This study investigated the potential of an innovative combinational therapy utilizing antimicrobial blue light (aBL) and cold atmospheric plasma (CAP) against Staphylococcus aureus, a clinically challenging gram-positive bacterium. For planktonic S. aureus, sequential treatment (1-3 min CAP followed by 270 J/cm2 aBL) demonstrated an increase of 3- to 6- log10CFU reduction compared to aBL alone, while simultaneous treatment (concurrent 90 J/cm2 aBL and 25 min CAP) led to an increase of 7-log10CFU reduction. For biofilms, a remarkable CFU reduction of 6.5- to 7- log10 was observed for sequential treatment (3-5 min CAP followed by 432 J/cm2 aBL) and a 3-log10CFU reduction for simultaneous treatment (concurrent application 108 J/cm2 aBL and 30 min CAP). These findings highlight the unprecedented efficacy of the aBL + CAP combinational therapy in enhancing the potency against S. aureus, offering a promising approach to overcome antibiotic resistance.
This study investigated the potentiation of KSeCN for both antimicrobial blue light (aBL,405nm) and antimicrobial photodynamic inactivation (aPDI) against different species of Gram-negative bacteria. For planktonic (108CFU/ml) E.coli, with KSeCN treatment (50mM concentration and 60J/cm2 aBL) demonstrated an increase of 4- to 5-log10 CFU reduction compared to aBL alone. E. coli, with KSeCN treatment (50mM,100uM Verteporfin and 20J/cm2 aBL) demonstrated an increase of 4- to 6-log10 CFU reduction compared to aPDI (100uM concentration of Verteporfin and 20J/cm2 aBL, 30min incubation time) alone. K. Pneumoniae and A. Baumannii show similar results. For biofilm (106 CFU/mL), an obvious CFU reduction of 5- to 7-log10 could be seen with KSeCN treatment (50mM and 100J/cm2 aBL) compared to aBL alone. CFU reduction of 6.5- to 8-log10 could be seen with KSeCN treatment (50mM,100uM Verteporfin and 40J/cm2 aBL) compared to aPDI alone (100uM concentration of Verteporfin and 40J/cm2). Confocal results (live/dead) made these findings solid which highlight the KSeCN function to potentiate aBL and aPDI against G-bacteria, offering a novel approach for antibiotic resistance infections.
Tympanostomy tube otorrhea (TTO) is a frequent complication associated with tympanostomy tube (TT) insertion in young children. Despite the standard treatment using topical antibiotics, TTO is often persistent or recurrent due to biofilm development on TT. In this study, we developed a novel antimicrobial blue light (aBL; 405nm) activatable Optical-TT, which emits aBL from the entire TT surface to kill major otopathogens in biofilms on TT. Our preliminary results showed that the Optical-TT activated by aBL led to over 2.5-log10 CFU reduction in 120 h-old H. influenzae films and 48 h-old P. aeruginosa biofilms formed on TT after 30 J/cm2 aBL and in 48 h-old S. aureus biofilms formed on TT after 60 J/cm2, implying the potential of aBL-activable Optical-TT in the management of TTO. Animal studies using a chinchilla model of TTO are currently underway.
Antibiotic resistance has posed a grand and rising threat to the global health. Blue light, specifically 400-430 nm range, has been shown as an attractive antimicrobial alternative considering its drug/agent-free nature, broad-spectrum antimicrobial effect, and no reported resistance. However, its clinical applications have been hampered by several major bottlenecks. Here, we present our translational development towards clinical application of blue light for managing wound infections via innovations in establishing a safe, effective treatment regimen built upon bacteriostatic and long-term illumination strategy (with therapeutical window identified between minimum inhibitory irradiance, MII, and maximum permissible irradiance, MPI), a wearable LED array-based device prototype, an in vivo testbed of free-moving rats with skin wound infections, and its integration with standard wound care procedures. These concepts, devices, safety, and effectiveness have been validated in vitro, ex vivo, and in vivo. This work paves a solid yet encouraging foundation for our follow-up clinical study on contaminated/infected wounds.
