Murine model to study brain, behavior and immunity during hepatic encephalopathy

Figure 1 Thioacetamide treatment triggered neuropsychomotor changes, diffuse brain edema and hyperammonemia, with electroencephalography spectrum compatible with of encephalopathy. A: Survival rate following repeated TAA injections (arrows); B: Neurological score was assessed through all experimental procedure. Neuropsychomotor deficit increases as higher is the score average, with 1 = normal and 5 = death; C: Mice treated with TAA also presented with significant weight loss in comparison to controls; D, E: Serum ammonemia was detected following 24 h of TAA injection, while brain ammonia concentration increased gradually, reaching significant higher values after 48 h (E); F: In agreement, MRI from TAA-treated mice brains showed discrete, but diffuse edema as pointed by arrow heads. G, H: EEG analysis revealed that while controls had coincident EEG records during the experimental protocol (G), brain waves spectrum of TAA-treated mice (H) was compatible with metabolic encephalopathy; I: 3D rendering of 60 minutes EEG record (Z axis) plotted comparing energy (in Y axis) with frequency (X axis). Note the increase in higher frequencies waves (theta and alpha; 4-8 and 8-13 Hz, respectively) with a concomitant increase of lower frequency ones (mainly delta; up to 4 Hz). Best case results were depicted here; N = 8 for each group; J: Total EEG energy was also reduced in TAA-treated group. aIndicates statistical significance in comparison to controls and bin comparison to 24 h group. P < 0.05, analysis of variance (Tukey’s post test). N≥ 5 for each group. TAA: Thioacetamide; EEG: Electroencephalography.

Figure 2 Thioacetamide caused severe liver necrosis and inflammation, which may explain mice metabolic encephalopathy, coagulopathy and remote lung injury. A: Liver intravital microscopy showing crescent number of necrotic cells (in red, propidium iodide) with concomitant neutrophil infiltration (in green, Lysm-eGFP mice); B: Liver sections stained by hematoxylin and eosin (4 × increase). Arrows indicate necrotic areas; C, D: Liver macroscopic analysis confirmed extensive and diffuse necrosis, which is also reflected by elevated serum transaminase activity (D); E, F: Liver failure was confirmed by prolonged prothrombin and partial thromboplastin times; G, H: Also, significant drop in mean arterial pressure (MAP; G) and increased heart rate (H), as assessed by tail cuff method, suggested that TAA-treated mice also evolved to hemodynamic shock; I: After TAA treatment hours, lungs from mice had increased cellularity in lung parenchyma, alveolar edema and hemorrhage in comparison to controls (arrows, 4 x increase) aIndicates statistical significance in comparison to controls. ^aP < 0.05, ANOVA (Tukey’s post test). N ≥ 5 for each group. TAA: Thioacetamide.