Hepatic encephalopathy is a neurologic condition associated with failure of the liver to detoxify inhibitory neurotoxins. Hepatic encephalopathy in dogs is most often seen with congenital portosystemic vascular abnormalities, but it may also be seen with hepatopathies associated with acquired portosystemic shunts or with acute fulminant hepatic failure.1
The pathogenesis of hepatic encephalopathy is complex and there are multiple contributing factors, including ammonia, short-chain fatty acids, γ-aminobutyric acid, aromatic amino acids, endogenous benzodiazepines, and manganese.1 There is general consensus that ammonia plays the central role in this pathogenesis.2–11 Ammonia is produced primarily in the gastrointestinal tract by intestinal urease-positive bacteria and amino acid metabolism. Typically, a high amount of ammonia is extracted by the liver via the urea cycle. Blood ammonia concentrations increase and cross the blood-brain barrier in cases of hepatocellular dysfunction or portosystemic shunting, which causes neurotoxicosis. Astrocytes are the site for ammonia detoxification in the brain. Ammonia is eliminated by the amidation of glutamate to glutamine by the enzyme glutamine synthetase.12
Two hypotheses have been proposed to explain the pathogenesis for hepatic encephalopathy: the osmotic gliopathy theory and the Trojan horse theory.2,8,13,14 The osmotic gliopathy theory proposes that glutamine synthesis is stimulated in response to hyperammonemia, which leads to accumulation of glutamine within the astrocytes and subsequent swelling of astrocytes.2,13 According to the Trojan horse theory, glutamine acts as a carrier and transfers ammonia across the mitochondrial membrane. In the mitochondria, glutamine is hydrolyzed by phosphate-activated glutamine synthetase to glutamate and ammonia. In this manner, excessive amounts of glutamine are transported from the cytoplasm to the mitochondria and serve as a carrier of ammonia. The glutamine-derived ammonia within the mitochondria interferes with mitochondrial function, which leads to excessive production of free radicals and induction of mitochondrial permeability transition (a Ca2+-dependent process associated with collapse of the inner mitochondrial membrane potential as a result of opening of the permeability transition pore). These 2 phenomena lead to mitochondrial and astrocyte dysfunction, including cell swelling and energy failure.8,14
There are numerous consequences of ammonia in the CNS. Deleterious effects include amino acid disturbances, alterations in neurotransmission, cerebral energy disturbances, alterations of nitric oxide synthesis and oxidative stress, impairments of axonal and dendritic growth, and alterations in signal transduction and channel transporter activities.2,5,9,10,15
Clinical signs of hepatic encephalopathy in dogs include those of diffuse cerebral disease, such as disorientation, seizures, stupor, and coma.1 In humans, hepatic encephalopathy is divided into 3 categories depending on the nature of the hepatic dysfunction, with further categorization depending on the pattern and severity of clinical signs.16 Type A encephalopathy is associated with acute liver failure, type B with portosystemic bypass without intrinsic liver disease, and type C with liver cirrhosis. Hepatic encephalopathy can be further described clinically as episodic, persistent, or minimal. The latter is associated with cognitive dysfunction but, if misdiagnosed, can lead to overt encephalopathy with a decreased survival rate, poor response to medical management, and irreversible brain damage.17
Proton magnetic resonance spectroscopy allows for determination of the biochemical composition of the brain in vivo and for accurate identification and quantification of metabolites in localized regions.18,19 The metabolites detected in physiologically normal conditions in the brain with long echo time sequences (typically ≥ 144 milliseconds) are N-acetyl aspartate, choline, and creatine. With short echo time sequences (typically 35 milliseconds), other smaller metabolites such as glutamate, glutamine, and myoinositol can be detected.18,19
N-acetyl aspartate resonates at 2.01 ppm. N-acetyl aspartate is considered a neuronal marker, and it is present only in neurons, axons, and dendrites. The choline signal represents a composite peak that consists of contributions from the trimethyl amine, groups of glycerophosphocholine and phosphocholine, and a small amount of free choline. Choline (which resonates at 3.20 ppm) is involved in membrane synthesis and degradation. Creatine (which resonates at 3.03 ppm) is a composite peak that consists of creatine and phosphocreatine, which are compounds involved in energy metabolism via the creatine kinase reaction to generate ATP. Myoinositol (which resonates at 3.5 to 3.6 ppm) is a pentose sugar, which is part of the inositol triphosphate intracellular second-messenger system. Glutamate and glutamine are key compounds in brain metabolism. Glutamate is an excitatory neurotransmitter that plays a role in mitochondrial metabolism, and glutamine plays a role in detoxification and regulation of neurotransmitter activity. At 1.5 T, there is almost complete overlap of the glutamate and glutamine peaks, and the composite peak is often referred to as the glutamine-glutamate complex peak (which resonates at 3.75 ppm and between 2.1 and 2.5 ppm). At 3 T, glutamate and glutamine can be reliably determined with an appropriate pulse sequence (short echo time) and curve fitting methods.20 In pathological conditions, there may be abnormal concentrations (total absence of or lower or higher concentrations) of these metabolites, and other metabolites (eg, lipids or lactate) not typically present in healthy brain tissue may be evident. Proton magnetic resonance spectroscopy is widely used in human medicine for the diagnosis of several intracranial diseases, such as tumors, inflammatory diseases, psychiatric disorders, and metabolic diseases.21–28
The use of 1H MRS for the diagnosis and investigation of pathophysiologic mechanisms of hepatic encephalopathy in humans and other animals has been reported.15,29–38 The main finding in those studies was the increased glutamine-glutamate complex peak and reduced myoinositol peak, which reflect amino acid disturbances and osmoregulation, respectively. The use of 1H MRS in veterinary medicine is not widespread. Recently, 2 studies39,40 have reported reference values for clinically normal Beagles. There are some reports41–44 of experiments in dogs. However, clinical use of 1H MRS has not been extensively reported in veterinary medicine.
The purpose of the study reported here was to investigate changes in the brains of dogs with hepatic dysfunction attributable to a portosystemic shunt, chronic cirrhosis, or acute liver disease by means of 1H MRS with single-voxel and short echo time sequences and to evaluate the clinical use of 1H MRS for the evaluation of dogs with hepatic encephalopathy. We hypothesized that dogs with hepatic encephalopathy would have brain bioprofiles that differed from those of control dogs.
Proton magnetic resonance spectroscopy
Philips Ingenia scanner, Philips AG, Zurich, Switzerland.
dStream HeadSpine coil solution, Philips AG, Zurich, Switzerland.
LCModel, version 6.3, S Provencher, Oakville, ON, Canada.
SPSS Statistics, version 18.104.22.168, 64-bit edition, IBM, Chicago, Ill.
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