This study investigated the aBL effects on bacterial species carrying β-lactamase genes and alterations in β-lactamase activity following aBL. In addition, we assessed the β-lactamase and protein damage induced by aBL and the bactericidal effect of aBL combined with a β-lactam antibiotic. Our finds demonstrate that aBL can effectively kill strains carrying β-lactamase genes and reduce the β-lactamase activity. We also demonstrated the role of porphyrins on the photoreaction promoted by aBL and the resulting protein damage. Additionally, ceftazidime activity was significantly improved by aBL in a Metallo-β-lactamase strain. Our finds support the aBL against the β-lactamases and encourage future outcomes.
Antimicrobial resistance is significant concern to public health that negatively impacts the worldwide populace. With the increasing incidences of multidrug-resistant (MDR) pathogens, treatment of infections is becoming more difficult than ever before. In recent years, our team has been involved with the use of blue light (BL) for the treatment of infection. BL is a potent microbicide that can kill a multiplicity of pathogenic microbes. A concern, however, is that the effects of BL are short lived, when the light switches off, so do the antimicrobial effects.Therefore, we considered, that we could increase the longevity of microbicidal activity of BL via its concomitant application with antibiotics. In this study, we tested our hypothesis that aBL can enhance antibiotic activity in MDR bacteria.
In this study, we first evaluated the effectiveness of antimicrobial blue light (aBL) in vitro against Vibrio vulnificus in planktonic and biofilm cultures. In addition, we assessed aBL for the prevention of potentially lethal burn infections caused by Vibrio vulnificus in mice. We found that aBL was highly effective in killing V. vulnificus in both planktonic and biofilm cultures. Moreover, aBL significantly reduced the bacterial burden in infected mouse burns and reduced the rate of fatal sepsis in mice.
Antimicrobial resistance has necessitated the investigation of novel approaches to prolong the use of conventional antibiotics. We hypothesized that using the innovative ‘drug-free’ approach, antimicrobial blue light (aBL), which is a selective generator of ROS in bacteria, we can exploit increases in intracellular ROS to synergize to conventional antibiotics, given that they share a parallel pathway of bactericidal activity. Studies from our group have also suggested the adjuvant potential of aBL that may further promote the effectiveness of antibiotics. Here, we explored the synergistic and adjuvant effects of aBL using different antibiotic classes against bacteria, in vitro and in vivo.
Antimicrobial resistance is a concern to public health, with methicillin resistant Staphylococcus aureus (MRSA) being particularly important. Blue light at 405 nm has demonstrated efficacy for the treatment of localized infections. With respect to MRSA, aBL is not effective enough to be developed into a stand-alone therapy. Findings demonstrated the antioxidant properties of the S. aureus pigment, staphyloxanthin (STX). We hypothesized that the efficacy of 405 nm light on MRSA may improve with STX photolysis using 460 nm light. We report an approach that exploits the STX photolysis effect of 460 nm light to sensitize MRSA to 405 nm light.
Otopathogens such as Moraxella Catarrhalis and Haemophilus influenzae are the major causes of pediatric chronic and recurrent otitis media (OM). This pilot study showed that both M. catarrhalis and H. influenzae were highly susceptible to antimicrobial blue light (aBL) inactivation at 405 nm, either in suspensions and biofilms. Transmission electron microscopy showed aBL-induced damage of cell membrane in M. catarrhalis cells. Ultra-performance liquid chromatography results revealed that protoporphyrin IX and coproporphyrin are the most abundant species of endogenous porphyrins in M. catarrhalis. Our findings suggest that aBL is potentially an effective alternative antimicrobial therapy for OM.
Antimicrobial resistance in Neisseria gonorrhoeae is a major issue of public health, and there is a critical need for the development of new anti-gonococcal strategies. In this study, we investigated the effectiveness of antimicrobial blue light (aBL; 405 nm wavelength), an innovative non-pharmacological approach, for the inactivation of N. gonorrhoeae. Our findings indicated that aBL preferentially inactivated N. gonorrhoeae, including antibiotic-resistant strains, over human vaginal epithelial cells in vitro. Furthermore, no genotoxicity of aBL to the vaginal epithelial cells was observed at the radiant exposure for inactivating N. gonorrhoeae. aBL also effectively inactivated N. gonorrhoeae that had attached to and invaded into the vaginal epithelial cells in their co-cultures. No gonococcal resistance to aBL developed after 15 successive cycles of sub-therapeutic aBL inactivation. Taken together, aBL represents a potent potential treatment for antibiotic-resistant gonococcal infection.
