ARTICLE |
Correspondence to: Christa J. Van Ginneken, Lab. of Veterinary Anatomy and Embryology, Slachthuislaan 68, 2060 Antwerp, Belgium..
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Summary |
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Methods that visualize subsets as well as the entire enteric neuron population are not readily available or have proved to be unreliable. Therefore, we attempted to combine NADPH-d histochemistry, AChE histochemistry, and CGRP immunohistochemistry, techniques that mark subsets of enteric neurons, with a technique that appeared to visualize the entire enteric neuron population, the cuprolinic blue staining method. To guarantee representative staining results, the individual staining methods were modified by using microwaves. In addition, this preserved the characteristics of each of the individual techniques. The distribution of NADPH-d, AChE, and CGRP corresponded well with previous morphological and physiological reports. Consequently, the different combinations gave rise to rapid, useful, and ready-to-use double labeling techniques. Their main advantage is that they simultaneously visualize the total population as well as subsets of enteric neurons. (J Histochem Cytochem 47:1321, 1999)
Key Words: enteric nervous system, cuprolinic blue, NADPH-d, acetylcholinesterase, CGRP, microwave, double labeling
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Introduction |
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The enteric nervous system (ENS) is very complex and its total number of neurons is believed to be of the same magnitude as the number of nerve cells in the spinal cord (
A variety of methods that stain the entire enteric neuron population have already been evaluated. However, they have proved to be nonselective, to have low reproducibility, to be not generally available, or to fall short of estimating the total number of enteric neurons (
Cuprolinic blue, an analogue of the well-known phthalocyanin dye Alcian Blue 8GX, has received renewed interest as a marker for the entire enteric neuron population (
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Materials and Methods |
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Tissue Preparation
Animals.
Two fetal pigs (Piétrain x White Large) from the second half of the gestational period were obtained from a local slaughterhouse. The time interval between the death of the sow and the removal of the fetuses was approximately 15 min and, at that moment, the fetuses were already dead. The age of the fetal pigs was estimated by measuring the crownrump length and by converting this into fetal age according to the method described by
Two neonatal pigs (Piétrain x White Large) of respectively 2 days and 4 days were used. These animals were sacrificed by severing the carotid arteries under deep barbiturate anesthesia (sodium pentobarbital).
Preparation. The intestinal tracts were removed through a midline ventral incision and were rinsed with 0.01 M PBS.
Fixation. The intestines were fixed by immersion using 4% freshly prepared phosphate-buffered (0.1 M) paraformaldehyde, pH 7.4 for 2 hr at 4C. The fixative was washed out with PBS over 24 hr. While awaiting further dissection, the intestines were stored at 4C in PBS enriched with 0.01% sodium azide.
Whole-Mount Preparation
Whole-mount preparations were made according to
In short, segments approximately 2 cm in length were cut from the intestine. These segments were cut open along the line of mesenteric attachment. The mucosa was scraped off and the residues of mesentery were removed. Then the slabs were rinsed in PBS. At this stage of the preparation, the slabs could be stored in PBS enriched with 0.01% sodium azide at 4C. The subsequent dissection was performed with watchmaker's forceps and very fine scissors. The submucosal layer, which contained the submucosal plexuses, was separated from the muscle layers and was transferred to PBS. From the muscle layers, the circular muscle layer's muscle bundles were peeled off. The remaining slab contained the serosal layer and the longitudinal muscle layer bearing the myenteric plexus and was placed in PBS.
Microwave Applications
Most of the subsequent incubations were performed in a 2,450-Hz 900-W Bosch microwave oven with multiple energy levels and a rotating plate (type HMT 862L/02; Bosch, Berlin, Germany), a microwave oven for domestic use.
All the parameters, i.e., surrounding water volume (SWV), fixed position, energy level, and time of irradiation, were experimentally set in function of the critical temperature of 45C of an incubation medium with a volume (IM) of 1 or 5 ml (
The standardization of the parameters resulted in the following set-up ( in case of Eppendorf tubes 10 cm;
in case of glass vials 15 cm), that was filled with 150 ml tapwater (SWV) on the rotating plate of the microwave. The Eppendorf tubes were situated 2.5 cm outside the center of the rotating plate and the glass vials 4 cm outside its center. This set-up is further referred to in the text as "standardized conditions."
