Saiqa Khan

Pulsed Electric Fields for Burn Wound Disinfection in a Murine Model

Emerging bacterial resistance renders many antibiotics ineffective, making alternative strategies of wound disinfection important. Here the authors report on a new, physical burn wound disinfection method: pulsed electric fields (PEFs). High voltage, short PEFs create nonthermal, permanent damage to cell membranes, possibly by irreversible electroporation. In medicine, PEF technology has recently been used for nonthermal ablation of solid tumors. The authors have expanded the spectrum of PEF applications in medicine to burn wound disinfection. A third-degree burn was induced on the dorsal skin of C57BL/6 mice. Immediately after the injury, the burn wound was infected with Acinetobacter baumannii expressing the luxCDABE operon. Thirty minutes after infection, the infected areas were treated with 80 pulses delivered at 500 V/mm, 70 &mgr;s, 1 Hz. The authors used bioluminescence to quantify bacteria on skin. Three animals were used for each experimental condition. PEFs were effective in the disinfection of infected burned murine skin. The bacterial load reduction correlated with the number of delivered pulses. Forty pulses of 500 V/mm led to a 2.04 ± 0.29 Log10 reduction in bacterial load; 80 pulses led to the immediate 5.53 ± 0.30 Log10 reduction. Three hours after PEF, the bacterial reduction of the skin treated with 500 V/mm, 80 pulses was 4.91 ± 0.71 Log10. The authors introduce a new method of wound disinfection using high voltage, short PEFs. They believe that PEF technology may represent an important alternative to antibiotics in addressing bacterial contamination of wounds, particularly those contaminated with multidrug-resistant bacteria.

Longitudinal, 3D Imaging of Collagen Remodeling in Murine Hypertrophic Scars In Vivo Using Polarization-Sensitive Optical Frequency Domain Imaging

Hypertrophic scars (HTS), frequently seen after traumatic injuries and surgery, remain a major clinical challenge because of the limited success of existing therapies. A significant obstacle to understanding HTS etiology is the lack of tools to monitor scar remodeling longitudinally and noninvasively. We present an in vivo, label-free technique using polarization-sensitive optical frequency domain imaging for the 3D, longitudinal assessment of collagen remodeling in murine HTS. In this study, HTS was induced with a mechanical tension device for 4-10 days on incisional wounds and imaged up to 1 month after device removal; an excisional HTS model was also imaged at 6 months after injury to investigate deeper and more mature scars. We showed that local retardation and degree of polarization provide a robust signature for HTS. Compared with normal skin with heterogeneous local retardation and low degree of polarization, HTS was characterized by an initially low local retardation, which increased as collagen fibers remodeled, and a persistently high degree of polarization. This study demonstrates that polarization-sensitive optical frequency domain imaging offers a powerful tool to gain significant biological insights into HTS remodeling by enabling longitudinal assessment of collagen in vivo, which is critical to elucidating HTS etiology and developing more effective HTS therapies.

Eradication of multidrug-resistant pseudomonas biofilm with pulsed electric fields.

Biofilm formation is a significant problem, accounting for over eighty percent of microbial infections in the body. Biofilm eradication is problematic due to increased resistance to antibiotics and antimicrobials as compared to planktonic cells. The purpose of this study was to investigate the effect of Pulsed Electric Fields (PEF) on biofilm‐infected mesh. Prolene mesh was infected with bioluminescent Pseudomonas aeruginosa and treated with PEF using a concentric electrode system to derive, in a single experiment, the critical electric field strength needed to kill bacteria. The effect of the electric field strength and the number of pulses (with a fixed pulse length duration and frequency) on bacterial eradication was investigated. For all experiments, biofilm formation and disruption were confirmed with bioluminescent imaging and Scanning Electron Microscopy (SEM). Computation and statistical methods were used to analyze treatment efficiency and to compare it to existing theoretical models. In all experiments 1500 V are applied through a central electrode, with pulse duration of 50 μs, and pulse delivery frequency of 2 Hz. We found that the critical electric field strength (Ecr) needed to eradicate 100–80% of bacteria in the treated area was 121 ± 14 V/mm when 300 pulses were applied, and 235 ± 6.1 V/mm when 150 pulses were applied. The area at which 100–80% of bacteria were eradicated was 50.5 ± 9.9 mm2 for 300 pulses, and 13.4 ± 0.65 mm2 for 150 pulses. 80% threshold eradication was not achieved with 100 pulses. The results indicate that increased efficacy of treatment is due to increased number of pulses delivered. In addition, we that showed the bacterial death rate as a function of the electrical field follows the statistical Weibull model for 150 and 300 pulses. We hypothesize that in the clinical setting, combining systemic antibacterial therapy with PEF will yield a synergistic effect leading to improved eradication of mesh infections. Biotechnol. Bioeng. 2016;113: 643–650.

