Hydrogen peroxide administered into the rat spinal cord at the level elevated by contusion spinal cord injury oxidizes proteins, DNA and membrane phospholipids, and induces cell death: attenuation by a metalloporphyrin

Abstract

We previously demonstrated that hydrogen peroxide concentration ([H2O2]) significantly increases after spinal cord injury (SCI). The present study explored (1) whether SCI-elevated [H2O2] is sufficient to induce oxidation and cell death, (2) if apoptosis is a pathway of H2O2-induced cell death, and (3) whether H2O2-induced oxidation and cell death could be reversed by treatment with the catalytic antioxidant Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP). H2O2 was perfused through a microcannula into the uninjured rat spinal cord to mimic the conditions induced by SCI. Protein and DNA oxidation, membrane phospholipids peroxidation (MLP), cell death and apoptosis were characterized by histochemical and immunohistochemical staining with antibodies against markers of oxidation and apoptosis. Stained cells were quantified in sections of H2O2-, or artificial cerebrospinal fluid (ACSF)-exposed with vehicle-, or MnTBAP-treated groups. Compared with ACSF-exposed animals, SCI-elevated [H2O2] significantly increased intracellular protein and DNA oxidation by threefold and MLP by eightfold in neurons, respectively. H2O2-elevated extracellular malondialdehyde was measured by microdialysis sampling. We demonstrated that SCI-elevated [H2O2] significantly increased extracellular malondialdehyde above pre-injury levels. H2O2 also significantly increased cell loss and the numbers of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-(dUTP)-biotin nick end labeling (TUNEL)-positive and active caspase-3-positive neurons by 2.3-, 2.8-, and 5.6-fold compared to ACSF controls, respectively. Our results directly and unequivocally demonstrate that SCI-elevated [H2O2] contributes to post-SCI MLP, protein, and DNA oxidation to induce cell death. Therefore, we conclude that (1) the role of H2O2 in secondary SCI is pro-oxidation and pro-cell death, (2) apoptosis is a pathway for SCI-elevated [H2O2] to induce cell death, (3) caspase activation is a mechanism of H2O2-induced apoptosis after SCI, and (4) MnTBAP treatment significantly decreased H2O2-induced oxidation, cell loss, and apoptosis to the levels of ACSF controls, further supporting MnTBAP's ability to scavenge H2O2 by in vivo evidence.

Keywords: Mn (III) tetrakis (4-benzoic acid) porphyrin; apoptotic cell death; hydrogen peroxide; membrane lipid peroxidation; proteins and DNA oxidation; spinal cord injury.

Fig. 1

Establishment of the SCI-elevated extracellular [H₂O₂]. Following anesthesia and laminectomy on vertebra T13 of the rat spinal cord, a microdialysis fiber and a microcannula were inserted together laterally through the gray matter of the cord. A shows the location of microcannula and microdialysis fiber in a cresyl violet-stained cross section of spinal cord. H₂O₂ in ACSF was perfused through the microcannula, diffused into the extracellular space of the cord, sampled through the microdialysis fiber, and measured by HPLC, as described in sections 2 and 3 in Experimental Procedures. Different [H₂O₂] were administered, and a series of time courses were measured from collected dialysates, corresponding to each [H₂O₂] administered. B shows the time course of [H₂O₂] measured from the dialysates (presented as [2,3-DHBA]) in responding to150 μM H₂O₂ perfused through the cannula. This time course replicates that of post-SCI [H₂O₂] and was subsequently administered through the cannula to test whether it induces oxidation and cell death.

