DNAzyme-functionalized porous carbon nanospheres serve as a fluorescent nanoprobe for imaging detection of microRNA-21 and zinc ion in living cells.

The present study shows that a dual-signal nanoprobe consisting of DNAzyme-functionalized porous carbon nanospheres (PCNs) responds to microRNA-21 and zinc ion (Zn2+). The fluorescent probe undergoes an increase in the fluorescence intensity of fluorescein isothiocyanate (FITC) (with excitation/emission wavelengths at 488/517 nm) and the fluorescence intensity of cyanine-5 (Cy5) (with excitation/emission wavelengths at 633/670 nm) in the presence of microRNA-21 and Zn2+. The recognition between microRNA-21 and its complementary strand in the PCNs induces the separation of Zn2+-specific DNAzyme from PCNs, thus resulting in the increase of green fluorescence, and the exogenous Zn2+ triggers the rupture of cleavage strand of DNAzyme and recovery of red fluorescence. This nanoprobe allows us to acquire in vitro the determination of microRNA-21 in the range of 2-300 nM with a detection limit of 0.57 nM and the determination of Zn2+ in the range 2-100 nM with a detection limit of 0.43 nM, and in situ simultaneous imaging in MCF-7 breast cancer cells. Therefore, this strategy permits to obtain the expression levels of different biomarkers in living cells, providing a useful tool for diagnosis of cancers and understanding their biological process. Graphical abstract Schematic representation of the DNAzyme-functionalized porous carbon nanospheres for the imaging analysis of microRNA-21 and Zn2+ in living cells.

Skin Sensitization to Fluorescein Isothiocyanate Is Enhanced by Butyl Paraben in a Mouse Model.

Contact hypersensitivity (CHS) to preservatives is receiving increased attention. Parabens are widely used in foods, pharmaceutics and cosmetics as preservatives. The skin sensitizing activity of parabens remains controversial but a few investigations have been made as to whether parabens could facilitate sensitization to other chemicals. We have shown that di-n-butyl phthalate (DBP), a phthalate ester, has an adjuvant effect in a fluorescein isothiocyanate (FITC)-induced CHS mouse model. We have also demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. Comparative studies of phthalate esters revealed that TRPA1 agonistic activity and the adjuvant effect on FITC-CHS coincide. Here we focused on two commonly used parabens, butyl paraben (BP) and ethyl paraben (EP), as to their adjuvant effects. BALB/c mice were epicutneously sensitized with FITC in acetone in the presence or absence of a paraben. Sensitization to FITC was evaluated as the ear-swelling response after FITC challenge. BP but not EP enhanced skin sensitization to FITC, but the effect of BP was much weaker than that of DBP. Mechanistically, BP enhanced the trafficking of FITC-presenting CD11c+ dendritic cells (DCs) from the skin to draining lymph nodes as well as cytokine production by draining lymph nodes. When the TRPA1 agonistic activity was measured with a cell line expressing TRPA1, BP exhibited higher activity than EP. The present study provides direct in vivo evidence that BP causes sensitization to other chemicals by means of a mouse FITC-CHS model.

Subcellular Fate of a Fluorescent Cholesterol-Poly(ethylene glycol) Conjugate: An Excellent Plasma Membrane Imaging Reagent.

