Temporal Changes in PTHrP, Bcl-2, Bax, Caspase, TGF-β, and FGF-2 Expression Following Growth Plate Irradiation With or Without Radioprotectant

Abstract

S U M M A R YThis study examined temporal changes in growth plate apoptosis molecules and growth factors in an animal model of radiation injury with and without a radioprotectant. Thirty weanling 5-week Sprague-Dawley rats underwent right knee irradiation with single-fraction 17.5 Gy while the left served as internal control. Six animals each were sacrificed at 0.5, 1, 2, 3, or 4 weeks after irradiation. Half of the animals received pretreatment with amifostine (WR-2721) radioprotectant. Immunohistochemical staining for PTHrP, Bcl-2, Bax, caspase-3, FGF-2, and TGF-␤ was performed. PTHrP decreased to a nadir at 1 week after irradiation but rebounded to above control levels at 2 weeks in the reserve and transitional zones. The radioprotectant amifostine blunted the decrease in PTHrP but kept PTHrP expression lower than controls during the rebound phase in untreated irradiated animals. Hypertrophic zone Bax expression was decreased by amifostine in both irradiated and non-irradiated limbs at 1 and 2 weeks. FGF, TGF-␤ , Bcl-2, and caspase levels generally decreased at 1 week and returned thereafter toward control levels. These findings underscore the importance of PTHrP in response to growth plate irradiation and show the novel finding of a decrease in Bax expression with amifostine pretreatment.

Restoration of growth plate function following radiotherapy is driven by increased proliferative and synthetic activity of expansions of chondrocytic clones

“…20 Specimens were dehydrated through graded concentrations of ethanol, and embedded at À208C in methylmethacrylate plastic resin using a technique shown to optimally preserve growth plate morphology 21 while preserving antigenicity. 7,9 Histology and Immunohistochemistry Serial 5-mm thick sections were cut on a rotary microtome and mounted on silanized glass slides as described previously. 8 Tissue sections for histomorphometric analysis were produced from PGR-fixed tissue that was deplasticized in xylene and stained with a pentachrome protocol described previously.…”

“…8 Tissue sections for histomorphometric analysis were produced from PGR-fixed tissue that was deplasticized in xylene and stained with a pentachrome protocol described previously. 9 Tissue sections for immunohistochemical detection of the injected BrdU label or proliferating cell nuclear antigen (PCNA) were cut from ethanol-fixed tissue hemispheres. PCNA participates as a critical component of the sliding clamp complex associated with DNA polymerase during genomic replication.…”

“…1 and 2). As these labels are restricted to nuclear localization during S-phase, and because not all cellular profiles in a given thin section contain a nucleus, a correction factor was calculated for each morphologically defined zone 9,17 to adjust the labeling percentage of cells to account for the probability of observing a nucleated cellular profile. Thus, the labeling indices presented represent the proportion of labeled cellular profiles per total nucleated cellular profiles in a section.…”

“…Using Image-Pro Plus, lines defining the proximal epiphyseal border, reserve-proliferative interface, proliferativetransitional interface, transitional-hypertrophic zone interface, the metaphyseal chondro-osseous junction, and leading edge of OTC-labeled mineralized tissue were drawn, according to morphological descriptions detailed elsewhere. 9,16,24 (See Table 1. )…”

“…8 Concurrently, evidence of elevated expression of pro-apoptotic indicators Bax and caspase-3, and decreased expression of the proliferation and differentiation regulatory molecules parathyroid hormone-related peptide (PTHrP), basic fibroblastic growth factor (bFGF), and transforming growth factor b (TGFb) have been observed in immunohistochemical studies. 9 By the second week following irradiation, there are initial signs of correction of the morphological and functional nadir that are coincident with the appearance of chondrocytic clones 9 that may serve to restore functional elongation. 10 It is hypothesized that a subpopulation of chondrocytes residing in the reserve zone of the growth plate serve as stem cells that periodically contribute daughter cells that proliferate in a clonal fashion to maintain growth plate cellularity opposing cell loss at the chondro-osseous junction.…”