In recent conflicts, battlefield injuries consist largely of extensive soft injuries from blasts and high energy projectiles, including gunshot wounds. Repair of these large, traumatic wounds requires aggressive surgical treatment, including multiple surgical debridements to remove devitalised tissue and to reduce bacterial load. Identifying those patients with wound complications, such as infection and impaired healing, could greatly assist health care teams in providing the most appropriate and personalised care for combat casualties.
Candidate technologies to enable this benefit include the fusion of imaging and optical spectroscopy to enable rapid identification of key markers. Hence, a novel system based on IR negative contrast imaging (NCI) is presented that employs an optical parametric oscillator (OPO) source comprising a periodically-poled LiNbO3 (PPLN) crystal. The crystal operates in the shortwave and midwave IR spectral regions (ca. 1.5 – 1.9 μm and 2.4 – 3.8 μm, respectively). Wavelength tuning is achieved by translating the crystal within the pump beam. System size and complexity are minimised by the use of single element detectors and the intracavity OPO design. Images are composed by raster scanning the monochromatic beam over the scene of interest; the reflection and/or absorption of the incident radiation by target materials and their surrounding environment provide a method for spatial location. Initial results using the NCI system to characterise wound biopsies are presented here.
In military medicine, one of the challenges in dealing with large combat-related injuries is the prevalence of bacterial infection, including multidrug resistant organisms. This can prolong the wound healing process and lead to wound dehiscence. Current methods of identifying bacterial infection rely on culturing microbes from patient material and performing biochemical tests, which together can take 2-3 days to complete. Surface Enhanced Raman Spectroscopy (SERS) is a powerful vibrational spectroscopy technique that allows for highly sensitive structural detection of analytes adsorbed onto specially prepared metal surfaces. In the past, we have been able to discriminate between bacterial isolates grown on solid culture media using standard Raman spectroscopic methods. Here, SERS is utilized to assess the presence of bacteria in wound effluent samples taken directly from patients. To our knowledge, this is the first attempt for the application of SERS directly to wound effluent. The utilization of SERS as a point-of-care diagnostic tool would enable physicians to determine course of treatment and drug administration in a matter of hours.
Combat wounds are sometimes confounded by healing complications that are not as prevalent in civilian wounds due to their high energy etiology. One complication of wound healing is dehiscence, where a surgically closed wound reopens after closure. This complication can have serious consequences for the patient, but knowledge about the molecular composition of the wound bed beyond what is readily visible may help clinicians mitigate these complications. It is necessary to develop techniques that can be used in vivo to assess and predict wound healing pointof- care so that care-takers can decide the best way to make informed clinical decisions regarding their patient’s healing. Raman spectroscopy is a perfect candidate for predicting wound healing due to its ability to provide a detailed molecular fingerprint of the wound bed noninvasively. Here, we study the spectral correlation index, a measure of orthogonality, with ten reference tissue components to stratify wounds based on how they heal. We analyze these indexes over time to show the modulation of these tissue components over the wound healing process. Results show that qualitative observation of the spectra cannot reveal major differences between the dehisced and normal healing wounds, but the spectral correlation index can. Analysis of the spectral correlations across the wound healing process demonstrates the changes throughout the wound healing process, showing that early differences in tissue components may portend wound healing. Furthermore, Raman spectroscopy coupled with the spectral correlation index presents as a possible point-of-care tool for enabling discrimination of wounds with impaired healing.
Traumatic heterotopic ossification (HO) is the pathological formation of bone in soft tissue and is a debilitating sequela
following acute trauma involving blast-related extremity musculoskeletal injuries, severe burns, spinal cord injury, and
traumatic brain injury. Over 60% of combat related injuries and severe burns develop HO; often resulting in reduced
mobility, chronic pain, ulceration, tissue entrapment, and reduced ambulation. Detection and prognosis is limited by
current clinical imaging modalities (computed tomography, radiography, and ultrasound). This study identifies Raman
spectral signatures corresponding to histological changes in a combat-trauma induced rat HO model at early time points
prior to radiographic evidence of HO. HO was induced in Sprague-Dawley rats via blast over pressure injury, mid-femoral
fracture, soft tissue crush injury, and limb amputation through the zone of injury. Rats were euthanized, and
amputated limbs were formalin fixed and embedded in paraffin; 10 μm sections were placed on gold slides, and paraffin
was chemically removed. Tissues from sham-treated animals served as controls. Tissue maps consisting of Raman
spectra were generated using a Raman microprobe system with an 80-90 μm spot size and 785 nm excitation in regions
exhibiting histological evidence of early HO development according to adjacent HE sections. Factors were extracted
from mapping data using Band-Target Entropy Minimization algorithms. Areas of early HO were highlighted by a
Raman factor indicative of the presence of collagen. Identification of collagen as an early marker of HO prior to
radiographic detection in a clinically relevant animal model serves to inform future clinical work.
