Muscle fiber diameter assessment in cleft lip using image processing.

OBJECTIVE: To pilot investigation of muscle fiber diameter (MFD) on medial and lateral sides of the cleft in 18 infants with cleft lip with or without cleft palate (CL/P) using image processing. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded (FFPE) tissue samples from the medial and lateral sides of the cleft were analyzed for MFD using an image-processing program (ImageJ). For within-case comparison, a paired Student's t test was performed. For comparisons between classes, an unpaired t test was used. RESULTS: Image processing enabled rapid measurement of MFD with majority of fibers showing diameter between 6 and 11 µm. There was no significant difference in mean MFD between the medial and lateral sides, or between CL and CLP. However, we found a significant difference on the medial side (p = .032) between males and females. CONCLUSION: The image processing on FFPE tissues resulted in easy quantification of MFD with finding of a smaller MFD on the medial side in males suggesting possible differences in orbicularis oris (OO) muscle between the two sexes in CL that warrants replication using larger number of cases. Moreover, this finding can aid subclinical phenotyping and potentially in the restoration of the anatomy and function of the upper lip.

MHC II-ß chain gene expression studies define the regional organization of the thymus in the developing bony fish Dicentrarchus labrax (L.).

MHC II-ß chain gene transcripts were quantified by real-time PCR and localised by in situ hybridization in the developing thymus of the teleost Dicentrarchus labrax, regarding the specialization of the thymic compartments. MHC II-ß expression significantly rose when the first lymphoid colonization of the thymus occurred, thereafter increased further when the organ progressively developed cortex and medulla regions. The evolving patterns of MHC II-ß expression provided anatomical insights into some mechanisms of thymocyte selection. Among the stromal cells transcribing MHC II-ß, scattered cortical epithelial cells appeared likely involved in the positive selection, while those abundant in the cortico-medullary border and medulla in the negative selection. These latter most represent dendritic cells, based on typical localization and phenotype. These findings provide further proofs that efficient mechanisms leading to maturation of naïve T cells are operative in teleosts, strongly reminiscent of the models conserved in more evolved gnathostomes.

Immune modulatory effects of Aloe arborescens extract on the piscine SAF-1 cell line.

The pharmacological potential of Aloe arborescens Miller leaf components was investigated, with special attention deserved to immune modulatory effects on the Sparus aurata fibroblast cell line SAF-1. The cells were treated with Aloe extract at different concentrations (1.2-4.8 mg ml(-1)) for various times (24-72 h). The lowest concentration did not provoke any cellular damage observable by SEM and did not affect ATP amounts after 24 and 48 h, while even induced a significant increase over controls after 72 h. We next examined the transcription kinetics of different immune-related genes (IL-1ß, TGF-ß, TNF-α, COX-2, IFN-I, Mx and MHCI-α) in SAF-1 cells stimulated with LPS or poly I:C. The Aloe extract (1.2 mg ml(-1)) acted as a powerful immune stimulant in LPS- or poly I:C-activated SAF-1 cells, inducing a synergic effect on interconnected genes that are expected to be involved in different aspects of the immune responses. These reports provide a new perspective for the use of A. arborescens to prevent or oppose bacterial and viral fish diseases and to face, as an alternative strategy based on natural plant extracts, the growing unwillingness to rely upon standard solutions involving antibiotics or antimicrobial chemicals.

Intestinal T cells of Dicentrarchus labrax (L.): gene expression and functional studies.

Cellular and molecular data have evidenced a gut-associated lymphoid tissue in a variety of teleost species, abundantly containing T cells, whose origin, selection and functions are still unclear. This study reports CD4, CD8-α, MHCI-α, MHCII-ß, rag-1 and TCR-ß gene transcription along the intestine (anterior, middle and posterior segments) and in the thymus of one year-old Dicentrarchus labrax (L.). Real-time PCR findings depicted a main role of the thymus in T-cell development, but also rag-1 and CD8-α transcripts are detected in the intestine, having significant expression in the posterior segment. In the whole intestine TCR-ß and CD8-α exceeded CD4 transcripts. RNA ISH confirmed these data and detailed that mucosal CD8-α+ cells were especially numerous in the epithelium and in aggregates in the lamina propria. Regional differences in T-cell-specific gene expressions are first described in the intestine of a bony fish. High non-specific cytotoxic activity against xenogeneic and allogeneic cells was found in lymphocytes purified from the intestinal mucosa, providing further insight into their local defence roles.

cAMP efflux from human trophoblast cell lines: a role for multidrug resistance protein (MRP)1 transporter.

