Revisiting definition and assessment of intestinal trans-epithelial passage.

This study aims to remind that Intestinal Passage (IP) measurement is a complex task that cannot be achieved by a unique measure of an orally given exogenous marker in blood or urine. This will be illustrated in the case of NOD mice. Indeed, various methods have been proposed to measure IP. Among them ex vivo measurement in Ussing chambers of luminal to serosal fluxes of exogenous markers and in vivo measurement of exogenous markers in blood or urine after oral gavage are the more commonly used. Even though they are commonly used indifferently, they do not give the same information and can provide contradictory results. Published data showed that diabetic status in female Non Obese Diabetic (NOD) mice increased FD4 concentration in blood after gavage but did not modify FD4 fluxes in Ussing chamber. We observed the same results in our experimental conditions and tracked FD4 concentrations in blood over a kinetic study (Area Under the Curve-AUC). In vivo measurements are a dynamic process and address not only absorption (IP and intestinal surface) but also distribution, metabolism and excretion (ADME). Diabetic status in NOD mice was associated with an increase of intestinal length (absorptive surface), itself positively correlated with AUC of FD4 in blood. We concluded that increased intestinal length induced by diabetic status will extend the absorptive surface and increase FD4 concentration in plasma (in vivo measurement) despite no modification on IP of FD4 (ex vivo measurement). In addition, this study characterized intestinal function in diabetic NOD mice. Diabetic status in NOD female mice increases intestinal length and decreases paracellular IP (FSS) without affecting transcellular IP (HRP, FD4). Histological studies of small and large intestine did not show any modification of intestinal circumference nor villi and crypt size. Finally, diabetic status was not associated with intestinal inflammation (ELISA).

In Vivo Assessment of Size-Selective Glomerular Sieving in Transplanted Human Induced Pluripotent Stem Cell-Derived Kidney Organoids.

BACKGROUND: The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell-derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation. METHODS: We transplanted kidney organoids under the renal capsule of the left kidney in immunodeficient mice followed by the implantation of a titanium imaging window on top of the kidney organoid. To assess glomerular function in the transplanted human pluripotent stem cell-derived kidney tissue 1, 2, and 3 weeks after transplantation, we applied high-resolution intravital multiphoton imaging through the imaging window during intravenous infusion of fluorescently labeled low and high molecular mass dextran molecules or albumin. RESULTS: After vascularization, glomerular structures in the organoid displayed dextran and albumin size selectivity across their glomerular filtration barrier. We also observed evidence of proximal tubular dextran reuptake. CONCLUSIONS: Our results demonstrate that human pluripotent stem cell-derived glomeruli can develop an appropriate barrier function and discriminate between molecules of varying size. These characteristics together with tubular presence of low molecular mass dextran provide clear evidence of functional filtration. This approach to visualizing glomerular filtration function will be instrumental for translation of organoid technology for clinical applications as well as for disease modeling.

Microbiological and environmental assessment of human oral dental plaque isolates.

Plaque-related diseases are amongst the most common ailments of the oral cavity. Streptococcus mutans is the causal agent of dental caries in animals and humans and is responsible for the formation and accumulation of plaques. This study aimed to identify and evaluate the role of the dental plaque isolates and its surrounding environment in plaque formation or inhibition. The study started with the identification of human dental plaque isolates from high caries index patients based on 16S rRNA and Mitis salivarius bacitracin agar (MSB) was used for S. mutans growing. Unexpectedly, the Streptococcus mutans was completely absent. The disc diffusion assay recorded that all the isolates had antimicrobial activity against the S. mutans growth. Enzymes assay revealed that the isolates produced dextransucrase, levansucrase and levanase activity with wide variation degrees. Also, the lactic acid production assay was done based in pH shift assessment. The highest pH shift and dextran yield were detected by the isolates Bacillus subtilis_AG1 and Bacillus mojavensis_AG3. The adherence test revealed that Lysinibacillus cresolivorans_W2 (MK411028) recorded the highest adhesion property (60%). Oligo- and polysaccharides were synthesized by the action of dextransucrase enzyme and their cytotoxicity tests were negative. Dextran with a molecular weight (117521 Da) recorded the highest antimicrobial efficacy against Bacillus subtilis_AG1 and Bacillusmojavensis_AG3 (65%, 63.5%) respectively. The results concluded that the dextran was the most important factor causing the dental plaque pathogenicity. Also, oral oligo- and polysaccharides might play a role in dental plaque control.

