α-Lipoic acid blocks the GMCSF induced protease/protease inhibitor spectrum associated with fetal membrane weakening in-vitro.

INTRODUCTION: We use an in-vitro human fetal membrane (FM) explant-based model to study inflammation-induced FM weakening, a prerequisite for PPROM. In this system, GMCSF is a critical intermediate, both necessary and sufficient for TNFα and thrombin induced FM weakening. α-Lipoic-acid (LA) blocks TNFα and thrombin, as well as GMCSF-induced weakening. Recently, we reported LA concomitantly blocks GMCSF-induction of MMPs 2, 9 and 10 and inhibition of TIMPs 1-3. The aim of this study was to show that LA blocks GMCSF-induced increases in additional proteases and reductions in additional protease inhibitors. METHODS: FM fragments were cultured±LA and then±GMCSF. In other experiments, weak versus strong, fresh FM were cultured without additions. Fragments were strength tested and media analyzed by multiplex protein ELISA for proteases and protease inhibitors. RESULTS: GMCSF induced FM weakening and concomitantly increased several Proteases (Cathepsin-S, Proteinase-3, Elastase-2) and decreased several protease inhibitors (NGAL, Cystatin-C, HE4 and Thrombospondin1). LA inhibited GMCSF-induced FM weakening and all enzymatic changes. Untreated weaker versus stronger regions of fresh FM showed comparable differences in proteases and protease inhibitor patterns to GMCSF-stimulated versus controls. CONCLUSION: LA blocks GMCSF-induced human FM weakening and associated protease increases and inhibitor decreases. The GMCSF-induced spectrum of protease/protease-inhibitor changes is similar to that in the natural weak FM fragments. In concert with previously reported GMCSF-induced changes in MMPs & TIMPs, these other protease and protease-inhibitor changes presumably facilitate FM weakening and rupture. LA blocks these GMCSF effects and therefore may be a useful agent to prevent PPROM.

Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.

INTRODUCTION: Inflammation/infection and decidual bleeding/abruption are highly associated with pPROM. As no animal model for pPROM exists, we have developed an in-vitro model system for the study of human fetal membrane (FM) weakening/rupture. Using it we have demonstrated that both TNF/IL-1 (modeling inflammation) and thrombin (modeling bleeding) weaken full thickness FM in a dose dependent manner concomitant with inducing biochemical changes similar to those seen in the FM physiological weak zone. METHODS: As the physiological site of infection and bleeding is the choriodecidua (CD), we modified our model system with full thickness FM tissue mounted on modified Transwell culture inserts to permit directional TNF/thrombin exposure on the decidua only (rather than both sides of the FM). After incubation, medium was sampled separately from the CD facing (maternal side) or from the amnion facing (fetal side) compartments and probed for cytokine release and confirmed with western blots. The FM was strength tested within the insert. RESULTS: Full-thickness FM fragments exposed to TNF or thrombin on CD side only showed dose dependent weakening and biochemical changes consistent with previous reports. Concomitantly, GM-CSF increased markedly on the CD but not the amnion side. Numerous proteases including MMP1 and MMP3 also increased on the CD side. Pre-incubation with GM-CSF antibody blocked both thrombin and TNF induced weakening. Finally, GM-CSF weakened FM in a dose dependent manner. DISCUSSION: GM-CSF is a critical common intermediate in the thrombin and TNF FM weakening pathways.

[Biomechanical characteristics of human fetal membranes. Preterm fetal membranes are stronger than term fetal membranes]. / Biomécanique de la rupture des membranes fœtales. Les membranes fœtales sont plus résistantes avant terme qu'à terme.

The purpose of this study was to determine the biomechanical characteristics of human fetal membranes (FM) throughout gestation. Biomechanical properties were determined for 115 FM of 23-41 weeks gestation using our previously described methodology. The areas of membrane immediately adjacent to the strongest and weakest tested spots were sampled for histomorphometric analysis. Clinical data on the patients whose FM were examined were also collected. FM less than 28 weeks gestation were associated with higher incidence of abruption and chorioamnionitis. Topographically FM at all gestations had heterogeneous biomechanical characteristics over their surfaces with distinct weak areas. The most premature membranes were the strongest. FM strength represented by rupture force and work to rupture decreased with increasing gestation in both weak and strong regions of FM. This decrease in FM strength was most dramatic at more than 38 weeks gestation. The FM component amnion-chorion sublayers were thinner in the weak areas compared to strong areas. Compared to term FM, preterm FM are stronger but have similar heterogeneous weak and strong areas. Following a gradual increase in FM weakness with increasing gestation, there is a major drop-off at term 38 weeks gestation. The FM weak areas are thinner than the stronger areas. Whether the difference in thickness is enough to account for the strength differences is unknown.

