Michael S. Chin

Hyperspectral imaging for early detection of oxygenation and perfusion changes in irradiated skin

Studies examining acute oxygenation and perfusion changes in irradiated skin are limited. Hyperspectral imaging (HSI), a method of wide-field, diffuse reflectance spectroscopy, provides noninvasive, quantified measurements of cutaneous oxygenation and perfusion. This study examines whether HSI can assess acute changes in oxygenation and perfusion following irradiation. Skin on both flanks of nude mice (n=20) was exposed to 50 Gy of beta radiation from a strontium-90 source. Hyperspectral images were obtained before irradiation and on selected days for three weeks. Skin reaction assessment was performed concurrently with HSI. Desquamative injury formed in all irradiated areas. Skin reactions were first seen on day 7, with peak formation on day 14, and resolution beginning by day 21. HSI demonstrated increased tissue oxygenation on day 1 before cutaneous changes were observed (p<0.001). Further increases over baseline were seen on day 14, but returned to baseline levels by day 21. For perfusion, similar increases were seen on days 1 and 14. Unlike tissue oxygenation, perfusion was decreased below baseline on day 21 (p<0.002). HSI allows for complete visualization and quantification of tissue oxygenation and perfusion changes in irradiated skin, and may also allow prediction of acute skin reactions based on early changes seen after irradiation.

Early Healing of Transcolonic and Transgastric Natural Orifice Transluminal Endoscopic Surgery Access Sites

BACKGROUND Natural Orifice Transluminal Endoscopic Surgery (NOTES) is a developing, minimally invasive surgical approach whose potential benefits are being investigated. Little is known about secure access site closure and early healing kinetics of transvisceral access. STUDY DESIGN Transvisceral access incisions were created in the colon (C-NOTES, n = 8) and stomach (G-NOTES, n = 8) for peritoneal exploration. Incisions were closed primarily with endoloops, endoclips, or t-tags. Macroscopic and histologic analyses performed on postoperative day 7 assessed gross appearance, granulation tissue, inflammation, ulceration, and complications. RESULTS Macroscopically, incisions appeared closed without intraperitoneal spillage. Incisions closed by endoloop and t-tags showed intense granulation tissue fill of defect despite partial (G-NOTES, n = 3) and transmural ulceration (C-NOTES, n = 8; G-NOTES, n = 3). Of the 30 t-tags applied, 40% broke or deployed into the peritoneal cavity. Endoclip closures (C-NOTES, n = 1; G-NOTES, n = 1) did not show histologic mucosal continuity. Healing complications included transmural necrosis (C-NOTES, n = 1; G-NOTES, n = 1), foreign body material (C-NOTES, n = 3; G-NOTES, n = 2), and microabscesses (G-NOTES, n = 1). CONCLUSIONS This study provides a reproducible model to assess noninvasive repair of planned visceral perforations. Of investigated technologies, endoloop closure was favored for transcolonic incisions, and t-tags with omental patch for transgastric incisions, although these have significant limitations. Endoclips were inadequate for primary closure, but may be useful as an adjunctive closure modality. Additional studies are needed to examine visceral repair at later time points, as they will help determine the quality and kinetics of repair of a variety of incision closure strategies. This study demonstrates the need for improved technologies to more reliably close visceral transluminal defects.

Induction of Adipogenesis by External Volume Expansion.

Background: External volume expansion by suction is used to prepare the recipient site for fat grafting by increasing its compliance and vascularity. The authors previously developed a mouse model for external volume expansion and demonstrated its pro-proliferative and angiogenic effects. Increased thickness of the subcutaneous tissue was also observed. This study was thus designed to assess the adipogenic potential of external volume expansion stimulation. Methods: A miniaturized external volume expansion device consisting of a rubber dome connected to a −25 mmHg suction source was applied to the dorsum of mice for a single 2-hour stimulation or for 2 hours daily for 5 days. Tissues were harvested up to 48 hours after the last stimulation and analyzed for edema, inflammation, and adipocyte content by staining for hematoxylin and eosin, CD45, and perilipin-A. Expression of peroxisome proliferator-activated receptor-&ggr; (proadipogenic factor) and preadipocyte factor 1 (preadipocyte marker) was evaluated by Western blot analysis. Results: Both a 2-hour stimulation and cyclical 2-hour stimulation for 5 days induced 1.5- and 1.9-fold increases in the number of adipocytes per millimeter. Edema was present in the immediate poststimulation period, and inflammation was seen 2 days later. Peroxisome proliferator-activated receptor-&ggr; was increased at the end of stimulation. Conclusions: Stretch is known to stimulate proliferation, whereas edema and inflammation are both emerging proadipogenic factors. Their combination in external volume expansion seems to produce proadipogenic effects, seen even after a single 2-hour stimulation.

