Histochem Cell Biol (2013) 140:157–167 DOI 10.1007/s00418-013-1078-9
ORIGINAL PAPER
Substance P- and choline acetyltransferase immunoreactivities in somatostatin-containing, human submucosal neurons Jakob Beyer • Samir Jabari • Tilman T. Rau Winfried Neuhuber • Axel Brehmer
•
Accepted: 10 January 2013 / Published online: 30 January 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract The submucous layers of human small and large intestines contain at least two separate neuron populations. Besides morphological features, they differ in their immunoreactivities for calretinin (CALR) and somatostatin (SOM), respectively. In this study, submucosal wholemounts of 23 patients or body donors (including all segments of small intestine and colon) were immunohistochemically quadruple stained for CALR and SOM as well as for substance P (SP) and choline acetyltransferase (ChAT). We found that all SOM-positive neurons co-stained for ChAT and the majority for SP [between 50 % in the small intestinal external submucosal plexus (ESP) and 75 % in the colonic ESP]. In contrast, a majority of CALR-neurons contained ChAT (between 77 % in the small intestinal ESP and 92 % in the large intestinal ESP) whereas less than 4 % of CALR-neurons were co-immunoreactive for SP. Another set of wholemounts was costained for peripherin, a marker enabling morphological analysis. Where identifiable, both SOM alone- and SOM/ SP-neurons displayed a uniaxonal (supposed pseudouniaxonal) morphology. We suggest that the chemical code of SOM-immunoreactive, human submucosal neurons may be ‘‘ChAT?/SOM?/SP±’’. In additional sections double stained for SOM and SP, we regularly found doublelabelled nerve fibres only in the mucosa. In contrast, around submucosal arteries mostly SOM alone- fibres were found and the muscularis propria contained numerous J. Beyer S. Jabari W. Neuhuber A. Brehmer (&) Institute of Anatomy I, University of Erlangen-Nuremberg, Krankenhausstraße 9, 91054 Erlangen, Germany e-mail:
[email protected] T. T. Rau Institute of Pathology, University of Erlangen-Nuremberg, Krankenhausstraße 8-10, 91054 Erlangen, Germany
SP-alone fibres. We conclude that the main target of submucosal SOM(/SP)-neurons may be the mucosa. Due to their morpho-chemical similarity to human myenteric type II neurons, we further suggest that one function of human submucosal SOM-neurons may be a primary afferent one. Keywords Enteric nervous system Gut Innervation Mucosa Submucosal plexus
Introduction Morphochemical identification of enteric neuronal subpopulations is a precondition for understanding both normal functions and pathological changes of the enteric nervous system (ENS). During the last three decades, this identification has been supplemented by co-application of several further (e.g. physiological, pharmacological) methods resulting in a comparatively complete understanding of enteric neuronal circuits and functions in the guinea pig, the major model species for ENS studies (Furness and Costa 1987; Costa et al. 1996; Furness 2006). Since species differences hamper direct transfer of data from animals to human, it is a prime challenge in human neurogastroenterology to establish a classification system that discriminates neuron populations as a basis for their further functional and pathophysiological characterisation. Recently, we analysed immunoreactivities for calretinin (CALR) and somatostatin (SOM) in human myenteric and submucosal nerve plexus. Whereas in the myenteric plexus, co-localisation of both substances regularly labels morphologically defined type II neurons (Brehmer et al. 2004b; Weidmann et al. 2007), in the two submucosal plexus, the external (ESP) and the internal (ISP) submucosal plexus (Brehmer et al. 2010), co-localisation of
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CALR and SOM is a rare exception as both substances label morphologically different neuron populations (Kustermann et al. 2011). In the guinea pig, myenteric and submucosal type II neurons act as intrinsic primary afferent neurons (IPANs; Brookes 2001; Furness 2006). Besides calbindin, a speciesspecific marker for IPANs in the guinea pig, they contain choline acetyltransferase (ChAT), tachykinins and other substances (Furness 2006). Both ChAT and substance P (SP; one member of the tachykinin family) were also found in human myenteric type II neurons. In the present study, we tested potential co-immunoreactivities for ChAT and SP in the two human submucosal neuron populations originally identified by their reactivities for CALR or SOM. For this purpose, we applied quadruple immunohistochemistry to human submucosal wholemounts and double immunolabelling to cryostat sections.