Cholesterol-containing molecules or nanoparticles play a significant role in achieving favorable plasma membrane imaging and efficient cellular uptake of drugs by the excellent membrane anchoring capability of the cholesterol moiety. By linking cholesterol to a water-soluble component (such as poly(ethylene glycol), PEG), the resulting cholesterol-PEG conjugate can form micelles in aqueous solution through self-assembly, and such a micellar structure represents an important drug delivery vehicle in which hydrophobic drugs can be encapsulated. However, the understanding of the subcellular fate and cytotoxicity of cholesterol-PEG conjugates themselves remains elusive. Herein, by using cholesterol-PEG2000-fluorescein isothiocyanate (Chol-PEG-FITC) as a model system, we found that the Chol-PEG-FITC molecules could attach to the plasma membranes of mammalian cells within 10 min and such a firm membrane attachment could last at least 1 h, displaying excellent plasma membrane staining performance that surpassed that of commonly used commercial membrane dyes such as DiD and CellMask. Besides, we systematically studied the endocytosis pathway and intracellular distribution of Chol-PEG-FITC and found that the cell surface adsorption and endocytosis processes of Chol-PEG-FITC molecules were lipid-raft-dependent. After internalization, the Chol-PEG-FITC molecules gradually reached many organelles with membrane structures. At 5 h, they were mainly distributed in lysosomes and the Golgi apparatus, with some in the endoplasmic reticulum (ER) and very few in the mitochondrion. At 12 h, the Chol-PEG-FITC molecules mostly aggregated in the Golgi apparatus and ER close to the nucleus. Finally, we demonstrated that Chol-PEG-FITC was toxic to mammalian cells only at concentrations above 50 µM. In summary, Chol-PEG-FITC can be a promising plasma membrane imaging reagent to avoid the fast cellular internalization and quick membrane detachment problems faced by commercial membrane dyes. We believe that the investigation of the dynamic subcellular fate of Chol-PEG-FITC can provide important knowledge to facilitate the use of cholesterol-PEG conjugates in fields such as cell surface engineering and drug delivery.

Dibutyl Maleate and Dibutyl Fumarate Enhance Contact Sensitization to Fluorescein Isothiocyanate in Mice.

Di-n-butyl phthalate (DBP), a phthalate ester, has been shown to have an adjuvant effect on fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse models. Di-n-butyl maleate (DBM), widely used as a plasticizer for industrial application, has been reported to cause dermatitis in humans. DBM is a butyl alcohol ester of di-carboxylic acid that represents a part of the DBP structure, while di-n-butyl fumarate (DBF) is a trans isomer of DBM. We examined whether DBM or DBF exhibits an adjuvant effect like DBP does. When BALB/c mice were epicutaneously sensitized with FITC in the presence of DBM or DBF, the FITC-specific CHS response was enhanced, as we have observed for DBP. As to underlying mechanisms, DBM and DBF facilitated the trafficking of FITC-presenting CD11c(+) dendritic cells (DCs) from skin to draining lymph nodes and increased the cytokine production by draining lymph nodes. In conclusion, DBM and DBF may have an effect that aggravates contact dermatitis through a skin sensitization process.

CXCR3 deficiency prolongs Th1-type contact hypersensitivity.

Sensitization and challenge using dinitrofluorobenzene (DNFB) induce contact hypersensitivity (CHS) with Th1 cell infiltration, whereas those using FITC generate CHS with Th2 cell infiltration. In this study, we attempted to determine the role of CXCR3, a chemokine receptor, in Th1- and Th2-type CHS induced by DNFB or FITC using CXCR3-deficient (CXCR3(-/-)) mice. Ear swelling was prolonged after DNFB challenge in CXCR3(-/-) mice, which was accompanied by increased Th1 cytokines and decreased TGF-ß and IL-10 expression at a late time point of CHS, whereas there was no significant difference between wild-type and CXCR3(-/-) mice in FITC-induced CHS. In Th1-type CHS, the number of regulatory T cells (Tregs) was decreased in the challenged ear of CXCR3(-/-) mice compared with that of wild-type mice, suggesting that CXCR3 would be important in migration of Tregs into the site of inflammation. Moreover, we examined the characteristics of CXCR3(+) Tregs both in vitro and in vivo, revealing that CXCR3(+) Tregs expressed high levels of TGF-ß and IL-10 as well as IFN-γ compared with CXCR3(-) Tregs. When CXCR3(-/-) mice were injected with CXCR3(+) Tregs, the prolonged ear swelling induced by DNFB was normalized. Taken together, our results suggest that CXCR3(+) Tregs play a key role for quenching Th1-type CHS.

