Changes in mRNA gene expression during growth in the femoral head of the young rat.

The rate of physeal growth slows as an animal matures with changes in mRNA gene expression due to the altered cellular activity. To measure the change in gene expression during the juvenile growth period, the femoral head, enclosing the proximal femoral physis, primary spongiosa, and articular cartilage, was collected from both femora of 16 female Sprague-Dawley rats between 4 and 10 weeks of age. One femur of each rat had had a mid-diaphyseal femoral fracture at 4 weeks of age. RNA was extracted and hybridized to 16 Affymetrix Rat Genomic 230 2.0 GeneChip microarrays with probe sets for 31,000 genes of which 18,200 were expressed. Of these, 8002 genes had a significant change in gene expression during growth, about half increasing and half decreasing. These changes included up-regulation with time of genes related to cartilage, blood vessels, osteoprotegerin, osteomodulin, and most ribosomal proteins. There was down-regulation with maturity of genes related to bone, growth-promoting cytokines, G proteins, GTPase-mediated signal transduction factors, cytokine receptors, mitosis, integrin-linked kinase, and the cytoskeleton. In summary, the slowing of growth with maturity was associated with changes in mRNA gene expression in the femoral head for a large number of genes. These changes in gene expression between young and mature rats suggest factors which are important for the support of the rapid linear growth during early life.

Genes with greater up-regulation in the fracture callus of older rats with delayed healing.

The rate of bone formation to bridge a fracture gap slows with age. To explore potential pathogenic mechanisms and possible negative-feedback responses by the skeleton to this reduced rate of healing, mRNA transcripts up-regulated more and/or longer were studied in older rats with delayed healing. Female rats at 6 (young), 26 (adult), and 52 (old) weeks of age received unilateral diaphyseal femoral fractures with intramedullary rod stabilization. At 0, 0.4, 1, 2, 4, and 6 weeks after fracture, the fracture site was harvested. Total RNA was extracted, cRNA was prepared, and the cRNA was hybridized to 54 Affymetrix U34A microarrays (three arrays/age/time point). Transcripts for 180 genes were identified as up-regulated more and/or longer in old rats with delayed fracture healing. Of these, 60 were selected for more intense review. Significantly more and/or longer expression was seen in genes related to myofibroblasts, cell proliferation, calcification inhibition, TGF-beta activity, lipid metabolism, cell adhesion, and the cytoskeleton. Further study is needed to determine if these up-regulated transcripts are related to the pathological processes which slow healing or are related to attempts by the fracture tissue to stimulate bone to bridge the fracture gap.

Evidence for overgrowth after midfemoral fracture via increased RNA for mitosis.

Middiaphyseal femoral fractures in children and young rats stimulate linear femoral growth, a phenomenon commonly attributed to increased vascularity. To test for changes in mRNA expression of genes related to blood vessels, nerve fibers, cartilage, bone, and cell metabolism, we measured mRNA gene expression for all known rat genes in the physis at various times after diaphyseal fracture. Female Sprague-Dawley rats, 4 weeks of age at surgery, were subjected to a unilateral, simple, transverse, middiaphyseal femoral fracture stabilized with an intramedullary rod. At 0 (intact), 0.1, 0.4, 1, 2, 3, 4, and 6 weeks after fracture, the femoral head with the proximal physis was collected from fractured and intact femora. The RNA was extracted, processed to biotinlabeled cRNA, and hybridized to Affymetrix Rat 230 2.0 GeneChip microarrays. Transcripts from fracture-induced lengthening of the injured femora were compared to those of the intact contralateral femur. In the proximal physis, transcripts related to blood vessels and cartilage formation were down-regulated by fracture. Transcripts related to bone remodeling, nerve axon elongation, cell division, and protein synthesis were up-regulated by fracture. The data support increased mitotic activity in the physis after a midshaft fracture and not increased vascularity.

Young, adult, and old rats have similar changes in mRNA expression of many skeletal genes after fracture despite delayed healing with age.

Genes active in fracture healing are not well understood. Because age slows skeletal repair, the change in gene expression between animals of differing ages may illuminate novel pathways important to this healing response. To explore this, 6-, 26-, and 52-week-old female Sprague-Dawley rats were subjected to mid-diaphyseal femoral fracture with intramedullary fixation. The fracture callus was collected at 0, 0.4 (3 days), 1, 2, 4, or 6 weeks after fracture. RNA was extracted and pooled between two animals for each sample. Three samples were done for each time point for each age for a total of 54 Affymetrix U34A GeneChip microarrays. Of the 8700 genes on each array, 3300 were scored as present. Almost all of these genes were affected by femoral fracture with either upregulation or downregulation in the 6 weeks after fracture. Upregulated genes included markers for matrix genes for both cartilage and bone, osteoblasts, osteocytes, osteoclasts, fibroblasts, and mast cells. Downregulated genes included genes related to blood cell synthesis. Nearly all genes presently associated with bone metabolism showed the same response to fracture healing regardless of the age of the animal. In conclusion, skeletal fracture led to similar changes in RNA expression for most skeletal genes despite the delay in the formation of bone to bridge the fracture gap in old rats. Defects in the healing of skeletal trauma in older rats may lie in systems not normally studied by skeletal biologists.