Materials and methods Tissue handling The use of human tissues was approved by the Ethics Committee of the University of Erlangen-Nuremberg. We used 23 samples derived from 21 tumour patients (only tissue gained from the non-tumour-infiltrated borders of the resected gut segments was used) and 2 body donors of the Institute of Anatomy (post mortem delay less than 6 h). The median age was 66 years (youngest 29, eldest 87; 14 females, 9 males). Intestinal segments were transported in physiological saline (pH 7.3) on ice to the laboratory. Upon arrival (in case of tumour patients up to 6 h after surgical resection), specimens were rinsed in Krebs solution at room
temperature and transferred to Dulbecco’s modified Eagle’s medium (DMEM/F12-Ham, Sigma Chemical Company, St. Louis, MO, USA) containing 10 mg/ml antibiotic–antimycotic (Sigma), 50 lg/ml gentamycin (Sigma), 2.5 lg/ml amphotericin B (Sigma), 10 % fetal bovine serum (Sigma), 4 lM nicardipine and 2.1 mg/ml NaHCO3, bubbled with 95 % O2 and 5 % CO2 at 37 °C for 1–2 h. For fixation, samples were divided. The larger pieces (dedicated for wholemount preparation) were pinned on a Sylgard-lined Petri dish and transferred to 4 % formalin in 0.1 M phosphate buffer (PB, pH 7.4) at room temperature for 2–4 h. The smaller pieces (dedicated for sections) were frozen at -70 °C in methylbutan after cryoprotection with 15 % sucrose in 0.1 M PB. For the following immunohistochemical incubations, two submucosal wholemounts (about 1 9 1.5 cm) together with two smaller pieces of mucosal wholemounts as well as cryostat sections parallel to the gut longitudinal axis were prepared from each. Additional wholemount material was used for negative controls and preabsorption tests (see below). Immunohistochemistry The two submucosal and mucosal wholemounts prepared from each sample were incubated differently applying antibodies listed in Table 1. One set (submucosa ? mucosa; for quantitative analysis) was quadruple stained for SOM, SP, ChAT and CALR, the other set (morphological analysis) for PER, SOM, SP and CALR. Sections were double stained for SOM and SP. Incubations included the following steps: preincubation of wholemounts for 2 h (sections 1 h) in 0.05 M TBS (pH 7.4) containing 1 % bovine serum albumin (BSA), 0.5 % Triton X-100, 0.05 %
Table 1 Antisera Antigen
Host
Dilution
Source
Calretinin (CALR)
Rabbit
1:500
7699/3H; Swant, Switzerland
Choline acetyl-transferase (ChAT) Peripherin (PER)
Goat Goat
1:40 1:200
AB144P; Millipore, Germany sc-7604; Santa Cruz, Germany
Primary antisera
Somatostatin (SOM)
Rat
1:200
sc-47706; Santa Cruz, Germany
Substance P (SP)
Guinea pig
1:200
NB 300-187; Novus Biologicals, CO/USA
CyTM 3
Donkey anti-rat
1:500
712-165-153; Dianova, Germany
CyTM 2
Donkey anti-guinea pig
1:200
706-225-148; Dianova, Germany
Secondary antisera
ALEXA Fluor 647
Donkey anti-goat
1:1,000
A-21447; Mobitec, Germany
DyLightTM 405
Donkey anti-rabbit
1:200
711-475-152; Dianova, Germany
ALEXA Fluor 488
Donkey anti-goat
1:1,000
A-11055; Mobitec, Germany
DyLightTM 647
Donkey anti-guinea pig
1:500
706-495-148; Dianova, Germany
