In-vivo Exposure Research Articles

Modelling the Time Evolution of Active Caspase-3 Protein in the Rat Lens after In Vivo Exposure to Ultraviolet Radiation-B

PurposeTo introduce a model for the time evolution of active caspase-3 protein expression in albino rat lens up to 24 hours after in vivo exposure to low dose UVR in the 300 nm wavelength region (UVR-300 nm).MethodsForty Sprague-Dawley rats were unilaterally exposed in vivo to 1 kJ/m2 UVR-300 nm for 15 minutes. At 0.5, 8, 16, and 24 hours after the UVR exposure, the exposed and contralateral not-exposed lenses were removed and processed for immunohistochemistry. The differences in the probability of active caspase-3 expression at four different time points after exposure were used to determine the time evolution of active caspase-3 expression. A logistic model was introduced for the expression of active caspase-3. The parameters for the exposed and the not exposed lenses were estimated for the observation time points.ResultsThe exposure to UVR-300 nm impacted on the parameters of the logistic model. Further, the parameters of the model varied with time after exposure to UVR-300 nm.ConclusionThe logistic model predicts the impact of exposure to UVR-300 nm on the spatial distribution of probability of active caspase-3 protein expression, depending on time.

Mouse protocadherin-1 gene expression is regulated by cigarette smoke exposure in vivo.

Protocadherin-1 (PCDH1) is a novel susceptibility gene for airway hyperresponsiveness, first identified in families exposed to cigarette smoke and is expressed in bronchial epithelial cells. Here, we asked how mouse Pcdh1 expression is regulated in lung structural cells in vivo under physiological conditions, and in both short-term cigarette smoke exposure models characterized by airway inflammation and hyperresponsiveness and chronic cigarette smoke exposure models. Pcdh1 gene-structure was investigated by Rapid Amplification of cDNA Ends. Pcdh1 mRNA and protein expression was investigated by qRT-PCR, western blotting using isoform-specific antibodies. We observed 87% conservation of the Pcdh1 nucleotide sequence, and 96% conservation of the Pcdh1 protein sequence between men and mice. We identified a novel Pcdh1 isoform encoding only the intracellular signalling motifs. Cigarette smoke exposure for 4 consecutive days markedly reduced Pcdh1 mRNA expression in lung tissue (3 to 4-fold), while neutrophilia and airway hyperresponsiveness was induced. Moreover, Pcdh1 mRNA expression in lung tissue was reduced already 6 hours after an acute cigarette-smoke exposure in mice. Chronic exposure to cigarette smoke induced loss of Pcdh1 protein in lung tissue after 2 months, while Pcdh1 protein levels were no longer reduced after 9 months of cigarette smoke exposure. We conclude that Pcdh1 is highly homologous to human PCDH1, encodes two transmembrane proteins and one intracellular protein, and is regulated by cigarette smoke exposure in vivo.

Blends of hydrophobic and swelling agents in the swelling layer in the preparation of delayed-release pellets of a hydrophilic drug with low MW: Physicochemical characterizations and in-vivo evaluations

In this study, a hydrophobic material, ethylcellulose, which was used as its aqueous suspension Surelease®, was combined with a swelling agent as the swelling layer to prepare delayed-release pellets for Danshensu, which is a hydrophilic drug with low MW. A rupturable, delayed-release pellet consists of a drug core, a swelling layer containing a swelling agent (cross-linked sodium carboxymethyl cellulose) with a hydrophobic agent (Surelease®), and a controlled layer composed by an insoluble, water-permeable polymeric coating (aqueous ethylcellulose dispersions) was developed in a fluidised bed. Results showed that blending Surelease® into the swelling layer could effectively extend the release of Danshensu from the pellets, which may be attributed to the slowed swelling rate by reduction of water penetration and improvement of mechanical integrity of the swelling layer. Drug in the delayed pellets showed sustained release in beagle dogs after oral administration with comparable in-vivo exposure to the uncoated drug pellets. In conclusion, blends of hydrophobic and swelling agents in the swelling layer in double-membrane pellets could achieve a delayed drug-release profile in vitro, as well as delayed and sustained absorption in vivo for highly soluble, low-MW drug. The present study highlighted the potential use of a delayed-release system for other hydrophilic, low-MW drugs to meet the formulation requirements for chronopharmacological diseases.

