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Urothelium and Lamina Propria (LP) are considered an integrate sensory system which is able to control the detrusor activity. Complete supra‐sacral spinal cord lesions cause Neurogenic Detrusor Overactivity (NDO) whose main symptoms are urgency and incontinence. NDO therapy at first consists in anti‐muscarinic drugs; secondly, in intra‐vesical injection of botulinum toxin. However, with time, all the patients become insensitive to the drugs and decide for cystoplastic surgery. With the aim to get deeper in both NDO and drug's efficacy lack pathogenesis, we investigated the innervation, muscular and connective changes in NDO bladders after surgery by using morphological and quantitative methodologies. Bladder innervation showed a significant global loss associated with an increase in the nerve endings located in the upper LP where a neurogenic inflammation was also present. Smooth muscle cells (SMC) anomalies and fibrosis were found in the detrusor. The increased innervation in the ULP is suggestive for a sprouting and could condition NDO evolution and drug efficacy length. Denervation might cause the SMC anomalies responsible for the detrusor altered contractile activity and intra‐cellular traffic and favour the appearance of fibrosis. Inflammation might accelerate these damages. From the clinical point of view, an early anti‐inflammatory treatment could positively influence the disease fate.

Irritable bowel syndrome (IBS) is a highly prevalent gastrointestinal disorder characterized by periods of remission and exacerbation. Among the risk factors to develop IBS, psychosocial stress is widely acknowledged. The water avoidance stress repeatedly applied (rWAS) is considered effective to study IBS etio‐pathogenesis. Otilonium bromide (OB), a drug with multiple mechanisms of action, is largely used to treat IBS patients. Orally administered, it concentrates in the large bowel and significantly ameliorates the IBS symptomatology. Presently, we tested whether rWAS rats developed neuro‐muscular abnormalities in the distal colon and whether OB treatment prevented them. The investigation was focussed on the nitrergic neurotransmission by combining functional and morphological methodologies. The results confirm rWAS as reliable animal model to investigate the cellular mechanisms responsible for IBS: exposure to one‐hour psychosocial stress for 10 days depressed muscle contractility and increased iNOS expression in myenteric neurons. OB treatment counteracted these effects. We hypothesize that these effects are due to the corticotropin‐releasing factor (CRF) release, the main mediator of the psychosocial stress, followed by a CRF1receptor activation. OB, that was shown to prevent CRF1r activation, reasonably interrupted the cascade events that bring to the mechanical and immunohistochemical changes affecting rWAS rat colon.

Background and aim Muscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine. Methods The distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy. Key results Immunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFRα-positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell-to-cell contacts with smooth muscle cells and TCs/PDGFRα-positive cells but not with ICCs and enteric neurons. Conclusion & inferences This study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell-to-cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFRα-positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions.

Botulinum neurotoxin (BoNT) can counteract the highly frequent involuntary muscle contractions and the uncontrolled micturition events that characterize the neurogenic detrusor overactivity (NDO) due to supra-sacral spinal cord lesions. The ability of the toxin to block the neurotransmitter vesicular release causes the reduction of contractions and improves the compliance of the muscle and the bladder filling. BoNT is the second-choice treatment for NDO once the anti-muscarinic drugs have lost their effects. However, the toxin shows a time-dependent efficacy reduction up to a complete loss of activity. The cellular mechanisms responsible for BoNT effects exhaustion are not yet completely defined. Similarly, also the sites of its action are still under identification. A growing amount of data suggest that BoNT, beyond the effects on the efferent terminals, would act on the sensory system recently described in the bladder mucosa. The specimens from NDO patients no longer responding to BoNT treatment displayed a significant increase of the afferent terminals, likely excitatory, and signs of a chronic neurogenic inflammation in the mucosa. In summary, beyond the undoubted benefits in ameliorating the NDO symptomatology, BoNT treatment might bring to alterations in the bladder sensory system able to shorten its own effectiveness.

Otilonium bromide (OB) is a spasmolytic drug successfully used for the treatment of irritable bowel syndrome (IBS). Its efficacy has been attributed to the block of L‐ and T‐type Ca2+ channels and muscarinic and tachykinin receptors in the smooth muscle. Furthermore, in healthy rats, repeated OB administration modified neurotransmitter expression and function suggesting other mechanisms of action. On this basis, we investigated whether repeated OB treatment prevented the functional and neurochemical changes observed in the colon of rats underwent to wrap restrain stress (WRS) a psychosocial stressor considered suitable to reproduce the main IBS signs and symptoms. In control, WRS and OB/WRS rats functional parameters were measured in vivo and morphological investigations were done ex vivo in the colon. The results showed that OB counteracts most of the neurotransmitters changes caused by WRS. In particular, the drug prevents the decrease in SP‐, NK1r‐, nNOS‐, VIP‐, and S100β‐immunoreactivity (IR) and the increase in CGRP‐, and CRF1r‐IR. On the contrary, OB does not affect the increase in CRF2r‐IR neurons observed in WRS rats and does not interfere with the mild mucosal inflammation due to WRS. Finally, OB per se increases the Mr2 expression in the muscle wall and decreases the number of the myenteric ChAT‐IR neurons. Functional findings show a significantly reduction in the number of spontaneous abdominal contraction in OB treated rats. The ability of OB to block L‐type Ca2+ channels, also expressed by enteric neurons, might represent a possible mechanism through which OB exerts its actions.

Urinary bladder activity involves central and autonomic nervous systems and bladder wall. Studies on the pathogenesis of voiding disorders such as the neurogenic detrusor overactivity (NDO) due to suprasacral spinal cord lesions have emphasized the importance of an abnormal handling of the afferent signals from urothelium and lamina propria (LP). In the LP (and detrusor), three types of telocytes (TC) are present and form a 3D‐network. TC are stromal cells able to form the scaffold that contains and organizes the connective components, to serve as guide for tissue (re)‐modelling, to produce trophic and/or regulatory molecules, to share privileged contacts with the immune cells. Specimens of full thickness bladder wall from NDO patients were collected with the aim to investigate possible changes of the three TC types using histology, immunohistochemistry and transmission electron microscopy. The results show that NDO causes several morphological TC changes without cell loss or network interruption. With the exception of those underlying the urothelium, all the TC display signs of activation (increase in Caveolin1 and caveolae, αSMA and thin filaments, Calreticulin and amount of cisternae of the rough endoplasmic reticulum, CD34, euchromatic nuclei and large nucleoli). In all the specimens, a cell infiltrate, mainly consisting in plasma cells located in the vicinity or taking contacts with the TC, is present. In conclusion, our findings show that NDO causes significant changes of all the TC. Notably, these changes can be interpreted as TC adaptability to the pathological condition likely preserving each of their peculiar functions.

Evidence indicate that adenosine A2A receptor subtype is of critical importance in stroke. An overexpression of A2A adenosine receptors occurs at central level on neurons and microglia of ischemic striatum and cortex after focal ischemia. Adenosine A2A receptor subtype is localized not only at central level but also peripherally on blood cells, where it is known to exert antiinflammatory effect. Purpose of the present work was to investigate the putative neuroprotective effect of the adenosine A2A receptor agonist CGS21680 in a rat model of transient medial cerebral artery occlusion (MCAo). Transient cerebral ischemia was induced by 1h occlusion of MCA. CGS21680 (0.01 and 0.1mg/kg, i.p.) was administered starting 4h after ischemia according to a chronic protocol (twice/day for 7 days). CGS21680, at the dose of 0.1mg/kg transiently increased heart frequency but did not modify blood pressure. At the dose of 0.01mg/kg the drug did not modify either heart frequency or blood pressure. Following transient MCAo, CGS21680 at both doses protected from neurological deficit from the first day up to 7 days thereafter. At this time, it has reduced microgliosis, astrogliosis and improved myelin organization in the striatum and cytoarchitecture of the ischemic cortex and striatum. Two days after transient MCAo, CGS21680 has reduced the number of infiltrated granulocytes into the ischemic tissue. Data indicate that CGS21680 systemically administered is protective by immunosuppressive effects.

