D-methionine administered as late as 36 hours post-noise exposure rescues from permanent threshold shift and dose-dependently increases serum antioxidant levels.

OBJECTIVE: To elucidate D-methionine's (D-met) dose and time rescue parameters from steady-state or impulse noise-induced permanent threshold shift (PTS) and determine D-met rescue's influence on serum and cochlear antioxidant levels. DESIGN: Five D-met doses at 0, 50, 100, or 200 mg/kg/dose administered starting at 1, 24, or 36 hours post steady-state or impulse noise exposure. Auditory brainstem responses at baseline and 21 days post-noise measured PTS. Serum (superoxide dismutase [SOD], catalase [CAT],, glutathione reductaseand glutathione peroxidase [GPx]) and cochlear (Glutathione [GSH] and glutathione disulphide [GSSG]) antioxidant levels measured physiological impact. STUDY SAMPLE: Chinchillas (10/study group; 6-8/confirmatory groups). RESULTS: D-met significantly reduced PTS for impulse noise (100 mg [2, 6, 14 and 20 kHz]; 200 mg [2, 14 and 20 kHz]) and steady-state noise (all dosing groups, time parameters and tested frequencies). PTS reduction did not significantly vary by rescue time. D-met significantly increased serum SOD (100 and 200 mg for 24 hour rescue) and GPx (50 mg/kg at 24 hour rescue) at 21 days post-noise. Cochlear GSH and GSSG levels were unaffected relative to control. CONCLUSION: D-met rescues from steady-state and impulse noise-induced PTS even when administered up to 36 hours post-noise and dose-dependently influences serum antioxidant levels even 21 days post-noise. D-met's broad and effective dose/time window renders it a promising antioxidant rescue agent.

D-methionine immediate and continued rescue after noise exposure does not prevent temporary threshold shift but alters cochlear and serum antioxidant levels.

OBJECTIVE: Determine if D-methionine (D-met) rescue prevents temporary threshold shift (TTS) from steady-state or impulse noise and determine D-met's impact on serum and cochlear antioxidant levels. DESIGN: D-met at 50, 100 or 200 mg/kg/doses were administered 0, 6 and 18 hours-post noise. ABRs at baseline and 24 hours post-noise measured TTS. Serum (SOD, CAT, GR, GPx) and cochlear (GSH, GSSG) antioxidant levels measured physiological influence. Three control groups, with impulse or steady-state or without noise, were saline-injected. STUDY SAMPLE: Ten Chinchillas/group. RESULTS: D-met rescue did not significantly reduce TTS or impact serum CAT, SOD, GPx or GR levels vs. noise-exposed control groups, but TTS was greater in all groups relative to no-noise controls. D-met significantly elevated CAT at 50 mg/kg vs. steady-state controls and SOD at 200 mg/kg vs. impulse noise controls. D-met significantly reduced cochlear GSH/GSSG ratios in the 100 mg/kg D-met group vs. impulse noise controls. CONCLUSIONS: While D-met rescue has reduced permanent threshold shift in previous studies, it did not reduce TTS in this study. However, D-met rescue did alter selective serum and cochlear oxidative state changes 24 hours post-noise relative to controls. Results demonstrate TTS studies do not always predict PTS protection in otoprotectant experimental designs.

Adipose-derived stem cell to epithelial stem cell transdifferentiation: a mechanism to potentially improve understanding of fat grafting's impact on skin rejuvenation.

