stimulation of tear production in mice and sample collection using schirmer strips

Protocollo per l'estrazione delle proteine lacrimali in modelli animali sperimentali

Tears are considered a plasma ultrafiltrate and have also been described as an intermediate fluid between plasma serum and cerebrospinal fluid due to a significant overlap in the biomolecules they share1. It has been reported that human tears contain proteins, tear lipids, metabolites, and electrolytes2. Recently, other biomolecules such as mRNAs, miRNAs, and extracellular vesicles have also been identified3,4,5,6,7.
In humans, basal tears are located in the tear film, which consists of three layers: the outer lipid layer, which maintains the tear surface smooth to allow us to see through it and prevent tear evaporation; the middle aqueous layer, which keeps the eye hydrated, repels bacteria, protects the cornea, and constitutes 90% of the tear film; and finally, the mucin layer, a family of high molecular weight proteins that are in contact with the cornea and allow the tear to adhere to the eye8. Tear distribution across the ocular surface begins with secretion from the lacrimal gland. This fluid is then guided through tear ducts to pass over the eye&#39;s surface and flow into drainage channels. Each blink allows tears to disperse evenly over the entire eye, keeping it moist9.
The tear film is a highly dynamic biofluid capable of reflecting molecular changes that occur not only on the ocular surface but also in other tissues and organs. The analysis of differential expression in this biofluid represents a promising approach for the discovery of biomarkers in human diseases10,11. The utilization of tear film as a source of biomarkers for early diagnosis in various pathologies has been greatly facilitated by the presence of non-invasive collection methods. The most common method for collecting tears in human and veterinary clinics involves membrane-based support (Schirmer&#39;s strip), which operates on the principle of capillary action, allowing the water in tears to travel along the length of a paper test strip or capillary tubes placed in the subject&#39;s lower conjunctival sac12,13,14. Despite the inherent limitation of obtaining a small sample volume through this method, the biochemical analysis of tear composition using various sensitive techniques has facilitated the identification of potential biomarker molecules11,15. Protocols for optimizing and evaluating the elution of tear proteins from Schirmer strips and capillaries in human patients are well documented16,17. However, complete descriptions of optimized protocols specifically designed to extract molecular information related to tears from experimental animal models are available but scarce. Existing methods, such as tear induction through direct stimulation of the lacrimal gland18, while allowing for the collection of larger volumes, are invasive and can cause discomfort to the animals. Non-invasive methods, such as collecting tears from the ocular surface, have been described as a way to isolate DNA and miRNAs19,21.
This protocol aims to establish a cost-effective and optimized method for collecting and processing tears in mice. The method prioritizes non-invasiveness while obtaining sufficient tear volumes suitable for molecular analysis through techniques like SDS-PAGE, qPCR, and dPCR. The extracted protein and mRNA content information can then be utilized to identify potential biomarkers in existing experimental models of disease.

Autore in primo piano: Isolamento di biomolecole dalle lacrime di topo - Una metodologia per l'analisi molecolare e la ricerca sui biomarcatori

Ottimizzazione della raccolta lacrimale nei topi per l'analisi di mRNA e proteine

We are interested in exploring the feasibility of isolating biomolecules such as proteins, DNA, RNAs from mouse tears, similar to what has been done in humans. This would allow us to develop a simple and efficient methodology to use in scientific basic research. In humans, a diverse range of technologies has been employed to unravel the molecular composition of tears, such as immunoassays like Western blot analyzer as well as mass spectrometry.These analyses have paved the way for the development of biosensors capable of identifying specific molecules of interest. We have developed an optimal methodology for obtaining a substantial volume of tears from mice, facilitating their molecular analysis in basic science laboratories. This protocol facilitates the identification of different proteins and messenger RNA of interest.Researching biomarkers present in eye liquid biopsy is in the public spotlight nowadays. Whether it is for eye diseases such as diabetic retinopathy or systemic diseases like Alzheimer or Parkinson, a minimally invasive sample, the tear film, poses an opportunity for diagnosis. As has been reported in humans, we have addressed the next question for our mirroring model.The tears reflect molecular changes that appear in diseases. Those tear composition harbor biomolecules that can be found in other liquid biopsies such as blood or saliva.

Stimolazione della produzione lacrimale nei topi e raccolta di campioni utilizzando strisce di Schirmer

stimulation-tear-production-mice-sample-collection-using-schirmer

Research

JoVE Journal

Biology

Stimulation of Tear Production in Mice and Sample Collection Using Schirmer Strips

104 Views. Per iniziare, preparare una soluzione madre di pilocarpina utilizzando acqua sterile. Quindi, taglia le strisce reattive di Schirmer a cinque millimetri di lunghezza. Quindi preparare 250 microlitri di acqua sterile con un inibitore della proteasi.Pesare ogni topo individualmente per calcolare il dosaggio appropriato dei trattamenti. Per stimolare la produzione di lacrime, utilizzando una siringa da insulina da sei millimetri, somministrare 30 milligra...

