Comparison of wholemount dissection methods for neuronal subtype marker expression in the mouse myenteric plexus

Abstract

Background:

Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression.

Methods:

We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation and a third rod-mounted peeling technique. We also tested a reversed orientation variation of flat sheet peeling, two step-by-step variations of the rod peeling technique, and whole-gut fixation as a tube. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis.

Key Results and Conclusions:

We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin or neuronal nitric oxide synthase (nNOS) as a proportion of total neurons in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased proportion of neurons labeled for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in ileum and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling. The step-by-step variations of this method point to mechanical manipulation of the tissue as the likely cause of decreased labeling. Our study thereby demonstrates a critical variability in wholemount muscularis dissection methods.

Introduction

The enteric nervous system (ENS) is an autonomous network of neurons residing in two mesh-like layers (plexuses) within the wall of the gastrointestinal (GI) tract. The submucosal plexus (SMP) lies just below the mucosa, regulating secretions and local blood flow, while the myenteric plexus (MP) resides between the circular (CM) and longitudinal (LM) muscle layers, controlling gut motility patterns¹. Distinct regions of the GI tract perform different functions, and we are beginning to understand how this is reflected in motility patterns and the structure of the ENS controlling them², especially by the involvement of many enteric neuronal subtypes^3–5. ENS subtypes have been previously defined by their morphology⁶, electrophysiology⁷, and marker expression³. Recently, multiple single cell RNA sequencing (scRNAseq) studies have also defined enteric neuronal subtypes by RNA expression^4,5,8,9. Associating these RNA signatures with previously defined ENS subtypes is of pressing interest to the field.

Comparing results between many of the studies reported on ENS subtypes can be difficult, however. Reports of the proportion of specific subtypes or marker expression in the ENS have sometimes varied, such as reports of 6–23% of small intestine myenteric neurons expressing enkephalin^3,10. Variability such as this can result from animal model³, background¹¹, age¹², or GI tract region analyzed¹³.

Tissue processing has also been shown to introduce variability in the quality of enteric marker visualization¹⁴. Many different GI dissection methods and adaptations thereof have been reported over the years, especially for wholemount preparations of the myenteric plexus (see Table 1). Here, we assessed this potential source of variability in myenteric neuron marker expression in a controlled set of experiments testing (i) the order of microdissection and fixation and (ii) the usage of a rod during the microdissection (see Dissection protocols) across two landmark-defined intestinal regions, ileum and distal colon. We find no significant differences in labeling between flat sheet methods differing in the order of dissection and fixation steps, but report significantly decreased labeling using the mechanical rod peeling method across most markers and regions tested.

Table 1:

Wholemount muscularis dissection methods and variations

Method	Variations
Fix segments pinned flat, then peel muscularis13	Muscle relaxants (nicardipine)6,22,23
	Amount of stretch22,24
	Layers peeled3,25
	Transcardial perfusion with fixative26
	Microdissection vs razor blade26
Peel muscularis, then fix27	Layers peeled2,28
	No fixation (for fluorescent reporters)21
	Transcardial perfusion with fixative21
Peel on rod, then fix29	Peeling longitudinal muscle – myenteric plexus (LMMP) only (in rats)30
Fix as tube, then cut open and peel	Distend gut with Krebs31 or fixative31
	No distension32

Materials and Methods

Mice

All procedures conformed to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Stanford University Administrative Panel on Laboratory Animal Care. Mice were group housed up to a maximum of five adults per cage. Food and water were provided ad libitum and mice were maintained on a 12:12 LD cycle.

Experiments used wild-type C57BL/6J mice and the reporter lines choline acetyltransferase(ChAT)-IRES-Cre (Δneo)¹⁵ and Tachykinin precursor 1(Tac1)-IRES2-Cre-Δ¹⁶, both crossed to Cre-dependent tdTomato (tdT) Ai14 mice¹⁷ (hereafter ChAT-Cre-tdT and Tac1-Cre-tdT, respectively). Mice aged 2–4 months from both sexes were used, except for ChAT-Cre-tdT (only males). Tac1-Cre-tdT mice used for live imaging were aged 11 months.

Dissection protocols

Intestinal segment harvest

Mice were culled by CO₂ and cervical dislocation. Retrieval of Ileum and distal colon segments was performed as in (Hamnett, Dershowitz et al., 2022)¹³ to the point of removing the mesentery from the intestines. The dissection then proceeded with Method 1, Method 2, Method 3, or Method 4, or a variation of these methods (Method 2R; Method 3a; Method 3b), described below. All dissections were performed in ice cold PBS (Corning 46013CM diluted in Milli-Q (MilliporeSigma) water and filtered through 0.22 μm SteriCup filters (Millipore)).