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thimerosal and 5 % normal donkey serum. After rinsing in TBS for 10 min, the wholemounts/sections were incubated in a solution containing BSA, Triton X-100, thimerosal (see above) and the primary antibodies (Table 2) for 72 h (4 °C; sections overnight). After an overnight rinse in TBS at 4 °C, specimens were incubated in the same solution as for the primary antibodies but with secondary instead of primary antibodies (Table 2; 4 h; room temperature; sections 1 h) followed by a rinse in TBS (overnight; 4 °C). In all specimens, we applied a lipofuscin reduction protocol: incubation in ammonium acetate buffer (pH 5.0) containing 1 mM CuSO4 for 120 min followed by a short rinse in distilled H2O (Schnell et al. 1999; Brehmer et al. 2004a) although, as described earlier, this was not as affective as in myenteric wholemounts. As mentioned earlier, only material was included in this study which did not display neuronal autofluorescence different from lipofuscin pattern and present without any kind of staining (Kustermann et al. 2011). Thereafter, specimens were mounted with TBS–glycerol (1:1; pH 8.6). Submucosal wholemounts were first mounted with their mucosal side up. After evaluation of the ISP, wholemounts were reversed and again mounted with the outer, abluminal side up for analysis of the ESP. Mucosal wholemounts were mounted with their abluminal side up, for analysis of the basal part of the mucosal plexus. Negative controls, preabsorption tests Negative controls for antibodies used in this study (omission of primary antibodies from incubation protocol as
described above) were carried out earlier (Brehmer et al. 2005; Beck et al. 2009; Kustermann et al. 2011). Preabsorption tests for antibodies against CALR, PER and SOM were described in a previous study (Kustermann et al. 2011). In this study, we tested the specificity of the antibodies ChAT (antigen: choline acetyltransferase recombinant protein AG220; Millipore, Germany) and SP (antigen: substance P peptide ab38217; Abcam, Germany). Preabsorptions with twofold excess of ChAT antigen (Fig. 1a) and with two- and fivefold excess of SP-antigen (Fig. 1b, c), respectively, were performed overnight at 4 °C. The antigen–antibody mixtures were spun at 20,000g for 20 min to sediment precipitating antigen–antibody complexes and avoid high background staining. The supernatants were then used in place of the primary antibodies, respectively. Image acquisition, quantification Wholemounts were evaluated using a confocal laser scanning microscope system (Nikon Eclipse E1000-M; Nikon Digital Eclipse C1; Tokyo, Japan) equipped with a quadruple laser configuration: 405 nm diode laser (coded blue in the figures), 488 nm argon laser (green), 543 nm helium–neon laser (red), 638 nm diode laser (yellow). The two diode laser originated from Coherent (Santa Clara, CA/ USA), the other two from Melles Griot Inc. (Carlsbad, CA/ USA). For reduction of unspecific background fluorescence, a BIO1-Filterset (DAPI/Cy5 for C1-Detector; AHF Analysentechnik, Tu¨bingen, Germany) was added. A 20 9 dry objective lens (numerical aperture 0.75) was
Table 2 Proportions of internal submucosal neurons Internal submucosal plexus Duodenum
CALR only (%) 2
ChAT CALR SP (%)
ChAT CALR (%)
ChAT CALR SOM SP (%)
ChAT SOM (%)
ChAT SOM SP (%)
ChAT SP (%)
ChAT only (%)
0
10
0
21
53
0
14
Neuron number n = patients 840 n=4
Jejunum
0
0
18
0
21
44
2
16
699 n=3
Ileum
16
2
29
4
13
27
2
6
5
0
65
0
10
16
1
3
817 n=3
Colon ascendens
902 n=3
Colon transversum
12
1
51
0
5
23
2
5
Colon descendens
20
0
52
0
8
12
2
6
Colon sigmoideum
8
892 n=3 1095 n=4
0
62
0
7
16
1
5
765 n=3
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about 1 mm2. Only neurons lying within the honeycomblike meshes (see below) of the mucosal plexus were considered (Kramer et al. 2011). We tried to carefully discriminate neurons lying at the same x-, y-position but at different z-positions to avoid false positive recordings of neurons (e.g. one double stained instead of two single-stained, overlapping neurons).
