Faculty Bibliography

BACKGROUND: Autogenous fat grafting has been observed to alleviate the sequelae of chronic radiodermatitis. To date, no study has replicated this finding in an animal model. METHODS: The dorsa of adult wild-type FVB mice were shaved and depilitated. The dorsal skin was then distracted away from the body and radiated (45Gy) using a Varian 2300 Linear Accelerator. Four weeks following radiation, 1.5cc fat or sham grafts were placed in the dorsal subcutaneous space. Gross results were analyzed photometrically. The animals were sacrificed at 4 and 8 weeks following fat or sham grafting and their dorsal skin was processed for histologic analysis. Inflammation was assessed by epidermal thickness measurements on H&E stained sections. Vascular density was assessed using CD31 staining. Fibrosis was assessed using Smad-3 and Picrosirius Red staining. RESULTS: Hyperpigmentation and ulceration were grossly improved in fat-grafted mice compared to sham-grafted controls. Epidermal thickness measurements demonstrated decreased thickness in fat-grafted animals at both timepoints (20.6+/-1.5mum vs 55.2+/-5.6mum, p=0.004; 17.6+/-1.1mum vs 36.3+/-6.1mum, p=0.039). Vascular density was decreased in fat-grafted mice compared to sham-grafted at both timepoints (17.5+/-1.3 vessels/hpf vs 29+/-3.5, p=0.055; 13.25+/-1.4 vs 17.0+/-1.6, p=0.003). Intensity of Smad3 staining was significantly decreased in fat-grafted animals at both timepoints (2.77+/-0.3% vs 4.98+/-0.9%, p=0.004; 3.05+/-0.2% vs 5.81+/-0.3%, p=0.011). Picrosirius red staining demonstrated a diminished scar-index in fat-treated animals at both timepoints (.54+/-0.05 vs .74+/-0.07, p=0.034; .55+/-0.06 vs .93+/-.07, p=0.001). CONCLUSIONS: Fat grafting attenuates inflammation in acute radiodermatitis and slows the progression of fibrosis in chronic radiodermatitis

Purpose: Cutaneous radiation injury occurs during the treatment of cancer, or in rare environmental exposure. As the acute wound heals, fibrosis is induced and extracellular matrix (ECM) is deposited. The fibrotic pathway is mediated by the transforming growth factor-beta (TGF-beta) cascade, and is dependent on Smad3, a transcription factor for ECM. We characterized gene expression of this cascade after radiation injury and performed in vitro and in vivo gene silencing of Smad3 in an attempt to reverse the fibrotic pathway. Methods: Wild-type murine dermal fibroblasts were irradiated with 20Gy and harvested at serial time-points. RT-PCR was performed for known regulators and mediators of fibrosis. Smad3 was silenced by transfection with siRNA. For the in vivo experiment, dorsal skin of wild-type mice was irradiated with 45 Gy. Five weeks later, siRNA was applied to the fibrotic areas for one week. Skin was harvested and tissue analyzed by RT-PCR and Western blotting, as well as tissue tensiometry, which quantitatively measures rigidity. Results: Following irradiation, there was a steady increase in mRNA expression of Smad3, TGFbeta, and ECM genes collagen 1A1, metalloprotease2, and tissue inhibitor of metalloprotease-1, with peak expression at 12-24 hours. Inhibition of Smad3 with siRNA significantly decreased expression of Smad3, TGFbeta, and ECM genes. In the mouse model, topical treatment with siRNA again significantly decreased expression of these genes. Tensiometry demonstrated decreased stiffness in Smad3 siRNA treated skin, with a Young's modulus nearer to normalcompared to untreated and nonsense siRNA treated skin. Conclusion: Following initiation of the fibrotic pathway by radiation, Smad3 siRNA treatment both in vitro and in vivo effectively reversed gene expression. Furthermore, cutaneous Smad3 inhibition mitigated radiation-induced fibrotic stiffening. These findings suggest a therapeutic role for Smad3 silencing for cancer patients treated with radiation as well as those accidentally exposed to radiation

OBJECTIVE: Radiotherapy, an essential modality in cancer treatment, frequently induces fibrotic processes in the skin, including accumulation of extracellular matrix. Transforming growth factor-beta is essential in regulating extracellular matrix gene expression and is dependent on Smad3, an intracellular mediator/transcription factor. Our study characterized the genetic expression involved in extracellular matrix accumulation during radiation-induced fibrosis. We performed Smad3 gene silencing in an attempt to abrogate the effects of radiation. STUDY DESIGN: Laboratory research. SETTING: University laboratory. SUBJECTS AND METHODS: C57 murine dermal fibroblasts were irradiated with 20 Gy RNA isolated (0, 6, 12, 24, 48, 72 hours postirradiation) and mRNA analyzed (reverse transcriptase polymerase chain reaction) for known regulators (Smad3, interleukin-13 [IL-13]), tumor necrosis factor-alpha [TNF-alpha]) and mediators of fibrosis (collagen 1A1 [Col1A1]), TGF-beta, matrix metalloprotease-1 and -2 (MMP-1, MMP-2), and tissue inhibitor of metalloprotease-1 (TIMP-1). Smad3 gene expression was silenced using siRNA in an effort to restore an unirradiated gene profile. RESULTS: Following irradiation, there was a steady increase in mRNA expression of Smad3, IL-13, TGF-beta, Col1A1, MMP-2, TIMP-1, with peak at 12 to 24 hours and subsequent decline by 72 hours. TNF-alpha expression remained elevated throughout. MMP-1 showed minimal expression initially, which decreased to negligible by 72 hours. Inhibition of Smad3 significantly decreased expression of Col1A1, TGF-beta, MMP-2, and TIMP-1. IL-13 and TNF-alpha expression was not affected by Smad3 silencing. CONCLUSION: We have characterized the early-phase mRNA expression profiles of the major mediators of radiation-induced fibrosis. Smad3 siRNA effectively abrogated the elevation of Col1A1, TGF-beta, TIMP-1, and MMP-2. IL-13 and TNF-alpha were unaffected by Smad3 silencing and appear to be minor regulators in fibrosis. These findings suggest a therapeutic rationale for Smad3 silencing in vivo