The effects of thrombin and cytokines upon the biomechanics and remodeling of isolated amnion membrane, in vitro.

Abruption-induced thrombin generation and inflammation/infection induced cytokine production have both been associated with fetal membrane (FM) weakening and preterm premature rupture of the fetal membranes (PPROM). Using our in vitro model system we have demonstrated that thrombin, and separately the cytokines, tumor necrosis factor-alpha (TNFα) and interleukin-1-beta (IL-1ß), remodel and weaken full thickness FM. Additionally, we have reported that the anti-oxidant and NFκB inhibitor, alpha-lipoic acid (LA), blocks these thrombin and cytokine induced effects. The purpose of these studies was to determine whether thrombin and cytokines directly weaken the amnion membrane (AM), the major load-bearing component of FM. Isolated AM or full thickness FM fragments from unlabored Cesarean deliveries were incubated with thrombin, TNFα, or IL-1ß, for 48 h. Rupture strength (breaking force) of each fragment was thereafter determined using our published methodology. Biochemical evidence of remodeling and apoptosis; immunoreactive Matrix Metalloproteinase 9 (MMP9), Tissue Inhibitor of Matrix Metalloproteinase 3 (TIMP3) and cleaved poly (ADP-ribose) polymerase (C-PARP) levels in tissue extracts, were determined by western blot and densitometry. Thrombin induced a dose-dependent weakening of isolated AM (P < 0.001) coupled with dose dependent increases in PARP cleavage, and reciprocal increases and decreases, respectively, in MMP9 and TIMP3 protein (all P < 0.01). Thrombin receptor activating peptide-6 (TRAP) also weakened isolated AM. Neither TNFα nor IL-1ß weakened isolated AM. However, both cytokines weakened AM when it was incubated together with the choriodecidua as part of full thickness FM (P < 0.001). Cytokine-conditioned choriodecidua medium also weakened isolated AM (P < 0.001). Under conditions in which cytokines weakened the AM, the changes in MMP9, TIMP3 and PARP cleavage were consistent with those seen after thrombin incubation. LA blocked the FM weakening and remodeling effects. In summary, thrombin weakens AM directly whereas cytokines weaken AM indirectly by causing the release of soluble intermediates from the choriodecidua.

Alpha-lipoic acid inhibits thrombin-induced fetal membrane weakening in vitro.

Cytokine-mediated inflammation and abruption-induced thrombin generation are separately implicated in matrix metalloproteinase (MMP)-mediated weakening of fetal membranes (FM) leading to preterm premature rupture of the fetal membranes (PPROM). At term, FM of both labored vaginal and unlabored Cesarean deliveries exhibit a weak zone overlying the cervix exhibiting ECM remodeling characterized by increased MMP9 protein and activity. We have reproduced these biochemical changes as well as FM weakening in vitro using tumor necrosis factor-alpha (TNF) and interleukin (IL)-1ß, inflammatory cytokines implicated in PPROM. Additionally, we have reported that the antioxidant and NFκB inhibitor alpha-lipoic Acid (LA) blocks these TNF-induced effects. We now present the first direct evidence that thrombin also can induce FM weakening in vitro, and LA treatment inhibits this thrombin-induced-weakening. Full thickness FM fragments from unlabored Cesarean deliveries were incubated with increasing doses of thrombin (0-100 u/ml) for 48 h. Fragments were then strength tested (breaking force and work to rupture) using our published methodology. MMP3 and 9 levels in tissue extracts were determined by Western blot and densitometry. To determine the effect of LA, FM fragments were incubated with control medium or 10 u/ml thrombin, with or without 0.25 mM LA. Strength testing and MMP induction were determined. Thrombin induced a dose-dependent decrease in FM strength (42% baseline rupture force and 45% work to rupture) coupled with a dose-dependent increase in MMP3 and 9 expression (all p < 0.001). Treatment of FM with 0.25 mM LA completely inhibited thrombin-induced FM weakening and MMP expression (all p < 0.001). Thrombin treatment of cultured FM induces mechanical weakening and increased MMP3 and 9. Treatment of FM with LA inhibits these thrombin-induced effects. We speculate LA may prove clinically useful in prevention of PPROM associated with abruption.

