An Inhalation Chamber Model for Controlled Studies of Tobacco Smoke Toxicity in Rodents

Introduction: Smoking is a serious worldwide public health problem. Animal models act as a bridge between laboratory and human studies. The models applied are difficult to reproduce because of the use of different types of inhalation chambers and mainly because of the lack of continuous monitoring of smoke concentration. Objective: To develop an inhalation chamber for rats (with only the nose exposed) in which the amount of carbon monoxide (CO) can be maintained and monitored constantly. Material and methods: Male Wistar rats weighing 250 g were exposed to 50 ppm CO produced by the smoke from a filter-free cigarette. The animals were submitted to a single 2-h exposure and then sacrificed at 0, 4, 24 and 48 h. The control group was left restrained inside the small perpendicular chambers, receiving only 5 L/min of compressed air. Results: The model was able to increase HbCO levels immediately after the end of exposure (p < 0.001). with a decrease being observed from 2 h onwards when compared to the levels of the control group. Plasma cotinine increased immediately after exposure, and showed still detectable levels at 2 and 4 h (p < 0.05). Conclusion: We conclude that the presented inhalation chamber system is able to maintain a controlled CO concentration in a model in which small animals are exposed to the inhalation of cigarette smoke, permitting well-controlled studies, as well as investigations involving other toxic gases and air pollutants. (C) 2008 SEPAR. Published by Elsevier Espana, S.L. All rights reserved.

Potassium restores vasorelaxation of resistance arterioles in non-hypertensive DOCA/salt fed mice.

BACKGROUND: Potassium-enriched diets exert renal and cardiovascular protective effects, but the underlying mechanisms are largely unknown. METHODS: Using the dorsal skinfold chamber model for intravital microscopy, we examined endothelium-dependent vasorelaxation of precapillary resistance arterioles in response to acetylcholine or the NO donor SNAP in awake mice. Experiments were performed in uni-nephrectomized one renin gene (Ren-1c) C57BL/6 mice (control group) and in mice having received a continuous administration of deoxycorticosterone acetate and a dietary supplementation of 1% sodium chloride for 8weeks (DOCA/salt group). An additional group of DOCA/salt treated animals received a dietary supplement of 0.4% KCl for 3weeks prior to the experiments (DOCA/salt + potassium group). RESULTS: DOCA/salt treatment for 8weeks resulted in hypokalemia, but blood pressure remained unchanged. In DOCA/salt mice, relaxation of resistance arterioles was blunted in response to acetylcholine, and to a lesser extent to SNAP, suggesting endothelial dysfunction. Endothelium-dependent vasorelaxation was restored by the potassium-enriched diet. CONCLUSION: This study is the first to demonstrate a protective effect of potassium on endothelium-dependent vasorelaxation in the absence of confounding anti-hypertensive effects, as observed in most animal models and the clinical situation. We propose that the known cardio- and nephro-protective effects of potassium might - at least in part - be mediated by the salutary effects on endothelium-dependent arteriolar relaxation.

Enterococcus faecalis leakage of root canal sealers: an ex vivo study

The aim of this ex vivo study was to evaluate bacterial penetration after filling root canals using 3 different techniques. Three experimental groups of 25 teeth each, obturated with lateral-warm-vertical condensation of gutta-percha, Microseal technique and EndoREZ (R) system, respectively, were tested in a split chamber model system using Enterococcus faecalis and monitored for 180 days to determine bacterial penetration. A statistical analysis was performed using the Kaplan-Meier method. Median survival time was 25 days for Microseal system, 41 for lateral-warm-vertical condensation and 81 for EndoREZ (R). Significant differences were demonstrated between Microseal and EndoREZ (R) (p<0.001) and between Microseal and lateral-warm-vertical condensation technique (p<0.05). No statistically significant differences were observed between EndoREZ (R) and lateral-warm-vertical condensation. After 180 days of assessment, 20% of the EndoREZ (R) samples resisted bacterial penetration and furthermore, the EndoREZ (R) system has the potential to be a filler system compatible with other currently used systems.

Estudo do comportamento dos gnaisses Casablanca, Juparaná Delicato e Giallo Falésia (Ceará - Brasil) em atmosfera salina

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

CLSM assessment of tubule penetration and bacterial leakage evaluation of two resin-based sealer

Aim: The aims of this study were to assess the penetration of two endodontic sealers (salicylate and epoxy resin-based sealers) into dentinal tubules using CLSM; and to evaluate the bacterial leakage of roots filled with the same sealers associated with gutta-percha. Material and Methods: For sealer penetrability assessment, thirty bovine roots were instrumented and divided into three groups: AHP: EDTA + filling with AH Plus and gutta-percha (n=10), MTAF: EDTA + filling with MTA Fillapex and gutta-percha (n=10), control group: canals were not irrigated with EDTA and were filled with gutta-percha and AH Plus (n=5) or MTA Fillapex (n=5). Rhodamine B was added to the sealers in order to provide adequate fluorescence. The roots were transversely sectioned 3mm from the apex to enable CLSM analysis. Leakage was evaluated for turbidity of the broth in a split chamber model system for 30 days, using Enterococcus faecalis as a microbial marker. Thirty roots were instrumented and divided in four grupos: AHP: filling with AH Plus and gutta-percha (n=10); MTAF: filling with MTA Fillapex and gutta-percha (n=10); positive control: filling with gutta-percha without sealer (n = 5); negative control: sealing with cyanoacrylate to test the seal of the system (n = 5). Results: The medians for dentinal tubule penetration were 6.8% (AHP) and 6.6% (MTAF) (P = 0.82). The average time for bacterial leakage was 8 days in both experimental groups (P = 0.79). Conclusion: MTA Fillapex and AH Plus presented similar behavior regarding dentinal tubule penetration and bacterial leakage.