Assessment of Carotid Plaque Inflammation in Diabetic and Nondiabetic Patients-An Exploratory Ultrasmall Superparamagnetic Iron Oxide-Enhanced Magnetic Resonance Imaging Study.

BACKGROUND: Ultrasmall superparamagnetic iron oxide (USPIO)-enhanced magnetic resonance (MR) imaging enables the identification of inflammation within the atheroma, predominantly by USPIO uptake by macrophages present in atherosclerotic tissue. Diabetic patients, however, may have dysfunctional macrophage activity, which may affect utilization of USPIO in identifying plaque inflammation in this patient cohort. METHODS: Fifteen diabetic and fifteen nondiabetic patients underwent USPIO-enhanced carotid MR imaging using 1.5T MR system. Pre- and post-USPIO carotid MR images were manually coregistered. The percentage decrease in the signal intensity after USPIO administration was calculated as a relative measure of the USPIO uptake. RESULTS: Diabetic and nondiabetic patients had comparable demographics and comorbidities. The mean global, maximum quadrant, and maximum slice changes showing change in relative signal intensity as a result of USPIO administration were comparable for the two patient cohorts (P > .05). CONCLUSIONS: USPIO can identify inflammatory burden with carotid atheroma in both diabetic and nondiabetic patients.

5-Hydroxytryptamine 1A receptors in the dorsomedial hypothalamus connected to dorsal raphe nucleus inputs modulate defensive behaviours and mediate innate fear-induced antinociception.

The dorsal raphe nucleus (DRN) is an important brainstem source of 5-hydroxytryptamine (5-HT), and 5-HT plays a key role in the regulation of panic attacks. The aim of the present study was to determine whether 5-HT1A receptor-containing neurons in the medial hypothalamus (MH) receive neural projections from DRN and to then determine the role of this neural substrate in defensive responses. The neurotracer biotinylated dextran amine (BDA) was iontophoretically microinjected into the DRN, and immunohistochemical approaches were then used to identify 5HT1A receptor-labelled neurons in the MH. Moreover, the effects of pre-treatment of the dorsomedial hypothalamus (DMH) with 8-OH-DPAT and WAY-100635, a 5-HT1A receptor agonist and antagonist, respectively, followed by local microinjections of bicuculline, a GABAA receptor antagonist, were investigated. We found that there are many projections from the DRN to the perifornical lateral hypothalamus (PeFLH) but also to DMH and ventromedial (VMH) nuclei, reaching 5HT1A receptor-labelled perikarya. DMH GABAA receptor blockade elicited defensive responses that were followed by antinociception. DMH treatment with 8-OH-DPAT decreased escape responses, which strongly suggests that the 5-HT1A receptor modulates the defensive responses. However, DMH treatment with WAY-100635 failed to alter bicuculline-induced defensive responses, suggesting that 5-HT exerts a phasic influence on 5-HT1A DMH neurons. The activation of the inhibitory 5-HT1A receptor had no effect on antinociception. However, blockade of the 5-HT1A receptor decreased fear-induced antinociception. The present data suggest that the ascending pathways from the DRN to the DMH modulate panic-like defensive behaviours and mediate antinociceptive phenomenon by recruiting 5-HT1A receptor in the MH.

Assessment of the Intestinal Barrier with Five Different Permeability Tests in Healthy C57BL/6J and BALB/cJ Mice.

BACKGROUND: Intestinal permeability is thought to be of major relevance for digestive and nutrition-related diseases, and therefore has been studied in numerous mouse models of disease. However, it is unclear which tools are the preferable ones, and how normal values should be defined. AIMS: To compare different in vivo permeability tests in healthy mice of commonly used genetic backgrounds. METHODS: We assessed the intestinal barrier in male and female C57BL/6J and BALB/cJ mice of different ages, using four orally administered permeability markers, FITC-dextran 4000 (FITC-D4000) and ovalbumin (OVA) measured in plasma, and polyethylene glycol (PEG) and lactulose/mannitol (Lac/Man) measured in urine, and by assessing lipopolysaccharide (LPS) in portal vein plasma. RESULTS: After gavage, FITC-D4000, OVA, Lac/Man, and PEG400, but not PEG4000, were detectable in plasma or urine. Female mice tended to have a higher permeability according to the FITC-D4000, OVA, and PEG400 tests, but the Lac/Man ratio was higher in males. No significant differences between the two mouse strains of young and old mice were observed except for mannitol recovery, which was higher in BALB/cJ mice compared to C57BL/6J mice (p < 0.05). Virtually no LPS was detected in healthy mice. For all markers, normal values have been defined based on 5th-95th percentile ranges of our data. CONCLUSION: Selected oral permeability tests, such as FITC-D4000, OVA, PEG400, and Lac/Man, as well as LPS measurements in portal vein plasma, could be suitable for the evaluation of the intestinal barrier in mice, if used in a standardized way.