Results In line with our previous study (Kustermann et al. 2011) and apart from very rare exceptions, CALR and SOM labelled different submucosal neuron populations. Results of counts in the two submucosal plexus are listed in Table 2 (ISP) and Table 3 (ESP) for each segment of the small and large intestine, respectively, and illustrated in aggregate in Fig. 2. Submucosal, SOM-positive neurons (SOM-neurons): ChAT and SP co-reactivities
Fig. 1 Preabsorption controls for antibodies against choline acetyltransferase (ChAT, yellow; a) and substance P (SP, green; b, c), counterstained for somatostatin (SOM, red) and depicted as single optical sections through submucosal ganglia (left side merged images; right side ChAT and SP images, respectively). Incubations using the supernatants after preabsorption with twofold antigen (ag) excess resulted in no staining for ChAT (a0 ). Whereas incubations using the supernatants after preabsorption with twofold antigen excess resulted in weak neuronal labelling for SP (b0 ), incubation after fivefold antigen excess resulted in no staining (c0 )
applied. Z-series dedicated for quantitative analysis were created using a zoom factor of 2.0 in all sessions, z-steps were 2 lM. The figures were prepared using Volocity Demo 6.1.1, Adobe Photoshop CS4 and CorelDRAW X6. In submucosal wholemounts, each 15 ganglia or single neurons lying outside of ganglia in interganglionic nerve strands were selected randomly in a meander-like fashion, first from the inner, mucosal side of the wholemount preparation (ISP), thereafter from the outer side of the wholemount (ESP). All counts were carried out on z-series of the ganglia, using the Nikon FreeViewer software (EZ-C1 3.30) and Volocity Demo 6.1.1. In mucosal wholemounts, each 10 z-series of consecutive viewfields (meander-like selection) of the mucosal plexus were recorded. These 10 viewfields corresponded to an area of
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All SOM-neurons co-stained distinctly, sometimes strongly for ChAT (Fig. 3). A considerable portion of SOM/ChATneurons co-stained for SP: about half in the ESP of the small intestine (21 % SOM/SP-neurons vs. 22 % SOMonly neurons), a majority each in the small intestinal ISP (42 vs. 18 %) and in the large intestine (ESP: 9 vs. 3 %; ISP: 17 vs. 8 %). Thus, between about 50 % (ESP, small intestine) and 75 % (ESP, large intestine) of all SOM/ ChAT-neurons co-stained for SP. In PER-co-stained specimens, morphological features of neurons could only be evaluated in small ganglia, single neurons or neurons lying at the margins of larger ganglia. If visible, both SOM/SP-neurons and SOM-only neurons displayed smooth somal surfaces and a single, long process (Fig. 4). Submucosal, CALR-positive neurons (CALR-neurons): ChAT co-reactivity Most CALR-neurons co-stained for ChAT although, with considerably different intensity (Figs. 3, 5). In the large intestine, CALR/ChAT-neurons were the largest population observed (ESP: 76 %, ISP: 57 %). In the small intestine, they ranged between 19 % (ISP) and 30 % (ESP). In a substantial proportion of CALR-neurons, ChAT staining could not be observed (between 6 and 12 % of all stained neurons, Fig. 5). Taken all CALR-neurons as 100 %, the relation of ChAT-positive to ChAT-negative CALR-neurons ranged between 77.5 and 22.5 % in the small intestinal ESP and 92.7 to 7.3 % in the large intestinal ESP (Fig. 6).