Cyclic adenosine 3'-5'-monophosphate (cAMP) is a second messenger, which exerts an important role in the control of human first-trimester trophoblast functions. In the present study we demonstrate the existence of a mechanism that is able to extrude cAMP from trophoblast-derived cell lines, and show evidence indicating the involvement of multidrug resistance protein (MRP) 1, a transporter belonging to the ATP-binding cassette family, in cAMP egress. MRP1 is expressed in trophoblast cell lines and cAMP efflux is highly reduced by the MRP1 inhibitor, MK-571. In addition, interleukin-1beta and estrone are able to enhance MRP1 gene expression and influence extracellular cAMP concentration. The occurrence of a MRP1-dependent cAMP efflux is also shown in human first-trimester placenta explants. Extracellular cAMP could represent a source for adenosine formation, which in turn could regulate cAMP-dependent responses in placental tissue. Evidence is provided that adenosine receptor subtypes are present and functional in human trophoblast-derived cells. A role for cAMP egress mechanism in the fine modulation of the nucleotide homeostasis is therefore suggested.

Somatostatin as a regulator of first-trimester human trophoblast functions.

We have tested the hypothesis that human early trophoblast is a target for somatostatin (SRIF) regulatory actions. We report for the first time that SSTR2A and 2B transcripts and proteins are present in first-trimester human chorionic villi and the trophoblast-derived HTR-8/SVneo and JAR cells. In both cell lines, SSTR are functional since SRIF inhibits cyclic AMP pathway, stimulates arachidonic acid release and enhances cell proliferation. Moreover, in HTR-8/SVneo cells, considered a good model of first-trimester EVT, SRIF also enhances migration. An involvement of the cyclic AMP pathway in mediating SRIF effects on proliferation and migration is suggested. Our data support the idea that SRIF regulates early trophoblast functions mainly through an interaction with SSTR2.

Relative contribution of sympathetic and sensory nerves to thermal nociception and tissue trophism in rats.

Neonatal injection of 6-hydroxydopamine (420 mg/kg s.c.) lowered thermal nociceptive threshold (hot plate and tail immersion tests) and increased levels of substance P-like immunoreactivity in the skin (paws, tail, area of vibrissae) of Wistar rats. Chemical ablation of primary afferents, induced in either neonatal or adult rats by systemic administration of capsaicin, increased thermal nociceptive threshold (hot plate), irrespective of 6-hydroxydopamine pretreatment, and reduced substance P-like immunoreactivity in the hind-paw skin of either control or sympathectomized rats. Capsaicin pretreatment of neonatal but not adult rats produced antinociceptive effect in the tail-immersion test and completely reversed the hyperalgesic effect of sympathectomy, without affecting levels of substance P-like immunoreactivity in the tail skin. These findings indicate that sympathetic nerves and different subsets of capsaicin-sensitive primary afferents are involved in the processing of thermal nociceptive input. Corneal and cutaneous lesions were induced by neonatal sensory denervation with capsaicin. Sympathectomy afforded protection against the development of corneal pathology, while it did not affect the occurrence of cutaneous lesions. It appears that a balance in the neuronal activity between sympathetic neurons and trigeminal sensory neurons is critical for maintaining the normal trophism of the cornea, and that sensory neuropeptides play a key role in the maintenance of normal trophism of the skin.

Rectospinal neurons: cell bodies, pathways, immunocytochemistry and ultrastructure.

