Mechanisms of Disease: the pathological basis of gastroparesis a review of experimental and clinical studies

Transcription

1 Mechanisms of Disease: the pathological basis of gastroparesis a review of experimental and clinical studies Harsha Vittal, Gianrico Farrugia, Guillermo Gomez and Pankaj J Pasricha* SUMMARY The pathogenesis of gastroparesis is complicated and poorly understood. This lack of understanding remains a major impediment to the development of effective therapies for this condition. Most of the scientific information available on the pathogenesis of gastroparesis has been derived from experimental studies of diabetes in animals. These studies suggest that the disease process can affect nerves (particularly those producing nitric oxide, but also the vagus nerve), interstitial cells of Cajal and smooth muscle. By contrast, human data are sparse, outdated and generally inadequate for the validation of data obtained from experimental models. The available data do, however, suggest that multiple cellular targets are involved. In practice, though, symptoms seldom correlate with objective measures of gastric function and there is still a lot to learn about the pathophysiology of gastroparesis. Future studies should focus on understanding the molecular pathways that lead to gastric dysfunction, in animal models and in humans, and pave the way for the development of rational therapies. KEYWORDS diabetes mellitus, gastroparesis, interstitial cells of Cajal, nitric oxide REVIEW CRITERIA PubMed was searched between January and September 2006, and then searched again after the peer-review process, for English-language papers containing the following terms alone and in combination diabetes, idiopathic, gastroparesis, gastropathy, pathogenesis, pathophysiology, gastric dysrhythmias, pylorospasm, nitric oxide, interstitial cells of Cajal, calcitonin gene-related peptide (CGRP), substance P, neurokinin A, somatostatin, serotonin, acetylcholine, vasoactive intestinal peptide, carbon monoxide, advanced glycation end products, reactive oxygen species, neurotrophins, stem cell factor, c-kit, vagus nerve, autonomic and anti-hud antibodies. H Vittal is an Advanced Endoscopy Fellow at the Maine Medical Center, G Gomez is Professor of Surgery, and PJ Pasricha is Bassel and Frances Blanton Distinguished Professor of Internal Medicine and Professor of Neuroscience and Cell Biology, at the University of Texas Medical Branch, Galveston, TX. G Farrugia is Professor of Medicine at the Mayo Clinic College of Medicine, Rochester, MN, USA. Correspondence *University of Texas Medical Branch, McCullough Building, 301 University Boulevard, Galveston, TX , USA jpasrich@utmb.edu Received 5 December 2006 Accepted 1 February doi: /ncpgasthep0838 INTRODUCTION Gastroparesis is the term used to describe a significant delay in the emptying of solids and liquids from the stomach. Such a delay in gastric emptying might be asymptomatic but can also be associated with nausea, vomiting, bloating, dyspepsia, early satiety and pain. The most common forms of gastroparesis are diabetic and idiopathic; other less common forms include postsurgical and medication-related gastroparesis. Published data from specialized centers suggest that gastroparesis can develop in 20 55% of patients with type 1 diabetes and up to 30% of patients with type 2 diabetes; however, the incidence and prevalence of gastroparesis might be significantly lower in the general community. 1,2 The pathogenesis of gastroparesis is multifactorial and poorly understood, in part because of a lack of comprehensive studies of the pathology of the stomach in affected patients. The lack of available data has resulted in therapy that is often empiric and suboptimal. This paper reviews the data that are available on the pathogenesis of gastroparesis in experimental animal models, in addition to those obtained from patients with gastroparesis. CLINICAL MANIFESTATIONS OF GASTROPARESIS Gastric emptying requires the coordination of events by several cell types, including extrinsic motor neurons, enteric motor neurons, interstitial cells of Cajal (ICC) and smooth-muscle cells (Box 1 and Figure 1). In healthy individuals, most stomach functions are generally unnoticed, although the ingestion and handling of a normal meal is often associated with a feeling of satisfaction. By contrast, patients with gastroparesis can have abnormal sensations that dominate their eating experience. The symptoms of gastroparesis can include any combination of nausea, vomiting, abdominal bloating, dyspepsia, early satiety and pain. The dominant symptom varies among patients. 1 One study that evaluated 146 patients with gastroparesis 336 NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY JUNE 2007 VOL 4 NO 6

2 revealed that nausea, vomiting, abdominal bloating and early satiety were experienced by 92%, 84%, 75% and 60% of patients, respectively. 3 Another study indicated that abdominal pain is a common symptom and that it occurs in 89% of patients with gastro paresis. 4 PATHOPHYSIOLOGIC CORRELATION Pathophysiologic alterations in gastric function caused by hyperglycemia and diabetes have been extensively reviewed elsewhere 5 7 and are, therefore, discussed only briefly here. A delay in gastric emptying is a necessary component of the definition of gastroparesis. In a prospective study of gastrointestinal symptoms and gastric emptying in 186 patients who attended a diabetes clinic over a 12-month period, gastric emptying was significantly slower in patients with diabetes than in healthy volunteers; nearly 22% of patients with diabetes had a prolonged T 1/2 emptying time and 28% of patients displayed increased retention of an ingested meal 2 h after eating. 