Skin Rejuvenation with Non-Invasive Pulsed Electric Fields

Degenerative skin diseases affect one third of individuals over the age of sixty. Current therapies use various physical and chemical methods to rejuvenate skin; but since the therapies affect many tissue components including cells and extracellular matrix, they may also induce significant side effects, such as scarring. Here we report on a new, non-invasive, non-thermal technique to rejuvenate skin with pulsed electric fields. The fields destroy cells while simultaneously completely preserving the extracellular matrix architecture and releasing multiple growth factors locally that induce new cells and tissue growth. We have identified the specific pulsed electric field parameters in rats that lead to prominent proliferation of the epidermis, formation of microvasculature, and secretion of new collagen at treated areas without scarring. Our results suggest that pulsed electric fields can improve skin function and thus can potentially serve as a novel non-invasive skin therapy for multiple degenerative skin diseases.

An automated image processing method to quantify collagen fibre organization within cutaneous scar tissue.

Standard approaches to evaluate scar formation within histological sections rely on qualitative evaluations and scoring, which limits our understanding of the remodelling process. We have recently developed an image analysis technique for the rapid quantification of fibre alignment at each pixel location. The goal of this study was to evaluate its application for quantitatively mapping scar formation in histological sections of cutaneous burns. To this end, we utilized directional statistics to define maps of fibre density and directional variance from Massons trichrome‐stained sections for quantifying changes in collagen organization during scar remodelling. Significant increases in collagen fibre density are detectable soon after burn injury in a rat model. Decreased fibre directional variance in the scar was also detectable between 3 weeks and 6 months after injury, indicating increasing fibre alignment. This automated analysis of fibre organization can provide objective surrogate endpoints for evaluating cutaneous wound repair and regeneration.

Eradication of multidrug-resistant A. baumannii in burn wounds by antiseptic pulsed electric field

Emerging bacterial resistance to multiple drugs is an increasing problem in burn wound management. New non-pharmacologic interventions are needed for burn wound disinfection. Here we report on a novel physical method for disinfection: antiseptic pulsed electric field (PEF) applied externally to the infected burns. In a mice model, we show that PEF can reduce the load of multidrug resistant Acinetobacter baumannii present in a full thickness burn wound by more than four orders of magnitude, as detected by bioluminescence imaging. Furthermore, using a finite element numerical model, we demonstrate that PEF provides non-thermal, homogeneous, full thickness treatment for the burn wound, thus, overcoming the limitation of treatment depth for many topical antimicrobials. These modeling tools and our in vivo results will be extremely useful for further translation of the PEF technology to the clinical setting, as they provide the essential elements for planning of electrode design and treatment protocol.

Photochemical Tissue Passivation Reduces Vein Graft Intimal Hyperplasia in a Swine Model of Arteriovenous Bypass Grafting

Background Bypass grafting remains the standard of care for coronary artery disease and severe lower extremity ischemia. Efficacy is limited by poor long‐term venous graft patency secondary to intimal hyperplasia (IH) caused by venous injury upon exposure to arterial pressure. We investigate whether photochemical tissue passivation (PTP) treatment of vein grafts modulates smooth muscle cell (SMC) proliferation and migration, and inhibits development of IH. Methods and Results PTP was performed at increasing fluences up to 120 J/cm2 on porcine veins. Tensiometry performed to assess vessel elasticity/stiffness showed increased stiffness with increasing fluence until plateauing at 90 J/cm2 (median, interquartile range [IQR]). At 90 J/cm2, PTP‐treated vessels had a 10‐fold greater Youngs modulus than untreated controls (954 [IQR, 2217] vs 99 kPa [IQR, 63]; P=0.03). Each pig received a PTP‐treated and untreated carotid artery venous interposition graft. At 4‐weeks, intimal/medial areas were assessed. PTP reduced the degree of IH by 66% and medial hypertrophy by 49%. Intimal area was 3.91 (IQR, 1.2) and 1.3 mm2 (IQR, 0.97; P≤0.001) in untreated and PTP‐treated grafts, respectively. Medial area was 9.2 (IQR, 3.2) and 4.7 mm2 (IQR, 2.0; P≤0.001) in untreated and PTP‐treated grafts, respectively. Immunohistochemistry was performed to assess alpha‐smooth muscle actin (SMA) and proliferating cell nuclear antigen (PCNA). Objectively, there were less SMA‐positive cells within the intima/media of PTP‐treated vessels than controls. There was an increase in PCNA‐positive cells within control vein grafts (18% [IQR, 5.3]) versus PTP‐treated vein grafts (5% [IQR, 0.9]; P=0.02). Conclusions By strengthening vein grafts, PTP decreases SMC proliferation and migration, thereby reducing IH.