Gene expression during fracture healing in rats comparing intramedullary fixation to plate fixation by DNA microarray.

OBJECTIVE: This study was designed to compare mRNA gene expression in healing diaphyseal femoral fractures between those injuries treated with intramedullary nails and those treated with internal plate fixation. DESIGN: RNA gene expression was measured at 1 day, 3 days, and 1, 2, 4, and 6 weeks after surgery in the fracture callus of rats randomized to femoral shaft fracture with intramedullary nail fixation, rigid plate fixation, or sham fracture. SETTING: AAALAC-accredited vivarium of an independent academic medical center. ANIMALS: Fifty-seven, adult, female, Sprague-Dawley rats at 16 weeks of age. INTERVENTION: Femoral fracture with intramedullary nail fixation, femoral fracture with plate and screw fixation, or sham surgery with no fracture. MAIN OUTCOME MEASUREMENTS: RNA expression for 8700 genes was measured with 19 Affymetrix U34A microarrays. The fracture callus was significantly larger with intramedullary nail fixation than with plate fixation. Most genes responded to fracture with a change in mRNA expression. Most of the responding genes followed the same time course for both fixation methods. This included genes related to growth factors, bone matrix, mast cells, most nerve factors, and hematopoiesis. The intramedullary nail group had significantly greater up-regulation for transcripts related to cartilage, cell division, inflammation, and the acetylcholine receptor. There was significantly greater up-regulation in the plate group for genes related to macrophage activity. CONCLUSIONS: There were differentially expressed genes present between the 2 surgical groups that may give insight into the control of fracture repair.

Altered expression of mitochondrial genes in response to fracture in old rats.

BACKGROUND: Old rats require more time for bone to bridge a fracture gap than young rats. To explore possible mitochondrial dysfunction in this delay, we measured levels of mRNA derived from mitochondrial genes in healing fractures of young, adult, and old rats. METHODS: Diaphyseal femoral fractures were induced in female rats at 6, 26, and 52 weeks of age (young, adult, and old rats, respectively). At baseline, at 3 days, and 1, 2, 4, and 6 weeks after fracture, the fracture site was harvested. Total RNA was extracted, and cRNA was prepared and hybridized to 54 Affymetrix U34A microarrays (2 rats/array and 3 arrays/age/time point). RESULTS: Radiographic union occurred progressively later with age. Of the 107 mitochondria-related genes reviewed, all 8 located within the mitochondrial DNA and some nuclear genes (of the electron transport chain and tricarboxylic acid cycle) showed a prolonged reduction in gene expression after fracture in the oldest rats, to about half the level of expression detected in young rats at 6 weeks after fracture. The transcript levels of apoptotic genes increased after fracture in rats of all three ages, while the transcript levels of 23 mitochondriarelated genes were largely unaltered after fracture. INTERPRETATION: Delayed fracture healing in old rats is associated with reduced mRNA expression of genes forming the mitochondrial energy pathways.

Altered mRNA expression of genes related to nerve cell activity in the fracture callus of older rats: A randomized, controlled, microarray study.

BACKGROUND: The time required for radiographic union following femoral fracture increases with age in both humans and rats for unknown reasons. Since abnormalities in fracture innervation will slow skeletal healing, we explored whether abnormal mRNA expression of genes related to nerve cell activity in the older rats was associated with the slowing of skeletal repair. METHODS: Simple, transverse, mid-shaft, femoral fractures with intramedullary rod fixation were induced in anaesthetized female Sprague-Dawley rats at 6, 26, and 52 weeks of age. At 0, 0.4, 1, 2, 4, and 6 weeks after fracture, a bony segment, one-third the length of the femur, centered on the fracture site, including the external callus, cortical bone, and marrow elements, was harvested. cRNA was prepared and hybridized to 54 Affymetrix U34A microarrays (3/age/time point). RESULTS: The mRNA levels of 62 genes related to neural function were affected by fracture. Of the total, 38 genes were altered by fracture to a similar extent at the three ages. In contrast, eight neural genes showed prolonged down-regulation in the older rats compared to the more rapid return to pre-fracture levels in younger rats. Seven genes were up-regulated by fracture more in the younger rats than in the older rats, while nine genes were up-regulated more in the older rats than in the younger. CONCLUSIONS: mRNA of 24 nerve-related genes responded differently to fracture in older rats compared to young rats. This differential expression may reflect altered cell function at the fracture site that may be causally related to the slowing of fracture healing with age or may be an effect of the delayed healing.

