Oxidative stress is related to the deleterious effects of heme oxygenase-1 in an in vivo neuroinflammatory rat model

Abstract

Heme oxygenase-1 (HO-1) induction is associated with beneficial or deleterious effects depending on the experimental conditions adopted and the neurodegenerative rodent models used. The present study aimed first to evaluate the effects of cerebral HO-1 induction in an in vivo rat model of neuroinflammation by intrastriatal injection of quinolinic acid (QA) and secondly to explore the role played by reactive oxygen species (ROS) and free iron (Fe(2+)) derived from heme catabolism promoted by HO-1. Chronic I.P. treatment with the HO-1 inductor and substrate hemin was responsible for a significant dose-related increase of cerebral HO-1 production. Brain tissue loss, microglial activation, and neuronal death were significantly higher in rats receiving QA plus hemin (H-QA) versus QA and controls. Significant increase of ROS production in H-QA rat brain was inhibited by the specific HO-1 inhibitor ZnPP which supports the idea that ROS level augmentation in hemin-treated animals is a direct consequence of HO-1 induction. The cerebral tissue loss and ROS level in hemin-treated rats receiving the iron chelator deferoxamine were significantly decreased, demonstrating the involvement of Fe(2+)in brain ROS production. Therefore, the deleterious effects of HO-1 expression in this in vivo neuroinflammatory model were linked to a hyperproduction of ROS, itself promoted by free iron liberation.

Figure 1

Dose effect of systemic hemin treatment on HO-1 protein expression in the brain (a) HO-1 and tubuline beta 3 (Tubb 3) western blotting bands for control (n = 3), hemin 10 (n = 3), and hemin 50 mg/kg (n = 3) groups. (b) DO of HO-1 normalized with Tubb-3. *P < 0.05.

Figure 2

Relative cerebral tissue loss in the ipsilateral hemisphere versus the contralateral hemisphere 3 days after injury in control (n = 6), H (n = 5), QA (n = 6), H-QA (n = 5), DFX-QA (n = 5) and DFX-H-QA (n = 5) groups. *P < 0.05; **P < 0.01.

Figure 3

Relative neuronal loss in the ipsi- versus contra-lateral striatum in control and H groups 3 days after striatal injection.

Figure 4

Microglial activation in the cortex (bregma −3 mm) 3 days after intrastriatal injection. (a) Sagittal rat brain representation [24]. The dotted and full lines symbolize, respectively, the site of injury (bregma +0.7 mm) and the area where Ox-42 immunohistochemistry was performed (bregma −3 mm). (b) Increasing of microglia activation in the ipsi- versus the contra-lateral hemisphere for each brain. Measurements were performed in the cortex (3 areas per hemisphere) at a distance of 3.7 mm posterior from the site of injury (bregma −3 mm). *P < 0.05; **P < 0.01. (c) Ox-42 immunochemistry in control, H, QA, H-QA, DFX-QA, and DFX-H-QA brains. 1: macroscopic view (bregma −3 mm). 2: microscopic view (×20) of activated microglia in the cortex of the ipsilateral hemisphere.

Figure 5

Immunofluorescence of NeuN and ethidium in rat striatum 1 h after QA injection. (a), (b), and (c) represent the same areas of contra- and ipsi-lateral striatum (1 and 2, resp.) of control (n = 5), QA (n = 5), H-QA (n = 5), ZnPP-H-QA (n = 5), and DFX-H-QA (n = 3) groups. Magnification was ×20 (×120 for inserts). Data are means  ±  SEM. Bars: 20 μm. (a) NeuN (green channel). Neurons staining in contra- and ipsi-lateral striatum (1 and 2) for each group. Data are expressed as relative neuronal loss in the ipsilateral hemisphere versus the contralateral hemisphere (d). *P < 0.05 compared to respective contralateral side. (b) ROS expression analyzed with dihydroethidium (DHE) in the contra- and ipsi-lateral striatum (1 and 2) for each group. ROS measurements were performed at the same location as NeuN counting. Data are expressed as relative augmentation of ROS level in neurons in the ipsilateral hemisphere versus the contralateral hemisphere (e). *P < 0.05. (c) Merge representing neurons and ROS expression in contra- and ipsi-lateral striatum (1 and 2) for each group.