Sildenafil reduces neuroinflammation in cerebellum, restores GABAergic tone, and improves motor in-coordination in rats with hepatic encephalopathy

Abstract

Aims: Patients with liver disease may develop hepatic encephalopathy (HE), with cognitive impairment and motor in-coordination. Rats with HE due to portacaval shunts (PCS) show motor in-coordination. We hypothesized that in PCS rats: (i) Motor in-coordination would be due to enhanced GABAergic tone in cerebellum; (ii) increased GABAergic tone would be due to neuroinflammation; (iii) increasing cGMP would reduce neuroinflammation and GABAergic tone and restore motor coordination. To assess these hypotheses, we assessed if (i) treatment with sildenafil reduces neuroinflammation; (ii) reduced neuroinflammation is associated with reduced GABAergic tone and restored motor coordination.

Methods: Rats were treated with sildenafil to increase cGMP. Microglia and astrocytes activation were analyzed by immunohistochemistry, extracellular GABA by microdialysis, and motor coordination in the beam walking.

Results: PCS rats show neuroinflammation in cerebellum, with microglia and astrocytes activation, increased IL-1b and TNF-a and reduced YM-1 and IL-4. Membrane expression of the GABA transporter GAT1 is reduced, while GAT3 is increased. Extracellular GABA and motor in-coordination are increased. Sildenafil treatment eliminates neuroinflammation, microglia and astrocytes activation; changes in membrane expression of GABA transporters; and restores motor coordination.

Conclusions: This study supports an interplay between cGMP-neuroinflammation and GABAergic neurotransmission in impairing motor coordination in PCS rats.

Keywords: GABAergic neurotransmission; GAT3; cGMP, neuroinflammation; hepatic encephalopathy; sildenafil.

Figure 1

Proposed model for an interplay between neuroinflammation‐cGMP and GABAergic/glutamatergic neurotransmission in the impairment of motor coordination and cognitive function in rats with MHE

Figure 2

Sildenafil restores motor coordination in PCS rats. Control (SM) and PCS rats treated with tap water or sildenafil (SIL) were subjected to the beam walking test. The number of slips is shown as a measure of in‐coordination. Values are the mean±SEM of 16 sham and PCS rats and 14 sham and PCS rats treated with sildenafil. Values significantly different from controls are indicated by asterisks. *P<.05

Figure 3

Sildenafil reduces GABAergic tone in cerebellum of PCS rats. Microdialysis guide was implanted in cerebellum as described in methods, in the position indicated in (A) and extracellular GABA concentration was measured by microdialysis in freely moving rats (B). Values are the mean±SEM of 14 sham and PCS rats and eight sham and PCS rats treated with sildenafil. Membrane expression of the alpha 5 subunit of GABA_A receptor (C) and of the GABA transporters GAT3 (D) and GAT1 (E) was analyzed using the BS3 cross‐linker procedure as described in methods. Samples, incubated in the absence or presence of BS3, were subjected to Western blotting using the corresponding antibodies. Samples in the absence of BS3 represent the total amount of each protein. Samples in the presence of BS3 represent the nonmembrane fraction. The intensities of the bands were quantified, and membrane expression was calculated as the difference of intensity between samples without and with BS3. Values are expressed as percentage of control rats and are the mean±SEM of five rats per group in (C) and 9‐13 rats per group in (D) and (E). Values significantly different from control rats are indicated by asterisks and from PCS rats by a. *P<.05;***P<.001; ^a P<.05

Figure 4

Sildenafil reduces microglial activation in cerebellum of PCS rats. Immunohistochemistry was performed as indicated in methods using antibody against IBA1 (A). The number of microglial cells (B) and their Perimeter (C) were quantified. Values are the mean±SEM of four rats per group. Values significantly different from controls are indicated by asterisks and from PCS rats by a. *P<.05; ***P<.001; ^aaa P<.005. Scale bar (A)=50 μm

Figure 5

Sildenafil reduces astrocyte activation and the number of IL‐1b‐positive cells in cerebellum of PCS rats. Immunohistochemistry was performed using antibody against GFAP (A) or IL‐1b (B). The area stained by GFAP antibody (C) and the number of IL‐1b‐positive cells (D) were quantified. Values are the mean±SEM of four rats per group. Values significantly different from controls are indicated by asterisks and from PCS rats by a. *P<=0.05; **P<.01; “aaa” P<.005. Scale bar (A)=50 μm

Figure 6

PCS rats show increased content of pro‐inflammatory markers IL‐1b and TNF‐a and reduced content of antiinflammatory YM‐1 and IL‐4 in cerebellum. Sildenafil normalizes their content. Cerebellar homogenates from control (SM) and PCS rats treated with tap water or sildenafil (SIL) were subjected to electrophoresis and Western blot using antibodies against IL‐1b (A); TNF‐a (B), YM‐1 (C), or IL‐4 (D). Representative images of the blots are shown for each protein. Values are the mean±SEM of 10‐12 rats per group. Values significantly different from control rats are indicated by asterisks. Values significantly different from hyperammonemic rats are indicated by “a.” *P<.05; **P<.01; ***P<.001; “a” P<.05; “aa” P<.01