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Table 3 Proportions of external submucosal neurons External submucosal plexus Duodenum
CALR only (%) 0
ChAT CALR SP (%)
ChAT CALR (%)
ChAT CALR SOM SP (%)
ChAT SOM (%)
ChAT SOM SP (%)
ChAT SP (%)
ChAT only (%)
0
18
0
36
20
1
24
Neuron number n = patients 320 n=4
Jejunum Ileum
2
2
22
0
27
31
2
14
20
1
42
1
9
17
4
7
258 n=3 457 n=3
Colon ascendens
2
0
84
0
4
9
1
1
322 n=3
Colon transversum
10
1
71
0
1
11
4
3
Colon descendens
8
0
71
0
3
8
3
7
Colon sigmoideum
5
352 n=3 575 n=4
0
78
0
2
10
1
4
417 n=3
Fig. 2 Proportions of neurons stained for the markers used in this study and their combinations observed. Percentages are related to the total number of stained neurons. Since no general neuronal marker has been applied, a small proportion of submucosal neurons may have been unstained. To illustrate this supposed population of uncoloured neurons, an overall segment of 5 % has been reserved (‘‘?’’) and the percentages were marked with ‘‘\’’
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Fig. 3 Immunoreactive versus non-reactive submucosal neurons (filled vs. empty markings: arrows indicate somatostatin (SOM)positive, arrowheads indicate calretinin (CALR)-positive neurons): SOM, red; substance P (SP), green; choline acetyltransferase (ChAT), yellow; CALR, blue. In a, two neurons contain SOM, one of them SP (a0 ), both contain ChAT (a00 ) but none of them CALR (a000 ).
In contrast, the CALR-positive neuron in a000 contains ChAT (a00 ) but neither SOM nor SP (a, a0 ). All-in-focus-projections; sample data: a external submucosal plexus (ESP) of jejunum, 87 years, female; b ESP of transverse colon, 76 years, female; c internal submucosal plexus of sigmoid colon, 72 years, male
Co-immunoreactivity for SP was detected only exceptionally (in 0–1 % of all stained neurons). Morphologically, in PER-co-stained specimens, CALR-neurons displayed several processes (Fig. 4).
between 1 % (ISP of small and large intestines) and 3 % (ESP of small intestine).
Submucosal, nonSOM-/nonCALR-immunoreactive neurons
The results of counts and the proportions are listed in Table 5. ChAT/CALR/SP co-reactivity, occurring in less than 1 % of submucosal neurons, amounted up to 60 % in colonic mucosal neurons whereas ChAT/CALR/SOM/SPneurons amounted to 10 % (in two colonic segments) or even 20 % (in the ileal specimens).
All nonSOM/nonCALR-neurons stained in our material were ChAT reactive. Their proportions ranged between 6 % in both plexus of the large intestine and 17 % in the small intestinal ESP (Table 4). The major proportion of nonCALR/nonSOM-neurons were positive for ChAT alone. These neurons amounted between 9 % (large intestine, ESP) and 17 % (large intestine ISP) of all neurons observed. The smaller portion co-stained for SP,
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Mucosal neurons
Distribution pattern of nerve fibres Nerve fibres co-reactive for SOM and SP were found regularly only in the mucosal layers (Fig. 7a). Submucosal
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Fig. 4 Morphology (demonstrated by immunoreactivity for peripherin, PER) of submucosal neurons co-reactive for somatostatin (SOM) and substance P (SP). In a, a neuron co-reactive for PER, SOM and SP (a–a00 ) displays one, single process (arrow). It is negative for CALR, in contrast to the arrowheaded neuron in (a000 ) which, in turn, is negative for both SOM and SP and only weakly positive for PER (arrowheads). In b, one PER-stained neuron displays a single process
(arrow), its soma is co-reactive for SOM and SP (b0 , b00 ). The other neuron (arrowhead) is co-reactive for calretinin (CALR in b000 ), negative for both SOM and SP (b0 , b00 ) and displays several, partly short processes. All-in-focus-projections; sample data: a internal submucosal plexus of duodenum, 70 years, female; b external submucosal plexus of sigmoid colon, 65 years, female
vessels (mainly arteries) were frequently surrounded by SOM alone- nerve fibres, only occasionally we found scattered SOM/SP-co-labelled nerve fibres (Fig. 7b). In the two external muscle layers, abundant SP-reactive nerve fibres were found, without co-staining for SOM (Fig. 7c).