Video: Stimolazione della produzione lacrimale nei topi e raccolta di campioni utilizzando strisce di Schirmer

Optimizing Tear Collection in Mice for mRNA and Protein Analysis

The tear film is a highly dynamic biofluid capable of reflecting pathology-associated molecular changes, not only in the ocular surface but also in other tissues and organs. Molecular analysis of this biofluid offers a non-invasive way to diagnose or monitor diseases, assess medical treatment efficacy, and identify possible biomarkers. Due to the limited sample volume, collecting tear samples requires specific skills and appropriate tools to ensure high quality and maximum efficiency. Various tear sampling methodologies have been described in human studies. In this article, a comprehensive description of an optimized protocol is presented, specifically tailored for extracting tear-related protein information from experimental animal models, especially mice. This method includes the pharmacological stimulation of tear production in 2-month-old mice, followed by sample collection using Schirmer strips and the evaluation of the efficacy and efficiency of the protocol through standard procedures, SDS-PAGE, qPCR, and digital PCR (dPCR). This protocol can be easily adapted for the investigation of the tear protein signature in a variety of experimental paradigms. By establishing an affordable, standardized, and optimized tear sampling protocol for animal models, the aim was to bridge the gap between human and animal research, facilitating translational studies and accelerating advancements in the field of ocular and systemic disease research.

The identification of RNA and protein biomarkers from tears in mouse models holds great promise for early diagnostics in various diseases. This manuscript provides a comprehensive protocol for optimizing the efficacy and efficiency of mRNA and protein isolation from mouse tears.

The tear film is a highly dynamic biofluid capable of reflecting pathology-associated molecular changes, not only in the ocular surface but also in other ...

Ricerca

Biologia

To begin, prepare a stock solution of pilocarpine using sterile water. Next, cut Schirmer's test strips to five millimeters in length. Then prepare 250 microliters of sterile water with a protease inhibitor.Weigh each mouse individually to calculate the appropriate dosage of treatments. To stimulate tear production, using a six millimeter insulin syringe, administer 30 milligrams per kilogram of pilocarpine intraperitoneally. For tears collection, place the end of each Schirmer strip fragment over the center of the lower eyelid with a pair of tweezers.Keep it in place until the tear reaches the strip limit, then replace the strip. Then place the Schirmer strip fragments in sterile tubes on ice to avoid degradation of components.

Isolating Mouse Tear Protein and its Analysis Using SDS-PAGE

Begin by using Schirmer strip fragments to collect the tear. Place these strips in a 600-microliter microcentrifuge tube containing 20 microliters of sterile water with a protease inhibitor and centrifuge at 600g for one hour at four degrees Celsius. Using a 7.5-centimeter stainless steel injector needle, puncture the tip of the 600-microliter tube containing the sample.Transfer this punctured tube to a new sterilized 1.5-milliliter tube and centrifuge the samples at 12, 000g for 10 minutes at four degrees Celsius to recover the volume of diluted tears. Combine the supernatant recovered from all the samples to create a protein pool of approximately 200 microliters. Heat the protein samples at 99 degrees Celsius for four minutes to denature the proteins.Vortex the samples briefly to mix the components. Then, load 30 micrograms of quantified protein content from each sample onto a 10%SDS-PAGE gel and run the samples at 90 volts for two hours using a standard method. After electrophoresis, cover the gel with staining solution and incubate for 30 minutes to stain the proteins.Then, rinse the gel with the destaining solution several times until the background becomes clear and protein bands are visible. Acquire images with a gel documentation system. Coomassie stained SDS-PAGE analysis of total protein extracts revealed patterns of protein expression in total retina and tears.

Isolamento della proteina lacrimale del topo e sua analisi mediante SDS-PAGE

Tear mRNA Extraction for qPCR to Evaluate the Efficacy and Efficiency of the Tear Collection Protocol

After collecting mouse tears using Schirmer strip fragments, place the strips in a 600 microliter micro centrifuge tube containing 20 microliters of sterile water. Puncture the tip of the tube containing the sample, and transfer it to a new sterilized 1.5 milliliter tube. Centrifuge the tube at 2, 000G for 20 minutes at 4 degrees Celsius.Then, pull the samples to get a total volume of approximately 22 microliters of supernatant per mouse. Place the tubes on dry ice to prevent biomolecule degradation. Next, add 200 microliters of a monophasic solution of phenol and guanidinium thiocyanate homogenized by pipetting, and allow the mixture to stand for five minutes at room temperature.Then, add 40 microliters of chloroform. Vortex for 15 seconds and incubate the mixture for two minutes at room temperature. After that, centrifuge at 10, 000G for 15 minutes at 4 degrees Celsius.Carefully transfer the aqueous phase to a new 1.5 milliliter tube without disturbing the interphase. Next, add 0.5 microliters of glycogen to 100 microliters of isopropanol and mix by pipetting. Allow the mixture to stand for 10 minutes at minus 20 degrees Celsius.Then, centrifuge at 10, 000G for 10 minutes at 4 degrees Celsius. Quickly decant the supernatant. