Spatial Frequency Domain Imaging (SFDI) of clinical burns: A case report.

While visual assessment by a clinician is the standard of care for burn severity evaluations, new technologies at various stages of development are attempting to add objectivity to this practice by quantifying burn severity. Assessment accuracy generally improves after the burn injury has progressed, but early assessments that correctly identify superficial partial and deep partial burns have the potential to lead to more prompt treatments and shorter recovery times. To date, Spatial Frequency Domain Imaging (SFDI) has only been used in animal models of burns, but has shown the potential to categorize burns accurately at earlier time points. Here we examine the potential for SFDI to assess burn severity in clinical patients. We also utilize Laser Speckle Imaging (LSI), an FDA cleared non-invasive imaging technology that typically measures blood perfusion in order to evaluate burns in clinical patients. We present a case series of two patients, both with partial thickness burns of varying severity. Partial thickness burns are often difficult for clinicians to categorize based on visual appearance alone. SFDI and LSI were both performed on each patient at approximately 24 and 72 h after their respective burn incidents. Each technique was able to render spatially resolved information that enabled improved assessment accuracy for each burn. This represents the first publication of SFDI applied to clinical burn patients after being successfully utilized in animal models, and highlights the potential for SFDI as a feasible tool for the timely categorization of burn severity.

Evaluating clinical observation versus Spatial Frequency Domain Imaging (SFDI), Laser Speckle Imaging (LSI) and thermal imaging for the assessment of burn depth.

While clinical examination is needed for burn severity diagnosis, several emerging technologies aim to quantify this process for added objectivity. Accurate assessments become easier after burn progression, but earlier assessments of partial thickness burn depth could lead to earlier excision and grafting and subsequent improved healing times, reduced rates of scarring/infection, and shorter hospital stays. Spatial Frequency Domain Imaging (SFDI), Laser Speckle Imaging (LSI) and thermal imaging are three non-invasive imaging modalities that have some diagnostic ability for noninvasive assessment of burn severity, but have not been compared in a controlled experiment. Here we tested the ability of these imaging techniques to assess the severity of histologically confirmed graded burns in a swine model. Controlled, graded burn wounds, 3cm in diameter were created on the dorsum of Yorkshire pigs (n=3, 45-55kg) using a custom-made burn tool that ensures consistent pressure has been employed by various burn research groups. For each pig, a total of 16 burn wounds were created on the dorsal side. Biopsies were taken for histological analysis to verify the severity of the burn. Clinical analysis, SFDI, LSI and thermal imaging were performed at 24 and 72h after burn to assess the accuracy of each imaging technique. In terms of diagnostic accuracy, using histology as a reference, SFDI (85%) and clinical analysis (83%) performed significantly better that LSI (75%) and thermography (73%) 24h after the burn. There was no statistically significant improvement from 24 to 72h across the different imaging modalities. These data indicate that these imaging modalities, and specifically SFDI, can be added to the burn clinicians' toolbox to aid in early assessment of burn severity.

Impact of hemoglobin breakdown products in the spectral analysis of burn wounds using spatial frequency domain spectroscopy.

Burn wounds and wound healing invoke several biological processes that may complicate the interpretation of spectral imaging data. Through analysis of spatial frequency domain spectroscopy data (450 to 1000 nm) obtained from longitudinal investigations using a graded porcine burn wound healing model, we have identified features in the absorption spectrum that appear to suggest the presence of hemoglobin breakdown products, e.g., methemoglobin. Our results show that the calculated concentrations of methemoglobin directly correlate with burn severity, 24 h after the injury. In addition, tissue parameters such as oxygenation (StO2) and water fraction may be underestimated by 20% and 78%, respectively, if methemoglobin is not included in the spectral analysis.

Burn wound classification model using spatial frequency-domain imaging and machine learning.