Recent studies have demonstrated the potential advantages of the use of Raman spectroscopy in the biomedical field due to its rapidity and noninvasive nature. In this study, Raman spectroscopy is applied as a method for differentiating between bacteria isolates for Gram status and Genusspecies. We created models for identifying 28 bacterial isolates using spectra collected with a 785 nm laser excitation Raman spectroscopic system. In order to investigate the groupings of these samples, partial least squares discriminant analysis (PLSDA) and hierarchical cluster analysis (HCA) was implemented. In addition, cluster analyses of the isolates were performed using various data types consisting of, biochemical tests, gene sequence alignment, high resolution melt (HRM) analysis and antimicrobial susceptibility tests of minimum inhibitory concentration (MIC) and degree of antimicrobial resistance (SIR). In order to evaluate the ability of these models to correctly classify bacterial isolates using solely Raman spectroscopic data, a set of 14 validation samples were tested using the PLSDA models and consequently the HCA models. External cluster evaluation criteria of purity and Rand index were calculated at different taxonomic levels to compare the performance of clustering using Raman spectra as well as the other datasets. Results showed that Raman spectra performed comparably, and in some cases better than, the other data types with Rand index and purity values up to 0.933 and 0.947, respectively. This study clearly demonstrates that the discrimination of bacterial species using Raman spectroscopic data and hierarchical cluster analysis is possible and has the potential to be a powerful point-of-care tool in clinical settings.
Over 30% of combat injuries involve peripheral nerve injury compared to only 3% in civilian trauma. In fact, nerve dysfunction is the second leading cause of long-term disability in injured service members and is present in 37% of upper limb injuries with disability. Identification and assessment of non-penetrating nerve injury in trauma patients could improve outcome and aid in therapeutic monitoring. We report the use of Raman spectroscopy as a noninvasive, non-destructive method for detection of nerve degeneration in intact nerves due to non-penetrating trauma. Nerve trauma was induced via compression and ischemia/reperfusion injury using a combat relevant swine tourniquet model (>3 hours ischemia). Control animals did not undergo compression/ischemia. Seven days post-operatively, sciatic and femoral nerves were harvested and fixed in formalin. Raman spectra of intact, peripheral nerves were collected using a fiber-optic probe with 3 mm diameter spot size and 785 nm excitation. Data was preprocessed, including fluorescence background subtraction, and Raman spectroscopic metrics were determined using custom peak fitting MATLAB® scripts. The abilities of bivariate and multivariate analysis methods to predict tissue state based on Raman spectroscopic metrics are compared. Injured nerves exhibited changes in Raman metrics indicative of 45% decreased myelin content and structural damage (p<<0.01). Axonal and myelin degeneration, cell death and digestion, and inflammation of nerve tissue samples were confirmed via histology. This study demonstrates the non-invasive ability of Raman spectroscopy to detect nerve degeneration associated with non-penetrating injury, relevant to neurapraxic and axonotmetic injuries; future experiments will further explore the clinical utility of Raman spectroscopy to recognize neural injury.
Ischemia and reperfusion injuries present major challenges for both military and civilian medicine. Improved methods for assessing the effects and predicting outcome could guide treatment decisions. Specific issues related to ischemia and reperfusion injury can include complications arising from tourniquet use, such as microvascular leakage in the limb, loss of muscle strength and systemic failures leading to hypotension and cardiac failure. Better methods for assessing the viability of limbs/tissues during ischemia and reducing complications arising from reperfusion are critical to improving clinical outcomes for at-risk patients. The purpose of this research is to develop and assess possible prediction models of outcome for acute limb ischemia using a pre-clinical model. Our model relies only on non-invasive imaging data acquired from an animal study. Outcome is measured by pathology and functional scores. We explore color, texture, and temporal features derived from both color and thermal motion imagery acquired during ischemia and reperfusion. The imagery features form the explanatory variables in a model for predicting outcome. Comparing model performance to outcome prediction based on direct observation of blood chemistry, blood gas, urinalysis, and physiological measurements provides a reference standard. Initial results show excellent performance for the imagery-base model, compared to predictions based direct measurements. This paper will present the models and supporting analysis, followed by recommendations for future investigations.
