LITHUANIAN UNIVERSITY OF HEALTH SCIENCES
FACULTY OF MEDICINE
INSTITUTE OF ANATOMY
Josef Göran Sundström
IMMUNOHISTOCHEMICAL STUDY OF CALCITONIN
GENE RELATED PEPTIDE IN THE SIGMOID COLON
OF DIVERTICULAR DISEASE PATIENTS
Master’s Thesis
Thesis Supervisor:
Professor Neringa Paužienė
TABLE OF CONTENTS
1.
SUMMARY ... 3
2.
CLEARANCE ISSUED BY ETHICS COMMITTEE ... 4
3.
ABBREVIATIONS ... 5
4.
INTRODUCTION ... 6
5.
AIM AND OBJECTIVES ... 7
6.
LITERATURE REVIEW ... 8
6.1 Etiology and pathogenesis of Diverticular disease ... 8
6.2 Calcitonin gene-related peptide ... 10
6.3 Possible involvement of CGRP in diverticular disease ... 12
7.
MATERIALS AND METHODS ... 14
8.
RESULTS ... 18
9.
DISCUSSION ... 25
10.
CONCLUSIONS ... 28
11.
REFERENCES ... 29
1. SUMMARY
Aim: To evaluate changes of sensory innervation in the sigmoid colon during diverticular disease with focus on calcitonin gene related peptide (CGRP) and its receptors calcitonin receptor like receptor (CRLR) and receptor modifying protein 1 (RAMP1).
Objectives: 1) To observe the morphological location of CGRP-IR, CRLR-IR and RAMP1-IR in the ganglia’s being examined. 2) To determine the difference in CGRP-IR (CGRP immunoreactivity) and its receptors CRLR-IR and RAMP1-IR expression in the colon of diverticular disease patients with comparison to control patients. 3) To determine if CGRP-IR, CRLR-IR and RAMP-IR is associated with the nitrergic neurons.
Material and Methods: 1) Colonic samples were obtained from patients who underwent surgery for colorectal carcinoma and symptomatic DD. 2) Cryo-sectioning of full thickness colon pieces followed by immunohistochemistry using antibodies against CGRP, CRLR, RAMP1 and nNOS. 3) Quantitative fluorescence microscopy of the intrinsic neural plexuses and subsequent image analysis by means of estimating the average densitometric fluorescence intensity.
Results: By direct visualization using microscopy the presence of CGRP-IR nerve fibers was mainly situated in the regions of enteric plexuses localization. The most abundant staining signal was found in the myenteric plexuses. The signal was noted to be weaker in the submucosal plexuses, the staining signal was scattered mainly along the internal and external surfaces of the submucosa. With morphologically similar appearance and location as the CGRP-IR signal, the CRLR-IR receptor signal was clearly stronger in the diverticular samples when comparing to the control samples. There was no observable difference in signal when comparing RAMP1-IR of diverticular diseased colons and control samples. The mean fluorescence intensity of CGRP-IR in the myenteric plexus is significantly decreased in DD samples in comparison to control patients. There is a statistically significant increase in the CRLR-IR mean fluorescence intensity in the myenteric and external submucosal plexuses in DD samples. No statistically significant difference was found between the plexuses of the DD and control samples with regard to RAMP1-IR mean fluorescence intensity. Clear association between CGRP-IR and nitrergic neurons where also demonstrated.
Conclusions: we can conclude the association of diverticular disease and a decrease in CGRP-IR and an increase in one of its receptors CRLR-IR in the sigmoid colon. With the fact that CRLR-IR had increased immunoreactivity, this can be explained by a compensatory mechanism due to decreased levels of calcitonin gene related peptide or due to other unknown cause.
2. CLEARANCE ISSUED BY ETHICS COMMITTEE
Approved by: Kaunas Regional Biomedical Research Ethics Committee, Kaunas, Lithuania Biomedical research name: Research Material Biobank of Digestive System Diseases. Number: BE-2-10Date 08/03/2011
3. ABBREVIATIONS
DD- Diverticular disease.CGRP- Calcitonin gene related peptide CRLR- Calcitonin receptor like receptor
RAMP1- Receptors activity modifying protein 1 MP- Myenteric plexus
ESP- External submucosal plexus ISP- Internal submucosal plexus IR- Immunoreactivity.
nNOS- Neuronal nitric oxide synthase NO- Nitric oxide
TRPV1- Transient receptor potential cation channel subfamily V member 1 NH2- Nitrogen hydrogen 2 terminus
RCP- Receptor component protein AU- Arbitrary units
4. INTRODUCTION
Colonic diverticular disease is a gastrointestinal disorder that usually manifests as a pouch like herniating sac through the different muscular layers of the descending and sigmoid colon [1]. It ranges from a mild asymptomatic condition which is very prevalent with aging, to a life threatening condition as in diverticulitis with complications [2].
Many pathogenetic factors have been found to contribute in the formation of DD, but the true cause of DD remains unknown [3]. Previous studies have found various factors to be involved including dietary deficiencies, anatomical and functional changes of the colon, as well as innervational changes [4].