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Nicotinamide Adenine Dinucleotidephosphate-Diaphorase (NADPH-d) Histochemistry
The whole-mount preparations were placed in glass vials filled with aNADPH-d incubation medium containing 10 mg NADPH (Serva; Polylab, Antwerp, Belgium), 1.25 mg nitroblue tetrazolium (NBT) (Sigma Aldrich; Bornem, Belgium) (50 µl stock solution 2.5% NBT, dissolved in a solution of methanol:dimethylformamide 1:1) and 50 µl stock solution 10% DMSO (Across Chimica; Beerse, Belgium) in 5 ml PBS containing 0.3% Triton X-100 (Merck Belgalabo; Overijse, Belgium). Once the open glass vials, filled with the NADPH-d incubation medium, were correctly placed under the standardized conditions, an irradiation burst of 90 W was given for 5 min. This burst was followed by a prolonged incubation for 10 min in the warm SWV. Afterwards, the whole-mount preparations were rinsed four times for 5 min each in PBS.
Acetylcholinesterase (AChE) Histochemistry
The protocol used, was based on the protocols of
Three stock solutions were made. Stock Solution A was made under continuous stirring and controlled pH. A starting volume of 800 ml distilled water was used. To this starting volume other reagents are added. First, 10 ml Triton X-100 (Merck Belgalabo) was added. Subsequently, 3.28 g sodium acetate anhydride was dissolved in the starting volume. Afterwards, 100% acetic acid was added until the pH reached 5.3. To increase the pH to 5.6, 1.468 g sodium citrate.2 H2O was dissolved in the starting volume. By adding dropwise 0.506 g CuSO4 anhydride, previously dissolved in 30 ml distilled water, the pH decreased to 5.31. Afterwards, 0.171 g Iso-OMPA (tetra-iso-propyl-pyro-phosphor-amide) (Koch-Light Lab. Interlaboratoire; Brussels, Belgium) was dissolved. Finally, the starting volume was adjusted to 1.000 liter with distilled water (pH 5.4). Stock Solution A could be stored for 1 year at 4C. Stock-Solution B consisted of a 5% S-acetylthiocholine iodide (Sigma Aldrich) solution in distilled water and remained stable for 1 month when stored at 4C. Stock Solution C consisted of a saturated (33%) K3Fe(CN)6 solution in distilled water and remained stable for 1 week when stored at 4C. After a rinse for 5 min in distilled water, the tissues were rinsed twice for 5 min each in 0.05 M sodium acetate buffer (pH 5.2). Then the whole-mount preparations were placed for 5 min in stock Solution A. Meanwhile, the incubation medium was prepared as follows: 5 ml stock Solution A, 50 µl stock Solution B, and 50 µl stock Solution C. The whole-mount preparations were brought into the glass vials filled with 5 ml of incubation medium. Once the open vessels were correctly placed under the standardized conditions, an irradiation burst of 90 W was given for 5 min. This burst was followed by standing for 15 min in the warm SWV, during which the IM temperature stabilized at room temperature (RT). Afterwards a second burst of 90 W for 5 min was given. Finally, the reaction was stopped by transferring the tissues for 5 min to 0.05 M sodium acetate buffer (pH 5.2), followed by a rinse in distilled water for 5 min.
