This work was supported by the Sichuan Provincial Department of Science and Technology (2021YJ0175).

The experimental protocol and procedures have received approval from the Administrative and Animal Research Committee of Chengdu University of Traditional Chinese Medicine (Record number: 2022DL-010). Eight-week-old male Sprague Dawley (SD) rats, weighing 180-200 g, were commercially obtained (see Table of Materials) and housed under a natural light/dark cycle with controlled temperature (23 &#177; 2 &#176;C) and relative humidity (55% &#177; 10%). Twelve rats were randomly divided into two groups: the control group and the AR group. All rats were acclimated to these conditions and provided with free access to food and water for one week before the trial.
1. Establishment of a rat model of AR
<ol>
	<li>Preparing the sensitizing solution: suspend 30 mg of ovalbumin (OVA) and 3 g of Al(OH)3 in 100 mL of saline (see Table of Materials)14,15.</li>
	<li>Basic sensitization: grasp and hold the fully awake rat with its abdomen facing up. Disinfect the abdomen using 75% alcohol-soaked cotton balls. Use a 1.5 mL syringe to inject 1 mL of the sensitizing solution prepared in step 1.1 into the left abdominal cavity of the rats in the AR group. Repeat this injection once every two days for 14 days (Figure 1A). 
	NOTE: Ensure that the rat&#39;s head is positioned lower than its tail to prevent damage to the large and small intestines when inserting the syringe. The injection site is located 1.5 cm from the ventral midline. Rats in the control group should receive intraperitoneal injections of 1 mL of normal saline once every two days for 14 days.</li>
	<li>Nasal challenge: Administer 50 &#181;L of 5% OVA via nasal drops using a 100 &#181;L pipette (Figure 1B) on the day following the basal sensitization, repeating this procedure for 7 days (Figure 1C). 
	&#8203;NOTE: Prepare the 5% OVA solution by mixing 5 g of OVA with 100 mL of saline. For the control group, administer drops of the same volume of saline.</li>
</ol>
2. Behavioral scoring of rats
<ol>
	<li>Within 30 min after completing the final nasal challenge, observe and record the rats using a digital camera to identify symptoms such as nose scratching, sneezing, and a runny nose, based on the scoring criteria outlined in Table 1 (Figure 1D). 
	&#8203;NOTE: The confirmation of successful AR modeling is based on achieving a total score of &#8805;5 points14.</li>
</ol>
3. Acquisition of nasal mucosa samples
<ol>
	<li>Sacrificing rats: sacrifice the rats by exposing them to an overdose of CO2. Afterward, disinfect the rats with 75% ethanol (see Table of Materials). Use a scalpel to make an incision along the two corners of the mouth to expose the muscle tissue (Figure 2A). Next, cut the joints of the zygomatic bone and the mandible on both sides of the skull using tissue scissors (Figure 2B), and remove the mandible (Figure 2C).</li>
	<li>Nasal cavity exposure: separate the skin of the maxilla using a hemostat (Figure 2D) to expose the nasal cavity (Figure 2E). Cut the bony connection between the nasal cavity and the maxilla with tissue scissors (Figure 2F). Then, sever the connection between the nasal cavity and the orbital bones on both sides at the infraorbital foramen (Figure 2G) and remove the nasal cavity (Figure 2H).</li>
	<li>Fixation: place the extracted nasal cavity in 4% paraformaldehyde for fixation and store it at room temperature for 24-72 h (see Table of Materials). 
	NOTE: Ensure that the amount of paraformaldehyde fixative used is sufficient to completely cover the sections for proper fixation.</li>
</ol>
4. Pre-processing of nasal mucosa samples
<ol>