Several cells are endowed in the interstitial space of the connective tissue; among them, a peculiar type has been recently described and named telocyte (TC). The increasing interest on this cell type has allowed identifying it in almost all the organs. All TCs have a proper ultrastructural feature that makes them undoubtedly recognizable under the transmission electron microscope (TEM). On the contrary, a complex often confusing picture comes out from the immunohistochemical investigations either due to the technical procedures used or, intriguingly, to the possibility that diverse subtypes of TC might exist.Among the several markers used to label the TC, the most common are the CD34 and the PDGFRalpha, and, in many organs, the TC expresses both these markers. An exception is represented by the human urinary bladder where none of the TC, as recognized under the TEM, was double labelled. All the data indicate that TCs show immunohistochemical differences depending on the organ where they are located and/or the animal species.On the basis of their ubiquitous distribution, TCs are unanimously considered organizers of the connective tissue because of their ability to form 3-D networks. Close to this common role, numerous other roles have been attributed to the TC. Indeed, each of the TC subtype likely plays an own organ-/tissue-specific role contributing to different aspects of physiological regulation in the various anatomical niches they occupy.

Impaired gastric motility ascribable to a defective nitric oxide (NO) production has been reported in dystrophic (mdx) mice. Since relaxin upregulates NO biosynthesis, its effects on the motor responses and NO synthase (NOS) expression in the gastric fundus of mdx mice were investigated. Mechanical responses of gastric strips were recorded via force displacement transducers. Evaluation of the three NOS isoforms was performed by immunohistochemistry and Western blot. Wild-type (WT) and mdx mice were distributed into three groups: untreated, relaxin pretreated, and vehicle pretreated. In strips from both untreated and vehicle-pretreated animals, electrical field stimulation (EFS) elicited contractile responses that were greater in mdx than in WT mice. In carbachol-precontracted strips, EFS induced fast relaxant responses that had a lower amplitude in mdx than in WT mice. Only in the mdx mice did relaxin depress the amplitude of the neurally induced excitatory responses and increase that of the inhibitory ones. In the presence of l-NNA, relaxin was ineffective. In relaxin-pretreated mdx mice, the amplitude of the EFS-induced contractile responses was decreased and that of the fast relaxant ones was increased compared with untreated mdx animals. Responses to methacholine or papaverine did not differ among preparations and were not influenced by relaxin. Immunohistochemistry and Western blotting showed a significant decrease in neuronal NOS expression and content in mdx compared with WT mice, which was recovered in the relaxin-pretreated mdx mice. The results suggest that relaxin is able to counteract the altered contractile and relaxant responses in the gastric fundus of mdx mice by upregulating nNOS expression.

Glucagon-like peptide-2 (GLP-2) reduces mouse gastric tone and small intestine transit, but its action on large intestine motility is still unknown. The purposes of the present study were 1) to examine the influence of GLP-2 on spontaneous mechanical activity and on neurally evoked responses, by recording intraluminal pressure from mouse isolated colonic segments; 2) to characterize GLP-2 mechanism of action; and 3) to determine the distribution of GLP-2 receptor (GLP-2R) in the mouse colonic muscle coat by immunohistochemistry. Exogenous GLP-2 (0.1–300 nM) induced a concentration-dependent reduction of the spontaneous mechanical activity, which was abolished by the desensitization of GLP-2 receptor or by tetrodotoxin, a voltage-dependent Na+-channel blocker. GLP-2 inhibitory effect was not affected by Nω-nitro-l-arginine methyl ester (a nitric oxide synthase inhibitor), apamin (a blocker of small conductance Ca2+-dependent K+ channels), or [Lys1,Pro2,5,Arg3,4,Tyr6]VIP7–28 (a VIP receptor antagonist), but it was prevented by atropine or pertussis toxin (PTX), a Gi/o protein inhibitor. Proximal colon responses to electrical field stimulation were characterized by nitrergic relaxation, which was followed by cholinergic contraction. GLP-2 reduced only the cholinergic evoked contractions. This effect was almost abolished by GLP-2 receptor desensitization or PTX. GLP-2 failed to affect the contractile responses to exogenous carbachol. GLP-2R immunoreactivity (IR) was detected only in the neuronal cells of both plexuses of the colonic muscle coat. More than 50% of myenteric GLP-2R-IR neurons shared the choline acetyltransferase IR. In conclusion, the activation of GLP-2R located on cholinergic neurons may modulate negatively the colonic spontaneous and electrically evoked contractions through inhibition of acetylcholine release. The effect is mediated by Gi protein.

Adenosine is a potent biological mediator, the concentration of which increases dramatically following brain ischaemia. During ischaemia, adenosine is in a concentration range (muM) that stimulates all four adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)). In recent years, evidence has indicated that the A(2A) receptor subtype is of critical importance in stroke. We have previously shown that 24 h after medial cerebral artery occlusion (MCAo), A(2A) receptors up-regulate on neurons and microglia of ischaemic striatum and cortex and that subchronically administered adenosine A(2A) receptor antagonists protect against brain damage and neurological deficit and reduce activation of p38 mitogen-activated protein kinase (MAPK) in microglial cells. The mechanisms by which A(2A) receptors are noxious during ischaemia still remain elusive. The objective of the present study was to investigate whether the adenosine A(2A) antagonist SCH58261 affects JNK and MEK1/ERK MAPK activation. A further aim was to investigate cell types expressing activated JNK and MEK1/ERK MAPK after ischaemia. We hereby report that the selective adenosine A(2A) receptor antagonist, SCH58261, administered subchronically (0.01 mg/kg i.p) 5 min, 6 and 20 h after MCAo in male Wistar rats, reduced JNK MAPK activation (immunoblot analysis: phospho-JNK54 isoform by 81% and phospho-JNK46 isoform by 60%) in the ischaemic striatum. Twenty-four hours after MCAo, the Olig2 transcription factor of oligodendroglial progenitor cells and mature oligodendrocytes was highly expressed in cell bodies in the ischaemic striatum. Immunofluorescence staining showed that JNK MAPK is maximally expressed in Olig2-stained oligodendrocytes and in a few NeuN stained neurons. Striatal cell fractioning into nuclear and extra-nuclear fractions demonstrated the presence of Olig2 transcription factor and JNK MAPK in both fractions. The A(2A) antagonist reduced striatal Olig 2 transcription factor (immunoblot analysis: by 55%) and prevented myelin disorganization, assessed by myelin-associated glycoprotein staining. Twenty-four hours after MCAo, ERK1/2 MAPK was highly activated in the ischaemic striatum, mostly in microglia, while it was reduced in the ischaemic cortex. The A(2A) antagonist did not affect activation of the ERK1/2 pathway. The efficacy of A(2A) receptor antagonism in reducing activation of JNK MAPK in oligodendrocytes suggests a mechanism of protection consisting of scarring oligodendrocyte inhibitory molecules that can hinder myelin reconstitution and neuron functionality.

The hippocampus (dentate gyrus DG plus Cornu Ammonis, CA) is vulnerable to neuropathological events such as ischemia. The DG is a region where neurogenesis takes place and it has been demonstrated that ischemia stimulates neurogenesis. Nitric oxide (NO) plays a major role in ischemic damage evolution and increases in rat hippocampus after ischemia. No information is available on the presence of nNOS-immunoreactive(IR) neurons in the hippocampus of ischemic animals; whereas, the presence of the iNOS protein has been reported in the DG after focal ischemia. We evaluated, immunohistochemically, the cell types expressing nNOS and iNOS in the rat hippocampus by 24 up to 144 h after transient middle cerebral artery occlusion to ascertain whether ischemia induces changes in nNOS or iNOS expression and whether a relationship exists between these changes and the animal survival. nNOS-IR interneurons were detected in control and ischemic rats; in the latter, their number was significantly decreased at all time points. iNOS-IR interneurons appeared in the hippocampus of ischemic rats at 24 h; their number was significantly higher in the animals with longer survival and did not change at later time points. More than 50% of the nNOS-IR interneurons co-expressed iNOS-IR. All these changes were seen both in the ipsilateral and contralateral hippocampus. In conclusion, the focal ischemia affects the hippocampus which responds bilaterally to the injury. We hypothesize that the decrease in the nNOS-IR neurons is likely due to either a neuronal loss or a switching towards the iNOS production which, by inducing neurogenesis, might compensate the neuronal loss.

