation mimics the duration of its elevation after SCI. The death of neurons and astrocytes was characterized at 24 h post-OH exposure and quantitated by counting surviving cells along the fiber track in sections stained with Cresyl Violet, or immunohistochemically stained with anti-neuron-specific enolase (anti-NSE) and anti-glial fibrillary acidic protein (anti-GFAP). DNA fragmentation in neurons was characterized by double staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and anti-NSE. Using a one way ANOVA followed by the Tukey test, we demonstrated that OH generated in the cord induced significant losses of neurons in both Cresyl Violet (P<0.001) and anti-NSE-stained sections (P<0.001), and of astrocytes in GFAP-stained sections (P=0.001). OH generated in the cord increased numbers of TUNEL-positive neurons compared with Ringer's solution administered as a control (P=0.001). Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic and a broad spectrum reactive species scavenger, significantly reduced OH-induced death of neurons (P<0.001 in anti-NSE stained sections and P=0.002 in the Cresyl Violet-stained sections) and astrocytes (P=0.03). It also reduced the numbers of TUNEL-positive neurons (P=0.01). Electron microscopy confirmed that generated OH induced neuronal and glial death with characteristic features of both necrosis and apoptosis. We conclude that 1) SCI-elevated OH is sufficient to induce both necrosis and apoptosis, criteria for identifying an endogenous secondary damaging agent; 2) MnTBAP reduces OH-induced cell death, perhaps by removing H2O2 administered in the tissue, thereby blocking formation of OH, and also by scavenging downstream reactive species.

Original languageEnglish (US)
Pages (from-to)285-295
Number of pages11
JournalNeuroscience
Volume126
Issue number2
DOIs
StatePublished - 2004

Fingerprint

Metalloporphyrins
Hydroxyl Radical
Spinal Cord
Spinal Cord Injuries
Cell Death
Necrosis
Phosphopyruvate Hydratase
Apoptosis
Neurons
In Situ Nick-End Labeling
Wounds and Injuries
Astrocytes
DNA Nucleotidylexotransferase
Glial Fibrillary Acidic Protein
Microdialysis
DNA Fragmentation
Biotin
Neuroglia
Hydrogen Peroxide
Superoxide Dismutase

Keywords

  • Cresyl Violet
  • CV
  • electron microscopy
  • EM
  • Fenton reaction
  • GFAP
  • glial fibrillary acidic protein
  • H O
  • hydrogen peroxide
  • Mn (III) tetrakis (4-benzoic acid) porphyrin
  • reactive oxygen species
  • secondary cell death
  • spinal cord injury

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

@article{63ab2627002b4ac4aab0e604a52463b9,
title = "Hydroxyl radicals generated in the rat spinal cord at the level produced by impact injury induce cell death by necrosis and apoptosis: Protection by a metalloporphyrin",
abstract = "We previously measured the time courses of hydrogen peroxide (H 2O2), hydroxyl radical (•OH), and catalytic iron increases following traumatic spinal cord injury (SCI). This study determines whether the SCI-elevated level of •OH causes cell death. OH was generated by administering H2O2 and Fe2+ at the concentrations attained following SCI, each through a separate microdialysis fiber inserted laterally into the gray matter of the cord. The duration of •OH generation mimics the duration of its elevation after SCI. The death of neurons and astrocytes was characterized at 24 h post-•OH exposure and quantitated by counting surviving cells along the fiber track in sections stained with Cresyl Violet, or immunohistochemically stained with anti-neuron-specific enolase (anti-NSE) and anti-glial fibrillary acidic protein (anti-GFAP). DNA fragmentation in neurons was characterized by double staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and anti-NSE. Using a one way ANOVA followed by the Tukey test, we demonstrated that •OH generated in the cord induced significant losses of neurons in both Cresyl Violet (P<0.001) and anti-NSE-stained sections (P<0.001), and of astrocytes in GFAP-stained sections (P=0.001). •OH generated in the cord increased numbers of TUNEL-positive neurons compared with Ringer's solution administered as a control (P=0.001). Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic and a broad spectrum reactive species scavenger, significantly reduced •OH-induced death of neurons (P<0.001 in anti-NSE stained sections and P=0.002 in the Cresyl Violet-stained sections) and astrocytes (P=0.03). It also reduced the numbers of TUNEL-positive neurons (P=0.01). Electron microscopy confirmed that generated •OH induced neuronal and glial death with characteristic features of both necrosis and apoptosis. We conclude that 1) SCI-elevated •OH is sufficient to induce both necrosis and apoptosis, criteria for identifying an endogenous secondary damaging agent; 2) MnTBAP reduces •OH-induced cell death, perhaps by removing H2O2 administered in the tissue, thereby blocking formation of •OH, and also by scavenging downstream reactive species.",
keywords = "Cresyl Violet, CV, electron microscopy, EM, Fenton reaction, GFAP, glial fibrillary acidic protein, H O, hydrogen peroxide, Mn (III) tetrakis (4-benzoic acid) porphyrin, reactive oxygen species, secondary cell death, spinal cord injury",
author = "F. Bao and D. Liu",
year = "2004",
doi = "10.1016/j.neuroscience.2004.03.054",
language = "English (US)",
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pages = "285--295",
journal = "Neuroscience",
issn = "0306-4522",
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TY - JOUR

T1 - Hydroxyl radicals generated in the rat spinal cord at the level produced by impact injury induce cell death by necrosis and apoptosis

T2 - Protection by a metalloporphyrin

AU - Bao, F.

