Enteric Reflexes Research Articles

Mast cells drive mesenteric afferent signalling during acute intestinal ischaemia

Acute intestinal ischaemia stimulates visceral afferent nerves but the mechanisms responsible for this excitation are not fully understood. Mast cells may participate in this process as they are known to signal to mesenteric afferents during intestinal anaphylaxis and contribute to early inflammation and neuronal damage in response to cerebral ischaemia. We therefore hypothesised that mast cells are early responders to acute intestinal ischaemia and their activation initiates rapid signalling to the CNS via the excitation of mesenteric afferents. Primary afferent firing was recorded from a mesenteric nerve bundle supplying a segment of jejunum in anaesthetized adult rats. Acute focal ischaemia was produced by clamping theme senteric vessels for 8 min, and reperfusion followed removal of the vessel clip. Two episodes of ischaemia–reperfusion (I–R) separated by a 30 min interval were performed. Drugs or their vehicles were administered 10 min before the 2nd I–R episode. Ischaemia caused a reproducible, intense and biphasic afferent firing that was temporally dissociated from the concomitantly triggered complex pattern of intestinal motor activity. The L-type calcium channel blocker, nifedipine, significantly attenuated this afferent firing by a mechanism independent of its action on intestinal tone. Ischaemia-induced afferent firing was also abrogated by the mast cell stabilizer, doxantrazole, and the H1 histamine receptor antagonist, pyrilamine. In contrast, the nicotinic receptor antagonist, hexamethonium, and the N-type calcium channel toxin, ω-conotoxin GVIA, each reduced the ischaemia-evoked motor inhibition but not the concurrent afferent discharge. Similarly, the cyclooxygenase inhibitor, naproxen, had no effect on the ischaemic afferent response but reduced the intestinal tone shortly from the onset of ischaemia to the early period of reperfusion. These data support a critical role for mast cell-derived histamine in the direct chemoexcitation of mesenteric afferents during acute intestinal ischaemia, whereas enteric reflex mechanisms and cyclooxygenase products contribute primarily to ischaemia-induced changes in intestinal motility. Therefore, targeting mast cells may provide benefits in patients with abdominal pain resulting from an ischaemic insult to the gastrointestinal tract.

The G‐protein on cholesterol‐rich membrane microdomains mediates mucosal sensing of short‐ chain fatty acid and secretory response in rat colon

Short-chain fatty acids (SCFA) stimulate colonic contraction and secretion, which are mediated by an enteric reflex via a mucosal sensing and cholinergic mechanisms. The involvement of G-protein signal transduction was examined in the secretory response to luminal propionate sensing in rat distal colon. Mucosa-submucosa and mucosa preparations were used to measure short-circuit current (I(sc)) and acetylcholine (ACh) release respectively. Cholesterol-rich membrane microdomains, lipid rafts/caveolae, were fractionated using a sucrose gradient ultra-centrifugation after detergent-free extraction of the isolated colonic crypt. Luminal addition of methyl-β-cyclodextrin (10 mm) and mastoparan (30 μm), lipid rafts/caveolae disruptors, significantly inhibited luminal propionate-induced (0.5 mm) increases in I(sc) , but did not affect increases in I(sc) induced by serosal ACh (0.05 mm) or electrical field stimulation (EFS). Luminal addition of YM-254890 (10 μm), a Gα(q/11) -selective inhibitor, markedly inhibited propionate-induced increase in I(sc) , but did not affect I(sc) responses to ACh and EFS. Both methyl-β-cyclodextrin and YM-254890 significantly inhibited luminal propionate-induced non-neuronal release of ACh from colonocytes. Real-time PCR demonstrated that in mRNA expression of SCFA receptors, GPR 43 was far higher than that of GPR41 in the colon. Western blotting analysis revealed that the cholesterol-rich membrane microdomains that fractionated from colonic crypt cells were associated with caveolin-1, flotillin-1 and Gα(q/11) , but not GPR43. Uncoupling of Gα(q/11) from flotillin-1 in lipid rafts occurred under desensitization of the I(sc) response to propionate. These data demonstrate that the secretory response to luminal propionate in rat colon is mediated by G-protein on cholesterol-rich membrane microdomains, provably via Gα(q/11) .

