Expression of glutamine transporter isoforms in cerebral cortex of rats with chronic hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that occurs due to acute and chronic liver diseases, the hallmark of which is the increased levels of ammonia and subsequent alterations in glutamine synthesis, i.e. conditions associated with the pathophysiology of HE. Under physiological conditions, glutamine is fundamental for replenishment of the neurotransmitter pools of glutamate and GABA. The different isoforms of glutamine transporters play an important role in the transfer of this amino acid between astrocytes and neurons. A disturbance in the GABA biosynthetic pathways has been described in bile duct ligated (BDL) rats, a well characterized model of chronic HE. Considering that glutamine is important for GABA biosynthesis, altered glutamine transport and the subsequent glutamate/GABA-glutamine cycle efficacy might influence these pathways. Given this potential outcome, the aim of the present study was to investigate whether the expression of the glutamine transporters SAT1, SAT2, SN1 and SN2 would be affected in chronic HE. We verified that mRNA expression of the neuronal glutamine transporters SAT1 and SAT2 was found unaltered in the cerebral cortex of BDL rats. Similarly, no changes were found in the mRNA level for the astrocytic transporter SN1, whereas the gene expression of SN2 was increased by two-fold in animals with chronic HE. However, SN2 protein immuno-reactivity did not correspond with the increase in gene transcription since it remained unaltered. These data indicate that the expression of the glutamine transporter isoforms is unchanged during chronic HE, and thus likely not to participate in the pathological mechanisms related to the imbalance in the GABAergic neurotransmitter system observed in this neurologic condition.

Expression of Glutamate Decarboxylase (GAD) mRNA in the brain of bile duct ligated rats serving as a model of hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neurologic disorder that involves different pathophysiological mechanisms, including disturbances in the GABAergic neurotransmitter system. Albeit an overall increase in the level of neurotransmitter GABA has not been found in HE, alterations in GABA receptors and metabolism have been described. Moreover, it has been reported that bile duct ligated (BDL) rats, an animal model for the study of HE, exhibited an altered GABA biosynthesis involving preferentially the tricarboxylic (TCA) cycle. In this context it should be noted that the GABA synthesizing enzyme glutamate decarboxylase (GAD) is expressed in the brain in two isoforms GAD67 and GAD65, GAD65 being related to the synthesis of GABA that occurs via the TCA cycle and coupled to the vesicular pool of the neurotransmitter. The aim of the present study was to investigate whether changes in mRNA expression of GAD67 and GAD65 were related to the altered GABA biosynthesis previously observed. To study this, cerebral cortices and hippocampi were dissected from control and BDL rats, total mRNA was isolated and cDNA was synthesized by reverse transcription reaction. Subsequently samples were analyzed for gene expression of GAD67 and GAD65 by qPCR multiplex assay, using GAPDH as endogenous control. No changes in GAD67 and GAD65 mRNA expression between control and BDL rats either in cerebral cortex or in hippocampus were observed indicating that the HE condition did not lead to changes in GAD mRNA expression. However, other regulatory mechanism might be affecting GAD activity and to clarify this additional studies need to be conducted.

Impairment of short term memory in rats with hepatic encephalopathy due to bile duct ligation.

Hepatic encephalopathy (HE) arises from acute or chronic liver diseases and leads to cognitive deficits. Different animal models for the study of HE have demonstrated learning and memory impairment and a number of neurotransmitter systems have been proposed to be involved in this. Recently, it was described that bile duct-ligated (BDL) rats exhibited altered spatio-temporal locomotor and exploratory activities and biosynthesis of neurotransmitter GABA in brain cortices. Therefore, the aim of this study was to evaluate cognition in the same animal model. Male adult Wistar rats underwent common bile duct ligation (BDL rats) or manipulation of common bile duct without ligation (control rats). Six weeks after surgery, control and BDL rats underwent object recognition behavioral task. The BDL rats developed chronic liver failure and exhibited a decreased discrimination index for short term memory (STM) when compared to the control group. There was no difference in long term memory (LTM) as well as in total time of exploration in the training, STM and LTM sessions between the BDL and control rats. Therefore, the BDL rats demonstrated impaired STM for recognition memory, which was not due to decreased exploration.

Blood collection for biochemical analysis in adult zebrafish.

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies. There are relatively few publications related to adult zebrafish physiology of organs and systems, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. Recently, Gupta et al. described how to dissect adult zebrafish organs, Kinkle et al. described how to perform intraperitoneal injections, and Pugach et al. described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish. The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.