Background Animal models proposed to reproduce some of the human irritable bowel syndrome (IBS) symptoms are based on the hypothesis that psychosocial stressors play a pivotal role in the IBS etio-pathology. We investigated the wrap restraint stress (WRS) model with the aim to analyze the morphological changes of the entire colonic wall of these animals that showed some of the human IBS symptoms such as visceral hypersensitivity. Methods Male Wistar rats were used and WRS was maintained for 2 h. Abdominal contractions (AC) were recorded in the colon-rectum by balloon distension. Fecal pellets were quantitated. Colonic specimens were examined by routine histology, immunohistochemistry and western blot. Key Results WRS animals were characterized by: (i) increase in AC number and fecal pellets mean weight; (ii) clusters of mononucleated cells, increase in eosinophilic granulocytes and mast cells in the mucosa; (iii) increase in CGRP-immunoreactive (IR) nerve fibers in the lamina propria; (iv) decrease in myenteric NK1r-IR and nNOS-IR neurons and in submucous nNOS-IR neurons; (v) decrease in SP-IR nerve fibers in the muscle wall; (vi) reduction in S100β-IR glia in the entire colonic wall; (vii) increase in CRF1r-IR myenteric neurons; (viii) no change in ChAT-IR neurons, smooth muscle cells and interstitial cells of Cajal. Conclusions and Inferences The present results support the consistency of the WRS as a potential model where part of the human IBS signs and symptoms are reproduced. The changes in glial cells and in excitatory and inhibitory neurotransmitters might represent the substrate for the dysmotility and hypersensitivity.

Ablation of the myenteric plexus in mouse colon with the detergent benzalkonium chloride (BAC) is followed by considerable recovery of the nerves, indicating that this plexus is capable of regeneration and has plasticity. Interstitial cells of Cajal (ICC) are closely associated with enteric nerves, and the acquisition and maintenance of their adult phenotype are nerve-dependent. Little is known about the regenerative processes of ICC or about the possible dependence of these processes on neurons. To address these questions, we ablated the myenteric plexus in the mouse colon with BAC and followed changes in the adjacent ICC (ICC-MP) from day 2 to day 70 after treatment, by using c-kit-immunohistochemistry and electron microscopy. In the untreated area, c-kit-positive cells and ICC-MP with normal ultrastructural features were always present. The region partially affected by BAC contained some c-kit-positive cells, and either normal or vacuolated ICC-MP were observed by electron microscopy. Moreover, at days 60-70, ICC-MP with particularly extended rough endoplasmic reticulum were present in this area. In the treated area, either denervated or reinnervated, c-kit-positive cells were always absent. By day 14 after BAC treatment, nerve fibers had started to grow back into the treated region and, in the reinnervated area, cells with fibroblast-like features appeared and were seen to contact both nerve endings and smooth muscle cells and to acquire some typical ICC features. Thus, ICC are vulnerable to external insult but appear to have some ability to regenerate.

The tachykinin substance P (SP) acts on the gut muscle coat via its preferred receptor, neurokinin 1 (NK1r). In the mouse ileum, NK1r-immunoreactivity (NK1r-IR) was detected in neurons, in the interstitial cells of Cajal at the deep muscular plexus (ICC-DMP) and the myoid cells of the villi. SP-IR was detected in neurons and varicose nerve fibers, which were especially numerous at the DMP and closely associated with the ICC-DMP. In mice with a mutation in the W locus (c-kit mutant animals), innervation is suggested to be normal although few studies have actually tested this hypothesis. Indeed, studies demonstrating ICC-DMP integrity are lacking and whether SP- and NK1r-IR are normal in these animals has not been investigated. Our aim was to perform an immunohistochemical study on the ileum of a strain of heterozygous mice with a mutation in the W locus, the We/+ mice, to test this hypothesis. SP-IR nerve fibers were significantly more numerous than in wild type mice; NK1r-IR was clustered on the plasma membrane and also intracytoplasmatic in the neurons, but absent in the ICC-DMP. The richness in SP-IR nerve fibers and the NK1r-IR distribution in the neurons, similar to that of activated cells, might be attempts to compensate for the SP preferred receptor absence at the ICC-DMP. In conclusion, SP content and NK1r expression are noticeably different in c-kit mutants with respect to wild type mice, and probably causing an anomalous tachykininergic control of intestinal motility. Physiological studies on W mutant mice have to take into account that innervation in this animal model is affected by the c-kit mutation.

The physiology and pathophysiology of the network of interstitial cells of Cajal associated with the deep muscular plexus (ICC-DMP) of the small intestine are still poorly understood. The objectives of the present study were to evaluate the effects of inflammation associated with Trichinella spiralis infection on the ICC-DMP and to correlate loss of function with structural changes in these cells and associated structures. We used immunohistochemistry, electron microscopy, and assessment of distention-inducing electrophysiological parameters in vitro. Damage to ICC-DMP was associated with a loss of distention-induced patterns of electrical activity normally associated with distention-induced peristalsis. Consistently, the timing of recovery of ICC-DMP paralleled the timing of recovery of the distention-induced activity. Nerve varicosities associated with ICC-DMP including cholinergic nerves, assessed by immunoelectron microscopy and whole mount double labeling, paralleled injury to ICC-DMP thus contributing to impaired excitatory innervation of smooth muscle cells. Major additional changes included a remodeling of the inner circular muscle layer, which may affect long-term sensitivity to distention after infection. In conclusion, transient injury to ICC-DMP in response to T. spiralis infection is severe and associated with a complete lack of distention-induced burst-type muscle activity.

Vanilloid receptor type 1 (TRPV1) is expressed in a capsaicin-sensitive and peptide-containing sub-population of primary sensory nerves that in the rat stomach seems involved in regulation of chlorhydropeptic secretion and gastroprotection. Our aim was to identify which cell types express TRPV1 in the human stomach in order to gain a better insight in the role of this receptor in the regulation of HCl secretion. Immunohistochemistry, by using three different commercially available anti-capsaicin antibodies, in situ hybridisation and Western blot analysis were performed on fragments surgically obtained from the gastric body on the large curvature. TRPV1 labelling was found in the parietal cells at the level of intra-cytoplasmatic granules matching mitochondrial features and distribution. Immunolabelled neurons and nerve fibres were also seen, the latter numerous in the submucosa and mucosa and often ending close to the parietal cells. TRPV1 presence was confirmed by Western blot analysis and in situ hybridisation. TRPV1 presence in nerve structures and parietal cells suggests the possibility of a combined effect of both neuronal and epithelial TRPV1 on chlorhydropeptic secretion. The presumed TRPV1 mitochondrial location inside parietal cells is in favour of the existence of a local pathway of auto-regulation of HCl secretion. Therefore, TRPV1 might modulate chlorhydropeptic secretion in the human stomach through more complex pathways than previously thought.

Adenosine A(2A) receptor antagonists have been proved protective in different ischemia models. In this study we verified if the protective effect of the selective A(2A) antagonist, SCH 58261, could be attributed to the reduction of the excitatory amino acid outflow induced by cerebral focal ischemia. A vertical microdialysis probe was inserted into the striatum of male Wistar rats and, after 24 h, permanent right intraluminal middle cerebral artery occlusion (MCAo) was induced. Soon after waking, rats showed a definite contralateral turning behavior, which persisted up to 7 h after MCAo. During 4 h after MCAo, glutamate, aspartate, GABA, adenosine and taurine outflow increased. SCH 58261 (0.01 mg/kg, i.p.), administered 5 min after MCAo, suppressed turning behavior and significantly reduced the outflow of glutamate, aspartate, GABA and adenosine. At 24 h after MCAo, the rats showed severe sensorimotor deficit and damage in both the striatum and cortex. SCH 58261 significantly reduced cortical damage but did not protect against the sensorimotor deficit. The protective effect of SCH 58261 against turning behavior and increased outflow of excitatory amino acids in the first hours after MCAo suggests the potential utility of selective adenosine A(2A) antagonists when administered in the first hours after ischemia. Furthermore, this study, for the first time, proposes that turning behavior after permanent intraluminal MCAo, be used as a precocious index of neurological deficit and neuronal damage.

Background Otilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well-characterized, little is known about the effects of chronic administration of OB on enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function. Methods Adult Sprague–Dawley rats were treated with OB in drinking water at a dose of 2 mg/kg for 30 days. The colons of OB-treated and age-matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology. Key Results Using immunohistochemistry, chronic OB administration did not change total neuron number, assessed by anti-Hu IR, but resulted in a significant increase in NK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. Chronic OB administration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonic ICC, detected by Kit IR, were not altered nor were slow waves or smooth muscle membrane potential. Conclusions & Inferences Chronic treatment with OB caused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.

A gene located on the X chromosome is responsible for the transcription of several mRNA and related dystrophin isoforms. Lack or truncated expression of the 427-kDa, full-length isoform in skeletal muscle results in Duchenne muscular dystrophy (DMD). Patients with DMD, as well as mdx mice, a mutant strain also lacking this isoform, show gastrointestinal dismotilities. The present aim was to identify the cell types that express full-length dystrophin in the gastrointestinal tract. An immunohistochemical study was performed using an antibody specific for this isoform, and double labelings were made for interstitial cells of Cajal (ICC) identification and to verify whether all neurons express full-length dystrophin. Three different fixation procedures were used. The results showed that ICC, enteric neurons, and smooth muscle and myoid cells expressed full-length dystrophin. In ICC and neurons, dystrophin-immunoreactive patches were irregularly distributed at the cell contour and within the cytoplasm. In smooth muscle and myoid cells, regularly spaced dystrophin-immunoreactive bars were located along the cell contour. Labeling intensity varied according to fixation procedure. The different subcellular distributions of dystrophin immunoreactivity might reflect diverse roles played by full-length isoforms in each cell type. Dystrophin loss in cells involved in gastrointestinal motility might explain the gastrointestinal symptomatology affecting DMD patients and mdx mice.

Injection into the nucleus basalis of the rat of preaggregated Abeta(1-42) produced a congophylic deposit and microglial and astrocyte activation and infiltration and caused a strong inflammatory reaction characterized by IL-1beta production, increased inducible cyclooxygenase (COX-2), and inducible nitric oxide synthase (iNOS) expression. Many phospho-p38MAPK-positive cells were observed around the deposit at 7 days after Abeta injection. Phospho-p38MAPK colocalized with activated microglial cells, but not astrocytes. The inflammatory reaction was accompanied by cholinergic hypofunction. We investigated the protective effect of the selective COX-2 inhibitor rofecoxib in attenuating the inflammatory response and neurodegeneration evoked by Abeta(1-42). Rofecoxib (3 mg/kg/day, 7 days) reduced microglia and astrocyte activation, iNOS induction, and p38MAPK activation to control levels. Cholinergic hypofunction was also significantly attenuated by treatment with rofecoxib. We show here for the first time in vivo the pivotal role played by the p38MAPK microglial signal transduction pathway in the inflammatory response to the Abeta(1-42) deposit.

Information on equipment and subcellular distribution of nitric oxide synthase (NOS) isoforms in myenteric neurons and pacemaker cells (ICC) might help to identify nitric oxide (NO) pathway(s) acting on gastrointestinal motility. In sections of mouse colon labelled with neuronal (n)NOS, endothelial (e)NOS and inducible (i)NOS antibodies, all myenteric neurons co-expressed eNOS and iNOS and a subpopulation of them co-expressed nNOS. ICC co-expressed nNOS and eNOS. In the neurons, nNOS-labeling was intracytoplasmatic, in the ICC at cell periphery. In both cell types, eNOS-labeling was on intracytoplasmatic granules, likely mitochondria. In conclusion, myenteric neurons and ICC co-express several NOS isoforms with specific subcellular distribution. Different nNOS splice variants are presumably present: intracytoplasmatic nNOSbeta and nNOSalpha producing neurogenic NO, plasma membrane-bound nNOSalpha producing ICCgenic NO. eNOS might be implicated in mitochondrial respiration and, in ICC, also in pacemaker activity. Neurons express iNOS also in basal condition.

Dystrophin, a membrane-associated protein, plays relevant roles in cell functions. Its lack or trunkated expression results in Duchenne muscular dystrophy (DMD), a pathology associated with alterations in gastrointestinal motility considered to be neural in origin. No data are available on the presence of dystrophin in myenteric neurones. We labelled mouse myenteric neurones with DYS1-, DYS2-, DYS3-antibodies; staining was located on the perikarya and processes, with no differences in distribution or intensity among the antibodies; the western immunoblot analysis indicated that myenteric neurones express several dystrophin isoforms; anti-dystrophins/anti-neuronal specific enolase double-labeling confirmed that all neurones express dystrophin. Dystrophin in myenteric neurones might play a role in cytoskeletal organization, axonal transport and signal pathways; its lack might cause the intestinal motor abnormalities reported in DMD patients.

Tachykinin receptors NK1r, NK2r and NK3r bind tachykinins with different affinities and share pharmacological and molecular differences among animal species. NK1r, NK2r, NK3r and tachykinin (SP/NKA) distribution was studied by immunohistochemistry in the ileum of mouse since no data are available for this species. The results were then compared to those obtained in the rat and guinea pig either by us or by others to ascertain interspecies similarities and/or differences. NK1r- and NK3r-immunoreactivity (IR) were detected in neurons and NK1r-IR in the interstitial cells of Cajal at the deep muscular plexus. At variance with rat and guinea pig, NK1r-IR was also found in the myoid cells of the villi, while NK2r-IR was never detected in nerve varicosities. This latter datum suggests that the NK2r does not play a presynaptic role in the mouse. Unexpectedly, a high NK2r-IR and the presence of NK3r-IR were observed at the inner portion of the circular muscle layer in the mouse as well as in the rat and guinea pig, demonstrating a subregional distribution of these receptors. Tachykinin distribution did not show noticeable species-related differences. The present findings show species-related differences in the tachykinin receptor distribution that might be related to a different tachykinin control of intestinal motility.

The distribution of NK2 tachykinin receptors-immunoreactivity (NK2r-IR) in the guinea-pig ileum and the co-distribution of NK2r-IR with substance P-immunoreactivity (SP)-IR were investigated. NK2r-IR was detected in varicose fibers of myenteric and submucous ganglia and nerve strands, in the longitudinal and circular muscle layers and at the deep muscular plexus (DMP). Except for the submucous plexus, some of the NK2r-IR varicose fibers were co-distributed with SP-IR ones and quantitative analysis showed significant regional differences in the percentages of these fibers. These results demonstrate that presynaptic NK2 receptors are located at varicose fibers likely originating from motor neurons projecting to muscle layers and DMP, and from interneurons. Furthermore, the NK2r/SP-IR co-distribution suggests that some of these receptors are autoreceptors on SP nerve endings.

We studied the effects of relaxin on ileal contractility in normal and dystrophic (mdx) mice. Ileal preparations from male normal and mdx mice showed spontaneous myogenic contractions whose amplitude was significantly higher in the latter ones. Relaxin added to the bath medium together with l-arginine depressed the amplitude of the spontaneous contractions in the mdx mice to a level similar to that of the normal mice. The nitric oxide synthase (NOS) inhibitor L-N(G)-nitroarginine reverted this effect. In mdx mice pretreated for 18 hours with relaxin, spontaneous motility was greatly reduced in amplitude, resembling that of the normal mice. Concurrently, iNOS expression in the muscle coat was markedly increased. Therefore, in mdx mice, relaxin can restore an ileal motility pattern similar to that of the normal mice by upregulating endogenous NO biosynthesis.

In the central nervous system (CNS) ATP and adenosine act as transmitters and neuromodulators on their own receptors but it is still unknown which part of extracellular adenosine derives per se from cells and which part is formed from the hydrolysis of released ATP. In this study extracellular concentrations of adenosine and ATP from the rat striatum were estimated by the microdialysis technique under in vivo physiological conditions and after focal ischemia induced by medial cerebral artery occlusion. Under physiological conditions, adenosine and ATP concentrations were in the range of 130 nmol/L and 40 nmol/L, respectively. In the presence of the novel ecto-ATPase inhibitor, PV4 (100 nmol/L), the extracellular concentration of ATP increased 12-fold to ~360 nmol/L but the adenosine concentration was not altered. This demonstrates that, under physiological conditions, adenosine is not a product of extracellular ATP. In the first 4 h after ischemia, adenosine increased to ~690 nmol/L and ATP to ~50 nmol/L. In the presence of PV4 the extracellular concentration of ATP was in the range of 450 nmol/L and a significant decrease in extracellular adenosine (to ~270 nmol/L) was measured. The contribution of extracellular ATP to extracellular adenosine was maximal in the first 20 min after ischemia onset. Furthermore we demonstrated, by immunoelectron microscopy, the presence of the concentrative nucleoside transporter CNT2 on plasma and vesicle membranes isolated from the rat striatum. These results are in favor that adenosine is transported in vesicles and is released in an excitation–secretion manner under in vivo physiological conditions. Early after ischemia, extracellular ATP is hydrolyzed by ecto-nucleotidases which significantly contribute to the increase in extracellular adenosine. To establish the contribution of extracellular ATP to adenosine might constitute the basis for devising a correct putative purinergic strategy aimed at protection from ischemic damage.

Salvianolic acid B (SalB) represents the most characteristic constituent of Salvia miltiorrhiza Bge. with a strong free radicals scavenger activity. This property may be useful in the treatment of some severe chronic diseases, where there is an imbalance of reactive oxygen species formation and where intracellular reactive oxygen and nitrogen species level can cause severe cell damage and even cell death. In particular, SalB can protect against the oxidative stress as well as the antioxidant superoxide dismutase and reduced activity of glutathione, important determinants of neuropathological and behavioural consequences in neuropathic pain. This is a chronic disease defined by the WHO as an untreatable illness because therapeutics are unsatisfactory in many cases and there is an urgent need to discover and develop novel active drugs. In the present work, SalB has been extracted and purified with an efficient and rapid method from the roots and rhizome of S. miltiorrhiza Bge. It was firstly submitted to pharmacological studies using the paw-pressure test, in an animal model of neuropathic pain where a peripheral mono neuropathy was produced by a chronic constriction injury of the sciatic nerve. SalB was effective against mechanical hyperalgesia when administered intraperitoneally at the dose of 100mg/kg, 15 min after administration. Due to the poor chemical stability and bioavailability of SalB, liposomes were developed as drug carriers for parental administration. SalB-loaded liposomes were characterised in terms of particle size, polydispersity index, encapsulation efficacy and morphology. According to the in vivo studies, encapsulation, especially into PEGylated liposomes, increased and prolonged the antihyperalgesic activity 30 min after i.p. administration and the effect was still significant at 45 min. Thus, PEGylated formulation ameliorated the performance of drug delaying, increasing and prolonging in time its antihyperalgesic effect.

Artemisinin (qinghaosu), a unique endoperoxide sesquiterpene lactone isolated from Artemisia annua L., is a very active antimalarial drug, including severe and cerebral malaria. However, its therapeutical efficacy is limited due to its scarce bioavailability. In this article, artemisinin-loaded conventional and polyethylene glycol (PEGylated) liposomes were proposed as carriers to increase biopharmaceutical properties of the drug. Encapsulation efficacy was determined by high-performance liquid chromatography/diode array detection/electrospray ionization-mass spectrometry, dimensional analysis was performed by dynamic light scattering, and morphology was performed by trasmission electron microscopy. After dialysis, both liposomal formulations showed an encapsulation efficacy of more than 70%; mean diameter of all the artemisinin-loaded vesicles was approximately 130-140 nm. The polydispersity index of the formulations ranged from 0.2 to 0.3 and resulted as suitable for intraperitoneal (i.p.) administration. Pharmacokinetic profile and the main pharmacokinetic parameters of the carriers were evaluated in healthy mice i.p. Free artemisinin was rapidly cleared from plasma and hardly detected 1 hour after administration. Conversely, both liposomal formulations showed much longer blood-circulation time than free artemisinin; artemisinin was still detectable after 3 and 24 hours of administration, respectively, for conventional and PEGylated liposomes. AUC(0-24 h) values were increased by approximately 6 times in both of the liposomal formulations, in comparison with free artemisinin. A strong effect of formulation on the half-life of artemisinin was enhanced by more than 5-fold by the incorporation of PEG into liposomes. Liposomes loaded with artemisinin, especially the long-circulating vesicles, could really represent a new strategy for developing smart, well-tolerated, and efficacious therapeutic nanocarriers to treat tumors, but could also be very useful to treat parasitic disease.

CD117 (or c-kit) is expressed by the interstitial cells of Cajal (ICC), which are located within the gastrointestinal (GI) muscle coat and directly involved in its motility. CD34 is expressed by several cell types some of which have features and location resembling the ICC; however, a sure identification of these cells is still lacking. In order to establish whether the CD34-positive cells of the human GI tract are to be considered as ICC subpopulation or a novel independent cell type, and to hypothesize their nature and role, we verified CD34 and CD117 receptor expression under light and fluorescence microscope and performed a routine and a CD34-immuno-electron microscopy. CD34-positive cells were seen in the entire human GI tract. In the muscularis propria, shared morphologies similar to the c-kit-positive cells, in the submucosa, resembled fibroblasts. Their ultrastructure resembled that of the fibrocytes/fibroblasts and of the interstitial Cajal-like cells (ICLC). Double labelling and immunoelectro-microscopy demonstrated that they are unequivocally different to the ICC and, due to the similarities with the ICLC, we identified them as ICLC. The novelty of these results is that two types of interstitial cells are present in the GI muscle coat of humans: the ICC and the ICLC. We hypothesize a mechanical role for the septal ICLC, those at the myenteric plexus level and those bordering the muscle layers; a helping role in neurotransmission is proposed for the ICLC intercalated with the intramuscular ICC, possibly in spreading the slow waves generated by the ICC. Furthermore, the possibility that the ICLC represent the adult mesenchymal stromal cells able to guarantee the ICC renewal deserves to be considered.

Medium spiny GABAergic projection neurons are progressively lost in Huntington's disease (HD), whereas there is preferential sparing of the few interneurons co-expressing NPY, somatostatin and neuronal nitric oxide synthase. We investigated the effect of the selective adenosine A(2A) receptor antagonist SCH58261 (0.01 mg/kg, acutely and chronically administered i.p.) on nNOS striatal expression and motor impairment in R6/2 transgenic mice in clearly symptomatic phase (10-11-week old). SCH58261 chronically administered increased the number of nNOS-immunoreactive neurons (nNOS-IR) in the striatum of R6/2 mice. No glial activation was detected in the striatum or cortex. SCH58261 also improved walking in the inclined plane test but not motor capability evaluated by the rotarod test. These findings demonstrate for the first time a role of adenosine A(2A) receptors in regulating nNOS expression in the striatum. We suggest that the protective effect of A(2A) antagonism in HD is related to the increase in striatal nNOS-IR neurons.

Altered nitric oxide (NO) production/release is involved in gastrointestinal motor disorders occurring in dystrophic (mdx) mice. Since the hormone relaxin (RLX) can upregulate NO biosynthesis, its effects on spontaneous motility and NO synthase (NOS) expression in the ileum of dystrophic (mdx) mice were investigated. Mechanical responses of ileal preparations were recorded in vitro via force-displacement transducers. Evaluation of the expression of NOS isoforms was performed by immunohistochemistry and Western blot. Normal and mdx mice were distributed into three groups: untreated, RLX pretreated, and vehicle pretreated. Ileal preparations from the untreated animals showed spontaneous muscular contractions whose amplitude was significantly higher in mdx than in normal mice. Addition of RLX, alone or together with l-arginine, to the bath medium depressed the amplitude of the contractions in the mdx mice, thus reestablishing a motility pattern typical of the normal mice. The NOS inhibitor NG-nitro-l-arginine (l-NNA) or the guanylate cyclase inhibitor ODQ reversed the effects of RLX. In RLX-pretreated mdx mice, the amplitude of spontaneous motility was reduced, thus resembling that of the normal mice, and NOS II expression in the muscle coat was increased in respect to the vehicle-pretreated mdx animals. These results indicate that RLX can reverse the altered ileal motility of mdx mice to a normal pattern, likely by upregulating NOS II expression and NO biosynthesis in the ileal smooth muscle.

Argininosuccinate-synthetase (ASS), argininosuccinate-lyase (ASL) and nitric oxide synthase (NOS) act in the l-arginine-NO-l-citrulline cycle. In the rat brain, ASS is expressed in neurons, ASL in neurons and astroglia in the striatum, both are co-expressed with nNOS in medium-sized neurons. Microglia cells express iNOS and ASS after activation but no information is available on ASL and on ASS/ASL/iNOS co-expression in this glial population. The present aim was to ascertain, by immunohistochemistry, whether the microglia cells of the rat striatum and fronto-parietal cortex express ASL and ASS in control conditions and after transient ischemia induced by middle cerebral artery occlusion, and whether ASL and ASS are co-expressed with iNOS. The study was conducted 24, 72 and 144 h after reperfusion in two groups of ischemic rats with different tissue damage and survival. ASS and ASL are not expressed by microglia cells in controls while are present in most of the activated microglia cells in the ischemic rats. In those animals with longer survival, ASS and ASL were no more detectable at 144 h, while, in the animals with shorter survival, they were co-expressed with iNOS, but only at 72 h. In the cortex, at variance with the striatum, almost all of nNOS-positive neurons co-expressed ASS and ASL. In conclusion, only activated microglia cells express ASS and ASL, this expression precedes that of iNOS and does not necessarily imply its appearance. Therefore, local factors such as the NO produced by nNOS/ASS/ASL-positive neurons, could influence ASS/ASL-positive microglia cells avoiding or allowing the induction, in these cells, of iNOS.

Chronic unexplained nausea and vomiting (CUNV) is a debilitating disease of unknown cause. Symptoms of CUNV substantially overlap with those of gastroparesis, therefore the diseases may share pathophysiologic features. We investigated this hypothesis by quantifying densities of interstitial cells of Cajal (ICCs) and mapping slow-wave abnormalities in patients with CUNV vs controls.Clinical data and gastric biopsy specimens were collected from 9 consecutive patients with at least 6 months of continuous symptoms of CUNV but normal gastric emptying who were treated at the University of Mississippi Medical Center, and from 9 controls (individuals free of gastrointestinal disease or diabetes). ICCs were counted and ultrastructural analyses were performed on tissue samples. Slow-wave propagation profiles were defined by high-resolution electrical mapping (256 electrodes; 36 cm(2)). Results from patients with CUNV were compared with those of controls as well as patients with gastroparesis who were studied previously by identical methods.Patients with CUNV had fewer ICCs than controls (mean, 3.5 vs 5.6 bodies/field, respectively; P.05), with mild ultrastructural abnormalities in the remaining ICCs. Slow-wave dysrhythmias were identified in all 9 subjects with CUNV vs only 1 of 9 controls. Dysrhythmias included abnormalities of initiation (stable ectopic pacemakers, unstable focal activities) and conduction (retrograde propagation, wavefront collisions, conduction blocks, and re-entry), operating across bradygastric, normal (range, 2.4-3.7 cycles/min), and tachygastric frequencies; dysrhythmias showed velocity anisotropy (mean, 3.3 mm/s longitudinal vs 7.6 mm/s circumferential; P.01). ICCs were less depleted in patients with CUNV than in those with gastroparesis (mean, 3.5 vs 2.3 bodies/field, respectively; P.05), but slow-wave dysrhythmias were similar between groups.This study defined cellular and bioelectrical abnormalities in patients with CUNV, including the identification of slow-wave re-entry. Pathophysiologic features of CUNV were observed to be similar to those of gastroparesis, indicating that they could be spectra of the same disorder. These findings offer new insights into the pathogenesis of CUNV and may help to inform future treatments.

The focal ischemia obtained in an animal model of middle cerebral artery occlusion (MCAo) causes the "core" of damage in the striatum and the "penumbra" of damage in the fronto-parietal cortex. The latter is mainly functionally affected and shows changes in nNOS and iNOS expression during the acute phase of ischemia. With the aim to study possible relationships between these changes and the affection entity during the animal recovery, we investigated from 24 up to 144 h after reperfusion the expression and content of these two NOS isoforms in the neurons and microglia and the degree of microglia reactivity in the fronto-parietal cortices of rats undertaken to transient MCAo. Evaluation of motor-sensory performances and survival allowed dividing the animals into two groups. Immunohistochemistry, western blot and quantitative analysis demonstrated, both in the ischemic and contralateral cortex of the rats with longer survival, wellness and significantly increased number of the nNOS-IR neurons at 24 h and moderately activated microglia up to 144 h. In the rats not recovering, injured and significantly decreased nNOS-IR neurons, intensely activated microglia and appearance of iNOS-IR were seen at all time points. In conclusion, since the recovery occurs when nNOS-IR neurons are greatly increased, we presume nNOS protect the tissue likely controlling the passage from the state of reactive to that of activated microglia. Moreover, the morphological signs of wellness and the two-fold increase in number of the nNOS-IR neurons appear to be characteristic of the "penumbra" area and could explain why this region is mainly functionally affected.

In neurodegenerative diseases, an increased number of neuronal nitric oxide synthase (nNOS)-positive neurons was reported, but nothing is known on which are the neurons induced to express nNOS. Argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and nNOS act in the L-arginine-NO-L-citrulline cycle permitting a correct NO production. In the brain, nNOS-positive neurons co-expressing ASS were known, while those co-expressing ASL were not demonstrated. We investigated by immunohistochemistry the presence of these types of neurons in the rat striatum to verify whether there was a correlation between their changes due to neurotoxic insults and animal survival. Transient ischemia, a neurodegenerative insult model, was induced in rat brain by 2 h of middle cerebral artery occlusion. The striatum, the core of ischemia, was examined at 24, 72 and 144 h after reperfusion and compared with that of rats in normal condition. ASS, ASL and nNOS-positive neurons, some of the latter also expressing ASS and ASL, were present both in normal and ischemic conditions. At 24 h after reperfusion, the number of the nNOS-positive neurons and the percentage of those co-expressing ASS and ASL were significantly increased in the animals with a longer survival and at 144 h after ischemia there was an almost complete restore of the number and/or percentage of these neurons. We hypothesize that the neurons induced to express nNOS were the ASS- and ASL-positive ones and that the neurons co-expressing nNOS, ASS and ASL, since having the enzymes necessary to maintain a correct NO production, might protect from neurotoxic insults.

We investigated the protective effect of subchronic treatment of the A2A receptor antagonist, SCH 58261 (0.01 mg/kg, i.p.), administered 5 min, 6 h and 15 h after permanent right middle cerebral artery occlusion (MCAo). Twenty-four hours after ischemia, an extensive pallid area, evaluated by cresyl violet staining, is evident in the vascular territories supplied by the MCA, the striatum and the sensory motor cortex. The pallid area reflects the extent of necrotic neurons. Soon after waking, rats showed a definite contralateral turning behavior which was significantly reduced by SCH 58261 treatment. Twenty-four hours after MCAo, SCH 58261 significantly improved the neurological deficit and reduced ischemic damage in the striatum and cortex. Phospho-p38 mitogen-activated protein kinase (MAPK), evaluated by Western Blot, increased by 500% in the ischemic striatum 24 h after MCAo. SCH 58261 treatment significantly reduced phospho-p38 MAPK by 70%. Microglia was immunostained using the OX-42 antibody. Phospho-p38 MAPK and OX-42-immunoreactive cells are localized in the ventral striatum and frontoparietal cortex. Furthermore, both OX-42 and phospho-p38 MAPK-immunoreactive cells have overlapping morphological features, typical of reactive microglia. SCH 58261 reduced phospho-p38 MAPK immunoreactivity in the striatum and in the cortex without changing the microglial cell morphology. These results indicate that the protective effect of the adenosine antagonist SCH 58261 during ischemia is not due to reduced microglial activation but involves inhibition of phospho-p38 MAPK and suggest that treatment with the A2A antagonist from the first hour to several hours after ischemia may be a useful therapeutic approach in cerebral ischemia.

