Acta Neuropathol DOI 10.1007/s00401-013-1099-4

REVIEW

Neural plasticity in the gastrointestinal tract: chronic inflammation, neurotrophic signals, and hypersensitivity

Ihsan Ekin Demir • Karl-Herbert Scha¨fer • Elke Tieftrunk • Helmut Friess • Gu¨ralp O. Ceyhan

Received: 11 September 2012 / Revised: 31 January 2013 / Accepted: 7 February 2013 Ó Springer-Verlag Berlin Heidelberg 2013

Abstract Neural plasticity is not only the adaptive inflammatory, infectious, neoplastic/malignant, or degen- response of the central nervous system to learning, struc- erative—neural plasticity in the GI tract primarily occurs in tural damage or sensory deprivation, but also an the presence of chronic tissue- and neuro-inflammation. It increasingly recognized common feature of the gastroin- seems that studying the abundant trophic and activating testinal (GI) nervous system during pathological states. signals which are generated during this neuro-immune- Indeed, nearly all chronic GI disorders exhibit a disease- crosstalk represents the key to understand the remarkable stage-dependent, structural and functional neuroplasticity. neuroplasticity of the GI tract. At structural level, GI neuroplasticity usually comprises local tissue hyperinnervation (neural sprouting, neural, and Keywords Neural plasticity Á Gastrointestinal tract Á ganglionic hypertrophy) next to hypoinnervated areas, a Enteric nervous system Á Neuro-inflammation Á switch in the neurochemical (neurotransmitter/neuropep- Hypersensitivity Á Pain tide) code toward preferential expression of neuropeptides which are frequently present in nociceptive neurons (e.g., substance P/SP, calcitonin-gene-related-peptide/CGRP) Introduction and of ion channels (TRPV1, TRPA1, PAR2), and con- comitant activation of peripheral neural glia. The In the peripheral nervous system, neuronal plasticity was functional counterpart of these structural alterations is described more than 100 years ago in the pioneering works altered neuronal electric activity, leading to organ dys- by Cajal and Langley [12, 56] after nerve transsection and function (e.g., impaired motility and secretion), together neuro-regeneration. In the past two decades, plasticity was with reduced sensory thresholds, resulting in hypersensi- also recognized as a striking feature of autonomic nerves in tivity and pain. The present review underlines that neural the enteric nervous system (ENS) owing to intensifying plasticity in all GI organs, starting from esophagus, stom- research in neurogastroenterology [34, 70, 110, 111]. ach, small and large intestine to liver, gallbladder, and Changes in innervation density, different neuropeptide pancreas, actually exhibits common phenotypes and release patterns, and the entailing functional disturbances mechanisms. Careful appraisal of these GI neuroplastic are detected at the same fascinating extent in the ENS as in alterations reveals that—no matter which etiology, i.e., the central nervous system (CNS) [110]. However, our knowledge on the functional implications of visceral neu- roplasticity is scarce. & I. E. Demir ( ) Á E. Tieftrunk Á H. Friess Á G. O. Ceyhan The present review aims at illustrating the various Department of Surgery, Klinikum rechts der Isar, Technische Universita¨tMu¨nchen, Ismaninger str. 22, structural and functional neuroplastic alterations in the 81675 Munich, Germany gastrointestinal (GI) tract and underlining the similarities e-mail: [email protected] between the reactions of peripheral autonomic nerves in different GI organs as a response to insult. Furthermore, the K.-H. Scha¨fer Department of Biotechnology, University of Applied Sciences so far identified functional aspects of neural plasticity in Kaiserslautern/Zweibru¨cken, Zweibrucken, Germany the GI tract are interpreted on the basis of their relevance 123 Acta Neuropathol

Liver cancer (HCC, CCC): • Loss of innervation in Central Achalasia: tumor-affected tissue reorganization • Loss of neurons and areas ICC Pain • Ganglionitis Liver cirrhosis: Nutcracker esophagus: • Hypoinnervation in Hypersensitivity • Imbalance of neuro- fibrotic areas transmitters • Hyperinnervation along portal veins GERD: • Mast cell infiltration • More nociceptive fibers • NGF, GDNF, NMDA Chronic pancreatitis & • Throacic spinal neuron Pancreatic cancer: hypersensitivity • Increased neural density & • Cross-sensitization in the hypertrophy spinal cord • Neural remodelling • NR1 (NMDA) upregulation • Pancretic neuritis • Enhanced excitability Gastric cancer: • Suppression of A-type • Increase in potassium currents nociceptive fibers • TRPV1 in DRG neurons Gastroparesis: Chronic cholecystitis: • Loss of neurons & ICC • Increased neural • Activation of ATP- density & sensitive K+-channels hypertrophy Chronic gastritis: Colon cancer: • Increased neural • Hypo- and hyper- density, peri-neuritis innervation • DRG & nodose neuron hyperexcitability Chagas enteropathy: • Neuro-degeneration IBS: IBD: • Myenteric ganglionitis • Ganglionic hyperplasia • Mast cell infiltration • Ganglionic hypertrophy • NGF, BDNF and TRPV1 • Hyperplasia of glia • Decreased descending • Myenteric plexitis pain inhibition • AH and S neuron hyperexcitability Diverticulosis: • Decreased neuron and Appendicitis: glia content • Neuronal hyperplasia • Fewer ganglia • Neural hypertrophy Chronic diverticulitis: • Neuroma-like bodies • Increased neural • NGF, GAP-43 density • Mast cell infiltration • Neuro-inflammation