Non‐neuronal release of ACh plays a key role in secretory response to luminal propionate in rat colon

Colonic chloride secretion is induced by chemical stimuli via the enteric nervous reflex. We have previously demonstrated that propionate stimulates chloride secretion via sensory and cholinergic systems of the mucosa in rat distal colon. In this study, we demonstrate non-neuronal release of ACh in the secretory response to propionate using an Ussing chamber. Mucosa preparations from the colon, not including the myenteric and submucosal plexuses, were used. Luminal addition of propionate and serosal addition of ACh caused biphasic changes in short-circuit current (Isc). TTX (1 μm) had no effects, while atropine (10 μm) significantly inhibited the Isc response to propionate and abolished that to ACh. In response to luminal propionate stimulation, ACh was released into the serosal fluid. A linear relationship was observed between the maximal increase in Isc and the amounts of ACh released 5 min after propionate stimulation. This ACh release induced by propionate was not affected by atropine and bumetanide, although both drugs significantly reduced the Isc responses to propionate. Luminal addition of 3-chloropropionate, an inactive analogue of propionate, abolished both ACh release and Isc response produced by propionate. RT-PCR analysis indicated that isolated crypt cells from the distal colon expressed an enzyme of ACh synthesis (ChAT) and transporters of organic cation (OCTs), but not neuronal CHT1 and VAChT. The isolated crypt cells contained comparable amounts of ACh to the residual muscle tissues including nerve plexuses. In conclusion, the non-neuronal release of ACh from colonocytes coupled with propionate stimulation plays a key role in chloride secretion, via the paracrine action of ACh on muscarinic receptors of colonocytes.

Getting a Handle on Cholera and the Circuits Controlling Intestinal Motility

Getting a Handle on Cholera and the Circuits Controlling Intestinal Motility

NaV‐igating excitement in the enteric nervous system

Na_V‐igating excitement in the enteric nervous system

Purinergic receptors and gastrointestinal secretomotor function

Secretomotor reflexes in the gastrointestinal (GI) tract are important in the lubrication and movement of digested products, absorption of nutrients, or the diarrhea that occurs in diseases to flush out unwanted microbes. Mechanical or chemical stimulation of mucosal sensory enterochromaffin (EC) cells triggers release of serotonin (5-HT) (among other mediators) and initiates local reflexes by activating intrinsic primary afferent neurons of the submucous plexus. Signals are conveyed to interneurons or secretomotor neurons to stimulate chloride and fluid secretion. Inputs from myenteric neurons modulate secretory rates and reflexes, and special neural circuits exist to coordinate secretion with motility. Cellular components of secretomotor reflexes variably express purinergic receptors for adenosine (A1, A2a, A2b, or A3 receptors) or the nucleotides adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), uridine 5'-triphosphate (UTP), or uridine diphosphate (UDP) (P2X(1-7), P2Y(2), P2Y(4), P2Y(6), P2Y(12) receptors). This review focuses on the emerging concepts in our understanding of purinergic regulation at these receptors, and in particular of mechanosensory reflexes. Purinergic inhibitory (A(1), A(3), P2Y(12)) or excitatory (A(2), P2Y(1)) receptors modulate mechanosensitive 5-HT release. Excitatory (P2Y(1), other P2Y, P2X) or inhibitory (A(1), A(3)) receptors are involved in mechanically evoked secretory reflexes or "neurogenic diarrhea." Distinct neural (pre- or postsynaptic) and non-neural distribution profiles of P2X(2), P2X(3), P2X(5), P2Y(1), P2Y(2), P2Y(4), P2Y(6), or P2Y(12) receptors, and for some their effects on neurotransmission, suggests their role in GI secretomotor function. Luminal A(2b), P2Y(2), P2Y(4), and P2Y(6) receptors are involved in fluid and Cl(-), HCO(3) (-), K(+), or mucin secretion. Abnormal receptor expression in GI diseases may be of clinical relevance. Adenosine A(2a) or A(3) receptors are emerging as therapeutic targets in inflammatory bowel diseases (IBD) and gastroprotection; they can also prevent purinergic receptor abnormalities and diarrhea. Purines are emerging as fundamental regulators of enteric secretomotor reflexes in health and disease.

