Julia G. Lyubovitsky

Cigarette Smoke Toxins Deposited on Surfaces: Implications for Human Health

Cigarette smoking remains a significant health threat for smokers and nonsmokers alike. Secondhand smoke (SHS) is intrinsically more toxic than directly inhaled smoke. Recently, a new threat has been discovered – Thirdhand smoke (THS) – the accumulation of SHS on surfaces that ages with time, becoming progressively more toxic. THS is a potential health threat to children, spouses of smokers and workers in environments where smoking is or has been allowed. The goal of this study is to investigate the effects of THS on liver, lung, skin healing, and behavior, using an animal model exposed to THS under conditions that mimic exposure of humans. THS-exposed mice show alterations in multiple organ systems and excrete levels of NNAL (a tobacco-specific carcinogen biomarker) similar to those found in children exposed to SHS (and consequently to THS). In liver, THS leads to increased lipid levels and non-alcoholic fatty liver disease, a precursor to cirrhosis and cancer and a potential contributor to cardiovascular disease. In lung, THS stimulates excess collagen production and high levels of inflammatory cytokines, suggesting propensity for fibrosis with implications for inflammation-induced diseases such as chronic obstructive pulmonary disease and asthma. In wounded skin, healing in THS-exposed mice has many characteristics of the poor healing of surgical incisions observed in human smokers. Lastly, behavioral tests show that THS-exposed mice become hyperactive. The latter data, combined with emerging associated behavioral problems in children exposed to SHS/THS, suggest that, with prolonged exposure, they may be at significant risk for developing more severe neurological disorders. These results provide a basis for studies on the toxic effects of THS in humans and inform potential regulatory policies to prevent involuntary exposure to THS.

Imaging corneal pathology in a transgenic mouse model using nonlinear microscopy

A transgenic mouse model with a Clim [co-factor of LIM (a combination of first letters of Lin-11 (C. elegans), ISL1 (rat), and Mec-3 (C. elegans) gene names) domain proteins] gene partially blocked in the epithelial compartment of its tissues is used to establish the sensitivity of intrinsic reflectance nonlinear optical microscopy (NLOM) to stromal and cellular perturbations in the cornea. Our results indicate dysplasia in the squamous epithelium, irregular collagen arrays in the stroma, and a compromised posterior endothelium in the corneas of these mice. As suggested by biochemical data, the collagen alterations are likely due to collagen III synthesis and deposition during healing and remodeling of transgenic mice corneal stromas. All of the topographic features seen in NLOM images of normal and aberrant corneas are confirmed by coregistration with histological sections. In this work, we also use ratiometric redox fluorometry based on two-photon excited cellular fluorescence from reduced nicotinamide adenine dinucleotide (NAD)(P)H and oxidized flavin adenine dinucleotide (FAD) to study mitochondrial energy metabolism. Employing this method, we detect higher metabolic activity in the endothelial layer of cornea compared to an epithelial layer located further away from the metabolites. The combination of two-photon excited fluorescence (TPF) with second harmonic generation (SHG) signals allows imaging to aid in understanding the relationship between alternation of specific genes and structural changes in cells and extracellular matrix.

A novel model of chronic wounds: importance of redox imbalance and biofilm-forming bacteria for establishment of chronicity.

Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼

Generating and Reversing Chronic Wounds in Diabetic Mice by Manipulating Wound Redox Parameters

25B/year in the US alone [1]. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.

Deletion of a tumor necrosis superfamily gene in mice leads to impaired healing that mimics chronic wounds in humans

By 2025, more than 500 M people worldwide will suffer from diabetes; 125 M will develop foot ulcer(s) and 20 M will undergo an amputation, creating a major health problem. Understanding how these wounds become chronic will provide insights to reverse chronicity. We hypothesized that oxidative stress (OS) in wounds is a critical component for generation of chronicity. We used the db/db mouse model of impaired healing and inhibited, at time of injury, two major antioxidant enzymes, catalase and glutathione peroxidase, creating high OS in the wounds. This was necessary and sufficient to trigger wounds to become chronic. The wounds initially contained a polymicrobial community that with time selected for specific biofilm-forming bacteria. To reverse chronicity we treated the wounds with the antioxidants α-tocopherol and N-acetylcysteine and found that OS was highly reduced, biofilms had increased sensitivity to antibiotics, and granulation tissue was formed with proper collagen deposition and remodeling. We show for the first time generation of chronic wounds in which biofilm develops spontaneously, illustrating importance of early and continued redox imbalance coupled with the presence of biofilm in development of wound chronicity. This model will help decipher additional mechanisms and potentially better diagnosis of chronicity and treatment of human chronic wounds.

