An Automated Technique for Double Staining Rat and Rabbit Fetal Skeletal Specimens to Differentiate Bone and Cartilage

Trueman, D; Jackson, Stuart W.; Trueman, Brian

doi:10.3109/10520299909066484

Cited by 10 publications

(7 citation statements)

References 5 publications

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“…After gross micrographs and micro‐CT images were generated, the organs of the fetuses were removed and tissue cleared with potassium hydroxide (KOH) using a modified technique of Trueman, Jackson, and Trueman (1999). Samples were subsequently stained with alizarin red.…”

Section: Methodsmentioning

confidence: 99%

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Oest

Jones

Hatfield

et al. 2008

Birth Defects Research Pt B

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Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear-staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro-CT) imaging is a versatile, well-documented tool traditionally used to generate high-resolution 3-D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro-CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro-CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro-CT measurement of fetal limb lengths correlates well with traditional clear-staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro-CT and 10.82% for clear-staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro-CT technique to generate 3-D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear-staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well.

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Section: Methodsmentioning

confidence: 99%

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Oest

Jones

Hatfield

et al. 2008

Birth Defects Research Pt B

View full text Add to dashboard Cite

show abstract

“…Fetuses are then harvested just before the expected natural delivery date, and evaluated in detail for external, visceral, or skeletal alterations (Taylor, 1986;USEPA, 1998). Examination of the fetal skeleton is accomplished by staining the bone with Alizarin Red S, and can be enhanced by staining with Alcian Blue for cartilage (Kimmel and Trammell, 1981;Igarashi et al, 1992;Webb and Byrd, 1994;Trueman et al, 1999;Young et al, 2000). Alterations in development are often classified as ''malformations,'' which imply more severe, permanent defects arising from basic changes to the normal pattern of development, or ''variations,'' which are less serious, often transient or repairable, and are seen commonly in control fetuses.…”

Section: Introductionmentioning

confidence: 99%

Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs

Carney

Kimmel²

2007

Birth Defects Research Pt B

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Delayed (or incomplete) ossification of developing fetal bones and wavy ribs are some of the most common skeletal variations encountered in regulatory guideline developmental toxicity studies. Although they tend to be regarded as minor effects, they can be quite sensitive and consequently may influence the study lowest-observed-adverse-effect levels (LOAELs), and thus, impact classification, labeling, and risk assessment. In this study, we review the underlying mechanisms of these skeletal variations, evaluate different scenarios in which they have been observed, offer guidance for their interpretation, and comment on their use for risk assessment. Both minor delays in ossification and wavy ribs seem to be readily repairable via postnatal skeletal remodeling, are not mechanistically linked to malformation, and often are seen in the presence of maternal or fetal toxicity. As such, these minor variations would not generally be considered adverse in and of themselves but should be interpreted in the context of other maternal and fetal findings, information available on normal skeletogenesis patterns, mode of action of the test agent, and historical control incidence using a weight of evidence approach. Birth Defects Res 80: 473-496, 2007. r 2007

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“…For calcification analysis, embryos were eviscerated and the skin was removed. The embryos were fixed in 95% ethanol and stained in Alcian blue solution and Alizarin red solution overnight as described previously[18]. …”

Section: Methodsmentioning

confidence: 99%

Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability

et al. 2011

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BackgroundAmplification of 3q26 is one of the most frequent genetic alterations in many human malignancies. Recently, we isolated a novel oncogene eIF-5A2 within the 3q26 region. Functional study has demonstrated the oncogenic role of eIF-5A2 in the initiation and progression of human cancers. In the present study, we aim to investigate the physiological and pathological effect of eIF-5A2 in an eIF-5A2 transgenic mouse model.MethodsAn eIF-5A2 transgenic mouse model was generated using human eIF-5A2 cDNA. The eIF-5A2 transgenic mice were characterized by histological and immunohistochemistry analyses. The aging phenotypes were further characterized by wound healing, bone X-ray imaging and calcification analysis. Mouse embryo fibroblasts (MEF) were isolated to further investigate molecular mechanism of eIF-5A2 in aging.ResultsInstead of resulting in spontaneous tumor formation, overexpression of eIF-5A2 accelerated the aging process in adult transgenic mice. This included decreased growth rate and body weight, shortened life span, kyphosis, osteoporosis, delay of wound healing and ossification. Investigation of the correlation between cellular senescence and aging showed that cellular senescence is not required for the aging phenotypes in eIF-5A2 mice. Interestingly, we found that activation of eIF-5A2 repressed p19 level and therefore destabilized p53 in transgenic mouse embryo fibroblast (MEF) cells. This subsequently allowed for the accumulation of chromosomal instability, such as errors in cell dividing during metaphase and anaphase. Additionally, a significantly increase in number of aneuploidy cells (p < 0.05) resulted from an increase in the incidences of misaligned and lagging chromosomal materials, anaphase bridges, and micronuclei in the transgenic mice.ConclusionThese observations suggest that eIF-5A2 mouse models could accelerate organismal aging by increasing chromosome instability.

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An Automated Technique for Double Staining Rat and Rabbit Fetal Skeletal Specimens to Differentiate Bone and Cartilage

Cited by 10 publications

References 5 publications

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs

Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability

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