Frontal Bone Insufficiency in Gsk3ß Mutant Mice.

The development of the mammalian skull is a complex process that requires multiple tissue interactions and a balance of growth and differentiation. Disrupting this balance can lead to changes in the shape and size of skull bones, which can have serious clinical implications. For example, insufficient ossification of the bony elements leads to enlarged anterior fontanelles and reduced mechanical protection of the brain. In this report, we find that loss of Gsk3ß leads to a fully penetrant reduction of frontal bone size and subsequent enlarged frontal fontanelle. In the absence of Gsk3ß the frontal bone primordium undergoes increased cell death and reduced proliferation with a concomitant increase in Fgfr2-IIIc and Twist1 expression. This leads to a smaller condensation and premature differentiation. This phenotype appears to be Wnt-independent and is not rescued by decreasing the genetic dose of ß-catenin/Ctnnb1. Taken together, our work defines a novel role for Gsk3ß in skull development.

Regional NAD(P)H:quinone oxidoreductase activity in Alzheimer's disease.

Converging evidence supports the role of oxidative stress in the pathology of Alzheimer's disease (AD). This notion is further supported by recent findings of increased NAD(P)H:quinone oxidodreductase (NQO1) activity, a potent antioxidant system, in association with hippocampal AD pathology. If increased NQO1 activity is truly related to the AD process, however, we would expect to see regional co-localization of NQO1 activity with AD pathology throughout affected brain regions and the absence of NQO1 activity in regions unaffected by AD. We examined this hypothesis by measuring NQO1 enzymatic activity and NQO1 immunohistochemical staining in regions commonly affected by the AD process such as frontal cortex and compared this to regions generally unaffected by the AD process such as occipital cortex, cerebellum, and substantia nigra for a group of AD patients and controls. The ratio of frontal to cerebellar NQO1 enzymatic activity was significantly increased in patients with AD (2.07 +/- 1.90) versus controls (0.60 +/- 0.31; P < 0.03). Moreover, regional immunohistochemical staining revealed specific localization of NQO1 staining to astrocytes and neurites surrounding senile plaques. The extent of immunohistochemical staining also closely correlated with the extent of local AD pathology across the various brain regions examined. Neuronal NQO1 staining seen in frontal cortex of AD patients was absent in frontal cortex of controls, but was found to the same extent in neurons of the substantia nigra of both AD patients and controls. We conclude that NQO1 activity co-localizes closely with AD pathology supporting a presumed role as an antioxidant system upregulated in response to the oxidative stress of the AD process. The antioxidant role for NQO1 is further supported by finding increased neuronal NQO1 activity in substantia nigra neurons of both AD patients and controls as this neuronal population is known to be under constant oxidative stress. While requiring further study, these findings, in conjunction with previous work, suggest that increased NQO1 activity may be neuroprotective, may offer novel insights into the pathophysiology of AD and may also provide possible avenues for future treatment.

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 enzymatic changes. Alkaline phosphatase, a marker of osteoblastic activity, and tartrate-resistant acid phosphatase (TRAP), a marker of osteoclastic activity, both increase significantly within fusing sutures and indicate changes in the control of bone synthesis and breakdown. Other enzymes not specifically related to bone formation or degradation also show activation within these fusing sutures. These enzymes include tartrate-sensitive acid phosphatase (TSAP), a marker of lysosomal activity; hexokinase, a glycolytic enzyme; glucose 6-phosphate dehydrogenase (G6PD), an enzyme of the pentose monophosphate shunt; and glutathione reductase, an enzyme of the antioxidant pathway. In the present study, we compared the enzymatic changes previously seen ex vivo with those occurring in vivo during the programmed closure of the posterior interfrontal suture of the rat. This suture fuses between postnatal days 10 and 30 in the rat. The sagittal suture, which remains patent during this period, was used to establish baseline enzymatic activities in a comparable 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 samples were isolated, dura mater was removed, and suture regions were assayed by microanalytical techniques. Activities of alkaline phosphatase, TRAP, TSAP, 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 performed per suture (posterior interfrontal or sagittal) for each time point assayed. Alkaline phosphatase and TRAP activities showed marked increases in fusing sutures compared with nonfusing controls, similar to the increases demonstrated ex vivo. TSAP and hexokinase also showed elevations in the fusing posterior interfrontal sutures, with the greatest differences predominantly during the period of fusion, comparable to the changes seen ex vivo. However, G6PD and glutathione reductase, enzymes of the antioxidant pathway, did not demonstrate the same degree of activation seen ex vivo in fusing sutures. In fact, the levels were actually higher in the patent sagittal samples for the majority of time points examined. Alkaline phosphatase and TRAP activity elevations indicated both osteoblastic and osteoclastic activation during fusion, as seen in the ex vivo phenomenon. 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, in contrast to the pattern seen ex vivo, was present. These differences may reflect the different environments of sutures in vivo and ex vivo. Alternatively, oxidative stress may play a more central role in the pathologic process of induced suture fusion ex vivo than in programmed suture fusion in vivo.

Regeneration of cranial suture and bone plate lesions in rabbits. Implications for positioning of osteotomies.

The positioning of osteotomies in intramembranous cranial bone was studied by exploring the pattern of bone regeneration in growth areas (the sutural region) as compared to that of the bone plate proper. Trephine defects in the left coronal suture area and the right parietal bone were produced in fifty-nine young rabbits. A pilot study to refine operative and analytical methods comprised 22 animals. The experiments were terminated at one, three, and six weeks after surgery. The bone regenerative response was assessed by x-ray planimetry, plain microscopy, enzyme histochemistry, and fluorescent labelling. Only minor divergences in healing capacity between the two defects were found. No adverse effects on the growth process were indicated. As to clinical management, the findings suggest that osteotomies designed to traverse sutural areas will, under normal circumstances, regenerate in a similar manner and rate to adjoining bone plates.

A study on the occurrence of alkaline phosphatase in the sutura interfrontalis of Wistar rats.

The aim of the present study was to determine the presence of alkaline phosphatase during various stages in development and closure of the sutura interfrontalis. The histological sections reveal that this enzyme could primarily be demonstrated in the dura mater of this suture. In further developmental stages, alkaline phosphatase could be observed within the intermediate zone as well as the pericranium. These findings are brought in relation with the occurrence of synostosis which can be induced under experimental conditions.