Addressing the fear of fat: extending imaginal exposure therapy for anxiety disorders to anorexia nervosa

Exposure therapy is fundamental for the treatment of core fears in anxiety disorders and it has been proposed that exposure therapy should be extended to Anorexia Nervosa (AN)[1]. Exposure therapy for AN has primarily focused on exercises such as mirror exposure to address body image anxiety and food exposure to address avoidant eating behavior[1]. Recently, exposure for mealtime anxiety was shown to be more efficacious than cognitive remediation therapy[1]. These types of exposure therapy show promise for the treatment of AN. However, they are limited because they only address stimuli that are accessible invivo (food, bodies). Therefore, they may not expose the patient to all of their fears, including the core fear of fatness, which can be conceptualized as an irrational belief driving avoidance behavior[1]. In imaginal exposure, patients are able to address hypothetical fears that cannot be replicated in everyday life (e.g., immediate fatness or abandonment). Imaginal exposure is highly effective for anxiety disorders and is the core feature of Prolonged Exposure Therapy for PTSD[PET;2]. In PET, patients create a script recounting their traumatic experience with the help of their therapist. The script is edited throughout treatment to focus on the most feared aspects of the trauma, which often elicits previously unidentified fears. Imaginal exposure is audio-recorded, and the patient listens to the recording as daily homework. Through this type of exposure therapy, patients learn to face their fears and learn that they can tolerate the anxiety elicited by discussing those fears. Imaginal exposure therapy draws on the avoidance-anxiety model[3]. In this model, avoidance is the chief maintaining factor of anxiety. For example, a patient with PTSD fears that she will re-experience her trauma. Motivated by the anxiety produced by this fear, she avoids leaving her house after dark because nighttime is associated with trauma. Therefore, she never learns that she can leave the house after dark and not re-experience trauma. Alleviation of anxiety becomes increasing paired with avoidance behavior via a process of negative reinforcement, promoting the maintenance of the disorder. Imaginal exposure is used in anxiety disorders to face fears that are not accessible or practical to address via in-vivo exposures. For example, a patient with PTSD cannot re-experience her trauma in real life, but she can imagine the past trauma and experience the subsequent anxiety. In AN, fear of fat is the central fear driving avoidance of eating[1]. Patients associate becoming fat with negative consequences, such as abandonment. Avoidance of eating prohibits learning that maintaining an optimal weight is not a predictor of such catastrophic outcomes. For patients with AN, catastrophic outcomes such as abandonment or immediate fatness are similarly impossible to recreate as in-vivo exposures. Patients cannot become fat solely for the purpose of the exposure, but they can imagine what it would be like to become fat. We theorize that using imaginal exposure to face the fear of hypothetical fatness can break the avoidance-anxiety cycle. We are unaware of any literature using imaginal exposure therapy to induce fears of fatness and conducted a case study to test whether imaginal exposure was feasible to (a)induce fears of fatness and (b)promote reduction in anxiety and eating disorder symptoms.