Accurate assessment of burn severity is critical for wound care and the course of treatment. Delays in classification translate to delays in burn management, increasing the risk of scarring and infection. To this end, numerous imaging techniques have been used to examine tissue properties to infer burn severity. Spatial frequency-domain imaging (SFDI) has also been used to characterize burns based on the relationships between histologic observations and changes in tissue properties. Recently, machine learning has been used to classify burns by combining optical features from multispectral or hyperspectral imaging. Rather than employ models of light propagation to deduce tissue optical properties, we investigated the feasibility of using SFDI reflectance data at multiple spatial frequencies, with a support vector machine (SVM) classifier, to predict severity in a porcine model of graded burns. Calibrated reflectance images were collected using SFDI at eight wavelengths (471 to 851 nm) and five spatial frequencies (0 to 0.2 mm - 1). Three models were built from subsets of this initial dataset. The first subset included data taken at all wavelengths with the planar (0 mm - 1) spatial frequency, the second comprised data at all wavelengths and spatial frequencies, and the third used all collected data at values relative to unburned tissue. These data subsets were used to train and test cubic SVM models, and compared against burn status 28 days after injury. Model accuracy was established through leave-one-out cross-validation testing. The model based on images obtained at all wavelengths and spatial frequencies predicted burn severity at 24 h with 92.5% accuracy. The model composed of all values relative to unburned skin was 94.4% accurate. By comparison, the model that employed only planar illumination was 88.8% accurate. This investigation suggests that the combination of SFDI with machine learning has potential for accurately predicting burn severity.

How does blood loss relate to the extent of surgical wound excision?

PURPOSE: We investigated a novel system that uses image-processing algorithms to accurately measure the hemoglobin content of discarded surgical sponges to determine how blood loss relates to the excised area when current methods to minimize bleeding are employed. The system was used during 130 procedures in adult patients having wound excisions ≥1% BSA (study group). An historic group of 105 similar cases in whom visually estimated blood was determined was also evaluated. RESULTS: Surgical blood loss was less than previous estimates. The correlation between blood loss and the excised area in the study group was poor (R2=0.3988 for all patients and R2=0.1439 for excisions ≥10% BSA). Moreover, the visual estimates of blood loss in the historic group were more closely related to excised area than the accurate measurements in the study group (R2=0.6017 (historic), R2=0.3988 (study), p<0.001 for both). The mean absolute unstandardized residuals were 140.18±158.52 (historic) vs. 307.99±317.03 (study), p<0.001. CONCLUSIONS: As demonstrated in the historic group, visual estimates of blood loss tend to be more related to the size of excision than the amount of bleeding. The actual blood loss is not well correlated with the extent of excision. Clinicians should not rely on traditional blood loss estimates. Accurate measurement is needed to inform transfusion decisions and guide care.

Racial Disparities in Access and Outcomes of Cholecystectomy in the United States.

Disparities in access to health care between white and minority patients are well described. We aimed to analyze the trends and outcomes of cholecystectomy based on racial classification. The Nationwide Inpatient Sample database was reviewed for all patients undergoing cholecystectomy from 2009 to 2012. Patients were stratified as white or non-white. A total of 243,536 patients were analyzed: 159,901 white and 83,635 non-white. Non-white patients had significantly higher proportions of Medicaid (25% vs 9.3%), self-pay (14% vs 7.1%), and no-charge (1.8% vs 0.64%). Non-white patients had significantly higher rates of emergent admission (84% vs 78%) compared with the white patients. Multivariate analysis revealed that non-whites had a significantly longer length of stay [mean difference of 0.14 days, 95% confidence interval (CI) 0.08-0.20] and higher total hospital charges (mean difference of $6748.00, 95% CI 5994.19-7501.81) than whites, despite a lower morbidity (odds ratio 0.94, 95% CI 0.90-0.98). Use of laparoscopy and mortality were not different. These differences persisted on subgroup analysis by insurance type. These findings suggest a gap in access to and outcomes of cholecystectomy in the minority population nationwide.

Opposing roles for p21(waf1/cip1) and p27(kip1) in enterocyte differentiation, proliferation, and migration.