Monosynaptic convergence of chorda tympani and glossopharyngeal afferents onto ascending relay neurons in the nucleus of the solitary tract: a high-resolution confocal and correlative electron microscopy approach.

Physiological studies suggest convergence of chorda tympani and glossopharyngeal afferent axons onto single neurons of the rostral nucleus of the solitary tract (rNTS), but anatomical evidence has been elusive. The current study uses high-magnification confocal microscopy to identify putative synaptic contacts from afferent fibers of the two nerves onto individual projection neurons. Imaged tissue is revisualized with electron microscopy, confirming that overlapping fluorescent signals in confocal z-stacks accurately identify appositions between labeled terminal and dendrite pairs. Monte Carlo modeling reveals that the probability of overlapping fluorophores is stochastically unrelated to the density of afferent label, suggesting that convergent innervation in the rNTS is selective rather than opportunistic. Putative synaptic contacts from each nerve are often compartmentalized onto dendrite segments of convergently innervated neurons. These results have important implications for orosensory processing in the rNTS, and the techniques presented here have applications in investigations of neural microcircuitry with an emphasis on innervation patterning.

Monitoring of single-cell responses in the optic tectum of adult zebrafish with dextran-coupled calcium dyes delivered via local electroporation.

The zebrafish (Danio rerio) has become one of the major animal models for in vivo examination of sensory and neuronal computation. Similar to Xenopus tadpoles neural activity in the optic tectum, the major region controlling visually guided behavior, can be examined in zebrafish larvae by optical imaging. Prerequisites of these approaches are usually the transparency of larvae up to a certain age and the use of two-photon microscopy. This principle of fluorescence excitation was necessary to suppress crosstalk between signals from individual neurons, which is a critical issue when using membrane-permeant dyes. This makes the equipment to study neuronal processing costly and limits the approach to the study of larvae. Thus there is lack of knowledge about the properties of neurons in the optic tectum of adult animals. We established a procedure to circumvent these problems, enabling in vivo calcium imaging in the optic tectum of adult zebrafish. Following local application of dextran-coupled dyes single-neuron activity of adult zebrafish can be monitored with conventional widefield microscopy, because dye labeling remains restricted to tens of neurons or less. Among the neurons characterized with our technique we found neurons that were selective for a certain pattern orientation as well as neurons that responded in a direction-selective way to visual motion. These findings are consistent with previous studies and indicate that the functional integrity of neuronal circuits in the optic tectum of adult zebrafish is preserved with our staining technique. Overall, our protocol for in vivo calcium imaging provides a useful approach to monitor visual responses of individual neurons in the optic tectum of adult zebrafish even when only widefield microscopy is available. This approach will help to obtain valuable insight into the principles of visual computation in adult vertebrates and thus complement previous work on developing visual circuits.

Riboflavin carrier protein-targeted fluorescent USPIO for the assessment of vascular metabolism in tumors.

Riboflavin (Rf) and its metabolic analogs flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential for normal cellular growth and function. Their intracellular transport is regulated by the riboflavin carrier protein (RCP), which has been shown to be over-expressed by metabolically active cancer cells. Therefore, FAD-decorated ultrasmall superparamagnetic iron oxide nanoparticles (FAD USPIO) were developed as the first carrier-protein-targeted molecular MR agents for visualizing tumor metabolism. FAD USPIO were synthesized using an adsorptive, fluorescent and non-polymeric coating method, and their physicochemical properties were characterized using TEM, SEM, FTIR, MRI and fluorescence spectroscopy. In vitro analyses showed the biocompatibility of FAD USPIO, and confirmed that they were strongly and specifically taken up by cancer (LnCap) and endothelial (HUVEC) cells. In vivo molecular MRI together with subsequent histological validation finally demonstrated that FAD USPIO efficiently accumulate in tumors and tumor blood vessels, indicating that RCP-targeted diagnostic nanoparticles are interesting new materials for the assessment of vascular metabolism in tumors.

Assessment of endothelial permeability and leukocyte transmigration in human endothelial cell monolayers.

