Modified Ussing Chamber Research Articles

The Influence of Retinol Ointment on Rabbit Skin (Oryctolagus cuniculus) Ion Transport-An In Vitro Study.

Retinoids are known to improve the condition of the skin. Transepithelial transport of sodium and chloride ions is important for proper skin function. So far, the effect of applying vitamin A preparations to the skin on ion transport has not been evaluated. In the study, electrophysiological parameters, including transepithelial electric potential (PD) and transepithelial resistance (R), of rabbit skin specimens after 24 h exposure to retinol ointment (800 mass units/g) were measured in a modified Ussing chamber. The R of the fragments incubated with retinol was significantly different than that of the control skin samples incubated in iso-osmotic Ringer solution. For the controls, the PD values were negative, whereas the retinol-treated specimens revealed positive PD values. Mechanical-chemical stimulation with the use of inhibitors of the transport of sodium (amiloride) or chloride (bumetanide) ions revealed specific changes in the maximal and minimal PD values measured for the retinol-treated samples. Retinol was shown to slightly modify the transport pathways of sodium and chloride ions. In particular, an intensification of the chloride ion secretion from keratinocytes was observed. The proposed action may contribute to deep hydration and increase skin tightness, limiting the action of other substances on its surface.

Hydraulic conductivity of human cancer tissue: A hybrid study.

Elevated tumor tissue interstitial fluid pressure (IFP) is an adverse biomechanical biomarker that predicts poor therapy response and an aggressive phenotype. Advances in functional imaging have opened the prospect of measuring IFP non-invasively. Image-based estimation of the IFP requires knowledge of the tissue hydraulic conductivity (K), a measure for the ease of bulk flow through the interstitium. However, data on the magnitude of K in human cancer tissue are not available. We measured the hydraulic conductivity of tumor tissue using modified Ussing chambers in surgical resection specimens. The effect of the tumor microenvironment (TME) on K was investigated by quantifying the collagen content, cell density, and fibroblast density of the tested samples using quantitative immune histochemistry. Also, we developed a computational fluid dynamics (CFD) model to evaluate the role of K on interstitial fluid flow and drug transport in solid tumors. The results show that the hydraulic conductivity of human tumor tissues is very limited, ranging from approximately 10-15 to 10-14 m2/Pa∙s. Moreover, K values varied significantly between tumor types and between different samples from the same tumor. A significant inverse correlation was found between collagen fiber density and hydraulic conductivity values. However, no correlation was detected between K and cancer cell or fibroblast densities. The computational model demonstrated the impact of K on the interstitial fluid flow and the drug concentration profile: higher K values led to a lower IFP and deeper drug penetration. Human tumor tissue is characterized by a very limited hydraulic conductivity, representing a barrier to effective drug transport. The results of this study can inform the development of realistic computational models, facilitate non-invasive IFP estimation, and contribute to stromal targeting anticancer therapies.

30-Min Exposure to Tobacco Smoke Influences Airway Ion Transport-An In Vitro Study.

Introduction: Smoking is one of the most important causes of cancer in humans. However, it has not been proven how long exposure to cigarette smoke is sufficient to induce cancerogenesis. Cigarette smoke can cause changes in ion and water transport and the maintenance of mucociliary transport. The conducted research concerned the assessment of changes in ion transport in rabbit tracheal specimens after 30 min of exposure to cigarette smoke. Materials and Methods: A modified Ussing chamber was used to measure the transepithelial electrical potential under stationary conditions (PD) and during mechanical stimulation (PDmin), and the transepithelial electrical resistance (R) in control and cigarette smoke-exposed tracheal fragments. Results: Significant changes in PD (-2.53 vs. -3.92 mV) and PDmin (-2.74 vs. -0.39 mV) were noted for the samples exposed to smoke, which can be associated with a rise in reactivity after applying a mechanical stimulus. In addition, the measured R (108 vs. 136 Ω/cm2) indicated no changes in the vitality of the samples, but an increase in their permeability to ions in the experimental conditions. Conclusions: A single 30-min exposure to cigarette smoke has been shown to be associated with increased permeability of the tracheal epithelium to ions and thus to substances emitted during smoking, which might be sufficient to create the possibility of initiating procarcinogenic processes.

