Quantitative immunofluorescence data suggest a permanently enhanced GAD67/GAD65 ratio in nerve endings in rat cerebral cortex damaged by early postnatal hypoxia-ischemia: a comparison between two computer-assisted procedures for quantification of confocal laser scanning microscopic immunofluorescence images

Abstract The aim of the present study was 2-fold: (1) to determine the ratio between the amount of GAD 67 and GAD 65 (two isoforms of the GABA synthetizing enzyme glutamic acid decarboxylase) in nerve endings in the mature rat cerebral cortex damaged by hypoxia-ischemia during early postnatal life; and (2) to compare two different computer-assisted procedures developed for quantitative analysis of immunofluorescence images obtained with a confocal laser scanning microscope (CLSM). One procedure was based on a program present in the standard Leica CLSM software packet for full-field analysis, the other on a specially written program for object-oriented analysis run on a Kontron IBAS-KAT image analysis system. To this end, rat pups were unilaterally exposed to hypoxic-ischemic conditions and, after a survival period of 6.5 months, sacrificed by perfusion fixation. After dissection of the brain and vibratome sectioning, three animals with substantial damage on one cortical side were selected. Sections of these animals were double-stained with primary antibodies against GAD 67 and GAD 65 and fluorophore-conjugated secondary antibodies and subsequently sampled with a CLSM. Analysis of the CLSM images with both computer-assisted procedures showed for all three animals a clear tendency to higher GAD 67 /GAD 65 ratios in cortical GABAergic nerve endings on the hypoxia-damaged side than in matched areas on the contralateral side. This outcome led to the following conclusions. (1) The correspondence between the outcome of both analysis procedures indicates that both procedures are valid for quantification of immunofluorescence images of nerve endings obtained with a CLSM. (2) The outcome lends further support to our view that hypoxic-ischemic encephalopathy, sustained during early postnatal life, may result in an unstable cortical network generating abnormal synchronizations and oscillations which can be amplified and propagated as true epileptic discharges. In such a network both excitatory and inhibitory processes are tonically enhanced, the latter probably as a homeostatic reaction tending to keep abnormal excitation within physiological limits.