123 Acta Neuropathol b Fig. 1 Structural and functional neural plasticity in the gastrointes- generation of neurogenic appendicopathy [43]. In a tinal (GI) tract. Morphological analysis of nerves and intrinsic ganglia pathomorphological study on acute appendicitis, the num- within GI organs reveals that all parts of the GI tract, starting from esophagus to the distal end of the large intestine, undergo in part very ber of Schwann cells, the number and size of ganglia, and similar structural alterations during disease processes. The upward the immunoreactivity for the nerve growth factor/NGF arrows denote upregulation of the preceding factors. In contrast to the were all elevated when compared to normal appendix. well-described pathomorphological alterations of GI nerves in disease Furthermore, a significant correlation between NGF states, the concomitant functional alterations (yellow-colored text)of peripheral GI neuronal networks have not yet been understood in expression and mast cell density was noted (Fig. 1)[19]. sufficient detail. The depicted alterations are all derived from Interestingly, the density of mast cells strongly correlated experimental models of the respective diseases. AH afterhyperpolar- to the degree of neuronal hyperplasia and hypertrophy in izing, DRG dorsal root ganglia, TRPV1 transient receptor potential acute appendicitis [19]. Interconnecting nerve bundles in vanilloid 1, ATP adenosine triphosphate, cAMP cyclic adenosine monophosphate, CREB cAMP response element binding protein, the myenteric plexus were detected to be enlarged in 55 % HCC hepatocellular cancer, CCC cholangiocellular cancer, NMDA of patients who underwent appendectomy for acute N-methyl-D-aspartate, NGF nerve growth factor, GDNF glial-cell- appendicitis and also in 41 % of patients with histologi- derived neurotrophic factor, GAP-43 growth-associated-protein 43, cally normal appendix [72]. Therefore, neuroplastic ICC interstitial cells of Cajal, IBD inflammatory bowel disease. Please refer to the manuscript for the respective references alterations in the appendix seem to result from repetitive bouts of inflammation together with chronic luminal obstruction [72]. Based on the extensive neural hypertro- phy, elevated growth-associated-protein-43 (GAP-43) for the pathophysiology of pain and the progression of the content, increased presence of substance P (SP)-, vasoac- diseases with which they are associated. Finally, it aims to tive intestinal peptide (VIP)-positive nerve fibers (Table 1) emphasize that these similarities in neuroplastic reactions and the concomitant perineural inflammatory cell infiltra- of different organs seem to be generated upon a common tion, and non-acute appendicitis were suggested to be of pathomechanistic background, i.e., their predominant ‘‘neuroimmune’’ origin [26]. occurrence in states of chronic tissue inflammation, in the presence of specific peripheral neuro-inflammation and the Small and large intestine corresponding neurotrophic signals. The part of the GI tract in which visceral neuroplasticity has probably been best studied is the intestine [110]. The Structural neuroplasticity in the GI tract: tissue ENS which represents an independently functioning com- hyperinnervation and altered chemical coding ponent of the autonomic nervous system can undergo profound structural plasticity in different contexts, e.g., Owing to advances in histopathology and increased atten- during inflammation, infection, aging, and (congenital) tion toward neural alterations, pathologists reported on enteric neuropathies [110]. In this regard, inflammatory numerous structural plastic alterations in the GI tract, from bowel diseases (IBD), i.e., ulcerative colitis and Crohn’s its very proximal (oral) end to its most distal parts, i.e., disease, harbor numerous alterations in different compo- from esophagus to rectum. The following sections provide nents of the ENS [110]. In particular, both entities an overview of the so far observed structural neuroplastic frequently demonstrate hypertrophy and/or hyperplasia of alterations and associated chemical codes during the ENS ganglia, of extrinsic and intrinsic nerve bundles, and pathogenesis of typical GI disorders. hyperplasia of ENS glia cells in the colon and/or ileum (Fig. 1)[110]. In Crohn’s disease, these plastic changes Appendix were mostly found in the mucosa, submucosa, and myen- teric plexus [101, 110]. Furthermore, there is a close In contrast with its acute occurrence, the presence of correlation between the extent of intestinal neuroplasticity ‘‘chronic appendicitis’’ remains an issue of debate. Histo- and the amount of perineural inflammatory cell infiltrates morphological examination of specimens from patients around myenteric ganglia (Fig. 2)[65, 70, 110]. In addition with ‘‘chronic appendicitis’’ or ‘‘neurogenic appendicopa- to these trophic alterations, ganglion/neural degeneration thy’’ frequently yields intramucosal, finely vacuolated and necrosis have been demonstrated in IBD both in nerve proliferations, and central neuromas, as well as inflamed and non-inflamed intestinal areas [28]. neuromuscular proliferations in the submucosa, with neu- Inflammation in the GI tract is often associated with the ropeptide-producing cells in the appendical stroma [43]. In preferential emergence of afferent (nociceptive) nerve particular, these stromal cells which were shown to pro- fibers which contain the pro-inflammatory (‘‘neurogenic duce 5-hydroxytryptamine, somatostatin, and substance P inflammation’’) neuropeptide substance P (SP) (Table 1) have long been considered as causal factors in the [101, 110]. Especially in ulcerative colitis, SP-positive 123 Acta Neuropathol

Table 1 Neurochemical code alterations in diseases of the gastrointestinal (GI) tract

TH ChAT SP NKR CGRP VIP NOS NPK Galanin NPY PACAP Appendix

Appendicitis

Intestine

Ulcerative colitis

Crohn’s disease

Trichinella spiralis-inf. Chagas’ disease * Diverticulitis

Colon cancer

Stomach

Chronic gastritis

Gastric cancer

Esophagus

Nutcracker esophagus

Inef. esophageal motility

Achalasia

Tracheoesophageal fist.

Reflux esophagitis

Esophageal cancer

Liver

Cirrhosis

HCC

Gallbladder

Chronic cholecystitis

Pancreas

Chronic pancreatitis

Pancreatic cancer

Nearly all disorders of the GI tract demonstrate relative up- and downregulation of certain neuropeptides as part of the remodeling of the organ innervation. In general, there is a tendency toward increased expression of the neuropeptides substance P (SP) and vasoactive intestinal peptide (VIP), and suppression of the sympathetic innervation (tyrosine hydroxylase/TH- containing nerve fibers), particularly within inflammatory disorders of the GI tract ChAT choline acetyltransferase, CGRP calcitonin-gene-related-peptide, NOS nitric oxide synthase, NPK neuropeptide K, NPY neuropeptide Y, PACAP pituitary adenylate cyclase-activating polypeptide, inf. infection, inef. esophageal motility ineffective esophageal motility, fist. fistula, HCC hepatocellular cancer * Indicates that SP and neurokinin receptors (NKR) are respectively up- and downregulated in the dilated chagasic colon, with contrasting changes in the non-dilated parts [21] neurons of enteric ganglia are upregulated both in inflamed neurons are not subject to any major alteration in their and non-inflamed portions of the colon [101]. Such an density in ulcerative colitis (Table 1)[74]. Intriguingly, upregulation seems to be specific for SP, since cholinergic this remodeling process as observed in ulcerative colitis is neurons or vasoactive intestinal peptide (VIP)-containing not equally encountered in Crohn’s disease, but similarly

123 Acta Neuropathol

abc

N

Normal N Ulcer. Col. Ulcer. Col. d e

Colon N

Colon-CA Colon-CA

f g N N N Liver Normal Liver-CA

h i j N

N Normal PCa PCa klm

N Pancreas

N N PCa PCa PCa N

Fig. 2 Histomorphology of neuroplasticity in the human gastroin- periportal fields of the healty liver, liver cancer (Liver-CA) tissues testinal (GI) tract. Inflammatory and neuroplastic diseases of the harbor enlarged nerves in fibrotic tissue areas. h In normal human colon, liver, and pancreas harbor neuroplastic alterations when pancreas, intrapancreatic nerves are encountered in interlobular septae compared to normal healthy state. a–b In ulcerative colitis (Ulcer. in proximity of vessels and ducts. i–j In pancreatic cancer (PCa), Col.), submucosal nerves (N) are enlarged and greater in number nerves (i) and ganglionic neurons (j, arrowheads) are frequently when compared to normal colon. c In ulcerative colitis, enteric (here infiltrated by inflammatory cells (arrows). k–l Nerves in PCa tissues myenteric) ganglia are frequently inflamed (‘‘ganglionitis’’). Arrows are enlarged, increased in number and are victims of neural invasion point to infiltrating inflammatory cells. d–e In colon cancer (Colon- by cancer cells (arrows). ‘‘N’’ denotes the immunostained nerve(s) on CA), subserosal nerves are frequently invaded by cancer cells each image. Immunostaining was performed against the pan-neural (arrows) and increased in size. The endoneural invasion results in marker protein gene product 9.5 (PGP9.5). The scale bars correspond disappearance of individual nerves fibers (arrowheads) between to 100 lm invading cancer cells. f–g When compared to the small nerves in observed in GI infections, e.g., during Trichinella spiralis jejunum exhibited decreased amounts of SP and VIP [77]. infection [110]. However, in such infection models, SP- A similarity between ulcerative colitis and Crohn’s disease upregulation was seen to be limited to colon, whereas in this context lies in the concomitant upregulation of SP

123 Acta Neuropathol receptors, i.e., neurokinin-1, -2, and -3 receptors (Table 1) experimental colon cancer, and Godlewski et al. demon- [86]. Moreover, a frequent finding is the parallel decrease strated that human colon cancer contained decreased of the sympathetic, tyrosine hydroxylase-positive nerve amounts of CGRP and NPY solely in tumor-affected nerve fibers in addition to SP-upregulation [101, 102]. Corre- fibers (Figs. 1, 2)[37, 97]. Importantly, neuritogenesis was spondingly, in Crohn’s disease, the sympathetic repellant shown to correlate to worse prognosis and disease pro- factor semaphorin 3C is increased in colonic mucosal gression in colon cancer [3]. Interestingly, intracolonic crypts with a major deficiency of sympathetic nerve fibers nerves which have greater NGF content were recently [102]. reported to demonstrate more severe nerve invasion, and As one of the most common disorders of the GI tract, thus be associated with worse prognosis of patients with diverticular disease similarly exhibits a prominent degree colon cancer [62]. of neuroplasticity (Fig. 1). Interestingly, colonic segments with diverticulosis demonstrate decreased neural density Stomach and diminished neuronal and glial cell numbers in myen- teric ganglia, smaller numbers of submucous ganglia, and The presence of neuroplastic alterations has been subject to an overall predominance of glia over neurons (‘‘higher glia morphological investigation in different disorders of the index’’) [115]. On the other hand, tissue specimens from stomach. Similar neurotrophic–neuroplastic alterations as patients with acute or chronic diverticulitis were detected seen in the intestine or appendix are encountered in chronic to bear increased neural density when compared to patients gastritis with Helicobacter pylori (H. pylori) infection [95]. with non-inflamed colon (Fig. 1; Table 1). Furthermore, Sipos et al. showed a clear increase in the density of SP, patients with symptomatic diverticular disease, i.e., those VIP, and neuropeptide Y (NPY)-immunoreactive inflamed with recurrent attacks of abdominal pain and visceral nerve fibers in chronic gastritis mucosa (Table 1)[95, 96]. hypersensitivity, demonstrate upregulation of SP, NPK, Immunoelectron-microscopic analysis of these immune pituitary adenylate cyclase-activating peptide (PACAP), cells revealed a heterogenous cell population including VIP, and galanin (Table 1)[94]. Recently, these alterations lymphocytes, NPY- and SP-containing mast cells, and were demonstrated to be associated with ongoing local macrophages [95, 96]. The longevity of such neuroplastic inflammation, which can be clinically discrete (e.g., low- alterations could be elegantly demonstrated in a mouse level inflammation around colonic diverticula) or apparent model of H. pylori infection where infected mice had just like in experimental colitis (e.g., TNBS-colitis) [46]. higher density of SP-, VIP-, and CGRP-immunoreactive Overall, these findings underscore that intestinal inflam- nerve fibers in the stomach (Table 1)[9]. This neuroplas- mation can cause local neuromuscular disturbance which is ticity process was accompanied by declined cholinergic accompanied by upregulation of neuropeptides mediating signaling as evidenced by diminished acetylcholine release visceral hypersensitivity and local foci of tissue after electric stimulation [9]. Strikingly, eradication of H. hyperinnervation. pylori resulted in restoration of normal gastric acetylcho- Irritable bowel syndrome (IBS) represents one of the line release, but the high density of gastric SP- and CGRP- best studied diseases in terms of intrinsic neuroplasticity. immunoreactive nerves persisted [9]. Hence, these findings Histopathological assessment of jejunal biopsies from IBS demonstrate that chronic inflammation in the stomach can patients revealed frequently infiltrated myenteric ganglia also trigger neuroplasticity and altered neuropeptide by lymphocytes [108]. Later studies ascribed a prominent expression which can be long-lasting even after clearance role to neuro-inflammation by infiltrating mast cells, since of the etiological agent. IBS patients were found to have significantly elevated mast The other major gastric disorder which harbors neuro- cells counts in colonic mucosa and around intramural plastic alterations is gastroparesis, which can be either due nerves [7]. Interestingly, in diarrhea-predominant IBS, to diabetes or idiopathic. Full-thickness gastric wall biop- mucosal mast cells counts correlated with the severity of sies from patients with either diabetic or idiopathic visceral hypersensitivity [79]. IBS-associated alterations in gastroparesis demonstrated a decrease in gastric innerva- the neuropeptide content of nerves have not yet been suf- tion and neuronal cell bodies, and particularly a reduction ficiently characterized. Nonetheless, in experimental IBS, in the density of neuronal nitric oxide synthase (nNOS)- there was increased SP immunoreactivity in the ileocecal containing neurons and tyrosine hydroxylase-immunor- junction, colon, the posterior horn of the spinal cord, and eactive sympathetic nerve fibers [29, 40]. Furthermore, the hypothalamus of rats [113]. there was loss of the normal anatomic association between Neoplastic diseases such as colon cancer have not yet ICCs and enteric nerve terminals [40]. This remodeling of been subject to investigation in terms of neuroplasticity. In ICC networks was accompanied by upregulation of heter- the very few studies on this topic, Sitohy et al. showed an ogeneous inflammatory cell deposits and increase in local increase in nerve fiber density in the muscularis propria in connective tissue [40]. Overall, local (neuro-) inflammation 123 Acta Neuropathol together with loss of ICC and neuronal networks were Due to the frequently observed dysphagia following suggested to play a causal role in the pathogenesis of surgical repair of esophageal atresia (EA) or tracheoe- diabetic gastroparesis [29, 40]. Interestingly, recent com- sophageal fistula (TEF), specimens from these patients parative studies showed that diabetic and idiopathic have been subject to structural analysis. Interestingly, gastroparesis indeed exhibit major histological differences, patients with EA demonstrated hyperplasia of myenteric e.g., thickened basal lamina around smooth muscle cells ganglia at both ends of the esophagus which was more and nerves in diabetic gastroparesis and pronounced peri- pronounced than in TEF (Fig. 1)[80]. Furthermore, TEF neural fibrosis in idiopathic gastroparesis [29, 40]. This was detected to harbor downregulation of SP-immunore- abnormal remodeling of gastric innervation is assumed to active innervation and increase in the density of VIP- or result in delayed gastric emptying which is the leading NOS-containing neurons (Table 1)[60]. Thus, altered symptom in gastroparesis [29, 40]. neuropeptide expression and ganglionic morphological The lack of studies on neuroplasticity in GI malignancies alterations were suggested to be pathomechanistically is similarly reflected in the small number of studies which involved in the esophageal dysfunction following repair of investigated the innervation pattern of gastric cancer tissues. EA or TEF [60, 80]. While comparative studies with normal gastric tissue are not Coming to the most common esophageal disorder, present to date, some studies demonstrated increased pres- neuroplasticity and nociception in reflux esophagitis (RE) ence of SP-containing nerve fibers in human gastric cancer have been studied extensively due to ‘‘heartburn’’ being the (Table 1;Fig.1)[31, 87]. The presence of these fibers was chief complaint of these patients. Indeed, esophagus of interpreted as potential accelerator of tumor growth due to patients with RE possess a greater density of nerve fibers the presence of NK-1-receptor on gastric cancer cells and (Fig. 1)[75] with a particular increase in VIP-containing the promotion of gastric cancer cell proliferation and nerve fibers (Table 1)[75], and sensory nerve fibers with migration after SP-administration [31, 87]. the nociceptor-activating ion channel transient receptor potential vanilloid 1 (TRPV1) [69]. Furthermore, experi- Esophagus mental models of RE showed a similar upregulation of TRPV1-immunoreactive and SP-immunoreactive neurons Proper analysis of neuroplasticity in the esophagus necessi- in the spinal cord, dorsal root ganglia, and nodose ganglia tates in-depth understanding of the complex esophageal [4]. More recently, this increase in the expression of innervation in its striated and smooth muscle cell layers [73]. TRPV1 was demonstrated to correlate to intraesophageal Imbalances in this complex circuit are assumed to result in upregulation of NGF and glial-cell-derived neurotrophic esophageal motility disorders such as achalasia and gastro- factor (GDNF) [92], highlighting the major pro-nociceptive esophageal reflux [73]. Similar to gastroparesis, ICCs were neuroplasticity associated with this painful disorder. shown to be lost to a major extent in the lower esophageal sphincter high-pressure zone in achalasia, accompanied by a Liver similarly severe reduction in nNOS-containing neurons den- sity (Fig. 1)[36]. Hence, achalasia was proposed to be related Liver with its most common diseases such as hepatitis and to loss of inhibitory neurotransmission in the lower esopha- cirrhosis has been studied for the structure of its intrinsic geal sphincter. In common with several other disorders in the innervation. In liver cirrhosis, intrinsic innervation was GI tract, also achalasia patients revealed in part high amounts reported to be reduced in the parenchyma in pre-cirrhotic of locally infiltrating T lymphocytes [52]. Interestingly, liver and nearly absent in the regenerating nodules in patients who did not demonstrate such inflammatory cell established cirrhosis (Fig. 1)[58, 90]. Importantly, nerves infiltration were found to bear an even more remarkable loss in pre-cirrhotic and cirrhotic livers were found to be pre- of nerve fibers in the muscularis propria [52]. Looking at the served along the fibrous septae and in portal tracts [58, 90], extent of these alterations, achalasia can in part be regarded as with predominance of SP- and NPY-positive nerve fibers a local inflammatory hypoinnervation, resulting in loss of over CGRP-containing ones (Table 1)[100]. Interestingly, inhibitory neurotransmission [36, 52]. in alcoholic hepatitis without overt clinicopathological Similar to other GI organs, esophagus also exhibits a features of portal hypertension, or among patients with disease-context-dependent neuroplasticity. In nutcracker psoriasis who received the potentially hepatotoxic drug esophagus and ineffective esophageal motility, there is methotrexate, there was hyperinnervation around portal impressive upregulation of cholinergic myenteric neurons vein branches [48]. In a large-scale study on 178 biopsy and nNOS-immunoreactive neurons (Fig. 1)[53], respec- specimens obtained from patients with primary biliary tively, suggesting a potential role for imbalance between cirrhosis, alcoholic liver disease, autoimmune hepatitis, these neuronal subtypes in the hyper- and hypomotility chronic hepatitis B, or chronic hepatitis C, Matsunaga et al. disorders of the esophagus [53]. [68] detected a parallel increase in the density of nerve 123 Acta Neuropathol

fibers and mast cells in cirrhotic livers, which were both abdominal pain severity of these patients [14, 24]. In these independent of the underlying etiology. Importantly, this enlarged nerves, the distribution of nerve fiber qualities is increase in inflammatory cells and nerve fibers correlated marked by suppression of autonomic nerve fiber qualities with the degree of liver fibrosis in these patients [68]. (especially sympathetic ones) and glial markers, collectively Similar findings were observed in a rat model of cirrhosis, termed ‘‘neural remodeling’’ [14]. This suppression is more in which nerve terminals were seen in close contact with remarkable within nerves which demonstrate one of the two myofibroblasts in periseptal sinusoids [2]. In contrast with further features of this ‘‘pancreatic neuropathy’’: pancreatic cirrhosis which is characterized by widespread hypoinn- neuritis and neural invasion by PCa cells [14]. Studies on the ervation, toxic hepatic injury was shown to specifically nerve-infiltrating inflammatory cells in CP revealed that induce proliferation of portal nerve fibers [47]. these cells frequently contain interleukin 8 (IL-8), suggest- In one of the very few studies on the innervation of pri- ing a potential IL-8-mediated cross-signaling between mary liver tumors, Terada et al. [106]performeda inflammatory cells and nerves which contain SP (Table 1) quantitative comparative analysis of nerve fibers in hepato- [27]. Furthermore, there is a close correlation between the cellular cancer (HCC) and intrahepatic cholangiocarcinoma expression of the neurotrophic factor artemin and the degree (CCC). There, the investigators found no nerve fibers in of neuroplasticity in CP [13]. tumoral areas and fibrous septa of the tumor, whereas some On the other hand, in PCa, much of the research on nerve fibers were detectable near the liver capsule (Figs. 1, 2) neuroplasticity has been shaped by studies on neural [106]. On the other hand, in CCC, tumor stroma contained a invasion [23]. Up to 100 % of patients with pancreatic small amount of fibers, and both entities showed nerve fibers adenocarcinoma exhibit neural invasion (NI) [6]. Interest- in non-affected tissue regions, particularly in pre-existing ingly, NI was reported to significantly correlate to portal tracts and sinusoids [106]. neuroplasticity in the pancreas [14]. Similar to CP, these neuroplastic alterations are closely correlated with the Gallbladder degree of pain sensation and worse survival in PCa [14]. These observations recently paved path for novel in vitro Despite the high prevalence of its inflammatory diseases and and in vivo models of NI in PCa, which underlined a key the associated pain syndrome, neuroplasticity of gallbladder pathomechanistic role for neurotrophic factors, their has not been subject to intensive investigation. In the single receptors, and neuronal chemokines in this process [23]. large-scale study on gallbladder innervation in acute and The study of neuroplasticity in PCa has recently been chronic cholecystitis, gallbladder tissue from patients with further elaborated by development of a novel in vitro chronic uncomplicated gallstone disease demonstrated a model designed for this phenomenon [24]. Neurons derived prominent increase in nerve density and neural hypertrophy from dorsal root ganglia (DRG) and myenteric plexus (MP) in the whole tissue area, particularly in the gallbladder neck neurons, i.e., neurons representing extrinsic and intrinsic (Fig. 1)[41]. In contrast, acute cholecystitis tissues showed pancreatic innervation, have been cultivated within tissue a major deficiency in the mean number and area of intra- extracts obtained from patients with PCa or CP and com- mural nerve fibers [41]. In accordance with these pared to those grown in tissue extracts of normal pancreas observations, Gonda et al. [39] reported intramural hyper- donors [24]. This model mimicked the key features of trophy of VIP-immunoreactive nerve fibers among patients pancreatic neuroplasticity at neuronal level owing to their with chronic cholecystitis around hypertrophied muscle increased neurite density, complex axonal branching, and bundles, Rokitansky Aschoff sinus, and in the gallbladder perikaryonal hypertrophy [24]. The same model also mucosa (Table 1). However, tissues with acute or gangre- allowed the analysis of specific cell types from the intra- nous cholecystitis were marked by reduction and pancreatic microenvironment, including PCa cells and disappearance of VIP-containing nerve fibers in the gall- pancreatic stellate cells, or of molecular agents for their bladder [39]. To date, further studies on neuroplasticity in capacity to induce neuroplasticity [16, 24]. other gallbladder diseases such as cancer are lacking.

Pancreas Functional neuroplasticity in the GI tract: inflammation and hyperexcitability Among all organs of the GI tract, pancreas occupies the special position of being the probably best studied one in Small and large intestine terms of its neuroplasticity in inflammation and cancer [24]. Chronic pancreatitis (CP) and pancreatic cancer (PCa) tis- Investigating neuroplasticity in the human GI tract at sues harbor a prominent degree of neural hypertrophy and functional level represents a technically greater challenge increased neural density (Figs. 1, 2), which correlate to the compared to structural alterations. Hence, our current 123 Acta Neuropathol knowledge on such functional alterations is mainly derived the pathogenesis of this multi-factorial disorder, they seem from animal models. A common denominator for func- to be imperative actors in the generation of IBS-associated tional neuroplasticity in the GI tract is inflammation- hypersensitivity. induced hyperexcitability of certain neuronal sub-classes At functional level, one of the most striking features of [63, 65]. Among these, the so-called after hyperpolarizing enteric neuroplasticity are concomitant external (out-of- neuron (AH neuron) has been repeatedly demonstrated to organ) neuroplastic alterations, e.g., in unaffected/non- be hyperexcitable in experimental gut inflammation, in inflamed organs and afferent nerve pathways. In TNBS- particular, in TNBS-colitis and in Trichinella spiralis induced ileitis, colonic enteric neurons and epithelial cells (T. spiralis) infection of guinea pig small intestine [63, 65]. were reported to undergo parallel alterations, including The mechanisms for this hyperexcitability seem to be hyperexcitability of submucosal AH neurons, increased complex and dependent on the investigated layer of intes- non-cholinergic synaptic transmission in S neurons, ele- tinal wall. For T. spiralis infection, there is evidence for vated colonic prostaglandin E(2) content, greater number decreased Ca2?-activated K? channel activity, enhanced of colonic 5-hydroxytryptamine (5-HT)-immunoreactive cAMP-pCREB signaling pathway activity, and more fre- enteroendocrine cells, and altered ion transport across quent evoked fast EPSPs [17]. For TNBS-colitis, colonic epithelium [76]. Furthermore, acute TNBS-colitis submucosal neurons were measured to have shorter action impaired gastric emptying via an extrinsic neural reflex potential duration, whereas myenteric neurons exhibited pathway over pelvic nerves [22]. Interestingly, functional spontaneous activity and increased hyperpolarization-acti- neuroplastic alterations as a result of intestinal inflamma- vated cation conductance [63, 70]. The other type of enteric tion can extend even beyond the GI tract. Experimental neuron which was reported to demonstrate altered activity TNBS-colitis was shown to be associated with bladder under inflammation is the S neuron which can be present as dysfunction and detrusor instability due to hyperexcitabil- a sensory, motor, or intermediate neuron [63, 65, 70]. For ity of spinal bladder neurons [67], colonic TRPV1-induced TNBS-colitis, it is known that S neurons with ascending SP and CGRP release [78], and voltage-gated sodium projections exhibit increased excitability (Fig. 2)[17, 63, channel upregulation [59] in the bladder. Moreover, colo- 65, 70]. At synaptic level, submucosal and myenteric nic afferent nerve pathways and especially DRG neurons neurons demonstrate fast EPSPs with higher average have been extensively studied in terms of their contribution amplitudes in TNBS-colitis and enhanced neurotransmitter to visceral hypersensitivity. In this context, TNBS-colitis release [63, 65, 70]. Interestingly, this is in contrast to was shown to induce increased numbers of voltage-gated T. spiralis infection where during the phase of acute sodium channels [54], increased expression of the receptor inflammation, cells were detected to release smaller amounts tyrosine TrkA and of the glial-cell-line-derived neurotro- of neurotransmitters and a suppression of EPSPs [70]. phic factor GFRa3[105], and decreased expression of the Importantly, part of these alterations is detectable up to mechanosensitive K(2P) receptor [55] in colon-specific several weeks after resolution of infection [70], suggesting DRG neurons. Therefore, the extra-intestinal neuroplas- the persistent character of functional neuroplasticity in GI ticity seems to contribute to intestinal dysfunction and inflammation. hypersensitivity at least as much as the intra-organ One of the best studied examples of functional neuro- neuroplasticity. plasticity in the GI tract is encountered in IBS. In this disorder, visceral hypersensitivity as the hallmark of IBS Stomach seems to be grounded on both peripheral and central neu- roplastic alterations. First, mast cells which are found in For stomach, it was shown that gastric ulcer, particularly close spatial contact with intramural nerves in human IBS kissing gastric ulcers, can sensitize vagal and spinal gastric specimens are assumed to activate sensory neurons via afferents (DRG and nodose neurons) (Fig. 1), as evidenced increased mast cell tryptase and histamine secretion [8]. In by doubling of P2X receptor activity to its agonists [20]. postinfectious IBS, persistent immune cell activity is con- Furthermore, Sugiura et al. [103] showed that gastric ulcers sidered responsible for sustained activation of sensory are associated with altered acid-elicited currents in DRG nerves [45]. Here, there is a 3.9-fold increase in the amount neurons, increasing their pH sensitivity, density, and of TRPV1-expression sensory nerve fibers in IBS tissues kinetics in gastric DRG neurons. In harmony with these than in healthy controls [1]. More recent studies demon- observations, Bielefeldt et al. [11] demonstrated that in rats strated that IBS tissues contain a neurotrophic milieu, with acetic-acid induced gastric ulcer, sodium currents characterized by increased tissue NGF and BDNF [116]. At recovered faster from inactivation in nodose and DRG CNS level, IBS patients had decreased descending pain neurons than control animals (Fig. 1). In a later study, the inhibition in the spinal cord [82]. While neuroplastic same group reported that NGF is upregulated during gas- alterations should not be considered sufficient to explain tritis and can induce increased tetrodotoxin-resistent 123 Acta Neuropathol sodium currents [11]. In gastroparesis, Zhou et al. [119] reflux and the associated electroesophagogram patterns showed that nodose ganglia undergo activation of their [91]. ATP-sensitive K?-channels upon exposure to hyperglyce- Important steps have, though, also been made in clinical mia (as in diabetic gastroparesis, Fig. 1), resulting in studies on humans in which acid-infusion-induced esoph- gastric smooth muscle relaxation. ageal and also distal (i.e., gastric) hypersensitivity have been repeatedly demonstrated [10, 42, 88]. A recent clin- Esophagus ical study addressed the effect of pregabalin, a centrally acting voltage-sensitive-calcium-channel modulator, on Due to the common association of pain and heartburn with acid-induced esophageal hypersensitivity, and showed that disorders of esophagus, the innervation of this organ has subjects pre-treated with pregabalin demonstrated recently been subject to more intensive investigation. In decreased esophageal hypersensitivity [18]. Thereby, the experimental acid-induced esophagitis, the four splice investigators presented one of the first pharmacological variants of the NMDA receptor subunits were shown to be functional studies on acid-induced esophageal hypersensi- differentially expressed, with significant upregulation of tivity in humans [18]. the NR1 subunit in DRG, NG neurons, and esophageal tissue (Fig. 2)[5]. In a study by Qin et al. [85], intra- Liver and gallbladder esophageal instillation of HCl, bradykinin, or capsaicin not only increased the activity of thoracic spinal neurons, but Studies on the functional neuroplasticity of liver are totally also elicited excitatory responses to esophageal distention lacking. Nonetheless, gallbladder stasis was shown as a in an even greater number of spinal neurons (Fig. 1). potential contributor to gallstone formation [49]. Microin- Thereby, they could provide evidence for cross-sensitiza- jection of prostaglandin E2 (PGE2) into the gallbladder tion of spinal neurons and for visceral hypersensitivity in wall resulted in hyperpolarization, in reduction of ampli- esophagitis (Fig. 1)[85]. In another study, Medda et al. tudes of the fast and slow postsynaptic potentials, and thus [71] showed that vagal afferent fibers, but not spinal neu- in the inhibition of intramural ganglionic neurons and rons, exhibited an increase in action potential firing upon decreased gallbladder motility [49]. In experimental acute esophageal distension or acid-pepsin infusion, with similar cholecystitis, gallbladder contractions and NO-mediated findings from Kang et al. [51] who detected increased neurotransmission were significantly attenuated. Here, sensitivity of afferents after exposure to acid or thermal emergence of contractions insensitive to tetrodotoxin and stimuli such as heated saline. In a subsequent study, the sensitive to atropine and omega-conotoxin suggested that group demonstrated that the acid-induced sensitization of intrinsic gallbladder innervation was the primarily affected esophageal neurons can be inhibited by TRPV1 antagonists neural component [38]. The decreased neurotransmission [81]. was also shown to be related to impaired calcium homeo- In one of the few studies on the neuro-immune crosstalk stasis, diminished smooth muscle contractility, and the in esophageal disorders, Gao et al. [35] showed that mast inflammatory actions of reactive oxygen species and his- cell activation led to increased phosphorylation of ERK1/2 tamine in the inflamed gallbladder [83]. in nodose neurons and enhanced mechano-excitability of esophageal nodose C-fibers. A similar activating role was Pancreas also demonstrated for TRPA1, which can similarly be induced by mast cells over a protease-activated-receptor-2 Currently, no experimental model has yet been found to be (PAR2)-dependent mechanism and result in vagal afferent suitable for studying functional neuroplasticity in PCa, and C-fiber hypersensitivity [118]. Therefore, mast cell- for CP, the only applied model remains CP as induced by induced vagal afferent C-fiber activation is considered a infusion of TNBS acid into the murine pancreatic duct [44, key mechanism in esophageal perception and hypersensi- 117]. While this model has not yet been tested for the tivity (Fig. 2). presence of structural neuroplastic alterations, it was pre- Regarding myoelectric properties of human esophagus viously shown that mast cell-deficient mice had during gastroesophageal reflux disease (GERD), Shafik significantly less pain sensation than wild-type mice during et al. [91] reported on three different patterns of electric TNBS-induced CP (Fig. 2)[44]. Using this model, Xu activity are encountered in GERD: First, reflecting a nor- et al. [117] demonstrated the enhanced excitability, sup- mal activity; the second, an irregular electric activity pression of A-type potassium currents, and increased suggesting decreased motility; and third, a silent stage expression of TRPV1 in pancreas-specific DRG neurons where no acid clearance occurred [91]. As ICCs are (Fig. 2). In TNBS-induced CP in rats, Qian et al. [84] assumed to be the electric potential generators in the showed that toll-like receptor 3 is upregulated in astrocytes esophagus, they may be causal in the generation of acid of the spinal cord, correlating to the degree of mechanical 123 Acta Neuropathol allodynia, and upregulation of inflammatory mediators and neuroepithelial stem cell marker nestin, suggesting such as IL-1b, TNF-a, IL-6, and monocyte chemotactic potential de-differentiation of intrapancreatic glia as a result protein-1 (MCP-1). In another study, the same model was of the activating environment [15]. demonstrated to induce astrocytic activation in the thoracic Unfortunately, further structural and functional altera- spinal cord of rats, as evidenced by upregulation of the tions of glia cells in disease states of the GI tract are largely glial fibrillary acidic protein (GFAP) and its reversibility absent. However, very recent intriguing results from by the intrathecal administration of specific inhibitor ontogenic studies on enteric glia revealed a novel role for l-alpha-aminoadipate [30]. In this regard, an important role these cells, which may be crucial for understanding neu- was also demonstrated for spinal microglia which was seen roplasticity in the GI tract. Tracing of embryonic and fetal to be activated during TNBS-induced CP, to overexpress Sox10-expressing cells in the murine GI tract revealed that phosphorylated p38 and to be further enhanced by these cells give rise to neurons and glia cells of myenteric administration of the neuronal chemokine fractalkine [64]. ganglia [57]. Postnatally, however, these cells ceased to These effects were reversible upon administration of the give rise to neurons [57]. Interestingly, glia-derived neu- microglia inhibitor minocycline [64]. rons could be generated from enteric glia under in vitro culture conditions or after chemical injury [50, 57]. Hence, enteric glia cells rather seem to be multipotent cells with Peripheral glia in the neuroplasticity of the GI tract: both gliagenic and neurogenic potential and have recently glial activation in inflammation emerged as major actors in enteric neuroplasticity [50, 57].

The remarkable extent of structural and functional neuro- plastic alterations in the GI tract seems in many cases to be Critical appraisal of GI neuroplasticity: the norm, accompanied by reactive alterations in glial cells, particu- the bystander or the defining characteristic? larly in enteric glia. Overall, these alterations can be considered to represent ‘‘glial activation’’, and like astro- Despite the growing number of studies on neuroplastic cytes of the central nervous system, enteric glia were alterations in several GI organs, the existence of genuine reported to increase their proliferation rate and expression neuroplasticity in GI disorders is not undebated. This of GFAP as their major intermediate filament during controversy is in part reflected by studies on intestinal inflammation, e.g., in ulcerative colitis and Crohn’s disease neuronal dysplasia type B (IND-B), a submucous plexus [112]. One can assume that activated glia cells may con- abnormality of infants characterized by giant enteric gan- tribute to the local reparative processes [89, 109], since the glia. Some studies, though, revealed that such giant ganglia selective depletion of GFAP-expressing enteric glia results can similarly be encountered in normal colon and may, in disruption of mucosa and severe inflammation in the therefore, be just part of the ‘‘normal variation’’ [66]. small intestine, characterized by hemorrhagic necrosis Looking at this controversy, one can consider three possi- [109]. In line with these observations, enteric glia cells were bilities for the true role of neuroplasticity in the GI tract: later found to modulate epithelial proliferation, differenti- ation, and inflammation via S-nitrosoglutathione [89]. This 1. Neuroplasticity as an ‘‘artificial entity’’: As in IND-B, key role of enteric glia for mucosal integrity is somewhat researchers should pay attention to the degree of reflected in observations from human IBD, since patients normal variations in GI neuromorphology. Do GI with Crohn’s disease were also reported to have smaller organs in their normal state contain areas with greater amounts of enteric glial cells in the intestinal wall before the density of innervation or larger intrinsic ganglia? In the onset of overt inflammation [112]. Their potential role in human and murine pancreas, the innervation density in local repair and structural maintenance is further supported the pancreatic head was reported to be greater than in by the fact that glial cells are an important source of growth the body and tail [107]. Similarly, murine colon factors such as pro-epidermal growth factor (proEGF), naturally harbors hypoganglionic areas which are NGF, GDNF, neurotrophin-3; of neurotransmitter precur- surrounded by densely clustered enteric neurons [93]. sors; of major histocompatibility complex class II In malignant GI tumors, there are possibly hypo- and molecules; and of pro-inflammatory cytokines such as IL-6 hyper-innervated areas in the same tissue [3]. and IL-1b and can, thus, modulate the function of peripheral 2. Neuroplasticity as ‘‘adaptive response’’: The frequent inflammatory cells and neurons [110]. A recent study on the occurrence of neuroplastic alterations in inflammatory state of intrapancreatic neural glia in PCa and CP showed GI disorders implicates that inflammatory cells may be that the hypertrophic intrapancreatic nerves lose their nor- releasing neurotrophic agents and, thereby, causing mally high content of Sox10-expressing glia, together with neural hypertrophy and neuro-sensitization. Such prominent upregulation of the glial intermediate filament enlarged and sensitized nerves may in turn signal 123 Acta Neuropathol

Fig. 3 Interaction of neuroplasticity with inflammation in the gastrointestinal (GI) tract. In the GI tract, inflammatory cells such as mast cells, lymphocytes, monocytes, and granulocytes secrete a battery of neurotrophic factors such as nerve growth factor (NGF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) which sensitize nociceptive nerve endings and at the same time foster neural growth. Mast cells additionally secrete tryptase and histamine which can also activate nociceptive fibers. On the other hand, immune cells can also release neurotoxic agents such as reactive oxygen species (ROS) which can promote local foci of tissue hypoinnervation. Importantly, epithelial cells in inflamed tissues were similarly shown to secrete neurotrophic factors such as NGF and brain- GI derived-neurotrophic factor INFLAMMATION (BDNF). Activated neurons not only signal to the central nervous system, but also release the pro-inflammatory neuropeptides substance P and calcitonin-gene-related-peptide (CGRP) in an antidromic fashion into the inflamed tissue and thereby aggravate local inflammation. Overall, inflammation-nerve interactions in GI inflammation are Hyperinnervation embedded in a vicious cycle INTRA -ORGAN (neurotrophic which promotes neuroplasticity INNERVATION factor -mediated ) in conjunction with local inflammation

Hypoinnervation (inflammatory damage , e.g. ROS- mediated )

more frequently to the CNS as part of an evolutionary 3. Neuroplasticity as ‘‘the defining characteristic’’: In this protective response to signify disease at the periphery third scenario, neuroplastic alterations can be consid- [32]. On other hand, in IBD, neuroplasticity currently ered to bear a crucial role in the pathophysiology of the seems to be a mere bystander, since an association respective GI disease. In IBS, achalasia and nutcracker between symptom promotion and neuroplasticity has esophagus, neuro-inflammation as well as degenerative not yet been convincingly reported. Still, in the and regenerative neuro-alterations belong to the scenario of neuroplasticity as adaptive response, one defining histopathological characteristics of these dis- would expect the reversal of neuroplasticity after orders. Hence, these neuroplastic alterations can be clearance of the initiating tissue injury (e.g., epithelial assumed to sustain the actual disease, and their lesions). In this case, one would refer to this adaptive reversal bears the potential of amending these response as ‘‘secondary neuroplasticity’’ (Table 2). disorders.

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Table 2 Visceral neuroplasticity: a bystander or the main actor? Primary neuro- Secondary neuro- Associated with disease/symptom Type of association with disease/ inflammation inflammation progression? symptoms

Appendix Appendicitis No Yes Yes Pain ‘‘Neurogenic Yes No Yes Pain appendicitis’’ Intestine Ulcerative colitis No Yes Unclear Neurogenic inflammation ? Pain? Dysmotility? Crohn’s disease No Yes Unclear Neurogenic inflammation ? Pain? Dysmotility? Irritable bowel Yes No Yes Hypersensitivity, dysmotility syndrome Trichinella spiralis- No Yes Yes Hypersensitivity inf. Chagas’ disease Yes No Yes Megacolon Diverticulitis No Yes Yes Hypersensitivity Colon cancer No Unknown Unknown Unknown Stomach Chronic gastritis No Yes Yes Hypersensitivity/pain Gastric cancer No Yes Yes Promotion of tumor growth Esophagus Nutcracker Yes No Yes Dysphagia/pain/dysmotility esophagus Achalasia Yes No Yes Dysphagia/pain/dysmotility Reflux esophagitis No Yes Yes Hypersensitivity Esophageal cancer No Unknown Unknown Unknown Liver Cirrhosis No Yes Unknown Unknown HCC No Unknown Unknown Unknown Gallbladder Chronic No Yes Yes Hypersensitivity/pain cholecystitis Pancreas Chronic No Yes Yes Increased inflammation and pain pancreatitis Pancreatic cancer No Yes Yes Promotion of tumor growth and pain Numerous disorders of the gastrointestinal (GI) tract exhibit neuroplasticity in the presence of (chronic) inflammation. In some diseases, inflammation is primarily directed to neurons or neural structures (‘‘primary’’ neuro-inflammation), whereas in the majority of other GI disorders, intra-organ nerves are inflamed in the presence of general tissue inflammation (‘‘secondary neuro-inflammation’’). Here, it should be noted that neuroplastic-neuroinflammatory alterations in the GI tract are frequently, but not unexceptionally, associated with disease progression or symptoms. However, functional studies which could show alleviation of disease or symptoms after reversal of GI neuroplasticity are so far lacking

But what criteria may help us identify the true role of neuroplasticity, resected tumors should be examined in neuroplasticity in a given GI disorder? First of all, studies their entirety, including intra-tumoral, peri-tumoral, and on GI neuroplasticity should ascertain the representative extra-tumoral regions. Concomitant peri-tumoral inflam- nature of the studied specimens. Samples from subjects mation should be recorded and considered as confounder. with matched demographic characteristics should be Once the observed neuroplasticity can be confirmed to obtained from several anatomic layers of the studied organ surpass the extent of the natural intra-organ and inter- and compared to the already known natural variation in subject variation, it can be classified as ‘‘genuine organ neuromorphology. In studies on tumor-associated neuroplasticity’’.

123 Acta Neuropathol

Second, conclusions on the implications and the true shown to produce considerable amounts of neurotrophic impact of GI neuroplasticity should be drawn primarily factors (Fig. 3)[98]. Here, particularly MC were shown to from functional studies which target at selected compo- release large amounts of neurotrophins such as NGF, NT-3, nents of the complex network between inflammation, and NT-4 upon activation [98]. These cells and factors can nerves, glia, and epithelial cells. Here, genetically engi- induce nerve hypertrophy (with increased presence of SP neered animal models allow by means of cell-specific and CGRP-containing nerve fibers) and specifically acti- promoters the over-expression, knock-down, or knock-out vate nociceptive signaling [104]. Furthermore, activated of defined molecular targets in each component of this nociceptive fibers which contain SP and CGRP can release network. The reversibility or augmentation of neuroplas- these neuropeptides and potentiate inflammation by the ticity in a certain GI disorder after modulation of disease- neurogenic inflammatory property of these neuropeptides associated molecular targets in non-neural cells would [61]. On the other hand, inflammatory cells can at the same allow the classification of neuroplasticity as ‘‘adaptive time be responsible for nerve damage and, thereby, cause response’’. Furthermore, if functional modulation of targets local hypoinnervation [114]. Such cells can release cyto- in neural or glial cells results in clearance or amelioration kines such as interleukin-6 (IL-6), IL-12, and TNF-alpha of the GI disease, neuroplasticity would qualify as ‘‘the and neuro-destructive agents such as reactive oxygen spe- defining characteristic’’. As of today, representative func- cies (ROS), phagocytose damaged neural components, and tional models are lacking for a large portion of the GI co-operate with cytotoxic T lymphocytes in the clearance disorders that are discussed in this review. In analogy with of damaged neural structures [114]. It is conceivable that Koch’s postulates for defining the causative role of during GI inflammation, such cells increasingly accumulate microbes in disease, we believe that these ‘‘postulates for around damaged neural structures and mediate their neuroplasticity’’ are going to be useful for future studies clearance for the subsequent neuroplastic regeneration, as trying to identify the true role and impact of neuroplasticity known from Wallerian degeneration. Therefore, tissue in GI disorders. hypoinnervation and hyperinnervation can be present in the same tissue, and the balance between the neuro-destructive and neurotrophic actions of inflammatory cells may Mechanisms of visceral neuroplasticity in GI determine the ultimate extent of neuroplasticity in GI inflammation: a vicious cycle? organs. Overall, there seems to be a vicious cycle com- prising inflammation (e.g., mast cell activation), increased Despite the frequent presence of inflammation in GI neu- neurotrophic/neurotoxic factor release from parenchymal roplasticity, the phenotypes of neuroplastic alterations are or inflammatory cells, hyper- and hypoinnervation, noci- very heterogeneous, e.g., tissue hyper- or hypoinnervation ceptive hyperexcitability, glial activation, and in turn (Fig. 1) and quite variable alterations in the neurochemical augmented inflammation (Fig. 3). This vicious cycle may code of visceral neurons (Table 1). The most plausible be central to understanding mechanisms of neuroplasticity explanation for this variability between different GI dis- in the GI tract. orders may be due to differences in the pattern of The specific subsets of inflammatory cells that compose inflammation between different GI disorders. In theory, one neuritis or ganglionitis lesions in the GI tract have not yet can assume that, regardless of its specifics, inflammatory been well characterized. The increased presence of MC in conditions could be associated with or even create a neu- IBS [7] and of lymphocytes, macrophages, and mast cells rotrophic milieu. In abundance of neurotrophic factors such in chronic gastritis [96] suggests that these cell types may as NGF, GDNF, and BDNF, typical traits of neuroplas- be involved in GI neuro-inflammation. Still, looking at all ticity, i.e., tissue hyperinnervation, hyperexcitability, and disorders in which inflammation-associated GI neuroplas- neurochemical code switch, may be induced. This ticity was reported (e.g., reflux esophagitis, gastritis, hypothesis is supported by the observation that colonic chronic pancreatitis, pancreratic cancer, IBD, IBS, liver mucosal cells of rats with TNBS-induced colitis secrete cirrhosis), it seems that GI neuroplastic alterations occur in increased amounts of NGF [99]. An association between the presence of chronic rather than acute inflammation. In NGF expression and GI inflammation was so far reported this context, future studies should correlate the extent of GI for esophagitis [92], appendicitis [19], colitis and IBD [25], neuroplasticity with the severity of ongoing inflammation and chronic pancreatitis [33]. Moreover, in GI cancer- (i.e., clinically apparent vs. silent) and both the timing and associated neuroplasticity, neural hypertrophy is most the severity of acute episodes. prominent in areas of perineural inflammation [14]. Inter- Currently, nearly the entire knowledge on GI neuro- estingly, another major source of such neurotrophic factors plasticity is derived from descriptive histomorphological are inflammatory cells per se. Mast cells (MC), B and T studies, and there is need for functional studies to elucidate lymphocytes, eosinophils, and basophils have all been the actual pathomechanism behind GI neuroplasticity. In 123 Acta Neuropathol particular, we propose in vivo models in which the con- disease-stage-dependent hypersensitivity and entailing tribution of neurotrophic factors such as NGF, GDNF, and pain. Unsurprisingly, this recent recognition of GI neuro- BDNF to neuroplasticity can be studied by driving their plasticity and the associated hypersensitivity and pain gave expression under the control of cell-specific promoters birth to novel studies on the efficacy of neuropathic pain (e.g., villin for enterocytes, p48 for pancreatic acinar cells, medications for treating visceral pain and hypersensitivity. H?-K? ATPase for gastric parietal cells, albumin for The repeatedly demonstrated pain-relief upon administra- hepatocytes) with subsequent induction of inflammation tion of neuropathic pain medications to visceral pain (e.g., TNBS-induced colitis or pancreatitis, H. pylori- underscores the presence of neuroplastic-neuropathic pain induced gastritis), and the study of organ neuroplasticity at mechanisms in GI disease states. When neural plasticity is histological level and organ function by means of e.g., considered as an adaptive response of the GI nervous ex vivo motility assays. Tissues that are isolated from such system to a pathological state, the majority of these adap- animals would also be accessible to detailed genomic and tive responses seem to be generated upon the same proteomic analysis for identifying further co-activated underlying condition, i.e., chronic tissue- and neuro- molecular pathways. In the same models, the contribution inflammation, which can be encountered in either inflam- of inflammatory cells and reversibility of neuroplasticity matory, infectious, neoplastic/malignant, or degenerative can be studied, e.g., by depletion of specific inflammatory disorders of the GI tract. Consequently, the key to in-depth cell subtypes, as previously performed in murine chronic understanding of neural plasticity in the GI tract seems to pancreatitis [44]. In addition, in vitro models in which lie at the intersection of chronic inflammation and the intrinsic GI neurons or glia cells can be cultured within associated trophic/excitatory signals. Future research shall, tissue extracts obtained from patients with different GI therefore, elucidate the peripheral neuromodulatory prop- disorders can be used to study the reaction of GI neurons to erties of chronic inflammation in the GI tract. disease microenvironment [24]. Neurons from such assays can be subjected to gene microarray analysis for identify- Acknowledgments The authors are grateful to Ms. Martina Scholle ing differentially regulated novel targets. We are convinced for her assistance in the generation of the figures. that only such functional models bear the potential to shed Conflict of interest None. light on the individual pathomechanistic steps in the gen- eration of GI neuroplasticity.

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