Evidence that TASK1 channels contribute to the background current in AH/type II neurons of the guinea-pig intestine

Neurons that have AH (designation of neurons with a prominent and prolonged afterhyperpolarizing potential that follows the action potential) electrophysiological characteristics and type II morphology (AH/type II neurons) are the first neurons in reflex circuits in the small intestine. Thus, the state of excitation of these neurons strongly influences the properties of enteric reflexes. The resting outward current in the type II neurons is reduced, causing depolarization and increased excitability, when protein kinase C (PKC) or synaptic inputs are activated, suggesting that regulation of background channels is an important determinant of the state of excitability of these neurons. However, the channels that carry the background current are not yet identified. We used intracellular microelectrodes to record from myenteric AH/type II neurons of the guinea-pig ileum, immunohistochemistry to localize channels and reverse transcriptase–polymerase chain reaction (RT-PCR) to characterize channel transcripts. The blockers of TASK1 channels, bupivacaine (1 mM) and methanandamide (10 μM), depolarized AH/type II neurons by 11.6 mV and 7.9 mV, respectively, and increased resting input resistance by about 30%. The reversal potential determined for the effect of bupivacaine was −92 mV, indicating that bupivacaine acts at K + channels, without significant action on other channel types that are open at rest. The membrane potential of type II neurons was depolarized by acidification to pH 6.4, but this depolarization was associated with decreased input resistance and was not reduced by bupivacaine. Thus an unidentified current that is activated by reduced pH masks effects on TASK channels. Slow excitatory post-synaptic potentials in the neurons were reduced in amplitude by methanandamide, suggesting that they are generated in part by closure of TASK1 channels. TASK1 immunoreactivity occurred in all type II neurons (determined by double labeling for IB4 and NeuN), but no type II neurons were immunoreactive for TASK2 or TASK3. These latter channels were localized to non-type II neurons. Transcripts for TASK1, TASK2, TASK3 and other two-pore-domain potassium channels were found in ganglion extracts. It is concluded that TASK1 channels contribute to the resting outward current in AH/type II neurons, and that neurotransmitters that evoke slow depolarizations in these neurons do so through the closure of resting K + channels that include TASK1 channels.

An Enteric Occult Reflex Underlies Accommodation and Slow Transit in the Distal Large Bowel

An Enteric Occult Reflex Underlies Accommodation and Slow Transit in the Distal Large Bowel

Purinergic receptors and synaptic transmission in enteric neurons.

Purines such as ATP and adenosine participate in synaptic transmission in the enteric nervous system as neurotransmitters or neuromodulators. Purinergic receptors are localized on the cell bodies or nerve terminals of different functional classes of enteric neurons and, with other receptors, form unique receptor complements. Activation of purinergic receptors can regulate neuronal activity by depolarization, by regulating intracellular calcium, or by modulating second messenger pathways. Purinergic signaling between enteric neurons plays an important role in regulating specific enteric reflexes and overall gastrointestinal function. In the present article, we review evidence for purine receptors in the enteric nervous system, including P1 (adenosine) receptors and P2 (ATP) receptors. We will explore the role they play in mediating fast and slow synaptic transmission and in presynaptic inhibition of transmission. Finally, we will examine the molecular properties of the native receptors, their signaling mechanisms, and their role in gastrointestinal pathology.

Identification of two-pore domain potassium (K2P) channels in myenteric neurons of the guinea-pigs ileum

The intrinsic primary afferent neurons (IPANs) in the enteric nervous system (ENS) are the neurons that receive information about the state of the intestine and provide outputs to the enteric reflex circuits. Changes in properties of IPANs affect the whole activity of ENS. We have already studied the major currents which regulate the excitability of IPANs — such as IAHP, Ih and IA. Recently, the K2P channels have been discovered and investigated, mainly in the central nervous system. These channels have important roles in regulating the resting K+ current, which is fundamental to the function of neurons.