BACKGROUND: Originating from proliferating stem cells of the intestinal crypt, enterocytes differentiate as they migrate up the crypt-villus axis. A regulatory role of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1) in these processes has been suggested by in vitro models. We sought to determine the effect of p21(waf1/cip1) and p27(kip1) deficiency on enterocyte differentiation, proliferation and migration. METHODS: Three strains of mice including control (C57Bl/6), p27(kip1)-null, and p21(waf1/cip1)-null were studied. Enterocyte differentiation was evaluated by immunostaining for intestinal alkaline phosphatase, by colorimetric assaying for intestinal alkaline phosphatase and sucrase enzyme activity, and by polymerase chain reaction for intestinal fatty acid-binding protein and villin-messenger RNA in enterocytes extracted by laser capture microdissection. Rates of enterocyte proliferation and migration were determined by 5-bromo 2-deoxyuridine immunostaining after a 50% small-bowel resection (SBR). RESULTS: Compared with controls, p27(kip1)-null mice demonstrated minimal differentiation but maintained a normal proliferative response to SBR. Contrarily, p21(waf1/cip1)-null mice demonstrated greater enterocyte differentiation without significant increases in enterocyte proliferation after SBR. CONCLUSIONS: These findings suggest that p21(waf1/cip1) and p27(kip1) have distinctive and opposing roles in the pathogenesis of enterocyte differentiation, proliferation, and migration.

Acute discrimination between superficial-partial and deep-partial thickness burns in a preclinical model with laser speckle imaging.

A critical need exists for a robust method that enables early discrimination between superficial-partial and deep-partial thickness burn wounds. In this study, we report on the use of laser speckle imaging (LSI), a simple, non-invasive, optical imaging modality, to measure acute blood flow dynamics in a preclinical burn model. We used a heated brass comb to induce burns of varying severity to nine rats and collected raw speckle reflectance images over the course of three hours after burn. We induced a total of 12 superficial-partial and 18 deep-partial thickness burn wounds. At 3h after burn we observed a 28% and 44% decrease in measured blood flow for superficial-partial and deep-partial thickness burns, respectively, and that these reductions were significantly different (p=0.00007). This preliminary data suggests the potential role of LSI in the clinical management of burn wounds.

Bax deficiency rescues resection-induced enterocyte apoptosis in mice with perturbed EGF receptor function.

BACKGROUND: Adaptation after massive smallbowel resection (SBR) is associated with increased cell turnover, increased rates of enterocyte proliferation, and apoptosis. Epidermal growth factor receptor (EGFR) inhibition attenuates adaptation and increases apoptosis. Intestinal levels of bax appear to correlate with EGFR signaling. This study tested the hypothesis that bax is required for the exaggerated postresection apoptosis induced by perturbed EGFR signaling. METHODS: Waved-2 mice with impaired EGFR signaling were crossbred with bax-null mice. Offspring were subjected to either 50% proximal SBR or sham operation (bowel transection and reanastomosis). After 7 days, parameters of adaptation (villus height, wet weight), proliferation (% Ki-67 immunostaining of crypt cells), and apoptosis (# apoptotic bodies per crypt) were recorded in the remnant ileum. RESULTS: Enterocyte apoptosis was increased in waved-2 mice and prevented in bax-null mice after SBR. The accelerated apoptosis in the waved-2 mice was rescued in the context of deficient bax expression. Other parameters of adaptation were restored in the bax-null/waved-2 mice. CONCLUSION: Bax is required for the induction of postresection enterocyte apoptosis. Defective EGFR signaling augments resection-induced enterocyte apoptosis via a mechanism that also requires bax expression. These data implicate a link between EGFR signaling and bax in the genesis of postresection apoptosis and adaptation.

Trends in 393 necrotizing acute soft tissue infection patients 2000-2008.