Recent studies have indicated the role of different neuromodulators and neurotransmitters such as serotonin, acetylcholine, nitric oxide and vasoactive intestinal peptide in DD [5]. However, no up to date studies has researched thoroughly about CGRP’s involvement in diverticular disease.
CGRP is a peptide composed of 37 amino acids, its exits in two isoforms named α and β CGRP. It is located in both the central and peripheral nervous system [6]. CGRP which is one of the most potent vasodilators, can induce both anti and pro-inflammatory, electrophysiological, functional and contractility changes in the colon. Thus it is reasonable to suspect CGRP of having a potential role in DD formation [6].
We would like to elaborate our basic understanding of CGRP and its receptors RAMP1 and CRLR in the colon by an immunohistochemical approach. As well as investigate the association of CGRP and its receptors to nitrergic neurons.
5. AIM AND OBJECTIVES
Aim: To evaluate changes of sensory innervation in the sigmoid colon during diverticular disease with focus on calcitonin gene related peptide (CGRP) and its receptors calcitonin receptor like receptor (CRLR) and receptor modifying protein 1 (RAMP1).
Objectives:
1) To observe the morphological location of CGRP-IR, CRLR-IR and RAMP1-IR in the enteric plexuses.
2) To determine the difference in CGRP-IR and its receptors CRLR-IR and RAMP1-IR expression in the colon of diverticular diseased patients with comparison to control patients.
6. LITERATURE REVIEW
Colonic diverticular disease implies diverticular formation and its various complications including diverticulitis, diverticular haemorrhage, abscess, phlegmon etc. Colonic diverticulosis is basically a sac like herniation of the colonic mucosa and submucosa through the deeper layers of the intestinal wall [1].
Being a very common condition in the west, it is well known that the prevalence of diverticular formation increases proportionally with age. The condition is uncommon amongst individuals younger than 40 years of age, but in the elderly aged 80 years statistics estimates 70% of having the condition [7].
About 10-25 % of patients which have colonic diverticulosis will have complications and the most prevalent complication is diverticulitis which is diverticular inflammation [8]. Asymptomatic diverticulosis does not require treatment, while symptomatic diverticular disease can be treated by proper hydration, fiber supplementation, antibiotics and surgery depending on the individuals case [9– 13]
6.1 Etiology and pathogenesis of Diverticular disease Anatomical changes of the colonic wall:
Microscopic studies have demonstrated that diverticula mainly are formed in areas of weakness in the colon where it is being penetrated by blood vessels [14]. The most common place of diverticula to develop is on the mesenteric side of the anti-mesenteric taeniae, and with further progression between the rows of anti-mesenteric taeniae. The exact explanation behind this finding is unclear but the most probable one is the fact that with mesentery comes the blood vessels which penetrates the circular layer of the muscle [14].
Other studies have shown colonic wall thickness to be increased in diverticular disease patients [14]. Elastin was increased 200% in comparison with controls, which led to increased thickness of the colon wall. Surprisingly that same isolated area is still more prone to herniation [14].
Neither with increased age nor in diverticular patients is the amount of collagen altered [4]. But studies have shown that the cross linking of collagen fibers increases in patients above 40 which is the same age diverticulosis begins to become more prevalent, a higher rate of collagen cross linking was present in diverticular patients [15]. The effect of this cross linking is thought to decrease resistance
and increase stiffness of the muscularis externa. When the submucosa fails to exhibit its compliance, it becomes weaker and more prone to herniate, especially with increased intraluminal pressure [15].
Functional changes:
Transit time of faecal material in the colon of DD patients have shown contradicting results. In one study, decreased colonic transit time in DD patients was demonstrated [16]. While another study has shown a quicker transit time in the large intestine up until the level of the sigmoid colon with the complete time of passage being equal to the control group [17].
It has been known for a longer period of time that intestinal motility is disturbed in DD patients. Older studies using organ bath technique of separated pieces of intestinal smooth muscles showed irregular increased contractility patterns and changed responses to excitatory and inhibitor stimuli [18]. Diminished relaxation capacity due to disordered nitrergic neurotransmitter system have been proven by some studies [19]. Other studies have also shown increased contractility due to cholinergic enhanced stimulation [20].
More recent studies support the earlier in vitro studies by using manometric techniques on DD patients [18]. Their study resulted in higher frequency of contractile activity as well as increased intraluminal pressure under both passive and active conditions of the colon [18].
Changes of the ENS:
A variety of innervational changes have been found in the colons of patients with diverticular disease. A whole mount study using histochemistry analysed the morphological pattern of the myenteric plexus revealed degradation of this plexus in diverticular colons, as well as some degree of interruption and thinning of interganglionic nerves when compared to control colons [21].
Another study performed by utilising immunohistochemistry and morphometric analysis of isolated segments of diverticular colon showed clear results of decrease in neuronal, ganglionic and glial cell density and content in most of the enteric nerve plexuses of the sigmoid colon [22]. Implying that diverticular disease is associated with enteric neuropathy. Other histopathological findings observed hypertrophy of the myenteric plexus [22]. These combined findings suggest oligo-neuronal hypoganglionosis that lead to the intestinal function abnormalities evident in DD patients [22].
A significant amount of chemical mediators has been found to have an impact on intestinal function and motility. But only a small amount of them have been thoroughly researched with regard to
diverticular disease. Serotonin, acetylcholine, nitric oxide and vasoactive intestinal peptide have all been shown to be involved in the pathogenesis of diverticular disease [23–25]. The imbalance of these chemical mediators some of which are excitatory and others inhibitory lead to increased motility and colonic pressure when altered and hence to the development of diverticular disease [5]. With that in mind, it is reasonable to research in more detail about other chemical mediators involvement in diverticular disease.
6.2 Calcitonin gene-related peptide Description
Calcitonin gene related peptide is a peptide modulator composed of 37 amino acids which is present in two isoforms, which are α and β CGRP. These isoforms are very similar in structure and activity but are expressed differently throughout the body [6]. αCGRP and βCGRP share >90% homology and differ only by three amino acids in the humans thus it is perhaps not surprising that they share similar biological activities [6].
The genes for CGRP are located on the human chromosome 11, with different genes involved for the respective isoform produced. CAL 1 gene is responsible for generating calcitonin or α-CGRP by alternative splicing, while β-CGRP is produced from a separate CAL 2 gene [6].
Studies suggest α-CGRP to be located in the central and peripheral nervous system, while β-CGRP is to be located principally in the enteric nervous system [26]. Animal studies done on rats suggests β-CGRP to be found in adventitia of mesenteric branch arteries as well as in the myenteric plexus and mucosa [27]. Other studies have shown β-CGRP to be secreted together with α-CGRP in the vascular system [26]. More so, β-CGRP has been recently discovered to be expressed centrally together with α-CGRP [6].
CGRP exists in neuronal cells and their fibers, with regard to the enteric nervous system they are found to be located intrinsically in the ganglionic cells and fibers of the myenteric and submucosal plexuses and extrinsically afferent fiber processes coming from the spinal ganglia. Their secretion is regulated by TRPV1 [28-29]
Receptors
CGRP receptor is formed by heterodimerization of two subunits: CRLR and RAMP1. The CRLR is a G protein coupled receptor, and the RAMP1 is a small membrane protein that possess a large extracellular NH2 terminus of 100 amino acids, a single transmembrane domain, and a short
intracellular domain of 10 amino acids [6]. One more component which is required for functioning of the CGRP receptor is the RCP which is not involved in the binding of CGRP to the receptor, but it increases the stimulation of cAMP production [30]-[31]. CRLR in conjunction with RAMP1 produces a receptor with strong affinity towards CGRP. When the CRLR subunit is combined with RAMP2 it results in a receptor with strong affinity for the peptide adrenomedullin. In the same manner RAMP3 and CRLR create a receptor that is responsive for the adrenomedullin 2 and also confers CGRP [6].
Signalling pathway
When CGRP binds extracellularly to its receptor, different downstream pathways intracellularly can take place. Of particular interest is the activated cAMP dependent pathway, in which the activated transmembrane receptor and RCP stimulates the G-alfa-s protein which in turn triggers the adenylate cyclase enzyme to convert ATP to cAMP and pyrophosphate. An increase in intracellular cAMP stimulates protein kinase A (PKA), leading to phosphorylation of several targets downstream [6]. By activating 1) ATP sensitive potassium channels which causes vasodilation, 2) extracellular signal-related kinases (ERKs) which mediates protective effects on smooth muscle cells, 3) Nitric oxide synthase activation, 4) cAMP response element- binding protein (CREB) influencing transcription factors [6].
Function
Vasodilation and effects on cardiovascular system. Studies have demonstrated CGRP of being a very
strong vasodilator, both in terms of its potency and its duration of action in the microvasculature. When its potency was compared to acetylcholine and substance P it was shown to be of multiple folds more potent. In certain experiments on animals when CGRP was administrated intravenously it decreased the blood pressure, in addition to influencing the heart by making it beat faster and stronger. This could be explained as a compensation mechanism to counter the lower blood pressure [6].
Nociception. The role of CGRP in nociception is still controversial. When CGRP was injected directly
into the skin of a human, there was no increase or change in nociception. However, when CGRP was injected systemically to rats it lead to hyperalgesia. Therefore its debated if CGRP plays a stronger role in central rather than peripheral nociception [6].
Other studies contemplate CGRP of having a role in nociception during abnormal pain processes, as in inflammatory and neuropathic pain. This was demonstrated when mice that did not exhibit RAMP1 receptor, did not acquire complex regional pain syndrome when associated with distal limb fracture [6].
Pro- and anti- inflammatory actions. According to the site of expression, CGRP has a dual function in
regard to inflammation, where it can act both in an anti- or pro- inflammatory way. CGRP takes part in neurogenic inflammation together with substance P [32]. CGRP with its vasodilatory effects and substance P acting on permeability allows influx of blood, inflammatory cells and edema to happen in the affected area [32]. By upregulating cAMP, CGRP has the ability to decrease inflammation by inhibiting different inflammatory mediators, such as lymphocyte differentiation and proliferation and IL-1β-induced reactive oxygen species [33].
CGRP and migraine. The etiology behind migraine is still not fully understood, although it is widely
accepted that CGRP has an active role in its pathogenesis[6]. During a migraine attack the afferent fibres of the trigeminal nerve which is innervating the meningeal arteries secretes peptides, this causes neurogenic inflammation, CGRP stimulates vasodilation and substance P induces increased permeability of capillaries [6, 34]. Studies have shown that if CGRP would be administrated intravenously in susceptible migraine patients it could elicit a migraine attack [35].
In recent years, advances in finding effective and safe treatment of migraine using CGRP antagonists such Olcegepant and Telcegepant has proved to be successful [36]. These antagonists bind to CGRP receptors and block them, hence they repress CGRP activation of cAMP responses, thus inhibiting CGRP action depending on location and cell type [29].
6.3 Possible involvement of CGRP in diverticular disease Inflammation
It has been known for a longer time that inflammation obviously occurs in complicated diverticular disease, called diverticulitis [37]. However recent studies also suggest inflammation in uncomplicated asymptomatic colonic diverticula [37].
Of particular interest are the effects of CGRP on colonic inflammation. A study demonstrated CGRP of mediating inflammation in the colon, by utilizing rats with TNBS (Trinitrobenzene sulfonic acid induced colonic inflammation), and intravenously administrating CGRP to them, this resulted in increased microscopic damage of the rat colons when compared to control rats infused with saline water [38].
Other experiments have shown CGRP to have anti-inflammatory properties as well, thus contradicting the previous study. Administration of CGRP subcutaneously reduced the lesions in TNBS colitis and
another interesting finding in the same study was that those rats that developed TNBS colitis had a reduction in CGRP immunoreactivity [39].
Enteric neuron excitability
Certain electrophysiological alterations have been reported with regard to colonic diverticular disease [20]. This was observed when utilizing organ bath studies and electrophysiological studies in vitro on diverticular samples of the colon [20]. It resulted in a steep decrease in the amplitude of rhythmic phasic contractions, however the frequency and tone were unchanged [20].
It has been demonstrated experimentally that CGRP excites myenteric neurones in longitudinal muscle [40]. When applied to neurons, CGRP lead to an increase in input resistance, suppression of post-spike hyperpolarizing potentials and enhanced excitability [40]. In the same calcitonin was also applied to the same preparations resulting in no change in excitability [40]. Thus it is reasonable to suspect CGRP of having a possible involvement in the earlier mentioned electrophysiological changes observed in diverticular patients [20][40].
Smooth muscle contractility and colonic motility
It may be useful to compare colonic motility with gastric motility as it was shown that in a healthy state CGRP increases smooth muscle relaxation and decreases smooth muscle contraction [41]. By knowing that CGRP slows gastric emptying it maybe be suspected to have a similar effect in the colon. Another study done in guinea pig small intestine has demonstrated CGRP to induce high-amplitude dose-dependent phasic contractions of intestinal smooth muscle. Beside this stimulatory response, the same study demonstrated CGRP to inhibit peristaltic contractions [42].
If we are to assume that CGRP has similar effects on the colon as it has on the gastric and small intestine contraction and motility, CGRP may induce both reduced relaxation of colonic smooth muscle and reduced peristaltic activity which are thought to be important pathogenetic factors leading to excessive intraluminal pressure in DD patients [4].
7. MATERIALS AND METHODS
Patients and Tissue Samples
Tissue specimens for the control group were obtained from patients undergoing surgery for non-obstructing colorectal carcinoma, who did not have symptoms of clinical motility disorders or previous episodes of symptomatic complicated or uncomplicated DD. This type of operation was a source of both normal (control) and DD, if diverticula were found to be present in these patients. Tissue specimens for the DD group were also obtained from patients who underwent sigmoid resection or left hemicolectomy for symptomatic DD. Patients were operated after recurrent attacks of diverticulitis by elective surgery.
Segments were taken from macroscopically normal regions of colon cancer patients or, in patients with diverticulitis, from the apparently normal area closest to the diverticulum. Diverticula containing areas displaying an altered anatomy of the colonic wall due to transmural mucosal/submucosal outpouchings or signs of inflammation and fibrotic scaring were excluded from tissue sampling. Colon segments were collected in the operating theatre, and immediately placed in a cold (4˚C) pre-aerated (95% O2, 5% CO2) Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 25 mM NaHCO3, 2.5 mM CaCl2, 11 mM glucose) and transported to the Institute of Anatomy laboratories within approximately 30 minutes.
Study groups and patient characteristics are described in Table 1. All the experimental procedures were approved by the Kaunas Regional Biomedical Research Ethics Committee, Kaunas, Lithuania (Code: BE-2-10).
Table 1. Table showing the age, gender, and BMI of the patients included in the study.
Group n Gender Age, years (range) BMI (range)
Control 14 8F/6M 60.14 (39–80) 26.11 (22.72–31.64)
DD 10 4F/6M 66.30 (35–87) 25.55 (21.19–31.11)
Immunohistochemistry
Tissue was submerged in 4% PFA solution (Sigma-Aldrich, Cas. No 30525-89-4) for 150 min at r.t. Remnants of the aldehydes were removed by repeated rinses in phosphate buffer saline (PBS, 0.01 M), and the tissue was sectioned into approximately 10 x 10 mm pieces. These segments were cryoprotected by immersion in PBS containing 20–25% sucrose (Sigma-Aldrich, Cas. No 57-50-1) and 0.05% sodium azide (Carl-Roth, Cas. No 26628-22-8) overnight at 4°C. Following
cryoprotection, the samples were embedded in OCT compound (Shandon™ Cryomatrix™, Thermo Fisher Scientific, USA), snap-frozen and stored at -80°C for later use.
Two pieces (10 x 10 mm) were randomly selected for each patient that would be later serially crosssectioned to obtain (16 µmthick) cryostat sections (CryoStar NX70, Thermo Scientific) at -23 °C. Cresyl violet staining was used to evaluate morphological appearance and the overall quality of the sections, in particular to check the orientation and to assess the degeneration of intestinal musculature. Sections were made both along longitudinal and circular muscle axes, changing the orientation of the mounted sample in the cryotome. Sections were made no less than 1 mm apart from one another to prevent measuring the same ganglia in adjacent sections. Sections were mounted onto Superfrost Plus standard microscope slides (Thermo Scientific, Germany), air-dried at room temperature for 1h and stored at -20°C until use.
The sections were rehydrated by successive rinsing in PBS solution and permeabilized for 1 hour at room temperature in PBS containing 0.5% Triton X-100 (Carl Roth, Cas. No9036-19-5)
and 10% DMSO (Carl Roth, Cas. No67-68-5). Then samples were successively washed in PBS for 3x10 min. and incubated in 5% NDS (Jackson ImmunoResearch Laboratories, Prod. No 017-000-121) for 1 hour at room temperature to block nonspecific binding. Preparations were subsequently washed (3 × 10 min) in PBS and incubated in primary antisera (Table 3) overnight in a dark humid staining tray at 4°C. After 3x20 min. washes in PBS, samples were incubated in an appropriate combination of secondary antibodies (Table 3) for 4 h in a dark humid chamber on a shaker stage at room temperature. Finally, the specimens were washed in PBS, cover-slipped (No.1) using antifade mounting medium (Vectashield, Vector Laboratories) with DAPI and sealed with clear nail polish.
For all antibodies used in this study, samples for internal negative controls were processed as outlined above, except that either the primary or the secondary antibody was omitted. In all trials this eliminated detection of histofluorescence. For anti-human CGRP primary antibodies internal positive controls (IPC) were used to identify specificity of antigen binding, i.e. samples were single stained for anti-CGRP. In all trials, signal of IPC was indistinguishable to that of experimental samples. Controls were performed in accordance to international guidelines [43-44].
Table 2. Primary and secondary antisera used in the study.
Antigen Host Type Dilution Source Cat. # Primary
CGRP Mouse Monoclonal 1:1000 Abcama Ab 10987
RAMP1 Rabbit Polyclonal 1:1000 Biossb BS-1567R
CRLR Rabbit Polyclonal 1:1000 Biossb BS-1860R
PGP 9.5 Mouse Monoclonal 1:1000 Abcama Ab 72911
PGP 9.5 Rabbit Polyclonal 1:1000 Bio-Radc 7863-0504
NOS1 Rabbit Monoclonal 1:1000 Abcama EP 1855Y
NOS1 Mouse Monoclonal 1:500 Santa
Cruzd SC-5302
Secondary
Anti-Rabbit
Cy3 Donkey Polyclonal 1:500 Milliporee AP182C
RabbitAF488 Donkey Polyclonal 1:500 Invitrogenf A21206 Mouse Cy3 Donkey Polyclonal 1:500 Milliporee AP192C Mouse FITC Donkey Polyclonal 1:500 Jackson
Imunoresg
715-095-151 aAbcam, Cambridge, UK.
b Bioss Antibodies Inc., Woburn, Massachusetts, USA; cBio-Rad (Formerly AbD Serotec), Kidlington, UK; d Santa Cruz biotechonology, Dallas, Texas, USA; eMillipore corp., Temecula, California, USA; f Invitrogen, Ltd., Paisley, UK;
gJackson ImmunoResearch, West Grove, Pennsylvania, USA.
2.1. Microscopy and Image Analysis
Quantitative microscopy experiments were designed in accordance with best practice recommendations [45]. Fluorescent images of intrinsic neural plexuses were taken using AxioImager Z1 equipped with AxioCam MRm Rev.3 digital camera and using 40x/.,9 EC Plan NeoFluar objective in the native Axiovision Rel. 4.8.2 environment (Carl Zeiss, Germany). The fluorescent light source was HXP 120V illuminator using 45HQ TR (EX 560/40, EM 630/75) filter set.
Images of myenteric (Auerbach), outer submucosal (Schabadasch) and inner submucosal (Meissner) plexuses ganglia were captured throughout the section, in three dimensions. 5-15 ganglia (N)(Table 3) were captured per plexus where each image consisted of 10 z-stack images at 1 µm intervals. Imaging of the samples was performed no later than 1 week following the sample
preparation to avoid fluorophore bleaching. Images were taken at a fixed exposure time (800ms) for the channel of interest for all images in the study.
Image analysis was performed using Fiji software [46]. In brief, images were loaded into Fiji as a stack and Z-projected using average intensity projection. Fluorescence intensity units was determined by selecting boundaries of the enteric ganglion (ROI) in PGP 9,5 signal view and measuring the densitometric sum of fluorescence intensity of that region in the channel of CGRP/ RAMP1/ CRLR signal. To correct for potential inconsistencies of the fluorescent lighting and optical shading effects, the resulting intensity values are reported as a fraction of a fluorescence standard as described by Model and Burkhardt (2001) [47]. Rose Bengal (Sigma-Aldrich, Cas. No 632-69-9) (0.25 g/ml) was used as a fluorescence standard and a series of images of this reference were made each time before imaging colonic sections. The original intensity values (with background values subtracted) were then divided by the reference values to obtain the final fluorescence intensity value. Immunofluorescence intensity is measured in arbitrary units.
Images for illustrations were generated using Zeiss LSM 700 laser-scaning confocal microscope equipped with dual T-PMT sensor and using 40x/1.4 Plan Apochromat and 60x/1.46 αPlan Apochromat oil immersion objectives in a native ZEN Black SP1 2010 software (Carl Zeiss, Germany). Confocal images were processed into final figures by adjusting image size, brightness, and contrast using Photoshop CS6 (Adobe Systems, San Jose, USA).
Table 3. Table showing the number of objects analysed for fluorescence intensity measurement
per marker.
Group CGRP CRLR RAMP1
MP eSP iSP MP eSP iSP MP eSP iSP
Control (N) 188 150 180 95 59 94 90 64 68
DD (N) 285 177 201 126 84 61 146 86 105
MP, myenteric (Auerbach) plexus; eSP, outer submucosal (Schabadasch) plexus; iSP, inner submucosal (Meissner) plexus;
2.2. Statistical Analysis
Data are presented as mean ± standard error of the mean (SEM). The Shapiro–Wilk and D’Agostino-Pearson omnibus tests were used to determine the normality of the data. Differences in the data were assessed using the t test or Mann–Whitney rank sum test as appropriate (GraphPad Prism 6, San Diego, USA). A P-value of < 0.05 was used as a criterion for statistical significance.
8. RESULTS
Immunohistochemical observations of CGRP, CRLR, RAMP1 in human sigmoid colon samples CGRP-IR, CRLR-IR and RAMP1-IR signal in the enteric ganglia.
We found the presence of CGRP-IR surrounding the cell bodies of all the plexuses that where examined including the myenteric and submucosal plexuses (Fig. 1-2). It was situated mainly in the regions of enteric plexuses localization. The most abundant CGRP signal was found in the myenteric plexuses which could be described as irregular shaped clusters of ganglia rich in nerve fibers laying horizontally between the circular and longitudinal muscle layers (Fig.1). More specifically CGRP signal location was found to be surrounding the cell bodies of the ganglia. The CGRP signal was noted to be weaker in the submucosal plexuses, the staining signal was scattered mainly along the internal and external surfaces of the submucosa. Appearing as single oval and ellipsoid shaped ganglia (Fig. 2). In very rare cases CGRP-IR was found in the cell bodies of the ganglia in the enteric plexuses (Fig. 3). CRLR-IR and RAMP1-IR was found in all enteric plexuses. CRLR and RAMP1 where found within the cell bodies of the ganglia (Fig. 4-5), alike CGRP which was found surrounding them. Apart from their location with regard to the respective ganglia their pattern of distribution within the plexuses was very similar to CGRP.
CGRP, CRLR and RAMP1 signal in the surrounding structures.
CGRP and its receptors where found with weak intensity in most parts of the sample, stronger signal was mainly located in the the apical surfaces in the glands of the mucosal layer, arborizing the most part of the mucosa to a lesser extent. It was also observed to be sharply demarcating the borders of the blood vessels in the submucosa and longitudinal muscle layers.
Fig. 1 Microscopical image of the myenteric plexus between the longitudinal and circular muscle with CGRP-IR and PGP 9.5 staining in the sigmoid colon of DD and control patients. This image shows the relation between CGRP-IR and the general neuronal tissue marker PGP 9.5 which are closely overlapping except for the cell bodies of the ganglia with obvious decrease in CGRP-IR in DD. PGP 9.5: protein gene product 9.5. CGRP: calcitonin gene related peptide. LM: longitudinal muscle. CM: circular muscle.
Fig. 2 Microscopical image of the external submucosal plexus with CGRP-IR and PGP 9.5 staining in the sigmoid colon. CGRP-IR is observed surrounding the cell bodies of the ganglia in an encapsulating manner. PGP 9.5: product gene product 9.5. CGRP: calcitonin gene related peptide.
Fig. 3 Microscopical image of the myenteric plexus with CGRP-IR and PGP 9.5 staining in the sigmoid colon. A rare occurrence where CGRP-IR was located within the cell bodies of the ganglia. PGP 9.5: protein gene product 9.5. CGRP: calcitonin gene related peptide.
Fig. 4 Microscopical image of the myenteric plexus with CRLR-IR, PGP 9.5 and DAPI staining in the sigmoid colon. CRLR-IR signal is observed to be scattered in similar locations as PGP 9.5 and is most abundantly located in the cell bodies of the ganglia. PGP 9.5: product gene product 9.5. CRLR: calcitonin receptor like receptor. DAPI: diamidino phenylindole.
Fig. 5 Microscopical image of the myenteric plexus with RAMP1-IR, PGP 9.5 and DAPI staining in the sigmoid colon. RAMP1-IR signal is observed to be scattered in similar locations as PGP 9.5 and is most abundantly located in the cell bodies of the ganglia PGP 9.5: product gene product 9.5. RAMP1: Receptor activity modifying protein. DAPI: diamidino phenylindole.
Quantitative microscopy results:
Table 4. Table illustrating different quantitive immunofourescence intensity units obtained by imaging analysis of the CGRP-IR, CRLR-IR and RAMP-IR in AU (arbitrary units) within the different plexuses of the sigmoid colon wall.
CGRP CRLR RAMP1
MP eSP iSP MP eSP iSP MP eSP iSP
DD 63± 4,5 12± 0,8 10± 0,9 26± 2,2 13± 1,2 10± 1 10± 0,7 8± 0,8 4± 0,5
Control 113±8,3 15± 1,4 12± 1,2 16± 1,3 7± 0,8 8± 1 11± 0,9 6± 0,7 3,75± 0,4
The mean fluorescence intensity units of CGRP-IR in the myenteric plexus is significantly decreased in DD samples in comparison to control patients (p<0,0001). The decrease was almost twofold in magnitude in the myenteric plexus. There was also an observable decrease of CGRP-IR signal in both submucosal plexuses, but we have found no significant statistical differences between the groups. There was a clear observable trend of DD values being lower than control values (Table 4).
We have observed that in the myenteric plexus the CGPR-IR signal was up to 6-10 greater than that of submucosal plexus. External submucosal plexus did not differ from the internal submucosal plexus when it came to CGRP-IR fluorescence intensity (Table 4).
There is a statistically significant increase in the CRLR-IR mean fluorescence intensity in the myenteric and external submucosal plexuses in DD samples (Fig. 7). No statistically significant difference is found between the plexuses of the DD and control samples with regard to RAMP1-IR mean fluorescence intensity (Fig. 8). In all barcharts there is a clear trend of CRLR-IR and RAMP1-IR being most abundant in the myenteric plexus (Fig. 7-8).
Fig. 6 Bar chart illustrating the mean fluorescence intensity of CGRP-IR in the enteric plexuses, comparing DD and control samples. Note the gap on the y axis.
Fig. 7-8 Bar charts illustrating the mean fluorescence intensity of CRLR-IR and RAMP1-IR in the enteric plexuses, comparing DD and control samples. Note the gap on the y axis.
When comparing the fluorescence intensity of CGRP-IR, CRLR-IR and RAMP1-IR in myenteric plexus,
we observed CGRP-IR signal to be 2-3 fold greater than the CRLR-IR signal and 6 fold greater than the RAMP1-IR signal in the diverticular group. While in the control group the CGRP-IR signal was greater than 6 fold when comparing to both CRLR and RAMP1 signal (Fig 8-10).
There were no significant differences between CGRP-IR, CRLR-IR and RAMP1-IR in the internal submucosal plexuses, except for RAMP1 signal was 2-3 times decreased when compared to the CGRP-IR and CRLR-IR signal in the respective plexuses (Table 4).
CGRP and nitrergic system.
We hypothesized about the potential role of CGRP in the sigmoid colon. It is known that the activation of CGRP receptor can result in the activation of nitric oxide synthase with subsequent release of nitric oxide. Thus we investigated the relationship between CGRP signalling pathway and gut nitrergic system in the enteric plexuses.
CGRP-IR signal was found most abundantly surrounding the nitrergic cell bodies in a encapsulating manner, it was also scattered close to nitrergic nerve fibers but with weaker signal. Meanwhile CRLR-IR and RAMP1-CRLR-IR where observed as multiple round clusters close to and within the nitrergic cell bodies (Fig. 9).
Fig. 9. Microscopical image of the myenteric plexus with CGRP-IR, CRLR-IR, RAMP1-IR, nNOS-IR and DAPI-IR staining in the sigmoid. Arrows and arrow heads showing nitrergic cell bodies. Dotted lines encircling the CRLR and RAMP1 richly stained areas. This image shows the clear association between CGRP-IR, CRLR-IR, RAMP1-IR and nNOS-IR. nNOS: neuronal nitric oxide synthase. CGRP: Calcitonin gene related peptide. CRLR: Calcitonin receptor like receptor RAMP1: Receptor activity modifying protein 1.DAPI: diamidino phenylindole.
9. DISCUSSION
Previous studies have been made about both enteric neuronal plexuses morphology changes, and certain neuropeptides and neurotransmitter with regard to the pathogenesis of DD [5, 21-22].
This is the first histochemical study to demonstrate the location and abundance of CGRP and its receptors CRLR and RAMP1 in the colon of DD patients. Thus this can pave way for a better understanding if this neuropeptide and its receptors has a role in the formation of colonic diverticula and its complications and potentially their involvement in other colonic diseases.
CGRP was of particular interest due to its known location in the colon, its physiological effects in different tissues [6, 48]. Bearing in mind that CGRP has a role in migraine pathogenesis, CGRP is released from the afferent nerve fibers in the trigeminovascular system leading to subsequent neurogenic inflammation, in which CGRP has the active role of vasodilation and Substance P causes increased permeability capillaries [6]. More so, CGRP antagonist medication are used for the treatment of migraine, such as Olcegepant and Telcegepant which have been proved in recent years to be successful and safe [36]. Therefore, we found it reasonable to hypothesize that a similar mechanism could be taking place in the colon worthy of further investigation.
It is evident from this study that CGRP-IR is decreased in the colon of DD patients while surprisingly one of its receptors CRLR-IR is increased and RAMP1-IR exhibits no differences when compared to control samples. The reduction of CGRP in these colons could be explained by decreased innervation due to aging [49]. On the other hand previous studies which also utilized immunhistochemistry on the colon of diverticular diseased samples, demonstrated no significant differences in the number of ganglionic cells in the myenteric plexus with regard to aging [21]. Thus there could potentially be a correlation between diverticular formation and its complications due to decreased amounts of CGRP and decline in its physiological effects.
The unexpected upregulation of CRLR is yet to be understood. One could assume that the increase in CRLR signal is due to a compensatory mechanism which is activated by decreasing levels of CGRP in DD. By a feedback mechanism the CRLR receptor could be produced to counteract the loss of CGRP found in DD. In other studies similar mechanisms have been explained with cholinergic innervation with regard to DD colons [50], In that study choline acetyltransferase IR nerve fibers where found to be decreased while upregulation of muscarinic M3 receptors where found in smooth muscles.
in the myenteric plexus than that of the submucosal plexuses could be due to both structural and functional differences between the enteric plexuses.
Previous studies have demonstrated altered levels of NOS in uncomplicated diverticular disease which led to relaxation of smooth muscles [19], thus we hypothesize CGRP signaling of counteracting the alternating levels of NOS. From animal studies it is known that CGRP relaxes smooth muscles [51]. Thus with the current results maintained in this study there is fair reason to assume that decreased levels of CGRP in DD patients may contribute to the reduced relaxation response. Furthermore, with double labeling for CRLR-NOS1 we have observed that some neurons that possessed CRLR-IR where not imunoreactive to NOS (Fig. 14). The mechanism behind this finding is still left unclear,
CGRP has been proven to stimulate spontaneous phasic contractions in guinea pig intestines, specifically the smooth muscles of the ileum [42]. Another study has demonstrated that these contractions are affected in DD patients [20]. Thus one could speculate that decreased levels of CGRP could potentially have a role in this. Therefore, it would be useful to further investigate the neurochemical profiles of neurons.
Due to the fact that both CRLR and RAMP1 are needed to form the heterodimeric receptor for CGRP, it could be speculated that an upregulation of RAMP1 could result in an increase in reactivity to CGRP. Therefore, we investigated the RAMP1 expression in DD patients. With the result of RAMP1 being unchanged it could potentially mean that the CRLR receptor is affected in a compensatory manner. Another possible explanation behind this could be that because CRLR is a functional receptor for both CGRP and adrenomedullin 1 and 2 depending on the variant of RAMP protein forming the receptor [6], thus it is fair to assume that an increase in CRLR could result in increased sensitivity to Adrenomedullin. That is known to have similar effects to CGRP.
If to assume that CGRP has the same effect on the colon in terms of muscle contractility and colonic motility as it has on other parts of the gastrointestinal tract [41], it would be useful to correlate this study with other studies that show CGRP to mediate smooth muscle relaxation and decreased peristalsis in the gastrointestinal tract [41]. Thus if CGRP was to be decreased as demonstrated this case, it could potentially lead to the long standing increased intraluminal pressure in the colon with subsequent herniation which is evident in DD.
If to assume that DD is a multifactorial disease, CGRP could possibly potentiate or exacerbate other factors as well, simply by being deficient it would increase the intraluminal pressure, the submucosa
would exhibit less compliance, thus the colon wall would become weaker and more prone to herniation superimposed by several other evident mechanisms involving vascular penetration and increased collagen cross linking which increased with aging and also weakens the colon wall [14-15].
Now that we have investigated CGRP, CRLR and RAMP1 in the intrinsic plexuses of the colons of DD as well as control patients, the results have given us a better understand of their location and role with regard to DD. Thus we have maybe come one step closer to understanding the pathogenesis of DD. For a more complete approach we recommend that further research would focus on adrenomedullin and adrenomedullin 2 with their corresponding receptors RAMP2 and RAMP3 [6]. Other mediators of interest would be those that take part in neurogenic inflammation such as Substance P, neurokinin A and neuropeptide Y. They could possibly have some important effect worthy of investigating that could have an impact on DD pathogenesis [34].
10. CONCLUSIONS
1) CGRP was mainly situated surrounding the cell bodies of the ganglia within the plexuses, alike CRLR and RAMP1 which were found within the cell bodies of the ganglia;
2) CGRP IR signal has been demonstrated to be decreased, CRLR increased and RAMP1 unchanged in patients with DD in comparison to control patients;
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