	<li>Decalcification: begin by fixing the removed tissues from step 3.1. Wash them three times with PBS for 20 min each. Follow this by washing them three times with distilled water, each time for 20 min. Subsequently, decalcify the tissues in a decalcification solution with a volume of 20-30 times that of the tissues at room temperature.
	<ol>
		<li>The decalcification solution (see Table of Materials) should maintain a pH of 7.2-7.4. Finally, place the decalcified tissue into a dehydration box. 
		NOTE: The decalcifying solution needs to be changed weekly. The decalcification process can be concluded when the tissue softens (approximately 2 months).</li>
	</ol>
	</li>
	<li>Dehydration and waxing: transfer the dehydration box to a dehydrator (see Table of Materials). Begin with 75% alcohol for 4 h, followed by 85% alcohol for 2 h, 90% alcohol for 2 h, and 95% alcohol for 1 h.
	<ol>
		<li>Subsequently, immerse the tissue in anhydrous ethanol for 30 min, repeat the process for another 30 min, use xylene for 5-10 min, repeat this step for another 5-10 min, immerse the tissue in wax for 1 h, repeat this step for another hour, and finally, immerse the tissue in wax for an additional 1 h.</li>
	</ol>
	</li>
	<li>Embedding: place the melted wax block into the embedding box (see Table of Materials) and store it in a -20 &#176;C freezer. Once the wax solidifies, remove it and trim the wax block.</li>
	<li>Slicing: insert the trimmed wax block into a paraffin microtome (see Table of Materials) and cut it into slices with a thickness of 3 &#181;m. Place the slices in a 40 &#176;C water bath on a slide spreader. Flatten the tissue, then lift it with a glass slide and bake it in a 60 &#176;C oven.
	<ol>
		<li>After the water has evaporated, and the wax is properly set, remove the slides and store them at a temperature of 25 &#176;C.</li>
	</ol>
	</li>
</ol>
5. H&#38;E staining of nasal mucosal tissue
<ol>
	<li>Dewaxing and hydration: place the slices in xylene for 20 min, repeat the process for another 20 min, absolute ethanol for 5 min, repeat this step for another 5 min, 75% alcohol for 5 min, and finally, wash for 5 min.</li>
	<li>Hematoxylin staining: dispense 100 &#181;L of hematoxylin staining solution (see Table of Materials) for 3-5 min using a 100 &#181;L pipette, rinse for 5-10 s, add 100 &#181;L of 0.5% hydrochloric acid ethanol for 10-30 s, rinse for 5-10 s, add 100 &#181;L of 0.2% ammonia water for 10-30 s, and rinse for 30 s. 
	NOTE: After hematoxylin staining, differentiation with 0.5% hydrochloric acid ethanol is necessary to remove excessive hematoxylin dye bound to the nucleus and absorbed by the cytoplasm. When eosin staining is performed, the nuclei and cytoplasm will be clearly stained. The slices will appear red or pink after differentiation with 0.5% hydrochloric acid ethanol, so rinse immediately after differentiation to remove the acid from the tissue slices to halt the differentiation. Then, use 0.2% ammonia water to enhance nuclear staining.</li>
	<li>Eosin staining: use a 100 &#181;L pipette to dispense 100 &#181;L of eosin staining solution (see Table of Materials) for 5 min and rinse for 30 s.</li>
	<li>Dehydration and sealing: immerse the slices in absolute ethanol for 5 min, repeat the process for another 5 min, repeat once more for 5 min, dimethyl for 5 min, and xylene for 5 min. Finally, seal with neutral resin (see Table of Materials).</li>
	<li>Placing the stained section on the microscope: position the stained section on the microscope, adjust the microscope&#39;s focus until the image is clearly visible, set the magnification to 40x, and capture the image.</li>
</ol>
6. Multicolor immunostaining of nasal mucosal tissue
<ol>
	<li>Dewaxing and hydration: follow the same as mentioned in step 5.1.</li>