Fig. 2

H₂O₂-induces and MnTBAP attenuates protein oxidation: The animal experiment procedures were similar to those in Fig 1, except two cannulas were inserted. Then, 150 μM H₂O₂ in ACSF was perfused through a microcannula into the gray matter of the cord for 10 h to mimic the concentration and duration produced in the extracellular space of the cord by SCI. ACSF (vehicle) or MnTBAP (2.5 mM in ACSF) was perfused in the second cannula in the vehicle- or MnTBAP-treated groups. ACSF was perfused through both cannulas in the ACSF control group. At 14 h post-H₂O₂ or ACSF exposure, the cord was collected and processed. The sections containing the cannula tracks were immuno-labeled with anti-DNP antibody and the sections 2-mm caudal from the tracks were cresyl violet-stained to serve as controls using the methods described in sections 5.1 and 6.1 in Experimental procedures. Upper panel, photomicrographs of anti-DNP antibody immuno-labeled cross sections. A-C: lower magnification; A’-C’: higher magnification of A-C. B’ shows the higher magnification of the dash box in B. The scale bars indicate 100 μm. A and A’, ACSF-exposed section; B and B’, H₂O₂-exposed and vehicle-treated section; C and C’, H₂O₂-exposed and MnTBAP-treated section. The arrow heads in A-C indicate the cannula tracks. The DNP-positive neurons and cresyl violet-stained neurons were counted and analyzed and are presented in the lower panel. Clearly, 150 μM H₂O₂ exposure significantly increased the density % of DNP-positive neurons compared to ACSF control (indicated by *), whereas MnTBAP treatment significantly reduced the density % of H₂O₂-induced DNP-positive neurons (indicated by #) compared with vehicle treatment.

Fig. 3

H₂O₂ induces and MnTBAP attenuates DNA oxidation. Sections obtained as in Fig. 2 were immuno-labeled with anti-8-OHdG antibody or stained with cresyl violet (2-mm from cannula tracks as control sections). Upper panel, photomicrographs of anti-8-OHdG antibody immuno-labeled cross sections. A-C, lower magnification; A’-C’, higher magnification of A-C, Scale bars: 100 μm. B’ shows the higher magnification of the dash box in B. The frame in B’ shows a typical 8-OHdG-positive neuron enlarged at high magnification. A and A’, ACSF control section; B and B’, H₂O₂-exposed with vehicle-treated section; C and C’, H₂O₂-exposed with MnTBAP-treated section. The arrow heads in A-C indicate the cannula tracks. The 8-OHdG-positive neurons and cresyl violet-stained neurons were counted and analyzed and are presented in the lower panel. H₂O₂ exposure significantly increased the % of 8-OHdG-positive neurons as indicated by *, whereas MnTBAP treatment significantly reduced the 8-OHdG-positive neurons (#).

Fig. 4

H₂O₂ induces and MnTBAP attenuates intracellular MLP. Sections obtained as in Fig. 2 were immuno-labeled with anti-HNE antibody or stained with cresyl violet. Upper panel: photomicrographs of anti-HNE antibody immuno-labeled cross sections. A-C, lower magnification; A’-C’, higher magnification of A-C, Scale bars indicate 100 μ m. B’ shows the higher magnification of the dash box in B. A and A’, ACSF control section; B and B’, H₂O₂-exposed with vehicle-treated section; C and C’, H₂O₂-exposed with MnTBAP-treated section. The hollow areas in center of section A-C are the cannula tracks as indicated by arrow heads in Figs. 2 and 3. Counting the HNE-positive neurons along the cannula tracks and cresyl violet-stained neurons in the 2-mm control sections from the tracks and comparing the density % demonstrated that H₂O₂ significantly increased the densities % of HNE-positive neurons (indicated by *), but this increase was significantly attenuated by MnTBAP (indicated by #).

Fig. 5

H₂O₂ induces and MnTBAP attenuates extracellular MLP. Two microcannulas were inserted laterally through the gray matter of the cord as described in section 2 in the Experimental Procedures. 150 μM H₂O₂ was perfused though one cannula and ACSF or MnTBAP (2.5 mM) was perfused though the second cannula in the vehicle- or MnTBAP-treated group. H₂O₂-elevated MDA in the perfusates sampled by the first microcannula was analyzed by HPLC with fluorescence detection. Perfusates collection and analysis were described in section 5.2 in the Experimental Procedures. H₂O₂ administration significantly increased extracellular [MDA] compared to pre-administration level, and MnTBAP significantly attenuated this increase.