8 The speed of gastric emptying differed according to sex women had slower gastric emptying than men but no correlations were noted with age, type of diabetes, duration of diabetes, fasting glucose concentration, glycosylated hemoglobin level or presence of other diabetic complications, such as peripheral neuropathy, retinopathy or nephropathy. Although a gastric emptying test is the gold standard for the diagnosis of gastroparesis, it has notable shortcomings. Occasionally, patients have all of the classic symptoms of gastroparesis but normal rates of gastric emptying. In fact, the correlation between individual symptoms and gastric-emptying abnormalities is poor, and high variability between and within individuals has been reported In prospective studies of patients with diabetic gastroparesis, delayed gastric emptying correlated with complaints of fullness, upper abdominal pain and reduced hunger in the 2 weeks preceding the gastricemptying test; however, the presence of the cardinal symptoms of gastroparesis nausea and vomiting were not related to gastric emptying. 8,12 The major known pathophysiologic abnormalities found in patients with gastroparesis are listed in Table 1, in addition to the symptoms that they might putatively cause. It should be noted, however, that, although the scheme presented in Table 1 is conceptually attractive and intuitively appealing, the link between Box 1 Normal gastric physiology. Normal gastric emptying requires the coordination of events by several cell types, including extrinsic neurons, enteric motor neurons, interstitial cells of Cajal, and smooth-muscle cells (Figure 1). The stomach functions as two separate regions the fundus and body. The fundus functions as a reservoir for ingested food and accommodates a meal without a significant increase in intragastric pressure. In addition, the fundus regulates the transfer of gastric contents to the body of the stomach and antrum. Trituration of gastric contents occurs in the antrum owing to powerful antral contractions that grind the gastric contents against the closed pylorus until food particles reach a size of 1 2 mm and can be passed into the duodenum. Gastric contractions and subsequent gastric emptying are controlled by several mechanisms. A pacemaker region, located in the body of the stomach on the greater curvature, provides a slow-wave rhythm of approximately three cycles per minute in humans and sets the frequency of smooth-muscle contraction. The pacemaker signal is generated by interstitial cells of Cajal, specialized mesenchymal cells that also participate in neurotransmission (by setting the smooth-muscle membrane potential) and mechanotransduction In this setting, motor neurons generate complex motor patterns, including peristalsis, segmentation and the migrating motor complex. These motor patterns are, in turn, modulated by signals from the central nervous system (CNS) and relayed predominantly by the vagus nerve. Contractions are coordinated not only between different regions of the stomach, but also between the stomach and small intestine. Duodenal feedback, both humoral and neuronal, can inhibit gastric motility and increase pyloric tone, which results in tight regulation of gastric emptying. Sensory information is relayed from the stomach to the CNS by both vagal and spinal afferent nerves. Spinal afferent nerves transmit nociceptive messages through spinal tracts to the CNS and are mediated by neuropeptides, such as calcitonin gene-related peptide, substance P and neurokinin A. 99 Vagal afferent nerves have an important role in the transmission of information about the physiologic state of the gastric wall from the gastric wall to the CNS. 100,101 A Wild type nnos / B 2 1 Impaired relaxation Delayed emptying Impaired relaxation Satiety Unregulated distal movement Figure 1 The importance of inhibitory nitrergic mechanisms in gastric emptying. (A) A comparison of stomachs from wild-type and Nos1 / knockout mice. Diffuse enlargement of the esophagus, stomach and duodenum is present in the Nos1 / adult mice (on the right) compared with the wild-type mice (on the left). Bezoars have been frequently found in stomachs of Nos1 / mice, despite fasting for 2 days. (B) The pathophysiologic and clinical consequences of impaired relaxation in the fundus (1) and pylorus (2) of the stomach. Permission obtained from the American Gastroenterological Association Mashimo et al. (2000) 25 Gastroenterology 119: Abbreviation: nnos, neuronal nitric oxide synthase. JUNE 2007 VOL 4 NO 6 VITTAL ET AL. NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY 337

3 Table 1 Potential pathophysiologic correlates in gastroparesis. Functional problem Contributing factors Possible clinical manifestation Delayed gastric emptying Impaired fundic relaxation Gastric hypersensitivity Electric dysrhythmia, antral hypomotility and distension, and pylorospasm Loss of nitrergic input to smooth muscle Possible vagal and spinal neuropathies Early satiety, nausea and vomiting, bloating, anorexia, discomfort, bezoar formation, unpredictable glycemic control and changes in drug pharmacokinetics Early satiety, postprandial discomfort and symptoms that arise from rapid gastric emptying of ingested nutrients, including abdominal cramps, diarrhea, dizziness and sweating after a meal Early satiety, pain and nausea these pathophysiologic abnormalities and their putative symptoms has yet to be evaluated and proven in a rigorous fashion. As stated above, the presence of abdominal bloating and early satiety, in the absence of other symptoms, has been shown to be predictive of delayed gastric emptying in patients with diabetes. 12 Proximal gastric function has mainly been studied in patients with functional dyspepsia and the data available provide conflicting results. Several reports suggest that there is a relationship between impaired gastric accommodation and early satiety, but other reports have been unable to confirm this association. 16,17 Similar findings have been demonstrated in patients with diabetes and autonomic neuro pathy a decrease in proximal gastric accommodation in response to a liquid meal correlated with bloating but not with nausea or pain. 18 Of note, impaired gastric accommodation might cause rapid gastric emptying, particularly of liquids, in patients in the early stages of type 2 diabetes. 19 Gastric dysrhythmias, identified by electrogastrography, are commonly found in patients with gastroparesis. Acute hyperglycemic events can cause gastric dysrhythmias, predominantly tachygastrias, which can resolve once euglycemia is achieved. 