Decreased adherence and spontaneous separation of fetal membrane layers--amnion and choriodecidua--a possible part of the normal weakening process.

INTRODUCTION: The fetal membrane (FM) layers, amnion and choriodecidua, are frequently noted to have varying degrees of separation following delivery. FM layers normally separate prior to rupture during in vitro biomechanical testing. We hypothesized that the adherence between amnion and choriodecidua decreases prior to delivery resulting in separation of the FM layers and facilitating FM rupture. METHODS: FM from 232 consecutively delivered patients were examined to determine the extent of spontaneous separation of the FM layers at delivery. Percent separation was determined by the weight of separated FM tissue divided by the total FM weight. Separately, the adherence between intact FM layers was determined. FM adherence was tested following term vaginal delivery (13), term unlabored cesarean section (10), and preterm delivery (6). RESULTS: Subjects enrolled in the two studies had similar demographic and clinical characteristics. FM separation was present in 92.1% of membranes. Only 4.3% of FM delivered following spontaneous rupture of the fetal membranes (SROM) had no detectable separation. 64.7% of FM had greater than 10% separation. FM from term vaginal deliveries had significantly more separation and were less adherent than FM of term unlabored, elective cesarean section (39.0+/-34.4% vs 22.5+/-30.9%, p=.046 and 0.041+/-0.018N/cm vs 0.048+/-0.019N/cm, p<.005). Preterm FM had less separation and were more adherent than term FM (9.95+/-17.7% vs 37.5+/-34.4% and 0.070+/-0.040N/cm vs 0.044+/-0.020N/cm; both p<.001). CONCLUSIONS: Separation of the amnion from choriodecidua at delivery is almost universal. Increased separation is associated with decreased adherence as measured in vitro. Increased separation and decreased adherence are seen both with increasing gestation and with labor suggesting both biochemical and mechanical etiologies. The data are consistent with the hypothesis that FM layer separation is part of the FM weakening process during normal parturition.

Plasma and pulmonary fluid endothelin in horses with seasonal recurrent airway obstruction.

BACKGROUND: Summer pasture-associated recurrent airway obstruction (SPA-RAO), a seasonal airway obstructive disease of horses, is characterized by clinical exacerbation after exposure to pasture during warm months of the year. Endothelin (ET)-1, potent bronchoconstrictor, mitogen, secretagogue, and proinflammatory mediator, has been implicated in the pathogenesis of asthma and equine heaves. HYPOTHESIS: Immunoreactive ET-1 concentrations increase during clinical exacerbation and return to basal values during periods of disease remission. ANIMALS: Twelve horses, 6 affected with SPA-RAO and 6 nonaffected. METHODS: Prospective, observational study. Bronchoalveolar lavage fluid (BALF), arterial and venous plasma samples, and clinical variables were obtained from affected horses during clinical exacerbation and remission. Samples and data of nonaffected horses were collected during the summer and winter on dates similar to affected horses. Immunoreactive ET-1 was determined using a commercial ELISA. RESULTS: The median and range ET-1 concentrations (pg/ml) in arterial (1.3, 0.7-1.8) and venous (1.3, 1.2-1.7) plasma and in BALF (0.3, 0.2-0.4), and pulmonary epithelial lining fluid (PELF) (25.5, 21-50) were greater in affected horses during clinical exacerbation compared with remission (P < .01). The concentrations of immunoreactive ET-1 were greater in affected horses during clinical exacerbation compared with nonaffected horses (P < .05). CONCLUSIONS AND CLINICAL IMPORTANCE: During clinical exacerbation of SPA-RAO, ET-1 is increased in circulation and pulmonary secretions. Intervention with ET receptor antagonists should provide further information on the role of ET-1 in SPA-RAO.

A new methodology to measure strength of adherence of the fetal membrane components, amnion and the choriodecidua.

