Multi-Scale Investigation of Human Renal Tissue in Three Dimensions.

Histopathology as a diagnostic mainstay for tissue evaluation is strictly a 2D technology. Combining and supplementing this technology with 3D imaging has been proposed as one future avenue towards refining comprehensive tissue analysis. To this end, we have developed a laboratory-based X-ray method allowing for the investigation of tissue samples in three dimensions with isotropic volume information. To assess the potential of our method for micro-morphology evaluation, we selected several kidney regions from three patients with cystic kidney disease, obstructive nephropathy and diabetic glomerulopathy. Tissue specimens were processed using our in-house-developed X-ray eosin stain and investigated with a commercial microCT and our in-house-built NanoCT. The microCT system provided overview scans with voxel sizes of [Formula: see text] and the NanoCT was employed for higher resolutions including voxel sizes from [Formula: see text] to 210 nm. We present a methodology allowing for a precise micro-morphologic investigation in three dimensions which is compatible with conventional histology. Advantages of our methodology are its versatility with respect to multi-scale investigations, being laboratory-based, allowing for non-destructive imaging and providing isotropic volume information. We believe, that after future developmental work this method might contribute to advanced multi-modal tissue diagnostics.

Hypnosis to Reduce Distress in Children Undergoing Anorectal Manometry: A Randomized Controlled Pilot Trial

Background/Aims@#To assess the effectiveness and feasibility of a brief session of hypnosis to reduce distress in children with functional constipationundergoing anorectal manometry (ARM). @*Methods@#A partially-blinded randomized controlled pilot trial was conducted in children 4-18 years old scheduled for ARM. Children were randomized to receive a brief session of hypnosis prior to ARM or standard care. Non-blinded and blinded observers rated the child’s level of distress using the Observation Scale of Behavioral Distress and a 4-point-Likert scale, respectively. Differences between groupswere analyzed using Fisher’s exact test or Mann-Whitney U test as appropriate. @*Results@#Data from 32 children (15 hypnosis and 17 standard care) were analyzed. Prior to insertion of the catheter, the observed mean levels of distress were lower in the hypnosis group according to both the non-blinded observer (median 0.0 [interquartile range {IQR} 0.0-0.3] vs 1.4 [IQR 0.3-2.4]; P = 0.009) and the blinded observer (median 0.0 [IQR 0.0-0.0] vs 0.5 [IQR 0.0-1.0]; P = 0.044). During ARM, observed and reported levels of distress did not differ significantly. In the hypnosis group, 92.9% of parents and childrenreported that hypnosis helped the child to relax. There were no significant differences in resting pressure, squeeze pressure, or duration of the procedure between both groups. @*Conclusion@#A brief session of hypnosis for children before ARM is an easily incorporable intervention that lowers distress levels prior to theprocedure and is positively perceived by children and parents.

Revealing the Microscopic Structure of Human Renal Cell Carcinoma in Three Dimensions.

For fully characterizing renal cell carcinoma (RCC), information about the 3D tissue microstructure is essential. Histopathology, which represents the current diagnostic gold standard, is destructive and only provides 2D information. 3D X-ray histology endeavors to overcome these limitations by generating 3D data. In a laboratory environment, most techniques struggle with limited resolution and the weak X-ray attenuation contrast of soft tissue. We recently developed a laboratory-based method combining nanoscopic X-ray CT with a cytoplasm-specific X-ray stain. Here, we present the application of this method to human RCC biopsies. The NanoCT slices enable pathological characterization of crucial structures by reproducing tissue morphology with a similar detail level as corresponding histological light microscopy images. Beyond that, our data offer deeper insights into the 3D configuration of the tumor. By demonstrating the compatibility of the X-ray stain with standard pathological stains, we highlight the feasibility of integrating staining based NanoCT into the pathological routine.