Method 1: Fix flat then peel

The intestinal segment was opened along the mesenteric border, flipped over (serosa-up) and pinned flat along the edges in a Sylgard-bottomed glass Petri dish, stretched under light tension. Time from CO₂ initiation to fixation was approximately 15–35 minutes per segment. Segments were fixed in 4% PFA in PBS at 4°C for 90 minutes with gentle rocking, followed by three washes with cold PBS for at least 10 minutes each. To obtain muscularis with myenteric plexus, the muscularis was carefully peeled up and away from the submucosa/mucosa, separating the layers along the first 2–3 mm. The segment was flipped over, mucosa side up, and re-pinned. The mucosa/submucosa was carefully pulled away, while a cotton-tipped swab was used to gently hold down the muscularis. Segments were stored in PBS with 0.1% NaN₃ at 4°C until use.

Method 2: Peel flat then fix

Each segment was prepared as in Method 1 with the following modifications: (i) the order of microdissection and fixation of the pinned segments was reversed and (ii) fine forceps were used to grasp the mucosa/submucosa and gently pull it away, avoiding the use of the cotton swab applying pressure to the muscularis before fixing. Time from CO₂ initiation to fixation was approximately 35–90 minutes, depending on the number of segments per mouse. Muscularis segments were fixed, washed, and stored as in Method 1.

Method 2R (Method 2, reversed):

As in Method 2, the tissue was peeled prior to fixation. The orientation of the tissue was reversed, such that the mucosa was facing down in the dish and remained pinned, while the muscularis, serosa side up, was peeled away. Time from CO₂ initiation to fixation was approximately 30 minutes per segment.

Method 3: Peel on rod

A glass rod was inserted through the lumen of the full-length intestinal segment. The diameter of the glass rod (2.5 mm) was chosen to fit the diameter of the segment and slightly distend it. Forceps were used to gently perforate the muscularis (but not the mucosa/submucosa) at the mesenteric attachment. Ice-cold PBS was applied frequently during dissection to maintain moisture and temperature of the segment. A cotton-tipped swab moistened with cold PBS was used to gently peel away the muscularis, swabbing circumferentially from the perforated mesenteric edge while the segment was manually held stable on the rod. Fully peeled muscularis segments were transferred to a Sylgard-bottomed glass Petri dish with cold PBS and pinned flat along the edges under light tension. Muscularis segments were fixed, washed, and stored as in Method 1. Time from CO₂ initiation to fixation was approximately 20–60 minutes, depending on the number of segments per mouse.

Method 3a (Rod, prior to fixation):

A glass rod was inserted through the lumen of the segment as in Method 3 and the tissue was kept on the rod, with ice-cold PBS applied frequently, for five minutes (the length of time equivalent to that of Method 3 peeling). The tissue was then unmounted from the rod and processed according to Method 1. Time from CO₂ initiation to fixation was approximately 25 minutes per segment.

Method 3b (Rod, with manipulation, prior to fixation):

As in Method 3a, with additionally manipulating the tissue by holding it gently with a finger against the rod, replicating holding the segment manually stable as in Method 3. After five minutes, the tissue was unmounted from the rod and processed according to Method 1. Time from CO₂ initiation to fixation was approximately 25 minutes per segment.

Method 4: Fix as tube

The intestinal segment was fixed whole immediately after harvest as a tube. Time from CO₂ initiation to fixation was approximately 30 minutes per segment. After fixation, the segment was opened longitudinally and the dissection proceeded according to Method 1.

Immunohistochemistry

Immunohistochemistry was performed as described previously¹³. Briefly, tissues were incubated with primary antibodies in PBT (PBS, 1% BSA, 0.1% Triton X-100) overnight at 4°C with shaking. Tissues were then washed three times in PBT at room temperature and incubated with secondary antibodies in PBT for two hours at room temperature with shaking. Tissues were then washed twice in PBT and twice in PBS, rinsed briefly (few seconds) in ddH2O, and mounted on glass slides using a paintbrush to smooth out any folds. The tissues were allowed to partially air-dry on the slides and coverslipped with Fluoromount-G (Southern Biotech). All wash steps were at least 10 minutes each. The following primary antibodies were used: rabbit anti-calretinin (1:4000; Millipore AB5054), goat anti-calretinin (1:8000; Swant SG1), rat anti-SST (1:500; Millipore MAB354), rabbit anti-nNOS (1:1000; Sigma N7280), and human anti-ANNA1 (HuC/D) (1:50,000; kind gift from Vanda Lennon, Mayo Clinic). The following secondary antibodies were used: donkey anti-goat Cy3 (1:1000; Jackson), donkey anti-rat AF488 (1:1000; Invitrogen), donkey anti-human AF405 (1:500; Jackson), donkey anti-rabbit Cy5 (1:500; Jackson), donkey anti-rabbit Cy3 (1:1000; Jackson).