Animal models of keloids and hypertrophic scars.

This communication is a commentary on the manuscript entitled “Is There an Ideal Animal Model to Study Hypertrophic Scarring?” by Ramos et al (J Burn Care Res 2008;29:363–8). We believe that this review will have an important impact on future keloid and hypertrophic scar study, but we would like to further elaborate on some issues regarding this topic. Animal models provide important translational vehicles for human treatment modalities. Keloids and hypertrophic scarring continue to plague large segments of our patient population, and both pathologies lack suitable animal models to aid in the discovery of better treatments. Many authors have attempted to construct suitable animal models as Ramos et al described in their manuscript. Although animal models are rarely a perfect replication of human pathophysiology, the current models described seem to be more related to an inflammatory response rather than actually hypertrophic scar or keloid processes, and they should be interpreted with caution. Thus, we would like to discuss some of the controversial issues surrounding animal models of keloids and hypertrophic scars. Human immature scars initially are normally slightly red, sometimes itchy or painful, and actively remodel. Conceptually, keloids and hypertrophic scars can be considered as immature scars that are slow to resolve. When normal scar maturation is complete, the remodeling process becomes quiescent, and the inflammatory process disappears. Generally, scars flatten out, but in certain locations and in certain genetic backgrounds, the mature scar sometimes becomes slightly elevated (Figure 1). However, an elevation in scar alone is not necessarily hypertrophic in nature, as it should have some degree of chronic inflammation. Both keloid and hypertrophic scars are reddish because of abnormal growth of capillary blood vessels, and should persist past several months in the human. Both hypertrophic scars and keloids have increased microcirculation compared with normal skin and mature scar tissue. Thus, “hypertrophic scars” in previous reports may be not true hypertrophic scars of the animal. There is a possibility that the scars in these models may actually be immature scars in the process of remodeling. Hence, the term “hypertrophic scar” should be used with caution in these studies. This remodeling period may be different according to animal species. In humans, hypertrophic scars occur within weeks after injury and may rapidly increase in size for 3 to 6 months and then, after a static phase, begin to regress. The full maturation process may take up to 2 years. We hope that additional work will provide us with a better model for hypertrophic scarring. We are encouraged by the work of Aarabi et al, who recently reported developing a hypertrophic scar model based on mechanical force loading. They demonstrated that there is decreased apoptosis in scars subjected to tension. Moreover, they illustrated the effect of inflamReprint requests: Rei Ogawa, MD, PhD, 1-1-5 Sendagi Bunkyo-ku, Tokyo, 113-8603, Japan. Copyright

Hyperspectral Imaging for Burn Depth Assessment in an Animal Model

Background: Differentiating between superficial and deep-dermal (DD) burns remains challenging. Superficial-dermal burns heal with conservative treatment; DD burns often require excision and skin grafting. Decision of surgical treatment is often delayed until burn depth is definitively identified. This study’s aim is to assess the ability of hyperspectral imaging (HSI) to differentiate burn depth. Methods: Thermal injury of graded severity was generated on the dorsum of hairless mice with a heated brass rod. Perfusion and oxygenation parameters of injured skin were measured with HSI, a noninvasive method of diffuse reflectance spectroscopy, at 2 minutes, 1, 24, 48 and 72 hours after wounding. Burn depth was measured histologically in 12 mice from each burn group (n = 72) at 72 hours. Results: Three levels of burn depth were verified histologically: intermediate-dermal (ID), DD, and full-thickness. At 24 hours post injury, total hemoglobin (tHb) increased by 67% and 16% in ID and DD burns, respectively. In contrast, tHb decreased to 36% of its original levels in full-thickness burns. Differences in deoxygenated and tHb among all groups were significant (P < 0.001) at 24 hours post injury. Conclusions: HSI was able to differentiate among 3 discrete levels of burn injury. This is likely because of its correlation with skin perfusion: superficial burn injury causes an inflammatory response and increased perfusion to the burn site, whereas deeper burns destroy the dermal microvasculature and a decrease in perfusion follows. This study supports further investigation of HSI in early burn depth assessment.