Optoacoustic imaging and staging of inflammation in a murine model of arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is one of the most frequent inflammatory diseases, causing pain and disability in the affected joints. Early diagnosis is essential for the efficiency of symptom-targeting treatments, but its diagnosis requires careful clinical, serologic, and imaging examinations, such as magnetic resonance imaging (MRI), which is both expensive and time consuming. In an effort to provide the biomedical community with a more accessible way to assess the advancement of arthritis, this study sought to investigate the use of multispectral optoacoustic tomography (MSOT) in a murine arthritis model, to visualize the extent of inflammation in vivo through an L-selectin/P-selectin-targeting contrast agent. METHODS: Mice with collagen-induced arthritis were studied as a model of RA. MSOT was performed using an L-selectin/P-selectin-targeting contrast agent, polyanionic dendritic polyglycerol sulfate (dPGS) labeled with a near-infrared (NIR) fluorophore, to increase the contrast of the arthritic joint. The signal intensity ratios between healthy legs and arthritic legs were calculated. Findings on contrast-enhanced MRI, clinical observations, the lymphocyte:granulocyte ratio, and histologic findings served as referents for comparison. RESULTS: MSOT using an inflammation-targeting contrast agent, dPGS-NIR, allowed for accurate diagnosis of inflammation in the mouse joints. In addition, use of this technique resulted in significant differentiation of the inflamed joints from the healthy joints (P = 0.023). The observed advancement of arthritis on the MSOT images was confirmed by clinical observation, blood analysis, contrast-enhanced MRI, and ex vivo histologic examinations. CONCLUSION: This study demonstrates that the combination of an inflammation-targeting contrast agent and optoacoustic tomographic imaging presents a promising means for the diagnosis of RA and the staging of arthritis-related inflammation.

Static compressive loading reduces the mRNA expression of type II and X collagen in rat growth-plate chondrocytes during postnatal growth.

The mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of loading on the mRNA expression pattern of key molecular components of the growth-plate related to the extracellular matrix (type II and type X collagen) and the PTH-PTHrP feedback loop. Short-term static compressive loading was applied to rat proximal tibial growth-plate explants. Four age groups at specific developmental stages were investigated. The spatial variation in the mRNA expression was compared among loaded explants, their contralateral sham controls, and uncultured growth plates from normal animals. Basic cell metabolism (18S rRNA) was unaffected by load. Results indicated a narrower spatial distribution of mRNA expression of type II collagen throughout the growth plate; similarly, a narrowed distribution of expression of type X collagen was noted in the lower hypertrophic zone of the growth-plate. This suggests that mechanical compression influences chondrocytes of the hypertrophic zone to alter their expression of specific genes encoding proteins of the extracellular matrix, while PTH-PTHrP receptor mRNA, a regulatory protein, remained unaffected by loading. The effects of compression were similar at the different stages of growth, suggesting that additional factors may be involved in the clinical progression of skeletal deformities observed during growth spurts. Although this study was done in vitro and limited to static loading, it furthers our understanding of growth-plate mechanobiology as a first step toward providing a scientific rationale for treating progressive musculoskeletal deformities.

The microsatellite polymorphism of heme oxygenase-1 is associated with baseline plasma IL-6 level but not with restenosis after coronary in-stenting / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Vascular smooth muscle cells (VSMCs) can express heme-oxygenase (HO), a rate-limiting enzyme in the degradation of heme to bilirubin, ferritin and carbon monoxide (CO). VSMC-derived CO can suppress VSMC proliferation and may serve as an antiproliferation factor. The promoter region of HO-1 shows a polymorphism with different (GT) n repeats that has been reported to differently induce gene expression. The objective of this study was to examine the effect of this variation on the occurrence of restenosis after in-stent treatment in patients with coronary artery disease.</p><p><b>METHODS</b>Candidates who underwent coronary stent implantation were genotyped for the HO-1 promoter polymorphism using polymerase chain reaction (PCR) and automated DNA capillary sequencer. Serum levels of IL-6 and C-reactive protein (CRP) were obtained at baseline, 24 hours and 48 hours after stenting. The primary end point for the study was angiographic evidence of in-stent restenosis at 6 months. All parameters for evaluation of restenosis were analysed by quantitative computer-assisted angiographic analysis (QCA).</p><p><b>RESULTS</b>One hundred and eighty-seven patients who underwent coronary stent implantation were studied of whom 27.8% showed > or = 50% restenosis after 6 months. The distribution of (GT) n repeats of all patients in the promoter region of HO-1 genotype ranged from 22 to 42, with (GT) 25 and (GT) 32 being the two most common alleles. The allelic repeats were divided into the short class (S) with 29 (GT) n, the middle class (M) with 30-37 (GT) n and the long class (L) with 38 (GT) n. There was no significant difference in the restenosis between the genotype groups or between post operation levels of inflammation markers, but carriers of the S allele (n = 120) had 33.3% lower baseline IL-6 compared with non-S carriers (n = 67, P = 0.0008).</p><p><b>CONCLUSIONS</b>Although no association was observed between the HO-1 promoter polymorphism and coronary in-stent restenosis following the stent procedure, the association with plasma IL-6 levels suggests that HO-1 S allele might protect from the atherosclerotic inflammatory process.</p>

The effects of mechanical loading on the mRNA expression of growth-plate cells.

