Enzymatic activation associated with programmed fusion of the posterior interfrontal sutures in rats

Fetal rat coronal sutures in culture undergo fusion in the absence of their dura mater. Coinciding with the period of fusion are marked cellular enzym atic changes. Alkaline phosphatase, a marker of osteoblastic activity, and tartrate-resistant acid phosphatase (TRAP), a marker of osteoclastic activi ty, both increase significantly within fusing sutures and indicate changes in the control of bone synthesis and breakdown. Other enzymes not specifica lly related to bone formation or degradation also show activation within th ese fusing sutures. These enzymes include tartrate-sensitive acid phosphata se (TSAP), a marker of lysosomal activity; hexokinase, a glycolytic enzyme; glucose 6-phosphate dehydrogenase (G6PD), an enzyme of the pentose monopho sphate shunt; and glutathione reductase, an enzyme of the antioxidant pathw ay. In the present study, we compared the enzymatic changes previously seen ex vivo with those occurring in vivo during the programmed closure of the post erior interfrontal suture of the rat. This suture fuses between postnatal d ays 10 and 30 in the rat. The sagittal suture, which remains patent during this period, was used to establish baseline enzymatic activities in a compa rable midline suture. Neonatal rats were killed at postnatal days 2, 4, 5, 8, 10, 12, 15, 20, and 30, and posterior interfrontal and sagittal sutures with bone plates on either side were removed. The suture regions of the sam ples were isolated, dura mater was removed, and suture regions were assayed by microanalytical techniques. Activities of alkaline phosphatase, TRAP, T SAP, hexokinase, G6PD, and glutathione reductase were measured. DNA content was also assayed, and enzyme activities were expressed per amount of DNA. Three pups were killed at each time point, and three to five assays were pe rformed per suture (posterior interfrontal or sagittal) for each time point assayed. Alkaline phosphatase and TRAP activities showed marked increases in fusing sutures compared with non-fusing controls, similar to the increases demonst rated ex vivo. TSAP and hexokinase also showed elevations in the fusing pos terior interfrontal sutures, with the greatest differences predominantly du ring the period of fusion, comparable to the changes seen ex vivo. However, G6PD and glutathione reductase, enzymes of the antioxidant pathway, did no t demonstrate the same degree of activation seen ex vivo in fusing sutures. In fact, the levels were actually higher in the patent sagittal samples fo r the majority of time points examined. Alkaline phosphatase and TRAP activity elevations indicated both osteoblast ic and osteoclastic activation during fusion, as seen in the ex vivo phenom enon. TSAP and hexokinase increases also reflected activation in lysosomes and in cellular metabolism during fusion, paralleling the ex vivo situation . However, a less clear pattern of activation in the antioxidant pathway, i n contrast to the pattern seen ex vivo, was present. These differences may reflect the different environments of sutures in vivo and ex vivo. Alternat ively, oxidative stress may play a more central role in the pathologic proc ess of induced suture fusion ex vivo than in programmed suture fusion in vi vo.