Gitika B. Kharkwal

Cell Death Pathways in Photodynamic Therapy of Cancer

Photodynamic therapy (PDT) is an emerging cancer therapy that uses the combination of non-toxic dyes or photosensitizers (PS) and harmless visible light to produce reactive oxygen species and destroy tumors. The PS can be localized in various organelles such as mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes and this sub-cellular location governs much of the signaling that occurs after PDT. There is an acute stress response that leads to changes in calcium and lipid metabolism and causes the production of cytokines and stress response mediators. Enzymes (particularly protein kinases) are activated and transcription factors are expressed. Many of the cellular responses center on mitochondria and frequently lead to induction of apoptosis by the mitochondrial pathway involving caspase activation and release of cytochrome c. Certain specific proteins (such as Bcl-2) are damaged by PDT-induced oxidation thereby increasing apoptosis, and a build-up of oxidized proteins leads to an ER-stress response that may be increased by proteasome inhibition. Autophagy plays a role in either inhibiting or enhancing cell death after PDT.

Low-Level Laser Therapy Activates NF-kB via Generation of Reactive Oxygen Species in Mouse Embryonic Fibroblasts

BACKGROUND Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear. METHODOLOGY/PRINCIPAL FINDINGS In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm(2) and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour) by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS) production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration. CONCLUSION We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

Photodynamic Therapy for Infections: Clinical Applications

Photodynamic therapy (PDT) was discovered over 100 years ago by its ability to kill various microorganisms when the appropriate dye and light were combined in the presence of oxygen. However it is only in relatively recent times that PDT has been studied as a treatment for various types of localized infections. This resurgence of interest has been partly motivated by the alarming increase in drug resistance amongst bacteria and other pathogens. This review will focus on the clinical applications of antimicrobial PDT.

Comparison of therapeutic effects between pulsed and continuous wave 810-nm wavelength laser irradiation for traumatic brain injury in mice.

Background and Objective Transcranial low-level laser therapy (LLLT) using near-infrared light can efficiently penetrate through the scalp and skull and could allow non-invasive treatment for traumatic brain injury (TBI). In the present study, we compared the therapeutic effect using 810-nm wavelength laser light in continuous and pulsed wave modes in a mouse model of TBI. Study Design/Materials and Methods TBI was induced by a controlled cortical-impact device and 4-hours post-TBI 1-group received a sham treatment and 3-groups received a single exposure to transcranial LLLT, either continuous wave or pulsed at 10-Hz or 100-Hz with a 50% duty cycle. An 810-nm Ga-Al-As diode laser delivered a spot with diameter of 1-cm onto the injured head with a power density of 50-mW/cm2 for 12-minutes giving a fluence of 36-J/cm2. Neurological severity score (NSS) and body weight were measured up to 4 weeks. Mice were sacrificed at 2, 15 and 28 days post-TBI and the lesion size was histologically analyzed. The quantity of ATP production in the brain tissue was determined immediately after laser irradiation. We examined the role of LLLT on the psychological state of the mice at 1 day and 4 weeks after TBI using tail suspension test and forced swim test. Results The 810-nm laser pulsed at 10-Hz was the most effective judged by improvement in NSS and body weight although the other laser regimens were also effective. The brain lesion volume of mice treated with 10-Hz pulsed-laser irradiation was significantly lower than control group at 15-days and 4-weeks post-TBI. Moreover, we found an antidepressant effect of LLLT at 4-weeks as shown by forced swim and tail suspension tests. Conclusion The therapeutic effect of LLLT for TBI with an 810-nm laser was more effective at 10-Hz pulse frequency than at CW and 100-Hz. This finding may provide a new insight into biological mechanisms of LLLT.

Dose response effects of 810 nm laser light on mouse primary cortical neurons.

In the past four decades numerous studies have reported the efficacy of low level light (laser) therapy (LLLT) as a treatment for diverse diseases and injuries. Recent studies have shown that LLLT can biomodulate processes in the central nervous system and has been extensively studied as a stroke treatment. However there is still a lack of knowledge on the effects of LLLT at the cellular level in neurons. The present study aimed to study the effect of 810 nm laser on several cellular processes in primary cortical neurons cultured from embryonic mouse brains.

Drug Discovery of Antimicrobial Photosensitizers Using Animal Models

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to antibiotics motivated by growing problems with multi-drug resistant pathogens. aPDT uses non-toxic dyes or photosensitizers (PS) in combination with harmless visible of the correct wavelength to be absorbed by the PS. The excited state PS can form a long-lived triplet state that can interact with molecular oxygen to produce reactive oxygen species such as singlet oxygen and hydroxyl radical that kill the microbial cells. To obtain effective PS for treatment of infections it is necessary to use cationic PS with positive charges that are able to bind to and penetrate different classes of microbial cells. Other drug design criteria require PS with high absorption coefficients in the red/near infra-red regions of the spectrum where light penetration into tissue is maximum, high photostability to minimize photobleaching, and devising compounds that will selectively bind to microbial cells rather than host mammalian cells. Several molecular classes fulfill many of these requirements including phenothiazinium dyes, cationic tetrapyrroles such as porphyrins, phthalocyanines and bacteriochlorins, cationic fullerenes and cationic derivatives of other known PS. Larger structures such as conjugates between PS and cationic polymers, cationic nanoparticles and cationic liposomes that contain PS are also effective. In order to demonstrate in vivo efficacy it is necessary to use animal models of localized infections in which both PS and light can be effectively delivered into the infected area. This review will cover a range of mouse models we have developed using bioluminescent pathogens and a sensitive low light imaging system to non-invasively monitor the progress of the infection in real time. Effective aPDT has been demonstrated in acute lethal infections and chronic biofilm infections; in infections caused by Gram-positive, Gram-negative bacteria and fungi; in infections in wounds, third degree burns, skin abrasions and soft-tissue abscesses. This range of animal models also represents a powerful aid in antimicrobial drug discovery.