Increased vascular permeability is the hallmark of inflammation. Here, we describe three methods to assess vascular permeability in cell culture: (1) Impedance measurements of endothelial cell monolayers that allow to monitor changes in cell shape in real time. (2) Diffusion of fluorescently labeled dextran across endothelial cell monolayers to identify openings large enough for bulky molecules. (3) Transmigration of neutrophils through confluent endothelial cell monolayers to study one major process that increases endothelial permeability in inflammation.

Assessment of glycoprotein interactions with 4-[(2-aminoethyl)carbamoyl]phenylboronic acid surfaces using surface plasmon resonance spectroscopy.

Reported here are analyses of the interactions between a select group of solution-phase glycoproteins and a unique boronic acid capture surface. The boronic acid derivative, 4-[(2-aminoethyl)carbamoyl]phenylboronic acid, AECPBA, was synthesized and then immobilized on carboxymethyl dextran surfaces using simple coupling methods. From surface plasmon resonance spectroscopy responses, it is found that model glycoproteins interact strongly with the AECPBA surface and subsequently can be readily released from the AECPBA surface using borate buffer. A striking difference between the glycoproteins fetuin and asialofetuin (desialylated fetuin), in terms of glycoprotein binding to the AECPBA surface, indicates that the interaction of glycoproteins with the immobilized AECPBA is dictated by the terminal saccharide of the heteroglycan chain. Surprisingly, secondary interactions of glycosylated and nonglycosylated proteins with the carboxymethyl dextran hydrogel matrix are observed. Importantly, it is demonstrated that use of tris(hydroxymethyl)aminomethane buffer allows for decreased secondary interactions of nonglycosylated proteins on the AECPBA/dextran surface, as noted with the model protein ExtrAvidin.

Assessment of human islet labeling with clinical grade iron nanoparticles prior to transplantation for graft monitoring by MRI.

Ex vivo labeling of islets with superparamagnetic iron oxide (SPIO) nanoparticles allows posttransplant MRI imaging of the graft. In the present study, we compare two clinical grade SPIOs (ferucarbotran and ferumoxide) in terms of toxicity, islet cellular uptake, and MRI imaging. Human islets (80-90% purity) were incubated for 24 h with various concentrations of SPIOs (14-280 µg/ml of iron). Static incubations were performed, comparing insulin response to basal (2.8 mM) or high glucose stimulation (16.7 mM), with or without cAMP stimulation. Insulin and Perl's (assessment of iron content) staining were performed. Electronic microscopy analysis was performed. Labeled islets were used for in vitro or in vivo imaging in MRI 1.5T. Liver section after organ removal was performed in the same plane as MRI imaging to get a correlation between histology and radiology. Postlabeling islet viability (80 ± 10%) and function (in vitro static incubation and in vivo engraftment of human islets in nude mice) were similar in both groups. Iron uptake assessed by electron microscopy showed iron inclusions within the islets with ferucarbotran, but not with ferumoxide. MRI imaging (1.5T) of phantoms and of human islets transplanted in rats, demonstrated a strong signal with ferucarbotran, but only a weak signal with ferumoxide. Signal persisted for >8 weeks in the absence of rejection. An excellent correlation was observed between radiologic images and histology. The hepatic clearance of intraportally injected ferucarbotran was faster than that of ferumoxide, generating less background. A rapid signal decrease was observed in rejecting xenogeneic islets. According to the present data, ferucarbotran is the most appropriate of available clinical grade SPIOs for human islet imaging.

Quantitative assessment of intestinal injury using a novel in vivo, near-infrared imaging technique.

Intestinal injury owing to inflammation, severe trauma, and burn is a leading cause of morbidity and mortality. Currently, animal models employed to study the intestinal response to injury and inflammation depend on outdated methods of analysis. Given that these classic intestinal assays are lethal to the experimental animal, there is no ability to study the gut response to injury in the same animal over time. We postulated that by developing an in vivo assay to image intestinal injury using fluorescent dye, it could complement other expensive, time-consuming, and semiquantitative classic means of detecting intestinal injury. We describe a novel in vivo, noninvasive method to image intestinal injury using a charge-coupled device (CCD) camera that allows for serial visual and quantitative analysis of intestinal injury. Our results correlate with traditional, time-consuming, semiquantitative assays of intestinal injury, now allowing the noninvasive, nonlethal assessment of injury over time.

Assessment of functional recovery and axonal sprouting in oligodendrocyte-myelin glycoprotein (OMgp) null mice after spinal cord injury.

Oligodendrocyte-myelin glycoprotein (OMgp) is a myelin component that has been shown in vitro to inhibit neurite outgrowth by binding to the Nogo-66 receptor (NgR1)/Lingo-1/Taj (TROY)/p75 receptor complex to activate the RhoA pathway. To investigate the effects of OMgp on axon regeneration in vivo, OMgp(-/-) mice on a mixed 129/Sv/C57BL/6 (129BL6) or a C57BL/6 (BL6) genetic background were tested in two spinal cord injury (SCI) models - a severe complete transection or a milder dorsal hemisection. OMgp(-/-) mice on the mixed 129BL6 genetic background showed greater functional improvement compared to OMgp(+/+) littermates, with increased numbers of cholera toxin B-labeled ascending sensory axons and 5-HT(+) descending axons and less RhoA activation after spinal cord injury. Myelin isolated from OMgp(-/-) mice (129BL6) was significantly less inhibitory to neurite outgrowth than wild-type (wt) myelin in vitro. However, OMgp(-/-) mice on a BL/6 genetic background showed neither statistically significant functional recovery nor axonal sprouting following dorsal hemisection.

A re-assessment of the effects of a Nogo-66 receptor antagonist on regenerative growth of axons and locomotor recovery after spinal cord injury in mice.

This study was undertaken as part of the NIH "Facilities of Research-Spinal Cord Injury" project to support independent replication of published studies. Here, we repeated a study reporting that treatment with the NgR antagonist peptide NEP1-40 results in enhanced growth of corticospinal and serotonergic axons and enhanced locomotor recovery after thoracic spinal cord injury. Mice received dorsal hemisection injuries at T8 and then received either NEP1-40, Vehicle, or a Control Peptide beginning 4-5 h (early treatment) or 7 days (delayed treatment) post-injury. CST axons were traced by injecting BDA into the sensorimotor cortex. Serotonergic axons were assessed by immunocytochemistry. Hindlimb motor function was assessed using the BBB and BMS scales, kinematic and footprint analyses, and a grid climbing task. There were no significant differences between groups in the density of CST axon arbors in the gray matter rostral to the injury or in the density of serotonergic axons caudal to the injury. Tract tracing revealed that a small number of CST axons extended past the lesion in the ventral column in some mice in all treatment groups. The proportion of mice with such axons was higher in the NEP1-40 groups that received early treatment. In one experiment, mice treated with either NEP1-40 or a Control Peptide (reverse sequence) had higher BBB and BMS scores than Vehicle-treated controls at the early post-injury testing intervals, but scores converged at later intervals. There were no statistically significant differences between groups on other functional outcome measures. In a second experiment comparing NEP-treated and Vehicle controls, there were no statistically significant differences on any of the functional outcome measures. Together, our results suggest that treatment with NEP1-40 created a situation that was slightly more conducive to axon regeneration or sprouting. Enhanced functional recovery was not seen consistently with the different functional assessments, however.

Modeling the release of proteins from degrading crosslinked dextran microspheres using kinetic Monte Carlo simulations.

To optimize and predict the release of proteins from biodegradable microspheres based on crosslinked dextran, a fundamental understanding of the mechanisms controlling their release is necessary. For that purpose, a mathematical model has been developed to describe the release of proteins from these hydrogel-based microspheres. A kinetic Monte Carlo scheme for the degradation of a small domain inside the microsphere was developed. The results from this were used in a second kinetic Monte Carlo scheme to model the diffusion and the subsequent release of proteins. The only processes included in this model are diffusion and degradation. The general effects of diffusion, crosslink density, protein loading, and clustering of proteins on the release were investigated. The model crosslink density (Xmodel) and the model diffusivity (Dmodel) were fitted to experimental release data of BSA monomer from hydroxyethyl methacrylated dextran (dex-HEMA) microspheres. By using the experimental release curves of liposomes and BSA monomer, it was found that (1) the model crosslink density (Xmodel) scales with the hydrodynamic diameter (dh) as dh(1.64) and (2) the diffusivity of the protein (Dmodel) scales approximately with 1/dh (Stokes-Einstein). Using these scaling relations, quantitative predictions of the release curves of BSA dimer, immunoglobulin G and human growth hormone were possible. In conclusion, this model may play an important role in the optimization, understanding and prediction of the release of various proteins from degradable hydrogels.

A novel approach for purification of recombinant proteins using the dextran-binding domain.