OBJECTIVE: To determine the outcomes effect of changing trends in patients with necrotizing acute soft tissue infections (NASTI) 2000-2008. METHODS: A single institution retrospective chart review of all patients treated for NASTI. RESULTS: There were 393 patients with mean age 50 years, diabetes 53%, % body surface area excised 3.5. Wounds were located on: extremity 57%, perineum 40%, trunk 26%. Wound cultures %: polymicrobial=62, Staphylococci=48, Streptococci=31. Patients developing complications %: Pulmonary=23, renal insufficiency/failure=27. During the study period, overall mortality rate remained unchanged: 30/393=7.6% (5.5% for patients first admitted by burn/trauma/acute care surgery vs. 29% for all other services, p=0.003). Significant annual increases were found in number of patients, p=0.03, male sex, p=0.000, transfer from outside hospital, p<0.001, BMI p=0.003, ventilator requirement >24h, p=0.0005, APACHE II p=0.002, and number of patients developing any complication, p=0.04. Statistically significant decreases annually were found in: days of antibiotic use, p=0.008, number of operations required for excision, p=0.02, development of non-wound infections, p=0.002, and length of stay in days (LOS), p=0.03. CONCLUSIONS: This is the largest cohort of NASTI patients from a single institution to date, demonstrating significantly shorter LOS and decreased non-wound infection rates in the face of increasing BMI and APACHE II scores. The increasing number of patients and BMI suggests a causal relationship between NASTI and obesity. Initial care by surgeons experienced in caring for these patients provides mortality rates well below the national average.

Cerebral and somatic near-infrared spectroscopy in normal newborns.

PURPOSE: Near-infrared spectroscopy has been used increasingly in the pediatric population as a continuous, noninvasive indicator of trends in organ perfusion and oxygenation. We studied healthy newborn babies to establish normal values during rest and feeding. METHODS: Forty-four term newborns were recruited. Near-infrared spectroscopy probes were placed on the forehead and over the right kidney to record cerebral (rSO(2)C) and renal-somatic (rSO(2)R) regional oxyhemoglobin saturation. Readings were collected continuously for 2 to 8 hours, spanning 1 to 3 feeding episodes. RESULTS: Data were available on 26 patients, with an average age of 44 +/- 28 hours. The overall average rSO(2)C was 77.9% +/- 8.5%, rSO(2)R was 86.8% +/- 8.1%, and DeltarSO(2)RC (somatic-cerebral rSO(2) difference) was 8.9% +/- 9.4%. During feeding, rSO(2)C was minimally decreased (78.6% +/- 8.4% versus 78.0% +/- 9.0%, P = .023), rSO(2)R did not change (87.0% +/- 8.1% versus 87.3% +/- 8.0%, P = .31), and DeltarSO(2)RC was minimally increased (8.5% +/- 9.5% versus 9.2% +/- 9.1%, P = .014). Over the first 120 hours after birth, average rSO(2)C decreased (P < .01), and rSO(2)R remained relatively unchanged. CONCLUSIONS: Clinical utility of near-infrared spectroscopy was partly limited by lack of normative data. These data demonstrate that regional oxygen extraction is greater across cerebral than across renal-somatic beds in normal newborns. Healthy newborns do not have clinically significant changes in organ oxygenation with feeding.

Roles for p21waf1/cip1 and p27kip1 during the adaptation response to massive intestinal resection.