A control strategy for bioavailability enhancement by size reduction: Effect of micronization conditions on the bulk, surface and blending characteristics of an active pharmaceutical ingredient

In a Quality by Design (QbD) development environment the effect of early process parameters on downstream manufacturing parameters, and the ultimate effect on drug product quality, need to be understood. For poorly soluble drugs, size reduction is frequently employed to obtain consistent in-vivo exposures. As a result, micronization is a key early stage processing step for many active pharmaceutical ingredients (APIs).This paper demonstrates the effect of varying micronization conditions on an API for which micronization is deemed necessary to ensure consistent drug delivery after human administration. Material micronized to different extents are confirmed as different by surface area, surface energy, particle size analysis, bulk density and surface adhesion measurements.These material characteristics can be correlated with the outcomes from a key processing step, blending. The evolution of the blending process is followed using PAT techniques, so that an overall understanding of the relationship between particle properties and blend uniformity can be demonstrated.Execution of such a study during drug development can enable selection of the appropriate control strategy to ensure production of API in the desired range where consistent optimal bioavailability and downstream processability are achieved.

Dose- and Time-Dependent Micronucleus Induction in Peripheral Erythrocytes of Catfish, <i>Heteropneustes fossilis</i> (Bloch) by Zinc

As far as the detection of metal genotoxicity in fish is concerned, micronucleus (MN) test is considered an extremely suitable measure. In this study, frequencies of micronucleated erythrocytes were scored in peripheral blood of catfish, Heteropneustes fossilis (bloch) after acute in-vivo exposure of zinc at different concentrations (5, 10 and 30 ppm) in the laboratory condition. These three concentrations of zinc were tested at different durations such as 24h, 48h, 72h and 96h respectively. Highly significant (P < 0.001) increased values were obtained for MN frequencies in the peripheral erythrocytes of exposed fishes compared to control groups of fishes. These results confirm that dose- and time-dependent micronucleation in the peripheral erythrocytes of fish after short-term exposure to zinc could provide valuable information regarding zinc containing effluent quality and also help in genetic biomonitoring with this test model. In this context safe concentration of zinc vis-a-vis genotoxicity range could be evaluated for future studies.

Prolonged Exposure Therapy for Toddlers With Traumas Following Medical Procedures

Traumatic events have potentially debilitating long-lasting effects on the child's normal development and, therefore, should be effectively treated. Prolonged Exposure (PE) therapy has been found to be effective in reducing posttraumatic stress disorder symptoms in adults and in adolescents (e.g., Gilboa-Schechtman et al., 2010; Nacasch et al., 2011). It has not yet been tested in toddlers.This paper presents a detailed description of four cases of PE therapy adapted to toddlers and their parents whose posttraumatic stress symptoms developed following invasive medical procedures. Treatment consisted of several active components that were tailored specifically for the treatment of toddlers: psychoeducation about trauma, recounting scenes from the traumatic events, and in-vivo exposures to associated feared stimuli and triggers. The treatments resulted in substantial improvement in the toddlers and in their parents. At the time of treatment termination all toddlers had lost the posttraumatic stress disorder diagnosis and resumed normal functioning. These case studies provide preliminary clinical evidence for the efficacy of PE in toddlers. Conclusions, limitations, and suggestions for future research regarding the treatment of toddlers with PTSD and their parents following medical procedures are discussed.

Dose- and Time-Dependent Micronucleus Induction in Peripheral Erythrocytes of Catfish, <i>Heteropneustes fossilis</i> (Bloch) by Zinc

As far as the detection of metal genotoxicity in fish is concerned, micronucleus (MN) test is considered an extremely suitable measure. In this study, frequencies of micronucleated erythrocytes were scored in peripheral blood of catfish, Heteropneustes fossilis (bloch) after acute in-vivo exposure of zinc at different concentrations (5, 10 and 30 ppm) in the laboratory condition. These three concentrations of zinc were tested at different durations such as 24h, 48h, 72h and 96h respectively. Highly significant (P < 0.001) increased values were obtained for MN frequencies in the peripheral erythrocytes of exposed fishes compared to control groups of fishes. These results confirm that dose- and time-dependent micronucleation in the peripheral erythrocytes of fish after short-term exposure to zinc could provide valuable information regarding zinc containing effluent quality and also help in genetic biomonitoring with this test model. In this context safe concentration of zinc vis-a-vis genotoxicity range could be evaluated for future studies.