	<li>Citrate-phosphate buffer treatment: place the citrate-phosphate buffer (pH 6.0) (see Table of Materials) into a microwave-safe container. Heat it until boiling, remove it, and then add the slides to the container. Microwave on medium heat for 8 min until boiling, turn off the heat for 8 min, and then switch to medium-low heat for 7 min.
	<ol>
		<li>After natural cooling, transfer the slices to PBS (pH 7.4), shake, and wash them three times using a decolorizing shaker, each time for 5 min. 
		NOTE: Ensure that the buffer does not evaporate during this process, and prevent the slices from drying out.</li>
	</ol>
	</li>
	<li>Blocking endogenous peroxidase: dry the slices, draw a circle around the tissue with an immunohistochemical pen (to prevent the antibody from flowing away), and apply 3% hydrogen peroxide solution (hydrogen peroxide: pure water = 1:9).
	<ol>
		<li>Incubate at room temperature in the dark for 15 min. After natural cooling, place the slices in PBS (pH 7.4) and shake them on a decolorizing shaker for three cycles of 5 min each.</li>
	</ol>
	</li>
	<li>Blocking: apply 5% goat serum within the circle and incubate for 30 min.</li>
	<li>Primary antibody addition: gently remove the blocking solution, add FOXP3 (dilution: 1:200, see Table of Materials) to the slice, and place it in a humid chamber. Incubate at 4 &#176;C overnight. 
	NOTE: Add a small amount of water to the humid chamber to prevent antibody evaporation.</li>
	<li>Secondary antibody addition: place the slices in PBS (pH 7.4) and shake them on a decolorizing shaker for three cycles of 5 min each. Gently dry the slices and apply the secondary antibody (Goat Anti-Rabbit IgG H&#38;L, see Table of Materials) within the circle. Incubate at room temperature for 50 min in the dark.</li>
	<li>Preparation of Tyramide dilution solution: combine 100 &#181;L of 3% H2O2 with 100 mL of 1x TBST (see Table of Materials).</li>
	<li>Addition of TSA working solution: Mix 1 mL of Tyramide diluent with 2 &#181;L of CY3-Tyramide to prepare TSA working solution. Apply 100 &#181;L of TSA working solution to each slice and incubate at room temperature in the dark for 10 min. Then, wash the slices with PBS three times, 5 min each time. 
	NOTE: Prepare and use the TSA working solution immediately, and store it at 4 &#176;C in the dark; it is valid for up to 24 h.</li>
	<li>Repeat Steps 6.2-6.8: switch to a 1:200 dilution of ROR&#947;t (FITC fluorescent dye) and then to a 1:200 dilution of TICAM1 (TYP620 dye) (see Table of Materials).</li>
	<li>DAPI counterstained nuclei: shake the slices gently to dry them, apply DAPI staining solution, and incubate at room temperature for 10 min in the dark.</li>
	<li>Quenching autofluorescence: place the slices in PBS (pH 7.4) and shake them on a decolorizing shaker for three cycles of 5 min each. Apply the autofluorescence quencher (see Table of Materials) to the slices, wait for 5 min, and then wash for 10 min.</li>
	<li>Blocking: place the slices in PBS (pH 7.4) and shake them on a decolorizing shaker for three cycles of 5 min each. Gently shake the slices to dry them and seal them with the anti-fluorescence quencher (see Table of Materials).</li>
	<li>Microscopy: place the stained section on the microscope, adjust the microscope&#39;s focus for clear visibility, set the magnification to 20x and 40x, and capture the image. 
	NOTE: DAPI has a UV excitation wavelength of 330-380 nm and an emission wavelength of 420 nm (blue light). FITC has an excitation wavelength of 465-495 nm and an emission wavelength of 515-555 nm (green light). CY3 has an excitation wavelength of 510-560 nm and an emission wavelength of 590 nm (red light). TYP-690 has an excitation wavelength of 630 nm and an emission wavelength of 690 nm (pink light).</li>
</ol>