Using the dextran-binding domain (DBD) of a type of glucosyltransferase (GTF) from Streptococcus sobrinus, we have developed a novel method for purifying recombinant proteins. DBD-tagged green and red fluorescent proteins as well as the parent GTF and DBD moiety were adsorbed well to commercially available cross-linked dextran (such as Sephadex beads and Sephacryl beads), and eluted efficiently with water-soluble dextran. The purity of the eluted proteins after this one-step affinity purification was approximately 90% or better. The results suggest that DBD can be used as a powerful carrier for purification of various recombinant proteins.

Effects of absorption enhancers on the transport of model compounds in Caco-2 cell monolayers: assessment by confocal laser scanning microscopy.

Three typical absorption enhancers, i.e., sodium caprate (Cap-Na), sodium deoxycholate (Deo-Na), and dipotassium glycyrrhizinate (Grz-K), were compared in terms of their permeability-enhancing effects on hydrophilic and hydrophobic model compounds in Caco-2 cell monolayers. The transepithelial electrical resistance (TEER) of the monolayers was reduced concentration-dependently by treatment with Cap-Na and Deo-Na, while treatment with Grz-K increased the TEER. Two patterns of TEER reduction were observed: one pattern indicated that Cap-Na had a rapid reducing effect, and another indicated that Deo-Na had a delayed reducing effect. These reductions in the TEER were accompanied by the increased transepithelial transport of two hydrophilic model compounds, sodium fluorescein (Flu-Na; MW = 376, log P = -1.52) and fluorescein isothiocyanate-dextran 4000 (FD-4; MW = 4400, log P = -2.0), and one hydrophobic model compound, rhodamine 123 hydrate (Rh123; MW = 381, log P = 1.13). The transport-enhancing effects of Cap-Na and Deo-Na on these model compounds decreased in the following order: FD-4 > Rh123 > Flu-Na, while Grz-K was found to have no effect on the transport of any of these model compounds. Confocal laser scanning microscopy (CLSM) of Caco-2 cell monolayers revealed that Cap-Na and Deo-Na enhanced the transepithelial transport of the hydrophilic model compounds via the paracellular route and that of the hydrophobic model compound via both paracellular and transcellular routes. Semiquantitative visual information obtained from CLSM images reflected the results of the transport experiment.

The effect of blood contact and reuse on the transport properties of high-flux dialysis membranes.

The effect of blood contact and reprocessing using bleach on the convective transport of both neutral and positively charged dextrans was determined for cellulose triacetate (CT), polyacrylonitrile (PAN), and polysulfone (PS) dialyzers (Fresenius USA, F60B, Concord, CA). For neutral dextrans, blood contact reduced the convective permeability, determined by differences in the sieving coefficient profile for both the PAN and PS, but not for CT dialyzers. Reprocessing of the dialyzers with bleach (up to 15 reuses) did not affect the convective transport of dextrans through CT or PAN, but did enhance the permeability of the blood contacted PS dialyzers. However, sieving coefficients for the blood contacted and reprocessed PS (F60B) dialyzers were significantly lower than those for the other dialyzers studied, approaching zero for dextrans larger than 18 k molecular weight. Sieving coefficients for positively charged, diethylaminoethyl (DEAE) dextrans were a function not only of solute size, but also of the membrane's capacity for adsorption of charged molecules. The majority of smaller, filtered DEAE dextrans adsorbed to the PAN membrane. Adsorption of DEAE dextrans to PAN was not observed for larger dextrans, or for DEAE dextrans of any size with CT, despite the lower permeability of both membranes for DEAE dextran compared to that for neutral dextrans.

Assessment of a model for measuring drug diffusion through implant-generated fibrous capsule membranes.

Fibrous tissue, which encapsulates subcutaneously implanted silastic, vinyl, polyurethane and Teflon discs in rats, has been isolated, characterized and tested for drug permeability in order to develop an in vitro model for determining the effect of this tissue on drug disposition from implant sites. With all materials, capsule tissue thickness and collagen content (approximately 59%) was consistent from 2 to 4 months after implantation. Silastic implants afforded the most consistent and usable tissue in terms of thickness and lack of vascularity, and these capsule membranes were used for determining the transport of three model compounds in an in vitro diffusion cell model. The rank ordering of permeability through these membranes was estrone (60.2 x 10(-6) cm s-1) > 3-O-methylglucose (18.7 x 10(-6) cm s-1) > dextran of molecular weight 70 000 (5.6 x 10(-6) cm s-1), which is consistent with expectations based on the molecular weights and partitioning behaviour of the model compounds. The results of these studies indicate that implant-generated encapsulating membranes can be successfully isolated and employed to study drug diffusion in an in vitro model, providing a direct assessment of the barrier properties of encapsulating membranes.