BACKGROUND: Recent evidence suggests that lipofilling improves overlying skin composition and appearance. Adipose-derived stem cells (ADSC) have been implicated. OBJECTIVE: The authors identify ADSC transdifferentiation into epithelial stem cells through coexpression of GFP+ (green fluorescent protein positive) ADSC with the epithelial stem cell marker p63 in an in vivo fat grafting model. METHODS: Six male, GFP+ mice served as adipose tissue donors. Twelve nude mice served as recipients. Recipients were subdivided into 2 arms (6 mice/each arm) and received either whole-fat specimen (group 1) or isolated and purified ADSC + peptide hydrogel carrier (group 2) engrafted into a 1-cm(2) left parascapular subdermal plane. The right parascapular subdermal plane served as control. Skin flaps were harvested at 8 weeks and subjected to (1) confocal fluorescent microscopy and (2) reverse transcriptase polymerase chain reaction (RT-PCR) for p63 mRNA expression levels. RESULTS: Gross examination of skin flaps demonstrated subjectively increased dermal vessel presence surrounding whole-fat and ADSC specimens. The GFP+ cells were seen within overlying dermal architecture after engraftment and were found to coexpress p63. Significantly increased levels of p63 expression were found in the ADSC + hydrogel skin flaps. CONCLUSIONS: We offer suggestive evidence that GFP+ ADSC are found within the dermis 8 weeks after engraftment and coexpress the epithelial stem cell marker p63, indicating that ADSC may transdifferentiate into epithelial stem cells after fat grafting. These findings complement current understanding of how fat grafts may rejuvenate overlying skin.

Preloaded D-methionine protects from steady state and impulse noise-induced hearing loss and induces long-term cochlear and endogenous antioxidant effects.

OBJECTIVE: Determine effective preloading timepoints for D-methionine (D-met) otoprotection from steady state or impulse noise and impact on cochlear and serum antioxidant measures. DESIGN: D-met started 2.0-, 2.5-, 3.0-, or 3.5- days before steady-state or impulse noise exposure with saline controls. Auditory brainstem response (ABRs) measured from 2 to 20 kHz at baseline and 21 days post-noise. Samples were then collected for serum (SOD, CAT, GR, GPx) and cochlear (GSH, GSSG) antioxidant levels. STUDY SAMPLE: Ten Chinchillas per group. RESULTS: Preloading D-met significantly reduced ABR threshold shifts for both impulse and steady state noise exposures but with different optimal starting time points and with differences in antioxidant measures. For impulse noise exposure, the 2.0, 2.5, and 3.0 day preloading start provide significant threshold shift protection at all frequencies. Compared to the saline controls, serum GR for the 3.0 and 3.5 day preloading groups was significantly increased at 21 days with no significant increase in SOD, CAT or GPx for any impulse preloading time point. Cochlear GSH, GSSG, and GSH/GSSG ratio were not significantly different from saline controls at 21 days post noise exposure. For steady state noise exposure, significant threshold shift protection occurred at all frequencies for the 3.5, 3.0 and 2.5 day preloading start times but protection only occurred at 3 of the 6 test frequencies for the 2.0 day preloading start point. Compared to the saline controls, preloaded D-met steady-state noise groups demonstrated significantly higher serum SOD for the 2.5-3.5 day starting time points and GPx for the 2.5 day starting time but no significant increase in GR or CAT for any preloading time point. Compared to saline controls, D-met significantly increased cochlear GSH concentrations in the 2 and 2.5 day steady-state noise exposed groups but no significant differences in GSSG or the GSH/GSSG ratio were noted for any steady state noise-exposed group. CONCLUSIONS: The optimal D-met preloading starting time window is earlier for steady state (3.5-2.5 days) than impulse noise (3.0-2.0). At 21 days post impulse noise, D-met increased serum GR for 2 preloading time points but not SOD, CAT, or GpX and not cochlear GSH, GSSG or the GSH/GSSG ratio. At 21 days post steady state noise D-met increased serum SOD and GPx at select preloading time points but not CAT or GR. However D-met did increase the cochlear GSH at select preloading time points but not GSSG or the GSH/GSSG ratio.

Transplantation of an LGR6+ Epithelial Stem Cell-Enriched Scaffold for Repair of Full-Thickness Soft-Tissue Defects: The In Vitro Development of Polarized Hair-Bearing Skin.

