Prospective observational study comparing burn surgeons' estimations of wound healing after skin grafting to photo-assisted methods.

INTRODUCTION: Appropriate graft healing after split-thickness skin graft and early recognition of complications (graft loss) are critical to burn patient management. Larger mesh ratio expansions and Meek micrografting may pose a greater challenge in estimating the percentage of wound healing. This study looks at the reliability of photograph assessments and the concordance of bedside evaluation to photograph assessments of wound healing after skin grafting. METHODS: Three assessment methods for percentage of wound healing after skin Grafting were assessed: (1) clinicians' bedside rating, (2) clinician assessment of high-definition photographs, and (3) digital image analysis through color subtraction using Adobe Photoshop. We compared each method using a mixed-effects model on absolute agreement using intra-class correlation (ICC) and Bland Altman (BA) plots. RESULTS: Fourteen burn patients were enrolled with 38 grafted wounds (100 sites). Bedside assessments had a mean ICC of 0.64 (compared to digital image analysis) and 0.69 (compared to photo assessment), with a wide range on BA-plots. Inter-rater reliability of photo assessment was excellent (0.96) among six clinicians. Repeated photo-assisted assessments had good intra-rater reliability (ICC: photo assessment: 0.88; digital analysis: 0.97). CONCLUSIONS: Bedside wound healing assessments show variability; photograph documentation of sequential wound progression could supplement active clinical management or studies for more reliable assessments.

Color subtractive-computer-assisted image analysis for quantification of cutaneous nerves in a diabetic mouse model.

Immunohistochemistry (IHC) is a valuable tool for labeling structures in tissue samples. Quantification of immunolabeled structures using traditional approaches has proved to be difficult. Manual counts of IHC-stained structures are inherently biased, require multiple observers, and generate qualitative data. Stereological methods provide accurate quantification but are complex and labor-intensive when staining must be compared among large numbers of samples. In an effort to quickly, objectively, and reproducibly quantify cutaneous innervation in a large number of counterstained tissue sections, we developed a color subtractive-computer-assisted image analysis (CS-CAIA) system. To develop and test the CS-CAIA method, tissue sections of diabetic (db/db) mouse skin and their wild-type (db/-) littermates were stained by IHC for the neural marker PGP 9.5. The brown-red PGP 9.5 peroxidase stain was colorimetrically isolated through a scripted process of color background removal. The remaining stain was thresholded and binarized for computer determination of nerve profile counts (number of stained regions), area fraction (total area of nerve profiles per unit area of tissue), and area density (total number of nerve profiles per unit area of tissue). Using CS-CAIA, epidermal nerve profile counts, area fraction, and area density were significantly lower in db/db compared to db/- mice.

Mitochondrial ultrastructure in embryos after implantation.

Information on the morphology of mitochondria during embryogenesis is scattered in the literature, but there appears to be a consistent pattern. During early organogenesis, the embryo is in a state of relative hypoxia associated with a major decrease in terminal electron transport system activity and a marked increase in anaerobic glycolysis. Ultrastructural studies of a 14-somite monkey embryo and day 10 and 12 rat embryos, together with a review of the literature, led us to determine that this hypoxic stage is characterized by vesiculation of the mitochondrial inner membranes, or cristae. Starting in the late morula stage and continuing during early postimplantation embryogenesis, the cristae increase but appear tubular or vesicular. After the end of neurulation, and with the onset of vascular perfusion of embryonic tissues, the cristae gradually become lamellated; by the limb bud stage they appear more mature. We suggest that new cristae derive from blebs of the inner mitochondrial membrane and that with maturation these blebs collapse, giving them a lamelliform appearance. The delamellated state of the cristae might inactivate oxidative phosphorylation to protect the embryo from toxic respiratory end-products that could accumulate in an embryo before there is vascular perfusion. Consistent with this hypothesis, mitochondrial diameters in the developing heart of monkey and rat embryos were approximately twice those found in skin and neural tube.

Ultrastructural study of mitochondria and their cristae in embryonic rats and primate (N. nemistrina).

Information on the morphology of mitochondria during embryogenesis is scattered in the literature but there appears to be a developmental pattern characterized by vesiculation of the mitochondrial cristae. During early organogenesis, the embryo is in a relative state of hypoxia and this is associated with decrease of terminal electron transport system activity and a marked increase in glycolysis. Ultrastructural studies of a 14 somite monkey embryo, and day 10 and 12 rat embryos, along with a review of the literature led us to determine that this hypoxic stage is characterized by vesiculation of the mitochondrial cristae. Starting in the late morula stage and continuing during early postimplantation embryogenesis the cristae increase and appear tubular or vesicular. After the end of neurulation, and with onset of vascular perfusion, the cristae gradually become lamellated and by the limb bud stage appear more mature. We suggest that new cristae form from blebs of the inner mitochondrial membrane and that subsequently with maturation these blebs collapse giving them a lamelliform appearance. The delamellated state of the cristae may protect the embryo from toxic respiratory end-products of oxidative respiration which could accumulate in an embryo lacking vascular perfusion. In the heart of monkey and rat embryos, the mitochondria had diameters which were approximately twice those found in skin and neural tube.