The inducible form of heme oxygenase, HO-1, was not expressed at detectable level in both Hx-null and wild-type mice

The inducible form of heme oxygenase, HO-1, was not expressed at detectable level in both Hx-null and wild-type mice. and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron-loaded oligodendrocytes in the basal ganglia and thalamus of Hx-null mice compared to wild-type controls. Interestingly, there was no increase in H- and L-ferritin expression in these regions. This condition is usually common to several human neurological disorders such as Alzheimers disease and Parkinsons disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx-null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and CuCZn superoxide dismutase-1 expression were higher in the brain of Hx-null mice than in that of wild-type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases. data have shown that this heme-Hx complex is able to induce the expression of heme oxygenase (HO)-1 and to activate cellular protection mechanisms [8, 9]. Thus, Hx provides Etofylline protection against free heme-mediated oxidative stress [10, 11], limits access by pathogens to heme [12] and contributes to iron homeostasis by recycling heme iron [13]. Consistently, Hx-null mice recover less efficiently than wild-type controls after acute haemolysis and suffer from severe renal damage due to iron overload and oxidative injury [14]. Moreover, compound mutant mice for Hx and its closely related protein, haptoglobin showed marked liver inflammation and fibrosis after haemolytic stress [15]. Other than in plasma and liver, Hx protein has been found in the sciatic nerve, skeletal muscle mass, retina and brain [16C19]. Moreover, human Hx promoter activity has been reported in several brain regions in transgenic mice, including cortex, hippocampus, thalamus, hypothalamus, cerebellum and brainstem nuclei [20, 21]. Recently, Hx has been identified in human cerebrospinal fluid (CSF), and a comparative proteomic analysis has demonstrated a significant increase SLCO2A1 in Hx levels in CSF from Alzheimers disease patients compared to non-demented elderly persons [22, 23]. However, it remains unclear whether Hx is usually locally synthesized in extra-hepatic tissues or it is taken up from plasma. Data on Hx expression in brain and its modulation under pathological conditions suggest that it might act as heme scavenger and protective factor in the nervous system as well. To test this hypothesis, we analysed iron deposits, ferritin expression and oxidative stress in the brain of Hx-null mice. Materials and methods Animals Hx-null mice were generated in our laboratory as previously explained [14]. Two-month-old Hx?/? and Hx+/+ mice utilized for experiments were in the 129Sv genetic background and were maintained on a standard diet. For the production Etofylline of an anti-Hx antibody, Hx?/? mice in C57BL/J6 background were immunized with a recombinant Hx protein (see next paragraph). All experiments were approved by the animal studies committee of the University or college of Torino (Italy). Production of anti-haemopexin antibodies Polyclonal and monoclonal antibodies were produced against a recombinant protein obtained in and corresponding to amino acids 81C459 of mouse Hx. This sequence was sub-cloned from a full-length murine Hx clone (“type”:”entrez-protein”,”attrs”:”text”:”NP_059067″,”term_id”:”160358829″,”term_text”:”NP_059067″NP_059067) purchased from Invitrogen (San Giuliano Milanese, MI, Italy). The recombinant Hx protein was injected in Hx-null mice of C57BL/J6 background, and the producing antibodies were characterized by Western blotting using plasma from wild-type mice. Plasma from Hx-null mice was used as unfavorable control. To test for antibody specificity, pre-adsorption with recombinant Hx was performed. Two monoclonal antibodies (1B5/D4 and Etofylline 3D6/E12) were thus obtained. Tissue and serum iron measurement Mice were anaesthetized with Avertin (2,2,2-tribromoethanol; Sigma-Aldrich, Milano, Italy) at a dose of 2 mg/kg body weight and transcardially perfused with 0.1 M phosphate-buffered.