BACKGROUND: Recent literature has shown that full-thickness wounds, devoid of the stem cell niche, can subsequently be reconstructed with functional skin elements following migration of the LGR6 epithelial stem cell into the wound bed. In this study, the authors use a variety of LGR6 epithelial stem cell-seeded scaffolds to determine therapeutic utility and regenerative potential in the immediate reconstruction of full-thickness wounds. METHODS: Isolated LGR6 epithelial stem cells were seeded onto a spectrum of acellular matrices and monitored in both in vitro and in vivo settings to determine their relative capacity to regenerate tissues and heal wounds. RESULTS: Wound beds containing LGR6 stem cell-seeded scaffolds showed significantly augmented rates of healing, epithelialization, and hair growth compared with controls. Gene and proteomic expression studies indicate that LGR6 stem cell-seeded constructs up-regulate WNT, epidermal growth factor, and angiogenesis pathways. Finally, the addition of stromal vascular fraction to LGR6 stem cell-seeded constructs induces polarized tissue formation, nascent hair growth, and angiogenesis within wounds. CONCLUSIONS: LGR6 stem cells are able to undergo proliferation, differentiation, and migration following seeding onto a variety of collagen-based scaffolding. In addition, deployment of these constructs induces epithelialization, hair growth, and angiogenesis within wound beds. The addition of stromal vascular fraction to LGR6 stem cell-containing scaffolds initiated an early form of tissue polarization, providing for the first time a clinically applicable stem cell-based construct that is capable of the repair of full-thickness wounds and hair regeneration. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Transplantation of the LGR6+ epithelial stem cell into full-thickness cutaneous wounds results in enhanced healing, nascent hair follicle development, and augmentation of angiogenic analytes.

BACKGROUND: The recently discovered leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6+) epithelial stem cell located within the follicular bulge of the adnexal compartment is capable of producing all cellular lineages of the skin. In this study, the authors sought to determine whether these cells can be transplanted for use as a type of cellular therapy for the repair of full-thickness wounds in which the native stem cell niche has been obliterated. METHODS: Full-thickness murine skin was harvested and LGR6(+GFP) epithelial stem cells were isolated using fluorescence-activated cell sorting. This enriched epithelial stem cell population was then transplanted by means of local injection into wound beds on the dorsum of nude mice. Viability, migration, healing, the development of nascent hair follicles, and gene and proteomic expression studies were performed to determine whether the engraftment of LGR6(+GFP) epithelial stem cells enhanced healing when compared with controls. RESULTS: Wound beds receiving LGR6(+GFP) epithelial stem cells showed enhanced healing; nascent follicle growth; and augmentation of the Wnt, vascular endothelial growth factor, epidermal growth factor, and platelet-derived growth factor pathways when compared with controls. CONCLUSIONS: The LGR6+ epithelial stem cells appear to hold great promise for the development of a clinically useful stem cell­based therapy for the repair of full-thickness wounds and hair regeneration. These results indicate that transplantation of LGR6+ epithelial stem cells promotes epithelialization, hair growth, and angiogenesis in tissues destined for scar formation.

Neurotrophin expression of laryngeal muscles in response to recurrent laryngeal nerve transection.

OBJECTIVE/HYPOTHESIS: The recurrent laryngeal nerve (RLN) commonly regenerates after injury; however, functional motion is rarely recovered. Animal experiments have documented aberrant reinnervation after nerve transection, with motor axons reaching inappropriate muscles. More recently, experimental results suggest that lack of vocal fold motion after RLN injury is due to preferential reinnervation of adductor muscles, with inadequate reinnervation of the posterior cricoarytenoid muscle (PCA), the only abductor muscle of the larynx. Information on factors that could influence the receptiveness of these muscles to reinnervation could be useful in developing new therapeutic strategies. It is hypothesized that the thyroarytenoid muscle (TA) and the PCA differ in expression of neurotrophins in response to denervation. STUDY DESIGN: Laboratory experiment. METHODS: Rats were sacrificed at 3 days, 6 weeks, or 4 months after unilateral RLN injury measure expression of brain-derived nerve growth factor (BDNF), nerve growth factor (NGF), and neurotrophin 4 (NT-4) in the TA and PCA muscles, using immunohistochemistry. We also assessed nerve regeneration. RESULTS: NGF was significantly diminished in the denervated TA muscle at 3 days after injury and increased at 6 weeks. BDNF expression was unchanged in the TA, but was diminished in both PCA muscles at 3 days and 6 weeks, returning to near-normal levels at 4 months after injury. Robust nerve regeneration of distal RLN was present at 4 months. CONCLUSIONS: Results suggest that the TA and PCA muscles respond differently to denervation.