Mar 15, 2023
Anterograde tracing of spinal afferent innervation in rat stomach flat-mounts
- Jichao Ma1,
- Duyen Nguyen1,
- Jazune Madas1,
- Andrew Kwiat1,
- Zulema Toledo1,
- Ariege Bizanti1,
- Nicole Kogut1,
- Anas Mistareehi1,
- Kohlton Bendowski1,
- Yuanyuan Zhang1,
- Jin Chen1,
- Terry Powley2,
- John Furness3,
- Zixi (Jack) Cheng1
- 1Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816;
- 2Department of Psychological Sciences, Purdue University, West Lafayette, IN 479062;
- 3Department of Anatomy & Physiology, Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia
Protocol Citation: Jichao Ma, Duyen Nguyen, Jazune Madas, Andrew Kwiat, Zulema Toledo, Ariege Bizanti, Nicole Kogut, Anas Mistareehi, Kohlton Bendowski, Yuanyuan Zhang, Jin Chen, Terry Powley, John Furness, Zixi (Jack) Cheng 2023. Anterograde tracing of spinal afferent innervation in rat stomach flat-mounts. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb9dxvx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 14, 2023
Last Modified: March 15, 2023
Protocol Integer ID: 78798
Keywords: stomach, myenteric plexus, smooth muscles, blood vessels, spinal afferent, anterograde labeling, axonal tracing
Funders Acknowledgement:
NIH
Grant ID: U01 HEAL/SPARC NS113867-01
NIH
Grant ID: 1R15HL137143-01A1
Abstract
This protocol describes the methods used to trace and digitize spinal afferent axons in the rat stomach. A mixture of dextran conjugates was injected into the dorsal root ganglia T7- T11 of adult Sprague-Dawley rats. 16 days after the tracer injection, the animals were sacrificed, ventral and dorsal stomachs were prepared and processed for avidin-biotin permanent labeling and Cuprolinic Blue labeling. All flat-mounts were examined using a Zeiss M2 Imager and MicroBrightField’s Neurolucida tracing and digitization system to determine the distribution and terminal structures of spinal afferent axons.
Protocol materials
Dextran-Biotin 3k, Lysine fixableThermo Fisher ScientificCatalog #D7135
Step 2
Thin Wall Glass CapillariesWorld Precision InstrumentsCatalog #TW150-4
Step 2
HeparinHenry Schein Animal HealthCatalog #049130
Step 3
DPXMerck Millipore (EMD Millipore)Catalog #1.00579.0500
Step 4
3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905
Step 4
Cuprolinic Blue (quinolinic phthalocyanine)American Elements
Step 4
VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)Vector LaboratoriesCatalog #PK-6100
Step 4
Sprague-DawleyEnvigo
Step 1
IsoThesia (Isoflurane) SolutionHenry Schein Animal HealthCatalog #029405
Step 2
10 µL SyringeHamilton CompanyCatalog ##80300
Step 2
Dextran-Biotin 10k, Lysine fixableThermo Fisher ScientificCatalog #D1956
Step 2
IsofluraneCovetrusCatalog #029404
Step 3
Animals
Animals
Four to five-month-old male Sprague-DawleyEnvigo rats (n = 20) were housed in an animal room at which the dark/light cycle was set to 12/12 hours and water and food were supplied ad libitum. All procedures were carried out under the ethical guidelines of the University of Central Florida and approved by the University of Central Florida’s Institutional Animal Care and Use Committee (IACUC).
Neural tracer injections
Neural tracer injections
Overnight-fasted rats were anesthetized with IsoThesia (Isoflurane) SolutionHenry Schein Animal HealthCatalog #029405 inhalation (3% in oxygen for induction, 2% for maintenance). Whilst under anesthesia, the rat was placed in a ventral decubitus position, and a 5~6 cm long incision was made along the midline of the dorsal surface. Left paraspinal muscles were separated from the transverse process by blunt dissection to expose the lateral aspect of the T7-T11 vertebrae. T7-T11 DRG were then exposed by drilling holes at T7-T11 intervertebral foramina.