Bone growth is a complex process involving proliferation, maturation and hypertrophy of chondrocytes in the growth plates. Mechanical forces applied to growing bones alter their longitudinal growth. However, the mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of mechanical loading on the mRNA expression pattern of key molecular components of the growth-plate. Short-term static loading was applied to rat proximal tibial growth-plate explants. Various age groups at specific developmental stages were investigated. In situ hybridization was used to assess the mRNA expression of the cells in different zones of the growth-plate. Four key components were investigated: 18s (basic cell metabolism), type II collagen (major extracellular matrix component), type X collagen (matrix component in hypertrophic zone) and PTH-PTHrP receptors (pre-hypertrophic chondrocytes). The spatial variation in the mRNA expression between loaded explants and their contralateral controls was compared to establish: -the sensitivity of the different growth-plate zones to mechanical loading; -the sensitivity of the different developmental stages to loading. Preliminary results indicated that static loading on the growth plate of 80 d.o. rats affects type II and X collagen gene expressions while PTH-PTHrP remains insensitive to static loading. Improved understanding of growth-plate mechanics and the underlying biology is required to provide a scientific basis for the treatment of progressive deformities.

Macromolecular transport across the rabbit proximal and distal colon.

BACKGROUND: Although many studies have investigated macromolecular uptake in the stomach and small intestine, little is known about macromolecular uptake in the colon. AIMS: To investigate the mechanisms involved in the transport of large antigenically intact macromolecules across the proximal and distal colonic epithelium in the rabbit. METHODS: The mucosal to serosal movement of bovine serum albumin (BSA) was examined in modified Ussing chambers under short circuited conditions. The mucosal surface was exposed to varying concentrations of BSA, and after a 50 minute equilibration period, the mucosal to serosal flux of immunologically intact BSA was determined by ELISA. Total BSA flux was determined by the transport of radiolabelled 125I-BSA. RESULTS: Intact BSA transport in proximal and distal colonic tissue showed saturable kinetics. Intact BSA transport in the proximal and distal segment was 7% and 2% of the total 125I-BSA flux respectively. Immunologically intact BSA transport in the distal segment was significantly less than that in the proximal segment. Intact BSA transport in the proximal colon was significantly reduced following treatment with sodium fluoride, colchicine, and tetrodotoxin. Cholinergic blockade had no effect on the uptake of intact BSA. CONCLUSION: The findings indicate that the transport of intact macromolecules across the proximal and distal large intestine is a saturable process. Further, intact BSA transport in the proximal colon is an energy dependent process that utilises microtubules and is regulated by the enteric nervous system.

Mast cell hyperplasia and increased macromolecular uptake in an animal model of giardiasis.

Giardiasis has been associated with an increase in allergic disease following infection suggesting an alteration in mucosal immune function. Jejunal in vivo and in vitro macromolecular transport, epithelial permeability, and mucosal and connective tissue mast cell counts were examined in Mongolian gerbils (35-45 g) orogastrically inoculated (I) with a pathogenic strain of Giardia lamblia and compared to age- and weight-matched, sham-treated controls (C) 6 and 21 days postinoculation. Macromolecular uptake was significantly increased in infected tissue at 6 days both in vivo (I 134 +/- 19 vs. C 74 +/- 17 ng/hr; n = 8; P < 0.05) and in vitro (I 125 +/- 17 vs. C 67 +/- 8 ng/hr/cm; n = 12; P < 0.05). Macromolecular uptake did not differ between groups at 21 days. Infection had no effect on mucosal permeability of [51Cr]EDTA. Mucosal mast cell counts did not differ at 6 days but were significantly elevated in infected tissue at 21 days (I 33.3 +/- 6.8 vs. C 2.7 +/- 0.4 per high magnification field; n = 5; P < 0.01) as were connective tissue mast cell counts (I 1.7 +/- 0.2 vs. C 1.0 +/- 0.1 per high magnification field; n = 13; P < 0.005). The findings indicate that during the peak phase of giardiasis, jejunal active antigen uptake is increased leading to a delayed recruitment of mucosal and connective tissue mast cells. These changes may play a role in the increased incidence of hypersensitivity reactions associated with Giardia infection.

Transport of albumin into the intestinal lumen of the rat.

