Blood–brain barrier disruption induced by hemoglobin in vivo: Involvement of up-regulation of nitric oxide synthase and peroxynitrite formation

• Three isoforms of NOS may all be related to the BBB disruption after ICH.
• Excessive ONOO− was formed and detected both in serum and rat brain after ICH.
• ONOO− plays a critical role in the BBB disruption after ICH.
• ONOO− is closely related to edema formation and neurological deficits after ICH.

Accumulating evidence has demonstrated that up-regulation of nitric oxide synthase (NOS) and subsequent peroxynitrite (ONOO−) formation exert a devastating effect on the damage of BBB in multiple diseases. However, considerably less attention has been focused on the role of NOS/ONOO− in BBB disruption after intracerebral hemorrhage (ICH). Using an experimental stroke model by injecting hemoglobin (Hb) into the caudate nucleus of male Sprague Dawley rats, we explored the role of NOS/ONOO− in BBB disruption after ICH. Brain edema content, behavioral changes, alterations of TJ proteins (claudin-5 and ZO-1), expression of neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), formation of 3-nitrotyrosine (3-NT), as well as NO production were investigated. Hb in the rat brain led to a significant brain edema production and neurological deficits. Overexpressed NOS was concomitant with large quantities of 3-NT formation. Moreover, sites of enhanced nNOS, iNOS, eNOS and 3-NT immunoreactivity were colocalized with diminished or discontinuous ZO-1 and/or claudin-5 staining as evidenced by Western blot and immunofluorescence, indicating the involvement of NOS and ONOO− in the BBB disruption. Meaningfully, levels of 3-NT in serum, which had a similar tendency with that of in brain tissues (r=0.934, P<0.001), had a marked correlation with brain edema content (r=0.782, P<0.001) and neurological deficits (r=0.851, P<0.001). We concluded that ONOO− formation by the upregulation of NOS may play a central role in promoting the BBB damage following ICH. Moreover, ONOO− may be a promising biomarker for the judgment or prediction of brain injury and clinical prognosis after ICH.