Cataract after repeated daily in vivo exposure to ultraviolet radiation.

Epidemiological data indicate a correlation between lifelong exposure to ultraviolet radiation and cortical cataract. However, there is no quantitative experimental data on the effect of daily repeated in vivo exposures of the eye to UVR. Therefore, this experiment was designed to verify whether the dose additivity for UVR exposures holds through periods of time up to 30 d. Eighty rats were conditioned to a rat restrainer 5 d prior to exposure. All animals were divided into four exposure period groups of 1, 3, 10, and 30 d of exposure to UVR. Each exposure period group of 20 animals was randomly divided into five cumulated UVR dose subgroups. Eighteen-wk-old non-anesthetized albino Sprague-Dawley rats were exposed daily to UVR-300 nm for 15 min. One week after the last exposure, animals were sacrificed. The lenses were extracted for macroscopic imaging of dark-field anatomy, and degree of cataract was quantified by measurement of the intensity of forward lens light scattering. Maximum tolerable dose (MTD(2.3:16)), a statistically defined standard for sensitivity for the threshold for UVR cataract, was estimated for each exposure period. Exposed lenses developed cataract with varying appearance on the anterior surface. Single low doses of UVR accumulated to cause cataract during periods up to 30 d. MTD(2.3:16) for 1, 3, 10, and 30 d of repeated exposures was estimated to 4.70, 4.74, 4.80, and 6.00 kJ m(-2), respectively. In conclusion, the lens sensitivity to UVR-B for 18-wk-old Sprague-Dawley rats decreases with the increasing number of days being exposed.

Disregulated Osteogenesis By Mesenchymal Stromal Cells (MSC) After In Vivo Exposure To Multiple Myeloma: Studies In a Novel Humanized Mouse Model For Bone Remodelling In Myeloma

Multiple myeloma (MM), a B-cell neoplasm characterized by a clonal expansion of malignant plasma cells in the bone marrow (BM), is accompanied by osteolytic lesions and/or diffuse osteopenia in up to 90% of the patients. Even after successful treatment, these MM-induced bone lesions do not normalize. We hypothesized that this might be caused by MM-induced irreversible impairment of the osteoblast function in the BM microenvironment. To study this bone remodeling processes in MM we used a recently developed, humanized mouse model of MM that allows engraftment and outgrowth of patient MM (pMM) cells in a humanized BM niche. To this end, ceramic scaffolds are seeded with culture-expanded human mesenchymal stromal cells (MSCs) from human BM, differentiated in vitro to osteoblasts for 1 week, then implanted subcutaneously in immune-deficient RAG2-/-gc-/--mice and after 6-8 weeks a layer of human bone is deposited on the surface of the scaffolds. Following the injection of luciferase-GFP gene marked primary MM cells (pMM), this results in homing and outgrowth of pMM in the scaffolds (Groen et al., Blood 2012). Here we describe a modification of this in vivo model, by co-implanting MSC loaded scaffolds, with pMM cells adhered to the hybrid scaffolds, at one side of the mouse, and with hybrid scaffolds only (without pMM) at the other side of the mouse. At this contra-lateral location bone formation can take place undisturbed (i.e., not affected by the presence of MM) and serves as an internal control for the osteogenic potential of the osteoblasts. Thus this model allows us to study bidirectional interactions between pMM cells and the osteoblast and the resulting inhibition of osteogenesis. Here we report that outgrowth pMM cells indeed resulted in on average 50-75% decrease in bone formation, and, using bioluminescence imaging, we found an inverse correlation between the size of the tumor and the amount of bone formation: with increasing tumor size, the amount of bone formed was less. Human AML growing in the scaffolds (serving as control) does not influence the bone forming process. At the end of the experiment when we analyzed gene expression in the human stromal cells (CD73+ CD90+ CD105+) that we cultured from scaffolds containing pMM tumors, we found a significant reduction in expression of transcripts for alkaline phosphatase (ALP), collagen1A1 (colA1), osteoglycin (OGN), osteomodulin (OMD), and abnormal spindle-like microcephaly associated (ASPM), genes that have been implicated in osteogenesis. These data suggest that pMM cells interfere with the osteogenic differentiation of MSCs in the context of an in vivo biocompatible scaffold engineered to simulate the human BM microenvironment. Taken together, our data show that co-implanting MSCs together with the pMM cells can serve as a model to study the effect of pMM cells on osteogenesis, which provides a tool to unravel the mutual interaction between MM cells and the bone marrow microenvironment. Disclosures:No relevant conflicts of interest to declare.