Fig. 6

H₂O₂ induces and MnTBAP attenuates cell loss. Sections obtained as in Fig. 2 were cresyl violet-stained along the cannula tracks and 2-mm caudal from the tracks. Upper panel, the photomicrographs of cresyl violet-stained sections along the cannula tracks. A-D, lower magnification; A’-D’, higher magnification of A-D, scale bars = 100 μm. B’ shows the higher magnification of the dash box in B. A and A’, control section 2-mm from the cannula tracks; B and B’, ACSF-control section; C and C’, H₂O₂-exposed with vehicle-treated section; D and D’, H₂O₂-exposed with MnTBAP-treated section. Lower panel, the quantitative results indicated that H₂O₂ significantly (*) increased the % of cell loss compared with ACSF control, and MnTBAP significantly (#) reduced such loss compared with vehicle-treated sections.

Fig. 7

H₂O₂ induces and MnTBAP attenuates DNA fragmentation. Sections obtained as in Fig. 2 were stained with TUNEL or double stained with TUNEL + NSE. Upper panel, photomicrographs of TUNEL-stained sections at three different magnifications to show the cannula tracks at lower magnification and TUNEL-positive cells at higher magnification. A-C, lower magnification; A’-C’, higher magnification of A-C; A”-C”, higher magnification of A’-C’; A-A”, ACSF-exposed control section; B-B” H₂O₂-exposed with vehicle-treated section; C-C” H₂O₂-exposed with MnTBAP-treated section. Middle panel, photomicrographs of TUNEL + NSE double-stained sections at three different magnifications as described for the upper panel. A’ shows the higher magnification of the dash box in A. D-F, TUNEL and NSE double-immunofluorescence-stained H₂O₂-exposed section. D, TUNEL florescence staining, the arrow heads indicate TUNEL-positive nuclei (green). E, the same section as in D with anti-NSE antibody immuno-fluorescence staining, the arrow heads indicate the same neurons (red) as in D. F, overlapping of D and E (yellow) indicates TUNEL-positive neurons. Scale bars indicate 100 μm. The quantitative results shown in the lower panel demonstrate that H₂O₂ significantly (*) increased the density % of TUNEL-positive neurons compared with ACSF control and MnTBAP treatment significantly (#) reduced neuronal apoptosis compared with vehicle treatment.

Fig. 8

H₂O₂ induces and MnTBAP attenuates caspase activation in neurons. Sections obtained as in Fig. 2 were immunohistochemically labeled with anti-active fragments of caspase-3 and caspase-8 antibodies. The upper panel shows photomicrographs of immuno-labeled sections with anti-active caspase-3 (A-C) and caspase-8 (D) fragment antibodies. A-D, lower magnification; A’-D’, higher magnification of A-D, scale bars indicate 100 μ m. B’ shows the higher magnification of the dash box in B. A-A’ ACSF-exposed control section; B-B’, H₂O₂-exposed with vehicle-treated section, C-C’, H₂O₂-exposed with MnTBAP-treated section, D and D’, active caspase-8-immuno-labeled H₂O₂-exposed section. The quantitative results (lower panel) reveal that H₂O₂ significantly (*) increased the density % of active caspase-3-positive neurons compared with ACSF control, and MnTBAP treatment significantly (#) reduced the H₂O₂-induced increase compared with vehicle-treated sections.

Fig. 9

TEM confirmation of neuronal apoptosis. H₂O₂ was perfused as in Figs. 2-8, and the cord was processed for TEM as described in section 6.4 of the Experimental Procedures. A, a normal neuron from a sham control (magnification: ×14,300); B, a neuron exposed to ACSF (×14,300); C-F, different stages of apoptotic neurons in H₂O₂-exposed sections (C, ×57,750, D, ×14,300, E, ×42,625 and F, ×31,625). H₂O₂ exposure induced specific morphological features indicative of neuronal apoptosis. No apoptotic neurons were found in sham or ACSF control sections. Symbols: N, nucleus; M, mitochondria; R, rough endoplasmic reticulum; G, Golgi complex; Ch, chromatin.