Width Of Intercellular Spaces Research Articles

The Potential Role for Impaired Mucosal Integrity in the Generation of Esophageal Pain Using Capsaicin in Humans: An Explorative Study.

INTRODUCTION:Esophageal pain is mediated by sensory nerves, most importantly by the activation of the transient receptor potential vanilloid 1 (TRPV1) capsaicin receptor. TRPV1 is activated and sensitized by a broad range of pungent compounds, as well as inflammatory mediators and tissue irritants. Luminal stressors are suggested to impair the barrier function, which results in consequent activation of these sensory nerve terminals and pain. In this study, we investigated the effect of the perfusion of capsaicin, a TRPV1 agonist, on mucosal impedance and pain in asymptomatic volunteers.METHODS:Thirteen asymptomatic volunteers completed a single-blind, saline-controlled, randomized crossover study. Capsaicin or saline was perfused for 30 minutes in the distal esophagus. Visual analog scale pain intensity scores and intraluminal impedance indicating mucosal integrity were determined. Distal and proximal biopsies were obtained 10 minutes later to measure TRPV1 messenger RNA and TRPV1 immunopositivity, as well as the intercellular space area.RESULTS:Capsaicin perfusion resulted in significantly greater pain intensity (P = 0.047) and impaired recovery of the mucosal impedance compared with saline-treated controls (P = 0.027). Pain response was significantly associated with decreased mucosal impedance. Similar dynamics were seen in the proximal esophagus, but mucosal impedance recovered entirely to the preinfusion values there. There was a significant association between mucosal impedance and intercellular space width in the distal esophagus. TRPV1 transcription and expression were not significantly altered within this observation period.DISCUSSION:Esophageal capsaicin perfusion results in pain, which is likely to be explained by impaired mucosal impedance and defective restoration capacity in the distal esophagus.

Association of Acute Gastroesophageal Reflux Disease With Esophageal Histologic Changes.

The histologic changes associated with acute gastroesophageal reflux disease (GERD) have not been studied prospectively in humans. Recent studies in animals have challenged the traditional notion that reflux esophagitis develops when esophageal surface epithelial cells are exposed to lethal chemical injury from refluxed acid. To evaluate histologic features of esophageal inflammation in acute GERD to study its pathogenesis. Patients from the Dallas Veterans Affairs Medical Center who had reflux esophagitis successfully treated with proton pump inhibitors (PPIs) began 24-hour esophageal pH and impedance monitoring and esophagoscopy (including confocal laser endomicroscopy [CLE]) with biopsies from noneroded areas of distal esophagus at baseline (taking PPIs) and at 1 week and 2 weeks after stopping the PPI medication. Enrollment began May 2013 and follow-up ended July 2015. PPIs stopped for 2 weeks. Twelve patients (men, 11; mean age, 57.6 year [SD, 13.1]) completed the study. Primary outcome was change in esophageal inflammation 2 weeks after stopping the PPI medication, determined by comparing lymphocyte, eosinophil, and neutrophil infiltrates (each scored on a 0-3 scale) in esophageal biopsies. Also evaluated were changes in epithelial basal cell and papillary hyperplasia, surface erosions, intercellular space width, endoscopic grade of esophagitis, esophageal acid exposure, and mucosal impedance (an index of mucosal integrity). At 1 week and 2 weeks after discontinuation of PPIs, biopsies showed significant increases in intraepithelial lymphocytes, which were predominantly T cells (median [range]: 0 (0-2) at baseline vs 1 (1-2) at both 1 week [P = .005] and 2 weeks [P = .002]); neutrophils and eosinophils were few or absent. Biopsies also showed widening of intercellular spaces (confirmed by CLE), and basal cell and papillary hyperplasia developed without surface erosions. Two weeks after stopping the PPI medication, esophageal acid exposure increased (median: 1.2% at baseline to 17.8% at 2 weeks; Δ, 16.2% [95% CI, 4.4%-26.5%], P = .005), mucosal impedance decreased (mean: 2671.3 Ω at baseline to 1508.4 Ω at 2 weeks; Δ, 1162.9 Ω [95% CI, 629.9-1695.9], P = .001), and all patients had evidence of esophagitis. In this preliminary study of 12 patients with severe reflux esophagitis successfully treated with PPI therapy, stopping PPI medication was associated with T lymphocyte-predominant esophageal inflammation and basal cell and papillary hyperplasia without loss of surface cells. If replicated, these findings suggest that the pathogenesis of reflux esophagitis may be cytokine-mediated rather than the result of chemical injury. clinicaltrials.gov Identifier: NCT01733810.