Confocal imaging

Images were acquired on a Leica SP8 confocal microscope using a 20x (NA 0.75) oil objective at 1024 × 1024 pixel resolution. Tiled images were acquired and stitched together using the Navigator mode within LASX (Leica). Z-stacks with 2.5 – 3 μm between each focal plane were acquired to capture the full depth of the myenteric plexus across the entire region. Imaged regions were located away from the mesenteric border, with an average area of 6.5 mm² and ~2000 neurons per region on average.

Live confocal imaging

Following dissection of Tac1-Cre-tdT intestines as described above, the distal colon was divided in two and placed on tissue paper wetted with ice-cold PBS atop a plastic 12-well plate lid. One half had a glass rod inserted through it, which remained for the duration of the experiment, while the other did not. Live images of tdTomato expression within a single field of view through the depth of the myenteric plexus were acquired on a Leica SP8 confocal microscope using a 10x air objective (NA 0.4). Images of approximately the same field of view for each tissue segment were acquired immediately after dissection (t = 0), and after 15 minutes (t = 15).

Neuronal and marker quantification

Image analysis was performed using ImageJ/FIJI (NIH, Bethesda, MD), as described previously¹³. HuC/D images (z-stack individual planes) were blurred and thresholded to create a mask, which was then applied to the raw image stacks of the marker expression using the Image Calculator function. The result was maximally projected and thresholded for marker expression, and cells counted using the Analyze Particles function. The HuC/D image mask was then also maximally projected and total neurons counted in the same fashion. Density of neurons was then calculated from the area of each image measured in FIJI. SST was counted manually after being combined with the thresholded HuC/D image stack and identified by its distinctive cytoplasmic expression pattern.

For analysis of live images, 3D reconstructions were performed in Imaris, using the Oblique Slicer function to exclude tdTomato expression from outside of the myenteric plexus. Image projections were then exported to ImageJ/FIJI, where a suitable region was identified that was present in both t = 0 and t = 15 conditions. tdTomato-positive neurons were manually counted and their locations marked in the t = 0 condition based on morphology and presence within ganglia. Non-neuronal cells marked by Tac1-Cre-tdT were not counted. Neuron locations were then overlaid on t = 15 images to determine if neurons had been lost.

Statistical analyses

Statistical tests and graphical representation of data were performed using Prism 9 software (GraphPad). Statistical comparisons were performed using one-way ANOVA followed by Tukey’s correction for multiple comparisons to assess if variations in dissection method were a significant factor (p<0.05) for calretinin and nNOS subtype marker proportion. Two-way ANOVAs followed by Šídák’s multiple comparisons tests were used to determine the effect of dissection method on ChAT-Cre-tdT and Tac1-Cre-tdT marker subtype proportion. Asterisks indicate significant differences.

Results

To probe possible effects of different tissue dissection methods on marker expression, we separately tested different aspects of three dissection methods described in the field (detailed in Methods). We focused on (i) the order, chronologically, of peeling and fixation, and (ii) mechanically different methods of peeling the muscularis, while preserving variables such as dissection solution and temperature, type of fixative, time to fixation and duration of fixation across the three methods. To delve further into these methods and address some more subtle differences commonly implemented, we also tested multiple variations (Methods 2R, 3a, and 3b) and a fourth method of whole-gut fixation (Method 4) (Fig 1a).

Figure 1 |. Proportion of ileum myenteric plexus neurons labeled immunohistochemically by seven dissection methods.

(a) Flowchart detailing the steps of each method and its variations.

(b, d, f, h, j, l, n) Representative images of ileum myenteric plexus wholemounts prepared via each of the dissection methods immunohistochemically labeled for calretinin (green) and neuronal label HuC/D (magenta).

(c, e, g, I, k, m, o) As in (b, d, f, h, j, l, n), labeled for nNOS (green).