BACKGROUND: Management of severe traumatic lower extremity injuries remains a considerable challenge. Free tissue transfer is now a standard part of reconstruction for Gustilo IIIB and IIIC injuries. There is limited information on arterial injury patterns in this population. We undertook a review of our experience to gain insight on vascular injury patterns and surgical outcomes. STUDY DESIGN: A 26-year retrospective analysis was performed of all lower extremity Gustilo IIIB and IIIC injuries requiring microvascular reconstruction at New York University Medical Center. Patient demographics, Gustilo classification, angiographic findings (conventional/computed tomographic angiography/magnetic resonance angiography), recipient vessels, elapsed time from injury, flap choices, and outcomes were examined. RESULTS: Two hundred twenty-two free flaps on 191 patients were performed from September 1982 until March 2008. There were 151 males and 40 females ranging in age from 4 to 83 years (median age 33 years). Patients sustained either Gustilo IIIB (170 patients) or IIIC (21 patients) open fractures. One hundred fifty-four patients had angiograms (78.2% IIIB, 100% IIIC). Sixty-six (42.9%) had normal 3-vessel runoff and 88 (57.1%) were abnormal. Sixty-one patients (31.9%) had anterior tibial injuries, 17 patients (8.9%) had posterior tibial injuries, and 30 (15.7%) had peroneal injuries. Sixty-three complications occurred (11 early thrombosis, 33 requiring secondary procedures, and 10 requiring amputation). CONCLUSIONS: Angiography of severe lower extremity injuries requiring free flap reconstruction usually revealed arterial injury and is generally indicated. In our experience, the anterior tibial artery is most commonly injured and the posterior tibial artery is most likely to be spared and used as a recipient

When free tissue transfer is employed for defects of the lower third of the leg, recipient anastomoses are typically performed to major vessels. The aim of this study was to assess soleal perforators located in the distal half of the leg as potential vessels for free flap recipient vessels. Six fresh cadavers (12 limbs) were dissected. Perforators of adequate size (>or=1 mm) were documented as was the location and ease of dissection. Lower extremity magnetic resonance angiograms (MRAs) of 18 extremities were retrospectively reviewed. Two free tissue transfers to lower extremity perforators were presented. Soleal perforators most reliably matched our recipient vessel requirements. Perforators were of adequate size to support free tissue transfer, easy to dissect, and were located at mid/distal fibula level. MRA evaluation confirmed these results. One free tissue reconstruction was performed for trauma (posterior tibial perforator) and one was performed for a chronic radiation wound (peroneal perforator). The soleus muscle is easily exposed and is supplied distally by perforators from both the posterior tibial and the peroneal artery systems. These perforating branches are more accessible than the major lower extremity arteries, making the exposure and anastomosis technically easier and sparing potential iatrogenic injury to critical vessels

OBJECTIVE: To attempt to mitigate the effects of irradiation on murine skin after high-dose radiation using a novel transcutaneous topical delivery system to locally inhibit gene expression with small interfering RNA (siRNA) against Smad3. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: Twenty-five wild-type C57 mice. INTERVENTION: In an isolated skin irradiation model, the dorsal skin of C57 wild-type mice was irradiated (45 Gy). Just before irradiation, Smad3 and nonsense siRNA were applied to 2 separate dorsal skin areas and then reapplied weekly. Skin was harvested after 1 and 4 weeks. Smad3 expression were assessed by immunohistochemistry, and collagen deposition and architecture was examined using picrosirius red collagen staining. MAIN OUTCOME MEASURES: Epidermal thickness was measured semiquantitatively at 4 weeks. Radiation-induced fibrosis was measured quantitatively via tensiometry. The Young modulus, a measure of cutaneous rigidity inversely related to elasticity, was determined, with normal irradiated skin serving as a control specimen. RESULTS: Murine skin treated with topical Smad3 siRNA demonstrated effective Smad3 inhibition at 1 week and persistent suppression at 4 weeks. Collagen deposition and epidermal thickness were significantly decreased in skin treated with Smad3 siRNA compared with control irradiated skin. Tensiometry demonstrated decreased tension in Smad3 siRNA-treated skin, with a Young modulus of 9.29 MPa (nonirradiated normal skin, 7.78 MPa) compared with nonsense (control) siRNA-treated skin (14.68 MPa). CONCLUSIONS: Smad3 expression can be effectively silenced in vivo using a novel topical delivery system. Moreover, cutaneous Smad3 inhibition mitigates radiation-induced changes in tissue elasticity, restoring a near-normal phenotype