KEYWORDS: In vivo imaging, Skin, In vitro testing, Raman spectroscopy, Tissues, Bacteria, Pathogens, Scanning electron microscopy, Laser therapeutics, Resistance
With the effectiveness of antimicrobials waning because of antimicrobial resistance, it is imperative that novel strategies are investigated for the treatment of infections. Antimicrobial blue light (aBL) is an innovative strategy that has proven efficacy against an array of pathogens, albeit, with different species having variable susceptibilities to the therapy. Quinine was discovered during the mid-17th century as a plant-derived potent antimalarial. More recently, its bactericidal properties were revealed, illustrating its potential as an antimicrobial adjuvant. Here we report a novel combination therapy, aBL+quinine hydrochloride (Q-HCL) for the treatment of multi-drug resistant infections. QHCL successfully potentiated the antimicrobial effects of aBL in numerous microbial pathogens of different etiologies, in vitro and in vivo. In addition, it synergistically improved the antimicrobial effects of aBL against bacterial biofilms. Raman spectroscopy revealed that concurrent exposure of aBL and Q-HCL improved uptake of Q-HCL into bacterial cells, when compared to the non aBL exposed sample. In addition, ultra-pure liquid chromatography (UPLC) revealed that Q-HCL increased the relative abundance of porphyrins in bacteria, suggesting the mechanism of this synergistic interaction is through increased production of intermediate photosensitizing porphyrins arising through perturbation of the heme biosynthesis pathway by Q-HCL. Genotoxic potential of the combination therapy against mouse skin tissue, was evaluated using the TUNEL assay, where it was revealed that a high dose exposure of aBL+Q-HCL (<3x the therapeutic dose) was not genotoxic to mouse skin tissue. In conclusion, the findings strongly suggest the potential of aBL+Q-HCL combination therapy as an alternative to traditional antibiotics for the treatment of localized infections.
With the increasing number of pathogenic microbes that are becoming resistant to routinely used antimicrobials, it is important to look to non-traditional approaches for the treatment of infections. Antimicrobial blue light (aBL;405 nm) is a novel strategy for the treatment of infections. Here we report an investigation into the potential for resistance development to aBL in three clinically important Gram-negative bacteria, through sequential exposure in vitro and in vivo. We found that 20 cycles of aBL exposure, in vitro, did not incur resistance development, in any of the species tested (Acinetobacter baumanii, Pseudomonas aeruginosaor Escherichia coli). In addition, sub-curative sequential aBL treatment of a wound infected with a bioluminescent variant of the P. aeruginosa PAO1 strain, did not influence sensitivity to aBL. In conclusion, it is unlikely that sequential treatment of aBL will result in resistance generation, suggesting that multiple treatments of aBL may be administered without resistance development becoming a concern.
Background and Objectives: Antimicrobial resistance is a rapidly evolving and emerging threat to modern medicine. Non-antibiotic approaches can be alternatives to minimize the development of antimicrobial resistance. The present study aims at investigation of synergistic effects of antimicrobial blue light (aBL) at 405 nm with oregano oil on bacterial inactivation, both of which are non-antibiotic approaches and possess different and multiple targets.
Catheter–associated urinary tract infections (CAUTIs) cause millions of infections in the US every year, with direct medical costs exceeding billions of dollars and resulting in > 1 million ER visits and hospitalizations, resulting in thousands of deaths. CAUTI is thought to be a major reservoir both containing and creating highly drug-resistant infections, due to the chronicity of infection, biofilm formation, and the setting of institutionalization with chronic exposure to antibiotics, thus enabling resistance.
To reduce antibiotic resistance developing in this setting, we are attempting to apply both antimicrobial photodynamic therapy (aPDT), antimicrobial blue light (aBL), and combinations of both with minimal use of antibiotics. To this end, we established a rat model of UTI. In this model, we catheterized female rats, infected them with a standard uropathogenic E.Coli (UPEC; UTI89), infused the bladder with methylene blue (MB) and potassium Iodide (KI), and illuminated the bladder once with a diffusing fiber connected to a 1W 660nm laser. Multilog killing was observed, but given the transient nature of ROS generation, regrowth of UPEC was seen 24 hours later. To this end, we are experimenting with the combination of illumination with antibiotics. When tetracyclines are illuminated by aBL, we have found > 6 log(10) steps of microbial killing in vitro, and significant drops in the antibiotic MIC effected by the combination of light and drug. Multiple treatments with aBL and aPDT both with and without limited intravesical application of antimicrobials may light the way to solving this problem.