Morphometric features of corneal epithelial basal cells, and their relationship with corneal nerve pathology and clinical factors in patients with type2 diabetes.

We compared the morphometric features of corneal epithelial basal cells between patients with type2 diabetes mellitus and healthy controls, and analyzed the relationship of these features with corneal nerve fiber pathology and clinical factors in the patients. Corneal epithelial basal cells and corneal nerve fibers were visualized by corneal confocal microscopy in 75 patients with type2 diabetes and 42 age-matched controls. Density, area and area variability of corneal epithelial basal cells, as well as the width of the intercellular space between neighboring cells, were evaluated for both groups. Patients showed decreased density (P<0.02) and area (P<0.0001), larger area variability (P<0.0001) and a wider intercellular space (P<0.0001) compared with controls. Density correlated inversely with area (P<0.0001), width of intercellular space (P<0.03) and beading frequency (P<0.03), whereas it correlated directly with prothrombin time (P<0.002) and activated partial thromboplastin time (P<0.03). Area correlated inversely with duration of diabetes (P<0.05) and coefficient of variation of area (P<0.01), whereas it correlated directly with beading frequency (P<0.05). Area variability correlated inversely with area (P<0.01) and prothrombin time (P<0.01), whereas it correlated directly with fibrinogen level (P<0.0001). Type2 diabetes induces morphometric changes in corneal epithelial basal cells; this seems to be related to the morbid period of diabetes, beading frequency of corneal nerve fibers and blood coagulation state.

Dilated intercellular space in chronic laryngitis and gastro-oesophageal reflux disease: at baseline and post-lansoprazole therapy

Dilation of intercellular spaces is reported to be an early morphological marker in gastro-oesophageal reflux. It remains unknown if this marker is useful in diagnosing reflux-related chronic laryngitis. To determine histopathology and electron microscopic changes in oesophageal and laryngeal epithelium in chronic laryngitis. In this prospective blinded study, we enrolled 53 participants: 15 controls, 20 patients with GERD and 18 patients with chronic laryngitis. The latter two groups were subsequently treated with lansoprazole 30 mg bid for 12-weeks. Baseline and postacid suppressive therapy biopsies were obtained from distal oesophagus and laryngeal postcricoid areas. Biopsy specimens were evaluated for histopathology and dilated intercellular space changes. There was no significant increase in oesophageal or laryngeal epithelium intercellular spaces among GERD or laryngitis patients compared with controls at baseline or postacid suppressive therapy. Only patients with GERD had significantly (P = 0.03) higher proportion of moderate-to-severe oesophageal spongiosis and basal cell hyperplasia, which normalized postacid suppressive therapy. There was no increase in the width of intercellular spaces in the oesophagus or larynx in GERD or chronic laryngitis at baseline or postacid suppressive therapy. Our findings question the uniform presence of dilated intercellular space in patients with GERD.