Nickel ions influence the transepithelial sodium transport in the trachea, intestine and skin

Measurements of transepithelial potential and resistance in tissue and organ model systems enable the evaluation of the Ni2+ effect on the epithelial sodium channels, aquaporin 3, and the sodium–potassium pump in the epithelial cells. The aim of the presented study was to assess the immediate and prolonged effect of nickel ions on the transport of sodium ions in tissues exposed to direct contact with nickel, including airways, digestive tract and the skin. The influence of 0.1 mM nickel solution was performed on the trachea (n = 34), intestine (n = 44), and skin (n = 51) samples descended from 16 New Zealand albino rabbits. The electrophysiological parameters were measured in a modified Ussing chamber in stationary conditions and during a 15-s mechanical-chemical stimulation. A statistically significant decrease in the electric resistance values and the smallest range of the measured potential were observed for the Ni-treated trachea specimens. The use of nickel solution did not affect the sodium transport in the intestine epithelium. The skin fragments showed altered sodium ion transport, as demonstrated by the lower range and intensity of the measured potential. The gastrointestinal tract seems to be an organ best adapted to contact with nickel ions. In airways, nickel ions most likely enter epithelial cells and the space between them, modifying proteins and the airway surface liquid. The skin turned out to be the most sensitive tissue to the intensification of sodium ion transport through nickel ions.

The Impact of Diclofenac Gel on Ion Transport in the Rabbit (Oryctolagus cuniculus) Skin: An In Vitro Study.

Diclofenac belongs to the non-steroidal anti-inflammatory drugs with analgesic, antipyretic and anti-inflammatory effects. Diclofenac administration on the skin may be associated with the appearance of side effects. The study aimed to evaluate the impact of diclofenac gel on transepithelial electrophysiological parameters of the 55 rabbit abdomen skin specimens. The electric parameters were analyzed in a modified Ussing chamber. The resistance (R) of the skin specimens treated with diclofenac gel significantly increased, which could be related to the reduction in the water content in intercellular spaces and, consequently, tighter adhesion of the cells. Increased electric potential (PD) was also observed in the skin specimens treated with diclofenac gel. The increase in both R and PD measured under stationary conditions was most likely caused by a transient and reversible increase in sodium ion transport, as the R and PD values decreased after the diclofenac gel was washed away. However, diclofenac gel did not affect the maximum and minimum PDs measured during stimulations. Therefore, it seems that diclofenac gel does not affect the perception of stimuli in the model system used.

Impact of a 24 h feed withdrawal on active nutrient transport, intestinal morphology and gene expression in the equine small and large intestine

Abstract Horses are often subjected to short term feed withdrawal (FW) pre- or post-surgery to reduce anesthetic complications. However, removing nutrients from the intestinal lumen may negatively impact intestinal health. Thirteen horses were used to determine the effects of a 24 h FW on gut barrier function, active nutrient transport, transporter gene expression and intestinal morphology. Following 0 or 24 h FW (0FW or 24FW, respectively), horses were euthanized via overdose of sodium- pentobarbital and –phenytoin, and segments of proximal jejunum (PJ), mid jejunum (MJ), ileum (Il) and right ventral colon (RVC) were harvested for histology (PJ and Il), gene expression, and active nutrient transport analysis. Active transport measurements were determined using modified Ussing chambers following the addition of glucose, phosphorus, glutamine and Gly-Sar. Carbachol induced chloride (Cl) ion secretion was measured to examine the diarrhetic response. Messanger RNA expression of the intestinal Na-dependent glucose cotransporter (SGLT1), fructose transporter (GLUT5), di- and tri-peptide transporter (PepT1) and neutral AA/glutamine transporter (ASCT2) were determined using RT-PCR. The GLM procedure of SAS was used to determine the effects of FW and responses among various intestinal sections. The horse served as the experimental unit. Villus heights (P < 0.002) and crypt depths (P < 0.02) in the Il were larger than in the PJ, though no differences were observed between 0FW and 24FW horses. Active glutamine absorption increased 82% in the PJ of 24FW horses compared to 0FW horses (P < 0.02). The mRNA expression of SGLT1 decreased (P < 0.05) moving aborally in the gastrointestinal tract. Horses subjected to 24FW had 82% less GLUT5 (P < 0.05) and 61% less PepT1 mRNA expression in the PJ, compared to 0FW horses. Interestingly, ASCT2 mRNA expression increased 164% from PJ to RVC (P = 0.05). However, a 36% decrease in ASCT2 mRNA expression was observed overall for 24FW horses. This data indicates that SGLT1, GLUT5, PepT1 and ASCT2 are expressed throughout the small intestine and RVC of the horse at varying concentrations, and that they can be differentially regulated by a 24 h FW. Data from this experiment also indicates that a 24 h FW results in up regulation of active glutamine absorption, presumably in an effort to supply glutamine as an energy substrate for enterocytes.