A novel class of enteric neurons projecting directly from the rectal wall to the spinal cord, "rectospinal neurons", was investigated in rats by combined retrograde neuronal tracing, immunocytochemistry and electron microscopy. Rectospinal neurons were almost confined to myenteric ganglia of the distal rectum below the pelvic diaphragm and were labeled preferentially by injections into spinal cord segments L6/S1. Injections into more rostral spinal cord segments resulted in hardly any labeled enteric neurons. Dorsal and ventral rhizotomy experiments indicated an almost exclusive projection of rectospinal neurons through dorsal roots L6/S1 to the respective spinal cord segments. Among various peptides immunostained, vasoactive intestinal polypeptide and calcitonin gene-related peptide were selectively found in rectospinal neurons, which were also shown to contain calbindin, neurofilament protein- and peripherin-immunoreactivity. Vasoactive intestinal polypeptide- and calbindin-immunostaining were frequently co-localized in the same perikarya, while calcitonin gene-related peptide-immunoreactive rectospinal neurons probably represented a separate population. Neonatal capsaicin treatment did not significantly reduce the number of rectospinal neurons. Electron microscopy revealed synaptic contacts on the surface of rectospinal neurons. Taken together, these results establish rectospinal neurons as an anatomically and neurochemically distinct class of enteric neurons. Synaptic contacts on rectospinal neurons suggest that these neurons may function as a direct link from the enteric to the central nervous system, thus indicating that connections between these two networks are reciprocal.

The contribution of sensory nerves to xylene-induced cystitis in rats.

The role of capsaicin-sensitive sensory nerves of the rat urinary bladder in xylene-induced cystitis was investigated. Instillation of xylene into the urinary bladder of female rats induced cystitis, e.g. detrusor hyperreflexia and increased vascular permeability. Detrusor hyperreflexia was also observed in rats desensitized to capsaicin as adults (50-125 mg/kg s.c., 4 days before) but only for a short period (1 h) after instillation. When a longer time lag (24 h) was allowed to elapse following instillation, reflex micturition was almost abolished. In rats desensitized to capsaicin as newborns (50 mg/kg s.c. on second day of life) reflex micturition was almost abolished and xylene (given 1 h before measurement) was ineffective. The xylene-induced plasma extravasation was greater in the bladder neck than in the dome. In the bladder neck the "early" response to xylene was reduced but not abolished in rats densensitized to capsaicin as adults or pretreated with compound 48/80 and was abolished in rats desensitized to capsaicin as newborns. The bladder content of substance P-like immunoreactivity decreased at various times following xylene instillation but this change occurred in parallel to the increase in bladder weight. These findings indicate that xylene-induced cystitis involves, at least in part, an irritation of capsaicin-sensitive sensory nerves in the bladder wall. The present results further suggest that xylene acts by stimulating at least two populations of sensory nerves which differ in their sensitivity towards capsaicin.

Topical versus systemic capsaicin desensitization: specific and unspecific effects as indicated by modification or reflex micturition in rats.

The aim of this study was to determine the acute and delayed effect of topical application of high concentrations of capsaicin on the rat urinary bladder on micturition reflex and compare the effects of "topical" bladder desensitization with those produced by systemic (subcutaneous administration) capsaicin desensitization. On acute application, capsaicin (1-3%) produced a transient bladder contraction, not observed in capsaicin-pretreated rats. After a transient increase in excitability of the micturition reflex, topical capsaicin suppressed micturition and overflow incontinence ensued which was reverted by intravenous injection of 4-aminopyridine. Topical capsaicin also abolished reflex micturition in rats which had been systemically treated with capsaicin as adults (50 mg/kg, 7 days before) and reduced significantly the neurogenic bladder contractions produced by intravenous dimethylphenylpiperazinium or neurokinin A, while the direct (myogenic) response to neurokinin A was unaffected. In rats whose bladder was pre-exposed to 1-3% topical capsaicin (7 days before) the micturition reflex was affected in a manner which is qualitatively and quantitatively similar to that observed in rats treated with capsaicin as adults, e.g. increase in bladder capacity with no change in voiding efficiency. Topical capsaicin desensitization of the rat urinary bladder was shown to produce a selective impairment of bladder sensory nerves without any sign of desensitization in other areas of the body using both functional (hot plate, wiping, plasma extravasation) and neurochemical (determination of substance P-like immunoreactivity) assays. Systemically administered capsaicin (7 days before) had little effect on reflex micturition at 12.5 mg/kg but the change in bladder capacity produced at a dose of 25 mg/kg was comparable with that produced at 350 mg/kg. These findings provide evidence that selective desensitization of peripheral terminals of capsaicin-sensitive nerves of the rat urinary bladder inactivates their sensory and "efferent" function in a manner similar to that observed after systemic capsaicin desensitization in adult rats. The functional deficit of reflex micturition produced in this way can be overcome by increasing the stimulus to void. By contrast, neonatal capsaicin desensitization produced a long lasting abolition of reflex micturition. These data are in keeping with the hypothesis that adult versus neonatal capsaicin desensitization may be used as a tool to distinguish between two sets of sensory nerves in the rat urinary bladder.(ABSTRACT TRUNCATED AT 400 WORDS)

