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BACKGROUND:
The accepted paradigm holds that regional differences in the dura are responsible for maintaining cranial suture patency and derangements in tissue signaling result in suture fusion. The role of dura mater in maintaining suture patency is not questioned, but its perceived preeminence may be due to the experimental tools previously available. Staining techniques are variable and subject to interpretation. Dura mater can easily be stripped from the calvaria and supplies sufficient tissue for cell culture and gene expression studies, while suture contents are only a small strip of fibrous tissue that cannot be sufficiently separated from the surrounding bone by dissection for the same analyses. Consequently we are biased to attribute signaling factor production to the dura. Laser capture microdissection (LCM) is a new technique which utilizes a microscope-mounted laser to isolate homogenous cell populations from tissues and allows precise quantification of gene expression through quantitative PCR (qPCR). Using this technique, it is possible to precisely isolate both samples of dura and suture and allow analyses of gene expression within these tissues so we can compare their expression between tissues and between noncraniosynostotic and synostotic animals.
METHODS:
Coronal sutures from 10 day old wild-type (wt) and craniosynostotic (cs) New Zealand White rabbits were frozen sectioned and stained with hematoxylin and eosin. An LCM microscope was used to isolate precisely selected samples of dura and suture contents. RNA was extracted from these LCM samples and linearly amplified before qPCR was performed with samples run in triplicate. Probes for FGF2, TGFβ-2, TGFβ-3, BMP-4, and Noggin were used to assess gene expression levels. Relative expression was determined using ΔΔCt calculations with 18s and GAPDH used as the endogenous control. Relative tissue-specific expression levels were calculated determined using SDS 2.2.2 software with comparisons made between wt and cs samples and between dura and suture samples.
RESULTS:
When comparing expression between tissues in wt rabbits, the suture contents were found to express 45x more TGFβ-3 than wt dura, while the two tissues expressed equal amounts of FGF-2 and Noggin. In the cs individual, TGFβ-3 signaling in the suture decreased to a level 16x less than dural expression; FGF-2 expression in the suture increased to 500x greater than dura; Noggin expression in the dura fell to 2000x less than the suture. These trends were confirmed with comparison of cs and wt tissues with suture contents showing 3x less TGFβ-3 and 10x more FGF-2 than wt. Cs dura made 50x less Noggin than wt.
CONCLUSIONS:
Our preliminary findings suggest within our craniosynostotic rabbit model suture fusion is accompanied by increased FGF-2 and decreased TGFβ-3 and Noggin. These findings are consistent with previous experimental data. The significant finding is that TGFβ-3 and FGF-2 are expressed by the suture contents, rather than dura, suggesting the suture is a dynamic tissue that actively signals to the surrounding tissues rather than passively receiving signals from the dura.