ANIMAL MODELS

Male Sprague‐Dawley rats (body weight, 350‐400 g) were obtained from Charles River Laboratories (Kent, UK).

Under general anesthesia (5% isoflurane in 100% oxygen for induction, 2% isofluorane in air for maintenance) 30 rats underwent triple ligation of the bile duct (way of a small laparotomy) to induce chronic liver injury and were studied 28 days after surgery.17

Thirty‐two rats were administered a hyperammonemic (HA) diet. The amino acid recipe used for a stock of approximately 100 g was: 15 g leucine, 7.7 g phenylalanine, 7 g glutamate, 10 g alanine, 4.4 g proline, 5.8 g threonine, 11 g aspartate, 5 g serine, 4.8 g glycine, 3.3 g arginine, 9.6 g lysine, 8.4 g histidine, 3 g tyrosine, 1.5 g tryptophan, and 10.6 g valine. 25 g of this mix (mixed 1:5 with standard rodent chow powder) was freshly prepared daily with water in a mash form and rats were given free access to it for 5 days. The recipe approximates the amino acid composition of a rodent haemoglobin,18 mimicking the effect of gastrointestinal bleeding, which is known to result in systemic hyperammonemia.19

Three weeks after surgery, 24 bile duct ligation (BDL)‐operated rats were given twice‐daily intraperitoneal injections of combined l‐ornithine and phenylacetate (0.3 g/kg; OP) approximately 7 hours apart for 5 days—a regimen that has been shown previously to effectively reduce plasma ammonia concentration.20 The rats were studied on day 28 after BDL surgery within 3 hours of the last OP injection.

Blood and brain tissue were collected under terminal isoflurane anesthesia. Plasma biochemistry was performed using a Cobas Integra II system (Roche Diagnostics, West Sussex, UK).

Rats were sacrificed humanely by way of isoflurane inhalation overdose. After cardiac perfusion with chilled (4 °C) artificial cerebrospinal fluid (aCSF; 124 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, 1 mM MgSO₄, 10 mM d‐glucose saturated with 95% O₂, 5% CO₂, pH 7.5, PCO₂ 35 mmHg), with an additional 9 mM Mg²⁺, the brains were rapidly removed and placed in a bath of chilled (4 °C‐6 °C) aCSF. Coronal cortical slices (300 µm thick) were cut using a vibrating microtome. The slices were recovered in oxygenated (95% O₂, 5% CO₂) aCSF at room temperature for 30 minutes.