We report a novel class of highly water-soluble decacationic methano[60]fullerene decaiodides C60[>M(C3N6+C3)2]-(I−)10 [1-(I−)10] capable of co-producing singlet oxygen (Type-II) and highly reactive hydroxyl radicals, formed from superoxide radicals in Type-I photosensitizing reactions, upon illumination at both UVA and white light wavelengths. The O2‒·-production efficiency of 1-(I−)10 was confirmed by using a O2‒·-reactive bis(2,4-dinitrobenzenesulfonyl)tetrafluorofluorescein probe and correlated to the photoinduced electron-transfer event going from iodide anions to 3C60*[>M(C3N6+C3)2] leading to C60‒·[>M(C3N6+C3)2]. Incorporation of a defined number (ten) of quaternary ammonium cationic charges per C60 in 1 was aimed to enhance its ability to target pathogenic Gram-positive and Gram-negative bacterial cells. We used the well-characterized malonato[60]fullerene diester monoadduct C60[>M(t-Bu)2] as the starting fullerene derivative to provide a better synthetic route to C60[>M(C3N6+C3)2] via transesterification reaction under trifluoroacetic acid catalyzed conditions. These compounds may be used as effective photosensitizers and nano-PDT drugs for photoinactivation of pathogens.
Due to the growing global threat of antibiotic resistance, there is a critical need for the development of alternative therapeutics for infectious diseases. Antimicrobial blue light (aBL), as an innovative non-antibiotic approach, has attracted increasing attention. This paper discussed the basic concepts of aBL and recent findings in the studies of aBL. It is commonly hypothesized that the antimicrobial property of aBL is attributed to the presence of endogenous photosensitizing chromophores in microbial cells, which produce cytotoxic reactive oxygen species upon light irradiation. A wide range of important microbes are found to be susceptible to aBL inactivation. Studies have also shown there exist therapeutic windows where microbes are selectively inactivated by aBL while host cells are preserved. The combination of aBL with some other agents result in synergistically improved antimicrobial efficacy. Future efforts should be exerted on the standardization of study design for evaluating aBL efficacy, further elucidation of the mechanism of action, optimization of the technical parameters, and translation of this technique to clinic.
Neisseria gonorrhoeae is a human-adapted, gram-negative diplococcus that infects human reproductive tracts and causes gonorrhea, a sexually transmitted disease, resulting in discharge and inflammation at the urethra, cervix, pharynx, or rectum. Over the years, N. gonorrhoeae has developed resistance to nearly every drug ever used to treat it, including sulfonamides, penicillin, tetracycline, and fluoroquinolones. Drug-resistant N. gonorrhoeae is now considered by the Centers for Disease Control and Prevention (CDC) as an urgent threat. The present study aimed to evaluate the efficacy of antimicrobial blue light (aBL) at 405 and 470 nm for inactivating N. gonorrhoeae and reveal the mechanism of action. Our results showed that an exposure of 45 J/cm2 aBL at 405 nm reduced the bacterial CFU by 7.16-log10. When the aBL exposure was increased to 54 J/cm2, eradication of bacterial CFU was achieved. When the bacteria were exposed to aBL at 470 nm, 3-log10 reduction of CFU was observed at an aBL exposure of higher than 126 J/cm2. Absorption and fluorescence spectroscopic analyses revealed the presence of endogenous porphyrins and flavins in N. gonorrhoeae cells. The present study indicated that aBL is a potential strategy to control N. gonorrhoeae infections. Endogenous porphyrins play a vital role in the killing effects of aBL. In vivo experiments are ongoing in our laboratory to treat genital tract infections in mice using aBL and explore the potential clinical applications.