Cavitation rheology as a potential method for in vivo assessment of skin biomechanics.

Evaluation of the biomechanical properties of skin is essential for characterization of wound healing kinetics and cutaneous diseases. Available techniques require tissue excision and preclude serial tissue assessment, which limit in vivo monitoring of biomechanical changes. Cavitation rheology (CR) is a method for focal assessment of the mechanical properties of materials.(1) An air cavity is created at the tip of a needle inserted into a material and pressurized until the material yields by rapid elastic deformation (cavitation) or irreversible fracture. An incisional wound model was utilized to determine if this technique could discern differences in the mechanical responses of skin in various states. In accordance with our Institutional Animal Care and Use Committee guidelines, a full-thickness incision was created in the dorsal midline of six Sprague-Dawley rats (The Jackson Laboratory, Bar Harbor, ME) and then closed with a subcuticular suture (4-0 Monocryl, Ethicon, Somerville, NJ). Rats were euthanized 28 days post-wounding and skin was harvested. Cavitation was achieved with a 34-gauge needle (PY2-72-0081, Harvard Apparatus, Holliston, MA) connected to a syringe pump (Nexus 6000, Chemyx, Stafford, TX) and real-time pressure sensor (Cajon Transducer 300psi, Swagelok, Solon, OH). (Figure 1) Air was injected in multiple incisional and surrounding unwounded areas and yielding pressures were recorded. The preferred pathway for yield is a product of material properties and length scale, which can be determined as previously described.(1) Cavitated tissues were excised and stained with hematoxylin and eosin in standard fashion. Statistical comparison of yielding pressures was made using a two-tailed t-test. Statistical significance was assumed when p < 0.05. Figure 1 A schematic representation of the experimental apparatus used for CR. The inset demonstrates the expanding cavity of air (blue circle) within the dermis. The deformational force generated by this cavity (blue arrows) is opposed by the intrinsic elastic ... Incisional skin yielded at a lower pressure than unwounded skin (Figure 2). Sampled sites (n=36, 18 incisional and 18 unwounded) demonstrated a significant difference (p<0.01) in the mean yielding pressure of 69 kPa and 79 kPa for incisional and unwounded skin, respectively. Histological analysis demonstrated a reliable cavitation plane at the dermal-subcutaneous junction, consistent with the realtime observation of maintenance in skin integrity. Figure 2 Box plot demonstrating yielding pressures for incisional (left) and unwounded (right) rat skin 28 days post-wounding. Error bars represent ± standard deviation. Non-destructive methods previously developed to measure cutaneous biomechanical properties have not achieved widespread application.(2, 3) The gold standard of tensiometry has several limitations.(3) Tissue must be excised and sectioned leading to shear stress on samples. Accurate measurements may also be confounded by tissue slippage in tensiometer clamps and imprecise timing of wound breakage. Additionally, methods that rely upon stretch cannot isolate individual layers of cutaneous tissues and their different biomechanical properties. CR has been used to describe the ex vivo biomechanical properties of other animal tissues.(4) We have demonstrated the ability to reproducibly measure the biomechanical properties of skin in various states, with results consistent with previous findings.(5) Ex vivo measurements of the biomechanical properties of skin may not accurately reflect in vivo properties,(3) and our future studies will use in vivo models to compare the ex vivo and in vivo biomechanical properties of skin as assessed by CR. We propose CR as a non-destructive method to assess cutaneous biomechanical properties. CR may provide accurate in vivo evaluation of biomechanical changes during cutaneous wound healing or disease evolution.

A simple method to facilitate full-thickness skin graft harvest.

We describe here a simple method that will make harvesting a full thickness skin graft an easy task. The procedure will allow the surgeon to take the graft with no fat attached to it, therefore saving the time taken to defat this type of grafts in the conventional way. Moreover, the technique is relatively bloodless rendering it more time saving.

External Volume Expansion in Irradiated Tissue: Effects on the Recipient Site.

