Muscularis macrophages establish cell-to-cell contacts with telocytes/PDGFRα-positive cells and smooth muscle cells in the human and mouse gastrointestinal tract.

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.

A novel intramuscular Interstitial Cell of Cajal is a candidate for generating pacemaker activity in the mouse internal anal sphincter.

The internal anal sphincter (IAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of Cajal (ICC) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the IAS where only intramuscular ICC (ICC-IM) are found, however the evidence linking ICC-IM to SWs is limited. This study examined the possible relationship between ICC-IM and SWs by recording Ca2+ transients in mice expressing a genetically-encoded Ca2+-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type Ca2+ channels (CavL) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous Ca2+ transients not dependent on CavL or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., Type II cells (64% of total) generated rhythmic, global Ca2+ transients at the SW frequency that were synchronised with neighbouring Type II cells and were abolished following blockade of either CavL or ANO1. Thus, the spatiotemporal characteristics of Type II cells and their dependence upon CavL and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the IAS.

Functional restoration of ex vivo model of pylorus: Co-injection of neural progenitor cells and interstitial cells of Cajal.

Transplantation of neural stem cells is a promising approach in treatment of intestinal dysfunctionality. The interstitial cells of Cajal (ICCs) are also critical in conditions such as pyloric dysfunctionality and gastroparesis. The objective of this study was to replenish neurons and ICCs in a dysfunctional pylorus as cell-based therapy to restore functionality. ICCs and enteric neural progenitor cells (NPCs) were isolated from rat duodenum and transduced with fluorescent proteins. Rat pylorus was harvested, and an ex-vivo neuromuscular dysfunctional model was developed by selective ablation of neurons and ICCs via chemical treatments. Cellular repopulation and restoration of motility were assessed by immunohistochemistry, qPCR, and functional analysis after delivery of fluorescently tagged cells. Chemical treatment of pylorus resulted in significant depletion of ICCs (67%, P = .0024; n = 3) and neural cells (83%, P = .0012; n = 3). Delivered ICCs and NPCs survived and integrated with host muscle layers. Co-injection of ICCs with NPCs exhibited 34.4% (P = .0004; n = 3) and 61.0% (P = .0003; n = 3) upregulation of ANO1 and ßIII tubulin, respectively. This regeneration resulted in the restoration of agonist-induced excitatory contraction (82%) and neuron evoked relaxation (83%). The functional studies with specific neuronal nitric oxide (NO) synthase blocker confirmed that restoration of relaxation was NO mediated and neuronally derived. The simultaneous delivery of ICCs observed 35.7% higher neuronal differentiation and functional restoration compared with injection of NPCs alone. Injected NPCs and ICCs integrated into the dysfunctional ex vivo pylorus tissues and restored neuromuscular functionality. The co-transplantation of NPCs and ICCs can be used to treat neurodegenerative disorders of the pylorus.

Interstitial cells of Cajal are diminished in critically ill patients: Autopsy cases.

OBJECTIVE: Gastrointestinal dysmotility in critically ill patients is important as enteral nutrition is crucial. However, normal gut motility is impaired under conditions of critical illness subsequent to severe insult. Interstitial cells of Cajal (ICC) form an extensive network associated with the myenteric plexus in the enteric nervous system. There are few reports about ICC distribution in critically ill patients. The aim of this study was to evaluate ICC in critically ill patients. METHODS: Postmortem colon harvest was obtained from critically ill patients. Control specimens were obtained from patients without bowel movement problems who underwent hemicolectomy. The tissues were stained with c-Kit for ICC. The number of ICC was identified by counting from 10 high-power fields (HPFs). RESULTS: Specimens from six patients were analyzed and compared with those from six control patients. All patients had abnormalities of crypt architecture and inflammatory cell infiltrations. Mucosal thickness tended to be lower in the critically ill patients than in the controls (147 ± 47 versus 231 ± 127 µm; P = 0.15). Muscle layer thickness tended to be higher in the critically ill patients than in the controls (494 ± 163 versus 394 ± 258 µm; P = 0.44). ICC in the critically ill patients were almost depleted in the colon compared with those in the controls. Significantly fewer ICC were present in the critically ill patients than in the controls (0.45 versus 7.25 cells/HPF; P < 0.05). CONCLUSIONS: Critical illness is associated with diminished numbers of ICC in the colon. This finding could have implications for dysmotility in critically ill patients.

Differentiation of human urine-derived stem cells into interstitial cells of Cajal-like cells by exogenous gene modification: A preliminary study.

Human urine-derived stem cells (hUSCs) show multipotential differentiation ability and can differentiate into mesodermal cell lineages. Interstitial cells of Cajal-like cells (ICC-LCs) are crucial for the pace-making function of spontaneous contraction in the bladder. However, the mechanisms by which hUSCs generate ICC-LCs have not been elucidated. In this study, we developed a strategy for directional differentiation of hUSCs into ICC-LCs. hUSCs were transfected with lentiviral vectors encoding c-Kit, stem cell factor (SCF), hyperpolarization activated cyclic nucleotide gated potassium channel 4 (HCN4), and 5-azacytidine induced 2 (AZI2) genes, and the cells were cultured for an additional 7 days in specific medium. The expression of the surface marker c-Kit on ICC-LCs was determined at 7 days after transfection. hUSCs were successfully expanded and transfected with the four lentiviral vectors. hUSCs transfected with lentiviral-c-Kit, lentiviral-HCN4, and lentiviral-AZI2 showed higher expression of c-Kit 7 days after transfection, but only the lentiviral-HCN4-transfected cells showed morphological alterations in ICC-LCs. These cells also displayed visible HCN current amplitude and density. This approach may provide a new strategy for the treatment of underactive bladder.

Comparison of different pathological markers in predicting pyeloplasty outcomes in children.

PURPOSE: To compare the efficacy of pathological markers like Interstitial cells of Cajal (ICC), neurons and Collagen to Muscle ratio (CM ratio), in predicting pyeloplasty outcomes. METHODS: Histological sections from 31 patients with UPJO were analyzed for ICC & neurons on immuno-histochemistry and CM ratio on Masson's trichrome staining. Post-operative outcomes were analyzed at 1-year follow up; expressed as excellent, moderate or mild improvement, static and deterioration based on the three factors: ultrasound grade, differential renal function and renogram drainage pattern. The pathological findings were correlated with clinical outcomes. RESULTS: The study group (n = 31) had a mean age 2.9 (0.6) years (M: F = 22:9). UPJ segment had significantly less ICC/neurons and more collagen compared to normal ureter (p = 0.001). Pathological parameters at the anastomosed end of ureter had a better correlation than those at UPJ with clinical outcome. CM ratio with a stronger correlation (r = - 0.94; p = 0.001) was a better predictor of prognosis than ICC (r = 0.76; p = 0.01) or neuron (r = 0.83; p = 0.01) density. ICC >10/HPF, neurons >6/HPF and CM ratio <1.2 at ureteric end anastomosed were predictors of success. CONCLUSIONS: CM ratio analysis at anastomosed ureter is a superior marker for predicting pyeloplasty outcomes. LEVEL OF EVIDENCE: Type 2: Development of diagnostic criteria in a consecutive series of patients.

c-Kit-stem cell factor signal-independent development of interstitial cells of Cajal in murine small intestine.

c-Kit receptor tyrosine kinase and its ligand stem cell factor (SCF) play critical roles in regulating the development and proliferation of various cells, including the interstitial cells of Cajal (ICC) in the gastrointestinal tract. Many subtypes of ICC are known to be lacking in c-Kit-SCF-insufficient mice, such as W/Wv and Sl/Sld, whereas ICC-deep muscular plexus (DMP) in small intestine are not lacking. In this study, we examine ICC-DMP development in normal and c-Kit-SCF signal-insufficient mice. In normal mice, numerous ICC-DMP labeled with c-Kit and neurokinin 1 receptor (NK1R) antibodies were observed only in the duodenum on the day of birth, in the duodenum and the jejunum on postnatal day 4 and throughout the small intestine after postnatal day 6. In W mutant mice (W/Wv, Wv/Wv, W/W), ICC-DMP investigated using c-Kit and NK1R immunoreactivities were similar to that in normal mice. c-Kit ligand SCF-deficient mice (Sl/Sl) also showed almost identical ICC-DMP development and proliferation as normal mice. These results show that the development and proliferation of ICC-DMP occur in the postnatal period independent of c-Kit-SCF signaling.

Effects of Morphine on Interstitial Cells of Cajal in Rabbit Colon and Small Intestinal Transit: An Experimental Study.

OBJECTIVE: This study aims to investigate the effect of morphine with naloxone on intestinal peristalsis and the number of interstitial cells of Cajal (ICC) in colon tissues of rabbits. METHODS: Thirty rabbits were randomly divided into five groups (n=6, each group): saline control group (NS group), low concentration of morphine group (L group), medium concentration of morphine group (M group), high concentration of morphine group (H group), medium concentration of morphine and naloxone mixed with antagonist group (NM group). Rabbits in these five groups were administered with an epidural puncture tube and dorsal epidural analgesia pump, and were continuously infused for seven days. Fecal characteristics were observed, and the ink propulsion rate was calculated. The expression level of ICC C-kit protein in colon tissues was tested by western blot. RESULTS: The stool characteristics in the L, M and H groups were more severe than those in the NS and NM groups. Furthermore, the intestinal propulsion rate in the L, M and H groups was lower than that in the NS and NM groups. The C-kit mRNA and protein expression in the colon of rabbits were significantly lower in the L, M and H groups, when compared to the NS and NM groups. CONCLUSION: Naloxone blocked the mRNA and protein expression of C-kit, and improved intestinal motor function.

Comparison of the ICC location in the gallbladder wall in patients with cholelithiasis and patients with non-calculous changes.

Interstitial cells of Cajal (ICC) were first described by Santiago Ramon y Cajal over 100 years ago. They are thought to play an important role in the regulation of gastrointestinal motility. There is increasing evidence that the decline in their number in the gallbladder wall contributes to the formation of concrements. The aim of the study was to determine the exact location of interstitial cells of Cajal in the gallbladder wall in patients with calculous and non-calculous cholecystitis. Sixty-eight patients were examined, of whom 50 were cases of cholelithiasis and 18 were of non-calculous cholecystitis. The technique of immunohistochemistry with the CD117 antibody was used to determine the cells of Cajal, while to distinguish them from mast cells the technique with mast cell tryptase (MCT) was applied. Redistribution of the interstitial cells of Cajal from the muscle membrane to lamina propria of mucous tissue was observed in the cases of cholelithiasis, while in the group of non-calculous cholecystitis most of the ICC was located within the muscle tissue.

Quantifying heart valve interstitial cell contractile state using highly tunable poly(ethylene glycol) hydrogels.

Valve interstitial cells (VIC) are the primary cell type residing within heart valve tissues. In many valve pathologies, VICs become activated and will subsequently profoundly remodel the valve tissue extracellular matrix (ECM). A primary indicator of VIC activation is the upregulation of α-smooth muscle actin (αSMA) stress fibers, which in turn increase VIC contractility. Thus, contractile state reflects VIC activation and ECM biosynthesis levels. In general, cell contraction studies have largely utilized two-dimensional substrates, which are a vastly different micro mechanical environment than 3D native leaflet tissue. To address this limitation, hydrogels have been a popular choice for studying cells in a three-dimensional environment due to their tunable properties and optical transparency, which allows for direct cell visualization. In the present study, we extended the use of hydrogels to study the active contractile behavior of VICs. Aortic VICs (AVIC) were encapsulated within poly(ethylene glycol) (PEG) hydrogels and were subjected to flexural-deformation tests to assess the state of AVIC contraction. Using a finite element model of the experimental setup, we determined the effective shear modulus µ of the constructs. An increase in µ resulting from AVIC active contraction was observed. Results further indicated that AVIC contraction had a more pronounced effect on µ in softer gels (72 ±â€¯21% increase in µ within 2.5 kPa gels) and was dependent upon the availability of adhesion sites (0.5-1 mM CRGDS). The transparency of the gel allowed us to image AVICs directly within the hydrogel, where we observed a time-dependent decrease in volume (time constant τ=3.04 min) when the AVICs were induced into a hypertensive state. Our results indicated that AVIC contraction was regulated by both the intrinsic (unseeded) gel stiffness and the CRGDS peptide concentrations. This finding suggests that AVIC contractile state can be profoundly modulated through their local micro environment using modifiable PEG gels in a 3D micromechanical-emulating environment. Moving forward, this approach has the potential to be used towards delineating normal and diseased VIC biomechanical properties using highly tunable PEG biomaterials. STATEMENT OF SIGNIFICANCE.

Clinicopathological study of intestinal smooth muscles, interstitial cells of Cajal, and enteric neurons in neonatal jejuno-ileal atresia with special reference to muscle morphometry.

PURPOSE: To assess the thickness of the intestinal smooth muscle layer and analyze the distribution and density of interstitial cells of Cajal (ICC) and enteric neurons in the proximal and distal segments of neonatal jejuno-ileal atresia. METHODS: This is an observational study done over a period of one year in which fifteen cases of jejuno-ileal atresia were included. All the cases underwent laparotomy and resection of the atretic segment with variable portions of the dilated proximal segment and distal segment. Histopathological analysis was done on the sections taken from proximal segments (at 3 cm, 5 cm & 8 cm) and the distal segment (at 2 cm) from the atretic portion. The mean thickness of the inner circular muscle layer (ICML) and outer longitudinal muscle layer (OLML) was assessed in the above segments using image morphometry. In addition, we also analyzed the distribution and density of the ICCs and enteric neurons in the different segments using immunohistochemistry for c-kit and S-100, respectively. Controls included normal jejuno-ileal segments resected from postmortem cases (n=7) and other nonrelated surgeries (n=3). The findings were then compared with each-other and with normal controls. RESULTS: Mean thickness of ICML and OLML of the proximal segments at 8 cm was significantly lower than at 3 cm and 5 cm of ileal and jejunal atresias (p≪ 0.5). The mean thickness of ICML and OLML of distal segments at 2 cm was similar to the controls in all the atretic cases (p≫ 0.5). The mean ICML thickness at proximal 8 cm segment was similar to the distal segment of both ileal & jejunal atresias (p= 0.06 & 0.37 respectively). The mean thickness of the OLML of the proximal 8 cm segments was significantly more than that at the distal segment (p=0.008) in ileal atresias but was similar in cases of jejunal atresias (p=0.07). Both the proximal and distal segments of ileal as well as jejunal atresias showed reduction in distribution and density of ICCs, as compared to normal controls. The density of ICCs in proximal segments at 3 cm and 5 cm was similar in both ileal (p=0.33) and jejunal segments (p=0.41) but was significantly lower than the proximal 8 cm segments (p≪0.05).The distribution of ICCs in the proximal segment at 8 cm was similar to the distal segments (p≪0.05). S-100 staining showed dense expression of neurons and glial cells with presence of submucosal giant ganglia within the proximal dilated segments as compared to the distal segments and the controls, which were more marked at 3 cm and 5 cm levels than at 8 cm level. CONCLUSION: Muscle morphometry using image analysis is a simple technique to assess the thickness of the intestinal smooth muscle layers. There is significant smooth muscle hypertrophy along with marked alteration in density and distribution of ICCs and ENS in the dilated proximal segments up to 5 cm, and relatively milder changes at 8 cm levels, as compared to the distal segments and the controls. TYPE OF STUDY: Prognosis study. LEVEL OF EVIDENCE: Level II.

Effects of epidural infusion of morphine combined with small-dose naloxone on gastrointestinal interstitial cells of Cajal in rabbits.

OBJECTIVE: To study the effect of epidural infusion of morphine combined with small-dose naloxone on gastrointestinal interstitial cells of Cajal (ICC) in rabbits. MATERIALS AND METHODS: A total of 80 healthy New Zealand rabbits were selected as objects of study, and divided into normal saline control group (Group NS, n=20), morphine group (Group M, n=20), naloxone group (Group N, n=20), and morphine + naloxone group (Group NM, n=20). Rabbits in four the groups received epidural catheterization for continuous drug infusion for 7 d, and epidural analgesia pump was connected. Visual analogue scale (VAS) score, intestinal propulsion rate, c-kit expression, and ICC count were detected and compared among four groups of rabbits. RESULTS: No statistical differences of occurrence rates regarding constipation as well as expressions of c-kit and ICC count in the proximal colon were shown among rabbits in Group NS, Group N, and Group NM during drug administration (p>0.05). However, the occurrence rates of constipation of rabbits in Group M at 3-7 d were statistically higher than those in Group NS, Group N, and Group NM, and the differences were statistically significant (p<0.05). Moreover, the VAS scores in Group NS and Group N were significantly higher than those in Group M and Group NM, while the scores in Group M were also significantly increased compared to that in Group NM (p<0.05). The intestinal propulsion rates, expressions of c-kit and ICC counts of rabbits in Group NS, Group N, and Group NM were statistically higher than that in Group M (p<0.05). CONCLUSIONS: Epidural infusions of morphine combined with small-dose naloxone effectively inhibit the gastrointestinal motility of rabbits via the reduction of ICC in the proximal colon of the gastrointestinal tract of rabbits. Moreover, small-dose of naloxone enhances the analgesic effect and reduces the risk of adverse reactions.

A Formal Approach for Scalable Simulation of Gastric ICC Electrophysiology.

OBJECTIVE: Efficient and accurate organ models are crucial for closed-loop validation of implantable medical devices. This paper investigates bio-electric slow wave modeling of the stomach, so that gastric electrical stimulator (GES) can be validated and verified prior to implantation. In particular, we consider high-fidelity, scalable, and efficient modeling of the pacemaker, Interstitial cells of Cajal (ICC), based on the formal hybrid input output automata (HIOA) framework. METHODS: Our work is founded in formal methods, a collection of mathematically sound techniques originating in computer science for the design and validation of safety-critical systems. We modeled each ICC cell using an HIOA. We also introduce an HIOA path model to capture the electrical propagation delay between cells in a network. The resultant network of ICC cells can simulate normal and diseased action potential propagation patterns, making it useful for device validation. RESULTS: The simulated slow wave of a single ICC cell had high correlation ( ≈ 0.9) with the corresponding biophysical models. CONCLUSIONS: The proposed model is able to simulate the slow wave activity of a network of ICC cells with high-fidelity for device validation. SIGNIFICANCE: The proposed HIOA model is significantly more efficient than the corresponding biophysical models, scales to larger networks of ICC cells, and is capable of simulating varying propagation patterns. This has the potential to enable verification and validation of implantable GESs in closed-loop with gastrointestinal models in the future.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ.

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Cajal Cell Counts are Important Predictors of Outcomes in Drug Refractory Gastroparesis Patients With Neurostimulation.

BACKGROUND AND AIMS: Cajal cells serve as the pacemaker cells of the gastrointestinal tract and regulates peristalsis. On the baisis of that fact, it has been hypothesized that a decrease in Cajal cells can lead to gastroparesis and other motility issues. Treatment with medications has a limited efficacy and most resort to gastric electrical stimulation (GES) devices for symptomatic relief. We believe that the number of Cajal cells present is directly proportional to symptomatic relief with GES. MATERIALS AND METHODS: Twenty-three (white female) subjects were recruited from the gastric motility clinic University of Mississipi for this study with the criteria of drug refractory gastropersis. Symptoms were measured using Likert scale and gastric emptying times were measured pre-GES and post-GES. Serosal electrogram measurements were recorded during surgical placement of permanent electrical stimulator under various modes. Cajal cell count scoring via immunohistochemistry were performed during the implantaion of the GES. RESULTS: The data were grouped in 2 categories based on the Cajal cells that is ≥2.00 and <2.00. Subjects with higher Cajal cells reported a statiscially improvement in gastroperesis symptoms. Significant differences were also noted in the first hour gastric emptying study. The mean group difference is 17.5 (95% confidence interval, 1.41-33.58; P=0.035). Serosal amplitude differences were noted being significantly higher in the group with ≥2 cajal cells. CONCLUSIONS: Electrograms obtained after GES demonstrates immediate improvement in gastric electrical activity and gastroparesis symptoms in patients with relatively higher Cajal cell counts when compared with patients with extensive loss of Cajal cells.