When molecular probes meet self-assembly: an enhanced quenching effect.

We demonstrate that the incorporation of one or two amino acids of phenylalanine (F) or 4-fluoro phenylalanine ((f)F) will greatly lower the background fluorescence intensities of conventional quenched probes with quenchers. This enhanced quenching effect was due to the synergetic effect of the aggregation caused quenching and the presence of a quencher. Such strategy will not greatly affect the enzyme recognition properties to the probes. We also demonstrated that our self-assembled nanoprobe with the enhanced quenching effect showed a better performance in cells for the detection of cell apoptosis than the unassembled probes. Our study demonstrates that using molecular self-assembly can optimize and improve the performance of molecular probes and it provides a simple but very useful strategy to boost the signal-to-noise ratios of fluorescence probes.

Comparison of adjuvant mechanisms of medium-chain triacylglycerol in a mouse FITC-induced contact hypersensitivity model.

The number of allergy sufferers has been increasing with the increase in chemicals to which we are potentially exposed. We have discovered that tributyrin, a short-chain triacylglycerol (TAG), enhanced fluorescein isothiocyanate (FITC)-induced contact hypersensitivity in a mouse model. Medium-chain triacylglycerols (MCTs) are used in cosmetics, with which we come into direct contact frequently, to maintain skin conditions and as a thickening agent for cosmetics. In this study, we examined whether MCTs with different side chain lengths enhanced skin sensitization to FITC in the mouse model. During skin sensitization to FITC, the presence of tributyrin (side chain carbon number, 4; C4) as well as that of each MCT, tricaproin (C6), tricaprylin (C8), or tricaprin (C10), resulted in enhanced skin sensitization, whereas that of trilaurin (C12) did not. As to the mechanism underlying the enhanced sensitization, three MCTs (C6, C8 and C10) facilitated migration of FTIC-presenting CD11c+ dendritic cells to draining lymph nodes. These results indicated that not only tributyrin but also MCTs, up to side chain carbon number 10, have an adjuvant effect on FITC-induced skin hypersensitivity in mice.

Investigations on the use of fluorescence dyes for labeling dendrimers: cytotoxicity, accumulation kinetics, and intracellular distribution.

Fluorescent dyes (e.g., dansyl, fluoresceine isothiocyanate, or naphthalimide groups) are widely used as markers to study biological properties of drugs. In order to evaluate possible mediated cytotoxicity, we attached three molecules each to 1,3,5-tris(3-propylamino)benzene initially synthesized as core molecule for the design of dendrimers. Cytotoxic effects were only observed for the NO(2)-substituted naphthalimide conjugate. The intracellular distribution was visualized via confocal fluorescence microscopy and pointed to an accumulation in the endosome or nucleus, dependent on the cell line used.

A novel cell-penetrating Janus nanoprobe for ratiometric fluorescence detection of pH in living cells.

pH regulates the function of many organelles and plays a pivotal role in requiring multitud cellular behaviors. Compared with single fluorescent probes, ratio fluorescent probes have higher sensitivity and immunity to interference. Herein, a novel Janus ratio nanoprobe was developed for intracellular pH detection. Modified rhodamine B probe and fluorescein isothiocyanate (FITC) were individually encapsulated in the independent hemispheres of Janus microparticles fabricated via Pickering emulsion. Moreover, it exhibits a satasified ratiometric detection of pH compared to the previous core-shell structure and organic small molecule probe. Accordingly, the Janus nanoprobe possesses many important features as an attractive sensor, including high anti-jamming capability, excellent stability, good reversibility and low cytotoxicity. Variations of the two fluorescence intensities (Fgreen/Fred) resulted in a ratiometric pH fluorescent sensor, which can respond to wide range of pH values from 3 to 8. To be more specific, with a single excitation wavelength of 488 nm, there are dual emission bands centered at 538 nm and 590 nm. Also the Janus nanoprobe displays a excellent linear relationship in the physiologically relevant pH range of 4.0-6.0. Consequently, detecting of pH and imaging was successfully achieved in living cells, which provides a simple and reliable method for detecting intracelluar pH and other similar substances.