Effects of natural winter pasture and commercial pellet on the ultrastructure of small intestinal epithelium in reindeer

Segments of small intestine (duodenum, jejunum and ileum) from slaughtered reindeer (Rangifer tarandus tarandus) grazing natural winter pastures (n=3) and reindeer fed commercially available pellets (RF-80) in winter (n=5) were collected and immediately fixed in McDowell's fixative. Transmission electron microscopy was employed to investigate the ultrastructural features of the epithelium and lamina propria along the small intestine and to relate these to the different diets. Major differences in ultrastructural features were observed between the small intestinal enterocytes of reindeer fed the two diets. Enterocytes in reindeer fed the natural diet displayed a normal appearance with a dense cytoplasm and distinct microvilli. In contrast, reindeer fed the commercial diet showed damaged enterocytes amongst the normal cells. Abnormal changes included disintegration and loss of microvilli, cytoplasmic swelling, loss of membrane integrity and increases in the width of intercellular spaces, especially in the jejunum.

Lack of Ultrastructural Detrusor Changes Following Endoscopic Injection of Botulinum Toxin Type A in Overactive Neurogenic Bladder

Endoscopical injections of Botulinum toxin type A into the detrusor muscle are gaining clinical acceptance in the treatment of neurogenic detrusor overactivity. Structural effects of Botulinum toxin type A are only known from studies on striated muscles, where a widespread nerve sprouting occurs temporarily. The aim of this study was to evaluate the ultrastructural effects of Botulinum toxin type A injections on the human detrusor. 30 detrusor biopsies were obtained from 24 patients with neurogenic detrusor overactivity. Patients were divided into two groups: Group I included 13 biopsies from patients before the first Botulinum toxin type A injection. Group II included 6 biopsies from patients within 3 months after the first injection and 11 biopsies at the time of decreasing efficacy of Botulinum toxin type A. The biopsies were processed by standard procedure for detailed electron microscopic study and evaluated by 2 examiners without prior knowledge of clinical/urodynamic data. No statistically significant detrusor changes have been found concerning muscle cell fascicle structure (p = 0.445), width of intercellular space (p = 0.482) and number/kind of muscle cell junctions (p = 0.443). A median of 70% of intrinsic axon terminals presented with signs of degeneration in group I, a median of 66% in group II (p = 0.840). Out of 309 evaluated axon terminals in both groups, 1 sprouting axon was found in group I, 3 sprouting axons in group II (p = 0.864). Specimen from group I and group II showed only limited collagen deposits within the detrusor. No changes in the ultrastructure of the detrusor have been observed in those biopsies obtained before and after the Botulinum toxin type A injection of the same patient. This study verifies our earlier report of severe intrinsic axon degeneration in the detrusor of patients with neurogenic detrusor overactivity. It also shows nearly no structural differences of the detrusor before and after Botulinum toxin type A injections. Contrary to reports of striated muscle, axonal sprouting within the detrusor was very limited after Botulinum toxin type A injections indicating pathophysiologically different reactions to the toxin either between striated muscle and smooth muscle or between different treated diseases.

Measurement of paracellular epithelial conductivity by conductance scanning.

A new method, conductance scanning, allows determination of local para- and transcellular conductivities in flat epithelia. Experiments were performed on kidney distal tubule cells, MDCK clone C11, which form monolayers on permeable supports. Above the apical surface, local voltage drops generated by a sinusoidal current clamp were recorded by means of a scanning microelectrode. Data were collected above cell centres and tight junctions. The scanning signal was always significantly higher above the tight junctions, but was uniformly distributed along the junctions. For determination of conductivities two procedures were applied. Method 1: the supraepithelial potential distribution was computed for given trans- and paracellular currents at all positions of the electrode. In a fit algorithm, the currents were varied until the calculated potential difference equalled the voltage measured. Method 2: after collecting scanning data in control Ringer's, intercellular space width was reduced by mucosal addition of 40 mM sucrose and a second set of data was obtained at decreased paracellular, but presumably unchanged transcellular, conductivity. From these data, trans- and paracellular conductivities were calculated. Results of both methods were in excellent agreement. Confluent MDCK-C11 monolayers exhibited a transepithelial conductivity of 13 mS/cm2. The transcellular pathway contributed 2.6 mS/cm2 (20%) and the paracellular pathway 10. 5 mS/cm2 (80%) to the total conductivity. Collapse of the lateral intercellular spaces decreased the paracellular conductivity to 4 mS/cm2 (60%). Confluent MDCK-C11 monolayers constitute true "leaky" epithelia with homogeneously distributed trans- and paracellular conductivities. In conclusion, conductance scanning fills a methodical gap, which hitherto impeded the functional characterization of tight junctions.