20 The relationship between abnormalities detected by electrogastrography and the symptoms of gastroparesis has not, however, been established, and normal electrogastrography findings do not predict normal gastric emptying. 21 Finally, the pathophysiologic basis of nausea and pain, the two most distressing symptoms experienced by patients with gastroparesis, remains mostly unknown. As in patients with dyspepsia, there is evidence of afferent neural pathway hypersensitivity with gastric distension, which results in increased nausea, pain and bloating in patients with diabetic gastroparesis compared with controls. 22 PATHOPHYSIOLOGY OF GASTROPARESIS: ANIMAL STUDIES Neuronal changes, nitric oxide and nitric oxide synthase Coordinated gastric motor function requires not only stimuli that promote contraction, such as those initiated by acetylcholine and substance P, but also stimuli that inhibit contraction, such as those initiated by vasoactive intestinal peptide (VIP) and nitric oxide. In addition, specialized functions, such as fundic accommodation and pyloric relaxation, are, by their nature, crucially dependent on inhibitory nitrergic nerves. 23,24 In the context of gastroparesis, it is, therefore, unsurprising that neuronal nitric oxide and the enzyme responsible for its synthesis, neuronal nitric oxide synthase (nnos), have emerged as the molecules of greatest interest in studies of the condition. The first evidence of a role for nnos in the pathogenesis of gastroparesis came from the observation that Nos1 / knockout mice develop grossly enlarged stomachs, with hypertrophy of the circular muscle layer and gastric stasis (Nos1 is the gene that encodes nnos; Figure 2). 25 Gastric emptying can also be delayed in healthy rodents by pharmacologic inhibition of NOS Several animal studies have shown that diabetes is associated with changes in gastric nnos expression and activity. In a rat model of spontaneous diabetes, impaired gastric relaxation was associated with a decrease in nnos expression and activity in the myenteric plexus; 29 similar results have been observed in rats with streptozotocin-induced diabetes. 30 In another study, nnos expression in the antrum 338 NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY VITTAL ET AL. JUNE 2007 VOL 4 NO 6

4 of rats with streptozotocin-induced diabetes was decreased compared with that in healthy controls, but, interestingly, no change in immunoreactivity was observed in the duodenum, ileum or colon. 31 An exception to the findings of these studies is the report by Adeghate et al., 32 who found that there was an increase in nnos expression in the tissue of the stomach and duodenum of rats with streptozotocin- induced diabetes at 4 and 32 weeks. The expression of other neuro transmitters has also been studied. For example, one study found that there was a concomitant decrease in the expression of VIP and nnos in the antrum of obese diabetic mice (a model of human type 2 diabetes). 33 In the antrum of nonobese diabetic (NOD) mice 34 (a model of human type 1 diabetes), however, there was an increase in the number of neurons that express VIP and a decrease in those that express nnos in the myenteric plexus. Substance P content has also been noted to be decreased in the antrum of both prediabetic and NOD mice. 35 Finally, several changes in endocrine cells and hormonal expression have been noted in diabetes, both in the stomach and in other regions of the gastro intestinal tract. Antral levels of somatostatin, VIP and galanin, in addition to duodenal secretin and jejunal motilin, were higher in NOD mice compared with controls, whereas duodenal gastric inhibitory poly peptide and colonic peptide YY concentrations were lower; furthermore, secretin and motilin levels correlated to gastric emptying in NOD mice. 36 Little is known about the factors that are responsible for the changes in gastric nnos expression in the setting of diabetes, but there are at least three theoretical mechanisms (which function either alone or in combination) that might be important. The first mechanism is neuronal loss or degeneration. The second mechanism is the inhibition of nnos tran scription owing to a lack of trophic support (e.g. a lack of insulin or vagal acetylcholine). The third mechanism is a post-translational modification that leads to impaired nnos function. In one study of NOD mice, nnos expression was found to be markedly decreased in gastric enteric neurons, but this decrease was reversed when the mice were treated with insulin (Figure 3). 37 These and other data suggest that the loss of nnos expression is not associated with neuronal loss. An increase in apoptosis of colonic myenteric neurons, dorsal root ganglion and Muscle Enteric nerves (excitatory) Enteric nerves (inhibitory) vagus nodose ganglion has, however, been noted in rats with streptozotocin-induced diabetes. 38 Another paper has also shown that apoptosis of enteric nitrergic neurons occurs in the proximal colon of mice with streptozotocin-induced diabetes, an effect that could be prevented by glialcell-line-derived neurotrophic factor through the phosphoinositide 3 kinase pathway. Despite their demonstration that these mice had delayed gastric emptying, the authors of this study did not report the effect of diabetes on gastric enteric neuronal apoptosis and it is, therefore, not clear whether these results add to our understanding of the pathogenesis of gastroparesis. The control of nnos expression is extremely complex, and the changes in nnos expression that are seen in various regions of the gastrointestinal tract might represent the production of multiple messenger RNA transcripts by a number of mechanisms. These mechanisms include the use of multiple promoter sites, alternative splicing, cassette insertions or deletions and varied cleavage and polyadenylation sites in the 3' untranslated region, which might be affected to different degrees by glucose, insulin or insulin-like growth factor Nitrergic neuropathy is suggested to occur in two phases, at least in other systems of the body. 43 First, after the Vagus nerve ICC Spinal nerve Figure 2 The main cellular elements involved in gastric motility and sensation. Enteric nerves (shown here) arise in the myenteric plexus and provide excitatory or inhibitory signals to smooth muscle in a process that also requires participation of the ICC. Two types of extrinsic nerves are also shown branches of the vagus, which can be both afferent (carrying information back to the brain) or efferent (modulating enteric nerves), and spinal nerve branches, which carry pain and other sensations. Not shown are branches of the sympathetic nerves, which also participate in regulation of gastric function. Abbreviation: ICC, interstitial cells of Cajal. JUNE 2007 VOL 4 NO 6 VITTAL ET AL. NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY 339