(p, q) Proportion of total HuC/D neurons (mean ± SEM) positive for each neuronal marker for each dissection method. (p) Calretinin: Method 1, n = 10; Method 2, n = 8; Method 2R, n = 6; Method 3, n = 9; Method 3a, n = 6; Method 3b, n = 6; Method 4, n = 6. (q) nNOS: Method 1, n = 6; Method 2, n = 4; Method 2R, Method 3, n = 11; Method 3a, n = 6; Method 3b, n = 6; Method 4, n = 6.

Scale bar represents 200 μm in all images. All tests one-way ANOVA. *p<0.05, **p<0.01, ****p<0.0001.

We first analyzed neuronal marker expression via immunohistochemistry. We chose three marker proteins for which antibody staining yields easily quantifiable cell body labeling: (1) calretinin, which is expressed in multiple subtypes of neurons including sensory neurons, interneurons, and excitatory motor neurons; (2) neuronal nitric oxide synthase (nNOS), expressed in inhibitory motor neurons and some interneurons; and (3) somatostatin (SST), expressed in a subpopulation of interneurons^18–20. For both calretinin and nNOS, no significant difference in the proportion of neurons labeled was found between the flat-sheet microdissection methods (Methods 1, 2, and 2R) (Fig 1). However, for both calretinin (Fig 1p) and nNOS (Fig 1q), the rod peeling method resulted in significantly fewer neurons labeled than any of the flat sheet methods (calretinin, ~30–35% (Methods 1, 2, and 2R) decreased to ~10% (Method 3); nNOS, ~23% (Methods 1, 2, and 2R) decreased to ~13% (Method 3)). For SST, due to the low proportion of neurons labeled by any of the methods (~2%), the experiment was insufficiently powered to detect any significant differences (Fig S1).

To test the potential contribution of mechanically distinct steps of Method 3 to marker expression, we also performed each step without peeling, before proceeding as in Method 1. For both calretinin and nNOS, placing the segment of ileum on the rod without further manipulation (Method 3a) had no effect on marker labeling compared to the flat sheet methods. Placing the segment on the rod and applying gentle pressure as in Method 3 but without peeling with the swab (Method 3b) had an intermediate effect on calretinin marker labeling (decreased to ~20%). Interestingly, whole-gut fixation as a tube (Method 4) also resulted in decreased calretinin labeling (decreased to ~20%).

We next asked whether the mechanical technique of wholemount peeling would also impact the reporting of genetically labeled neurons in transgenic mice. Transgenic mice that express a fluorescent reporter protein in a gene-dependent manner can be used to quantify neuronal subtype marker expression for markers where antibody staining does not yield easily quantifiable cell body labeling, such as ChAT and substance P. Like calretinin, ChAT and substance P are both expressed in excitatory motor neurons. We compared the two mechanically different peeling methods, flat-sheet (Method 1) and rod mounted (Method 3) in ChAT-Cre-tdT and Tac1-Cre-tdT mice, and expanded our analysis to two regions, ileum and distal colon. In Tac1-Cre-tdT ileum, the proportion of neurons labeled by tdT was similar between the two methods tested (Fig 2 f–g, j); however, in ChAT-Cre-tdT ileum, the proportion of neurons labeled was significantly decreased with the rod peeling method as compared to the flat-sheet method, from ~62% (Method 1) to ~26% (Method 3) (Fig 2 a–b, e). In the distal colon, the rod peeling method resulted in decreased proportion of neurons labeled in ChAT-Cre-tdT (~38% (Method 1) decreased to ~14% (Method 3)) and Tac1-Cre-tdT (~36% (Method 1) decreased to ~3% (Method 3)) myenteric plexus, with neuronal cell body labeling in Tac1-Cre-tdT colon almost completely abolished (Fig 2 c–e, h–j). Importantly, we did not note any differences in overall neuronal density (Fig 2o), which if found might have suggested neuronal cell death as an underlying cause for decreased labeling.

Figure 2 |. Proportion of ileum and distal colon myenteric plexus neurons labeled genetically by two distinct dissection methods.

(a-d) Representative images of ileum (a, b) and distal colon (c, d) myenteric plexus prepared via Method 1 (a, c), or Method 3 (b, d), genetically labeled for ChAT-Cre-tdT (green) and neuronal label HuC/D (magenta).

(f-i) As in (a-d), labeled for Tac1-Cre-tdT (green).

(e, j) Proportion of total HuC/D neurons (mean ± SEM) positive for each neuronal marker for each region and each dissection method. (e) ChAT-Cre-tdT: Method 1 ileum, n = 3; Method 3 ileum, > fix, n = 9; Method 1 distal colon, n = 3; Method 3 distal colon, n = 9. (j) Tac1-Cre-tdT: Method 1 ileum, n = 3; Method 3 ileum, n = 6; Method 1 n = 3; Method 3, n = 6.