Quantitative analysis of intercellular changes in the short‐term partially obstructed rabbit detrusor

AbstractIn a previous qualitative ultrastructural study, we developed the impression that muscle cell changes following partial outlet obstruction of the rabbit bladder are associated with an increase in the width of intercellular spaces [Elbadawi et al., 1989]. In the present study, we tested that impression by direct measurement of intercellular distances using a simple method that we devised for that purpose. The material for study was obtained from 25 New Zealand White male rabbits, including three normal controls, 15 with partial bladder outlet obstruction, and seven with 7‐day obstruction followed by deobstruction. Specimens from the body detrusor were obtained from each animal and processed for electron microscopy by standard methods. Multiple sections from each sample were studied. A total of 3,889 individual measurements made in 163 electron photomicrographic prints were evaluated statistically. A statistically significant progressive increase in the width of intercellular spaces was observed over the 14‐day period of obstruction, confirming our previous qualitative impression. On the other hand, there were no significant differences in the width of spaces between the deobstructed and the 7‐day obstructed detrusor. Our observations underscore the fact that evaluation of the intercellular compartment by measuring its volume density does not reflect the true width of spaces between muscle cells of the pathologically altered detrusor. The herein verified progressively wider separation of detrusor muscle cells following partial bladder outlet obstruction probably hampers both electrotonic and mechanical coupling of muscle cells, thus preventing efficient and coordinated detrusor muscular responses to neural stimuli.

Ultrastructural changes in the obstructed pig detrusor: A preliminary report

AbstractInterest in using the pig to study the effects of urinary tract obstruction on the kidney has been established for some time. The present study was intended as a pilot investigation to find out whether or not structural changes in the detrusor occur in a chronic obstructive pig model. Because of the pilot nature of the study, and the high cost of purchase and maintenance of pigs, it was limited to one normal and one obstructed animal. Obstruction was created by allowing a young pig to grow for 7 weeks with an inactive inflatable urethral cuff in place. Samples from the bladder body detrusor were obtained from this and from a normal pig. These were then processed for electron microscopy by standard procedures. Multiple sections of each sample were examined and photographed to determine the structural status of muscle cells, the width of intercellular spaces and the relative area of muscle cell junctions. In samples from the obstructed pig, we found (1) no evidence of degeneration or regeneration of muscle cells; (2) hypertrophy of detrusor muscle cells; (3) widening of intercellular spaces (from 237 nm in the normal to 503 nm in the obstructed pig); (4) intercellular deposits of basal lamina‐like material; and (5) reduction of junctional sarcolemmal area relative to total sarcolemmal area as well as total muscle cell volume. All morphometric data were statistically significant. The observed reduction of muscle cell junctional area may be a “dilution” effect secondary to muscle cell hypertrophy, and/or represent a true loss of junctions. Despite the small sample size, our findings suggest that ultrastructural obstructive detrusor changes occur and indicate the need for further investigation to define the scope and chronologic evolution of these changes.

Ultrastructure of the tubular nephron of the lizard Podarcis (= Lacerta) Taurica.

