Reduced bone length, growth plate thickness, bone content, and IGF-I as a model for poor growth in the CFTR-deficient rat

<div><p>Background</p><p>Reduced growth and osteopenia are common in individuals with cystic fibrosis (CF). Additionally, improved weight and height are associated with better lung function and overall health in the disease. Mechanisms for this reduction in growth are not understood. We utilized a new CFTR knockout rat to evaluate growth in young CF animals, via femur length, microarchitecture of bone and growth plate, as well as serum IGF-I concentrations.</p><p>Methods</p><p>Femur length was measured in wild-type (WT) and SD-<i>CFTR</i>^{<i>tm1sage</i>} (<i>Cftr-/-</i>) rats, as a surrogate marker for growth. Quantitative bone parameters in <i>Cftr-/-</i> and WT rats were measured by micro computed tomography (micro-CT). Bone histomorphometry and cartilaginous growth plates were analyzed. Serum IGF-I concentrations were also compared.</p><p>Results</p><p>Femur length was reduced in both <i>Cftr-/-</i> male and female rats compared to WT. Multiple parameters of bone microarchitecture (of both trabecular and cortical bone) were adversely affected in <i>Cftr-/-</i> rats. There was a reduction in overall growth plate thichkness in both male and female <i>Cftr-/-</i> rats, as well as hypertrophic zone thickness and mean hypertrophic cell volume in male rats, indicating abnormal growth characteristics at the plate. Serum IGF-I concentrations were severely reduced in <i>Cftr-/-</i> rats compared to WT littermates.</p><p>Conclusions</p><p>Despite absence of overt lung or pancreatic disease, reduced growth and bone content were readily detected in young <i>Cftr-/-</i> rats. Reduced size of the growth plate and decreased IGF-I concentrations suggest the mechanistic basis for this phenotype. These findings appear to be intrinsic to the CFTR deficient state and independent of significant clinical confounders, providing substantive evidence for the importance of CFTR on maintinaing normal bone growth.</p></div

<i>Cftr</i>-/- rats have reduced size and femur length.

<p><b>(</b>A) <i>Cftr-/-</i> rats are notably smaller in size compared to their wild type (WT) littermates. Animal total body weights are demonstrated versus age of the animals in <i>Cftr-/-</i> rats (•) compared to WT rats (▯). These findings are consistent with previously published data by Tuggle et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.ref013" target="_blank">13</a>]. (B) Excised femurs obtained at sacrifice were measured by digital calipers. Femur length is reduced in <i>Cftr-/-</i> rats (•) compared to WT rats (▯) regardless of age or gender (p<0.0001).</p

Micro CT imaging demonstrates reduced bone content.

<p>Representative 3D micro CT images of cortical and trabecular bone structures from young <i>Cftr-/-</i> and WT rats. MicroCT images demonstrate reduced bone content in <i>Cftr-/-</i> rats (more predominantly in females and detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.t001" target="_blank">Table 1</a>). MicroCT images obtained from 38 day old WT and 42 day old <i>Cftr-/-</i> male rats. Female rat microCT images are from 42 day old <i>Cftr-/-</i> and WT rats. MicroCT images are from the male and female rat femurs pictured in histology images (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g004" target="_blank">4</a>).</p

Micro CT of femurs.

<p>Micro CT of femurs.</p

IGF-I is reduced in <i>Cftr-/-</i> rats.

<p>Serum IGF-I concentrations were reduced in <i>Cftr-/-</i> (•) male (261.5 ± 65.7 vs. 850.8 ± 61.1 ng/mL, p<0.0001, (A) and female (512.6 ± 70.1 vs. 1139 ± 149.7 ng/mL, p = 0.001, (B) rats compared to WT (▯). Age and weight adjusted IGF-I concentrations are found in the supplemental figure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.s001" target="_blank">S1 Fig</a>).</p

Femur histomorphometry.

<p>Femur histomorphometry.</p

Cartilaginous growth plate analysis.

<p>Cartilaginous growth plates were evaluated based on overall growth plate thickness, average proliferating zone thickness, average number of proliferating cells/column, average hypertropic zone thickness, mean volume of hypertrophic cells, and mean hypertrophic cell volume standard deviation. Pictured growth plates are from the rats included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g003" target="_blank">Fig 3</a> (as well as microCT images in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g002" target="_blank">Fig 2</a>). They include 38 day old WT (A) and 42 day old <i>Cftr-/-</i> (C) male rats. Female bone images are from 42 day old <i>Cftr-/-</i> (B) and WT (D) rats. Each larger image is at 2X, with inserts demonstrating areas of measurement at 20X. Green outlined cells represent the proliferative zone and blue outlined cells are the hypertrophic zone. Both male and female <i>Cftr-/-</i> rats demonstrated a reduction in overall growth plate thickness (43% reduction in males and 35% in females). However, in males there was also a reduction in the hypertrophic zone thickness, mean volume of hypertrophic cells and hypertrophic cell volume standard deviation in the <i>Cftr-/-</i> rats, but not in the proliferative zone thickness or average number of proliferating cells/column. These findings are suggestive of a difference in the maturation from the proliferative zone into the hypertrophic zone, or differences in cellular activity of the hypertrophic zone, between the <i>Cftr-/-</i> and the WT rats.</p

Histomorphometric analysis.

<p>Representative histology images demonstrate histomorphologic findings of reduced bone content in <i>Cftr-/-</i> rats (more predominantly in females and detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.t002" target="_blank">Table 2</a>). Each figure demonstrates a blue line of 1000 microns in length for size consistency and photographed at 1.25X magnification. Pictured images obtained from 38 day old WT (A) and 42 day old <i>Cftr-/-</i> (C) male rats. Female bone images are from 42 day old <i>Cftr-/-</i> (B) and WT (D) rats. Histology images are from the male and female rat femurs pictured in microCT images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g002" target="_blank">Fig 2</a>).</p