Nov 11, 2024
Anterograde tracing of spinal afferent innervation in rat atria flat-mounts
- Jichao Ma1,
- Ariege Bizanti1,
- Andrew Kwiat1,
- Kayla Barton1,
- Duyen Nguyen1,
- Jazune Madas1,
- Zulema Toledo1,
- Kohlton Bendowski1,
- Jin Chen1,
- Zixi (Jack) Cheng1
- 1Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816
- SPARCTech. support email: info@neuinfo.org
Protocol Citation: Jichao Ma, Ariege Bizanti, Andrew Kwiat, Kayla Barton, Duyen Nguyen, Jazune Madas, Zulema Toledo, Kohlton Bendowski, Jin Chen, Zixi (Jack) Cheng 2024. Anterograde tracing of spinal afferent innervation in rat atria flat-mounts. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo95kbv4o/v1
Manuscript citation:
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: November 11, 2024
Last Modified: November 11, 2024
Protocol Integer ID: 111916
Keywords: spinal afferent, anterograde labeling, dorsal root ganglia, heart, axon tracing
Funders Acknowledgement:
NIH
Grant ID: U01 HEAL/SPARC NS113867-01
Abstract
This protocol describes the methods used to trace and digitize spinal afferent axons in the rat atria. A mixture of dextran conjugates was injected into the dorsal root ganglia C8-T3 of adult Sprague-Dawley rats. 16 days after the tracer injection, the animals were sacrificed, the left and right atria 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
DPXMerck Millipore (EMD Millipore)Catalog #1.00579.0500
Step 5
HeparinHenry Schein Animal HealthCatalog #049130
Step 4
IsoThesia (Isoflurane) SolutionHenry Schein Animal HealthCatalog #029405
Step 2
10 µL SyringeHamilton CompanyCatalog ##80300
Step 2
FluorogoldFluorochrome
Step 3
IsofluraneCovetrusCatalog #029404
Step 4
BuprenorphineMcKesson CorporationCatalog #42023017905
Step 2
Dextran-Biotin 10k, Lysine fixableThermo Fisher ScientificCatalog #D1956
Step 2
Dextran-Biotin 3k, Lysine fixableThermo Fisher ScientificCatalog #D7135
Step 2
Thin Wall Glass CapillariesWorld Precision InstrumentsCatalog #TW150-4
Step 2
VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)Vector LaboratoriesCatalog #PK-6100
Step 5
3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905
Step 5
Cuprolinic Blue (quinolinic phthalocyanine)American Elements
Step 5
Sprague-DawleyEnvigo
Step 1
Animals
Animals
Three to five-month-old male Sprague-DawleyEnvigo rats (n = 15) 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). Hind paw pinch withdrawal reflex was checked as an indicator of the depth of anesthesia. While 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 vertebrae by blunt dissection to expose the lateral aspect of the C8-T3 vertebrae. Throughout the dissection, special attention was given to avoid large blood vessels and other critical anatomical features that are located close to the vertebral bodies. This delicate procedure was carried out under a Leica surgical microscope, which provided magnification and enhanced visualization of the surgical field. DRGs (C8-T3) were then exposed by drilling holes at their corresponding intervertebral foramina using
Equipment
Micro drill
NAME
Burr for micro drill
TYPE
Fine Science Tools
BRAND
19007-21
SKU
LINK
Shaft Diameter: 2.3mm, Length: 44mm, Tip Diameter: 2.1mm, Alloy/Material: Carbon Steel, Autoclave Safe: No
SPECIFICATIONS
. To locate the correct foramina for each DRG, the spinous processes of the vertebrae directly before and after the target DRG were first identified. These landmarks were used to guide the positioning of the drill and ensure that the procedure was both accurate and minimally invasive. During drilling, extreme care was taken to avoid any damages of targeted DRGs.
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 5-10 μm.
The micropipette was inserted at a 45-80 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 each DRG (C8-T3) was injected, cotton-tipped applicators (Puritan, USA) were immediately used to remove any possible leakage of tracers at the injection sites ensuring the confinement of the tracer within the ganglia.
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 normal reflexes prior to being transferred to their home cages. To reduce post-surgical discomfort and pain, BuprenorphineMcKesson CorporationCatalog #42023017905 (0.01 mg/kg body weight; s.c., Par Pharmaceuticals, Chestnut Ridge, NY, USA) was administered during surgery and once a day for the following 72 h post-surgery. Also, to prevent infection, a single dose of penicillin (50,000 IU/kg body weight; i.m., NorBrook, Newry, UK) was given. Both body weight and intake of food and water were monitored throughout the duration of the recovery period to ensure that rats did not experience any postsurgical complications.
Fluoro-Gold counterstaining
Fluoro-Gold counterstaining
Twelve days after survival surgery, seven animals were intraperitoneally (i.p.) injected withFluorogoldFluorochrome (1 mL of 2 mg/mL per rat) to counterstain neurons in the peripheral ganglia. The animals were perfused 4 days after the FG injection.
Tissue fixation and heart dissection
Tissue fixation and heart 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.
The heart was pinned to a dissecting dish containing PBS (pH 7.4), and the specimen was further dissected under a Leica surgical microscope. In brief, the lungs and trachea were removed with fine forceps, the atria were separated from the ventricles, and the aortic arch was removed from the atria. The right and left atria were separated along the interatrial septum. The right atrium tissue also contained the great vessels, including the IVC, superior vena cava (SVC), and left precaval vein (LPCV), whereas the junctions of all pulmonary veins (PVs) remained attached to the left atrium.
In this study, we have identified structures corresponding to the right cranial vein (RCV), left cranial vein (LCV), and caudal vein (CV), and we consistently referred to them here as the SVC, LPCV, and IVC in both our current study and in prior research.
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.
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.
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).
Equipment
TCS SP5 Laser Scanning
NAME
Confocal Microscope
TYPE
Leica
BRAND
TCSSP5
SKU
LINK
with 40x oil immersed lens (NA. 1.25) and a 405 nm laser (excitation) was used to detect FG-labeled neurons in the flat-mounts of the atria. Simultaneously, DIC optics were used to detect DB-labeled spinal afferents. Single optical section, partial, and maximum projection images were used to show the innervation on cardiac targets.
The distribution map of spinal afferent axons in the atria
The distribution map of spinal afferent axons in the atria
Each left and right atria were contoured using Neurolucida. One left and one right atria image were selected to serve as reference images. Then all contours were averaged using the interactive landmark-based deformable image alignment BigWarp tool provided by the open source ImageJ Fiji plugin.
To align the landmarks with the reference images, similar fiducial points were selected for each image. Following image alignments, the tracing data were added using the calculator tool of ImageJ to show the total traced axons of the aligned images. The first bifurcation of each parent axon was resembled by a dot in the reference images.
Quantification
Quantification
Morphometric analysis was performed automatically using
Software
Neurolucida Explorer
NAME
MicroBrightField Bioscience
DEVELOPER
on traced axons, and information, including axon diameter, receptive fields, terminals, and node (bifurcation point) numbers, were obtained. Values are expressed in the table as mean values ± standard error of mean (SEM). In categorizing the size of small and large axons, the distribution of axon diameters was considered. Given that the majority of axons fell either below 0.7 or above 0.9 μm in diameter with few intermediary values, a threshold of 0.9 was selected for categorization purposes.
Receptive field was calculated using convex hull spatial analysis. A convex polygon is generated by connecting distal branches of each axon, and the surface area is reported as the receptive field parameter for each axon. An independent t-test was performed using
Software
Prism
NAME
GraphPad
DEVELOPER
. Significance was considered at p < 0.05.