Multicolor Immunofluorescence of the Nasal Tissue in an Allergic Rhinitis Rat Model

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+unsupervised+pixel-based+clustering+algorithm+for+compartmentalization+of+immunohistochemical+expression+using+automated+quantitative+analysis.">Development of an unsupervised pixel-based clustering algorithm for compartmentalization of immunohistochemical expression using automated quantitative analysis.</a> Applied Immunohistochemistry &amp; Molecular Morphology. 17 (4), 329-337 (2009).</li><li>Lu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+biomarker+modalities+for+predicting+response+to+PD-1/PD-L1+checkpoint+blockade:+a+systematic+review+and+meta-analysis.">Comparison of biomarker modalities for predicting response to PD-1/PD-L1 checkpoint blockade: a systematic review and meta-analysis.</a> JAMA Oncology. 5 (8), 1195-1204 (2019).</li><li>Hernandez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+immunofluorescence+tyramide+signal+amplification+for+immune+cell+profiling+of+paraffin-embedded+tumor+tissues.">Multiplex immunofluorescence tyramide signal amplification for immune cell profiling of paraffin-embedded tumor tissues.</a> Frontiers in Molecular Biosciences. 8, 667067 (2021).</li></ol>

The authors have nothing to disclose.

Allergic Rhinitis (AR) is a non-infectious inflammatory disease of the nasal mucosa resulting from a combination of environmental and genetic factors. It has become a global health concern, impacting work efficiency, diminishing the quality of life, impairing sleep, cognitive function, and causing irritability and fatigue. AR affects 10%-20% of the world&#39;s population&#185; and carries substantial economic costs, causing annual losses of up to 30-50 billion euros in EU countries18. Moreover, se...

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa primarily mediated by specific immunoglobulin E (IgE)1,2. It is characterized by symptoms such as sneezing, a runny nose, nasal congestion, and nasal itching. With industrialization and urbanization, the prevalence of AR is gradually increasing, affecting approximately 10%-20% of the world&#39;s population1. The immunofluorescence (IF) technique is a fluorescent staining method that utilizes an antibody-antigen binding reaction. It can be employed to detect and quantify the distribution and expression levels of specific proteins in biological tissues or cells. In AR research, IF can simultaneously detect multiple targets, including AR-related cytokines, inflammatory cells, receptors, and more, facilitating the exploration of AR pathogenesis and the effects of drugs3,4,5,6.
The multicolor immunofluorescence (mIF) staining process closely resembles traditional IF, with the addition of an antibody elution step during each round of staining. This modification enables the simultaneous detection of multiple biomarkers on the same tissue section through sequential single-labeling and multiple rounds of re-staining. mIF is based on tyramide signal amplification (TSA), allowing for repeated cycles of TSA fluorescent staining and the use of microwave heating to remove antibodies while retaining fluorescent signals7,8. In comparison to conventional IF, mIF offers several advantages: (1) it can detect weakly expressed antigens that are challenging to identify with conventional IF9,10; (2) it provides high-quality staining with an improved signal-to-noise ratio; (3) it allows for the quantification of tissue-specific structures and regions of interest11; (4) multiplexing multiple pathways efficiently utilizes tissues and conserves limited pathological resources12; (5) multi-parameter analysis through mIF offers deeper insights into tissues, uncovering hidden biological information13.
Overall, mIF allows the observation of different antigen expressions and distributions within the same sample, facilitating the study of target proteins. In the future, researchers seeking to understand the expression and distribution of multiple target proteins will find this technique a valuable choice. This study demonstrates the application of mIF for staining nasal mucosa samples from rats with AR and evaluate the establishment of a rat model of AR.

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immunofluorescent labeling in nasal mucosa tissue sections of allergic rhinitis rats via multicolor immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

Six SD rats were successfully induced into the AR model through OVA intraperitoneal injection and nasal challenge. AR was induced in all rats in the AR group, accounting for 100% of the group. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose. All behavioral observations scored &#8805;5 points (Table 2).
H&#38;E staining results on the 21st day of modeling revealed that in the control rats, the nasal mucosa&#39;s e...

Watch this Scientific Journal Video about Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence at JoVE.com

Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

Our main research is based on a study of allergic rhinitis. Our research intends to analyze what proteins are associated with allergic rhinitis. Multicolor immunofluorescence is currently used to advance research in our field.In future studies, we will focus on the possible mechanisms of TCM in the treatment of allergic rhinitis.

immunofluorescent-labeling-nasal-mucosa-tissue-sections-allergic

Research

JoVE Journal

Medicine

1.5K Views.  Hospital of Chengdu University of Traditional Chinese Medicine. Our main research is based on a study of allergic rhinitis. Our research intends to analyze what proteins are associated with allergic rhinitis. Multicolor immunofluorescence is currently used to advance research in our field.In future studies, we will focus on the possible mechanisms of TCM in the treatment of allergic rhinitis.

