Implications of Aging in Plastic Surgery.

Given the rapidly aging population, investigating the effect of age on plastic surgery outcomes is imperative. Despite this, the topic has received relatively little attention. Furthermore, there appears to be little integration between the basic scientists investigating the mechanisms of aging and the plastic surgeons providing the majority of "antiaging" therapies. This review first provides a description of the effects and mechanisms of aging in 5 types of tissue: skin, adipose tissue, muscles, bones and tendons, and nervous tissue followed by an overview of the basic mechanisms underlying aging, presenting the currently proposed cellular and molecular theories. Finally, the impact of aging, as well as frailty, on plastic surgery outcomes is explored by focusing on 5 different topics: general wound healing and repair of cutaneous tissue, reconstruction of soft tissue, healing of bones and tendons, healing of peripheral nerves, and microsurgical reconstruction. We find mixed reports on the effect of aging or frailty on outcomes in plastic surgery, which we hypothesize to be due to exclusion of aged and frail patients from surgery as well as due to outcomes that reported no postsurgical issues with aged patients. As plastic surgeons continue to interact more with the growing elderly population, a better appreciation of the underlying mechanisms and outcomes related to aging and a clear distinction between chronological age and frailty can promote better selection of patients, offering appropriate patients surgery to improve an aged appearance, and declining interventions in inappropriate patients.

CRISPR Craft: DNA Editing the Reconstructive Ladder.

The clustered regularly interspaced short palindromic repeats (CRISPR) system of genome editing represents a major technological advance spanning all areas of genetics and downstream applications. CRISPR's potential impact on treating human disease encompasses all clinical specialties, including areas important to the plastic surgeon such as oncology, wound healing, immunology, and craniofacial malformations. Plastic surgeons should gain familiarity with this gene editing technology, and become active contributors and leaders in applying CRISPR to their respective areas of expertise. This review describes the history and basic mechanism of CRISPR genome editing, highlights current and future applications, and discusses limitations. The authors will consider CRISPR's potential impact and use in plastic and reconstructive surgery.

Age-related dystrophic changes in corneal endothelium from DNA repair-deficient mice.

The corneal endothelium (CE) is a single layer of cells lining the posterior face of the cornea providing metabolic functions essential for maintenance of corneal transparency. Adult CE cells lack regenerative potential, and the number of CE cells decreases throughout life. To determine whether endogenous DNA damage contributes to the age-related spontaneous loss of CE, we characterized CE in Ercc1(-/Δ) mice, which have impaired capacity to repair DNA damage and age prematurely. Eyes from 4.5- to 6-month-old Ercc1(-/Δ) mice, age-matched wild-type (WT) littermates, and old WT mice (24- to 34-month-old) were compared by spectral domain optical coherence tomography and corneal confocal microscopy. Histopathological changes in CE were further identified in paraffin tissue sections, whole-mount immunostaining, and scanning electron and transmission electron microscopy. The CE of old WT mice displayed polymorphism and polymegathism, polyploidy, decreased cell density, increased cell size, increases in Descemet's thickness, and the presence of posterior projections originating from the CE toward the anterior chamber, similar to changes documented for aging human corneas. Similar changes were observed in young adult Ercc1(-/Δ) mice CE, demonstrating spontaneous premature aging of the CE of these DNA repair-deficient mice. CD45(+) immune cells were associated with the posterior surface of CE from Ercc1(-/Δ) mice and the tissue expressed increased IL-1α, Cxcl2, and TNFα, pro-inflammatory proteins associated with senescence-associated secretory phenotype. These data provide strong experimental evidence that DNA damage can promote aging of the CE and that Ercc1(-/Δ) mice offer a rapid and accurate model to study CE pathogenesis and therapy.

Quantitative assessment of ultrastructure and light scatter in mouse corneal debridement wounds.

PURPOSE: The mouse has become an important wound healing model with which to study corneal fibrosis, a frequent complication of refractive surgery. The aim of the current study was to quantify changes in stromal ultrastructure and light scatter that characterize fibrosis in mouse corneal debridement wounds. METHODS: Epithelial debridement wounds, with and without removal of basement membrane, were produced in C57BL/6 mice. Corneal opacity was measured using optical coherence tomography, and collagen diameter and matrix order were quantified by x-ray scattering. Electron microscopy was used to visualize proteoglycans. Quantitative PCR (Q-PCR) measured mRNA transcript levels for several quiescent and fibrotic markers. RESULTS: Epithelial debridement without basement membrane disruption produced a significant increase in matrix disorder at 8 weeks, but minimal corneal opacity. In contrast, basement membrane penetration led to increases in light scatter, matrix disorder, and collagen diameter, accompanied by the appearance of abnormally large proteoglycans in the subepithelial stroma. This group also demonstrated upregulation of several quiescent and fibrotic markers 2 to 4 weeks after wounding. CONCLUSIONS: Fibrotic corneal wound healing in mice involves extensive changes to collagen and proteoglycan ultrastructure, consistent with deposition of opaque scar tissue. Epithelial basement membrane penetration is a deciding factor determining the degree of ultrastructural changes and resulting opacity.