For the DRG injection, a 10 µL SyringeHamilton CompanyCatalog ##80300 was used to aspirate 2-μl of lysine-fixable Dextran Biotin (DB) solution consisting of a 1:1 mixture of 3K and 10K MW dextrans in ultrapure water (final concentration 15% DB consisting of 7.5% Dextran-Biotin 3k, Lysine fixableThermo Fisher ScientificCatalog #D7135 and 7.5%Dextran-Biotin 10k, Lysine fixableThermo Fisher ScientificCatalog #D1956 ). This 2-μl solution was then transferred to a Thin Wall Glass CapillariesWorld Precision InstrumentsCatalog #TW150-4 (outer diameter: 1.5 mm, inner diameter: 1.12 mm) that was pulled on a
Equipment
P-87 Flaming/Brown
NAME
micropipette puller
TYPE
Sutter Instruments
BRAND
Rev. 0299c
SKU
LINK
with tip diameter of approximately 5 μm.
The micropipette was inserted at a 45-degree angle relative to the spine into the posterior side of DRG, and the tracer was slowly injected as the needle gradually retracted in a stepwise fashion within the ganglion. The micropipette was kept in place for 1 minute during each injection before withdrawal to ensure the infusion pressure within the DRG had dissipated.
After the injection, the paraspinal muscles and skin were closed using interrupted sutures. The rats were placed on a heating pad at 37°C for recovery and regaining of their righting reflexes prior to being transferred to their home cages.
Tissue fixation and GI dissection
Tissue fixation and GI dissection
16 days following post-op, the rats were weighed and deeply anesthetized withIsofluraneCovetrusCatalog #029404 (5%). Upon the absence of response to hind-paw pinching, 0.3 mL of HeparinHenry Schein Animal HealthCatalog #049130 was injected into the left ventricle to prevent blood coagulation followed by a cut to the inferior vena cava.
The animal was perfused through the left ventricle of the heart with 500 mL of physiological saline at 37°C and then with 500 mL of 4% paraformaldehyde in phosphate-buffered saline (0.1 M PBS, pH 7.4) at 4°C.
In order to shape the stomach in normal distension at the time of fixation, a catheter was slowly intubated into the stomach and 10 mL of physiological saline at 37°C was infused into the stomach.
After the perfusion, the whole stomach was removed from the abdominal cavity by introducing cuts to the distal esophagus and the proximal duodenum. Next, a midline cut along the lesser and greater curvature was made to yield two equal halves. The chyme was rinsed off to expose the mucosa.
8 hours into post-fixation with paraformaldehyde, the whole mount muscle wall of the stomach was separated from the gastric mucosa and submucosa using forceps. The muscle wall includes the longitudinal muscle, myenteric plexus, and circular muscle.
DAB and Cuprolinic Blue staining
DAB and Cuprolinic Blue staining
Similar to the protocol that was previously used for the rat stomach (Walter et al., 2016), all steps of tracer processing and neuronal counterstaining of whole mount tissue were done in room temperature (~22°C), on a shaker, and with the tissue free floating.
The samples were washed 6 times x 5 minutes each in phosphate-buffered saline (PBS; 0.1 M, pH = 7.4), followed by a 30-minute soaking in methanol:hydrogen peroxide block (4:1 ratio) to inactivate endogenous peroxidase.
Following additional PBS washes, the tissues were blocked with a PBS solution containing 0.5% Triton X-100 and 0.08% sodium azide NaN3 for 5 days to facilitate penetration of the reagent.
The samples were rinsed in PBS 6 times x 5 minutes each, followed by a one-hour incubation in VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)Vector LaboratoriesCatalog #PK-6100 . The DB-filled spinal afferent axons were visualized using 3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905 , a traditional dye that produces a golden-brown color on labeled axons.
Samples were then rinsed in PBS 6 times x 5 minutes each, soaked in DAB solution for 5 minutes, and rinsed in distilled water 6 times x 5 minutes. The pan-neuronal marker Cuprolinic Blue (quinolinic phthalocyanine)American Elements which binds to RNA in the cytoplasm of neurons (Phillips et al., 2004) was used to counterstain the enteric neurons in another group of samples (n = 6).
Briefly, the samples were rinsed in distilled water and then incubated in 0.5% Cuprolinic Blue solution which dissolved in 0.05 M sodium acetate buffer containing 1.0 M MgCl₂ (pH 4.9) for 2 hours in a humidified slide warmer (38°C).
Next, the samples were rinsed in distilled water and incubated in 0.05 M sodium acetate buffer containing 1.0 M MgCl₂ (pH 4.9) for 2 minutes, followed by a repeating rinse in distilled water.
