Protein Synthesis Inhibition in Neocortical Grafts Evaluated by Systemic Amino Acid Uptake Autoradiography

Abstract

The temporal pattern of protein synthesis inhibition was examined in grafted neocortical neurons using [3H]valine in vivo autoradiography. Neuronal uptake levels of systemically administered 3H-labeled amino acids which cross the blood–brain barrier (BBB) via endothelial cell neutral carriers have long been a hallmark in studies of experimental ischemic pathology; there is likely a strong correlation between persistent protein synthesis inhibition and the progression of cell damage. Because the grafting procedure involves the loss of blood flow and the subsequent reperfusion of the donor tissue there are, mechanistically, important similarities to reversible ischemia models. The effects of ischemic injury on grafted CNS neurons are not fully understood. Quantitative analysis of grain distribution in individual graft or control (adjacent host cortex) neurons indicated an initial breakdown of the amino acid barrier system, subsequent recovery, and progressive reduction of amino acid uptake by 1 year. Up to 3 weeks after surgery grafts were flooded with the [3H]valine tracer but individual neurons contained relatively few silver grains. After this time, the tracer was normally distributed within graft neurons but at significantly lower levels than in controls. Grain density gradually decreased over time such that 12-month grafted neurons had approximately half that compared to control and only 58% of that in 2-month grafts; the 12-month levels were comparable to those observed at early (10 days) postoperative times. Autoradiography of immunostained sections for MAP-2, SMI 311 (neurofilament marker), and neuron-specific enolase showed reduced expression of these proteins in neurons coupled with weak amino acid tracer uptake. The results further suggest that grafted neurons bear intriguing similarities to neurons placed at ischemic risk, particularly “penumbral” neurons, which are affected by reduced blood flow and are metabolically weakened. The loss of BBB properties in early grafts may also extend to the endothelial cell amino acid carrier system, and the delayed revascularization process could affect neuronal uptake mechanisms.