Initial in vitro investigation of the human immune response to corneal cells from genetically engineered pigs.

PURPOSE: To compare the in vitro human humoral and cellular immune responses to wild-type (WT) pig corneal endothelial cells (pCECs) with those to pig aortic endothelial cells (pAECs). These responses were further compared with CECs from genetically engineered pigs (α1,3-galactosyltransferase gene-knockout [GTKO] pigs and pigs expressing a human complement-regulatory protein [CD46]) and human donors. METHODS: The expression of Galα1,3Gal (Gal), swine leukocyte antigen (SLA) class I and class II on pCECs and pAECs, with or without activation by porcine IFN-γ, was tested by flow cytometry. Pooled human serum was used to measure IgM/IgG binding to and complement-dependent cytotoxicity (CDC) to cells from WT, GTKO, and GTKO/CD46 pigs. The human CD4(+) T-cell response to cells from WT, GTKO, GTKO/CD46 pigs and human was tested by mixed lymphocyte reaction (MLR). RESULTS: There was a lower level of expression of the Gal antigen and of SLA class I and II on the WT pCECs than on the WT pAECs, resulting in less antibody binding and reduced human CD4(+) T-cell proliferation. However, lysis of the WT pCECs was equivalent to that of the pAECs, suggesting more susceptibility to injury. There were significantly weaker humoral and cellular responses to the pCECs from GTKO/CD46 pigs compared with the WT pCECs, although the cellular response to the GTKO/CD46 pCECs was greater than to the human CECs. CONCLUSIONS: These data provide the first report of in vitro investigations of CECs from genetically engineered pigs and suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.

Impact on the corneal endothelium of mitomycin C during photorefractive keratectomy.

PURPOSE: This brief review examines both basic science and clinical studies to evaluate the potential impact on the health of the corneal endothelium of mitomycin C (MMC) usage during photorefractive keratectomy (PRK). METHODS: The mechanism of action and consequences of MMC are reviewed within the context of in vitro, animal, and clinical studies and a hypothesis of how this vital cell layer responds to MMC at both the cellular and clinical levels is formed. RESULTS: Seven basic science studies were reviewed demonstrating significant MMC toxicity to corneal endothelial cells. Of the five clinical studies reviewed, three demonstrated no effect on corneal endothelial density, whereas two studies found significant cell loss after MMC usage. CONCLUSIONS: Although all of the basic science studies reviewed highlight the toxicity of MMC on the corneal endothelium, current clinical studies are less conclusive. Given the corneal penetration of MMC and the fragile nature of the corneal endothelium, additional follow-up studies are needed to determine the long-term impact of MMC usage during PRK on the corneal endothelium.

DNA cross-linking, double-strand breaks, and apoptosis in corneal endothelial cells after a single exposure to mitomycin C.

PURPOSE: To investigate the cellular effects of mitomycin C (MMC) treatment on corneal endothelial (CE) cells at clinically relevant applications and dosages. METHODS: Radial and posterior diffusion of MMC was determined by an Escherichia coli growth inhibition bioassay. A modified version of the comet assay (single cell gel electrophoresis) was used to detect DNA cross-linking. Immunostaining detected the nuclear phosphorylated histone variant H2AX (gamma-H2AX) indicating DNA double-strand breaks. Apoptosis in MMC-treated cells was detected with annexin V staining. RESULTS: Topical application of 0.02% MMC to intact goat globes resulted in MMC in the CE at 0.37 microg/mL and produced a significant increase in CE DNA cross-linking with as little as 6 seconds of topical MMC treatment. DNA cross-linking was also demonstrated in cultured CE cells by using MMC exposures similar to those detected in CE of intact eyes. Such MMC treatment of CE produced elevated and persistent gamma-H2AX-positive cells indicative of DNA double-strand breaks. Similarly, there was an increase in the proportion of apoptotic CE cells, evidenced by positive annexin V staining. CONCLUSIONS: The results demonstrate that exposure to MMC at times and concentrations commonly used in refractive surgery produces cross-linking of corneal endothelial DNA, persistent DNA damage, and endothelial death via apoptosis. Current practices of MMC application during refractive surgeries may increase the potential for long-term and permanent deleterious effects on the health of the corneal endothelium.