Acute psychological stress increases paracellular permeability and modulates immune activity in rectal mucosa of healthy volunteers.

Psychological stress and increased permeability are implicated as contributing factors in the initiation and worsening of gastrointestinal diseases. A link between stress and intestinal permeability has been shown in animal models as well as in human small intestine, but stress effects on the human colorectal mucosal barrier has not been reported. To investigate the potential effects of acute psychological stress on colorectal mucosal barrier function and to explore stress-induced molecular events in the rectal mucosa under healthy conditions. Endoscopic biopsies were taken from the rectosigmoid region of healthy volunteers, who had been subjected to dichotomous listening stress and after a control session, respectively. Paracellular and transcellular permeability were assessed in modified Ussing chambers. RNA expression (microarray technology confirmed by quantitative real-time polymerase chain reaction) and biological pathway analysis were used to investigate the local mucosal response to acute stress. Dichotomous listening stress induced a subjective and objective stress response, and significantly increased paracellular but not transcellular permeability. We also identified a stress-induced reduction in RNA expression of genes related to immune cell activation and maturation (CR2, CD20, TCLA1, BANK1, CD22, FDCSP), signaling molecules of homing of immune cells to the gut (chemokines: CCL21, CXCL13, and CCL19, and receptors: CCR7, CXCR5), and innate immunity (DUOX2). Eight of the 10 top down-regulated genes are directly involved in B cell activation, signaling and migration. The systemic stress response correlated positively with paracellular permeability and negatively with DUOX2 expression. Dichotomous listening stress increases paracellular permeability and modulates immune cell activity in the rectal mucosa. Further studies are warranted to identify the primary mechanisms of stress-mediated reduction of mucosal defensive activity and barrier dysfunction, and their potential implications for gastrointestinal disorders.

268 In-feed Antibiotics Elicit Intestinal Integrity Modifications Early in Post-weaning Life

Abstract The physiological mechanisms by which in-feed antibiotics improve pig growth performance are largely unknown. One proposed mode of action is improvements in intestinal integrity and function. Therefore, the objective of this study was to test the hypothesis that in-feed therapeutic and sub-therapeutic antibiotics would improve intestinal integrity and function in nursery pigs. Twenty-four weaned pigs (6.1±1.1 kg BW) were randomly allotted to individual pens and assigned one of three dietary treatments as follows (n = 8 pigs/trt): 1) control, no antibiotics (CON), 2) CON + sub-therapeutic chlortetracycline [40 ppm in feed (sCTC)], and 3) CON + chlortetracycline-tiamulin [400 ppm + 35 ppm, respectively (CTCDen)]. The study consisted of two consecutive 14 d phases. Chlortetracycline-tiamulin was only fed in phase 1, sCTC was fed in both phases. Phase 1 and 2 ADG, ADFI, and G:F were determined. After 28 d, ileal and colonic ex vivo intestinal integrity was assessed via transepithelial resistance (TER) and macromolecule flux (FD4) in modified Ussing chambers. All data were analyzed for the fixed effects of treatment and start BW as a covariate. In phase 1, compared with CON and sCTC, CTCDen tended to have greater ADG (0.28, 0.31, and 0.33 kg/d, respectively, P = 0.10) and ADFI (0.28, 0.30, and 0.35 kg/d, respectively, P = 0.09). No differences in phase 1 G:F were observed (P = 0.11). Phase 2 ADG, ADFI, and G:F did not differ (P > 0.10). Further, ileal TER and FD4 did not differ (P > 0.10). Colonic TER tended to be increased in sCTC compared with CON and CTCDen (78, 56, and 59 Ω/cm2, respectively, P = 0.07). Compared with CON, colonic FD4 flux was decreased in sCTC and CTCDen by 35–40% (P = 0.03). Altogether, these data indicate that in-feed antibiotics improve colon integrity early in production which may contribute to improved growth performance.