We have previously shown that separation of the amnion from choriodecidua occurs as an integral part of the fetal membranes (FM) rupture process. We have also reported that spontaneous separation of FM is nearly universal with term vaginal delivery. The etiology of this spontaneous FM separation is unknown. If biochemical degradation at the amnion-choriodecidua interface is a factor, decreased adhesive force between the FM components prior to their complete separation would be expected. The purpose of this project was to develop and validate machinery and procedures to measure the adhesive force between amnion and choriodecidua. Commercial tensile testing equipment was adapted to perform a standard T-peel test, per the American Society for Testing and Materials (ASTM) guidelines. FM test strip dimensions, peel speed, and peel force data measurements from force versus displacement curves were optimized for reproducibility. Test system validation was performed using Shurtape CP 60 (slow release painter's masking tape) as the standard. Equipment and procedures for a standard T-peel test on FM were developed. Shurtape CP 60 of decreasing widths showed reproducible, linear changes in the adhesive force range for FM (r(2)=0.96). The adhesive force between FM components ranged from 0.017 to 0.262 N/cm. Reproducibility was optimal with FM test strips of 4 x 6 cm and a peel speed of 25.4 cm/min. FM showed greater adhesive force adjacent to the placental disc than distal from the disc (p<0.05). We have developed equipment and procedures to accurately and reproducibly measure adhesive force between the FM amnion and choriodecidua.

Differential expression of fibulin family proteins in the para-cervical weak zone and other areas of human fetal membranes.

OBJECTIVE: Human fetal membranes (FM) at term have been shown to contain a weak zone in the region overlying the cervix which exhibits characteristics of increased collagen remodeling and apoptosis. It has been hypothesized that the FM rupture initiation site is within this weak zone. Although the FM weak zone has been partially characterized, it is unclear what structural differences in the extracellular matrix result in its decreased rupture strength. A screen for differentially expressed proteins in the amnion of the weak zone versus other FM areas demonstrated that fibulin 1 was decreased. We investigated potential regional differences in all fibulin protein family members. METHODS: FM fibulins were localized by immunohistochemistry. Detected fibulins were screened by Western blot for differences in abundance in the amnion of the weak zone versus non-weak zone FM regions. Amnion epithelial and mesenchymal cells were also screened for fibulin production. RESULTS: Fibulins 1 and 5 were detected in the cytoplasm of and in a pericellular pattern surrounding all FM cells, and in a dense extracellular pattern in the amniotic compact zone. Fibulin 3 was detected within the cytoplasm of amnion epithelial and chorion trophoblast cells. Fibulins 2 and 4 were not detected. Fibulins 1, 3 and 5 demonstrated decreased abundance of 33%, 63% and 58% (all P<0.01) in amnion of the weak zone relative to other FM regions. Amnion cells produced all three detected fibulins. Furthermore, TNF inhibited amnion cell fibulin production in a dose dependent manner. CONCLUSION: Fibulins 1, 3 and 5 were localized coincident with major microfibrillar networks in amnion. Each showed decreased abundance in the amnion component of the FM weak zone. Amnion epithelial and mesenchymal cells produced all three fibulins and their abundance was inhibited by TNF. We speculate that the amnion microfibrillar layer undergoes significant remodeling with the development of the FM weak zone.

The effects of Strongylus vulgaris parasitism on eosinophil distribution and accumulation in equine large intestinal mucosa.

REASONS FOR PERFORMING STUDY: Eosinophilic granulocytes have been associated with parasite or immune-mediated diseases, but their functions in other disease processes remain unclear. Cause and timing of eosinophil migration into the equine gastrointestinal mucosa are also unknown. OBJECTIVE: To determine the effects of intestinal parasitism on eosinophils in equine large intestinal mucosa. METHODS: Large intestinal mucosal samples were collected from horses and ponies (n = 16) from the general veterinary hospital population, ponies (n = 3) raised in a parasite-free environment, ponies experimentally infected with 500 infective Strongylus vulgaris larvae and treated with a proprietary anthelmintic drug (n = 14), and a similar group of ponies (n = 7) that received no anthelmintic treatment. Total eosinophil counts and eosinophil distribution in the mucosa were determined by histological examination. A mixed model analysis was performed and appropriate Bonferroni adjusted P values used for each family of comparisons. P<0.05 was considered significant. RESULTS: There was no difference in large intestinal mucosal eosinophil counts and eosinophil distribution between ponies infected with S. vulgaris and those raised in a parasite-free environment. Experimental infection with S. vulgaris, with or without subsequent anthelmintic treatment, did not change eosinophil counts, and counts were similar to those for horses from the general population. CONCLUSIONS: Migration of eosinophils to the equine large intestinal mucosa appears to be independent of exposure to parasites. Large intestinal mucosal eosinophils may have more functions in addition to their role in defence against parasites.