5 WT NOD NOD + insulin nnos immunohistochemistry nnos / STZ + insulin Figure 3 Effects of diabetes on gastric nnos. Expression of nnos is depleted in the pyloric myenteric neurons of mice with experimental diabetes, but this effect can be reversed by insulin administration. Immunohistochemical analysis of nnos protein expression shows that nnos is present in the pyloric myenteric neurons of WT mice but not in those of Nos1 / mice (top row). By contrast, nnos expression is lost in both NOD mice and mice with STZinduced diabetes. Insulin administration for 1 week to NOD mice and mice with STZ-induced diabetes reverses the loss of nnos expression. Permission obtained from the American Society for Clinical Investigation Watkins et al. (2000) 37 J Clin Invest 106: Abbreviations: nnos, neuronal nitric oxide synthase; NOD, nonobese diabetic; STZ, streptozotocin; WT, wild-type. STZ induction of diabetes, there is a decrease in nnos expression and activity in the pylorus, which can be reversed by insulin administration. This decrease in nnos expression and activity might be caused by a defect in axonal nnos transport and is not accompanied by apoptotic cell loss. Second, if treatment is delayed for 12 weeks after the induction of diabetes, nnos expression and activity do not seem to recover, despite an adequate insulin level; this effect might be caused by apoptotic cell loss. 44 These studies suggest that nitrergic neuropathy results from the accumulation of toxic components that function in a synergistic fashion with endogenous nitric oxide. 45 Candidate toxic compounds include advanced glycation end products, receptors for which are expressed by myenteric neurons and which, if activated, result in decreased nnos expression. 46 Although this study was performed in the duodenum, it is conceivable that similar mechanisms might operate in the stomach. The expression of nnos in the gastric myenteric plexus also seems to be regulated, in part, by vagal innervation. In rats, for example, truncal vagotomy significantly reduced the expression and activity of nnos in gastric tissue. 47 Further more, the same study demonstrated that nnos expression in cultured gastric myenteric nerves was increased by nicotinic receptor stimulation and decreased by a protein kinase C antagonist. In combination, these results suggest that acetylcholine release from vagal nerve endings is a regulator of nnos expression in enteric motor neurons. Autonomic neuropathy is relatively common in patients with diabetes, so this model might explain, at least in part, the changes in gastric nnos expression. Although most experimental work has focused on examining the effects of diabetes on levels of gastric nnos expression, this approach might be too simplistic. The catalytic activity of the nnos enzyme depends on its ability to maintain a dimerized state, which results in the creation of high-affinity binding sites for arginine and other molecules and enables electron transfer between the flavin (reductase domain) and heme (oxygenase domain) groups. This electron transfer, in turn, causes the heme iron to bind oxygen and catalyzes the mono- oxygenation of arginine, which results in nitric oxide production. 48 Our own study suggests that the inhibition of gastric relaxation in the setting of diabetes does not correlate with absolute nnos levels, but instead correlates with the amount of dimerized enzyme, which is markedly reduced in female diabetic rats. 49 A related mechanism of neuronal damage might involve the formation of reactive oxygen species (ROS), large amounts of which are generated in hyperglycemic states. ROS have been demonstrated to activate protein kinase C, which causes vascular abnormalities in rats with experimental diabetes. 50 In this regard, the use of the antioxidant α-lipoic acid prevents a decrease in gastric relaxation in rats with streptozotocininduced diabetes, which supports a potential role for ROS in gastroparesis. 51 The downstream effects of nitric oxide on smooth-muscle relaxation are mediated by cyclic GMP (cgmp). Theoretically, agents that augment cgmp levels in neurons (e.g. by blocking the activity of phosphodiesterase, the enzyme that metabolizes cgmp) could counter act the effects of low levels of nnos activity following gastric emptying, as has been demonstrated in diabetic mice. 37 Trials of agents in this class, such as sildena fil, in patients with gastro paresis have, however, been disappointing. 52 In summary, impaired neuronal and, in particular, nitrergic function seems to be the 340 NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY VITTAL ET AL. JUNE 2007 VOL 4 NO 6

