EFFECT OF VEGF EXPRESSION ON VERTEBRAL GROWTH PLATES FOLLOWING HYPOXIC STRESS

Abstract

The purpose of the study was to examine the effects of vascular-targeted photodynamic therapy (PDT) using benzoporhyrin derivative (BPD) on growth plates in spine and long bones. Specifically we wish to determine whether the ipsilateral up-regulation of VEGF in the thoracic and/or lumbar spine following treatment with leads to onset of scoliosis morphologically similar to idiopathic adolescent scoliosis. And secondly confirm growth plate closure in long bones following BPD-PDT resulting in leg length discrepancy.

A 0.2 mm fiber was placed through an 18g needle onto one side of the distal femoral epiphysis (n=24) or lower thoracic/upper lumbar vertebral bodies of four-week old mice (n=18). Mice are genetically modified to emit bioluminescence upon activation of the vascular endothelial growth factor gene (VEGF). Accurate placement was confirmed using fluoroscopy. BPD (2 mg/Kg, i.v.) was administered systemically and the growth plates were stimulated with 690nm laser light five minutes later. Range of light dose regimens were tested. Animals were followed for a total of seven-twelve weeks post treatment. Faxitron imaging and bioluminescent imaging were obtained to determine leg length or curve progression and VEGF activity. Histology and immunohistochemistry including H& E, HIF-1á, CD31 and VEGF immunohistochemistry was performed.

PDT was able to up-regulate VEGF for up to four weeks following treatment following a percutaneous treatment using a 0.2mm treatment fiber both in the femur and vertebrae. Femoral shortening occurred with histological evidence of bone formation across the growth plate. We were able to identify using faxitron abnormal curvature in a number of the animals that received 5J, 10 mW regimen.

This study confirms that that the epiphyses of vertebrae and long bones are similarly susceptible to the effects of a hypoxic insult resulting in VEGF up-regulation. We are proposing that this stress response can lead to premature closure of epiphyseal growth plates of long bones resulting in limb growth arrest or asymmetric growth of vertebrae and the development of scoliosis in an animal model.