Over 70% of military casualties resulting from the current conflicts sustain major extremity injuries. Of these the majority are caused by blasts from improvised explosive devices. The resulting injuries include traumatic amputations, open fractures, crush injuries, and acute vascular disruption. Critical tissue ischemia—the point at which ischemic tissues lose the capacity to recover—is therefore a major concern, as lack of blood flow to tissues rapidly leads to tissue deoxygenation and necrosis. If left undetected or unaddressed, a potentially salvageable limb may require more extensive debridement or, more commonly, amputation. Predicting wound outcome during the initial management of blast wounds remains a significant challenge, as wounds continue to “evolve” during the debridement process and our ability to assess wound viability remains subjectively based. Better means of identifying critical ischemia are needed. We developed a swine limb ischemia model in which two imaging modalities were combined to produce an objective and quantitative assessment of wound perfusion and tissue viability. By using 3 Charge-Coupled Device (3CCD) and Infrared (IR) cameras, both surface tissue oxygenation as well as overall limb perfusion could be depicted. We observed a change in mean 3CCD and IR values at peak ischemia and during reperfusion correlate well with clinically observed indicators for limb function and vitality. After correcting for baseline mean R-B values, the 3CCD values correlate with surface tissue oxygenation and the IR values with changes in perfusion. This study aims to not only increase fundamental understanding of the processes involved with limb ischemia and reperfusion, but also to develop tools to monitor overall limb perfusion and tissue oxygenation in a clinical setting. A rapid and objective diagnostic for extent of ischemic damage and overall limb viability could provide surgeons with a more accurate indication of tissue viability. This may help reducing the number of surgical interventions required, by aiding surgeons in identifying and demarcating areas of critical tissue ischemia, so that a more adequate debridement may be performed. This would have obvious benefits of reducing patient distress and decreasing both the overall recovery time and cost of rehabilitation.
Renal ischemia that occurs intraoperatively during procedures requiring clamping of the renal artery (such as renal procurement for transplantation and partial nephrectomy for renal cancer) is known to have a significant impact on the viability of that kidney. To better understand the dynamics of intraoperative renal ischemia and recovery of renal oxygenation during reperfusion, a visible reflectance imaging system (VRIS) was developed to measure renal oxygenation during renal artery clamping in both cooled and warm porcine kidneys. For all kidneys, normothermic and hypothermic, visible reflectance imaging demonstrated a spatially distinct decrease in the relative oxy-hemoglobin concentration (%HbO 2 ) of the superior pole of the kidney compared to the middle or inferior pole. Mean relative oxy-hemoglobin concentrations decrease more significantly during ischemia for normothermic kidneys compared to hypothermic kidneys. VRIS may be broadly applicable to provide an indicator of organ ischemia during open and laparoscopic procedures.
The care of modern traumatic war wounds remains a significant challenge for clinicians. Many of the extremity
wounds inflicted during Operation Enduring Freedom and Operation Iraqi Freedom are colonized or infected with
multi-drug resistant organisms, particularly Acinetobacter baumannii. Biofilm formation and resistance to current
treatments can significantly confound the wound healing process. Accurate strain identification and targeted drug
administration for the treatment of wound bioburden has become a priority for combat casualty care. In this study,
we use vibrational spectroscopy to examine wound exudates for bacterial load. Inherent chemical differences in
different bacterial species and strains make possible the high specificity of vibrational spectroscopy.
Wound care and management accounted for over 1.8 million hospital discharges in 2009. The complex nature of wound physiology involves hundreds of overlapping processes that we have only begun to understand over the past three decades. The management of wounds remains a significant challenge for inexperienced clinicians. The ensuing inflammatory response ultimately dictates the pace of wound healing and tissue regeneration. Consequently, the eventual timing of wound closure or definitive coverage is often subjective. Some wounds fail to close, or dehisce, despite the use and application of novel wound-specific treatment modalities. An understanding of the molecular environment of acute and chronic wounds throughout the wound-healing process can provide valuable insight into the mechanisms associated with the patient's outcome. Pathologic alterations of wounds are accompanied by fundamental changes in the molecular environment that can be analyzed by vibrational spectroscopy. Vibrational spectroscopy, specifically Raman and Fourier transform infrared spectroscopy, offers the capability to accurately detect and identify the various molecules that compose the extracellular matrix during wound healing in their native state. The identified changes might provide the objective markers of wound healing, which can then be integrated with clinical characteristics to guide the management of wounds.