6 best-characterized finding in experimental studies of diabetic gastropathy. A tempting hypothesis has been promulgated that this impairment is responsible for several of the pathophysiological features observed in gastroparesis, including loss of proximal relaxation and delayed gastric emptying (as a combination of pylorospasm, that is, a prolonged, intense contraction of the pylorus, and dis ordered, nonpropulsive antral motility). A substantial amount of work is, however, still required before these experimental findings from animal studies can be extra polated to humans, in whom the situation might be more complex. Pharmacological blockade of nnos activity, therefore, enhances antral contractions and gastric emptying in humans, 53 whereas the opposite is observed in rodents. By contrast, systemic nitroglycerin administration inhibits liquid gastric emptying in healthy adults, despite a reduction in pyloric contractility and tone, probably by slowing proximal gastric motility. Changes in interstitial cells of Cajal Attention is increasingly being paid to the role of ICC in the pathogenesis of gastro paresis. Numbers of ICC are reduced in NOD mice, especially in the antrum, which is associated with significant disruption of slow-wave activity and attenuated neuronal responses. 54 The mechanism underlying ICC depletion in the setting of diabetes is unknown, but might reflect deprivation of the trophic benefits of insulin. Insulinopenic states can result in smooth-muscle atrophy, which might contribute to ICC depletion. 55 In organotypic cell cultures from the mouse stomach, chronic hyperglycemia resulted in the loss of ICC networks, an effect that was prevented by insulin and insulin-like growth factor Nitric oxide might also be required for ICC maintenance, and the loss of nnos could make additional contributions to the depletion of ICC in diabetes. 57 Changes in extrinsic innervation Little information is available about the effect of diabetes on the extrinsic innervation of the gastrointestinal tract. In rats with streptozotocininduced diabetes, gastric acid secretion decreases in response to vagal electrical stimulation, which suggests that a vagal neuropathy is present. 58,59 A reduction in pyloric relaxation has been noted after gastric distension if acute hyperglycemia is induced in rats; pyloric sphincter dysfunction has been suggested to be mediated by vagal damage because this effect can also be induced by subdiaphragmatic vagotomy and hexamethonium. 60 In addition, acute hyperglycemia impairs antral contractions and antropyloric coordination, which is caused, at least in part, by impaired vagal activity. 61 A number of studies have also demonstrated morphologic abnormalities of both motor and sensory components of the vagus nerve in rats with chronic diabetes. In the BB rat model of spontaneous diabetes, myelinated and unmyelinated fibers of the vagus nerve were smaller than those in controls. 62,63 Changes (degeneration and apoptosis) have also been demonstrated in both sensory 38,64 and motor nuclei of the vagus 65 in streptozotocin-induced diabetes. Some of these changes might result from deprivation of trophic factors. In rats with streptozotocin-induced diabetes, retrograde transport of nerve growth factor and neurotrophin 3 to the nodose ganglia is impaired, and this impairment is related, in part, to impaired phosphoinositide 3 kinase protein kinase B signaling Neurotrophins are essential for the survival and maintenance of somatic and autonomic neurons, and a defect in the uptake of neurotrophins might lead to neural pathology. Sympathetic nerves are also affected by diabetes in a process that has been termed sympathetic axonal dystrophy, which is characterized by pathologically distinct changes in axons and dendrites within the prevertebral sympathetic ganglia of both animals and patients with diabetes The significance and clinical relevance of these findings remains to be established. Little or no information is available about changes to gastric spinal nerves in the setting of diabetes. It is, however, reasonable to assume that these nerves will be affected in patients with diabetes, because somatic sensorimotor and autonomic neuropathies are common in this population. 74,75 If these nerves are involved, the pain that often accompanies diabetic gastroparesis can be explained as neuropathic in origin; however, further studies need to be performed to test this hypothesis. Changes in smooth muscle In general, the stomachs of patients with diabetes are stiffer and have lower compliance (the ability to distend to accommodate a meal) than those of healthy individuals. 76 Studies of gastric smooth-muscle tissue from rats with experimental diabetes have demonstrated that there JUNE 2007 VOL 4 NO 6 VITTAL ET AL. NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY 341

7 A B pathologic changes in the stomach of patients with gastroparesis is generally outdated and sparse, and reports conflicting results. A subset of patients with long-term diabetes might have pylorospasm, presumably because of a lack of inhibitory modulation, which might contribute to the symptoms of gastroparesis by impeding gastric emptying. 81 Figure 4 Effects on gastric smooth muscle in a patient with type 1 diabetes. (A) A section through the external muscle layer of stomach from a healthy individual. (B) A section through the external muscle layer of stomach from a patient with diabetic gastroparesis. Atrophic muscle fibers and an increase in interstitial collagenous connective tissue can be seen. (Picro Mallory stain, original magnification 120). Data from Ejskjaer et al. 84 is a disruption in protein kinase C activation, which results in smooth-muscle dysfunction, as shown by a poor response to direct stimulation of gastric myocytes. 77 As previously discussed, lack of (or resistance to) insulin can cause smooth-muscle atrophy and depletion of ICC, with a concomitant decrease in the expression of stem-cell factor (the ligand for c-kit, which is the tyrosine kinase receptor located on ICC). 55 Further evidence of muscle dysfunction comes from a study of rats with streptozotocininduced diabetes that showed specific changes in the postsynaptic muscarinic control of antral smooth-muscle contraction. 78 Muscle function in the diabetic stomach might be affected in a region-specific manner. In a mouse model of spontaneous diabetes, fundic hypomotility and pyloric hyper contractility were noted in response to bethanochol administration, but there was no change in antral motility. 79 PATHOPHYSIOLOGY OF GASTROPARESIS: HUMAN STUDIES Studies of volunteer patients with diabetes have shown that acute hyperglycemia can delay gastric emptying, possibly because of alterations in autonomic function or other neurohormonal effects. 6,80 Whether changes to the various cellular components of gastric motility as a direct result of hyperglycemia are permanent is, however, unclear. In contrast to animal studies, human studies provide little definitive evidence that there is a selective or predominant involvement of gastric myenteric nnos in gastroparesis. This lack of evidence is largely attributable to the fact that the literature on Structural changes One study that examined patients with gastroparesis and controls determined that there was no structural difference in the vagus nerve, myenteric nerves and smooth muscle between the two groups. 82 By contrast, another study, which examined the vagus nerve from two patients with gastroparesis who had undergone partial gastrectomy, found a marked reduction in the density of unmyelinated nerves. 83 This variability in the structural changes found in patients with diabetic gastroparesis was illustrated by histopathologic analysis of the stomach of four patients who had a partial gastrectomy as therapy for treatment-refractory diabetic gastroparesis. 84 Severe collagen deposition and fibrosis of the smooth-muscle layers was seen, without any significant changes to the myenteric plexus or vagus nerve (Figure 4). In combi nation, these varied results suggest that there might be a spectrum of pathologic changes associated with diabetes that affect the gut. This spectrum is, perhaps, a reflection of the varied causes of diabetes, its progression and ease of control that is observed in different individuals. Increasingly, investigators are focusing on a potential role for ICC in gastroparesis. A single case study, in which a full- thickness gastric biopsy was performed in a patient with idiopathic gastroparesis, revealed markedly reduced numbers of myenteric neurons and ICC (confirmed by staining with protein gene product 9.5 and c-kit, respectively), with minimal inflammatory changes to the smooth muscle and minimal fibrosis of the gastric submucosa (Figures 5 and 6). 85 In another study, full-thickness antral wall biopsies were obtained surgically from 14 patients with treatment-refractory gastroparesis (nine patients with diabetic gastroparesis, four patients with idio pathic gastroparesis and one patient with postsurgical gastroparesis) who underwent gastric electrical stimulator placement. 86 Five patients (four with diabetic gastroparesis and one with idiopathic gastroparesis) were 342 NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY VITTAL ET AL. JUNE 2007 VOL 4 NO 6

8 found to have almost no ICC or a significantly decreased number of ICC (a finding that correlated with an increase in tachygastrias) and worse symptom scores before placement of the gastric electrical stimulator compared with the other nine patients. Gastric specimens from 51 controls and 36 diabetic men with gastric cancer have been used for immunohistochemistry, to look for changes in c-kit, nnos and substance P expression. 87 ICC density in the inner circular muscle layer, but not in the myenteric plexus, was found to be lower in patients with severe diabetes compared with controls, in addition to decreased expression of nnos and substance P. Although it is not known whether these patients had gastroparesis, these results do suggest that diabetes can induce significant changes in gastric tissue that could be predicted to impair gastric function. Autoimmunity Increasing evidence suggests that there is an autoimmune component to gastroparesis. 88 The link between autoimmunity and gastro paresis has been best established for paraneoplastic gastroparesis. Patients with paraneoplastic gastroparesis, usually the result of small-cell lung cancer, lymphoma or ovarian cancer, have an inflammatory infiltrate in the myenteric plexus and a circulating antibody known as anti-hud. Whether this antibody is a cause or effect of neuronal injury has not yet been established; however, isolated anti- HuD can cause neuronal apoptosis in SH-SY5Y neuroblasts and cultured myenteric neurons. 89 In another study of patients with type 2 diabetes and peripheral neuropathy, calciumdependent apoptosis was observed when the sera from these patients were incubated with neuronal cell lines; 90 these studies support a role for autoantibodies in the development of peri pheral neuropathies. A marked loss of ICC was also seen in a jejunal biopsy sample obtained from a patient with severe paraneoplastic gastroparesis, which suggests that ICC might also be a target for circulating autoantibodies. 91 In addition to morphologic changes, evidence suggests that circulating autoantibodies can cause a functional disturbance that disrupts gastric motility. In a study of 16 patients with type 1 diabetes, eight patients had various gastrointestinal symptoms and a novel autoantibody that activated smooth-muscle L-type calcium channels at the dihydropyridine binding site. This autoantibody caused disruption of the migrating A Figure 6 Enteric neurons and gastroparesis. (A) Protein gene product 9.5 immunoreactivity in the corpus of a healthy individual. Arrows show individual enteric neurons in the myenteric ganglion of control tissue. Arrowheads show protein gene product 9.5 immunoreactivity in nerve fibers in both circular and longitudinal muscle layers. (B) Protein gene product 9.5 immunoreactivity in the stomach of a patient with gastroparesis. Arrows show individual enteric neurons in the myenteric ganglion of control tissue. The total amount of nerve structures in (A) is greater than that in (B). Much less immunoreactivity is seen in the gastroparesis than in the control tissue. No nerve cell bodies are found in the myenteric ganglia in (B) and very little reaction is displayed in muscle layers. Permission obtained from BMJ Publishing Group Ltd Zarate et al. (2004) 85 Gut 52: motor complex in mouse colons. 92 In another case report, a 60-year-old woman with idiopathic gastroparesis was found to have circulating levels of autoantibodies against both the ganglionic neuronal acetylcholine receptor and the N-type voltage-gated calcium channel. 88 The patient responded favorably to acetyl cholinesterase inhibitors, which suggests that these auto antibodies have an immuno pharmacologic, rather than a cytotoxic, effect. 30 μm Figure 5 Interstitial cells of Cajal and gastroparesis. (A) KIT immunoreactivity, a marker for interstitial cells of Cajal, in a healthy individual. Immunoreactivity is distributed throughout the myenteric plexus (arrows) and muscle layers (arrowheads) (B) KIT immunoreactivity in a patient with idiopathic gastroparesis. Less immunoreactivity is seen in all muscle layers, except for a few scattered reactive cells (arrowheads). Permission obtained from BMJ Publishing Group Ltd Zarate et al. (2004) 85 Gut 52: A B B 30 μm JUNE 2007 VOL 4 NO 6 VITTAL ET AL. NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY 343

9 Postinfectious and inflammatory causes No cause for the disease can be found in a significant subset of patients with gastroparesis. Clinical experience suggests that a number of these patients might develop gastric dysfunction after an acute viral illness. In a study of 11 children with idiopathic gastroparesis, eight children tested positive for rotavirus; all the children experienced complete recovery within 6 24 months. 93 Cases of patients who developed gastroparesis after vaccination (e.g. for hepatitis B, anthrax or tetanus) or contracting Lyme disease have also been reported, which suggests an immune-related etiology. 94 Being able to demonstrate that these patients have inflammation in the gastric wall is, however, rare. In one case report, a young man who developed acute symptoms of gastro paresis was evaluated by full-thickness gastric wall biopsy. 95 Histopathology demonstrated the presence of an inflammatory infiltrate of T lymphocytes (CD4 + and CD8 + ) and a marked decrease in the amount of substance P (as detected by tachykinin immuno reactive staining) in nerve fibers and myenteric neurons. This patient responded to steroid therapy, which further supports the role of inflammation in the development of gastro paresis. CONCLUSIONS The pathogenesis and pathology of gastroparesis remain poorly understood for many reasons. The disease process is multifactorial and can include dysfunction at various levels, including autonomic neurons, enteric neurons, ICC, and muscle cells. It is also difficult to distinguish the effects of hyperglycemia per se from those of insulinopenia in patients with type 1 diabetes. There is a lack of systematic studies of gastric tissue from patients with gastroparesis, especially studies using modern techniques that enable sophisticated investigation of diverse cell types, antibodies and inflammation. Experimental studies have been performed principally in animal models of diabetes, and whether their results can be extrapolated to humans is, therefore, unclear because of the probability that there are substantial differences in the physiology of humans and rodents. Despite these problems, it is clear that scientists and clinicians have begun to unravel the cellular basis for the development of gastro paresis, and it is imperative that research efforts are continued in this area if meaningful strides are to be taken in the treatment of this condition. KEY POINTS The pathogenesis of gastroparesis is complex; disruption of gastric function occurs at multiple cellular levels Evidence suggests that the pathogenesis of gastroparesis might involve nerves (particularly those producing nitric oxide, but also the vagus nerve), interstitial cells of Cajal and smooth muscle Most research into the pathogenesis of gastroparesis has been performed in animals; human studies are limited Further studies in animal models and humans are needed to increase our understanding of the molecular pathways that lead to gastric dysfunction so that viable treatment options can be developed References 1 Parkman HP et al. (2004) American Gastroenterological Association medical position statement: diagnosis and treatment of gastroparesis. Gastroenterology 127: Maleki D et al. (2000) Gastrointestinal tract symptoms among persons with diabetes mellitus in the community. Arch Intern Med 160: Soykan I et al. (1998) Demography, clinical characteristics, psychological and abuse profiles, treatment, and long-term follow-up of patients with gastroparesis. Dig Dis Sci 43: Hoogerwerf WA et al. (1999) Pain: the overlooked symptom in gastroparesis. Am J Gastroenterol 94: Rayner CK et al. (2001) Relationships of upper gastrointestinal motor and sensory function with glycemic control. Diabetes Care 24: Horowitz M et al. (2002) Gastric emptying in diabetes: clinical significance and treatment. Diabet Med 19: Horowitz M et al. (2004) Gastric function in diabetes. In Gastrointestinal Function in Diabetes Mellitus, 117 (Eds Horowitz M and Samsom M) Chichester: John Wiley & Sons 8 Samsom M et al. (2003) Prevalence of delayed gastric emptying in diabetic patients and relationship to dyspeptic symptoms: a prospective study in unselected diabetic patients. Diabetes Care 26: Guo JP et al. (2001) Extending gastric emptying scintigraphy from two to four hours detects more patients with gastroparesis. Dig Dis Sci 46: Lartigue S et al. (1994) Inter- and intrasubject variability of solid and liquid gastric emptying parameters. A scintigraphic study in healthy subjects and diabetic patients. Dig Dis Sci 39: Thomforde GM et al. (1995) Evaluation of an inexpensive screening scintigraphic test of gastric emptying. J Nucl Med 36: Jones KL et al. (2001) Predictors of delayed gastric emptying in diabetes. Diabetes Care 24: Tack J et al. (1998) Role of impaired gastric accommodation to a meal in functional dyspepsia. Gastroenterology 115: Kim DY et al. (2001) Noninvasive measurement of gastric accommodation in patients with idiopathic nonulcer dyspepsia. Am J Gastroenterol 96: Piessevaux H et al. (2003) Intragastric distribution of a standardized meal in health and functional 344 NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY VITTAL ET AL. JUNE 2007 VOL 4 NO 6

10 dyspepsia: correlation with specific symptoms. Neurogastroenterol Motil 15: Boeckxstaens GE et al. (2002) The proximal stomach and postprandial symptoms in functional dyspeptics. Am J Gastroenterol 97: Bredenoord AJ et al. (2003) Gastric accommodation and emptying in evaluation of patients with upper gastrointestinal symptoms. Clin Gastroenterol Hepatol 1: Samsom M et al. (1998) Proximal gastric motor activity in response to a liquid meal in type I diabetes mellitus with autonomic neuropathy. Dig Dis Sci 43: Frank JW et al. (1995) Mechanism of accelerated gastric emptying of liquids and hyperglycemia in patients with type II diabetes mellitus. Gastroenterology 109: Jebbink RJ et al. (1994) Hyperglycemia induces abnormalities of gastric myoelectrical activity in patients with type I diabetes mellitus. Gastroenterology 107: Chen JD et al. (1996) Abnormal gastric myoelectrical activity and delayed gastric emptying in patients with symptoms suggestive of gastroparesis. Dig Dis Sci 41: Samsom M et al. (1995) Compliance of the proximal stomach and dyspeptic symptoms in patients with type I diabetes mellitus. Dig Dis Sci 40: Takahashi T and Owyang C (1995) Vagal control of nitric oxide and vasoactive intestinal polypeptide release in the regulation of gastric relaxation in rat. J Physiol 484 (Part 2): Shah V et al. (2004) Nitric oxide in gastrointestinal health and disease. Gastroenterology 126: Mashimo H et al. (2000) Gastric stasis in neuronal nitric oxide synthase-deficient knockout mice. Gastroenterology 119: Anvari M et al. (1998) Role of nitric oxide mechanisms in control of pyloric motility and transpyloric flow of liquids in conscious dogs. Dig Dis Sci 43: Orihata M and Sarna SK (1994) Inhibition of nitric oxide synthase delays gastric emptying of solid meals. J Pharmacol Exp Ther 271: Plourde V et al. (1994) Delayed gastric emptying induced by inhibitors of nitric oxide synthase in rats. Eur J Pharmacol 256: Takahashi T et al. (1997) Impaired expression of nitric oxide synthase in the gastric myenteric plexus of spontaneously diabetic rats. Gastroenterology 113: Jenkinson KM and Reid JJ (1995) Effect of diabetes on relaxations to non-adrenergic, non-cholinergic nerve stimulation in longitudinal muscle of the rat gastric fundus. Br J Pharmacol 116: Wrzos HF et al. (1997) Nitric oxide synthase (NOS) expression in the myenteric plexus of streptozotocindiabetic rats. Dig Dis Sci 42: Adeghate E et al. (2003) Increase in neuronal nitric oxide synthase content of the gastroduodenal tract of diabetic rats. Cell Mol Life Sci 60: Spangeus A and El-Salhy M (2001) Myenteric plexus of obese diabetic mice (an animal model of human type 2 diabetes). Histol Histopathol 16: Spangeus A et al. (2000) Diabetic state affects the innervation of gut in an animal model of human type 1 diabetes. Histol Histopathol 15: El-Salhy M and Spangeus A (1998) Substance P in the gastrointestinal tract of non-obese diabetic mice. Scand J Gastroenterol 33: El-Salhy M and Spangeus A (2002) Gastric emptying in animal models of human diabetes: correlation to blood glucose level and gut neuroendocrine peptide content. Ups J Med Sci 107: Watkins CC et al. (2000) Insulin restores neuronal nitric oxide synthase expression and function that is lost in diabetic gastropathy. J Clin Invest 106: Guo C et al. (2004) Diabetic autonomic neuropathy: evidence for apoptosis in situ in the rat. Neurogastroenterol Motil 16: Hall AV et al. (1994) Structural organization of the human neuronal nitric oxide synthase gene (NOS1). J Biol Chem 269: Wang Y and Marsden PA (1995) Nitric oxide synthases: gene structure and regulation. Adv Pharmacol 34: Wang Y et al. (1999) Neuronal NOS: gene structure, mrna diversity, and functional relevance. Crit Rev Neurobiol 13: Wang Y et al. (1999) RNA diversity has profound effects on the translation of neuronal nitric oxide synthase. Proc Natl Acad Sci USA 96: Cellek S (2004) Point of NO return for nitrergic nerves in diabetes: a new insight into diabetic complications. Curr Pharm Des 10: Cellek S et al. (2003) Two phases of nitrergic neuropathy in streptozotocin-induced diabetic rats. Diabetes 52: Cellek S et al. (2004) Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: a new insight into selective nitrergic neuropathy in diabetes. Diabetologia 47: Korenaga K et al. (2006) Suppression of nnos expression in rat enteric neurones by the receptor for advanced glycation end-products. Neurogastroenterol Motil 18: Nakamura K et al. (1998) Nicotinic receptor mediates nitric oxide synthase expression in the rat gastric myenteric plexus. J Clin Invest 101: Ghosh DK et al. (1996) Domains of macrophage N(O) synthase have divergent roles in forming and stabilizing the active dimeric enzyme. Biochemistry 35: Gangula PR et al. (2007) Diabetes induces sexdependent changes in neuronal nitric oxide synthase dimerization and function in the rat gastric antrum. Am J Physiol Gastrointest Liver Physiol 292: G725 G Koya D and King GL (1998) Protein kinase C activation and the development of diabetic complications. Diabetes 47: Gibson TM et al. (2003) Effects of alpha-lipoic acid on impaired gastric fundus innervation in diabetic rats. Free Radic Biol Med 35: Dishy V et al. (2004) The effect of sildenafil on gastric emptying in patients with end-stage renal failure and symptoms of gastroparesis. Clin Pharmacol Ther 76: Sun WM et al. (1998) Effects of nitroglycerin on liquid gastric emptying and antropyloroduodenal motility. Am J Physiol 275: G Ordog T et al. (2000) Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis. Diabetes 49: Horvath VJ et al. (2006) Reduced stem cell factor links smooth myopathy and loss of interstitial cells of cajal in murine diabetic gastroparesis. Gastroenterology 130: Horvath VJ et al. (2005) Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetesassociated depletion of interstitial cells of Cajal in the murine stomach. Diabetes 54: Choi K et al. (2005) Loss of neuronal nitric oxide synthase results in altered volume and distribution of interstitial cells of Cajal in mouse stomach [abstract]. Neurogastroenterol Motil 17: Tashima K et al. (2000) Gastric acid secretion in streptozotocin-diabetic rats different responses to various secretagogues. J Physiol Paris 94: JUNE 2007 VOL 4 NO 6 VITTAL ET AL. NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY 345