271 Investigating the Relationship Between Nursery Pig Performance and Markers of Intestinal Morphology and Integrity

Abstract Weaning induces major structural and function changes to the small intestine of pigs and they transition from milk to solid feedstuffs. Thus, the objective of this study was to determine how intestinal morphology and function markers relate to feed intake and growth rates of nursery pig. Forty-eight weaned pigs (5.63 ± 0.50 kg) were randomly selected, individually penned and fed a common diet. Pig bodyweights and feed intake were determined at d 2, 7, and 21. At each time point, 16 pigs were randomly selected and euthanized. Sections of ileum were assessed for morphology [villus height (VH), crypt depth (CD) and VH:CD] and ex vivo transepithelial resistance (TER), macromolecule permeability (FD4), and active transport of glucose and glutamine via modified Ussing chambers. Within each period (d 0–2, 0–7, and 0–21), Pearson correlations were performed between ADG, ADFI, VH, VH:CD, TER, FD4 and active transport of glucose and glutamine. At d 2 post-weaning, no correlations (P > 0.05) were observed between performance and intestinal variables. By d 7, moderate positive correlations between VH and ADFI (r = 0.69, P = 0.005), VH and ADG (r = 0.68, P = 0.006) were reported. At 21 d post-weaning, moderate positive correlations were still observed for VH and ADFI (r = 0.55, P = 0.026) and between VH and ADG (r = 0.51, P = 0.042). Interestingly, ADFI and ADG tended to be negatively correlated with active glucose transport (r = -0.45, P = 0.083 and r = -0.47, P = 0.064, respectively) and active glutamine transport (r = -0.45, P = 0.083 and r = -0.46, P = 0.073, respectively). Markers of ileal integrity (TER and FD4) were not correlated with ADG or ADFI at any time point. Altogether, these data highlight the importance of intestinal morphology on early nursery pig performance.

Basolateral activation with TLR agonists induces polarized cytokine release and reduces barrier function in RPE in vitro

PurposeSystemic inflammation may be of importance in the development of AMD. RPE cells can recognize danger signals with toll-like receptors (TLR) and may react in a pro-inflammatory manner. In this study, we evaluated the basal and apical secretions of TNFα, IL-6, and IL-1β in primary RPE cells and RPE/choroid explant cells under basolateral stimulation of TLR2, 3, and 4; the effects on barrier function; and their influence on neuronal cell viability.MethodsRPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers; primary porcine RPE cells on transwell plates. Cells were basally stimulated with agonists Pam2CSK4 (Pam; TLR2), polyinosinic/polycytidylic acid (Poly I:C; TLR3), and lipopolysaccharide (LPS; TLR4) for 24 h. Supernatants were evaluated with ELISA for cytokines TNFα, IL-6, and IL-1β. Apical supernatants were applied to SHSY-5Y cells, and cell viability was evaluated in MTT assay. Barrier function was tested by measuring transepithelial electrical resistance (TER) and occludin immunostaining.ResultsNone of the tested TLR agonists was toxic on RPE cells after 24 h of exposure. Unstimulated RPE cells secreted hardly any cytokines. Pam induced IL-6, IL-1ß, and TNFα on the basal and apical sides at all concentrations tested. Poly I:C induced IL-6 and TNFα primarily at the basal side at lower but on both sides at higher concentrations. LPS induced IL-6, IL-1ß, and TNFα apically and basally at all concentrations tested. In the RPE/choroid, a strong difference between apical and basal secretions could be found. IL-6 was constitutively secreted basally, but not apically, but was induced by all agonists on both sides. IL-1ß and TNFα alpha were strongly induced on the basal side by all agonists. TER was reduced by all agonists, with Pam and LPS being effective in all concentrations tested. Occludin expression was unaltered, but the distribution was influenced by the agonists, with a less distinct localization at the cell borders after treatment. None of the agonists or supernatants of treated RPE and RPE/choroid organ cultures exerted any effect on viability of SHSY-5Y cells.ConclusionsDanger signals activating TLRs can induce polarized cytokine expression and contribute to the loss of barrier function in the RPE.

Response of Retinal Pigment Epithelium (RPE)-Choroid Explants to Thermal Stimulation Therapy of the RPE (TSR).