Ruxolitinib Cream Has Dual Efficacy on Pruritus and Inflammation in Experimental Dermatitis.

The goal of this study was to elucidate the anti-pruritic and anti-inflammatory efficacy of ruxolitinib cream in experimentally-induced dermatitis. Atopic dermatitis (AD), the most common chronic relapsing inflammatory skin disease, significantly impairs patients' quality of life, with pruritus being a common complaint. The sensation of itch results from the interplay between epidermal barrier dysfunction, upregulated immune signaling and the activation of the central nervous system. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway plays a central role in pro-inflammatory cytokine signaling in AD. Ruxolitinib cream is a potent and selective JAK1/2 inhibitor currently undergoing clinical evaluation in adults with mild-to-moderate AD (NCT03745638, NCT03920852 and NCT03745651). The efficacy of ruxolitinib cream was tested in murine models of acute and chronic dermatitis and was also characterized in an ex vivo human skin dermatitis model. Ruxolitinib cream was highly effective at ameliorating disease symptoms in multiple murine dermatitis models through downregulation of T helper (Th)2-driven inflammation, resulting in reduced skin thickening and decreased itch. Pathway analysis of mouse ear tissue and human skin explants underscored the role for ruxolitinib in ameliorating inflammation and reducing itch via modulation of the JAK-STAT pathway. Together, the data offer a strong rationale for the use of ruxolitinib cream as a potent therapeutic agent for the clinical management of atopic dermatitis.

Evaluation of FITC-induced atopic dermatitis-like disease in NC/Nga mice and BALB/c mice using computer-assisted stereological toolbox, a computer-aided morphometric system.

BACKGROUND: The NC/Nga mouse spontaneously develops eczematous atopic dermatitis (AD)-like skin lesions when maintained under conventional conditions, but not when kept under specific pathogen-free (SPF) conditions. Hence, there is a need for an AD model in mice housed under SPF conditions, as this is mandatory for research animals in many countries. METHODS: We evaluated the use of the hapten FITC as an inducer of AD-like disease in NC/Nga and BALB/c mice maintained under SPF conditions. Mice were either untreated or treated with tacrolimus or betamethasone. Using the software Computer Assisted Stereological Toolbox as a stereological method, the mice were sensitized to FITC and the histological efficiency of disease induction with regard to inflammation and CD4+ and CD8+ lymphocytes, in addition to mast cells, was evaluated. The method was validated by comparison to a conventional semiquantitative observer-dependent method. RESULTS: Our findings prove that FITC does indeed induce AD-like lesions in NC/Nga mice with regard to the histological appearance of the mice. However, when evaluating the immunological response in the affected areas of the mice with regard to the CD4/CD8 ratio and the effect of treatment, we found that the immune response in the NC/Nga mice differed from AD skin lesions in humans in certain aspects. CONCLUSIONS: These results emphasize the importance of an assessment of not only the histological but also the immunological appearance of the skin when evaluating AD-like disease in mice as a model for AD in humans.

Enhancement of mouse contact hypersensitivity appears with a short chain triacylglycerol but not with a long chain one.