Video: Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact and tangible object.
However, preparation and printing of such a fully colored anatomical model remains a difficult task. Therefore, a step-by-step guide is provided, demonstrating the fusion of different cross-sectional imaging data sets, segmentation of anatomical structures, and creation of a virtual model. In a second step the virtual model is printed with volumetrically colored anatomical structures using a plaster-based color 3D binder jetting technique. This method allows highly accurate reproduction of patient-specific anatomy as shown in a series of 3D-printed petrous apex chondrosarcomas. Furthermore, the models created can be cut and drilled, revealing internal structures that allow for simulation of surgical procedures.

The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. ...

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

Hypoparathyroidism (HypoPT) is a rare disease involving the parathyroid glands that is characterized by a reduced secretion or potency of the parathyroid hormone (PTH), which leads to high serum phosphorus levels and low serum calcium levels. HypoPT most commonly results from accidental damage to the glands or their removal during thyroid or other anterior neck surgery. Parathyroid/thyroid surgery has become more common in recent years, with a corresponding rise in the occurrence of HypoPT as a postoperative complication. There is a critical need for a HypoPT animal model to better understand the mechanisms underlying the effects of HypoPT on mineral ion homeostasis and to verify the therapeutic effectiveness of novel treatments. Here, a technique is reported to create acquired HypoPT in male rats by performing parathyroidectomy (PTX) using carbon nanoparticles. The rat model shows great promise over the mouse models of hypoparathyroidism.&#160;Importantly, the human PTH receptor binding region has an 84.2% sequence similarity with that of the rat, which is higher than the 73.7% similarity shared with mice. Moreover, the effects of estrogen, which can affect the PTH/PTHrP receptor signaling pathway, have not been fully investigated in male rats. Carbon nanoparticles are lymphatic tracers that stain the thyroid lymph nodes black without affecting their function, but they do not stain the parathyroid glands, which makes them easy to identify and remove. In this study, serum PTH levels were undetectable after PTX, and this resulted in significant hypocalcemia and hyperphosphatemia. Thus, the clinical state of postoperative HypoPT can be remarkably represented in the rat model. Carbon-nanoparticle-assisted PTX can, therefore, serve as an extraordinarily effective and readily implementable model for studying the pathogenesis, treatment, and prognosis of HypoPT.

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

An animal model of acquired hypoparathyroidism (HypoPT) is crucial to understanding how HypoPT affects mineral ion homeostasis and to verify the effectiveness of novel treatments. Here, a technique is presented to generate an acquired hypoparathyroidism (AHypoPT) rat model by parathyroidectomy (PTX) using carbon nanoparticles.

Hypoparathyroidism (HypoPT) is a rare disease involving the parathyroid glands that is characterized by a reduced secretion or potency of the parathyroid ...

Innovative Lung Imaging Using Agarose-Embedded Vibratome Sections

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

Author Spotlight: Studying Spatial Protein Expression Using Agarose Embedded Lung Tissue Sections 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...

Tracheotomy-Based Endotracheal Intubation Protocol for Non-Survival Rat Models

Endotracheal Intubation via Tracheotomy and Subsequent Thoracotomy in Rats for Non-Survival Applications

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that require controlled or high ventilation pressures or access to the thoracic cavity and organs. Conventional endoorotracheal intubation using the anatomically existing route through the mouth is well suited for survival experiments. However, this procedure poses some challenges, including generally higher levels of the required experience and technical skill, more advanced equipment, and greater time effort with relevant intubation failure rates and complications such as tracheal perforation, temporary systemic hypooxygenation, and relevant aerial leakage.
This manuscript, therefore, presents a detailed step-by-step protocol for endotracheal intubation through tracheotomy in non-survival rat models when guaranteed intubation success, constant oxygenation levels, high ventilation pressures, or open thoracotomy are required.
The protocol emphasizes the importance of meticulous surgical technique to ensure consistent and reliable outcomes, especially for researchers who are inexperienced or lack routine in the technique of endoorotracheal intubation via direct laryngoscopy. This procedure is, therefore, expected to minimize animal suffering and unnecessary animal losses.