After the staining process, samples were mounted on slides, flattened under lead blocks for 6 hours, air-dried overnight in a fume hood, dehydrated in an ascending concentration of alcohol (75%, 95%, 100%, and 100%), and cleared in xylene.
Coverslips were used to cover the tissue after applying DPXMerck Millipore (EMD Millipore)Catalog #1.00579.0500 mounting medium.
Spinal afferent axon screening and tracing
Spinal afferent axon screening and tracing
Equipment
Eclipse 80i
NAME
Fluorescent Microscope
TYPE
Nikon
BRAND
M318E
SKU
LINK
(Lens: 20X, NA 0.5) with brightfield optics was first used to systematically examine all flat-mount stomachs. Once a DB-labeled spinal axon was identified, three criteria were used to inspect the quality of the axon: (a) adequate labeling, (b) completeness of the axon, and (c) minimal tissue artifacts such as folds and tears.
All spinal axons were categorized based on their relative locations (e.g., fundus, corpus, antrum) and gastric targets (e.g., myenteric ganglia, longitudinal muscle sheet, circular muscle sheet, blood vessels).
The spinal axons that satisfied all criteria were re-evaluated using a higher magnification lens (40X, NA 0.75) to assess the morphology of any intertwined neighboring individuals that could potentially lead to the failure of distinction and digitization of the entire tracing of a single spinal afferent axon. Such neighboring spinal axons were removed from the inventory, and the axons that satisfied all qualifications were marked and ready to be digitized.
Axon tracing, digitization, and analysis were performed using
Software
Neurolucida
NAME
MBF BioScience
DEVELOPER
(RRID:SCR_001775). The software controlled the motorized stage of
Equipment
Axio Imager.M2
NAME
Upright microscope
TYPE
Zeiss
BRAND
Zeiss
SKU
LINK
(RRID:SCR_018876, Oberkochen, Germany) equipped with brightfield optics and a long-working-distance 40X objective lens (NA 0.75). All spinal afferent axons were traced in real time and in their original three dimensional space. The tracing file was then saved as .xml.
The Neurolucida system was also used to locate and outline the myenteric ganglia on which the spinal afferent axons formed terminals or where the axon processes coursed in close proximity with and on the same layer without making direct innervation. The criteria for identifying myenteric ganglia and myenteric plexus connectives were established (Walter et al., 2016; Bar-Shai et al., 2004; Berthoud et al., 1997), where at least two myenteric neurons clustered together were considered a ganglion. Two clusters of neurons were considered two ganglia if they were separated by a distance of at least three average neuron long-axis diameters, with or without the presence of a string of consecutive single-file neurons.
The digital reconstruction of spinal afferent axons in the stomach was performed on a 2D montage image instead of the live tissues like we mentioned above. In order to do this, we scanned the whole stomach in bright field using a 20X (NA 0.8) objective lens on the Zeiss Axio Imager M2 and performed a maximum projection for all image stacks using
Software
Zen Digital Imaging for Light Microscopy
NAME
Zeiss
DEVELOPER
(RRID:SCR_013672). The image tiles were stitched together using
Software
Adobe Photoshop
NAME
Adobe
DEVELOPER
(RRID: SCR_014199) to create a montage of the whole stomach. The montage was then loaded into
Software
Neurolucida 360
NAME
MBF Bioscience
DEVELOPER
(RRID:SCR_016788) for axon tracing, digitization, and analysis.
Image acquisition
Image acquisition
Single-field and multiple-field (or mosaic) photomicrographs were acquired using Zeiss Axio Imager M2 microscope with a 20X (NA 0.8) objective lens and a 63X oil immersion (NA 1.4) objective lens. The AxioCam 208 color camera was mounted on the microscope with BrightField and Differential Interference Contrast (DIC) optics. Mosaic photomicrographs consisted of the whole innervating field of a spinal afferent axon and were assembled via Photoshop. To capture the varying depths of a spinal afferent axon within a smooth muscle whole mount, each image consisted of multiple focal z-planes that were stacked using Photoshop to generate a partial projection image. Modifications, including brightness and contrast adjustments, and scale bar additions were conducted utilizing Photoshop or
Software
ImageJ/Fiji
NAME
Windows 7
OS
National Institutes of Health
DEVELOPER
(RRID: SCR_003070).