Preventing Scars after Injury with Partial Irreversible Electroporation

Preventing the formation of hypertrophic scars, especially those that are a result of major trauma or burns, would have enormous impact in the fields of regenerative and trauma medicine. In this report, we introduce a noninvasive method to prevent scarring based on nonthermal partial irreversible electroporation. Contact burn injuries in rats were treated with varying treatment parameters to optimize the treatment protocol. Scar surface area and structural properties of the scar were assessed with histology and non-invasive, longitudinal imaging with polarization-sensitive optical coherence tomography. We found that partial irreversible electroporation using 200 pulses of 250 V and 70 μs duration, delivered at 3 Hz every 20 days during a total of five therapy sessions after the initial burn injury, resulted in a 57.9% reduction of the scar area compared with untreated scars and structural features approaching those of normal skin. Unlike humans, rats do not develop hypertrophic scars. Therefore, the use of a rat animal model is the limiting factor of this work.

Prevention of vein graft intimal hyperplasia with photochemical tissue passivation.

Objective: Saphenous vein is the conduit of choice for bypass grafting. Saphenous vein grafts have poor long‐term patency rates because of intimal hyperplasia (IH) and subsequent accelerated atherosclerosis. One of the primary triggers of IH is endothelial injury resulting from excessive dilation of the vein after exposure to arterial pressures. Photochemical tissue passivation (PTP) is a technology that cross‐links adventitial collagen by a light‐activated process, which limits dilation by improving vessel compliance. The objective of this study was to investigate whether PTP limits the development of IH in a rodent venous interposition graft model. Methods: PTP is accomplished by coating venous adventitia with a photosensitizing dye and exposing it to light. To assess the degree of collagen cross‐linking after PTP treatment, a biodegradation assay was performed. Venous interposition grafts were placed in the femoral artery of Sprague‐Dawley rats. Rats were euthanized after 4 weeks, and intimal thickness was measured histologically. Vein dilation at the time of the initial procedure was also measured. Results: Time to digestion was 63 ± 7 minutes for controls, 101 ± 2.4 minutes for rose bengal (RB), and 300 ± 0 minutes for PTP (P < .001 PTP vs control). A total of 37 animals underwent the procedure: 12 PTP, 12 RB only, and 13 untreated controls. Dilation of the graft after clamp release was 99% for control, 65% for RB only, and 19% for PTP‐treated (P < .001 PTP vs control). Intimal thickness was 77 ± 59 &mgr;m in controls, 60 ± 27 &mgr;m in RB only, and 33 ± 28 &mgr;m in PTP‐treated grafts. There was a statistically significant 57% reduction in intimal thickness after treatment with PTP compared with untreated controls (P = .03). Conclusions: PTP treatment of venous interposition grafts in a rat model resulted in significant collagen cross‐linking, decreased vessel compliance, and significant reduction in IH. Clinical Relevance: Long‐term patency rates are poor after coronary or lower extremity bypass with autologous vein. This is largely due to intimal hyperplasia (IH), superimposed accelerated atherosclerosis, and graft thrombosis. A key inciting event is graft overdistention and endothelial denudation after exposure to arterial pressure. Limiting overdistention by external mechanical support has been shown to reduce IH. Photochemical tissue passivation involves cross‐linking adventitial collagen, a novel approach to limit overdistention of the vein without an external prosthesis. Photochemical tissue passivation resulted in a 57% reduction of IH. Minimizing vein graft IH would greatly improve long‐term outcomes after arterial bypass.

Skin regeneration with all accessory organs following ablation with irreversible electroporation.

Skin scar formation is a complex process that results in the formation of dense extracellular matrix (ECM) without normal skin appendages such as hair and glands. The absence of a scarless healing model in adult mammals prevents the development of successful therapies. We show that irreversible electroporation of skin drives its regeneration with all accessory organs in normal adult rats. Pulsed electric fields at 500 V, with 70 μs pulse duration and 1000 pulses delivered at 3 Hz, applied through two electrodes separated by 2 mm lead to massive cell death. However, the ECM architecture of the skin was preserved. Six months after the ablation, the epidermis, sebaceous glands, panniculus carnosus, hair follicles, microvasculature and arrector pili muscle were altogether re‐formed in the entire ablated area. These results suggest a key role of the ECM architecture in the differentiation, migration and signalling of cells during scarless wound healing. Copyright