The intestine is considered a major site for the breakdown and clearance of serum proteins. The mechanism of transport of macromolecules from the serosa into the lumen is unclear. The present study was designed to characterize the serosal to mucosal movement of albumin. Transport of bovine serum albumin (BSA) was assessed in short-circuited Ussing chambers, using stripped rat jejunum devoid of Peyer's patches. To define the kinetics of serosal to mucosal albumin transport the serosal surface was exposed to BSA at varying concentrations (0.5-5 mg.mL-1). Fluids from the mucosal compartment were sampled over time and assayed for immunologically intact BSA by ELISA. All subsequent experiments utilized a concentration of cold BSA (2 mg.mL-1) that produced maximal levels of intact BSA transport. To assess total BSA transport (intact BSA plus degraded BSA), 10 microCi (1 Ci = 37 GBq) 125I-labelled BSA was added to the serosal surface in addition to 2 mg.mL-1 cold BSA. To further characterize BSA transport tissues were treated with sodium fluoride (NaF) (metabolic inhibitor) or colchicine (an inhibitor of microtubule polymerization) or with the nerve blocker tetrodotoxin (TTX). All experiments using inhibitors were performed in paired tissues obtained from the same animal. Transport of intact BSA into the intestinal lumen was a saturable process, with a Vmax of 251 +/- 13 ng.cm-2.h-1 and a Km of 0.72 +/- 0.1 mg.mL-1, and represented 7% of the total BSA flux into the intestinal lumen. In the presence of NaF (2 x 10(-3) M), transport of both intact and total BSA was significantly inhibited (intact: control 374 +/- 80 vs. NaF 46 +/- 11, 88% inhibition; total: control 3288 +/- 296 vs. NaF 2550 +/- 235 ng.cm-2.h-1, 22% inhibition; p < 0.05). In addition, colchicine significantly inhibited intact BSA transport (control 339 +/- 15 vs. colchicine 206 +/- 13 ng.cm-2.h-1, 39% inhibition; p < 0.05). TTX had no effect on intact BSA flux. The findings suggest that transport of intact BSA from the serosa into the intestinal lumen is a saturable, energy-dependent process, which involves microtubules but is not under neural regulation.

The role of nitric oxide in the regulation of macromolecular transport in rat jejunum.

1. Nitric oxide is known to affect epithelial and microvascular permeability and is a major non-adrenergic non-cholinergic neurotransmitter in the intestine. We have previously demonstrated neuronal regulation of macromolecular transport in the intestine. To define this regulation further the role of nitric oxide was investigated. 2. Stripped rat jejunum was mounted in Ussing chambers exposing the mucosal surface to bovine serum albumin (BSA; 2 mg ml-1), or BSA (2 mg ml-1) plus [125I]BSA (10 microCi). Following a 50 min equilibration, serosal fluids were sampled for four 10 min periods, and fluxes determined for intact BSA by enzyme-linked immunosorbent assay (ELISA) and total BSA by [125I]BSA under basal conditions, and after treatment with NG-nitro-L-arginine-methyl ester (L-NAME) alone or in conjunction with L-arginine or decarboxylated molsidomine (SIN 1). 3. L-NAME significantly increased intact BSA uptake. Total (intact + degraded) BSA flux was not altered. The L-NAME effect was reversed by L-arginine and SIN 1. Additional experiments were performed by adding the nitric oxide donors sodium nitroprusside and SIN 1 directly to control tissue. Nitric oxide donors did not further decrease intact BSA flux below levels obtained from control tissue. The L-NAME enantiomer D-NAME had no effect. Sodium-free bathing solutions also had no effect on intact BSA uptake. Non-specific permeability, as assessed by the serosal to mucosal movement of [51Cr]ethylene-diamine-tetraacetate ([51Cr]EDTA), was decreased with L-NAME. 4. The findings indicate that nitric oxide downregulates intact macromolecular flux in the small intestine.

Transport of bovine serum albumin across rat jejunum: role of the enteric nervous system.

To assess the mechanisms for movement of antigenically intact macromolecules across small intestinal mucosa, transport kinetics of bovine serum albumin (BSA) uptake and the effect of neural and metabolic inhibition were examined in stripped short-circuited rat jejunum. The mucosa was exposed to BSA, and, after a 50-min equilibration, mucosal-to-serosal movement of immunologically intact BSA was determined by enzyme-linked immunosorbent assay and total BSA by radiolabeled 125I-BSA. Intact BSA uptake demonstrated saturable kinetics. Immunologically intact BSA crossed the intestinal mucosa as 4.5% of total 125I-BSA flux. Colchicine and 4 degrees C significantly reduced uptake of immunologically intact BSA. NaF significantly reduced uptake of immunologically intact BSA and 125I-BSA. Treatment with tetrodotoxin significantly reduced intact BSA uptake, but did not significantly alter total BSA uptake. The muscarinic cholinoceptor antagonist atropine also significantly inhibited transport of intact BSA, whereas the nicotinic cholinoceptor antagonist hexamethonium had no effect. These findings indicate that transport of intact macromolecules across small intestinal mucosa is a saturable energy-dependent process that utilizes the microtubular network and is regulated by the enteric nervous system primarily through cholinergic nerves acting on muscarinic receptors.