The management of modern traumatic war wounds remains a significant challenge for clinicians. This is a
reflection of the extensive osseous and soft-tissue damage caused by blasts and high-energy projectiles. The ensuing
inflammatory response ultimately dictates the pace of wound healing and tissue regeneration. Consequently, the
eventual timing of wound closure or definitive coverage is often subjectively based. Some wounds require an
extended period of time to close or fail to remain closed, despite the use and application of novel wound-specific
treatment modalities. Aside from impaired wound healing, additional wound complications include wound infection,
biofilm formation, and heterotopic ossification (the pathological mineralization of soft tissues). An understanding of
the molecular environment of acute wounds throughout the debridement process can provide valuable insight into
the mechanisms associated with the eventual wound outcome. The analysis of Raman spectra of ex vivo wound
biopsy tissue obtained from serial traumatic wound debridements reveals a decreased 1665 cm-1/1445 cm-1 band area
ratio in impaired healing wounds, indicative of an impaired remodeling process, in addition to a decreased 1240
cm-1/1270cm-1. The examination of debrided tissue exhibits mineralization during the early development of
heterotopic ossification. Finally, preliminary results suggest that Fourier transform infrared (FT-IR) images of
wound effluent may be able to provide early microbiological information about the wound.
Many surgical techniques are currently shifting from the more conventional, open approach towards minimally invasive laparoscopic procedures. Laparoscopy results in smaller incisions, potentially leading to less postoperative pain and more rapid recoveries . One key disadvantage of laparoscopic surgery is the loss of three-dimensional assessment of organs and tissue perfusion. Advances in laparoscopic technology include high-definition monitors for improved visualization and upgraded single charge coupled device (CCD) detectors to 3-CCD cameras, to provide a larger, more sensitive color palette to increase the perception of detail.
In this discussion, we further advance existing laparoscopic technology to create greater enhancement of images obtained during radical and partial nephrectomies in which the assessment of tissue perfusion is crucial but limited with current 3-CCD cameras. By separating the signals received by each CCD in the 3-CCD camera and by introducing a straight forward algorithm, rapid differentiation of renal vessels and perfusion is accomplished and could be performed real time. The newly acquired images are overlaid onto conventional images for reference and comparison. This affords the surgeon the ability to accurately detect changes in tissue oxygenation despite inherent limitations of the visible light image. Such additional capability should impact procedures in which visual assessment of organ vitality is critical.
Craniosynostosis is a severe craniofacial disease where one or more sutures, the fibrous tissue that lies between the cranial bones, fuses prematurely. Some craniosynostosis syndromes are known to be caused by mutations in fibroblast growth factor (FGF) receptors. Mutated FGF receptors are thought to cause constitutive signaling. In this study, heparin acrylic beads released fibroblast growth factor 2 (FGF2) to mimic constitutive signaling by mutated receptors, delivering FGF2 in addition to already existing normal tissue amounts. Fetal day 18.5 mouse sutures were treated with FGF2-soaked beads and cultured in serum free media for 48 h. We have shown previously that this treatment leads to fusion and increased Msx2 expression, but here we use near-infrared Raman imaging to simultaneously examine the mineral components and matrix components of cranial tissue while providing light microscopic spatial information. FGF2-treated mouse sutures show increased v1 phosphate and v1 carbonate bandwidths, indicating a slightly chemically modified mineral being rapidly deposited. In addition, FGF2-treated mouse sutures show a marked increase in mineral-to-matrix ratios compared to control mouse sutures, typical of increased mineralization.
Raman spectral imaging provides the means to study spatially localized response to controlled external perturbations to tissue specimens with light microscopy resolution. We discuss use of heparin-acrylic microbeads soaked with the protein fibroblast growth factor-2 (FGF2). Microbeads containing FGF2 are placed in murine calvarial tissue and stimulate abnormally rapid mineralization. The tissue response simulated the effects of craniosynostosis, a birth defect occurring in 1 in 2400 live births. We describe Raman imaging measurements of the spatial distribution of apatitic mineral and matrix (predominantly type I collagen) from normal murine calvarial tissue and murine calvarial tissue modeling craniosynostosis. We also discuss spectroscopic evaluation of the state of the mineral induced by the FGF2 beads.
Craniosynostosis, the premature fusion of the skull bones at the sutures, is the second most common human birth defect that affects the face and skull. The top most flat bones that comprise the skull, or calvaria, are most often affected. We previously showed that treatment of mouse calvaria with FGF2-soaked beads leads to craniosynostosis. In this study we treated mouse calvaria with FGF2-soaked beads and then used Raman imaging to demonstrate the spatial distribution of apatitic mineral and matrix in the sutures. There was no difference between FGF2 treated and control calvaria in the type of mineral produced (a lightly carbonated apatite), however we did observe increased mineral deposition in FGF2 treated calvaria. Raman imaging has great promise to detect the earliest mineral and matrix changes that occur in craniosynostosis.
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