Short-term CO2-C emissions from soil prior to sugarcane (Saccharum spp.) replanting in southern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Einfluss von Hilfsstoffen auf die Bioverfügbarkeit von Substanzen der BCS Klasse III

In dieser Arbeit wurde der Effekt verschiedener Hilfsstoffe auf die Permeabilität von Substanzen der BCS Klasse III untersucht. Drei pharmazeutische Hilfsstoffe wurden hinsichtlich der Möglichkeit ihres Einsatzes als Permeationsverbesserer in Arzneistoffformulierungen untersucht. Außerdem wurde die Beteiligung von Gallensalzen an der Nahrungsmittel-Interaktion von Trospium untersucht.rnEs wurden Komplexe aus Trospium und λ-Carrageen hergestellt. Eine verbesserte Permeation, die höchstwahrscheinlich durch Mukoadhäsion zustande kam, war im Ussing-Kammer-Modell sehr gut reproduzierbar. In vivo war der Effekt nur bei einigen Tieren zu sehen und es kam zu hohen Standardabweichungen.rnTrospium bildet Ionenpaare mit Gallensalzen, welche zu einer besseren Permeabilität des Wirkstoffes führten. In Gegenwart von Nahrungsfetten blieb dieser Effekt aus. Eine Beteiligung der Interaktion von Trospium und Gallensalzen am Food-Effekt kann auf Basis dieser Ergebnisse als wahrscheinlich gelten.rnIm Caco-2-Modell konnte bereits eine Verbesserung der Permeabilität von Trospium durch Zusatz von Eudragit E gezeigt werden. Nun konnte gezeigt werden, dass durch den Hilfsstoff auch in vivo in Ratten eine verbesserte Permeation erreicht werden kann.rnDie Permeationsverbesserung von Aciclovir durch Zusatz von Chitosan-HCl sollte untersucht werden. Im Caco-2-Modell kam es zu einer signifikanten Permeationsverbesserung. Im Ussing-Kammer-Modell wurde die Permeation nicht verbessert. In Loop-Studien konnte nur bei hohen Hilfsstoff-Konzentrationen eine Tendenz zur Permeationsverbesserung erkannt werden.rn

Lowered microvascular vessel wall oxygen consumption augments tissue pO2 during PgE1-induced vasodilation

Continuous infusion of intravenous prostaglandin E1 (PgE1, 2.5 mug/kg/min) was used to determine how vasodilation affects oxygen consumption of the microvascular wall and tissue pO(2) in the hamster window chamber model. While systemic measurements (mean arterial pressure and heart rate) and central blood gas measurements were not affected, PgE1 treatment caused arteriolar (64.6 +/- 25.1 microm) and venular diameter (71.9 +/- 29.5 microm) to rise to 1.15 +/- 0.21 and 1.06 +/- 0.19, respectively, relative to baseline. Arteriolar (3.2 x 10(-2) +/- 4.3 x 10(-2) nl/s) and venular flow (7.8 x 10(-3) +/- 1.1 x 10(-2)/s) increased to 1.65 +/- 0.93 and 1.32 +/- 0.72 relative to baseline. Interstitial tissue pO(2) was increased significantly from baseline (21 +/- 8 to 28 +/- 7 mmHg; P < 0.001). The arteriolar vessel wall gradient, a measure of oxygen consumption by the microvascular wall decreased from 20 +/- 6 to 16 +/- 3 mmHg (P < 0.001). The arteriolar vessel wall gradient, a measure of oxygen consumption by the vascular wall, decreased from 20 +/- 6 to 16 +/- 3 mmHg (P < 0.001). This reduction reflects a 20% decrease in oxygen consumption by the vessel wall and up to 50% when cylindrical geometry is considered. The venular vessel wall gradient decreased from 12 +/- 4 to 9 +/- 4 mmHg (P < 0.001). Thus PgE1-mediated vasodilation has a positive microvascular effect: enhancement of tissue perfusion by increasing flow and then augmentation of tissue oxygenation by reducing oxygen consumption by the microvascular wall.