have not used a general, pan-neuronal marker in the present investigation. The slightly lower proportions of nonCALR/ nonSOM-neurons of the present study as compared to those of Kustermann et al. (2011) indicate that there may be a small number of neurons not stained here. Therefore, we illustrated this potential group of neurons within the circle diagrams of Fig. 2 by adding a segment marked with ‘‘?’’. Furthermore, the percentages in these diagrams were marked with ‘‘\’’ to indicate that their real proportions as related to the whole submucosal populations may be slightly lower. The identity of nonCALR/nonSOM-neurons is presently not clear. This study showed that a subpopulation of them contains ChAT (between 4 and 14 % of neurons labelled in this study), a smaller portion ChAT and SP (between 1 and 3 %). Anyway, results of both studies are in line with each other as these as yet unidentified neurons are more numerous in the small than in the large intestine.
Discussion In contrast to CALR and SOM which were suggested to label two separate submucosal neuron populations (Kustermann et al. 2011), neither ChAT nor SP appear to do so (this study). Whereas ChAT immunoreactivity seems to be present in the great majority of human submucosal neurons, SP was mainly found in a majority of SOM-neurons. The general proportions of CALR- and SOM-neurons as related to the whole submucosal neuron populations in small and large intestines estimated in this and our previous study are comparable (Table 4) although, the present percentages are slightly higher than those of Kustermann et al. (2011). This will be discussed first. No pan-neuronal marker In contrast to our previous study applying the human neuronal protein Hu C/D (Kustermann et al. 2011), we
Submucosal, non-dendritic SOM?/ChAT?/SP± neurons: identification Almost all SP-immunoreactive neurons found in this study co-stained for SOM, only between 1 % and 4 % of SPneurons were SOM negative. However, there are a substantial proportion of SOM-neurons non-reactive for SP
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Fig. 5 Immunoreactivity for choline acetyltransferase (ChAT) in submucosal neurons (filled vs. empty markings: arrows indicate three somatostatin (SOM)-positive neurons, arrowheads indicate 3 selected calretinin (CALR)-positive neurons). Both SOM- and SP- (a, a0 ) and CALR-positive neurons (a000 ) are co-stained for ChAT (a00 ). In contrast (b–b000 ), three SOM-positive neurons (one of them is coreactive for SP, b0 ) are positive for ChAT (b00 ) whereas numerous
CALR-positive neurons (three are marked with arrowheads in b000 ) are non-reactive for ChAT (b00 ). One neuron (asterisk) is ChAT positive (b00 ) but non-reactive for the other three markers. All-infocus-projections; sample data: a internal submucosal plexus of transverse colon, 76 years, female; b external submucosal plexus of descending colon, 53 years, male
Fig. 6 Co-immunoreactivities of somatostatin (SOM)-containing, mucosal neurons. Of the three SOM-stained neurons (filled arrows in a), two are co-reactive for substance P (filled vs. empty arrows in a0 ), all three contain choline acetyltransferase (ChAT, in a00 ) but none
is co-reactive for calretinin (CALR, in a000 ). All-in-focus-projections; sample data: basal part of mucosal plexus of duodenum, 58 years, female
(up to 22 %). Accili et al. (1995) reported on a complete co-localisation of both peptides in human jejunum but these authors did not focus on a quantitative evaluation. Morphologically, both SOM alone- (Kustermann et al. 2011) and SOM/SP-neurons (this study) appear as ‘‘unipolar’’ (suggested non-dendritic/pseudouniaxonal) neurons. Thus, we conclude that there exists a human submucosal neuron population combining morphological (non-
dendritic/supposed pseudouniaxonal) with obligatory (SOM/ChAT co-reactivity) and facultative (SP reactivity) immunohistochemical features. As far as we are aware, only SOM is suitable as type-specific marker for these neurons in the human submucosa. This is in contrast to the myenteric plexus where at least two different neuron types contain SOM (morphological type II neurons and unidentified non-type II neurons; Weidmann et al. 2007).
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Table 4 Comparison of percentage values of neurons immunoreactive for either SOM or CALR with those of neurons negative for both markers Kustermann et al. (2011)
Present study
SOM? or CALR? (%)
SOM- and CALR- (%)
SOM? or CALR? (%)
SOM- and CALR- (%)
Small intestine ISP
61
40
87
13
Small intestine ESP
66
34
83
17
Large intestine ISP
80
20
94
6
Large intestine ESP
81
19
94
6
Table 5 Proportions of mucosal neurons Mucosal Plexus
CALR only (%)
ChAT CALR SP (%)
ChAT CALR (%)
Duodenum
0
0
27
Jejunum
0
0
Ileum
7
0
ChAT CALR SOM SP (%)
ChAT SOM (%)
ChAT SOM SP (%)
ChAT SP (%)
ChAT only (%)
Neuron number n = patients
0
27
46
0
0
11
59
4
27
5
0
5
22 n=2
34
20
16
22
2
6
67
n=3
n=3 Colon ascendens
0
6
82
0
0
0
12
0
Colon transversum
2
42
16
10
3
13
0
16
Colon descendens
0
60
8
10
3
19
0
0
Colon sigmoideum
0
17 n=3 63 n=3 37 n=3
40
60
0
Submucosal, non-dendritic SOM?/ChAT?/SP± neurons: possible functions Recently, we have discussed the possible functional role(s) of the human submucosal SOM-neurons as possible primary afferent (Kustermann et al. 2011), antisecretory (Hens et al. 2001), vasoactive (De Fontgalland et al. 2008) and/or interneurons (Kustermann et al. 2011). In both laboratory animals and human, SP is abundant in both enteric and extrinsic neurons (Christofi et al. 2001; Engel et al. 2011; Gross and Pothoulakis 2007; Holzer and Holzer-Petsche 1997b). To this peptide, which is widely co-localised with SOM in human submucosal neurons (this study), a number of functions have been attributed including stimulation and inhibition of intestinal motor activity (Holzer and Holzer-Petsche 1997a; Ho¨kfelt et al. 2001), involvement in sensory processes including visceral pain and the development and progress of intestinal inflammation (Ho¨kfelt et al. 2001; Harmar 2004), influence on the secretory activity of the intestinal epithelium as well
0
0
0
0
62 n=2
as on (arterial) vasodilation (Holzer and Holzer-Petsche 1997b). In the sections of our present study, numerous nerve fibres co-reactive for SOM and SP have been found (outside the submucosal plexus) only in the mucosa. They may have been derived from the above mentioned, submucosal neurons and from myenteric, multiaxonal type II neurons. The latter have only been analysed in detail in the human small intestine (Brehmer et al. 2004b; Weidmann et al. 2007). However, we suggest that neurons displaying the axonal projection pattern specific for myenteric type II neurons (i.e. both within the myenteric plexus and vertically, to submucosa/mucosa) combined with a chemical code including both SOM and SP, may also be present in the large intestine. SOM-/SP-co-reactive fibres were not observed in the muscularis propria (here only SP-alone fibres, likely derived from myenteric neurons) and only scarcely around arteries (here mostly SOM alone- fibres, maybe at least partly derived from extrinsic sources) (Quartu et al. 1993; Holzer and Holzer-Petsche 1997b).