Recent advances in understanding the role of serotonin in gastrointestinal motility in functional bowel disorders: alterations in 5‐HT signalling and metabolism in human disease

Serotonin (5-hydroxytryptamine, 5-HT) is present in abundance within the gut, most stored in enterochromaffin cell granules. It is released by a range of stimuli, most potently by mucosal stroking. Released 5-HT stimulates local enteric nervous reflexes to initiate secretion and propulsive motility. It also acts on vagal afferents altering motility and in large amounts induces nausea. Rapid reuptake by a specific transporter (serotonin transporter, SERT) limits its diffusion and actions. Abnormally increased 5-HT is found in a range of gastrointestinal disorders including chemotherapy-induced nausea and vomiting, carcinoid syndrome, coeliac disease, inflammatory bowel disease and irritable bowel syndrome (IBS) with diarrhoea (IBS-D), especially that developing following enteric infection. Impaired SERT has been described in IBS-D and might account for some of the increase in mucosal 5-HT availability. 5-HT(3) receptor antagonists inhibit chemotherapy-induced nausea and diarrhoea associated with both carcinoid syndrome and IBS. While IBS-D is associated with increased 5-HT postprandially, IBS with constipation (IBS-C) is associated with impaired 5-HT response and responds to 5-HT(4) agonists such as Prucalopride and 5-HT(4) partial agonists such as Tegaserod.

Effects of NMDA receptor antagonists on visceromotor reflexes and on intestinal motility, in vivo

Antagonists of NMDA receptors can inhibit both the transmission of pain signals from the intestine and enteric reflexes. However, it is unknown whether doses of the NMDA antagonist, ketamine, that are used in anaesthetic mixtures suppress motility reflexes and visceromotor responses (VMRs). In fact, whether intestinal motility is affected by NMDA receptor blockers in vivo has been little investigated. We studied the effects of ketamine and memantine, administered intravenously or intrathecally. Rats were maintained under alpha-chloralose plus xylazine or pentobarbitone anaesthesia; VMR and jejunal motility were measured. Under alpha-chloralose/xylazine anaesthesia, i.v. ketamine inhibited VMRs at 6 mg kg h(-1), but not at 3 mg kg h(-1). It did not inhibit propulsive reflexes in the jejunum at 10 mg kg h(-1), but reduced them by 30% at 20 mg kg h(-1). Under alpha-chloralose/pentobarbitone anaesthesia, i.v. ketamine reduced propulsive reflexes at 40 mg kg h(-1) and VMR at 10 mg kg h(-1). Memantine inhibited VMRs at 20 mg kg h(-1) and propulsion at 2 mg kg h(-1). Ketamine and memantine, intrathecally, prevented VMRs, but not jejunal propulsion. We conclude that peripherally administered ketamine reduces both VMR and motility reflexes, but not at doses used in anaesthetic mixes (1.8-2.4 mg kg h(-1)). Effects on motility reflexes are likely to be due to non-NMDA receptor actions, possibly on nicotinic receptors.

An Enteric Occult Reflex Underlies Accommodation and Slow Transit in the Distal Large Bowel

Transit of fecal material through the human colon takes > or =30 hours, whereas transit through the small intestine takes 24 hours. The mechanisms underlying colonic storage and slow transit have yet to be elucidated. Our aim was to determine whether an intrinsic neural mechanism underlies these phenomena. Recordings were made from circular muscle (CM) cells and myenteric neurons in the isolated guinea pig distal colon using intracellular recordings and Ca(2+) imaging techniques. Video imaging was used to determine the effects of colonic filling and pellet transit. Circumferential stretch generated ongoing oral excitatory and anal inhibitory junction potentials in the CM. The application of longitudinal stretch inhibited all junction potentials. N-omega-nitro-L-arginine (100 micromol/L) completely reversed the inhibitory effects of longitudinal stretch suggesting that nitric oxide (NO) inhibited interneurons controlling peristaltic circuits. Ca(2+) imaging in preparations that were stretched in both axes revealed ongoing firing in nNOS +ve descending neurons, even when synaptic transmission was blocked. Inhibitory postsynaptic potentials were evoked in mechanosensitive interneurons that were blocked by N-omega-nitro-L-arginine (100 micromol/L). Pellet transit was inhibited by longitudinal stretch. Filling the colon with fluid led to colonic elongation and an inhibition of motility. Our data support the novel hypothesis that slow transit and accommodation are generated by release of NO from descending (nNOS +ve) interneurons triggered by colonic elongation. We refer to this powerful inhibitory reflex as the intrinsic occult reflex (hidden from observation) because it withdraws motor activity from the muscle.