Arteriolar vasoconstrictive response: comparing the effects of arginine vasopressin and norepinephrine

INTRODUCTION: This study was designed to examine differences in the arteriolar vasoconstrictive response between arginine vasopressin (AVP) and norepinephrine (NE) on the microcirculatory level in the hamster window chamber model in unanesthetized, normotonic hamsters using intravital microscopy. It is known from patients with advanced vasodilatory shock that AVP exerts strong additional vasoconstriction when incremental dosage increases of NE have no further effect on mean arterial blood pressure (MAP). METHODS: In a prospective controlled experimental study, eleven awake, male golden Syrian hamsters were instrumented with a viewing window inserted into the dorsal skinfold. NE (2 microg/kg/minute) and AVP (0.0001 IU/kg/minute, equivalent to 4 IU/h in a 70 kg patient) were continuously infused to achieve a similar increase in MAP. According to their position within the arteriolar network, arterioles were grouped into five types: A0 (branch off small artery) to A4 (branch off A3 arteriole). RESULTS: Reduction of arteriolar diameter (NE, -31 +/- 12% versus AVP, -49 +/- 7%; p = 0.002), cross sectional area (NE, -49 +/- 17% versus AVP, -73 +/- 7%; p = 0.002), and arteriolar blood flow (NE, -62 +/- 13% versus AVP, -80 +/- 6%; p = 0.004) in A0 arterioles was significantly more pronounced in AVP animals. There was no difference in red blood cell velocities in A0 arterioles between groups. The reduction of diameter, cross sectional area, red blood cell velocity, and arteriolar blood flow in A1 to A4 arterioles was comparable in AVP and NE animals. CONCLUSION: Within the microvascular network, AVP exerted significantly stronger vasoconstriction on large A0 arterioles than NE under physiological conditions. This observation may partly explain why AVP is such a potent vasopressor hormone and can increase systemic vascular resistance even in advanced vasodilatory shock unresponsive to increases in standard catecholamine therapy.

Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na+ and K+ and urinary Na+ and K+ excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO2−/NO3− (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-d,l-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60–70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to ≈70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.

Ethanol prevents NMDA receptor reduction by maternal separation in neonatal rat hippocampus

We measured the effects of ethanol on glutamate receptor levels in the hippocampus of neonatal Wistar rats using a vapor chamber model. Two control groups were used; a normal suckle group and a maternal separation group. Levels of NMDA receptors were not significantly altered in ethanol-treated animals compared to the normal suckle control group, as shown by [H-3]MK-801 binding and Western blot analysis. However, MK-801 binding and NR1 subunit immunoreactivity were greatly reduced in the hippocampus of separation control animals. Neither ethanol treatment nor maternal separation altered levels of GluR1 or GluR2(4). These results have serious implications for the importance of maternal contact for normal brain development.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract ( Matrigel (TM)) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 mu L collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Management of allergic conjunctivitis and dry eye

Ocular allergy is a common eye condition encountered in clinical practice. However, little is known how seasonal allergic conjunctivitis (SAC), the most common subtype, is managed in clinical practice. Further, dry eye, another common eye condition, may be misdiagnosed as SAC and vice-versa as they share similar signs and symptoms. In addition, despite the frequent recommendation of non-pharmacological treatments for SAC, evidenceto support their use has not been identified in the scientific literature. The aim of this thesis was therefore to determine the actual diagnosis and management of SAC and dry eye in clinical practice and investigate the efficacy of non-pharmacological treatments for these conditions. The diagnostic and management strategies for SAC and dry eye employed by pharmacy staff are found to be inconsistent with current guidelines and scientific evidence based upon a mystery shopper design. Cluster analysis of tear film metrics in normal and dry eye patients identified several clinically relevant groups of patients that may allow for targeted treatment recommendations. Using a novel environmental chamber model of SAC, the use of artificial tears and cold compresses, either alone or combined is an effective treatment modality for acute and symptomatic SAC, on a par with topical anti-allergic medication, and has been demonstrated for the first time. In addition, eyelid warming therapy with a simple, readily available, seed filled device is an effective method of treating meibomian gland dysfunction (MGD) related evaporative dry eye, perhaps the most common dry eye subtype. A greater focus on ophthalmology must be implemented as part of the formal education and training of pharmacy staff, while greater professional communication between community pharmacists, optometrists and the population they serve is required. Artificial tears and cold compresses may be considered as front line agents for acute SAC by pharmacy staff and optometrists, to whom pharmacological treatment options are limited.

Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

NMR imaging of fluid pathways during drainage of softwood in a pressure membrane chamber

An experiment was implemented to study fluid flow in a pressure media. This procedure successfully combines nuclear magnetic resonance imaging with a pressure membrane chamber in order to visualize the non-wetting and wetting fluid flows with controlled boundary conditions. A specially designed pressure membrane chamber, made of non-magnetic materials and able to withstand 4 MPa, was designed and built for this purpose. These two techniques were applied to the drainage of Douglas fir sapwood. In the study of the longitudinal flow, narrow drainage fingers are formed in the latewood zones. They follow the longitudinal direction of wood and spread throughout the sample length. These fingers then enlarge in the cross-section plane and coalesce until drainage reaches the whole latewood part. At the end of the experiments, when the drainage of liquid water in latewood is completed, just a few sites of percolation appear in earlywood zones. This difference is a result of the wood anatomical structure, where pits, the apertures that allow the sap to flow between wood cells, are more easily aspirated in earlywood than in latewood. (C) 2007 Elsevier Ltd. All rights reserved.