We presently investigated the time-course of neuronal nitric oxide synthase and inducible nitric oxide synthase expression and content in the rat striatum up to 6 days after ischemia induced by transient middle cerebral artery occlusion, a condition that potentially allows functional recovery, with the aim to identify the cell types expressing these two enzymes and to correlate neuronal nitric oxide synthase and inducible nitric oxide synthase changes in order to verify whether and how these changes are related to tissue damage, motor-sensory performances and survival. Before and after surgery, the animals underwent neurological evaluation. The results demonstrated that the rats with a score ≥12 at the neurological evaluation 24 h after ischemia showed a significant increase in neuronal nitric oxide synthase-immunoreactive neurones and absence of inducible nitric oxide synthase-immunoreactive cells and survived up to the sixth day; conversely, the rats with a score

Interest is growing in the role of adenosine triphosphate (ATP) on P2 receptors during hypoxic/ischemic events in the brain. However, there is no direct evidence of an increase in extracellular ATP levels during cerebral ischemia in vivo. The aim of the present study was to evaluate ATP outflow from the rat striatum by the microdialysis technique associated with focal cerebral ischemia in vivo by intraluminal occlusion of the right middle cerebral artery (MCA). Between 1 and 4h after ischemia, rats showed a clear turning behavior contralateral to the ischemic side. Twenty-four hour after MCA occlusion, ischemic rats had definite neurological deficit and striatal and cortical damage. The ATP concentration (mean+/-S.E.M.) in the striatum of normoxic rats (n = 8) was 3.10+/-0.34 nM. During 220 min after MCA occlusion, the extracellular ATP levels significantly increased two-fold, being 5.90+/-0.61 nM (p < 0.01 versus normoxic level). ATP outflow showed a tendency to increase over time during the 220 min of ischemia. Since extracellular ATP is rapidly metabolized to adenosine, we also assessed ATP outflow in the presence of the ecto-5'-nucleotidase inhibitor, alpha,beta-methylene-adenosine diphosphate (AOPCP, 1 mM) directly perfused into the striatum. The ATP concentration in normoxic rats (n = 8) was increased three-fold in the presence of the ecto-5'-nucleotidase inhibitor (9.57+/-0.26 nM). During 220 min of ischemia, extracellular ATP levels significantly increased 1.3-fold in AOPCP-treated rats (12.62+/-0.65 nM, p < 0.01 versus normoxic level). The present study confirms that ATP is continuously released in the brain and demonstrates for the first time that ATP outflow increases during ischemia in vivo. These results confirm that ATP may be an important mediator in brain ischemia.

At least two populations of c-kit positive interstitial cells of Cajal (ICC) lie in the gastric wall, one located at the myenteric plexus level has a pace-making function and the other located intramuscularly is intermediary in the neurotransmission and regenerates the slow waves. Both of these ICC sub-types express full-length dystrophin. Mdx mice, an animal model lacking in full-length dystrophin and used to study Duchenne muscular dystrophy (DMD), show gastric dismotilities. The aim of the present study was to verify in mdx mice whether: (i) gastric ICC undergo morphological changes, through immunohistochemical and ultrastructural analyses; and (ii) there are alterations in the electrical activity, using intracellular recording technique. In control mice, ICC sub-types showed heterogeneous ultrastructural features, either intramuscularly or at the myenteric plexus level. In mdx mice, all of the ICC sub-types underwent important changes: coated vesicles were significantly more numerous and caveolae significantly fewer than in control; moreover, cytoskeleton and smooth endoplasmic reticulum were reduced and mitochondria enlarged. c-Kit-positivity and integrity of the ICC networks were maintained. In the circular muscle of normal mice slow waves, which consisted of initial and secondary components, occurred with a regular frequency. In mdx mice, slow waves occurred in a highly dysrhythmic fashion and they lacked a secondary component. We conclude that the lack of the full-length dystrophin is associated with ultrastructural modifications of gastric ICC, most of which can be interpreted as signs of new membrane formation and altered Ca(2+) handling, and with defective generation and regeneration of slow wave activity.

Recent reports suggest a far greater plasticity in nerve tissue than previously believed. As the digestive tract is exposed to a variety of insults, this question is relevant to enteric nerves, but little is known about their ability to recover from damage. To address this problem, we ablated the myenteric plexus of the mouse colon with the detergent benzalkonium chloride (BAC) and followed the ensuing morphologic changes for up to 60 days by using light- and electron microscopy. We found that, 2 days after BAC application, the treated area was essentially devoid of intact nerve elements. From day 7, new nerve fibers were observed within the denervated region. This growth progressed until, at days 30-60, newly grown nerve fibers were present in most of this region, and the pattern of muscle innervation was similar to the normal one. At least part of these fibers originated at neurons within intact ganglia surrounding the denervated region. The cross-sectional area of neurons near the denervated region at day 14 was 52% greater than controls. Glial cells were closely associated with the regenerating nerve fibers. From day 14 onward, we observed undifferentiated cells and differentiating neurons in ganglia surrounding the denervated region, and by day 30, new neurons were present in the myenteric region, along with regenerating nerve fibers. We conclude that the myenteric plexus is endowed with a considerable ability of regeneration and plasticity. The results provide evidence for the presence of stem cells and for an adult neurogenesis in this plexus.

The aim of the present study was to evaluate whether alterations in the distribution and/or function of nitric oxide synthase (NOS) could be involved in the development of the spontaneous mechanical tone observed in colon from dystrophic ( mdx) mice. By recording the intraluminal pressure of isolated colon from normal mice, we showed that Nω-nitro- l-arginine methyl ester (l-NAME) increased the tone, even in the presence of tetrodotoxin. The effect was prevented by l-arginine, nifedipine, or Ca2+-free solution. In colon from mdx mice, l-NAME was ineffective. Immunohistochemistry revealed that the presence and distribution of neuronal (nNOS), endothelial, and inducible NOS isoforms in smooth muscle cells and neurons of colon from mdx mice were the same as in controls. However, the expression of myogenic nNOS was markedly reduced in mdx mice. We conclude that there is a myogenic NOS in mouse colon that can tonically produce nitric oxide to limit influx of Ca2+through L-type voltage-dependent channels and modulate the mechanical tone. This mechanism appears to be defective in mdx mice.

The substance P (SP)-containing nerves at the deep (DMP) and myenteric (MP) plexuses and the related interstitial cells of Cajal (ICC-DMP and ICC-MP) were immunohistochemically studied in rat and guinea-pig ileum. All the ICC expressed SP-preferred receptor NK1r: the ICC-DMP showed an intense and the ICC-MP a faint NK1r-immunoreactivity(IR). c-kit-labeling confirmed that they were ICC. The SP-IR nerves at the DMP were significantly more numerous in the guinea-pig than in the rat, and more numerous than those at the MP in both animal species. All the ICC-DMP in the guinea-pig and half of them in the rat were close to SP-IR nerves. The ICC-MP were rarely near to SP-IR nerves in either species. The SP-innervation shows interspecies differences at the DMP that imply a different tachykinergic control of the local ICC.

Neuron density and distribution of the NADPH-diaphorase positive neurons were studied in the fundus, corpus and antrum of adult human stomach using cresyl violet staining and NADPH-diaphorase histochemistry. The submucous plexus contained significantly less neurons than the myenteric plexus. Submucous NADPH-d positive neurons were mostly located in ganglia close to the circular muscle layer. Myenteric NADPH-d positive neurons represented 50-60% of the neurons in all the three regions; their density, however, was significantly lower in the fundus. NADPH-d positive fibers formed a rich plexus in the innermost portion of the circular muscle layer of the corpus.