Evidences of submucosal neuronal plasticity in distal colon of chronic restraint stress rat

Chronic stress is involved in the pathogenesis of gastrointestinal disorders. The phenotype and function of neurons in submucosal reflex may be subjects for this evidence. We investigated whether enteric neuronal reflex was changed during chronic stress. Male Wistar rats were immobilized for 14 days (1-hr/day). Distal colonic epithelium was isolated to measure Isc and Rt by Ussing chamber techniques. Isc and Rt of stressed-rat colonic segments were lower than those in control rats. The secretory effects of vanilloid capsaicin (3 μM) mediated by VR-1 on sensory neurons was decreased by 70% in stressed-rat colon. Basolateral addition of DAMGO, DSLET or U-50,488 (1 μM), μ-, δ- or μ-opioid receptor (OR) agonists respectively, diminished the basal Isc of control rat colon by 20–30%, whereas only U-50,488 decreased the baseline Isc of stress rat colon by 20%. Substance P (SP, 1 μM) produced the same magnitude of Isc response in both control and stress rat colon. Pretreatment with DAMGO potentiated the SP response by 2-fold in stress rats. These findings suggested that the submucosal neuronal reflex mediated through VR-1 and opioid receptors was modified in distal colon of chronic stress rat. The changes in submucosal neuronal plasticity may be the cause of stress induced gastrointestinal disorders. This work was supported by the Thailand Research Fund (RSA 4780010) and Chulalongkorn University.

Sensory elements within the circular muscle are essential for mechanotransduction of ongoing peristaltic reflex activity in guinea‐pig distal colon

We have recently identified a population of mechanosensory myenteric S-interneurons in the distal colon of guinea-pigs. However, the role of the longitudinal (LM) and circular muscle (CM) in transducing these mechanosensory signals into enteric reflexes is unclear. In this study, we have investigated whether the LM or CM layer is necessary for activation of ascending excitatory and descending inhibitory neuronal pathways by static stretch of the paralysed isolated guinea-pig distal colon. Simultaneous intracellular recordings were made from pairs of CM cells at either end of isolated sheet preparations of distal colon that were devoid of mucosa and submucous plexus; and were maintained under circumferential stretch. In the presence of nifedipine (1 microm), an ongoing discharge of excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs) were recorded simultaneously at the oral and anal ends of the preparation. When the LM was sharp dissected off the myenteric plexus, the synchronized discharge of ascending EJPs and descending IJPs in the CM layer was unaffected. In contrast, when the majority of CM was sharp dissected off the myenteric plexus, ongoing neural activity was absent, or substantially decreased in both the LM and CM. In these preparations, immunohistochemical staining, together with transmural electrical stimuli confirmed that the myenteric plexus was always present and intact in these preparations. When full-thickness strips of CM were removed from progressively longer lengths of myenteric plexus, a graded reduction in the correlation of coordinated oral EJPs and anal IJPs occurred. However, removing approximately 40% of the thickness of CM layer from the entire preparation did not significantly disrupt, nor reduce the degree of correlation between oral EJPs and anal IJPs, suggesting that critical sensory elements did not lie adjacent to the submucosal plexus. It is concluded that mechanosensory transmission that underlies repetitive firing of ascending excitatory and descending inhibitory neuronal pathways is critically dependent upon sensory elements within the CM layer. These elements are likely to activate stretch-sensitive interneurons in the myenteric plexus. No evidence was found to suggest that the connectivity between the LM and the myenteric plexus was required for mechanotransduction.

Serotonin and sensory signalling from the gastrointestinal lumen

Serotonin and sensory signalling from the gastrointestinal lumen

Gastrointestinal secretions and vomiting

The two main gastrointestinal secretions are mucus and digestive enzymes. There are numerous pits or crypts throughout the gastrointestinal tract containing specialized secretory cells. Tubular glands are present in the stomach and proximal duodenum. Specialized glands include the salivary glands, pancreas and liver. The presence of food in the gastrointestinal tract causes direct stimulation of mucous (goblet) cells and nearby glands, which release digestive enzymes. The mucus protects the epithelium and acts as a lubricant. Mucous cells and glands are also stimulated by local enteric reflexes in response to distension or irritation. Parasympathetic stimulation increases secretion from salivary, oesophageal, gastric, pancreatic, duodenal Brunner's and distal large intestinal glands. Secretion from the rest of the small intestine and proximal large intestine is controlled predominantly by local neural and hormonal stimuli. Sympathetic stimulation alone slightly increases gastrointestinal secretion, but in the presence of parasympathetic or hormonal stimulation can reduce secretion because it induces constriction of splanchnic blood vessels. Nausea is a conscious awareness of arousal of the vomiting centre or related areas of the brain. It often precedes vomiting and may be initiated by higher CNS centres or local irritation in the gastrointestinal tract. Vomiting can be initiated by overdistension or irritation of the upper gastrointestinal tract, resulting in afferent impulses transmitted via the parasympathetic (vagus) and sympathetic nervous systems to the vomiting centre in the medulla. It can also be initiated by stimulation of other areas of the brain, especially the chemoreceptor trigger zone.

