Rats with minimal hepatic encephalopathy due to portacaval shunt show differential increase of translocator protein (18 kDa) binding in different brain areas, which is not affected by chronic MAP-kinase p38 inhibition

Abstract

Neuroinflammation plays a main role in neurological deficits in rats with minimal hepatic encephalopathy (MHE) due to portacaval shunt (PCS). Treating PCS rats with SB239063, an inhibitor of MAP-kinase-p38, reduces microglial activation and brain inflammatory markers and restores cognitive and motor function. The translocator protein-(18-kDa) (TSPO) is considered a biomarker of neuroinflammation. TSPO is increased in brain of PCS rats and of cirrhotic patients that died in hepatic coma. Rats with MHE show strong microglial activation in cerebellum and milder in other areas when assessed by MHC-II immunohistochemistry. This work aims were assessing: 1) whether binding of TSPO ligands is selectively increased in cerebellum in PCS rats; 2) whether treatment with SB239063 reduces binding of TSPO ligands in PCS rats; 3) which cell type (microglia, astrocytes) increases TSPO expression. Quantitative autoradiography was used to assess TSPO-selective (3)H-(R)-PK11195 binding to different brain areas. TSPO expression increased differentially in PCS rats, reaching mild expression in striatum or thalamus and very high levels in cerebellum. TSPO was expressed in astrocytes and microglia. Treatment with SB239063 did not reduces (3)[H]-PK11195 binding in PCS rats. SB239063 reduces microglial activation and levels of inflammatory markers, but not binding of TSPO ligands. This indicates that SB239063-induced neuroinflammation reduction in PCS rats is not mediated by effects on TSPO. Also, enhanced TSPO expression is not always associated with cognitive or motor deficits. If enhanced TSPO expression plays a role in mechanisms leading to neurological alterations in MHE, SB239063 would interfere these mechanisms at a later step.

Fig. 1

Brain regions in which binding of the TSPO-selective ligand ³[H]-PK11195 was quantified. Binding of the TSPO-selective ligand ³[H]-PK11195 was quantified by autoradiography in striatum (STR), frontal cortex (FrCx), parietal cortex (ParCx), thalamus (Tal), hippocampus (HP) and regions 2,3 (CB 2,3) and 8,9 of cerebellum (CB 8,9). The areas quantified are shown in (a). To analyze the ³[H]-PK11195 binding to TSPO the brain was divided in the 4 neuroanatomical levels shown in (b). Level 1 is the most dorsal and the level 4 is the most ventral

Fig. 2

Binding of ³[H]-PK11195 in different regions of neuroanatomical level 1 of control and PCS rats treated or not with the inhibitor of p38 SB239063. a Shows representative autoradiography images for each group. b Shows the quantification of the binding of 3[H]-PK11195 to cerebellar region 2,3 (Cb 2,3) and 8,9 (Cb 8,9), hippocampus (HP), striatum (STR), thalamus (Tal), Frontal cortex (Fr Ctx) and Parietal cortex (Par Ctx). Values are the mean ± SEM of the number of rats indicated in Table 1 for each group. SM = control rats with “sham” operation treated with vehicle, SM + SB = control rats with “sham” operation and treated with SB239063, PCS = PCS rats treated with vehicle, PCS + SB = PCS rats treated with SB239063

Fig. 3

TSPO is expressed in astrocytes and microglia in the cerebellum. Co-expression of TSPO and markers for astrocytes (GFAP) or microglia (Iba-1) was analyzed by double immunofluorescence. All of the experimental groups co-expressed both GFAP and Iba-1 (green) with TSPO (red). Co-expression of GFAP positive cells (astrocytes) with TSPO (arrows) and of the iba-1 positive cells (microglia) with TSPO (arrow heads) is illustrated in the bottom pannels for PCS rats. Scale bar: 25 μm