The thermal stimulation therapy of the retinal pigment epithelium (TSR) is a sublethal laser technique for thermal stimulation of the retinal pigment epithelium (RPE)-Bruch's membrane (BrM)-complex. The aim of this study was to investigate the influence of TSR on the release of age-related macular degeneration (AMD)-relevant cell mediators. Porcine RPE-BrM-choroid explants were irradiated with a 532 nm continuous wave laser using different spot sizes (100-300 µm, duration 100 milliseconds, 15-100 mW). Cell death was investigated by calcein staining. Explants were treated with grids of sublethal spots and cultivated in modified Ussing chambers. The effect on matrix metalloproteinase-2 (MMP-2) and -9 was investigated by zymography and quantitative reverse transcription polymerase chain reaction. Secretion of vascular endothelial growth factor (VEGF), pigment epithelium derived factor (PEDF), and transforming growth factor-β (TGF-β) was analyzed by enzyme-linked immunosorbent assay and expression of HSP70 was examined by western blot. Integrity of the RPE/BrM-complex was analyzed by scanning electron microscopy. Laser powers of 15 mW (100 µm) and 45 mW (300 µm) did not induce RPE cell death. The integrity of the RPE/BrM-complex was not impaired after TSR. After TSR with 300 µm spot size, we observed a significant increase of active MMP-2 in the basal compartments. The content of PEDF significantly increased in treated explants in both compartments with 100 and 300 µm spot sizes. VEGF and TGF-β secretion was not triggered by TSR. TSR represents a possible RPE stimulating treatment for dry AMD. TSR increases the basal release of active MMP-2, which might reverse age-related thickening of BrM. VEGF secretion was not triggered by TSR while anti-angiogenic PEDF was increased, indicating an induction of an anti-angiogenic and neuroprotective environment. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Altered Structural Expression and Enzymatic Activity Parameters in Quiescent Ulcerative Colitis: Are These Potential Normalization Criteria?

Mucosal healing determined by endoscopy is currently the remission standard for ulcerative colitis (UC). However, new criteria for remission are emerging, such as histologic normalization, which appears to correlate better to the risk of relapse. Here, we study mucosal healing on a molecular and functional level in quiescent UC. We obtained endoscopic biopsies from 33 quiescent UC patients and from 17 controls. Histology was assessed using Geboes score. Protein and mRNA levels were evaluated for the tight junction proteins claudin-2, claudin-4, occludin, and tricellulin, as well as Cl−/HCO3− exchanger DRA, and cyclo-oxygenase enzymes (COX-1, COX-2). The mucosal activity of COX-1 and COX-2 enzymes was assessed in modified Ussing chambers, measuring electrogenic ion transport (short-circuit current, SCC). Chronic inflammation was present in most UC patients. The protein level of claudin-4 was reduced, while mRNA-levels of claudin-2 and claudin-4 were upregulated in UC patients. Surprisingly, the mRNA level of COX-1 was downregulated, but was unaltered for COX-2. Basal ion transport was not affected, while COX-2 inhibition induced a two-fold larger decrease in SCC in UC patients. Despite being in clinical and endoscopic remission, quiescent UC patients demonstrated abnormal mucosal barrier properties at the molecular and functional level. Further exploration of mucosal molecular signature for revision of current remission standards should be considered.

Ussing Chamber Methods to Study the Esophageal Epithelial Barrier.

The Ussing chamber was developed in 1949 by Hans Ussing and quickly became a powerful tool to study ion and solute transport in epithelia. The chamber has two compartments strictly separating the apical and basolateral sides of the tissue under study. The two sides of the tissue are connected via electrodes to a modified electrometer/pulse generator that allows measurement of electrical parameters, namely, transepithelial voltage, current, and resistance. Simultaneously, permeability of the tissue to specific solutes or markers can be monitored by using tracers or isotopes to measure transport from one side of the tissue to the other. In this chapter, we will describe the use of the Ussing chamber to study the barrier properties of the mouse esophageal epithelium. We will also briefly describe the use of the modified Ussing chamber to simultaneously study transepithelial and cellular electrophysiology in the rabbit esophageal epithelium. Lastly, we will cover the use of the Ussing chamber to study bicarbonate secretion in the pig esophagus. These examples highlight the versatility of the Ussing chamber technique in investigating the physiology and pathophysiology of epithelia including human biopsies.