The physiology of fetal membrane rupture: insight gained from the determination of physical properties.

Premature rupture of the fetal membranes is a major cause of preterm birth and its associated infant morbidity and mortality. Recently, it has become clear that rupture of the fetal membranes, term or preterm, is not merely the result of the stretch and shear forces of uterine contractions, but is, in significant part, the consequence of a programmed weakening process. Work in the rat model has demonstrated that collagen remodeling, with activation of matrix metalloproteinases (MMPs), and apoptosis increase markedly in the amnion at end-gestation, suggesting that these processes are involved in fetal membrane weakening. We have developed fetal membrane strength testing equipment and a systematic tissue sampling methodology that has allowed us to demonstrate that term, non-labored, fetal membranes have a zone of weakness overlying the cervix, which contains biochemical markers of both collagen remodeling and apoptosis. These findings provide strong support for the concept of programmed fetal membrane weakening prior to labor. Our model has also been used to establish the physical properties of individual fetal membrane components (amnion, chorion), determine the sequence of events during the fetal membrane rupture process, and demonstrate that treatment of fetal membranes with TNF or IL-1beta, in vitro, induces weakness and the identical biochemical markers of collagen remodeling and apoptosis seen in the physiological weak zone. The ability to simultaneously correlate macroscopic physical properties with histological and biochemical fetal membrane characteristics, presents a unique perspective on the physiology of fetal membrane rupture.

A pilot study of methyl chloride emissions from tropical woodrot fungi.

Flux chamber measurements made in a rainforest provide evidence that methyl chloride is emitted from rotting wood. However, its net flux was found to be into the soil, probably due to competing production and consumption processes within the soil. Evidence was found for a regional source, possibly vegetation, since its concentration above the canopy was substantially greater than reported average equatorial values.

Term human fetal membranes have a weak zone overlying the lower uterine pole and cervix before onset of labor.

The etiology of fetal membrane (FM) rupture is unknown. A hypothesis that the FM weakens by a process of collagen remodeling and apoptosis to facilitate rupture has been proposed. Human FMs reportedly exhibit a zone of altered histology, postulated to be the FM rupture site, but concomitant FM weakness has not been demonstrated. We hypothesized that a discrete zone of FM with marked weakness, histological change, and evidence of remodeling and apoptosis, develops in late gestation in the FM overlying the cervix. FM tissue from women undergoing prelabor cesarean delivery were perioperatively marked to identify the FM overlying the cervix, cut with a procedure that facilitates remapping the rupture strength of FM pieces to their former location and orientation on a three-dimensional model, and tested for strength. A 10-cm FM zone centered at the cervical mark was compared with the remaining FM. Mean rupture strength within the cervical zone was 55% of the remaining FM. The cervical zone also exhibited increased MMP-9 protein, decreased tissue inhibitor of metalloproteinases-3 (TIMP-3) protein, and increased PARP cleavage coincident with the previously reported zone of altered histology. A discrete zone of weakness is present in term prelabor FMs overlying the cervix and has biochemical characteristics consistent with tissue remodeling and apoptosis.

Vitamin C exacerbates hydrogen peroxide induced apoptosis and concomitant PGE2 release in amnion epithelial and mesenchymal cells, and in intact amnion.