(k-n) Representative images of unfixed distal colon myenteric plexus genetically labeled for Tac1-Cre-tdT either without (k, l) or with (m, n) a glass rod inserted through the intestines immediately following dissection (t = 0 min; k, m) or 15 minutes later (t = 15 min; l, n).

(o) Density of HuC/D neurons (mean ± SEM) for each region by each dissection method. All tests two-way ANOVA.

Scale bar represents 200 μm in (a-d), (f-i); 50 μm in (k-n). All tests two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001.

tdTomato expression being present without requiring immunostaining allowed us to image the tissue without fixation to directly visualize if the presence of the rod inside of the intestine resulted in loss of endogenous fluorescence. We observed no loss of tdTomato-positive Tac1-Cre cells in the 15 minutes after dissection, regardless of the absence (percentage neuron survival (mean ± SEM): 99.44% ± 0.56) or presence (99.38% ± 0.62) of a glass rod.

Taken together, our results demonstrate that while the order of dissection and fixation steps in the flat-sheet methods does not impact marker labeling, for both native protein detection and transgenic reporter fluorescent protein expression, and in multiple intestinal regions, the rod peeling method can result in decreased marker labeling compared to the flat-sheet microdissection methods. These results suggest that mechanical handling of the tissue, especially peeling with the swab, is responsible for the observed decrease in proportions of neurons labeled, rather than distension from introduction of the rod.

Discussion

In this study we performed a controlled and comparative investigation of the effect of different wholemount mouse myenteric plexus preparations on neuronal subtype marker expression, both native proteins and fluorescent transgenic reporters, as a proportion of total neurons. We evaluated whether marker expression is dependent on either (i) the chronological order of the dissection and fixation steps, or (ii) the mechanical method of wholemount dissection (peeling from flat tissue or peeling on a rod). We found no significant differences in neuronal labeling when varying the order of the peeling and fixation steps or the orientation of peeling (mucosa peeled away, Method 2; muscularis peeled away, Method 2R) in the flat-sheet preparations. By contrast, in our hands, we found that neuronal markers (both native proteins and transgenic reporter proteins) are affected by the mechanical method of wholemount peeling, and the proportion of neurons labeled was significantly decreased in the rod peeling method. This was particularly striking in the case of ChAT-Cre-tdT, where the proportion of neurons labeled was decreased from about two-thirds (~62%) to about one-quarter (~26%) in ileum myenteric plexus. ChAT and nNOS together are known to cover the vast majority of enteric neurons [between them]²¹, but the decrease in both ChAT-Cre-tdT and nNOS labeling with the rod peeling method would suggest a large proportion of myenteric neurons unlabeled for either marker.

We included variations of the rod peeling method to test the potential cumulative contribution of each of the three different steps in Method 3 compared to the flat sheet peeling methods: (1) distension of the rod (Method 3a), (2) additionally applying gentle pressure to hold the segment in place (Method 3b), and (3) peeling of the muscularis with the swab (Method 3). Distension had no effect on the proportion of neurons labeled for either calretinin or nNOS, while gentle pressure against the rod had an intermediate effect on the proportion of neurons labeled for calretinin. Consistent with this, live imaging of Tac1-Cre-tdT expression revealed no loss of tdTomato expression due to the presence of a glass rod. These results suggest that mechanical manipulation of the tissue, especially the peeling with the swab and to a lesser extent pressure on the segment, is the main contributor to a cellular response leading to the observed decrease in the proportion of neurons labeled in Method 3. In this study we did not explicitly test for any effect of time elapsed before fixation. However, in our hands the time to fixation between the methods was comparable, suggesting that any decrease in marker labeling with the rod peeling method is not due to the time required to process the tissue prior to fixation.

Our results suggest care should be taken when evaluating neuronal marker expression in samples prepared with Method 4, as it showed decreased proportion of neurons labeled compared to Method 1 (for both calretinin and nNOS) and Method 2 (for calretinin). This may potentially be due to decreased access and penetration of the fixative to the tissue through the lumen. Further investigation would be required to determine the cause of decreased proportion of neurons labeled with this method. In our hands, Method 4 also resulted in the most difficult tissue to image, as the tube fixation prevented the tissue from lying as completely flat on the slide as with the other methods in which the tissue was fixed flat. Finally, we note that our comparative study is not exhaustive and that future experiments are required to uncover the exact mechanism underlying the observed differences in marker expression.