English

Toad urothelium barrier is the model to mimic and investigate urothelium permeability. Thiazide blocked ionic transportation in polarized membrane state. Jellyfish venom causes pores to be adjusted to urothelium permeability which improved polarization. This study aimed at CfTX-1 peptide in urothelium permeability evoked polarizations. Thiazide pretreated toad urothelium permeability to ions were investigated in modified Ussing chamber. Thiazide urothelium were further intervened by CfTX-1 peptide and treated with G-protein receptor agonists. 0.1 mol CaCl2 activated transurothelium potential differences were recorded by unipolar lead and computerized by fast Fourier transform technique. Apical chamber was settled as anode. The amplitude of potential differences were evaluated to determine the urothelium polarizations. The results indicated that CaCl2 activation induced a positive monophasic wave in thiazide urothelium, which suggested the urothelium was slightly polarized and significantly enhancive in adrenergic receptor treated urothelium. Furthermore, CfTX-1 peptide enhanced transurothelium potential difference in thiazide urothelium, therefore, urothelium were supra polarized. NPPB treatment significantly attenuated this supra polarization, which suggested that the Cl- influx was the main stream ionic compound of this polarization. It is concluded that CfTX-1 peptide was considered to generate supra polarization in thiazide urothelium. This mechanism is useful to study the improvement of drug delivery crossing urothelium barrier. Key words: CfTX-1 partial sequence, toad urothelium, supra polarization, Cl- permeability.

PSIV-18 Effect of sub-therapeutic antibiotics and pharmacological zinc oxide on nursery pig performance and intestinal integrity and function

Abstract The objective of this study was to evaluate the impact of sub-therapeutic chlortetracycline (CTC) and pharmacological zinc oxide on nursery pig performance and intestinal integrity and function. Twenty-four newly weaned pigs (4.51 ± 0.21 kg BW) were randomly assigned to one of three dietary treatments for 21 d (n = 8/treatment): 1) Control diet (NC), 2) NC + Zinc oxide at 3000 ppm for 7 d followed by 2000 ppm for 14 d (ZnO), and 3) NC + 40 ppm CTC for 21 d (sCTC). Pig ADG, ADFI, and G:F were determined for the overall 21 d test period. At d 21, all pigs were euthanized for tissue collection. Jejunum and ileum ex vivo transepithelial resistance (TER), macromolecule (FD4) flux, and active glucose and glutamine transport were assessed in modified Ussing Chambers. Additionally, jejunum brush border enzyme and Na+/K+ ATPase activities were determined. Morphology and T-cell abundance were determined in all ileum sections by H&E staining and immunohistochemistry, respectively. Compared to the NC, sCTC and ZnO increased ADG (0.14, 0.20 and 0.23 kg/d, respectively, P = 0.020). Overall ADFI was increased by 150% due to sCTC compared to the NC and ZnO pigs (P < 0.05). G:F was increased due to ZnO and sCTC compared to the NC (0.82, 0.63 and 0.47, respectively, P < 0.01). Jejunum and ileum ex vivo function and integrity did not differ. Ileal villus height was increased in the NC compared to sCTC treatment (P < 0.01), but not different from the ZnO treatment. In the ileum, total T-cell number per unit area was significantly lower in the ZnO segments compared to sCTC and NC (0.95, 2.29 and 2.59 count per μm2, respectively, P = 0.05). These data indicate that ZnO and sCTC improved pig performance but did not directly alter small intestinal integrity and function. However, mucosal T cell abundance was altered.

364 Impact of porcine reproductive and respiratory syndrome virus challenge and deoxynivalenol on intestinal integrity and nutrient transport