This study was undertaken to investigate the effect of hydrogen peroxide (HP), a reactive oxygen species, and vitamin C, an antioxidant, on apoptosis and prostaglandin (PGE(2)) release in human amnion epithelial and mesenchymal cells, and intact amnion. Amnion cells and explants were incubated with and without HP and vitamin C. Cytoproliferation assay for viability, DNA fragmentation and PARP cleavage for apoptosis, EIA for PGE(2), and western blots for cyclooxygenases (COX) were performed. In amnion cells and explants, HP (0-5 mm) induced dose dependent apoptosis as per DNA fragmentation and PARP cleavage. HP (0-0.5 mm) also induced PGE(2)release concomitant with apoptosis in both cell types. In amnion explants, HP (0-10 mm) induced COX-2 protein and PGE(2)release concomitant with apoptosis. Vitamin C (0.01-10 mm), alone, enhanced epithelial but inhibited mesenchymal cell viability. It induced PGE(2)release in amnion explants. Vitamin C (1 mm) failed to inhibit HP induced apoptosis, but instead exacerbated it in epithelial and mesenchymal cells, and amnion explants. Vitamin C (0-10 mm) enhanced HP induced PGE(2)in mesenchymal cells. HP induces concomitant apoptosis and PGE(2)release in amnion epithelial and mesenchymal cells, and in intact amnion explants. HP induced apoptosis is not inhibited but enhanced by vitamin C.

Evidence for vascular and enzymatic events in the pathophysiology of acute laminitis: which pathway is responsible for initiation of this process in horses?

To date, there is a substantial amount of data to support the hypotheses that vascular and enzymatic changes are ongoing in experimental laminitis. Furthermore, there is substantial in vitro evidence that the enzymatic changes weaken the dermo-epidermal attachments leading to mechanical failure of the hoof-bone interface of the equine digit. However, investigators of both the vascular and enzymatic theories have, to date, been unable to substantiate the effects of these pathophysiological changes in vivo on laminar tissues of horses afflicted with experimentally induced or naturally acquired laminitis. In addition, the effects of laminitis-inducing treatment have not been prevented or reversed by treatment with an MMP inhibitor or a vasoactive antagonist. It is possible that there is simultaneous activation of the vascular and enzymatic pathways and/or other inflammatory processes. Moreover, the third theory involving mechanical factors cannot be discounted simply because strong evidence for vascular and enzymatic changes exists. It is common for horses with severe musculoskeletal disease affecting weightbearing on a limb to develop laminitis in the contralateral limb. It remains to be determined what factors are responsible for initiation of laminitis in these individuals. Evidence has not been presented that precludes the possibility of coincident occurrence of vascular and enzymatic changes. In fact, many of the inflammatory mediators (e.g. interleukin-1beta) found in laminitic tissues can concurrently stimulate synthesis of vasoactive substances and activate MMPs. Because enzymatic action on proteins is largely dependent on the concentrations of proteins and enzyme, the enzymatic theory is not dependent upon increased delivery of enzymes via increased capillary flow. Likewise, because vascular changes can alter tissue function via increased capillary flow and oedema formation, the vascular theory is not dependent upon decreased capillary flow. It is true that naturally acquired laminitis is widely variable in severity and predisposing diseases. Therefore, most probably there are multiple mechanisms involved in the initiation and propagation of the pathophysiologic cascade(s) and, therefore, successful intervention will necessitate multiple treatment modalities.

Hydrogen peroxide induced apoptosis in amnion-derived WISH cells is not inhibited by vitamin C.

Increased reactive oxygen species (ROS) have been identified as a potential cause of remodelling and apoptotic change in fetal membrane. Vitamin C has been suggested as a therapeutic agent to prevent ROS induced chorio-amnion apoptosis. The purpose of this study was to determine whether hydrogen peroxide (HP), a ROS, initiates apoptosis in the WISH cell model and whether vitamin C would inhibit HP induced apoptosis. HP induced apoptosis in WISH cells; as assessed by cytochrome-c release from mitochondria, Poly-(ADP-ribose)-Polymerase (PARP) cleavage, nuclear matrix protein (NMP) release and DNA fragmentation analysis. HP induced dose dependent release of cytochrome-c, PARP cleavage, NMP release, and DNA fragmentation. HP also increased PGE(2)release in parallel with apoptosis in WISH cells, in a manner similar to that reported with other apoptotic agents. Vitamin C pre-incubation caused cytochrome-c release earlier, and at lower HP doses, than HP alone. It had no effect on HP induced PARP cleavage, but enhanced DNA fragmentation, and induced NMP release on its own. Vitamin C partially suppressed dose dependent HP induced PGE(2)release. We conclude that HP causes apoptosis in WISH cells and vitamin C pre-incubation does not inhibit, and may accelerate and exacerbate, HP induced apoptosis.

Articular chondrocyte apoptosis in equine osteoarthritis.