The magnitude of gut adaptation is a decisive factor in determining whether patients are able to live independent of parenteral nutrition after massive small bowel loss. We previously established that the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) is necessary for enterocyte proliferation and a normal adaptation response. In the present study, we have further elucidated the role of this CDKI in the context of p27(kip1), another member of the Cip/Kip CDKI family. Small bowel resections (SBRs) or sham operations were performed in control (C57/BL6), p21(waf1/cip1)-null, p27(kip1)-null, and p21(waf1/cip1)/p27(kip1) double-null mice. Morphological (villus height/crypt depth) alterations in the mucosa, the kinetics of enterocyte turnover (rates of enterocyte proliferation and apoptosis), and the protein expression of various cell cycle-regulatory proteins were recorded at various postoperative times. Enterocyte compartment-specific mRNA expression was investigated using laser capture microdissection. Resection-induced adaptation in control mice coincided with increased protein expression of p21(waf1/cip1) and decreased p27(kip1) within 3 days postoperatively. Identical changes in mRNA expression were detected in crypt but not in villus enterocytes. Adaptation occurred normally in control and p27(kip1)-null mice; however, mice deficient in both p21(waf1/cip1) and p27(kip1) failed to increase baseline rates of enterocyte proliferation and adaptation. The expression of p21(waf1/cip1) protein and mRNA in the proliferative crypt compartment is necessary for resection-induced enterocyte proliferation and adaptation. The finding that deficient expression of p27(kip1) does not affect adaptation suggests that these similar CDKI family members display distinctive cellular functions during the complex process of intestinal adaptation.

Absent STAT-1 expression perturbs adaptation and apoptosis after massive intestinal resection.

BACKGROUND: We have previously established the significance of epidermal growth factor receptor (EGFR) activity and the cyclin-dependent kinase inhibitor p21waf1/cip1 (p21) for the adaptive response of the intestine to massive small bowel resection (SBR). In this study, we tested the role of the signal transducer and activator of transcription 1 (STAT-1) as this transcription factor is activated by the EGFR and known to induce p21 expression. METHODS: Control (n = 40; C57/Bl6) and STAT-1-null mice (n = 40) underwent 50% proximal SBR or sham operation. After 3 days, the remnant ileum was harvested and the villus and crypt morphology was measured along with changes in rates of enterocyte proliferation and apoptosis. RESULTS: The magnitude of resection-induced adaptation was greater in STAT-1-null animals as verified by taller villi and deeper crypts. The expected increase in enterocyte apoptosis did not occur after SBR in the background of STAT-1 deficiency. Western blotting revealed elevated expression of p21 protein in both STAT-1-null and controls after SBR. CONCLUSION: Increased p21 expression after SBR in the absence of STAT-1 suggests an alternate mechanism for resection-induced regulation of p21. Enhanced adaptation in STAT-1-null animals suggests that this transcription factor serves an inhibitor to the process of adaptation, perhaps via regulation of enterocyte apoptosis.

Combined pharmacotherapy that increases proliferation and decreases apoptosis optimally enhances intestinal adaptation.

BACKGROUND: Adaptation after massive small bowel resection (SBR) is associated with increased rates of enterocyte proliferation (P) and apoptosis (A). In the present study, we sought to determine the effect of dual therapy designed to increase P and simultaneously reduce A. METHODS: C57Bl/6 mice underwent a 50% small bowel resection (SBR) or sham operation, and then received an inhibitor of apoptosis (pan-caspase inhibitor), a stimulus for proliferation (epidermal growth factor; EGF), a combination, or vehicle control. After 3 days, adaptive morphology (villus height, crypt depth) and rates of enterocyte turnover (proliferation and apoptosis) were measured in the remnant ileum. RESULTS: Adaptation in controls and treated with the inhibitor was similar. EGF-treated mice demonstrated an even greater adaptive response. Combined therapy with the inhibitor and EGF resulted in maximal adaptation as gauged by the greatest increases in villus height and crypt depth and ratio of rates of P to A. CONCLUSION: The capacity for adaptation following massive SBR is maintained via tight regulation of cell production and death. Pharmacologic intervention directed at increasing enterocyte proliferation while simultaneously decreasing apoptosis augments adaptation greater than either intervention alone and may provide a useful strategy to clinically amplify adaptation.

Epidermal growth factor receptor signaling regulates Bax and Bcl-w expression and apoptotic responses during intestinal adaptation in mice.