Abstract Porcine reproductive and respiratory syndrome (PRRS) virus infections and mycotoxin contaminated feedstuffs are two common issues facing the pork industry as both act, independently, to antagonize pig intestinal function, growth performance, and health. However, the combined impact of PRRS and mycotoxin exposure on pig intestinal health and function has not been investigated. Therefore, the objective of this experiment was to examine the impact of the mycotoxin deoxynivalenol (DON) on the intestinal integrity and function of PRRS infected pigs, employing an ex vivo model. Jejunum samples were collected from PRRS naïve (control; n = 6) and 21 day post inoculated PRRS virus infected (n = 8) gilts (10 weeks of age). In duplicate, explants were mounted into modified Ussing Chambers and incubated with or without 25 mM DON for 45 minutes prior to assessment of transepithelial resistance (TER), mucosal to serosal macromolecule (FD4) flux, and active glucose and glutamine transport. Data were analyzed using the mixed model procedure of SAS and a split plot design to examine the fixed effects of PRRS, DON, and their interaction. There were no PRRS by DON interactions for any ex vivo parameters assessed. Irrespective of DON, PRRS jejunum segments had decreased TERs (57%, P < 0.001), increased FD4 flux (P = 0.005), and tended to have increased active glucose transport (P = 0.093) compared with uninfected controls. Irrespective of PRRS status, DON increased FD4 flux (113%, P = 0.013), and tended to increase active glucose transport (P = 0.088) compared with jejunal explants not treated with DON. However, TERs and active glutamine transport did not differ due to DON treatment. Taken together, these data highlight the impacts that viral health challenge and mycotoxin contamination have on aspects of intestinal integrity and function. However, it does not appear that PRRS challenged pigs are more sensitive to the effects of DON than their healthy counterparts.

Effect of Selective Retina Therapy (SRT) on the Inflammatory Microenvironment of the Subretinal Space

To investigate the effect of Selective Retina Therapy (SRT) on inflammatory key factors such as complement factor-C3 (CC3), tumor growth factor-beta2 (TGF-β2), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). Porcine RPE-Bruch's membrane-choroid explants were irradiated with two SRT laser systems, SRTYLF and SRTYAG (Nd : YLF laser, wave length 527 nm, pulse duration 1.7 µs and Nd : YAG laser, wave length 532 nm, pulse duration 2.4 - 3 µs). Laser irradiation was performed on a spot size of 200 × 200 µm, 30 pulses, with a repetition rate of 100 Hz, and a radiant exposure of 140 (threshold RPE death) and 180 mJ/cm2 per pulse (above threshold RPE death). Explants were cultivated in modified Ussing chambers and culture viability was assessed by calcein-AM cell staining. Secretion of inflammatory factors was analyzed by ELISA. Protein expression of tissue explants was assessed by Western blot. Regeneration of RPE was observed after 4 days. One day after SRT with 140 mJ/cm2 per pulse the secretion of basal CC3 decreased in ELISA. Following 180 mJ/cm2 radiant exposure, the level of IFN-γ decreased at day 4. SRT does not induce the release of the pro-inflammatory factors analyzed in this in-vitro study.

Mycoplasma hyopneumoniae-Lawsonia intracellularis dual challenge modulates intestinal integrity and function1.

Lawsonia intracellularis (LI) and Mycoplasma hyopneumoniae (Mh) are 2 globally distributed pathogens that cause significant morbidity and mortality in grow-finish pigs. However, mechanisms that reduce growth and feed efficiency during LI and Mh infection are poorly defined. We hypothesized that reductions in performance are partially due to declines in intestinal function and integrity; thus, this study aimed to evaluate intestinal function and integrity of pigs during a 21-d Mh and LI dual challenge (MhLI). Littermate pairs of barrows (48.1 ± 6.7 kg BW) were selected; 1 pig from each pair was assigned to either MhLI challenge or nonchallenge treatments (n = 12). Pigs were individually housed, fed a corn-soybean diet, and allowed to acclimate for 21 d prior to inoculation. On days postinoculation (dpi) 0, MhLI pigs were dual inoculated with LI and Mh. On dpi 21, all pigs were euthanized for ileal and colon tissue collection. Formalin-fixed tissues were clinically scored and morphology analyzed, frozen tissues assayed for digestive enzyme activities, and fresh tissues mounted into modified Ussing Chambers to assess active nutrient transport, barrier integrity, and bacterial translocation. Data were analyzed using the Mixed Procedure of SAS with treatment as a fixed effect, age and start BW as covariates, and litter as a random effect. Compared with controls, MhLI pigs had decreased ADG (38%, P < 0.001), ADFI (25%, P < 0.001), and G:F (19%, P = 0.012). The MhLI dual challenge did not alter ileum morphology or transepithelial resistance (P > 0.10); however, ex vivo mucosal to serosal translocation of S. Typhimurium in the colon was increased (60%, P = 0.003) in MhLI pigs compared with controls. Additionally, MhLI pigs had increased ileal glucose transport (30%, P = 0.05) and decreased sucrase activity (30%, P = 0.049) compared with controls. This MhLI challenge antagonized intestinal function and integrity, and this may be a contributing factor to reduced pig performance.