Acrylamide-induced visceral neuropathy: evidence for the involvement of capsaicin-sensitive nerves of the rat urinary bladder.

The mechanisms underlying the severe urinary retention induced by acrylamide intoxication were studied in detail in the rat. Subcutaneous treatment with acrylamide monomer (50 mg/kg daily for 10 days) almost completely impaired the micturition reflex, resulting in urinary retention. In fact, the ability to eliminate an oral water load was virtually abolished, while bladder filling with saline (transvesical cystometrogram) failed to activate reflex micturition. Instead, a picture of overflow incontinence resulted in urethane-anaesthetized rats, which was not reversed by intravenous administration of 4-aminopyridine. The nerve-mediated contractile response to field stimulation (0.1-20 Hz, 0.5 ms, 60 V) of the isolated bladder was unaffected, thus suggesting the integrity of bladder efferent innervation, and no evidence was found from in vitro experiments that the myogenic contractility of the bladder was depressed by acrylamide treatment. Conversely, the sensory nerve-mediated response to capsaicin was abolished and sensory nerve fibres of the bladder were selectively depleted of their content of substance P- and calcitonin gene-related peptide immunoreactivity following acrylamide treatment. In fact, concentrations of the same neuropeptides in other organs, including the adjoining ureters, were unaffected. As to the urethral segment, including the striated sphincter, the D-tubocurarine (0.2 mM)-sensitive urethral response to electrical stimulation (0.1 Hz, 0.1 ms, 20 V) was significantly reduced in acrylamide-treated animals. At the same level, neurofilament protein immunostaining revealed striking accumulations of neurofilament protein-like material in motor end-plates, thus indicating that neuromuscular junctions of the urethral striated sphincter were severely affected. Thus, the afferent arm of the micturition reflex was shown to be severely deranged by acrylamide intoxication, especially in its capsaicin-sensitive component. Since twitch-like contractions of the urethral striated sphincter are probably involved in promoting bladder voiding, a decreased efficiency of this mechanism could participate in the picture of urinary retention induced by acrylamide.

The immune system of sea bass, Dicentrarchus labrax, reared in aquaculture.

The sea bass Dicentrarchus labrax is one the most important seawater fish species of south Europe and Mediterranean aquaculture, and studies on its immune system are important for both scientific and applied purposes. In this paper, we summarise the results obtained in studies of the immune system in this species, and present original data on cell-mediated acquired immune response.

Ontogeny of the thymus in a teleost fish, Cyprinus carpio L.: developing thymocytes in the epithelial microenvironment.

A monoclonal antibody, WCL9, specific for membrane molecules of a thymocyte subpopulation was used to detect these cells in situ during the ontogeny of thymus. Cryo-sections revealed WCL9+ cells in the rudiment of the thymus (day 4 post fertilization); thereafter, the positive cells were observed exclusively in the cortex from the first appearance of thymic regionalization (week 4 post fertilization) until adult age. Whole-mount immunostaining of the thymus with WCL9 revealed the three-dimensional structure of the cortex by specific staining. The presence and distribution of apoptotic cells during thymus development was studied by in situ end-labelling of fragmented DNA. From week 4 post fertilization onwards, apoptotic cells were more frequently detected in the cortex than medulla, suggesting a continuous selection of thymocytes in the cortex. Ultrastructural studies confirmed the presence of numerous cortical apoptotic cells inside macrophages. Electron microscopy provided evidence for the existence of epithelial heterogeneity in the thymus. During the ontogeny, the differentiation of epithelial cells was followed from the first weeks until the juvenile age. Cell types were classified on the basis of their localization and cytological characteristics as: i) limiting epithelial cells located in subcapsular, perivascular and peritrabecular zones; ii) reticular epithelial cells situated in medullary and cortical zones; iii) nurse-like cells at the border between the cortex and medulla, iiii) Hassall's body-like structures localized in the medulla. This study could suggest the occurrence of a wide range of lympho-epithelial interactions throughout thymocytes differentiation.