Notch-1 Signaling Regulates Microglia Activation via NF-κB Pathway after Hypoxic Exposure In Vivo and In Vitro

Neuroinflammation mediated by the activated microglia is suggested to play a pivotal role in the pathogenesis of hypoxic brain injury; however, the underlying mechanism of microglia activation remains unclear. Here, we show that the canonical Notch signaling orchestrates microglia activation after hypoxic exposure which is closely associated with multiple pathological situations of the brain. Notch-1 and Delta-1 expression in primary microglia and BV-2 microglial cells was significantly elevated after hypoxia. Hypoxia-induced activation of Notch signaling was further confirmed by the concomitant increase in the expression and translocation of intracellular Notch receptor domain (NICD), together with RBP-Jκ and target gene Hes-1 expression. Chemical inhibition of Notch signaling with N-[N-(3,5-difluorophenacetyl)-1-alany1- S-phenyglycine t-butyl ester (DAPT), a γ-secretase inhibitor, effectively reduced hypoxia-induced upregulated expression of most inflammatory mediators. Notch inhibition also reduced NF-κB/p65 expression and translocation. Remarkably, Notch inhibition suppressed expression of TLR4/MyD88/TRAF6 pathways. In vivo, Notch signaling expression and activation in microglia were observed in the cerebrum of postnatal rats after hypoxic injury. Most interestingly, hypoxia-induced upregulation of NF-κB immunoexpression in microglia was prevented when the rats were given DAPT pretreatment underscoring the interrelationship between Notch signaling and NF-κB pathways. Taken together, we conclude that Notch signaling is involved in regulating microglia activation after hypoxia partly through the cross talk between TLR4/MyD88/TRAF6/NF-κB pathways. Therefore, Notch signaling may serve as a prospective target for inhibition of microglia activation known to be implicated in brain damage in the developing brain.

Effects of Poly-MVA on the rheological properties of blood after in-vivo exposure to gamma radiation

This study aims to examine the radio-prophylactic effects of poly-MVA against exposure to acute dose of gamma radiation. Adult male rats, weighing 200 gm, were exposed to 6 Gy gamma radiation from Cs-137 source. The animals received daily oral administration of 2 ml/kg body weight of poly-MVA for different time intervals. The prophylactic effect was examined by two modes of administration: two weeks before irradiation and another group which received continuous administration for two weeks before and two other weeks after irradiation (total time of administration 28 days). Different parameters were performed, which include determination of cellular antioxidant enzymes (Glutathione (GSH), catalase and superoxide dismutase) in hepatic cells, the rheological properties of blood, osmotic fragility and scanning electron microscope photography of red blood cells. Exposure to radiation resulted in a significant decrease in cellular antioxidant enzymes (GSH, Catalase and SOD) and decrease in Bingham viscosity, yield stress and aggregation index of blood. Furthermore it induced slightly increase in average osmotic fragility of red blood cells accompanied by decrease in osmotic dispersion and remarkable modification of red blood cell morphology. Administration of Poly-MVA showed markedly elevation in GSH, Catalase and SOD content in liver cells in all treated groups. It also showed improvement in all observed parameters. The obtained results showed that oral uptake of poly MVA posses a radio-prophylactic effect that might be used in planned radiation exposure in diagnosis and radiotherapy.