BACKGROUND & AIMS: Normal intestinal adaptation to massive small-bowel resection requires intact epidermal growth factor receptor signaling and consists of increased enterocyte proliferation and apoptosis. Although emphasis has been placed on understanding the regulation of proliferation, few studies have evaluated the mechanism and contribution of apoptosis to the adaptation response. We sought to test the hypothesis that epidermal growth factor receptor signaling regulates specific Bcl-2 family members (Bax and Bcl-w) to direct apoptosis and adaptation after massive small-bowel resection. METHODS: Laser capture microdissection microscopy permitted measurement of Bax and Bcl-w messenger RNA expression in crypt and villus enterocytes in control conditions and under epidermal growth factor receptor-inhibited (waved-2 mice) or stimulated (epidermal growth factor transgenic mice) conditions after a 50% small-bowel resection or sham operation. Resection-induced adaptation was then studied in Bax-null and Bcl-w-null mice under control circumstances and after epidermal growth factor receptor stimulation. RESULTS: When compared with Bcl-w, the most significant expression changes were observed with Bax and took place within crypt enterocytes. Epidermal growth factor receptor stimulation resulted in a decreased ratio of Bax to Bcl-w expression and decreased rates of apoptosis. Bax-null mice had no apoptosis response to small-bowel resection and displayed an amplified adaptation response to the administration of epidermal growth factor. Bcl-w-null mice had poor survival and impaired adaptation to small-bowel resection, an effect that was rescued by crossbreeding these mice with epidermal growth factor transgenic mice. CONCLUSIONS: The crypt expression of Bax and Bcl-w is influenced by epidermal growth factor receptor signaling and is key for the regulation of apoptosis. Epidermal growth factor receptor stimulation, coupled with apoptosis inhibition, may provide a novel strategy to amplify adaptation responses in patients after massive intestinal loss.

Evidence for active Wnt signaling during postresection intestinal adaptation.

BACKGROUND: In the intestine, Wnt proteins are powerful regulators of cell proliferation, differentiation, and adhesion. Mutations of the adenomatous polyposis coli (APC) gene elevate nuclear beta-catenin and provoke intestinal tumor formation. We sought to determine whether Wnt signaling is involved in adaptive response to massive small bowel resection (SBR). METHODS: Male Min mice with a mutation of the APC gene and wild-type controls underwent a 50% proximal SBR or sham operation. After 3 days, villus height, crypt depth, and rates of proliferation and apoptosis were recorded in the remnant ileum. RESULTS: After SBR, villus height and enterocyte proliferation were significantly greater in the Min mice. Western blotting demonstrated resection-induced increases in beta-catenin, c-Myc, and E-cadherin after SBR, which was more pronounced in Min mice. CONCLUSIONS: Mutation of the APC gene and augmented Wnt signaling in the intestine results in an enhanced adaptive response to massive SBR. These data, for the first time, implicate an important role for Wnt signaling during the pathogenesis of resection-induced intestinal adaptation.

Epidermal growth factor receptor signaling regulates goblet cell production after small bowel resection.

BACKGROUND/PURPOSE: Intestinal adaptation is a compensatory response to massive small bowel loss in which there are increased numbers of absorptive enterocytes. However, the generation of secretory epithelial cell subtypes in this process has not been investigated. The purpose of this study was to examine the adaptive changes of several small intestinal cell lineage changes in response to massive small bowel resection (SBR). METHODS: A 75% SBR or sham operation was performed on male Sprague-Dawley rats. On postoperative day 7, the remnant ileum was harvested and immunohistochemical staining for goblet, Paneth, and enteroendocrine cells was performed. Cell subtypes were evaluated as cells per micrometer of villus/crypt length and compared among operations. RESULTS: A significant increase in goblet cell density occurred after SBR. Intestinal resection did not alter the number of Paneth and enteroendocrine cells. In additional experiments, inhibition of epidermal growth factor receptor signaling was associated with a diminished goblet cell density. CONCLUSIONS: The adaptive response of the intestine to massive bowel loss results in an expansion of the goblet cell population in addition to greater numbers of absorptive enterocytes. Although the mechanism and purpose for selective expansion of these stem cell-derived lineages are not presently known, epidermal growth factor receptor signaling appears to be a common pathway.