The proximal, intermediate, and distal convoluted tubules of the neprhon of Podarcis (= Lacerta) taurica were examined by electron microscopy. Proximal tubule cells have large, apical cytoplasmic protrusions and microvilli interpreted to function in urate secretion. Adjacent cells are bound apically by tight junctions and desmosomes but interdigitate in their basal region. This situation is repeated in the other tubules with significant differences in intercellular space width. The basal surfaces bear numerous cytoplasmic processes. The intermediate tubule has proximal and distal segments each with dark, ciliated, and light cells, the cuboidal dark cells with dense cytoplasm constituting the main bulk of the wall. As the cells of the proximal and distal segments resemble those of the proximal and distal convoluted tubules, respectively, the intermediate tubule is considered as a transition region. The ciliated cell body has two broad processes extending from the lumen, one to the basement membrane and one to a foot process of a light cell. The light cell is surrounded by dark and ciliated cells. It does not reach the lumen, but contacts the basement membrane through a process running below a ciliated cell to form a mushroom-shaped structure in tubule cross-section, the light cell process forming the stalk and a ciliated cell the cap. The cilia probably propel the glomerular filtrate towards the distal convoluted tubule. This latter tubule has initial, middle, and terminal zones, all nonciliated but with different lumen widths and cell shapes.

Cell contacts in the kidney epithelium of Helisoma (mollusca: gastropoda)—Effects of osmotic pressure and brain extracts: A freeze-fracture study

Kidney epithelial cell contacts of Helisoma have been studied under different osmotic conditions (hyposmotic, isosmotic, control). In hyposmotic medium 20% of the observed zonulae adherentes appear opened, and septate junctions appear short with few septa or long with many septa showing irregular alignment and periodicity; intercellular spaces are wide. Snails from isosmotic medium show long septate junctions with compact regular septal periodicity and greatly reduced intercellular spaces. The length of septate junctions and the number of septa appear to be related to intercellular space width. Lanthanum-impregnated septa appear pleated and punctate. In freeze-fracture replicas septate junctions from hyposmotic medium have fewer rows or irregular sinuous rows of junctional particles. Osmotically induced changes in the junctional complexes are reversible. In vitro treatment with crude brain extract dramatically increases intercellular space width with significant changes in the junctional complexes. It is proposed that the septate junctions are involved in osmoregulation and a brain factor may regulate them. Gap junctions are found in various places in the kidney cell membranes; they may provide additional structural integrity in the epithelium. Rhombic particle arrays and desmosome-like particles have been observed in freeze-fracture replicas but their identity is unknown and no clear correlation for these particles in thin sections has been possible.

Osmotic water flow in leaky epithelia

I review three currently unsolved and controversial problems in understanding solute-linked water transport in epithelia. 1. Values of osmotic water permeability (Posm) calculated from steady-state osmotic flow in response to a gradient of a probe molecule tend to be underestimates, because of three unstirred-layer (USL) effects. These are: dissipation of the probe's gradient by diffusion in USL's; reduction of the probe's gradient, due to the sweeping-away effect of water flow generated by the probe itself; and solute polarization (creation of an opposing gradient of an initially symmetrically distributed solute by the sweeping-away effect). These errors increase with probe permeability, USL thickness, Posm, and concentration ratio of symmetrically distributed solute to probe, and vary inversely as the fractional area available for water flow (e.g., lateral intercellular space width). The form of an osmotic transient, and the possibility of extracting a true Posm value from the transient, depend on the relative values of three time constants: those for solute diffusion in USL's, for solute polarization by water flow in USL's and for measuring water flow. Sweeping-away effects cause major underestimates (by one or more orders of magnitude) in epithelial Posm determinations, as shown by apparent streaming potentials during osmotic flow and by transiently reversed flows after removal of the proble. True Posm values for leaky epithelia probably exceed 10(-3) or 10(-2) cm/sec.osm. The necessary conditions for resolving osmotic transients are set out. 2. I illustrate the difficulties in deciding what fraction of transepithelial water flow is via the cells, and what fraction via the junctions. There is no existing method for answering this question. 3. Controversies about the validity, or need for modification, of the standing-gradient theory are discussed. Progress in this field requires new methods: to resolve osmotic transients; to separate transcellular and transjunctional water flows; and to measure solute concentrations in lateral intercellular spaces directly.