15 Feb 2016

Changes in GABAA Receptor Expression and Brain Cytoskeleton in Normally Grown and Intrauterine Growth Restricted Piglets Across the Perinatal Period

Viskasari Pintoko Kalanjati, August 2010
The University of Queensland

Abstract
Intrauterine growth restriction (IUGR)  is one of the commonest causes of perinatal mortality and neuromorbidity, including a higher propensity to develop seizures and epilepsy. Impairment of GABA (-aminobutyric acid) and glutamate systems and abnormalities of the brain cytoskeleton have been reported in humans, particularly in association with seizures and epilepsy. Whether impairments to the GABA and glutamatergic systems and brain cytoskeleton take place in spontaneously occurring IUGR has yet to be elucidated.

This thesis is directed at understanding the pathophysiology of the neuromobidities which occur in IUGR infants.  GABAA  (-aminobutyric acid type A)  receptor  1,  3  and  2  subunit  protein expression  levels  and distribution  in  parietal cortex and hippocampus  of normally grown (NG) piglets (n=34) were  analysed and compared to those  of  spontaneously occurring IUGR piglets (n=31) at 14 days preterm (P-14), 10 days preterm (P-10), birth (P0) and post-natal 7 days (P7) by western blotting and immunohistochemistry. The four time-points were chosen because GABAA receptor subunit expression is known to be developmentally regulated around birth. Immunohistochemistry of GAD67 (glutamic acid decarboxylase 67) was used to compare GABAergic-positive cell expression in NG and IUGR piglet brains in the four age groups; with GLAST1b (glutamate-aspartate transporter 1b) performed to label dysfunctional neurons. Gray and white matter brain cytoskeleton was studied by MAP2 (microtubule-associated protein 2) and MBP (myelin basic protein) immunolabelling to allow visualisation of any impairments to neuronal somatodendrites and myelinated axonal fibres respectively.

In NG piglets,  significantly higher 1 expression in P7 cortex was observed compared to all other age groups (p<0.05); whilst in hippocampus it was significantly higher in P7 compared to the P-14 and P-10 groups and in P0 compared to the P-14 group (p<0.05). In IUGR piglets, 1 expression in cortex increased in preterm groups to P7 and a similar trend was seen in hippocampus with significant differences found only in P7 compared to the P-10 group (p<0.05).  In NG piglets, 3 subunit expression in cortex peaked at P0, when it was significantly higher than at P7 (p<0.05); a similar trend was seen  in hippocampus. In IUGR cortex and hippocampus however, 3 expression was highest at P-14 compared to the other age groups but this was not statistically significant. In P7 IUGR cortex, the 3 expression was significantly higher than in P7 NG cortex (p<0.05) whilst the 1  subunit expression tended to be lower (p=0.1). There was a  significantly lower  1/3  in P7 IUGR cortex than P7 NG cortex  (p<0.05).  In NG and IUGR piglets, 2 expression in cortex and hippocampus did not differ from the P-14 to the P7 group.

There were no obvious differences on the laminar and cellular distributions of GABAA receptor 1, 3 and 2 subunits  in NG and IUGR piglet parietal cortex and hippocampus across the age groups. Immunolabelling of  the 1, 3  and 2  subunits were shown throughout all  cortical  layers with an intense 2 labelling observed in layer IV, whilst the 3 subunit was predominantly observed in layer V-VI.  The 1 and 2 subunits were widely distributed in all strata in the hippocampus of NG and IUGR piglets, whereas the 3 subunit was minimally expressed. At the cellular level, these subunits were observed in the neuropiles, cell bodies and processes of pyramidal and non-pyramidal neurons in both NG and IUGR cortex and hippocampus. No obvious differences in GLAST1b expression were found in NG and IUGR piglets. However neuronal somatodendrite labelled by MAP2 was impaired in P7 IUGR parietal cortex and CA1 hippocampus compared to the P7 NG. An obvious reduction in myelinated axonal fibres labelled by MBP was observed in subcortical parietal white matter of P-14, P-10 and P0 IUGR piglets when compared to NG piglets at each age group. However, there was no clear difference in the P7 group or in hippocampal MBP labelling between the IUGR and NG piglets in each age group.

In conclusion, GABAA  receptor  1,  3  and  2  subunit protein expression is developmentally regulated in a specific manner in NG piglet cortex and hippocampus; with significant impairments observed in P7 IUGR cortex when compared to P7 NG cortex. Brain cytoskeleton abnormalities detected using MAP2 and MBP in IUGR piglet cortex and hippocampus, were observed during the perinatal time period. These findings collectively contribute to understanding the underlying mechanisms involved in IUGR neuromorbidity and may partly explain the propensity to seizures and epilepsy found in the clinical condition of IUGR. 

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