Background: External volume expansion prepares recipient sites to improve outcomes of fat grafting. For patients receiving radiotherapy after mastectomy, results with external volume expansion vary, and the relationship between radiotherapy and expansion remains unexplored. Thus, the authors developed a new translational model to investigate the effects in chronic skin fibrosis after radiation exposure. Methods: Twenty-four SKH1-E mice received 50 Gy of &bgr;-radiation to each flank and were monitored until fibrosis developed (8 weeks). External volume expansion was then applied at −25 mmHg to one side for 6 hours for 5 days. The opposite side served as the control. Perfusion changes were assessed with hyperspectral imaging. Mice were euthanized at 5 (n = 12) and 15 days (n = 12) after the last expansion application. Tissue samples were analyzed with immunohistochemistry for CD31 and Ki67, Masson trichrome for skin thickness, and picrosirius red to analyze collagen composition. Results: All animals developed skin fibrosis 8 weeks after radiotherapy and became hypoperfused based on hyperspectral imaging. Expansion induced edema on treated sides after stimulation. Perfusion was decreased by 13 percent on the expansion side (p < 0.001) compared with the control side for 5 days after stimulation. Perfusion returned to control-side levels by day 15. Dermal vasculature increased 38 percent by day 15 (p < 0.01) in expansion versus control. No difference was found in collagen composition. Conclusions: External volume expansion temporarily reduces perfusion, likely because of transient ischemia or edema. Together with mechanotransduction, these effects encourage a proangiogenic and proliferative environment in fibrotic tissue after radiotherapy in the authors’ mouse model. Further studies are needed to assess these changes in fat graft retention.

Hyperspectral Imaging as an Early Biomarker for Radiation Exposure and Microcirculatory Damage.

Background Radiation exposure can lead to detrimental effects in skin microcirculation. The precise relationship between radiation dose received and its effect on cutaneous perfusion still remains controversial. Previously, we have shown that hyperspectral imaging (HSI) is able to demonstrate long-term reductions in cutaneous perfusion secondary to chronic microvascular injury. This study characterizes the changes in skin microcirculation in response to varying doses of ionizing radiation and investigates these microcirculatory changes as a possible early non-invasive biomarker that may correlate with the extent of long-term microvascular damage. Methods Immunocompetent hairless mice (n = 66) were exposed to single fractions of superficial beta-irradiation in doses of 0, 5, 10, 20, 35, or 50 Gy. A HSI device was utilized to measure deoxygenated hemoglobin levels in irradiated and control areas. HSI measurements were performed at baseline before radiation exposure and for the first 3 days post-irradiation. Maximum macroscopic skin reactions were graded, and histological assessment of cutaneous microvascular densities at 4 weeks post-irradiation was performed in harvested tissue by CD31 immunohistochemistry. Results CD31 immunohistochemistry demonstrated a significant correlation (r = 0.90, p < 0.0001) between dose and vessel density reduction at 4 weeks. Using HSI analysis, early changes in deoxygenated hemoglobin levels were observed during the first 3 days post-irradiation in all groups. These deoxygenated hemoglobin changes varied proportionally with dose (r = 0.98, p < 0.0001) and skin reactions (r = 0.98, p < 0.0001). There was a highly significant correlation (r = 0.91, p < 0.0001) between these early changes in deoxygenated hemoglobin and late vascular injury severity assessed at the end of 4 weeks. Conclusion Radiation dose is directly correlated with cutaneous microvascular injury severity at 4 weeks in our model. Early post-exposure measurement of cutaneous deoxygenated hemoglobin levels may be a useful biomarker for radiation dose reconstruction and predictor for chronic microvascular injury.

Abstract 125: adipogenesis by external volume expansion.

Jorge R Lujan-Hernandez, MD1; Luca Lancerotto, MD1; Christoph Nabzdyk, MD1; Kazi Z Hassan, BS1; Roger K Khouri, Jr, BS1; Hamed Zartab, MD, MSc1; Michael S Chin, MD2; Franco Bassetto, MD3; Janice F Lalikos, MD2; Dennis P Orgill, MD, PhD1 1Brigham and Women’s HospitalHarvard Medical School, Boston, MA, University of Massachusetts Medical School, Worcester, MA, Institute of Plastic Reconstructive and Aesthetic Surgery, University of Padova, Padova, Italy