Rescue of coronal suture fusion using transforming growth factor-beta 3 (Tgf-beta 3) in rabbits with delayed-onset craniosynostosis.

Craniosynostosis results in cranial deformities and increased intracranial pressure, which pose extensive and recurrent surgical management problems. Developmental studies in rodents have shown that low levels of transforming growth factor-beta 3 (Tgf-beta 3) are associated with normal fusion of the interfrontal (IF) suture, and that Tgf-beta 3 prevents IF suture fusion in a dose-dependent fashion. The present study was designed to test the hypothesis that Tgf-beta 3 can also prevent or "rescue" fusing sutures in a rabbit model with familial craniosynostosis. One hundred coronal sutures from 50 rabbits with delayed-onset, coronal suture synostosis were examined in the present study. The rabbits were divided into five groups of 10 rabbits each: 1) sham controls, 2) bovine serum albumin (BSA, 500 ng) low-dose protein controls, 3) low-dose Tgf-beta 3 (500 ng), 4) high-dose BSA (1,000 ng) controls, and 5) high-dose Tgf-beta 3 (1,000 ng). At 10 days of age, radiopaque amalgam markers were implanted in all of the rabbits on either side of the coronal suture to monitor sutural growth. At 25 days of age, the BSA or Tgf-beta 3 was combined with a slow-absorbing collagen vehicle and injected subperiosteally above the coronal suture. Radiographic results revealed that high-dose Tgf-beta 3 rabbits had significantly greater (P < 0.05) coronal suture marker separation than the other groups. Histomorphometric analysis revealed that high-dose Tgf-beta 3 rabbits also had patent coronal sutures and significantly (P < 0.01) greater sutural widths and areas than the other groups. The results suggest that there is a dose-dependent effect of TGF-beta 3 on suture morphology and area in these rabbits, and that the manipulation of such growth factors may have clinical applications in the treatment of craniosynostosis.

Comparison of hedgehog and patched-1 protein expression in the cranial sutures of craniosynostotic and wild-type rabbits.

Craniosynostosis is characterized by premature fusion of the cranial sutures. At the molecular level, mutations in homeobox genes, transcription factors, and growth factor receptors have been implicated in the pathogenesis of this disorder, but the specific etiologic pathways have not yet been elucidated. To further study the molecular biology behind craniosynostosis, perisutural tissues in a unique rabbit model with congenital delayed-onset coronal craniosynostosis were examined for the presence of the hedgehog family of growth factors and their receptor, patched-1. Expression of desert hedgehog, Indian hedgehog, sonic hedgehog, and patched-1 was evaluated in four areas: suture, endosteum, periosteum, and osteocytes, using immuno-histochemistry (n = 8). Protein levels in affected animals were compared with protein levels in wild-type control rabbits (n = 8). Overall, sonic hedgehog, Indian hedgehog, and patched-1 protein levels were greater in affected animals. Specifically, areas of increased staining were seen along the bony interface of the endosteum and periosteum and in the osteocytes of the synostotic rabbits. Interestingly, in the suture, increased levels of Indian hedgehog and sonic hedgehog, but not patched-1, were seen. There was minimal expression of desert hedgehog in both rabbit types. The increased overall presence of hedgehog and patched-1 proteins in synostotic rabbits may be a reactive change to the disorder or part of the pathogenic process. Although the specific cause cannot be determined from the data, it is clear that the molecular milieu of the cranial sutures in synostotic rabbits is markedly different from that of wild-type rabbits.

Changes in the protein expression of hedgehog and patched-1 in perisutural tissues induced by cranial distraction.