The genomic response of the mouse kidney to low-phosphate diet is altered in X-linked hypophosphatemia.

The mechanism for the renal adaptation to low-phosphate diets is not well understood. Whether the Hyp mutation of the Phex gene blocks this adaptation is also not clear. To gain further insight into this, 5-wk-old normal and Hyp mice were fed a control (1.0% P) or low-phosphate diet (0.03% P) for 3-5 days. Renal RNA was hybridized to Affymetrix U74Av2 microarrays (5 arrays/group). Of the 5,719 detectable genes on each array, 290 responded significantly (P < 0.01) to low-phosphate diet in normal mice. This was reduced significantly (P < 0.001) to 7 in the Hyp mice. This suggested that the adaptations of the normal kidney to a low-phosphate environment were blocked by the Hyp mutation. The Npt2 phosphate transporter, vitamin D 1alpha- and 24-hydroxylases, and calbindins D9K and D28K responded in the expected fashion. Genes with significant (P < 0.05) diet-by-genotype interaction were analyzed by GenMAPP and MAPPFinder. This revealed a cluster of differentially expressed genes associated with microtubule-based processes. Most alpha- and beta-tubulins and most kinesins had responses to low-phosphate diet in normal mice which were abolished or reversed in Hyp mice. In summary, renal adaptation to low-phosphate diet involved changes in the mRNA expression of specific genes. Disruption of these responses in Hyp mice may contribute to their abnormal phosphate homeostasis.

Comparison of mRNA gene expression by RT-PCR and DNA microarray.

Few studies have compared the quantification of mRNA by DNA microarray to the results obtained by reverse transcription PCR (RT-PCR). In this study, mRNA was collected from the healing femoral fracture callus of adult and juvenile rats at various times after fracture. Ten samples were measured by both methods for 26 genes. For RT-PCR, mRNA was reverse transcribed, amplified, electrophoresed, blotted, and probed with 32P-labeled internal oligonucleotides, which were quantified. For DNA microarray, the mRNA was processed to biotin-labeled cRNA, hybridized to 10 Affymetrix Rat U34A microarrays, and quantified. Correlation coefficients (r) for each gene for the agreement between RT-PCR and microarray ranged from -0.48 to +0.93. This variation made the interpretation gene-specific. Genes with moderate expression levels gave the highest r values. Increased numbers of absent calls by the microarray software and increased separation between the location of the PCR primers and the microarray probes both led to reduced agreement. Microarray analysis suggested a floor effect in expression levels measured by RT-PCR for two genes. In conclusion, moderate mRNA expression levels with overlap in the location of PCR primers and microarray probes can yield good agreement between these two methods.

Evaluation of a cleanser for petroleum-contaminated skin.

BACKGROUND: Extremity injuries contaminated with petroleum products pose clinical dilemmas. This project was designed to evaluate the efficacy of a dioctyl sulfosuccinate (DS) solution for cleansing petroleum-contaminated skin. METHODS: One hundred Sprague-Dawley rats were subjected to a contamination protocol followed by a cleansing procedure. Four petroleum contaminants and five cleansing solutions were selected. The protocol consisted of shaving, initial punch biopsy, contamination, precleansing punch biopsy, standardized scrub protocol, and postcleansing punch biopsy. Biopsy samples were analyzed for petroleum residue using fluorometry. RESULTS: The 10% DS solution had the highest reduction of crude oil, grease, and tar: 99.6 +/- 0.4% (mean +/- SD) contaminant reduction for crude oil, 99.8 +/- 0.2% for grease, and 99.8 +/- 0.2% for tar. The other cleansers showed less efficacy (p < 0.05). CONCLUSION: Concentrated DS appears to be significantly more effective at cleaning petroleum products from skin than the commonly chosen surgical and commercial cleansers.

The effect of age on gene expression in adult and juvenile rats following femoral fracture.