Cuprolinic Blue Staining
The protocol used was based on the protocol described by
The whole-mount preparations were rinsed three times for 5 min each in distilled water, followed by rinsing twice for 5 min each in 0.05 M sodium acetate buffer (pH 5.6). Then, the whole-mount preparations were incubated in 5 ml of 0.05 M sodium acetate buffer (pH 5.6) enriched with 100 µl of a saturated (33% in distilled water) K3Fe(CN)6 solution for 30 min at RT. After triple rinses in distilled water for 1 min each, the whole-mount preparations were placed in 5 ml of 0.05 M sodium acetate buffer (pH 5.6) enriched with 1 M MgCl2, Triton X-100 (final concentration 0.3%) (Merck Belgalabo), and DMSO (final concentration 0.1%) (Across Chimica) for 10 min at RT. Subsequently, the whole-mount preparations were blotted onto filter paper. From this moment on, direct contact between metal tools and the 0.5% cuprolinic blue incubation medium was avoided. This 0.5% incubation medium consisted of 20 ml 0.05 M sodium acetate buffer (pH 5.6), 0.1 g quinolinic phthalocyanin (cat. no. 17052; Polysciences, Warrington, PA), and 4.07 g MgCl2 (final pH becomes 4.6). It could be stored for several months at 4C as 1-ml portions in Eppendorf tubes or as 5-ml portions in glass vials and could be reused several times. The whole-mount preparations were placed in these vessels (Eppendorf tubes or glass vials) for the incubation. The open vessels were correctly placed under the standardized conditions and an irradiation burst of 90 W was given for 5 min. This burst was followed by standing for 15 min in the warm SWV. Differentiation was carried out by transferring the tissues to 0.05 M sodium acetate buffer (pH 5.6), enriched with 1 M MgCl2 for 2 min. Finally, the whole-mount preparations were rinsed three times for 5 min each in distilled water.
Calcitonin Gene-related peptide (CGRP) Immunohistochemistry
Polyclonal CGRP antiserum raised in rabbits was purchased from Amersham (Gent, Belgium). All other immunoreagents were obtained from Dako (Glostrup, Denmark): normal swine serum (X0901 065), biotinylated F(ab')2 fragment of swine anti-rabbit serum (E0431), and peroxidase-conjugated streptavidin (P0397).
After three rinses in PBS for 5 min each, the whole-mount preparations were incubated in 3% H2O2 in PBS for 30 min to block endogenous peroxidase. After four rinses in PBS for 5 min each, nonspecific tissue reactivity was blocked with 5% normal swine serum (Dako) in PBS containing 1% Triton X-100 and 5% nonfat dry milk powder (Nestle). Once the open vessels that contained the whole-mount preparations and the incubation medium were correctly placed under the standardized conditions, an irradiation burst of 90 W was given for 5 min. After standing for 15 min in the warm SWV, the whole-mount preparations were blotted on filter paper. Next, the tissues were incubated in the different antisera. The polyclonal primary antiserum against CGRP (dilution 1:400), the secondary swine anti-rabbit biotinylated antiserum (dilution 1:200), and the tertiary peroxidase-conjugated streptavidin antiserum (dilution 1:200) were diluted in PBS containing 1% Triton X-100, 5% normal swine serum, and 5% nonfat dry milk powder. The incubations in the different antisera consisted of three irradiation bursts of 90 W for 5 min each under the standardized conditions. After each burst, the whole-mount preparations were left to incubate for 15 min in the warm SWV. Before another irradiation burst was given, the whole-mount preparations were slightly stirred in the tubes to improve homogeneous penetration of the medium and the SWV was replaced with fresh SWV RT. After the third incubation in the warm SWV, antisera were removed by rinsing six times for 5 min each in PBS, followed by blotting of the whole-mount preparations on filter paper. After removal of the tertiary antiserum with PBS, the whole-mount preparations were rinsed twice for 5 min each in 0.05 M sodium acetate buffer, pH 5.2. Subsequently, the sites of antibody binding were visualized with a 0.05% AEC (aminoethylcarbozol; Sigma Chemie, Deisenhofen, Germany) solution made with 5 ml 0.05 M sodium acetate buffer (pH 5.2), 50 µl 5% AEC stock solution in dimethylformamide, and 50 µl 3% H2O2. Before the incubation, the AEC solution was filtered through Millipore 0.22 µm. The incubation in the AEC solution consisted of an irradiation burst for 5 min at 90 W under the standardized conditions, followed by standing in the warm SWV for 2 to a maximum of 5 minutes (determined empirically by microscopic examination). The reaction was stopped by rinsing the whole-mount preparations three times for 5 min each in 0.01 M PBS.
Combinations
NADPH-d Histochemistry/Cuprolinic Blue Staining.
Immediately after performing NADPH-d enzyme-histochemistry, overnight fixation in 4% freshly prepared paraformaldehyde solution in 0.1 M PBS, pH 7.4, was carried out at 4C. Then, the whole-mount preparations were rinsed three times for 5 min each in distilled water. Subsequently, cuprolinic blue staining was performed according to the above-mentioned protocol. Finally, the whole-mount preparations were mounted on slides in a 3:1 glycerin:PBS mixture.