Here we introduce a standardized procedure for endotracheal intubation via tracheotomy followed by thoracotomy in rats, aimed at enhancing the precision and reproducibility of non-survival applications requiring invasive ventilation and exposure of thoracic organs in in vivo rat models.

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that ...

Rat Model for Visceral Organ Malperfusion Studies

Controlled Reversible Visceral Arterial Ischemia, Venous Congestion and Combined Malperfusion via Midline Laparotomy in Rats

Besides sepsis and malignancy, malperfusion is the third leading cause of tissue degradation and a major pathomechanism for various medical and surgical conditions. Despite significant developments such as bypass surgery, endovascular procedures, extracorporeal membrane oxygenation, and artificial blood substitutes, tissue malperfusion, especially of visceral organs, remains a pressing issue in patient care. The demand for further research on biomedical processes and possible interventions is high. Valid biological models are of utmost importance in enabling this kind of research. Due to the multifactorial aspects of tissue perfusion research, which include not only cell biology but also vascular microanatomy and rheology, an appropriate model requires a degree of biological complexity that only an animal model can provide, rendering rodents the obvious model of choice. Tissue malperfusion can be differentiated into three distinct conditions: (1) isolated arterial ischemia, (2) isolated venous congestion, and (3) combined malperfusion. This article presents a detailed step-by-step protocol for the controlled and reversible induction of these three types of visceral malperfusion via midline laparotomy and clamping of the abdominal aorta and caval vein in rats, underscoring the significance of precise surgical methodology to guarantee uniform and dependable results. Prime examples of possible applications of this model include the development and validation of innovative intraoperative imaging modalities, such as Hyperspectral Imaging (HSI), to objectively visualize and differentiate malperfusion of gastrointestinal, gynecological, and urological organs.

Author Spotlight: Advancing Spectral Characterization of Physiological and Malperfused Tissues

This article introduces a standardized procedure for controlled, reversible malperfusion of visceral organs in rat models. The aim is to induce these malperfusion states with a high degree of methodological certainty and control while maintaining technical simplicity and error resilience.

Besides sepsis and malignancy, malperfusion is the third leading cause of tissue degradation and a major pathomechanism for various medical and surgical ...

Standardized 5/6 Nephrectomy in Rats for Renal Insufficiency and Malperfusion

5/6 Nephrectomy Using Sharp Bipolectomy Via Midline Laparotomy in Rats

Chronic kidney disease (CKD) affects over 10% of the global population, amounting to more than 800 million individuals worldwide. The advances in the treatment of CKD have had a significant impact on patient outcomes. While in the past, CKD was often considered a death sentence, with many patients succumbing to the complications of end-stage renal disease, it is now increasingly being managed as a chronic condition with the availability of dialysis and kidney transplantation, as well as new pharmaceutical developments such as SGLT2 inhibitors or nonsteroidal mineralocorticoid receptor antagonists. 
Yet, there continues to be a growing demand for further exploration into the pathophysiological processes and potential therapeutic interventions. Reliable biological models play a crucial role in facilitating this research. Given the multifaceted nature of renal disease, which encompasses not only cell biology but also vascular microanatomy and endocrine signaling, an appropriate model must possess a level of biological complexity that only an animal model can offer, rendering rodents an obvious choice.
This manuscript, therefore, provides an intricate, systematic protocol for surgically reducing renal parenchyma through midline laparotomy and combined total and partial nephrectomy in rats for survival as well as non-survival applications. It emphasizes the critical role of precise surgical techniques in ensuring consistent and reliable outcomes. Prime examples of potential applications for this model include biomolecular and pharmaceutical studies as well as the development of innovative intraoperative imaging modalities, such as hyperspectral imaging, to objectively visualize and differentiate renal malperfusion.

This manuscript introduces a standardized protocol for a 5/6 nephrectomy by sharp bipolectomy via midline laparotomy in a rat model, aiming to induce a state of renal insufficiency via renal parenchyma reduction with a great degree of methodical precision and low risk of technical error.

Chronic kidney disease (CKD) affects over 10% of the global population, amounting to more than 800 million individuals worldwide. The advances in the ...

Methods Article

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay