Advances in Anatomic Pathology:
Histopathology in Gastrointestinal Neuromuscular Diseases: Methodological and Ontological Issues
Bernardini, Nunzia MD*; Ippolito, Chiara PhD*; Segnani, Cristina PhD*; Mattii, Letizia PhD*; Bassotti, Gabrio MD, PhD†; Villanacci, Vincenzo MD‡; Blandizzi, Corrado MD§; Dolfi, Amelio MD*
*Unit of Histology
§Division of Pharmacology, Department of Clinical and Experimental Medicine, University of Pisa, Pisa
†Section of Gastroenterology and Hepatology, Department of Clinical and Experimental Medicine, University of Perugia, Perugia
‡Second Section of Pathology, Spedali Civili, Brescia, Italy
Supported by an institutional research grant issued by the Interdepartmental Center for Research in Clinical Pharmacology and Experimental Therapeutics, University of Pisa, Pisa, Italy.
All figures can be viewed online in color at http://www.anatomicpathology.com.
The authors have no conflicts of interest to disclose.
Reprints: Nunzia Bernardini, MD, Unit of Histology, Department of Clinical and Experimental Medicine, University of Pisa, Via Roma, 55, I-56126 Pisa, Italy (e-mail: firstname.lastname@example.org).
Gastrointestinal neuromuscular diseases (GINMDs) comprise a heterogenous group of chronic conditions associated with impaired gut motility. These gastrointestinal (GI) disorders, differing for etiopathogenic mechanisms, pathologic lesions, and region of gut involvement, represent a relevant matter for public health, because they are very common, can be disabling, and determine major social and economic burdens. GINMDs are presumed or proven to arise as a result of a dysfunctioning GI neuromuscular apparatus, which includes myenteric ganglia (neurons and glial cells), interstitial cells of Cajal and smooth muscle cells. Despite the presence of symptoms related to gut dysmotility in the clinical phenotype of these patients, in the diagnostic setting scarce attention is usually paid to the morphologic pattern of the GI neuromuscular apparatus. It is also objectively difficult to collect full-thickness gut tissue samples from patients with GINMDs, because their disease, which can be only functional in nature, may not justify invasive diagnostic procedures as a first-line approach. As a consequence, whenever available, bioptic gut specimens, retrieved from these patients, must be regarded as a unique chance for obtaining relevant diagnostic information. On the basis of these arguments, there is an urgent need of standardized and validated histopathologic methods, aiming at overcoming the discrepancies affecting current approaches, which usually lead to conflicting definitions of normality and hamper the identification of disease-specific pathologic patterns. This review article intends to address current methodological and ontological issues in the histopathologic diagnosis of GINMDs, to foster the debate on how to discriminate normal morphology from abnormalities.
The term gastrointestinal neuromuscular disease (GINMD) applies to a heterogenous group of chronic conditions associated with impaired gut motility. In these disorders, the gastrointestinal (GI) motor dysfunction may result from morpho/functional alterations affecting the enteric nervous system (ENS) and/or the muscularis propria, with histopathologic pictures ranging from severe to mild abnormalities, up to a lack of evident microscopic changes.
GINMDs differ largely in their etiopathogenic mechanisms, histopathologic patterns, and region of gut involvement. Fortunately, the number of patients with severe forms of such motor disorders is relatively low, even though the overall incidence of GINMDs certainly climbs when considering other pathologic conditions, such as enteric neuropathies (eg, achalasia, slow-transit constipation), inflammatory bowel disease (IBD) chronic constipation, irritable bowel syndrome, or colonic diverticular disease.1–3 Thus, it seems that these entities may actually encompass a wide range of pathologic conditions, some of which (irritable bowel syndrome, colonic diverticular disease, chronic constipation) are often encountered in the daily clinical practice.
The matter of GINMDs is still quite complex and highly debated, due to the fact that no established and widely accepted classifications are available, and that these disorders may be viewed from different perspectives (ie, clinical, pathologic, etc.) by different investigators and health professionals. A classification of GINMDs, mostly based on morphologic findings, has been recently proposed by an International Working Group,4 as summarized in Table 1. However, as we have previously pointed out,25 this classification is somewhat scholastic and rigid. Indeed, it leaves out some important abnormalities, found in commonly occurring diseases [such as the consistent decrease in enteric glial cells (EGCs) in several conditions characterized by constipation], while giving emphasis to rare or very rare conditions, which have been regarded as single well-defined nosologic entities, while they very often display intermingled/overlapping features (for instance, syndromes associated with multiple morphologic abnormalities, which might be more appropriately classified as neuro-gliopathies, or neuro-glio-mesenchimopathies).26
Despite the heterogenous nature of GINMDs, these disorders are characterized mainly by clinical symptoms related to alterations of the gut functions deputed to ensuring adequate emptying/propulsion/excretion of luminal contents, with different degrees of dysmotility and a variable negative impact on health-related quality of life. In this respect, GINMDs have relevant implications for public health, because some are quite common, many are disabling, and most of them are associated with high social and economic burdens.27,28
Although gut dysmotility is suggestive of morphologic and/or functional alterations of the GI neuromuscular compartment, scarce attention has been paid to the rearrangements of this apparatus in GINMDs. It must be also acknowledged that it is very difficult to collect full-thickness tissue samples from the gut of GINMD patients, because most of them rarely undergo surgery, and those being operated are usually managed in an emergency setting. Thus, the rare, often unique, tissue samples, obtained from very small series or even individual patients, which should be processed very carefully, in full accordance with standardized techniques to obtain relevant and consistent diagnostic information, are often wasted or processed only by conventional, routine methods, which seldom allow to obtain reliable data.
The present article was aimed at reviewing the most updated guidelines and recent advances in the histopathologic diagnosis of GINMDs. Methodological issues and controversies in the interpretation of data, concerning the histopathologic assessment of gut neuromuscular compartment in patients with intestinal dysmotility, are discussed. Special attention has been paid to the lack of consistent morphologic data on smooth muscle cells (SMCs) in the majority of GINMDs, resulting in an inadequate characterization of this cell population, which represent the final effectors in the GI neuromuscular unit, and are therefore of pivotal importance in the implementation of digestive motor programs.
MORPHOLOGIC BASES OF GASTROINTESTINAL MOTILITY
In GINMDs, symptoms related to digestive dysmotility occur as the consequence of abnormalities in the functions of GI neuromuscular system, which, under physiological conditions, ensures coordinated patterns of motor activity of enteric smooth muscles. Schematically, the morphologic architecture of GI neuromuscular apparatus consists of 3 highly integrated cellular components: (a) intrinsic neurons of the ENS and EGCs; (b) interstitial cells of Cajal (ICCs); (c) SMCs of the tunica muscularis, acting as final effectors of gut motility.
The ENS is a complex neuronal network which, in contrast with the innervation of other organs, is deputed to the control of gut functions, independently from central nervous inputs. This network contains over 100 millions of neurons and EGCs distributed along the whole GI tract.29 Within the GI neuromuscular compartment, the ENS is arranged into several interconnecting plexi, the main being the myenteric (or Auerbach) plexus, which is formed by nerve strands and ganglia located between the circular and longitudinal muscle layers. Most of the enteric neurons, involved in the control of gut motor functions, are located within the myenteric plexus, whereas few primary afferent neurons belong to the submucous (Meissner) plexus. Similarly to other nervous systems involved in the control of sensory-motor functions, the ENS contains primary afferent neurons, which are sensitive to chemical and/or mechanical stimuli, interneurons and motor neurons, which act on different effector cells, including SMCs and ICCs.30
Enteric ganglia contain both neurons and glial cells, which have been found to express the same markers as the neurons and astrocytes of the central nervous system, respectively. In particular, neurofilament, neuron-specific enolase, protein gene product 9.5 (PGP9.5), or type 1 neuronal nuclear (HuC/D) antigens can be used to identify neurons, whereas S-100β or glial fibrillary acidic protein (GFAP) are regarded as specific markers of glial cells. Of note, by analogy with their counterparts in the brain, EGCs outnumber neurons by 4 to 10 times in humans and are likely to act as major regulators of the mucosal barrier and neuronal functions in the gut.31,32
The control of intestinal motility is accomplished through the release of neuro/glial factors that can target SMCs, either directly or indirectly, through the activation of intervening ICCs. The latter are important targets of the enteric motor innervations, because they can function as intermediaries between neural inputs arising from the varicosities of enteric nerve endings and the contractile responses of SMCs, thus contributing significantly to the transmission and propagation of stimuli directed from enteric neurons to SMCs. In support of the concept that ICCs are functionally innervated by the ENS, immunohistochemical and functional studies on ICCs have concurred in demonstrating that these cells do express receptors for neurotransmitters released from myenteric motor neurons and that changes in their intracellular levels of second messengers do occur after field stimulation of intrinsic myenteric neurons.33 Of note, ICCs are also known to act as pacemakers of the gut rhythmic motor activity and as conductors of electrical activity throughout the enteric smooth muscle syncytium, as suggested by morphologic studies and electrophysiology experiments, showing that specific pacemaker areas are populated by networks of ICCs connected with SMCs by means of gap junctions.34 These particular subtypes of ICCs account for the generation and propagation of electrical slow waves, which determine the characteristic frequency of phasic GI contractions. Slow waves determine also the direction and propagation rate of peristaltic activity, in concert with regulatory inputs from the ENS. On the basis of this knowledge, intensive efforts are being made to characterize the receptors and ion channels expressed on ICC membranes. Indeed, the manipulation of slow-wave ICC activity might represent a suitable way to elicit remarkable changes in the propulsion of contents in different gut regions. In addition, there is evidence to suggest that a loss of ICCs can contribute to dysmotility in some human GI diseases.35–40
SMCs are the final effectors of GI neuromuscular apparatus, because they represent the target cells of both enteric neurons and ICCs. Although not all SMCs are directly innervated or connected to ICCs, the presence of gap junctions ensures an adequate intercellular transmission of regulatory signals throughout the SMC network, thus allowing the generation of patterns of coordinated contractions.29
MORPHOLOGIC CORRELATES OF GASTROINTESTINAL DYSMOTILITY
Although it is fairly expected that a noxious stimulus to the GI neuromuscular apparatus translates into gut dysmotility, the architecture and morphologic remodeling of the enteric neuromuscular components have been scarcely studied under pathologic conditions.41 This is particularly true with regard for SMCs, which have received very scarce attention.42 As a consequence, despite in the clinical practice a GI dysmotility can be demonstrated or suspected in patients with GINMDs, it is rarely proven that such dysfunction results from a derangement of GI neuromuscular morphology.2,27 Thus, the concept that the clinical diagnosis of GINMDs must be substantiated by a careful histopathologic analysis of gut tissues is becoming increasingly recognized.
Enteric neuroplasticity and/or ICC abnormalities have been demonstrated under different pathologic conditions, ranging from enteric neuropathies to intestinal or extraintestinal diseases.1 In particular, histopathologic abnormalities of myenteric ganglia have been reported in functional disorders such as irritable bowel syndrome,7,43 intestinal neuronal dysplasia,44 obstructed defecation,42,45 Hirschsprung disease (Fig. 1),46 slow-transit constipation,42,47–50 as well as idiopathic and chagasic megacolon.51 Furthermore, abnormal cellular patterns (eg, duodenal eosinophilia, colonic mastocytosis, etc.) have been described in various functional GI disorders.27,52 Consistent evidence indicates the presence of enteric neuromuscular changes also in extraintestinal conditions, such as Parkinson disease53,54 or diabetes.55,56
All together, current data support the notion that gut dysmotility in functional GI disorders can be also characterized by histopathologic correlates. In this respect, there is mounting evidence that several (if not all) GI disorders, previously regarded as “functional” in nature, may actually harbor 1 or more organic (ie, mucosal, neuroenteric, muscular) abnormalities. Interestingly, some of these abnormalities can be considered as responsible for the motor derangement, as it happens in severe slow-transit constipation, where marked reductions in ICCs, neurons, and EGCs contribute, all together, to the severe decrease (up to the loss) of colonic motor activity.16 In this regard, we have previously shown that, as compared with controls, severely constipated patients display a significant decrease in neurons, EGCs (Figs. 2A–D), and ICCs. In addition, our patients displayed a significantly higher proportion of apoptotic enteric neurons than controls (Figs. 2E, F). Of note, some of these abnormalities may result from a genetic background, because 45.5% of patients displayed significant (>10%) aneusomy of chromosome 1 in enteric neurons. In our series, aneusomy <10% for the same chromosome, but less than the suggested cutoff (10%), was found in EGCs in 45.4% of the same patients, whereas no abnormalities were detected in controls.57 Therefore, it is likely that, in the next future, terms, such as “functional” or “idiopathic,” will be banned in favor of more appropriate definitions (eg, enteric neuro-gliopathies, neuro-myopathies, etc.).16,48
Looking at literature data, it seems that histopathologic studies of the gut neuromuscular compartment in GI motor disorders have been mainly focused on myenteric ganglionic cells and ICCs. By contrast, although the possible role of SMCs in gut motor dysfunctions has been extensively investigated by means of electrophysiological approaches,50 to date scarce attention has been paid to the morphologic abnormalities of GI musculature in GINMDs, and most of the available data pertain to single case reports or small series of patients with inflammatory or degenerative gut myopathies (Fig. 3). It is also worth noting that abnormalities of contractile proteins in the SMCs of muscularis propria in patients with colorectal motor disorders have been poorly studied. Moreover, the following few data on the expression of classic SMC markers have been reported in GINMDs: (1) abnormalities of enteric SMC histologic phenotype, causing functional bowel obstruction in childhood58; (2) reduced α-smooth muscle actin (α-SMA) expression in chronic intestinal pseudo-obstruction59,60 and its idiopathic form61; (3) reduced α-SMA expression in association with a decrease in SMC cytoskeleton proteins, in children affected by megacystis-microcolon-intestinal-hypoperistalsis syndrome21,62; (4) SMC inclusion bodies in slow-transit constipation.22 By contrast, a homogenous expression of α-SMA has been reported in children with severe defecation disorders,63 as well as in patients with idiopathic megacolon, slow-transit constipation, and Hirschsprung disease.42 On the basis of literature data, it seems that α-SMA, the most studied and commonly used marker for SMCs, can be regarded as a good biomarker only in subsets of GINMDs, as suggested by the heterogenous and often conflicting results reported in Table 2.
Interestingly, slow-transit constipation affects with higher incidence and severity females than males.69 Although serum progesterone levels are normal in these female patients, they have been found to overexpress progesterone B receptors on their colonic SMCs, and this feature might partly account for the alterations in cell-signaling pathways regulating the excitation-contraction coupling in the responses of enteric SMCs to progesterone.70,71 Among the GI dysmotility disorders, Hirschsprung disease is certainly one of the most studied SMC pathology. Under this pathologic condition, the correct evaluation of submucosal nervous plexus, with particular regard for ganglionic cells (present or absent, mature or immature), and the evaluation of the extension of the agangliar zone, based on adequate and correctly oriented bioptic material, are of paramount importance (Fig. 1). In these patients, despite the reported normal α-SMA levels, abnormalities of the intestinal musculature have been identified by demonstrating decreased levels of smoothelin, histone deacetylase 8, and smooth muscle miosin heavy chain, which have been proposed as novel SMC markers.42 Furthermore, other molecular factors have been found to be deranged in the aganglionic bowel muscle layers of patients with Hirschsprung disease, such as, for instance, an overexpression of semaphoring-3A,72 and a loss of sarcoglycan (e-SG)73 and cytoskeleton proteins (eg, dystrophin, vinculin, desmin).74 Abnormalities of the immunopositivity pattern in SMCs of the GI muscularis propria have been described also in IBD. In particular, an altered expression of proteins involved in SMC contraction, such as CPI-1775 and motilin receptor binding,76 has been observed in ulcerative colitis.
Studies on SMCs in the context of GINMDs are clearly needed, because the identification of novel SMC markers may unravel alterations of the gut musculature, which have not been previously appreciated by conventional histologic stainings and immunostainings, and might contribute to demonstrate that specific defects of SMCs are involved in the pathogenesis and/or pathophysiology of GI motor disorders.42
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An important issue, related to the histopathologic diagnosis of GINMDs, is that data reported by different investigators are often hardly comparable, or even conflicting.77,78 These discrepancies can be ascribed to a number of factors, which open ontological and dialectical discussions about the “sense” (ie, the “reason of being”) of investigations in this area, without prior achieving a widespread consensus on standard criteria for the methodological approaches and the interpretation of data regarding histologic abnormalities. With regard for this point, it is not surprising that the lack of histopathologic standards and, as a consequence, the lack of homogenous methodological procedures account for conflicting results in GINMDs. Besides the importance of careful tissue examination by dedicated investigators,38 comparable morphologic examinations can be obtained only by a close adherence to standardized and validated histologic procedures. Thus, in an attempt of overcoming these difficulties, detailed methodological approaches, guidelines and recommendations for the histologic analysis of gut tissues in GINMDs have been recently proposed by the Gastro International Working Group.52 Starting from tissue collection and handling (eg, mucosal biopsies, full-thickness and seromuscular biopsies), this report provides useful advices to pathologists on a wide array of technical notes (eg, sample size fixation, orientation and sectioning, etc.).
With regard for fixation and inclusion, only well-prepared tissues should be considered and then processed in full compliance with gold standard procedures. Samples with poor or delayed fixation should be discarded to avoid artefacts (ie, shrinking, tearing, or vacuolization), which may mimic some features of neuronal/smooth muscle degeneration or interfere with successful staining and data interpretation, resulting in pitfall specimens (Fig. 4). As far as paraffin block sectioning is concerned, the recommended 10 µm thickness has the advantage of minimizing artefacts due to cutting (ie, slice tearing or folding), and, above all, of obtaining reliable counts of small-sized cells, with particular regard for EGCs and ICCs. According to our experience, cell counting of myenteric ganglia is not significantly influenced by section thickness, at least in the range of 5 to 10 µm for HuC/D-S100β double immunostaining. Indeed, the number of ganglionic cells, estimated on 10-µm thick-sections,79 are fairly comparable with those counted in 5-µm sections.80 Moreover, thin sections can be useful to spare tissue specimen, obtain more brightly stained sections, and, above all, to perform comparative studies on serial sections, where the thinness allows to visualize the same architecture of the morphologic structures of interest in adjacent sections.
The guidelines by Knowles et al52 provide also detailed information on traditional immunomarkers suitable for cell identification (eg, labeling of neurons, EGCs, ICCs, SMCs, inflammatory cells, etc.). In this context, special attention deserves the estimation of cell counts, with particular regard for neuron and glial density, within myenteric ganglia. The usefulness of assessing these parameters, taking the ganglionic size as reference, has been recently appreciated, and studies on this issue, which has previously received scarce attention, particularly in the human gut, have been strongly encouraged. In studies on preclinical models, the estimation of neuron density has been referred to the overall ganglionic structures both under normal81 and inflammatory conditions,82 whereas in the human bowel this evaluation has been usually normalized to the sectioned surface area.83,84
In our opinion, referring cell counts to the respective individual ganglia can be the best choice in studies conducted on 3-dimensional whole-mount preparations, but not in the case of bidimensional histologic sections, such as those obtained from fresh biopsy specimens or archival materials. Indeed, in the latter setting, different sections of the same ganglion can give rise to multiple serial areas, which thus appear as a sequence of distinct ganglionic areas. In this regard, we have shown that a limited number of sequential colonic sections can be suitable for a reliable quantitative estimation of myenteric ganglionic cells by appropriate HuC/D-S100β immunostaining (Fig. 5).79,80 Moreover, we have noted that referring cell counts to the respective ganglionic area, rather than to the whole microscopic field,85,86 may offer the advantage of excluding extraganglionic neurons, the number of which is known to progressively increase with aging,81 in contrast to ganglionic ones,87 thus obtaining more accurate estimations.
Additional interesting issues deal with the careful retrieval of tissue samples and interpretation of findings. As anticipated above, the collection of full-thickness bowel specimens from such patients can be rarely accomplished, because most of them suffer of functional symptoms and, as a consequence, the only available bioptic material comes from individual patients or small case series. For these reasons, the highest level of care and methodological accuracy must be taken when processing these samples, and standardized procedures must be applied to carry out accurate morphologic examinations.
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The choice of markers selected for labeling specific cell lineages must be carefully taken into account when comparing results from different laboratories. Indeed, disregarding this aspect may lead to severe misinterpretations, as documented also by our experience in the morphometric analysis of colonic samples by immunohistochemistry.79,80 For instance, different estimations of the density of ganglionic cells have been obtained by means of different pan-neural or glial markers (eg, NSE, PGP9.5, HuC/D, or GFAP, S100, etc.), with consequent difficulties and discrepancies in the comparison of enteric neuron and glial cell counts, and the respective glial/neuron ratios, under both normal and pathologic conditions.79,80,86,91,92 Similar difficulties can be encountered also while evaluating specific cellular processes (eg, apoptosis).93,94
When comparing data from different laboratories, the specificity of primary antibodies deserves a careful attention. Thus, an appreciable variability in staining patterns may stem from the use of antibodies raised against different epitopes of the same antigen. Consistently with this notion, different immunostaining patterns can be obtained by using different unmasking protocols, primary antibodies, or detection systems in the morphometric analysis of bowel neuromuscular compartment.95 As an example, according to our experience, when carrying out the detection of cyclooxygenase-2 immunoreactivity in bowel tissues by means of different techniques and antibody sources, conflicting results can be obtained (Fig. 6) (Bernardini N, unpublished data 2010). Furthermore, in the final step of the immunostaining procedure, to highlight at the best, a specific cellular phenotype can be also a critical task. In this regard, the use of specific chromogens and counterstaining can make a great difference, as shown for ICC detection by c-Kit (CD117) immunostaining: in our laboratory, the best result was achieved by using 3,3′-diaminobenzidine-nickel chromogen enhancement followed by nuclear fast red counterstaining, allowing to carry out reliable counts of ICCs as black cells equipped with red nuclei (Table 3). It is important to note that a reduction in ICC density has been considered as pathologically relevant if it overcomes the threshold of 50% (Fig. 7).52
An important point of concern deals with the interpretation and evaluation of histologic abnormalities. These issues are closely related with the lack of a reliable definition of normality and the peculiarity of pathologic changes specifically associated with each disease. Both these aspects have been addressed in a recent review focused on chronic constipation, but the discussion can be extended to the whole body of GINMDs, because it refers to the bases of histopathologic diagnosis.92 In that article, the questions “Do findings actually deviate from normality?” (ie, are there qualitative or quantitative differences, as compared with adequately studied controls?), and “Are the examined tissues representative of the “average” patient population affected by a given pathology?” rise a number of additional issues (ie, patient selection, delineation of abnormality, diagnostic criteria, etc.), which deserve careful consideration to properly plan histopathologic studies and ensure correct data interpretation. The concept of “normality,” when considering human GI neuromuscular tissues, is far from being clarified and defined, and exhaustive studies on accurate morphologic and quantitative analysis, together with immunophenotype characterization, are expected to define robust normal values. Indeed, as recently discussed, these ranges of normality have not yet been defined at least with regard for the quantitative estimates of neurons, nervous fibers, EGC, or ICC density. Such evaluations would be highly useful for defining normal ranges of the ganglionic cell (ie, glia/neuron ratio) or ICC density and distribution within the neuromuscular layers. On the basis of unanimously accepted normal reference values, which should be established for each pathologic condition, the normality ranges and the limits of abnormality should be clearly defined and proposed as diagnostic criteria. For instance, Table 4 displays normality values for the main cellular ENS components of the human colon, as obtained in our setting. The main point to consider is that obtaining “normal” human intestinal tissue is extremely difficult, for both technical and ethical reasons. For instance, autoptic material would be useful, but, unfortunately, the rapid degradation of gut tissues by bacteria makes these samples less than optimal for subsequent morphologic analysis. The main viable alternative is to use, where possible, resection margins or healthy tissue at a distance of 3 to 5 cm from neoplastic lesions during surgery. Although, from a conceptual point of view, even this strategy might be regarded as not adequate to obtain “normal” tissue samples, it is common experience that this is usually the most practical (and practised) approach. In most studies, another point of weakness deals with the number of tissue specimens, because even in relatively large series the controls usually average 10. Thus, to obtain reliable data on normality, large cohorts of “normal” specimens would be necessary. However, with regard for pathologic abnormalities, some indications have been already published. For example: ganglionitis in IBD is diagnosed if the mean number of lymphocytes is ≥2 lymphocytes/ganglion; inflammatory neuropathies are defined on the basis of the number of lymphocytes/ganglion (≥1 intraganglionic and/or>5 periganglionic) (Fig. 8); intestinal neuronal dysplasia is diagnosed when a number >8 neurons is found in >20% of 25 submucosal ganglia; ICC density is considered as reduced if a decrease in ICCs over 50% is found in comparison with normal values.4,8,52 Other studies in constipated patients have pointed out that an ICC/high-power field cutoff value of 7 in the inner circular muscle layer can be used as a further confirmation to the clinical diagnosis of slow-transit constipation in resected specimens.96
It would be important to have a grading of established histopathologic parameters against which to score the severity of disease. An accurate assessment of disease severity is also pivotal to estimate the impact of GINMDs on public health and, whenever possible, design adequate therapeutic approaches and implement novel pharmacological treatments. Overall, a well-conducted histopathologic analysis may contribute to reliably assessing and quantifying the degree of tissue lesion, and immunohistochemistry could be considered as a tool to perform accurate quantitative immunoassays applied to tissue sections. However, it must be also acknowledged that, very often, the analysis of sections from individual (and often unique) subjects requires a particular histopathologic tailoring, meaning that specific pathologies require specific immunohistochemical or different methodological approaches (eg, electron microscopy). Unfortunately, the same considerations and relative concerns discussed above for “normal” tissues apply here, with the aggravating circumstance that most of such patients often undergo surgery in primary or nonacademic hospital settings, which are scarcely or not at all equipped to deal with the complex immunohistochemical procedures required to assess correctly these tissue samples. Finally, a further confounding aspect is represented by the fact that some degree of GINMD standardization has been attempted only in recent years, and therefore, some pathologists may not be sufficiently experienced to be able to recognize even simple abnormalities in the ENS. In this regard, the present review might help in spreading current knowledge and awareness of pending issues well outside from the restricted group of scientists and health professionals dedicated specifically to neurogastroenterology. Moreover, a better understanding of the morphologic and pathophysiological bases of the abnormalities associated with GINMDs is of paramount importance to develop more targeted therapeutic approaches, even though this presently remains a quite distant goal.
On the basis of the above discussion, adequate histopathologic studies of GI neuromuscular morphology, by means of standardized and validated histopathologic procedures, are critical to shed light on the pathogenesis and pathophysiology of GINMDs, allowing to achieve a correct clinical diagnosis. In particular, a better characterization of SMCs, in terms of cytoskeleton and contractile protein expression, cellular receptors, and intracellular signaling molecules, is likely to contribute to improve the understanding of the pathophysiological bases of GINMDs. Of course, methodological accuracy and customized studies should be reserved to individual cases or small series of patients and to specific pathologies, respectively. In addition, the combination of basic morphofunctional studies with clinical research may help to translate results from bench to bed side, allowing improved or novel therapeutic approaches for restoring or repairing malfunctioning GI neuromuscular units in several GINMDs. For instance, the exciting perspectives, arising from neural stem cell transplantation for the treatment of some gut disorders, are opening new, and until now unimaginable roads, toward a preventive approach of at least some of these too often frustrating (from a therapeutic point of view) entities.
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