AChE Histochemistry/Cuprolinic Blue Staining. Cuprolinic blue staining could be performed immediately after AChE enzyme histochemistry without any modifications in either of the two protocols described above. Finally, the whole-mount preparations were mounted on slides in a 3:1 glycerin:PBS mixture.
Cuprolinic Blue Staining/CGRP Immunohistochemistry. After the cuprolinic blue staining according to the standardized protocol, the whole-mount preparations were rinsed three times for 5 min each in PBS. Subsequently, CGRP immunohistochemistry could be performed according to the above-mentioned protocol. Finally, the whole-mount preparations were mounted on slides in a 3:1 glycerin:PBS mixture.
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Results |
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The elaboration of combinations among cuprolinic blue, NADPH-d enzyme histochemistry, AChE enzyme histochemistry, and CGRP immunohistochemistry resulted in useful double labeling techniques. Furthermore, the staining results did not differ between the late fetal and neonatal jejunal whole-mount preparations.
In each of these combinations, the enteric neurons that stained for cuprolinic blue alone appeared as blue-green cells with nonreacting nuclei (Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13). At high magnification, the cytoplasm contained a fine blue-green granulation and in the nucleus, the nucleolus or nucleoli appeared as blue-iridescent round structures (Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 and Figure 13). The neuronal processes lacked staining because of lower RNA concentrations.
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In each of the combinations, a light blue background staining occurred. However, this did not prevent the recognition of the distinct meshworks of the different enteric plexuses. In the whole-mount preparation of the myenteric plexus, more or less rectangular ganglia that ran parallel to the circular muscle bundles were clearly visible (Figure 2, Figure 6, and Figure 10). The background staining was mainly due to the cuprolinic blue-stained fibroblasts, which appeared as spindle-shaped cells along the muscle cells of the longitudinal muscle layer. However, the staining result in the fibroblasts was much weaker. On the basis of their location and appearance, fibroblasts were easily distinguished from perikarya. In the whole-mount preparation of the submucosal plexuses, background staining was generally more pronounced owing to the higher fibroblast content of this tissue layer (Figure 7, Figure 9, and Figure 11 Figure 12 Figure 13). Nevertheless, a distinction between the inner and outer submucous plexus could be made. The outer submucous plexus consisted of large ganglia that were situated far apart and contained rather large cuprolinic blue-stained perikarya (Figure 3, Figure 5, Figure 7, Figure 9, Figure 11, and Figure 13). The inner submucous plexus consisted of small polygonal ganglia that were situated close to each other and contained rather small cuprolinic blue-stained perikarya (Figure 4, Figure 8, and Figure 12).
NADPH-d Histochemistry/Cuprolinic Blue Staining
Only a small number of the enteric neurons population were stained with NADPH-d histochemistry, appearing as dark blue-stained perikarya that gave rise to dark blue neuronal processes. In the nonreacting nucleus, the nucleolus or nucleoli were stained blue-green by the cuprolinic blue staining technique. In each of the enteric plexuses NADPH-d-expressing neurons were easily distinguishable from the cuprolinic blue-stained perikarya by the difference in color and staining-intensity (Figure 2, Figure 3, and Figure 5). In ganglia of the myenteric plexus, NADPH-d-expressing neurons of variable size and shape were scattered (Figure 2), whereas they appeared to be clustered in those of the outer submucous plexus (Figure 3 and Figure 5). In the inner submucous plexus, rather small NADPH-d-expressing neurons were very rarely seen (Figure 4).
The nerve fibers revealed by NADPH-d enzyme histochemistry facilitated the recognition of the cuprolinic blue-stained perikarya. These NADPH-d-expressing varicose nerve fibers were observed in the paravascular plexus, in the interconnecting nerve strands, in the ganglia, and parallel to smooth muscle bundles. In the ganglia, NADPH-d-expressing nerve fibers encircled both NADPH-d-expressing neurons and cuprolinic blue, non-NADPH-d-expressing perikarya (Figure 2 and Figure 3).
