Volume 139, Issue 2, Pages 675-684 (August 2010) Hyperammonemia Induces Neuroinflammation That Contributes to Cognitive Impairment in Rats With Hepatic Encephalopathy  Regina Rodrigo, Omar Cauli, Ulises Gomez–Pinedo, Ana Agusti, Vicente Hernandez–Rabaza, Jose–Manuel Garcia–Verdugo, Vicente Felipo  Gastroenterology  Volume 139, Issue 2, Pages 675-684 (August 2010) DOI: 10.1053/j.gastro.2010.03.040 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Chronic hyperamonemia or bile-duct ligation (BDL) induce microglial activation, which is reversed by chronic treatment with ibuprofen. Expression of major histocompatibility complex class II (MHCII), a marker of microglia activation, was analyzed in sections from control (C) and hyperammonemic rats (HA) (A) and from BDL rats and sham-operated controls (SM) (B), treated with vehicle (VH) or ibuprofen (IBU). Control rats (C-VH), sham controls (SM-VH), and hyperammonemic (HA-IBU) or BDL (BDL-IBU) rats treated with ibuprofen show typical morphology of resting microglia (ramified). Untreated hyperammonemic (HA-VH) or BDL (BDL-VH) rats show reactive microglia with ameboid shape. Gastroenterology 2010 139, 675-684DOI: (10.1053/j.gastro.2010.03.040) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Time course of hyperammonemia-induced microglial activation in cerebellum. Sections from control (C) and hyperammonemic rats (HA) were used to analyze the expression of major histocompatibility complex class II (MHCII). (A) Five virtual rings were drawn around each microglial cell, and the number of intersections of microglial processes with the rings was quantified. (B–D) show the mean number of intersections of microglial processes with rings in control (C) and hyperammonemic (HA) rats at 1 (B), 7 (C), or 28 days (D) of hyperammonemia. Total numbers of intersections/cell are shown in (E). Values are the mean ± standard error of mean of 6 rats. Values significantly different from controls are indicated by asterisks. (Unpaired t test, *P < .01; ** P < .001). Gastroenterology 2010 139, 675-684DOI: (10.1053/j.gastro.2010.03.040) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Chronic hyperammonemia or bile-duct ligation (BDL) increases the amount of inducible nitric oxide (iNOS) and major histocompatibility complex class II (MHCII) in cerebellum. Cerebella of control (C) or hyperammonemic (HA) rats and of BDL rats and its controls (SM), treated with vehicle (VH) or ibuprofen (IBU), were homogenized and the homogenates (80 μg protein) subjected to electrophoresis. iNOS (A, C) and MHCII (B, D) content were analyzed by immunoblotting. Representative immunoblots are shown. The intensities of the bands were quantified and are expressed as a percentage of control rats (C-VH or SM-VH). Values are the mean ± standard error of mean of 8 rats. Values significantly different from control rats are indicated by asterisks and from hyperammonemic rats by “a”. *P < .05; aP < .05). Gastroenterology 2010 139, 675-684DOI: (10.1053/j.gastro.2010.03.040) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Chronic hyperammonemia or bile-duct ligation (BDL) increases extracellular levels of prostaglandin E2 (PGE2) in cerebellum. Treatment with ibuprofen normalizes PGE2 levels. Prostaglandin E2 concentrations were determined by in vivo brain microdialysis in the extracellular fluid of cerebellum of control (C) and hyperammonemic (HA) rats (A) and of BDL rats and their controls (SM) (B), treated with vehicle (VH) or ibuprofen (IBU). Values are mean ± standard error of mean of 10 rats in (A) and 8 rats in (B). Values significantly different from control rats are indicated by asterisks and from hyperammonemic or BDL rats by “a”. *P < .01; aP < .001. Gastroenterology 2010 139, 675-684DOI: (10.1053/j.gastro.2010.03.040) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Chronic treatment with ibuprofen restores learning ability in hyperammonemic and bile-duct ligation (BDL) rats. Control (C) and hyperammonemic (HA) rats (A) and BDL rats and their controls (SM) (B), treated with vehicle (VH) or ibuprofen (IBU), were subjected to the learning test after 10 days of treatment. Values are the mean ± standard error of mean of 8–9 rats and are given as the number of trials needed to learn. Values significantly different from control rats are indicated by asterisks and from hyperammonemic or BDL rats by “a”. *P < .05; aP < .05). Gastroenterology 2010 139, 675-684DOI: (10.1053/j.gastro.2010.03.040) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Chronic treatment with ibuprofen restores motor activity in hyperammonemic and bile-duct ligation (BDL) rats. Motor activity was analyzed in control (C) and hyperammonemic (HA) rats (A) and BDL rats and their controls (SM) (B), treated with vehicle (VH) or ibuprofen (IBU). Values are the mean ± standard error of mean of 9 rats and are given as ambulatory counts in 60 minutes. Values significantly different from control rats are indicated by asterisks and from hyperammonemic rats by “a”. *P < .05; aP < .05). Gastroenterology 2010 139, 675-684DOI: (10.1053/j.gastro.2010.03.040) Copyright © 2010 AGA Institute Terms and Conditions