Osteoarthritis (OA) is the most common joint disease in horses. Chondrocyte apoptosis has been implicated as a major pathological OA change in humans and experimental animals but no studies have been performed on equine OA. Articular cartilage was collected from three normal and five OA horses. Histopathological changes were scored by a modified Mankin grading system. A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed to identify chondrocyte apoptosis. Nitric oxide (NO) production from chondrocytes was indirectly evaluated by immunohistochemistry with polyclonal antibody to nitrotyrosine. The histopathological score and percentage of chondrocyte apoptosis from the OA cartilages were significantly higher than from normal cartilages. There was a significant correlation between histopathological grade and the percentage of chondrocyte apoptosis. OA cartilages exhibited stronger immunoreactivity to nitrotyrosine than normal cartilage. Topographical distributions of chondrocyte apoptosis, cartilage matrix degeneration, and NO production overlapped in equine OA cartilages, suggesting that these pathological phenomena are closely interrelated.

Cardiopulmonary, blood and peritoneal fluid alterations associated with abdominal insufflation of carbon dioxide in standing horses.

REASONS FOR PERFORMING STUDY: Abdominal insufflation is performed routinely during laparoscopy in horses to improve visualisation and facilitate instrument and visceral manipulations during surgery. It has been shown that high-pressure pneumoperitoneum with carbon dioxide (CO2) has deleterious cardiopulmonary effects in dorsally recumbent, mechanically ventilated, halothane-anaesthetised horses. There is no information on the effects of CO2 pneumoperitoneum on cardiopulmonary function and haematology, plasma chemistry and peritoneal fluid (PF) variables in standing sedated horses during laparoscopic surgery. OBJECTIVES: To determine the effects of high pressure CO2 pneumoperitoneum in standing sedated horses on cardiopulmonary function, blood gas, haematology, plasma chemistry and PF variables. METHODS: Six healthy, mature horses were sedated with an i.v. bolus of detomidine (0.02 mg/kg bwt) and butorphanol (0.02 mg/kg bwt) and instrumented to determine the changes in cardiopulmonary function, haematology, serum chemistry and PF values during and after pneumoperitoneum with CO2 to 15 mmHg pressure for standing laparoscopy. Each horse was assigned at random to either a standing left flank exploratory laparoscopy (LFL) with CO2 pneumoperitoneum or sham procedure (SLFL) without insufflation, and instrumented for measurement of cardiopulmonary variables. Each horse underwent a second procedure in crossover fashion one month later so that all 6 horses had both an LFL and SLFL performed. Cardiopulmonary variables and blood gas analyses were obtained 5 mins after sedation and every 15 mins during 60 mins baseline (BL), insufflation (15 mmHg) and desufflation. Haematology, serum chemistry analysis and PF analysis were performed at BL, insufflation and desufflation, and 24 h after the conclusion of each procedure. RESULTS: Significant decreases in heart rate, cardiac output and cardiac index and significant increases in mean right atrial pressure, systemic vascular resistance and pulmonary vascular resistance were recorded immediately after and during sedation in both groups of horses. Pneumoperitoneum with CO2 at 15 mmHg had no significant effect on cardiopulmonary function during surgery. There were no significant differences in blood gas, haematology or plasma chemistry values within or between groups at any time interval during the study. There was a significant increase in the PF total nucleated cell count 24 h following LFL compared to baseline values for LFL or SLFL at 24 h. There were no differences in PF protein concentrations within or between groups at any time interval. CONCLUSIONS: Pneumoperitoneum with CO2 during standing laparoscopy in healthy horses does not cause adverse alterations in cardiopulmonary, haematology or plasma chemistry variables, but does induce a mild inflammatory response within the peritoneal cavity. POTENTIAL RELEVANCE: High pressure (15 mmHg) pneumoperitoneum in standing sedated mature horses for laparoscopic surgery can be performed safely without any short-term or cumulative adverse effects on haemodynamic or cardiopulmonary function.

Physiological apoptotic agents have different effects upon human amnion epithelial and mesenchymal cells.