Antibiotic resistance is one of the most serious threats to public health. It is estimated that at least 23,000 people die each year in the USA as a direct result of antibiotic-resistant infections. In addition, many antibiotic-resistant microorganisms develop biofilms, surface-associated microbial communities that are extremely resistant to antibiotics and the immune system. A light-based approach, antimicrobial blue light (aBL), has attracted increasing attention due to its intrinsic antimicrobial effect without the involvement of exogenous photosensitizers. In this study, we investigated the effectiveness of this non-antibiotic approach against biofilms formed by multidrug-resistant (MDR) microorganisms. MDR Acinetobacter baumannii, Escherichia coli, Candida albicans, and Pseudomonas aeruginosa biofilms were grown either in 96-well microtiter plates for 24 h or in a CDC biofilm reactor for 48 h, and then exposed to aBL at 405 nm emitted from a light-emitting diode (LED). We demonstrated that, for the biofilms grown in the CDC biofilm reactor, approximately 1.88 log10 CFU reduction was achieved in A. baumannii, 2.78 log10 CFU in E. coli and 3.18 log10 CFU in P. aeruginosa after 162 J/cm2 , 576 J/cm2 and 500 J/cm2 aBL were delivered, respectively. For the biofilms formed in the 96-well microtiter plates, 5.67 and 2.46 log10 CFU reduction was observed in P. aeruginosa and C. albicans polymicrobial biofilm after an exposure of 216 J/cm2 . In conclusion, aBL is potentially an alternative non-antibiotic approach against MDR biofilm-related infections. Future studies are warranted to investigate other important MDR microorganisms, the mechanism of action of aBL, and aBL efficacy in vivo.
Background: With the increasing emergence of multidrug-resistant (MDR) bacterial strains, there is a pressing need for the development of alternative treatment for infections. Antimicrobial blue light (aBL) has provided a simple and effective approach.
Methods: We first investigated the effectiveness of aBL (415 nm) inactivation of USA300 LAClux (a communityacquired Methicillin-resistant Staphylococcus aureus strain) both in the planktonic and biofilm forms. The survival of the bacteria in suspensions was determined by serial dilution and that of the biofilm-embedded bacteria was determined by bioluminescence quantification. Using a mouse model of thermal burn infected with USA300 LAClux, we further assessed the effectiveness of aBL for treating localized infections. Bioluminescence imaging was performed to monitor in real time bacterial viability in vivo.
Results: In vitro study showed that, for the planktonic counterpart of the bacteria or the 24-h-old biofilms, an irradiance of 55 mW/cm2 for 60 min resulted in a 4.61 log10 or 2.56 log10 inactivation, respectively. In vivo study using infected mouse burns demonstrated that a 2.56-log10 inactivation was achieved after 100-mW/cm2 irradiation for 62 min. Conclusions: aBL is a potential alternative approach for treating Methicillin-resistant Staphylococcus aureus infections.
KEYWORDS: Phototherapy, Luminescence, Resistance, In vitro testing, Bioluminescence, Statistical analysis, In vivo imaging, Light emitting diodes, Real time imaging, Pathogens
In this preclinical study, we investigated the utility of antimicrobial blue light therapy for Candida albicans infection in acutely burned mice. A bioluminescent strain of C. albicans was used. The susceptibilities to blue light inactivation were compared between C. albicans and human keratinocyte. In vitro serial passaging of C. albicans on blue light exposure was performed to evaluate the potential development of resistance to blue light inactivation. A mouse model of acute thermal burn injury infected with the bioluminescent strain of C. albicans was developed. Blue light (415 nm) was delivered to mouse burns for decolonization of C. albicans. Bioluminescence imaging was used to monitor in real time the extent of fungal infection in mouse burns. Experimental results showed that C. albicans was approximately 42-fold more susceptible to blue light inactivation in vitro than human keratinocyte (P=0.0022). Serial passaging of C. albicans on blue light exposure implied a tendency for the fungal susceptibility to blue light inactivation to decrease with the numbers of passages. Blue light reduced fungal burden by over 4-log10 (99.99%) in acute mouse burns infected with C. albicans in comparison to infected mouse burns without blue light therapy (P=0.015).