[Santiago Ramón y Cajal - Neuroanatomist, Artist and Writer]. / Santiago Ramón y Cajal ­ Neuroanatom, Künstler und Literat.

Santiago Ramón y Cajal came from North Spain and was very talented in painting and writing. Actually he wanted to become a painter but his father, who was a medical doctor himself, forced him to study medicine. In the end, he became a famous neuroanatomist, developed the neuron theory and, together with Camillo Golgi, was awarded the Nobel Price for medicine in 1906. In his career, he was very much supported by German scientists, especially by the Würzburg anatomist von Koelliker. He also displayed a lively literary activity. But most of his works are little known to us. Of his 5 "Cuentos de vacaciones" (vacation stories) there is a translation into English only since 2001.

Telocytes role during the postnatal development of the Mongolian gerbil jejunum.

Telocytes are recently categorised CD34-positive interstitial cells that comprise the cells which were previously called interstitial Cajal-like cells (ICLCs). These were detected in the stroma of various organs such as the prostate, lungs, mammary glands, liver, gallbladder, and jejunum, among others. Several functions have been proposed for telocytes, such as a supportive role in smooth muscle contraction and immune function in adult organs, and tissue organisation and paracrine signalling during development, as well as others. In the jejunum, little is known about the function of telocytes in the adult organ, or is there any information about when these cells develop or if they could have an auxiliary role in the development of the jejunum. The present study employed histological, immunohistochemical and immunofluorescence techniques on histological sections of the jejunum of Mongolian gerbil pups on two different days of postnatal development of the jejunum, covering the maturation period of the organ. By immunolabelling for CD34, it was observed that telocytes are already present in the jejunum during the first week of postnatal life and exist in close association with the developing muscularis mucosae, which are therefore TGFß1-positive. The telocytes are still present at the end of the first month of life, and a portion of them present co-localisation with c-Kit. Fibroblast-like cells, which are exclusively c-Kit-positive, are also observed, which may indicate the presence of interstitial Cajal cells (ICCs). Finally, it can be hypothesised that a portion of the telocytes may give rise to ICCs, which are c-Kit-positive but CD34 negative.

SOCE mediated by STIM and Orai is essential for pacemaker activity in the interstitial cells of Cajal in the gastrointestinal tract.

Electrical pacemaker activity generates phasic contractions and motility patterns such as segmentation and peristalsis in the gastrointestinal tract. Pacemaker currents are generated in interstitial cells of Cajal (ICC), which release Ca2+ from intracellular stores that stimulates Ca2+-activated Cl- channels (CaCCs) in the plasma membrane. Thus, Ca2+ stores must be maintained to sustain pacemaker activity. Store-operated Ca2+ entry (SOCE) facilitates the refilling of Ca2+ stores by a mechanism dependent upon interactions between STIM and Orai proteins. We investigated the role of SOCE in ICC pacemaker activity. Reintroduction of extracellular Ca2+ in store-depleted ICC resulted in CaCC activation. Blocking CaCCs revealed an inwardly rectifying current with properties of a Ca2+ release-activated current (ICRAC). An inhibitory peptide that interfered with the STIM-Orai interaction blocked ICRAC in HEK 293 cells expressing STIM1 and Orai1 and blocked spontaneous transient inward currents (STICs) and slow wave currents in ICC. STICs, which are fundamental pacemaker events in ICC, were blocked by an Orai antagonist. Imaging of Ca2+ transients linked to pacemaker activity in ICC in intact muscles showed that the Orai antagonist blocked Ca2+ transients in ICC. These data suggest that Ca2+ recovery through STIM-Orai interactions is necessary to maintain ICC pacemaker activity.

Expression of TRPV1 channels by Cajal-Retzius cells and layer-specific modulation of synaptic transmission by capsaicin in the mouse hippocampus.