Brain acetylcholine release and memory performance were investigated in young (three- to six-months) and old (20- to 24-months) rats. Acetylcholine release was measured in vivo in the cortex and hippocampus of freely-moving animals, under basal conditions and in the presence of the following muscarinic antagonists: scopolamine, (+/-)-5,11-dihydro-11-[[(2-[2-[(dipropylamino) methyl]-1-piperidinyl]ethyl) amino] carbonyl]-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one (AFDX 384) and pirenzepine. The amount of acetylcholine released from the cortex and hippocampus of old rats was significantly reduced. In the presence of scopolamine and AFDX 384 but not of pirenzepine, the acetylcholine release was significantly higher in the old than the young rats, suggesting that changes in presynaptic M2/M4 muscarinic receptor function occur with ageing in the two brain regions. Cognitive capacities were evaluated using two different behavioural tasks: object recognition and passive avoidance response. Old rats were unable to discriminate between familiar and novel objects and had impaired performance in the passive avoidance test. AFDX 384 restored the performance in both tests. Furthermore, in young rats AFDX 384 reversed the impairment of both object recognition and passive avoidance response induced by scopolamine. The effect of AFDX 384 on acetylcholine release and behaviour in the old rats offers further support to a relationship between the age-related cholinergic hypofunction and cognitive impairment and indicates the blockade of presynaptic muscarinic receptors as a possible selective target for therapeutic strategies aimed at improving age-associated memory deficits.

The effect of intraperitoneal administration of scopolamine (1 mg/kg) on acetylcholine (ACh) release in vivo in 3- and 24-month-old freely behaving rats was investigated in the cerebral cortex, hippocampus and striatum by means of transverse microdialysis. In the parietal cortex, the increase in ACh release after scopolamine administration was significantly greater in the old than in the young rats, reaching a maximum increase of about 600 and 300% in the old and young animals, respectively. In the hippocampus, scopolamine caused a larger increase in ACh release in the young (+900%) than in the old rats (+600%). In the striatum of aged rats, a 40% increase occurred only at 40 min after scopolamine administration while in the striatum of young animals the increase lasted for at least 2 h, reaching a maximum of about 100%. These findings demonstrate that the modulation of ACh release in vivo is affected in a different manner in the cerebral cortex than in the hippocampus and striatum by aging.

An attempt to identify the muscarinic receptor subtypes involved in presynaptic modulation of acetylcholine (ACh) release from cortical and hippocampal slices was made by means of several muscarinic antagonists. Cortical and hippocampal slices prepared from adult rats were superfused with Krebs solution containing physostigmine; ACh content of the superfuse at rest and after electrical stimulation (1 Hz) was quantified by high performance liquid chromatography. The antagonists were added to the Krebs at the concentration of 1μM. ACh release at rest was enhanced only in the cortex by (±)-5,11-dihydro-11-{[(2-[2-[(dipropylamino)methyl]-1-piperidinyl}ethyl)amino]carbonyl}-6 H -pyrido[2,3- b ](1,4)-benzodiazepine-6 (AFDX384), an M 2 M 4 selective antagonist. The evoked ACh release from the cerebral cortex was significantly increased by AFDX384, methoctramine, pirenzepine, M 2 M 4 , M2 and M1 selective antagonists, respectively, and scopolamine. This finding suggests that M1, M2 and M4 presynaptic receptor subtypes could regulate evoked ACh release in the cortex. In hippocampal slices, the evoked ACh release was enhanced by AFDX384, pirenzepine and scopolamine but not by methoctramine. In this region ACh release seems therefore regulated only by M1 and M4 receptor subtypes. The M3 antagonist (±)- p -fluorohexahydro-sila-difenidol hydrochloride did not affect ACh release.

Background & Aims: Recent advances have raised the possibility of treating enteric nervous system (ENS) disorders with transplanted progenitor cells (ENSPC). Although these cells have been shown to migrate and differentiate after transplantation, no functional effects have been demonstrated. We therefore aimed to investigate whether embryonic mouse and neonatal human ENSPC can regulate the contractility of aganglionic bowel. Methods: Embryonic mouse and neonatal human ENSPC were grown as neurospheres before transplantation into aganglionic embryonic mouse hindgut explants and culture for 8–12 days. Engraftment and neural differentiation were confirmed using immunofluorescence and transmission electron microscopy. The contraction frequency of transplanted bowel was measured and compared with that of embryonic day 11.5 embryonic ganglionic and aganglionic bowel cultured for the same period. Calcium movement was measured at spatially defined points in bowel wall smooth muscle. Neural modulation of bowel contractility was assessed using tetrodotoxin. Results: Both mouse and human ENSPC migrated and differentiated after neurosphere transplantation. Transmission electron microscopy demonstrated the existence of synapses. Transplantation restored the high contraction frequency of aganglionic bowel to the lower rate of ganglionic bowel. Calcium imaging demonstrated that neurosphere transplantation coordinates intracellular free calcium levels. Both these effects were reversed by the addition of tetrodotoxin, indicating the functional effect of neurosphere-derived neurons. Conclusions: Neonatal human gut is a source of ENSPC that can be transplanted to restore the contractile properties of aganglionic bowel by a neurally mediated mechanism. This may aid development of a stem cell-based treatment for Hirschsprung's disease.

The release of total acetylcholine (ACh) and [3H]ACh was investigated in electrically stimulated cortical slices prepared from 4- and 18-month-old male Wistar rats. The slices were prelabeled with [3H]choline ([3H]Ch) and perfused with Krebs solution containing physostigmine. Total ACh was measured and the nature of the tritium efflux identified by HPLC. The total tritium content in the slices at the end of the incubation period was half as great in the old as in young rats. A linear relationship was found between stimulation frequencies (2, 5, and 10 Hz) and fractional [3H]ACh release in both young and old rats. In the latter the release was significantly smaller. At 10 Hz stimulation frequency the ratio between the two 2-min stimulation periods, S2/S1, was higher in the 18-month-old rats than in the young rats. Specific activity of the evoked ACh release was significantly smaller in S2 than in S1 in 4-month-old rats only. These findings indicate that the young synthetize ACh from endogenous unlabeled Ch more than older rats. In 18-month-old rats both the evoked total ACh and [3H]ACh release, expressed as picograms per minute, showed an approximately 50% decrease in both S1 and S2 stimulation periods, with no significant difference in specific activity. Phosphatidylserine (PtdSer) administration (15 mg/kg, i.p. daily) for 1 week to 18-month-old rats prevented the reduction in total evoked ACh release but not the reduction in evoked [3H]ACh release. The specific activity of ACh release was therefore significantly smaller than that of the young and untreated old rats. This finding indicates that PtdSer is able to increase the availability of endogenous choline for de novo ACh synthesis and release. PtdSer treatment also prevented impairment of the passive avoidance conditioned response observed in old rats.

Cortical slices were prepared from male rats 3 to 28 months old. The slices were superfused with choline-enriched Krebs solution containing physostigmine and electrically stimulated at frequencies of 1, 2 and 5 Hz for 5 min periods preceded and followed by rest periods. The amount of acetylcholine released during the stimulation periods was quantified by bioassay. In some experiments acetylcholine content was measured at the end of the superfusion period in stimulated and unstimulated slices. The evoked acetylcholine release was constant between 3 and 11 months of age at each f frequency tested and showed a 50% decrease between 11 and 14 months of age with no further decrease up to 28 months. No difference in the evoked acetylcholine release was detected between 3 and 16 month old rats if the old rats were pretreated with phosphatidylserine 15 mg/kg IP for at least 7 days. The effect of phosphatidylserine lasted for 5 days after interruption of the treatment. There was no difference in acetylcholine content between the stimulated and unstimulated slices in 3 month old rats. In 16 month old rats stimulation brought about a 44% decrease in acetylcholine content. This decrease did not occur in rats pretreated with phosphatidylserine for 7 days. Phosphatidylserine appears to restore acetylcholine release in aging rats by maintaining an adequate acetylcholine supply in the slices.