OBJECTIVE: To compare mRNA gene expression during fracture healing in young and adult rats. DESIGN: Gene expression was measured at zero, 1, 2, 4, 6, 8 and 10 weeks after fracture (6 rats/age/time point) in rats at 6 and 26 weeks of age at surgery. SETTING: AAALAC-accredited vivarium of an independent academic medical center. ANIMALS: Female Sprague-Dawley rats at 6 and 26 weeks of age. INTERVENTION: An intramedullary rod was placed retrograde in the left femur, and a simple transverse closed middiaphyseal fracture was induced. MAIN OUTCOME MEASUREMENTS: mRNA gene expression was measured for 34 genes for extracellular matrix, osteoblasts, bone morphogenic protein, inflammation, cytokine, and receptor genes. RESULTS: The young rats reached radiographic union by 4 weeks after fracture, whereas the adult rats took 8 to 10 weeks to unite. All genes studied increased in mRNA expression with a peak at 1 to 2 weeks after fracture. All genes in the young rats then subsided to baseline by 4 weeks after fracture. However, during the longer period needed for radiographic union in the adult rats, only genes related to bone matrix, osteoblastic markers, angiogenesis, and the fibroblast growth factors remained significantly up-regulated at 4 and 6 weeks after fracture. Genes related to cartilage, Indian hedgehog, the bone morphogenetic proteins, and transforming growth factor-beta came to undetectable baseline values in the adult rats prior to radiographic union. CONCLUSIONS: Most stimulators of bone healing are not expressed during the later stages of fracture repair in adult rats. Other genes must control bone growth to bridge the fracture gap.

Gene expression in older rats with delayed union of femoral fractures.

BACKGROUND: Fracture-healing slows with age. While six-week-old rats regain normal bone biomechanics at four weeks after a fracture, one-year-old rats require more than twenty-six weeks. The objective of this study was to examine the possible role of altered mRNA gene expression in this delayed union. METHODS: Closed midshaft femoral fractures were created in six-week-old and one-year-old Sprague-Dawley female rats. The animals were killed at zero-time (unfractured) or at 0.4, one, two, three, four, or six weeks after the fracture. mRNA levels were measured by reverse transcription-polymerase chain reaction in the fracture callus for twenty-seven matrix, cytokine, and cytokine-receptor genes for the seven animals per time-point per age-group. RESULTS: The younger rats healed radiographically by four weeks after the fracture, whereas none of the older rats had healed by the sixth week. Despite the difference in healing rates, the levels of mRNA gene expression, in general, followed the same pattern in both age-groups. The mRNA expression levels increased to a peak at one to two weeks after the fracture and then decreased to very low or undetectable levels at four and six weeks after the fracture for both age-groups. Significantly lower levels of mRNA for Indian hedgehog (Ihh) and bone morphogenetic protein-2 (BMP-2) were detected in the fracture calluses of the older rats (p < 0.01 and p < 0.05, respectively). CONCLUSIONS: All genes studied were up-regulated by the fracture in both age-groups. Thus, the failure of the older rats to heal promptly was not due to the lack of expression of any of the studied genes. The increase in mRNA for Ihh and BMP-2 in the older rats was smaller than that in the younger rats, which may contribute to slower fracture repair. The return of mRNA gene expression to baseline in the older rats prior to healing may contribute to the delayed union. The slower healing response of the older rats did not stimulate a negative-feedback increase in the mRNA expression of stimulatory cytokines.

Na/P(i) cotransporter ( Npt2) gene disruption increases duodenal calcium absorption and expression of epithelial calcium channels 1 and 2.

Mice homozygous for the disrupted type-II Na/P(i) cotransporter gene ( Npt2(-/-)) exhibit hypophosphataemia, increased serum concentration of 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) and calcium (Ca) and elevated urinary Ca excretion. To determine whether the hypercalcaemia and hypercalciuria are secondary to 1,25-(OH)(2)D-stimulated intestinal Ca absorption, we examined the effect of Npt2 gene disruption on serum Ca and urinary Ca excretion after an overnight fast, and on duodenal Ca absorption. We also compared the duodenal expression of the epithelial Ca channels, ECaC1 and ECaC2, and calbindinD(9K) mRNAs, relative to that of beta-actin mRNA, in Npt2(+/+) and Npt2(-/-) mice. Both serum Ca and urine Ca/creatinine were significantly decreased in Npt2(-/-) mice after an overnight fast and were no longer different from that in wild-type mice. Absorption of (45)Ca from isolated duodenal segments in vivo and (45)Ca appearing in the plasma were significantly increased in Npt2(-/-) compared with Npt2(+/+) mice. In addition, the duodenal abundance of ECaC1, ECaC2 and calbindinD(9K) mRNAs was significantly elevated in mutant mice relative to that in wild-type mice. In contrast, both duodenal Ca absorption and ECaC1 and ECaC2 mRNA abundance were lower in mice with X-linked hypophosphataemia ( Hyp) than in normal littermates. In summary, we provide evidence for increased duodenal Ca absorption in Npt2(-/-) mice and suggest a role for ECaC1, ECaC2 and calbindinD(9K) in mediating this response.