Adverse effects induced by ecgonine methyl ester to the zebra mussel: A comparison with the benzoylecgonine

Cocaine and its metabolites are the prevalent psychotropic substances in aquatic environment. However, to date the knowledge on their adverse effects to non-target organisms is inadequate. The aims of this study were to investigate sub-lethal effects induced by the ecgonine methyl ester (EME) to the freshwater bivalve Dreissena polymorpha and to compare its toxicity to that by benzoylecgonine (BE), the other main cocaine metabolite. EME sub-lethal effects were investigated by 14 days in-vivo exposures and a multi-biomarker approach. Slight variations in biomarker responses were found at 0.15 μg/L treatment. 0.5 μg/L EME treatment induced destabilization of lysosome membranes, an overall inactivation of defense enzymes, increases in lipid peroxidation, protein carbonylation and DNA fragmentation, but no variations in fixed genetic damage. The use of a biomarker response index (BRI) showed that at 0.5 μg/L both cocaine metabolites had the same toxicity to zebra mussels specimens.

Assessment of Genotoxicity in Gonads, Liver and Gills of Zebrafish (Danio rerio) by Use of the Comet Assay and Micronucleus Test after In Vivo Exposure to Methyl Methanesulfonate

Since generative tissues are a link between the generations, the detection of genetic damage in testis and ovary of fish is conductive to elucidating the relationship between genotoxicity and impairment of reproduction. In the current study, exposure of zebrafish to methyl methanesulfonate over two weeks caused concentration dependent genotoxic effects in gonads, liver and gills using the alkaline comet assay. Likewise, the micronucleus frequency was elevated in all of these organs. Thus, the comet assay and the micronucleus test proved appropriate for the detection of genotoxicity in primary male and female gonad cells and histological sections of the gonads from zebrafish, respectively.

Evolution of TUNEL-labeling in the Rat Lens After In Vivo Exposure to Just Above Threshold Dose UVB

Purpose/Aim: To quantitatively analyse the evolution of TUNEL-labeling, after in vivo exposure to UVB.Methods: Altogether, 16 Sprague Dawley rats were unilaterally exposed in vivo for 15 min to close to threshold dose, 5 kJ/m2, of ultraviolet radiation in the 300 nm wavelength region. Animals were sacrificed in groups of 4 at 1, 5, 24 and 120 h after exposure. For each animal, both eye globes were removed and frozen. The frozen eye was cryo-sectioned in 10 µm thick midsagittal sections. From each globe, three midsagittal sections with at least five sections interval in between were mounted on a microscope slide. Sections were TUNEL-labeled and counter stained with DAPI. For quantification of apoptosis, a fluorescence microscope was used. In sections with a continuous epithelial cell surface, the number of lens epithelial cell nuclei and the number of TUNEL-positive epithelial cell nuclei was counted. The total number of TUNEL-positive epithelial cell nuclei for all three sections of one lens in relation to the total number of epithelial cell nuclei for all three sections of the same lens was compared between exposed and contralateral not exposed lens for each animal.Results: The relative difference of the fraction of TUNEL-positive nuclei between exposed and contralateral not exposed lens increased gradually, peaked in the time interval 5–120 h after exposure, and then declined.Conclusions: Close to threshold dose of UVB induces TUNEL-labeling that peaks in the time window 5–120 h after exposure to UVB.

How Technology Influences the Therapeutic Process: Evaluation of the Patient-Therapist Relationship in Augmented Reality Exposure Therapy and In Vivo Exposure Therapy

New technologies have slowly become a part of psychologists' therapeutic office. However, many therapists still have doubts about the possibility of creating a good therapeutic relationship with patients in the presence of technology. This study evaluates the development of the therapeutic alliance in individuals with small animal phobia disorder who were treated with Augmented Reality Exposure Therapy or In Vivo Exposure Therapy. Twenty-two participants received an intensive session of cognitive behavioural therapy in either a technology-mediated therapeutic context or in a traditional therapeutic context. The results show no significant difference for the therapeutic alliance between two conditions. The results seem to show that technologies such as Augmented Reality do not represent a danger to negatively influence the therapeutic alliance.