AU - Liu, D.

PY - 2004

Y1 - 2004

N2 - We previously measured the time courses of hydrogen peroxide (H 2O2), hydroxyl radical (•OH), and catalytic iron increases following traumatic spinal cord injury (SCI). This study determines whether the SCI-elevated level of •OH causes cell death. OH was generated by administering H2O2 and Fe2+ at the concentrations attained following SCI, each through a separate microdialysis fiber inserted laterally into the gray matter of the cord. The duration of •OH generation mimics the duration of its elevation after SCI. The death of neurons and astrocytes was characterized at 24 h post-•OH exposure and quantitated by counting surviving cells along the fiber track in sections stained with Cresyl Violet, or immunohistochemically stained with anti-neuron-specific enolase (anti-NSE) and anti-glial fibrillary acidic protein (anti-GFAP). DNA fragmentation in neurons was characterized by double staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and anti-NSE. Using a one way ANOVA followed by the Tukey test, we demonstrated that •OH generated in the cord induced significant losses of neurons in both Cresyl Violet (P<0.001) and anti-NSE-stained sections (P<0.001), and of astrocytes in GFAP-stained sections (P=0.001). •OH generated in the cord increased numbers of TUNEL-positive neurons compared with Ringer's solution administered as a control (P=0.001). Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic and a broad spectrum reactive species scavenger, significantly reduced •OH-induced death of neurons (P<0.001 in anti-NSE stained sections and P=0.002 in the Cresyl Violet-stained sections) and astrocytes (P=0.03). It also reduced the numbers of TUNEL-positive neurons (P=0.01). Electron microscopy confirmed that generated •OH induced neuronal and glial death with characteristic features of both necrosis and apoptosis. We conclude that 1) SCI-elevated •OH is sufficient to induce both necrosis and apoptosis, criteria for identifying an endogenous secondary damaging agent; 2) MnTBAP reduces •OH-induced cell death, perhaps by removing H2O2 administered in the tissue, thereby blocking formation of •OH, and also by scavenging downstream reactive species.

AB - We previously measured the time courses of hydrogen peroxide (H 2O2), hydroxyl radical (•OH), and catalytic iron increases following traumatic spinal cord injury (SCI). This study determines whether the SCI-elevated level of •OH causes cell death. OH was generated by administering H2O2 and Fe2+ at the concentrations attained following SCI, each through a separate microdialysis fiber inserted laterally into the gray matter of the cord. The duration of •OH generation mimics the duration of its elevation after SCI. The death of neurons and astrocytes was characterized at 24 h post-•OH exposure and quantitated by counting surviving cells along the fiber track in sections stained with Cresyl Violet, or immunohistochemically stained with anti-neuron-specific enolase (anti-NSE) and anti-glial fibrillary acidic protein (anti-GFAP). DNA fragmentation in neurons was characterized by double staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and anti-NSE. Using a one way ANOVA followed by the Tukey test, we demonstrated that •OH generated in the cord induced significant losses of neurons in both Cresyl Violet (P<0.001) and anti-NSE-stained sections (P<0.001), and of astrocytes in GFAP-stained sections (P=0.001). •OH generated in the cord increased numbers of TUNEL-positive neurons compared with Ringer's solution administered as a control (P=0.001). Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic and a broad spectrum reactive species scavenger, significantly reduced •OH-induced death of neurons (P<0.001 in anti-NSE stained sections and P=0.002 in the Cresyl Violet-stained sections) and astrocytes (P=0.03). It also reduced the numbers of TUNEL-positive neurons (P=0.01). Electron microscopy confirmed that generated •OH induced neuronal and glial death with characteristic features of both necrosis and apoptosis. We conclude that 1) SCI-elevated •OH is sufficient to induce both necrosis and apoptosis, criteria for identifying an endogenous secondary damaging agent; 2) MnTBAP reduces •OH-induced cell death, perhaps by removing H2O2 administered in the tissue, thereby blocking formation of •OH, and also by scavenging downstream reactive species.

KW - Cresyl Violet

KW - CV

KW - electron microscopy

KW - EM

KW - Fenton reaction

KW - GFAP

KW - glial fibrillary acidic protein

KW - H O

KW - hydrogen peroxide

KW - Mn (III) tetrakis (4-benzoic acid) porphyrin

KW - reactive oxygen species

KW - secondary cell death

KW - spinal cord injury

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U2 - 10.1016/j.neuroscience.2004.03.054

DO - 10.1016/j.neuroscience.2004.03.054

M3 - Article

C2 - 15207346

AN - SCOPUS:2942657443

VL - 126

SP - 285

EP - 295

JO - Neuroscience

JF - Neuroscience

SN - 0306-4522

IS - 2

ER -