The rats were randomly distributed into four groups: the control, control+infliximab, hyperammonemic, and hyperammonemic+infliximab group. Infliximab is an anti-TNF-a antibody that does not cross the blood-brain barrier [40 (link)], binds to peripheral TNF-a, and reduces peripheral inflammation in rats with MHE or hyperammonemia [9 (link), 10 (link), 22 ]. Infliximab (Remicade; Merck Sharp & Dohme, Spain) was dissolved in saline (0.9% sodium chloride) and administered by i.v. injection (5 mg/kg) in the tail vein as described previously [41 (link)]. The first administration of infliximab was performed 2 days before the start of the ammonium-containing diet, and injections were repeated weekly for 4 weeks. Control rats were injected i.v. with saline. At the end of the 4th week, the rats were sacrificed by perfusion for in vivo immunohistochemical analysis of microglial and astrocyte activation (blue box), the protein expression of nuclear NF-kB and TNF-a in glia and Purkinje neurons (green box), and the mRNA expression of TNF-a in glia and Purkinje neurons (violet box) in the cerebellum (Fig. 1a).
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