The effect of dietary vitamin D supplementation on sodium-dependent phosphate uptake and expression of NaPi-IIb in the small intestine of weanling pigs.

Two experiments were conducted to investigate the effects of 1,25(OH)2D3 to stimulate Na+-dependent phosphate uptake in Caco-2 cells, and the effects of dietary vitamin D supplementation to vitamin D-deficient nursery pigs on Na+-dependent nutrient uptake and mRNA expression of NaPi-IIb cotransporter and calbindin D(9k) in the jejunum. In Exp. 1, 250,000 Caco-2 cells were seeded on Costar 12 mm Snapwell inserts with a 0.40 µm polycarbonate filter and a seeding density of 0.25 × 106 and studied at 15 d postconfluence. Cells were treated with 10 nM of either 1,25(OH)2D3 or vehicle for 48 h and then mounted in modified Ussing chambers for transepithelial measurements. In Exp. 2, pigs (n=32) were removed from sows at 3 d of age, placed on a vitamin D-deficient milk replacer diet and housed in a room devoid of sunlight and UV light in the range of 280 to 300nm. On d 28, serum 25(OH)D3 concentrations were measured to verify low vitamin D status. Pigs (BW 10.10 ± 0.38 kg) were then individually housed d 28 post-weaning and allotted to 1 of 2 dietary treatments. Dietary treatments consisted of corn-soybean based diets with vitamin D supplementations of 0 or 1,500 IU/kg diet for 12 d. Blood samples were taken from the brachiocephalic vein on the initial (d 0) and final day (d 10,11, or 12) of the study for analysis of serum 25(OH)D3 , P, and Ca. Pigs were euthanized and jejunal segments were harvested and used in modified Ussing chambers and for RNA isolation and subsequent quantitative RT-PCR analysis. In Exp. 1, treating Caco-2 cells with 10 nM 1,25(OH)2D3 resulted in a 52% increase (P < 0.005) in Na+-dependent phosphate uptake compared with cells treated with a vehicle. In Exp. 2, Na+-dependent phosphate and glucose transport did not differ (P > 0.10) among treatment groups. Additionally, NaPi-IIb and calbindin D(9k) mRNA expression were not different (P > 0.10) between treatment groups. No differences (P > 0.10) were detected in final serum P or 25(OH)D3 concentrations between treatments. However, serum Ca linearly increased (P < 0.05) as the concentration of supplemental vitamin D increased in the diet. Overall, while 1,25(OH)2D3 stimulated Na+-dependent phosphate uptake in Caco-2 cells, supplementing diets with 1,500 IU/kg Vitamin D3 from cholecalciferol did not increase jejunal Na+-dependent phosphate uptake or NaPi-IIb mRNA expression over that of pigs fed diets with no supplemental cholecalciferol.

Electrophysiological evidence for light-activated cation transport in calcifying corals.

Light has been demonstrated to enhance calcification rates in hermatypic coral species. To date, it remains unresolved whether calcifying epithelia change their ion transport activity during illumination, and whether such a process is mediated by the endosymbiotic algae or can be controlled by the coral host itself. Using a modified Ussing chamber in combination with H+ sensitive microelectrode measurements, the present work demonstrates that light triggers the generation of a skeleton positive potential of up to 0.9 mV in the hermatypic coral Stylophora pistillata. This potential is generated by a net flux of cations towards the skeleton and reaches its maximum at blue (450 nm) light. The effects of pharmacological inhibitors targeting photosynthesis 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and anion transport 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) were investigated by pH microelectrode measurements in coral tissues demonstrating a rapid decrease in tissue pH under illumination. However, these inhibitors showed no effect on the electrophysiological light response of the coral host. By contrast, metabolic inhibition by cyanide and deoxyglucose reversibly inhibited the light-induced cation flux towards the skeleton. These results suggest that ion transport across coral epithelia is directly triggered by blue light, independent of photosynthetic activity of algal endosymbionts. Measurements of this very specific and quantifiable physiological response can provide parameters to identify photoreception mechanisms and will help to broaden our understanding of the mechanistic link between light stimulation and epithelial ion transport, potentially relevant for calcification in hermatypic corals.