The prevalence of skin allergies could be partly due to the increased exposure to chemicals from consumer products. Chemicals that can enhance hypersensitivity caused by other chemicals are the focus of this study. We have demonstrated that phthalate esters with short chain alcohols enhance fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) in a mouse model. We have also found that tributyrin, a triacylglycerol (TAG) with three butyric acids, enhances sensitization to FITC. To elucidate such an enhanced skin sensitization might be based on a general feature of TAG, we compared tributyrin and triolein, a natural TAG, as to an adjuvant effect on FITC-CHS. Triolein is the dominant TAG in olive oil and contains long chain mono-unsaturated fatty acids. Unlike tributyrin and dibutyl phthalate (DBP), triolein did not exhibit an adjuvant effect. With triolein, enhancement of FITC-presenting CD11c+ dendritic cell trafficking to draining lymph nodes was weak, and the activation status of DC, as revealed as CD86 expression, was low. We found a difference in the pattern of skin cytokine production, i.e., that thymic stromal lymphopoietin was produced with DBP and interleukin-1ß with tributyrin. Triolein did not induce either of these cytokines. This illustrates that the adjuvant effect of tributyrin on FITC-CHS is not a general phenomenon for TAGs. Although beneficial effects may be expected through oral administration of tributyrin, the effect on skin immune systems should be considered.

Chinese medicine Yu-Ping-Feng-San attenuates allergic inflammation by regulating epithelial derived pro-allergic cytokines.

This study aimed to investigate the mechanisms of Yu-Ping-Feng-San (YPFS) on attenuating allergic inflammation in the initial stage of atopic dermatitis (AD). AD mouse model was established with fluorescein isothiocyanate (FITC) sensitization and elicitation. Epithelial barrier structure was observed with transmission electron microscope. The populations of dendritic cells (DCs) and group 2 innate lymphoid cells (ILC2s) were detected by flow cytometry. Human immortalized keratinocyte (HaCaT) cells were stimulated with Poly(I:C)/TNF-α in vitro to assessthymic stromal lymphopoietin (TSLP), interleukin (IL)-33 and nuclear factor-κB (NF-κB) levels or expressions by immunofluorescence, enzyme linked immunosorbent assay (ELISA) and western blot. In the initial stage of AD, ear swelling and infiltration of inflammatory cells in ear tissues were markedly attenuated with YPFS treatments. The damaged structures of ear epithelium and the increased levels of Th2-cytokines induced by FITC were significantly rescued in YPFS-treated mice. The production of pro-allergic cytokines, TSLP and IL-33, as well as the cell populations of their target cells DCs and ILC2s were decreased in AD model, respectively. Likewise, the levels of TSLP and IL-33 in Poly(I:C)/TNF-α-stimulated HaCaT cells showed the same results. Lower levels of p-NF-κB were detected with YPFS treatment, and the expressions of TSLP and IL-33 could be further decreased with inhibiting of NF-κB. Therefore, YPFS attenuates allergic inflammation in the initial stage of AD probably through regulating NF-κB-TSLP/IL-33 pathway, which may provide a novel effective target for the prevention and treatment of allergic diseases.

IL-21 Attenuates FITC-Induced Contact Hypersensitivity Response via Regulation of Dendritic Cell Function.

IL-21 is mainly produced by activated CD4+ T cells and is involved in the activation of immune cells such as T cells and macrophages. In contrast, IL-21 suppresses dendritic cell maturation. We studied the effect of IL-21 in a mouse model of FITC-induced contact hypersensitivity using IL-21 isoform transgenic (IL-21iso-Tg) mice. Tissue inflammation at 24 hours after elicitation in IL-21iso-Tg mice was significantly weaker than that in wild-type mice. In agreement with tissue inflammation, recruitment of CD4+ and CD8+ T cells, neutrophils, and macrophages into the inflamed tissue was decreased in IL-21iso-Tg mice. In addition, both mRNA expression and protein production of inflammatory cytokines were lower in IL-21iso-Tg mice. In the skin, T cells were activated at inducible skin-associated lymphoid tissue, which is likely a gut-associated lymphoid tissue. The mRNA level of CXCL2, an essential chemokine for inducible skin-associated lymphoid tissue formation, was significantly lower in IL-21iso-Tg mice, and histological analysis showed that dendritic cell clustering, a preliminary step in inducible skin-associated lymphoid tissue formation, was impaired. Our study showed that IL-21 down-regulated inducible skin-associated lymphoid tissue formation and reduced contact hypersensitivity response.