Risk for Eating Disorders Modulates Startle-Responses to Body Words

Body image disturbances are core symptoms of eating disorders (EDs). Recent evidence suggests that changes in body image may occur prior to ED onset and are not restricted to in-vivo exposure (e.g. mirror image), but also evident during presentation of abstract cues such as body shape and weight-related words. In the present study startle modulation, heart rate and subjective evaluations were examined during reading of body words and neutral words in 41 student female volunteers screened for risk of EDs. The aim was to determine if responses to body words are attributable to a general negativity bias regardless of ED risk or if activated, ED relevant negative body schemas facilitate priming of defensive responses. Heart rate and word ratings differed between body words and neutral words in the whole female sample, supporting a general processing bias for body weight and shape-related concepts in young women regardless of ED risk. Startle modulation was specifically related to eating disorder symptoms, as was indicated by significant positive correlations with self-reported body dissatisfaction. These results emphasize the relevance of examining body schema representations as a function of ED risk across different levels of responding. Peripheral-physiological measures such as the startle reflex could possibly be used as predictors of females’ risk for developing EDs in the future.

A Kind of D-Amino Acid Oxidase Inhibitor, Sodium Benzoate, Might Relieve Panic Symptoms in a First-Episode, Drug-Naïve Panic-Disorder Patient

A Kind of D-Amino Acid Oxidase Inhibitor, Sodium Benzoate, Might Relieve Panic Symptoms in a First-Episode, Drug-Naïve Panic-Disorder Patient

Bactericidal and wound disinfection efficacy of nanostructured titania

Infections are caused due to the infiltration of tissue or organ space by infectious bacterial agents, among which Staphylococcus aureus bacteria are clinically most relevant. While current treatment modalities are in general quite effective, several bacterial strains exhibit high resistance to them, leading to complications and additional surgeries, thereby increasing the patient morbidity rates. Titanium dioxide is a celebrated photoactive material and has been utilized extensively in antibacterial functions, making it a leading infection mitigating agent. In view of the property amelioration in materials via nanofication, free-standing titania nanofibers (pure and nominally doped) and nanocoatings (on Ti and Ti6Al4V implants) were fabricated and evaluated to assess their efficacy to mitigate the viability and growth of S. aureus upon brief (30 s) activation by a portable hand-held infrared laser. In order to gauge the effect of exposure and its correlation with the antibacterial activities, both isolated (only titania substrate) and simultaneous (substrate submerged in the bacterial suspension) activations were performed. The bactericidal efficacy of the IR-activated <TEX>$TiO_2$</TEX> nanocoatings was also tested against E. coli biofilms. Toxicity study was conducted to assess any potential harm to the tissue cells in the presence of photoactivated materials. These investigations showed that the photoactivated titania nanofibers caused greater than 97% bacterial necrosis of S. aureus. In the case of titania-coated Ti-implant surrogates, the bactericidal efficacy exceeded 90% in the case of pre-activation and was 100% in the case of simultaneous-activation. In addition to their high bactericidal efficacy against S. aureus, the benignity of titania nanofibers and nanocoatings towards tissue cells during in-vivo exposure was also demonstrated, making them safe for use in implant devices.