KEYWORDS: Photodynamic therapy, Bacteria, Tissues, Defense and security, Image processing, Light emitting diodes, In vivo imaging, Statistical analysis, Data analysis, Surgery
Photodynamic therapy (PDT) for cancer is known to induce an immune response against the tumor, in addition to its
well-known direct cell-killing and vascular destructive effects. PDT is becoming increasingly used as a therapy for
localized infections. However there has not to date been a convincing report of an immune response being generated
against a microbial pathogen after PDT in an animal model. We have studied PDT as a therapy for bacterial arthritis
caused by Staphylococcus aureus infection in the mouse knee. We had previously found that PDT of an infection
caused by injection of MRSA (5X107 CFU) into the mouse knee followed 3 days later by 1 μg of Photofrin and 635-
nm diode laser illumination with a range of fluences within 5 minutes, gave a biphasic dose response. The greatest
reduction of MRSA CFU was seen with a fluence of 20 J/cm2, whereas lower antibacterial efficacy was observed
with fluences that were either lower or higher. We then tested the hypothesis that the host immune response
mediated by neutrophils was responsible for most of the beneficial antibacterial effect. We used bioluminescence
imaging of luciferase expressing bacteria to follow the progress of the infection in real time. We found similar
results using intra-articular methylene blue and red light, and more importantly, that carrying out PDT of the noninfected
joint and subsequently injecting bacteria after PDT led to a significant protection from infection. Taken
together with substantial data from studies using blocking antibodies we believe that the pre-conditioning PDT
regimen recruits and stimulates neutrophils into the infected joint which can then destroy bacteria that are
subsequently injected and prevent infection.
Multi-drug resistant Acinetobacter baumanii infections represent a growing problem, especially in traumatic wounds and
burns suffered by military personnel injured in Middle Eastern conflicts. Effective treatment using traditional antibiotics
can be extremely difficult and new antimicrobial approaches are being investigated. One of these antimicrobial
alternatives could be the combination of non-toxic photosensitizers (PS) and visible light known as photodynamic
therapy (PDT). We report on the establishment of a new mouse model of full thickness thermal burns infected with a
bioluminescent derivative of a clinical Iraqi isolate of A. baumannii and its PDT treatment by topical application of a PS
produced by covalent conjugation chlorin(e6) to polyethylenimine followed by illumination of the burn surface with red
light. Application of 108A. baumannii cells to the surface of 10-second burns made on the dorsal surface of shaved
female BALB/c mice led to chronic infections that lasted on average 22 days characterized by a remarkably stable
bacterial bioluminescence. PDT carried out on day 0 soon after applying bacteria gave over three logs of loss of bacterial
luminescence in a light exposure dependent manner, while PDT carried out on day 1 and day 2 gave approximately a
1.7-log reduction. Application of PS dissolved in 10% or 20% DMSO without light gave only modest reduction in
bacterial luminescence from mouse burns. Some bacterial regrowth in the treated burn was observed but was generally
modest. It was also found that PDT did not lead to inhibition of wound healing. The data suggest that PDT may be an
effective new treatment for multi-drug resistant localized A. baumannii infections.
It has been known for many years that low level laser (or light) therapy (LLLT) can ameliorate the pain, swelling
and inflammation associated with various forms of arthritis. Light is absorbed by mitochondrial chromophores
leading to an increase in ATP, reactive oxygen species and/or cyclic AMP production and consequent gene
transcription via activation of transcription factors. However, despite many reports about the positive effects of
LLLT in medicine, its use remains controversial. Our laboratory has developed animal models designed to
objectively quantify response to LLLT and compare different light delivery regimens. In the arthritis model we
inject zymosan into rat knee joints to induce inflammatory arthritis. We have compared illumination regimens
consisting of a high and low fluence (3 J/cm2 and 30 J/cm2), delivered at a high and low irradiance (5 mW/cm2 and 50 mW/cm2) using 810-nm laser light daily for 5 days, with the effect of conventional corticosteroid
(dexamethasone) therapy. Results indicated that illumination with 810-nm laser is highly effective (almost as good
as dexamethasone) at reducing swelling and that longer illumination time was more important in determining
effectiveness than either total fluence delivered or irradiance. Experiments carried out using 810-nm LLLT on
excisional wound healing in mice also confirmed the importance of longer illumination times. These data will be of
value in designing clinical trials of LLLT.