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Fig. 7 Nerve fibres immunoreactive for somatostatin (SOM, red) and/or substance P (SP, green) in different intestinal layers. Co-immunoreactive nerve fibres are marked with arrows and appear yellow in the right column, nerve fibres reactive for only one marker are indicated with (filled vs. empty) arrowheads. In the mucosa (a), nerve fibres regularly display co-reactivity of SOM and SP. The red spots in the upper half of a and a00 are SOM-containing enteroendocrine cells, on the margin below there is an internal submucosal ganglion. In b, a submucosal artery is well visible due to the
autofluorescent elastica interna (asterisk). Nerve fibres, located at the outside of the media (unstained), were mostly SOM reactive (three arrowheads), only infrequently they were double labelled (arrow). Intramuscular nerve fibres (c) displayed SP but no SOM reactivity (below longitudinal muscle; above circular muscle; in between a myenteric ganglion; across the circular muscle two small external submucosal ganglia). Single optical sections; sample data: a jejunum, 63 years, male; b ascending colon, 77 years, male; c ileum, 50 years, female
Thus, submucosal SOM/ChAT/SP-neurons may have their main innervation target in the mucosa. Due to their morphological (they are non-dendritic neurons) and immunohistochemical similarities (co-reactivities for SOM, SP, ChAT) to myenteric type II neurons, we suggest that at least one of their functions may be a primary afferent one. Interestingly, in chagasic megacolon, SOM-reactive submucosal neurons and mucosal nerve fibres had almost completely disappeared in contrast to CALR-neurons and -fibres (Jabari et al. 2012). Since chagasic patients suffering from megacolon survive for decades, this may indicate that SOM(/SP)-neurons are not of vital importance.
Submucosal, multidendritic CALR-neurons
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This study focused on submucosal neurons co-reactive for ChAT and SP, which were shown to label only subpopulations of CALR-neurons, respectively. While SP coimmunoreactivity was found in less than 1 % of all neurons (this corresponds to less than 4 % of all CALR-neurons), more than 75 % of CALR-neurons were ChAT co-reactive. The functional meaning of this difference in ChAT-staining is presently unknown. The further chemical coding of CALR-neurons including discussion of the possible functional meaning of their striking quantitative preponderance
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in the colonic submucosa will be addressed in a forthcoming study. Mucosal neurons and conclusions Recently (Kramer et al. 2011), we announced to address a parallel morphochemical identification of submucosal and mucosal neurons in order to clarify the issue whether mucosal neurons may be displaced by submucosal neurons or rather represent a separate entity. Besides their nonobligatory presence (discussed by Kramer et al. 2011), the results of the present study argue for the former option. As yet, we found no mucosal neuron morphologically or immunohistochemically strikingly different from submucosal neurons. Although the percentages differ (e.g. ChAT/ CALR/SP-neurons and ChAT/CALR/SOM/SP-neurons were found more frequently in the mucosal than in the two submucosal plexus), the significance of the proportions estimated from the mucosal plexus is restricted due to the much smaller total number of mucosal neurons, as compared to the submucosal plexus. In this study, we have specified the chemical coding of the submucosal (pseudo-)uniaxonal SOM-neurons; we suggest they are SOM?/ChAT?/SP± neurons. Future studies will address the more precise immunohistochemical definition of the CALR-neurons as well as of the remainder, submucosal nonSOM/nonCALR-neurons, which account for up to 40 % in the small and up to 20 % in the large intestine (Kustermann et al. 2011). Acknowledgments The excellent technical assistance of Karin Lo¨schner, Stefanie Link, Anita Hecht, Andrea Hilpert and Hedwig Symowski is gratefully acknowledged.
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