A Cell-Surface-Specific Ratiometric Fluorescent Probe for Extracellular pH Sensing with Solid-State Fluorophore.

Extracellular acidity is correlated with the development of various pathological states and bulk pH measurements could not report surface acidity. In this study, we have developed a ratiometric fluorescent probe that aggregates upon interaction with cells, allowing persistent labeling of cells and in situ measurement of cell surface pH. The ternary nanoplatform is constructed by a convenient noncovalent combination of bovine serum albumin protected gold nanoclusters (BSA-AuNCs), fluorescein isothiocyanate (FITC) labeled cationic peptides (CPs), and FITC-free CPs. The red fluorescent AuNCs serve as reference fluorophore, while FITC labeled peptides act as specific recognition element for H+ and FITC unlabeled peptides are used for delivery. The probe displays a sensitive fluorescence ratiometric response for pH in the range of 5.0-9.5 with calculated p Ka of 7.2. Further studies have demonstrated that this nanosensor also has properties of high selectivity, reversibility to pH fluctuations, as well as low cytotoxicity. The new surface pH-measurement tool was validated in mapping extracellular pH and monitoring acidification regarding cell metabolism, demonstrating its potential for bioimaging and biosensing.

Adjuvant effect of short chain triacylglycerol tributyrin on a mouse contact hypersensitivity model.

Little attention has been paid to chemicals that can enhance hypersensitivity caused by other chemicals. We have demonstrated that phthalate esters with short chain alcohols enhance fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) in a mouse model. Furthermore, phthalate esters with such an enhancing effect were found to activate transient receptor potential ankyrin 1 (TRPA1) cation channels, which are expressed on a part of sensory neurons, using a TRPA1-expressing cell line. In this study, we examined these activities of esters comprising glycerol and a short chain fatty acid, i.e. dibutyrin and tributyrin. We carried out chemical synthesis of dibutyrin isomers. Each dibutyrin isomer weakly activated TRPA1 and slightly enhanced skin sensitization to FITC. Unexpectedly, TRPA1 activation and enhancement of FITC-CHS were much more evident in the presence of tributyrin. Mechanistically, tributyrin induced increased dendritic cell trafficking from the skin to draining lymph nodes. Tributyrin enhanced interferon-γ (IFN-γ) production by draining lymph nodes, while its effect on interleukin-4 (IL-4) production was relatively less prominent. These results suggested that tributyrin concomitantly caused TRPA1 activation and an adjuvant effect on FITC-CHS.

Direct fluorescent labeling for efficient biological assessment of inhalable particles.

Labeling of aerosol particles with a radioactive, magnetic, or optical tracer has been employed to confirm particle localization in cell compartments, which has provided useful evidence for correlating toxic effects of inhaled particles. However, labeling requires several physicochemical steps to identify functionalities of the inner or outer surfaces of particles, and moreover, these steps can cause changes in size, surface charge, and bioactivity of the particles, resulting in misinterpretations regarding their toxic effects. This study addresses this challenging issue with a goal of introducing an efficient strategy for constantly supplying labeled aerosol particles in a single-pass configuration without any pre- or post-physicochemical batch treatments of aerosol particles. Carbon black (CB, simulating combustion-generated soot) or calcium carbonate (CC, simulating brake-wear fragments) particles were constantly produced via spark ablation or bubble bursting, respectively. These minute particles were incorporated with fluorescein isothiocyanate-poly(ethylene glycol) 2-aminoethyl ether acetic acid solution at the orifice of a collison atomizer to fabricate hybrid droplets. The droplets successively entered a diffusion dryer containing 254-nm UV irradiation; therefore, the droplets were dynamically stiffened by UV to form fluorescent probes on particles during solvent extraction in the dryer. Particle size distributions, morphologies, and surface charges before and after labeling were measured to confirm fluorescence labeling without significant changes in the properties. In vitro assays, including confocal imaging, were conducted to confirm the feasibility of the labeling approach without inducing significant differences in bioactivity compared with untreated CB or CC particles.