KEY POINTS: By taking advantage of calcium imaging and electrophysiology, we provide direct pharmacological evidence for the functional expression of TRPV1 channels in hippocampal Cajal-Retzius cells. Application of the TRPV1 activator capsaicin powerfully enhances spontaneous synaptic transmission in the hippocampal layers that are innervated by the axons of Cajal-Retzius cells. Capsaicin-triggered calcium responses and membrane currents in Cajal-Retzius cells, as well as layer-specific modulation of spontaneous synaptic transmission, are absent when the drug is applied to slices prepared from TRPV1- /- animals. We discuss the implications of the functional expression of TRPV1 channels in Cajal-Retzius cells and of the observed TRPV1-dependent layer-specific modulation of synaptic transmission for physiological and pathological network processing. ABSTRACT: The vanilloid receptor TRPV1 forms complex polymodal channels that are expressed by sensory neurons and play a critical role in nociception. Their distribution pattern and functions in cortical circuits are, however, much less understood. Although TRPV1 reporter mice have suggested that, in the hippocampus, TRPV1 is predominantly expressed by Cajal-Retzius cells (CRs), direct functional evidence is missing. As CRs powerfully excite GABAergic interneurons of the molecular layers, TRPV1 could play important roles in the regulation of layer-specific processing. Here, we have taken advantage of calcium imaging with the genetically encoded indicator GCaMP6s and patch-clamp techniques to study the responses of hippocampal CRs to the activation of TRPV1 by capsaicin, and have compared the effect of TRPV1 stimulation on synaptic transmission in layers innervated or non-innervated by CRs. Capsaicin induced both calcium responses and membrane currents in ∼50% of the cell tested. Neither increases of intracellular calcium nor whole-cell currents were observed in the presence of the TRPV1 antagonists capsazepine/Ruthenium Red or in slices prepared from TRPV1 knockout mice. We also report a powerful TRPV1-dependent enhancement of spontaneous synaptic transmission onto interneurons with dendritic trees confined to the layers innervated by CRs. In conclusion, our work establishes that functional TRPV1 is expressed by a significant fraction of CRs and we propose that TRPV1 activity may regulate layer-specific synaptic transmission in the hippocampus. Lastly, as CR density decreases during postnatal development, we also propose that functional TRPV1 receptors may be related to mechanisms involved in CR progressive reduction by calcium-dependent toxicity/apoptosis.

Acute Cholecystitis Reduces Interstitial Cells of Cajal in Porcine Gallbladder Through Decreased mRNA Synthesis.

BACKGROUND/AIMS: Acute cholecystitis is a common gastrointestinal disorder, often characterized by acute cholecystitis with gallbladder motility disorder. Interstitial cells of Cajal (ICCs) are the pacemaker cells of gut motility in the gastrointestinal tract. Disruption of ICC function is related to motility disorders. The aim of this study was to explore the cellular and molecular mechanisms of ICCs in acute cholecystitis and after the resolution of acute inflammation. MATERIALS AND METHODS: Fifty adult guinea pigs were randomly divided into five groups: a sham-administered group (control group); two groups that were intraperitoneally administered an anti-polyclonal neutrophil (PMN) antibody 24 h before common bile duct ligation (CBDL); and two groups of guinea pigs that were subjected to CBDL without receiving the PMN antibody. Guinea pigs that underwent CBDL were held for 24 h or 48 h after surgery before being subjected to laparotomy and cholecystectomy. Immunohistochemistry, TUNEL assays, western blotting, and real-time PCR were performed to determine ICC morphology and density, to detect ICC apoptosis, and to examine stem cell factor (SCF) and c-kit protein expression and SCF and c-kit mRNA levels, respectively. RESULTS: Both hematoxylin-eosin staining and histological inflammation scores in the PMN groups were lower than those in the control groups (P < 0.01). No differences were observed in ICC morphology between groups. During acute cholecystitis, ICCs numbers were reduced. Conversely, the density of ICCs increased after inflammation was relieved (P < 0.01). In addition, SCF and c-kit protein and mRNA expression levels decreased during acute cholecystitis (P < 0.05) and increased after inflammation was relieved (P < 0.05). Furthermore, ICC apoptosis increased during acute cholecystitis and decreased after resolution of acute cholecystitis (P < 0.01). CONCLUSIONS: In acute cholecystitis, ICC injury may be related to gallbladder motility disorder.