With the modern emphasis on minimally invasive therapies, the concept of distraction is being applied in the treatment of craniosynostosis. Although specific genetic mutations have been identified in craniosynostotic patients, changes in the gene expression induced by cranial distraction have not yet been explored. The effects of cranial distraction on hedgehog and patched-1 expression were evaluated in a rabbit model for craniosynostosis. Rabbits (n = 8) were divided into four groups: affected rabbits, wild-type rabbits, affected rabbits subject to cranial distraction, and wild-type rabbits subject to distraction. Perisutural tissue was examined using immunohistochemistry in four areas: suture, endosteum, periosteum, and osteocytes, for the expression of Indian hedgehog, sonic hedgehog, and desert hedgehog and their receptor, patched-1. Two experimental groups were compared: (1) wild-type before distraction to wild-type after distraction, and (2) synostotic before distraction to synostotic after distraction. Distraction produced several variable and interesting changes in hedgehog protein presence. In wild-type rabbits, the predominant effect was a mild decrease in Indian hedgehog levels. Sonic and desert hedgehog and patched-1 protein levels were unchanged. In synostotic rabbits, the predominant effect of distraction was to decrease Indian hedgehog, sonic hedgehog, and patched-1 protein levels. This was especially true in the periosteum and endosteum. Cranial distraction of normal and affected rabbits differentially changed both the expression levels and patterns of the hedgehog and patched-1 proteins in the cranial tissues examined. These results suggest that molecular and genetic parameters of distraction and bone response may be different in craniosynostotic individuals, which may influence treatment protocols in these patients.

Intracranial pressure changes in craniosynostotic rabbits.

Cranial vault and brain deformities in individuals with craniosynostosis are thought to result, in part, from changes in intracranial pressure, but clinical findings are still inconclusive. The present study describes intracranial pressure changes in a rabbit model with naturally occurring, uncorrected coronal suture synostosis. Longitudinal and cross-sectional intracranial pressure data were collected from 241 New Zealand White rabbits, divided into four groups: normal controls (n = 81); rabbits with delayed-onset coronal suture synostosis (n = 78); rabbits with early-onset unilateral coronal suture synostosis (n = 32); and rabbits with early-onset bilateral coronal suture synostosis (n = 50). Epidural intracranial pressure measurements were obtained at 10, 25, 42, and 84 days of age using a NeuroMonitor microsensor transducer. Normal rabbits and rabbits with delayed-onset coronal suture and early-onset unilateral coronal suture synostosis showed a similar oscillating pattern of age-related changes in normal and head-down intracranial pressure from 10 to 84 days of age. In contrast, rabbits with early-onset bilateral coronal suture synostosis showed markedly elevated normal and head-down intracranial pressure levels from 10 to 25 days and showed a different pattern through 84 days. Results from one-way analysis of variance revealed significant (p < 0.01) group differences only at 25 days of age. Rabbits with early-onset bilateral coronal suture synostosis had significantly (p < 0.05) greater normal and head-down intracranial pressure (by 42 percent) than the other three groups. These results showed differing intracranial pressure compensations in rabbits with uncorrected multiple-suture synostosis compared with normal rabbits or rabbits with uncorrected single-suture synostosis, possibly through progressive cerebral atrophy and decreased intracranial volume, abnormal intracranial vascular patterns and blood volume, and/or differing cranial vault compensatory changes.

Early neuromotor behavior in craniosynostotic rabbits.

OBJECTIVE: Clinical studies have shown both abnormal and normal mental and psychomotor development in patients with craniosynostosis. However, a number of confounding variables make study comparisons difficult. For these reasons, the present study describes early neuromotor development in an homogeneous rabbit model of craniosynostosis. DESIGN: Fifty-three newborn New Zealand white rabbit kits were used: 13 were wild-type, normal control rabbits; 23 had delayed-onset coronal suture synostosis (onset is approximately 57 to 74 days post conception); and 17 had early-onset coronal suture synostosis (onset is approximately 21 to 25 days post conception). All rabbits were observed individually and blindly in an open field, daily for 2 minutes, from birth through the first 14 days of life. The first day of emergence of 10 different mature behaviors and developmental events (in developmental order of appearance: falling, righting, cliff avoidance, first sign of fur, body elevation, head elevation, circling, dragging, eye opening, and hopping) was recorded for each kit. Daily activity levels (grid crossing), and body weights were also recorded. RESULTS: Significant group (p <.05) differences were observed in 9 of 11 measures. Both synostosed groups had significantly (p <.05) accelerated onset of behavior in 8 of 9 measures, compared with wild-type controls. The early-onset synostosis group had significantly (p <.05) accelerated onset in five of eight measures, compared with wild-type controls, and three of eight measures, compared with the delayed-onset synostosis group. CONCLUSIONS: Synostotic rabbits showed precocious neuromotor development possibly through frontal lobe constrictions and altered brain activity from increased intracranial pressure, although primary genetic effects cannot be ruled out.