Foetal membrane rupture is thought to follow from gene-controlled tissue remodelling and apoptosis. We reported previously that staurosporine, cycloheximide, actinomycin D, as well as more physiological apoptotic agents (lactosylceramide, 15d-PGJ(2)) increase prostaglandin release in parallel with induction of apoptosis in WISH and amnion epithelial cells. Also, inhibition of prostaglandin release by cyclooxygenase inhibitors or PKA activators is accompanied by a parallel decrease in apoptosis. We hypothesize that amnion prostaglandin metabolism is linked with apoptosis in amnion epithelial cells and thus to membrane rupture. Amnion mesenchymal cells are also critical for membrane integrity. Their susceptibility to apoptotic agents is unknown and is the subject of this report. In amnion epithelial cells, lactosylceramide (125 microM) induced 6.5-fold, 20-fold increases in PGE(2) and NMP production (apoptosis), respectively. Conversely, in mesenchymal cells, lactosylceramide doses up to 200 microM had no effect on PGE(2) or NMP release. In both cell types, incubation with 15d-PGJ(2) (5-100 microM) demonstrated dose and time dependent increases in PGE(2) and NMP. PKA activators inhibited 15d-PGJ(2) induced PGE(2) release and apoptotis in epithelial cells, but not in mesenchymal cells, however. Major amnion cell types have different sensitivities to physiological apoptotic agents. Prostaglandin release occurs coincident with apoptosis in both amnion epithelial and mesenchymal cells.

Non-adrenergic non-cholinergic excitatory innervation in the airways: role of neurokinin-2 receptors.

1 The hypothesis that the non-adrenergic, non-cholinergic excitatory (NANC-e) innervation is involved in the induction of asthma and that antagonists of NANC-e neurotransmitter could reduce bronchoconstriction during asthma was tested. 2 The first objective was to identify the neurotransmitter(s) of NANC-e innervation from a group of selected putative neurotransmitters. The second objective was to use the antagonist of the identified neurotransmitter(s) to determine its effectiveness against bronchoconstriction to ovalbumin (OVA) in sensitized guinea-pigs. 3 Neurotransmitter identification was performed using the "tracheal pouch"', a surgical preparation established for demonstrating NANC innervation, in anaesthetized guinea-pig airways. A segment of trachea was cannulated and clamped at one end and the other end was connected to a pressure transducer. The stump of the trachea was connected to a ventilator to keep the blood gas values within the normal range. The vagus nerve and the sympathetic nerves were isolated bilaterally and cut. The left carotid artery was cannulated to monitor blood pressure and for sampling blood for blood gas analysis. The jugular vein was cannulated for administration of test agents. 4 Both NANC-e and NANC-i (inhibitory) control responses of airways were obtained by bilateral vagal stimulation after complete autonomic blockade with atropine, propranolol and prazosin. The relaxation of the tracheal pouch was indicative of the NANC-i response and the increase in insufflation pressure of the ventilated peripheral airways was due to NANC-e stimulation. 5 The involvement of the putative neurotransmitters such as neurokinin-A (NK-A), histamine, serotonin and endothelin (ET) was investigated by using the respective antagonists, MEN-10376, pyrilamine maleate, cyproheptadine hydrochloride, and two ET receptor antagonists (BQ-123 and IRL-1038), respectively. The antagonists were administered at the dose rate of 4 mg kg-1 i.v. which was determined from preliminary studies by testing against the respective agonists. 6 MEN-10376 (neurokinin-2 receptor antagonist) significantly inhibited the insufflation pressure (peripheral airway pressure) increase caused by NANC-e stimulation. MEN-10376 also inhibited the fall in blood pressure caused by bilateral vagal stimulation. The 5-HT antagonist, cyproheptadine, significantly enhanced the NANC-e response. 7 After identifying the NANC-e neurotransmitter as NK-A, the effectiveness of its antagonist, MEN-10376, was evaluated for its ability to attenuate the increase in insufflation pressure (bronchoconstriction) induced in guinea-pigs sensitized by OVA. Guinea-pigs were sensitized to OVA (200 mg i.p.) and 10 days later prepared for the determination of tracheal pouch and insufflation responses to 100 microg of OVA administered i.v. (challenge dose). This caused an increase in insufflation pressure in the presence of adrenergic and cholinergic blockade, which was significantly attenuated by MEN-10376. 8 These studies indicated that neurokinin-2 receptors were involved in the vagally mediated efferent neurotransmission of NANC-e and that NANC-e plays a role in allergen-induced bronchoconstriction.