AChE Histochemistry/Cuprolinic Blue Staining
In this sequential staining technique, the cuprolinic blue-stained neurons appeared to be more pronounced, compared to the combination with NADPH-d enzyme histochemistry. Furthermore, there was a remarkable color shift from blue to green in the cells that were stained with cuprolinic blue alone.
AChE histochemistry revealed a subset of the enteric neuron population larger than that revealed by NADPH-d histochemistry. AChE-expressing neurons were stained with different intensities. In the perikaryon and in the nerve fibers of these enteric neurons a brown granulation filled the cytoplasm (Figure 7 Figure 8 Figure 9). However, the AChE histochemical reaction did not reveal the fine structure of the processes. The nucleus remained unstained, except for the nucleolus or nucleoli, which were stained by the cuprolinic blue staining technique (Figure 6, Figure 7, and Figure 9).
The slightly colored background did not affect the evaluation of the plexuses because the brownish color of the AChE-expressing perikarya and nerve fibers contrasted well with the background and with the blue-green cuprolinic blue-stained perikarya. AChE-expressing neurons of variable size and shape were scattered throughout the ganglia of the different enteric plexuses. In these ganglia, AChE-expressing nerve fibers encircled both AChE-expressing neurons and cuprolinic blue, non-AChE-expressing perikarya. Furthermore, AChE-expressing varicose nerve fibers were observed in the paravascular plexus, in the interconnecting nerve strands, and parallel to smooth muscle bundles.
Cuprolinic Blue Staining/CGRP Immunohistochemistry
CGRP immunohistochemistry revealed a small portion of the enteric neuron population. In the perikaryon and in the nerve fibers, the immunohistochemical reaction resulted in a red granulation of the cytoplasm. However, the reaction was unable to reveal the fine structure of the neuronal processes. The nucleus remained unstained, except for the cuprolinic blue-stained nucleolus or nucleoli.
Evaluation of the enteric plexuses was facilitated by the contrast between the red immunohistochemical reaction product and the cuprolinic blue staining. In the ganglia of the myenteric and outer submucous plexus, CGRP-expressing neurons were located mainly in clusters on the outside of the ganglia (Figure 10 and Figure 11). Some of them were observed in the interconnecting nerve strands (Figure 10). In the inner submucous plexus, scattered CGRP-expressing neurons were rarely found (Figure 12). These sparse CGRP-expressing neurons were rather large compared with the cuprolinic blue-stained perikarya that were seen in the outer submucous plexus (Figure 13).
In the ganglia, CGRP-expressing nerve fibers encircled both CGRP-expressing neurons and cuprolinic blue, non-CGRP-expressing perikarya. In addition to ganglia, CGRP-expressing varicose nerve fibers were observed in the paravascular plexus, in the interconnecting nerve strands, and parallel to smooth muscle bundles.
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Discussion |
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The individual staining techniques, i.e., NADPH-d histochemistry, AChE histochemistry, and CGRP immunohistochemistry, proved to be highly compatible with the cuprolinic blue staining. Nevertheless, some modifications were carried out to optimize the staining results: the incorporation of microwave applications into the different staining methods and an additional fixation in combining NADPH-d histochemistry. Encouraged by the knowledge that microwaves have a beneficial effect on chemical reactions, on the retrieval of antigens, and on the penetration of substances, we incorporated microwave applications into the different staining methods (
A one-to-one co-localization of NADPH-d and bNOS in enteric plexuses has been demonstrated, implying that both enzyme histochemistry and immunohistochemistry can be used to visualize nitrergic neurons (
AChE histochemistry revealed the largest subpopulation of enteric neurons compared with NADPH-d histochemistry and CGRP immunohistochemistry. AChE-expressing neurons are abundant in the myenteric and outer submucous plexus. This is in agreement with previous reports on AChE expression in the porcine small intestine (
Our demonstration of CGRP expression corresponds well with previous reports that describe the distribution of CGRP-expressing nerve cell bodies and fibers in the porcine small intestine (
By incorporating the above-mentioned modifications, the elaboration of combinations among cuprolinic blue, NADPH-d histochemistry, AChE histochemistry and CGRP immunohistochemistry resulted in rapid, useful, and ready-to-use double labeling techniques. Their main advantage is that they simultaneously visualize the total population as well as subsets of enteric neurons.
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