Animal models of external traumatic wound infections.

Background: Despite advances in traumatic wound care and management, infections remain a leading cause of mortality, morbidity, and economic disruption in millions of wound patients around the world. Animal models have become standard tools for studying a wide array of external traumatic wound infections and testing new antimicrobial strategies. Methods: This review covers experimental infections in animal models of surgical wounds, skin abrasions, burns, lacerations, excisional wounds, and open fractures. Results: Animal models of external traumatic wound infections reported by different investigators vary in animal species used, microorganism strains, the number of microorganisms applied, the size of the wounds, and, for burn infections, the length of time the heated object or liquid is in contact with the skin. Conclusions: As antibiotic resistance continues to increase, more new antimicrobial approaches are urgently needed. These should be tested using standard protocols for infections in external traumatic wounds in animal models.

Ultraviolet-C Light for Treatment of Candida albicans Burn Infection in Mice

Burn patients are at high risk of invasive fungal infections, which are a leading cause of morbidity, mortality, and related expense exacerbated by the emergence of drug resistant fungal strains. In this study, we investigated the use of UVC light (254 nm) for the treatment of yeast Candida albicans infection in mouse third degree burns. In vitro studies demonstrated that UVC could selectively kill the pathogenic C. albicans compared with a normal mouse keratinocyte cell line in a light exposure dependent manner. A mouse model of chronic C. albicans infection in non‐lethal third degree burns was developed. The C. albicans strain was stably transformed with a version of the Gaussia princeps luciferase gene that allowed real‐time bioluminescence imaging of the progression of C. albicans infection. UVC treatment with a single exposure carried out on day 0 (30 min postinfection) gave an average 2.16‐log10‐unit (99.2%) loss of fungal luminescence when 2.92 J cm−2 UVC had been delivered, while UVC 24 h postinfection gave 1.94‐log10‐unit (95.8%) reduction of fungal luminescence after 6.48 J cm−2. Statistical analysis demonstrated that UVC treatment carried out on both day 0 and day 1 significantly reduced the fungal bioburden of infected burns. UVC was found to be superior to a topical antifungal drug, nystatin cream. UVC was tested on normal mouse skin and no gross damage was observed 24 h after 6.48 J cm−2. DNA lesions (cyclobutane pyrimidine dimers) were observed by immunofluorescence in normal mouse skin immediately after a 6.48 J cm−2 UVC exposure, but the lesions were extensively repaired at 24 h after UVC exposure.

Photodynamic inactivation of bacteria using polyethylenimine–chlorin(e6) conjugates: Effect of polymer molecular weight, substitution ratio of chlorin(e6) and pH

Antimicrobial photodynamic therapy (APDT) is a novel technique to treat local infections. Previously we reported that the attachment of chlorin(e6) to polyethylenimine (PEI) polymers to form PEI‐ce6 conjugates is an effective way to improve ce6 PDT activity against bacteria. The aim of this work was to explore how the polymer molecular weight, substitution ratio (SR) of ce6 and pH value affect the PDT efficacy.

Low-level light therapy aids traumatic brain injury

Traumatic brain injury (TBI) caused by falls, motor vehicle accidents, and violence leads to skull fractures, intracranial hemorrhages, elevated intracranial pressure, and cerebral contusion. Severe and moderate TBI, accidental or inflicted, is a major health and socio-economic problem throughout theworld, especially in children and young adults. Despite promising preclinical data, most therapeutic trials for TBI performed in recent years have not demonstrated any significant improvement in outcomes.1 Because of this disappointing state of affairs, a plethora of experimental therapies that are not based on standard pharmaceutical agents have been investigated,2 including several physical treatments.3 Low-level laser therapy (LLLT), also known as photobiomodulation, is an emerging therapeutic approach in which cells or tissues are exposed to low-levels of red and near-IR light. Its experimental applications have broadened to include serious diseases such as heart attack,4 stroke,5 and spinal cord injury.6 LLLT may have beneficial effects in the acute treatment of TBI by increasing mitochondrial respiration, activating transcription factors, reducing key inflammatory mediators, inhibiting apoptosis (programmed cell death), stimulating angiogenesis, and increasing neurogenesis7 (see Figure 1). We studied the effect of an 810nm laser on several cellular processes in primary cortical neurons cultured from mouse embryonic brains. We found that at low fluences (0.3–3Jcm2/ mitochondrial respiration was stimulated, as shown by the increase in adenosine triphosphate (ATP), Ca2C; and mitochondrial membrane potential. This, in turn, generated low amounts of reactive oxygen species (ROS) and nitric oxide (NO) that activated signaling pathways and gene transcription without causing cytotoxicity (see Figure 2). At 10J/cm2; the stimulation of these parameters was reduced because instead of activating mitochondrial respiration, they damaged Figure 1. Possible mechanisms of transcranial low-level laser therapy (LLLT) for traumatic brain injury (TBI). Mitochondrial signaling causes increased neuronal survival; lowered edema, inflammation and excitotoxicity; and increased angiogenesis, neurotrophins, and neural progenitor cells. ROS: Reactive oxygen species. NO: Nitric oxide. NGF: Nerve growth factor. BDNF: Brain-derived neurotrophic factor. NT-3: Neurotrophin-3.