KEYWORDS: Skin, Cryogenics, Injuries, Blood vessels, Temperature metrology, Ear, Laser irradiation, Laser therapeutics, In vivo imaging, In vitro testing
Cutaneous laser treatment in dark skin patients is challenging due to significant light absorption by the melanin at the basal layer of epidermis, which can result in irreversible nonspecific thermal injury to the epidermis. Cryogen spray cooling (CSC) with R-134a (boiling point –26.2°C at 1 atm), which is currently used during cutaneous laser treatment, has shown poor efficacy in protecting dark human skin. We investigated the potential of CSC with R-404a (boiling point –46.5°C at 1 atm), which has a lower boiling point than R-134a, for improved therapeutic outcome in dark human skin at three levels: in vitro (epoxy resin skin phantom), ex vivo (normal dark human skin sample), and in vivo (skin of the rabbit external ear). The skin phantom was used to acquire the surface and internal temperature profiles in response to CSC with R-134a or R-404a at various spurt durations, based upon which CSC-induced heat removal from the skin phantom was estimated using an algorithm that solved a one-dimensional inverse heat conduction problem. CSC with R-404a increased the temperature reductions within the phantom and subsequently the amount of heat removal from the phantom in comparison to that with R-134a. Normal ex vivo Fitzpatrick types V-VI human skin samples were used to investigate the thermal response of dark human skin epidermis to CSC (R-134a or R-404a) at various spurt durations in conjunction with 595-nm pulsed dye laser irradiation at various radiant exposures. Cryogen R-404a increased the threshold radiant exposures for irreversible thermal injury to the epidermis in dark pigmentation skin. No obvious CSC-induced morphological changes to human skin was observed when sprayed with R404-a spurts using durations up to 300 ms. In vivo rabbit ear vasculature was used as a model of cutaneous anomalies to assess the influences of CSC (with R-134a or R-404a) on the photothermolysis of dermal blood vessels. CSC (R-134a or R-404a) with the spurt durations of 100 to 300 ms increased the most superficial depth of ...
Near-infrared wavelengths are absorbed less by epidermal melanin mainly located at the basal layer of epidermis (dermo-epidermal junction), and penetrate deeper into human skin dermis and blood than visible wavelengths. Therefore, laser irradiation using near-infrared wavelength may improve the therapeutic outcome of cutaneous hyper-vascular malformations in moderately to heavily pigmented skin patients and those with large-sized blood vessels or blood vessels extending deeply into the skin. A mathematical model composed of a Monte Carlo algorithm to estimate the distribution of absorbed light followed by numerical solution of a bio-heat diffusion equation was utilized to investigate the thermal response of human skin to near-infrared laser irradiation, and compared it with that to visible laser irradiation. Additionally, the effect of skin surface cooling on epidermal protection was theoretically investigated. Simulation results indicated that 940 nm wavelength is superior to 810 and 1064 nm in terms of the ratio of light absorption by targeted blood vessel to the absorption by the basal layer of epidermis, and is more efficient than 595 nm wavelength for the treatment of patients with large-sized blood vessels and moderately to heavily pigmented skin. Dermal blood content has a considerable effect on the laser-induced peak temperature at the basal layer of epidermis, while the effect of blood vessel size is minimum.
Improved laser treatment of port wine stains is expected by utilizing higher incident dosages, longer pulse durations, and longer wavelengths than those currently used in clinical settings. However, higher incident dosages also increase the risk of nonspecific thermal injury to the epidermis. Using ex-vivo human skin samples, we investigated the thermal respone of human skin epidermis in different skin types ot 595-nm wavelength laser irradiation at high incident dosages (up to 20 J/cm2) and long pulse durations (1.5 to 40 milliseconds) in conjunction with cryogen spray cooling (CSC). Human skin samples (Fitzpatrick types I-VI) from consenting adult females undergoing trans-rectus abdominis myocutaneoues flap procedures were irradiated at the incident dosages D0=4, 6, 10, 15, and 20 J/cm2, pulse durations τlaser=1.5, 10, and 40 milliseconds without and with CSC (Refrigerant-134A, spurt duration τCSC=100 milliseconds). Thermal injury to the epidermis was evaluated by histological observations. Experimental results showed that thermal injury to the epidermis could not be avoided in skin type VI even at D0 = 4 J/cm2 in conjunction with CSC. However, CSC allowed utilization of high incident dosages (15 - 20 J/cm2) in skin types I-IV. Under the same incident dosage, longer pulse durations led to decreased degree of thermal injury to the epidermis. Threshold values for irradiation parameters that resulted in thermal injury to the epidermis for each skin type were obtained.
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