Chitosan nanoparticles as a potential drug delivery system for the ocular surface: toxicity, uptake mechanism and in vivo tolerance.

PURPOSE: To study the in vitro and in vivo interaction of chitosan nanoparticles (CSNPs), a new particulate drug carrier, with epithelial cells on the ocular surface. METHODS: CSNPs labeled with fluorescein isothiocyanate-bovine serum albumin were produced by ionotropic gelation. Human conjunctival epithelial cells (IOBA-NHC) were exposed for 15, 30, 60, and 120 minutes to three different CSNP concentrations. Immediately after treatment and after a 24-hour recovery period in culture medium, cell survival, and viability were measured. The association of CSNPs with IOBA-NHC cells was investigated by confocal microscopy. The influence of temperature and the effect of metabolic inhibition were studied by fluorometry. The in vivo uptake and acute tolerance of the ocular surface to CSNPs were evaluated in rabbits. RESULTS: Cell survival and viability of CSNP-exposed cells were equivalent to that of the control. Uptake of CSNPs was continuous for the 2-hour duration of these experiments and was temperature dependent. Metabolic inhibition by sodium azide had no effect on CSNP uptake. The rabbit ocular surface showed no signs of inflammation or alteration after CSNP exposure compared with the control. Fluorescence microscopy of rabbit eyeball and lid sections confirmed in vivo uptake by conjunctival and corneal epithelia. CONCLUSIONS: CSNPs were internalized by IOBA-NHC cells by an active transport mechanism that did not compromise cell viability. Moreover, these nanoparticles were well tolerated by the ocular surface tissues. These facts add further support for the potential use of these colloidal systems to delivery drugs to the ocular surface.

Animal models of pulmonary fibrosis.

In general, there are two types of animal models: natural and experimental. Because there are no natural models for pulmonary fibrosis, an experimental model that reproduces key aspects of the human disease would be useful for the study of this form of lung disease, the natural history of which is not always known. To date, a variety of animal models have been used to investigate mechanisms of pulmonary and other types of fibrosis, including the intratracheal instillation of bleomycin, fluorescein isothiocyanate, or particulate matter, such as silica and asbestos. This chapter will describe two commonly used techniques, namely bleomycin and silica-induced, which have been developed for the study of pulmonary fibrosis in animal models.

Prolonged convection-enhanced delivery into the rat brainstem.

OBJECTIVE: Prolonged convection-enhanced delivery was used in an attempt to achieve large volumes of distribution (V(d)) in the rat brainstem. Clinical assessment and histological analysis were performed to establish the safety of this approach. METHODS: For evaluation of V(d,), 10 rats underwent stereotactic cannula placement into the brainstem. Five rats underwent a 24-hour infusion (volume of infusion [V(i)], 200 microl), and 5 rats underwent a 7-day infusion (V(i), 2 ml) of fluorescein isothiocyanate-dextran. Serial brainstem sections were imaged with ultraviolet illumination, and V(d) was assessed. For assessment of clinical tolerance, 30 additional rats underwent chronic infusions of an isotonic saline solution into the brainstem. Serial neurological examinations were performed, followed by histological analysis after the animals' death. RESULTS: No animal demonstrated clinically recognized neurological deficits. Foci of organizing necrosis were limited to the site of infusion and cannula tract. V(d) increased linearly with increasing V(i) (range, 24.8-130.6 mm(3)). Maximal cross sectional area of fluorescence and craniocaudal extent of fluorescence increased with increasing V(i). Fluorescence was detected throughout the entire brainstem beyond the compact area of highly concentrated tracer. CONCLUSION: Prolonged convection-enhanced delivery can be applied safely in the rat brainstem with no recognized limitations of V(d) and minimal histological changes beyond the site of infusion. Chronic brainstem infusions may enhance the potential application of convection-enhanced delivery for therapeutic purposes in treating diffuse pontine gliomas.