Capsaicin-like effect of resiniferatoxin in the rat stomach.

Neurochemical and functional studies were performed to investigate and to compare the effects of resiniferatoxin and capsaicin in the rat stomach. Neonatal administration of resiniferatoxin (0.6-1.6 mumol/kg subcutaneously (s.c.)) produced a marked decrease in gastric calcitonin gene-related peptide-like immunoreactivity in both secretory and non-secretory region of the stomach. Almost complete depletion of the peptide was determined by neonatal administration of capsaicin (164 mumol/kg s.c.). Vasoactive intestinal polypeptide-like immunoreactivity was concomitantly unaffected by resiniferatoxin or capsaicin, thus showing the selectivity of action of the neurotoxins on gastric afferent fibers. Oral administration of an equimolar dose (0.3 nmol/kg) of resiniferatoxin or capsaicin together with 50% ethanol reduced at a similar extent gastric haemorrhagic lesions produced by the mucosal barrier-breaker agent. These findings provide evidence that resiniferatoxin and capsaicin may act on a common neuronal target in the rat stomach and that the acute exciting (protective) effect is of the same magnitude.

Neuronal intermediate filaments in rat dorsal root ganglia: differential distribution of peripherin and neurofilament protein immunoreactivity and effect of capsaicin.

Two major neuronal populations were revealed in rat dorsal root ganglia, immunoreactive for either peripherin, or neurofilament triplet proteins (adult L2 ganglia: 66.2% and 25.6%, mainly small and large diameter cells, respectively), together with a minor, double-immunostained population (L2: 8.1%, mainly intermediate-size neurons). After capsaicin treatment, a striking expansion in the latter population was seen (L2: 22.0%) together with a significant increase in size, restricted to the same population and the (remaining) peripherin-only immunoreactive neurons. Calcitonin gene-related peptide (CGRP) immunoreactivity was revealed in neurons of all 3 groups, in both normal and capsaicin-treated rats.

Visceromotor responses to calcitonin gene-related peptide (CGRP) in the rat lower urinary tract: evidence for a transmitter role in the capsaicin-sensitive nerves of the ureter.

Either intra-arterial or topical administration of calcitonin gene-related peptide (CGRP) had little effect on motility of the urinary bladder in urethane-anaesthetized rats. Only a high concentration (50 microM) of topical CGRP activated the micturition reflex and potentiated the response to exogenous substance P (SP). In the isolated rat bladder CGRP had inconsistent effects on spontaneous or field-stimulated contractions. CGRP neither produced any significant plasma extravasation (Evans blue leakage) in the rat lower urinary tract, nor potentiated the response to exogenous SP. CGRP inhibited motility in the rat isolated proximal urethra and ureters and counteracted the contractile response to neurokinins. An inhibitory effect of capsaicin on stimulated motility of the urethra was observed in all preparations and a small contractile response was evident in about 40% of cases. Lack of desensitization to the action of CGRP prevented the study of its interaction with capsaicin. The inhibitory effect of CGRP in the ureter exhibited a specific desensitization: if the preparations were pre-exposed to exogenous CGRP, the inhibition of motility produced by antidromic activation of the capsaicin-sensitive nerve terminals (field stimulation) as well as the response to capsaicin (1 microM) was prevented but the inhibitory response to isoprenaline was unaffected. These findings indicate that CGRP is able to influence markedly the motility of the rat lower urinary tract, but exhibits marked regional differences in its action. Endogenous CGRP could be the inhibitory transmitter which, when released from capsaicin-sensitive fibers, participate in the control of ureteral motility.

The motor effect of the capsaicin-sensitive inhibitory innervation of the rat ureter.

Neurokinins activate a series of tetrodotoxin (TTX)-insensitive rhythmic contractions of the rat isolated ureter. Field stimulation or capsaicin (1-3 microM) produced a transient inhibition of neurokinin-activated ureteral motility in preparations from control but not from capsaicin-pretreated (50 mg/kg s.c.) rats. The inhibitory action of field stimulation but not that of capsaicin was prevented by TTX. It is concluded that a capsaicin-sensitive inhibitory innervation exists in the rat ureter.