Enteric nervous system

The purpose of this review is to provide a synopsis of how the field of enteric neurobiology has advanced during the past 2 years. With more than 500 studies from which to choose, the authors have focused on several themes that illustrate recent progress. There has been an explosion of interest in the development of the enteric nervous system driven by the need to understand development abnormalities, particularly in Hirschsprung disease, and fueled by technical advances for investigating how neural crest-derived cells migrate, proliferate, and differentiate into enteric neurons and glia. The use of neural stem cells as a therapeutic strategy aimed at repopulating regions of bowel, where enteric neurones are reduced or absent, is on the horizon. Enteric reflexes involve interactions between sensory neurons, interneurons, and motor neurons. Recent findings suggest this distinction may be blurred, with neurons having multifunctional properties, perhaps because enteric neurons, unlike their central nervous system counterparts, are directly exposed to mechanical forces that they regulate. Another topic the authors have highlighted is pharmacology, with new tools for investigating ion channels, receptors, and transporters, leading to an expanding list of molecular mechanisms that regulate neuronal excitability. Long-term alterations in the expression of these molecules during disease or injury may underlie many gastrointestinal disorders that currently have unknown etiology. The authors finish with a look to the future and what may be the subject of this review next time. Basic science information gathered during the past 2 years provides insight into pathophysiologic processes and will pave the wave for improved understanding of both organic and 'functional' gastrointestinal disorders.

A quantitative approach to recording peristaltic activity from segments of rat small intestine in vivo

We have developed methods that allow correlation of propulsive reflexes of the intestine with measurements of intraluminal pressure, fluid movement and spatio-temporal maps of intestinal wall movements for the first time in vivo. A segment of jejunum was cannulated and set up in a Trendelenburg recording system while remaining connected to the vascular and nerve supply of the anaesthetized rat. The resting intraluminal pressure in intact intestine was 2-4 mmHg. Hydrostatic pressures of 2, 4, 8 and 16 mmHg were imposed. At a baseline pressure of 4 mmHg, propulsive waves generated pressures of 9 +/- 1 mmHg, that progressed oral to anal at 2-5 mm s(-1). Individual propulsive waves propelled 0.8 +/- 0.4 mL of fluid. The frequency of propulsive waves increased with pressure, but peristaltic efficiency (mL per contraction) decreased with pressure increase between 4 and 16 mmHg. Atropine, as a bolus, transiently blocked peristalsis, but caused maintained block when infused. Hexamethonium blocked propulsive contractions. Inhibition of nitrergic transmission converted regular peristalsis to non-propulsive contractions. These studies demonstrate the utility of an adapted Trendelenburg method for quantitative investigation of motility and pharmacology of enteric reflexes in vivo.

Origin and mediation of secretion induced by oral phytohaemagglutinin (PHA) in rats

After oral administration several gut-binding lectins induce accumulation of liquor and amylase in the proximal small intestine. Orally administered Phaseolus vulgaris phytohaemagglutinin (PHA) was used to study the mediation of these effects in rats. The regulation of amylase secretion clearly differed from that of the liquor. The amylase activity was of pancreatic origin, in agreement with the known cholecystokinin-releasing effect of PHA. It appears that CCK exerts its effect both directly and by facilitating neural stimulatory pathways. Intestinal secretion was identified as the source of the liquor, without a contribution by other secretions. It was mediated by a local cholinergic reflex with the involvement of both muscarinic and nicotinic acetylcholine receptors. It is speculated that the observed enteric reflex may enable the gut to transport secreted antibacterial peptides or secretory antibodies from the crypts to adherent bacteria on adjacent villi.