In situ multiphoton optical tomography of hair follicles in mice

Proper healing of cutaneous wounds progresses through a series of overlapping phases. Nonhealing wounds are defective in one or more of these processes and represent a major clinical problem. A critical issue in developing treatments for chronic wounds is the paucity of animal models to study the mechanisms underlying the defects in healing. Here we show that deletion of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT) leads to impaired wounds in mice that have the characteristics of nonchronic and chronic ulcers. These wounds show: (1) excessive production of cytokines, in particular three chemokines (KC/CXCL8, MCP‐1/CCL2, IP‐10/CXCL10), that may be key to the abnormal initiation and resolution of inflammation; (2) defective basement membranes, explaining blood vessel leakage and disruption of dermal/epidermal interactions; and (3) granulation tissue that contains high levels of Coll III, whereas Coll I is virtually absent and does not form fibrils. We also see major differences between nonchronic and chronic wounds, with the latter populated by bacterial films and producing eotaxin, a chemokine that attracts leukocytes that combat multicellular organisms (which biofilms can be considered to be). This new mouse model captures many defects observed in impaired and chronic human wounds and provides a vehicle to address their underlying cell and molecular mechanisms.

Release of insulin from PLGA-alginate dressing stimulates regenerative healing of burn wounds in rats.

We report multiphoton in situ optical sectioning of hair follicles in mice and a preliminary investigation of the pathological hair follicles in a transgenic mouse model. Using this imaging technology, we rapidly obtain detailed three-dimensional (3-D) reconstructions of individual hair follicles. No staining or mechanical sectioning is involved, since multiphoton microscopy coregisters two-photon excited fluorescence (TPF) from cells and second harmonic generation (SHG) signals from the extracellular matrix (ECM). These signals are ideally suited for estimating molecularly encoded hair follicular 3-D geometries, including sizes of the follicular orifices and their angles relative to the skin surface. In the normal hair follicles, spectral separation of SHG signals generated by the ECM of the hair follicle from that of intrinsic cellular fluorescence revealed intricate spatial interaction of the cellular components with the surrounding connective tissue. In the pathological hair follicles, these were clearly modified. In particular, in the transgenic mice, we observed lack of cellular fluorescence and significantly shallower angles of follicular orifices with respect to the skin surface. The combination of TPF with SHG is sensitive to structural changes in cells and extracellular matrix brought on by normal hair follicle physiology and specific gene alterations.

Multiphoton optical image guided spectroscopy method for characterization of collagen-based materials modified by glycation.

Burn wound healing involves a complex set of overlapping processes in an environment conducive to ischaemia, inflammation and infection costing

The structural analysis of three-dimensional fibrous collagen hydrogels by Raman microspectroscopy.

7.5 billion/year in the U.S.A. alone, in addition to the morbidity and mortality that occur when the burns are extensive. We previously showed that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization and stimulates angiogenesis. More recently, we developed an alginate sponge dressing (ASD) containing insulin encapsulated in PLGA [poly(D,L-lactic-co-glycolic acid)] microparticles that provides a sustained release of bioactive insulin for >20 days in a moist and protective environment. We hypothesized that insulin-containing ASD accelerates burn healing and stimulates a more regenerative, less scarring healing. Using heat-induced burn injury in rats, we show that burns treated with dressings containing 0.04 mg insulin/cm(2) every 3 days for 9 days have faster closure, a higher rate of disintegration of dead tissue and decreased oxidative stress. In addition, in insulin-treated wounds, the pattern of neutrophil inflammatory response suggests faster clearing of the burned dead tissue. We also observe faster resolution of the pro-inflammatory macrophages. We also found that insulin stimulates collagen deposition and maturation with the fibres organized more like a basket weave (normal skin) than aligned and cross-linked (scar tissue). In summary, application of ASD-containing insulin-loaded PLGA particles on burns every 3 days stimulates faster and more regenerative healing. These results suggest insulin as a potential therapeutic agent in burn healing and, because of its long history of safe use in humans, insulin could become one of the treatments of choice when repair and regeneration are critical for proper tissue function.

Collagen hydrogel characterization: multi-scale and multi-modality approach

The cross-linking with reducing sugars, known as glycation, is used to increase stiffness and strength of tissues and artificial collagen-based scaffolds. Nondestructive characterization methods that report on the structures within these materials could clarify the effects of glycation. For doing this nondestructive evaluation, we employed an in situ one-photon fluorescence as well as multiphoton microscopy method that combined two-photon fluorescence and second harmonic generation signals. We incubated collagen hydrogels with glyceraldehyde, ribose, and glucose and observed an increase in the in situ fluorescence and structural alterations within the materials during the course of glycation. The two-photon fluorescence emission maximum was observed at about 460 nm. The fluorescence emission in the one-photon excitation experiment (λ(ex) = 360 nm) was broad with peaks centered at 445 and 460 nm. The 460 nm emission component subsequently became dominant during the course of glycation with glyceraldehyde. For the ribose, in addition to the 460 nm peak, the 445 nm component persisted. The glucose glycated hydrogels exhibited broad fluorescence that did not increase significantly even after 6 weeks. As determined from measuring the fluorescence intensity at the 460 nm maximum, glycation with glyceraldehyde was faster compared to ribose and generated stronger fluorescence signals. Upon excitation of glycated samples with 330 nm light, different emission peaks were observed.