Further studies on the mechanisms of the tachykinin-induced activation of micturition reflex in rats: evidence for the involvement of the capsaicin-sensitive bladder mechanoreceptors.

The relative ability of substance P, neurokinin A, neurokinin B and kassinin to activate the micturition reflex was investigated in urethane-anaesthetized rats. When administered topically neurokinin A, neurokinin B and kassinin were 14, 36 and 280 times, respectively, more potent than substance P to activate micturition. On the other hand substance P, neurokinin A and kassinin were practically equipotent (and neurokinin B was about 3-4 times less potent than substance P) to stimulate the contraction of the rat isolated bladder and to potentiate the contractions induced by electrical field stimulation. This indicates that neither a direct action on muscle cells nor a potentiating effect on efferent neurotransmission can account for the rank order of potency of tachykinins for activation of the micturition reflex. The ability of topical tachykinins to activate the micturition reflex was largely impaired in 2 months old rats pretreated with capsaicin (50 mg/kg s.c.) on their second day of life, indicating that integrity of the capsaicin-sensitive bladder mechanoreceptors is essential for the production of this effect. These findings indicate that an NK-B receptor, possibly located on sensory nerves in the bladder wall, participates in the tachykinin-induced activation of reflex micturition.

Isbufylline, a new xanthine derivative, inhibits airway hyperresponsiveness and airway inflammation in guinea pigs.

The pharmacological actions of the new xanthine, isbufylline, were evaluated in several models of airway hyperresponsiveness and airway inflammation in guinea pigs. At a dose (106 mumol kg-1 i.p.) providing complete protection against acetylcholine aerosol-induced dyspnea in the guinea pig, isbufylline inhibited platelet activating factor (PAF)- and antigen-induced eosinophil infiltration into bronchoalveolar lavage fluid 24 h after challenge of normal and actively immunized guinea pigs, respectively. In addition, this dose of isbufylline also inhibited capsaicin-induced extravasation of protein into bronchoalveolar lavage fluid. Isbufylline, 4.2 mumol kg-1 i.v., significantly inhibited PAF-induced bronchial hyper-responsiveness to i.v. histamine, without exerting evident bronchodilator activity. On the other hand the bronchodilator, salbutamol, at a dose (10.4 mumol kg-1 i.p.) shown to be equieffective to isbufylline (106 mumol kg-1 i.p.) for blocking acetylcholine aerosol-induced dyspnea, had no protective action against PAF- or antigen-induced eosinophil recruitment in bronchoalveolar lavage fluid, or against capsaicin-induced plasma protein extravasation. Furthermore, salbutamol (3.5 mumol kg-1) significantly potentiated allergen-induced cell infiltration and PAF-induced bronchial hyperresponsiveness. The results suggest that isbufylline can exert significant anti-inflammatory actions in guinea pig airways, in addition to its bronchodilator activity. These pharmacological activities are not shared by the beta 2-adrenoceptor agonist, salbutamol.

In vivo pharmacology of [beta Ala8]neurokinin A-(4-10), a selective NK-2 tachykinin receptor agonist.

We studied the effect of [beta Ala8]neurokinin A-(4-10), a newly developed selective NK-2 tachykinin receptor agonist, on various parameters in anaesthetized rats (blood pressure, urinary bladder motility, plasma extravasation) and guinea-pigs (salivation, increase of pulmonary insufflation pressure) as compared to the response produced by tachykinins. [beta Ala8]Neurokinin A-(4-10) was as active as, or more active than, neurokinin A (NKA) or NKA-(4-10) in producing rat bladder contraction or bronchospasm in guinea-pigs, two effects known to involve activation of NK-2 receptors. On the other hand, the synthetic peptide was weakly active, if active at all, in producing hypotension or plasma extravasation in the rat bladder as well as salivation in guinea-pigs, effects known to involve activation of NK-1 receptors. These findings provide evidence that [beta Ala8]NKA-(4-10) acts as a selective NK-2 agonist in vivo and that it can be used to explore the distribution and function of NK-2 receptors.