A Novel View of Bone Marrow As a “stem Cell sensor” of Tissue/Organ Damage -Evidence That in Vivo Exposure to the Neurotoxin Kainic Acid (KA) Induces Proliferation and Neural Specification of Developmentally Early Stem Cells Directly in Bone Marrow Before They Are Mobilized Into Peripheral Blood

Abstract 1192 Background.It is well known that various stem cells become mobilized into peripheral blood (PB) in response to tissue/organ injuries (e.g., heart infarct, stroke, or bleeding); however, the data on the immediate response of stem cells in BM during organ injuries are somewhat limited. We and others have demonstrated the presence of developmentally early stem cells in BM that we have named very small embryonic-like stem cells (VSELs). These Oct-4+SSEA-1+Sca-1+Lin–CD45– cells are kept quiescent in BM in the G0 phase of the cell cycle by erasure of the somatic imprint in the differentially methylated regions (DMRs) of some crucial paternally imprinted genes, (Igf2-H19, RasGRF1, and p57Kip2) that regulate proliferation of embryonic stem cells (Leukemia 2009;23:2042). These cells are mobilized into peripheral blood, for example, during heart infarct (J Am Coll Cardiol 2009;6:1–9.), stroke (Stroke 2009;40:1237–44.), or skin burns (Stem Cell Rev. 2012;8:184–94.). Hypothesis.We hypothesized that this population of BM-residing, small, quiescent, pluripotent cells should be able to respond to organ injury induced by a known neurotoxin, kainic acid (KA), in a brain damage model. We hypothesized that these quiescent cells would began to proliferate, expand, and become specified into the neural lineage. Experimental strategies.C57Bl6 mice were injected with increasing doses of KA and at various time intervals mice were sacrificed to harvest BM, PB samples, and brains for analysis. Brain damage was confirmed by histological analysis. The number of Sca-1+Lin–CD45– VSELs and Sca-1+Lin–CD45+ HSPCs was evaluated in BM and PB by FACS. The cell cycle status of VSELs and HSPCs was evaluated by FACS in cells isolated from mice that received bromodeoxyuridine (BrdU) after KA injection. By employing RQ-PCR, we also measured the expression of genes that regulate stem cell pluripotency (Oct-4, Nanog, Sox2, and Rex1) and regulate neuronal development (Nestin, βIII-tubulin, Olig1, Olig2, and GFAP). The expression of these genes was subsequently confirmed in sorted cells by immunohistochemical staining. The numbers of clonogenic CFU-GM and BFU-E progenitors residing in BM and circulating in PB were tested in methylcellulose cultures. Results.We found that 12 hrs after administration of KA (25 mg/kg bw) quiescent VSELs residing in BM enter the cell cycle: ∼2 ± 1% for control vs. 37 ± 6% for KA-treated cells. Interestingly, at the same time we did not observe significant changes in the proliferation rate of HSPCs (15±5% for control vs. 17±4% for KA-treated cells). The elevated number of VSELs in the cell cycle remained detectable for a few days and returned to control values (∼2%) after 1 week after KA administration. Furthermore, an increase in the number of cycling VSELs correlated with an increase in expression of pluripotent markers, according to RQ-PCR analysis. In parallel, 48 hrs after KA administration we observed the release from BM into PB of Sca-1+Lin–CD45–VSELs highly enriched for mRNAs characteristic of neural differentiation. Interestingly, while we observed a significant increase in VSEL number in BM and PB after KA-induced brain damage, no significant changes were observed for both BM-residing and circulating HSPCs. Conclusions.For the first time, we provide evidence that the compartment of developmentally early stem cells residing in BM responds robustly to brain damage induced by a neurotoxin. This effect seems to be specific for VSELs, as no significant changes were observed for HSPCs. The kinetics of changes in BM revealed that BM VSELs enter the cell cycle and, after they become specified into the neural lineage, egress from BM and enter the PB. Thus, our data provide novel evidence that developmentally early stem cells in BM “sense” the damage to brain tissue and respond to this type of organ injury. In parallel, we are studying the specificity of the response of BM-residing VSELs and HSPCs to other types of organ damage, such as heart infarct and acute limb ischemia. Disclosures:Ratajczak:Neostem Inc: Member of SAB Other.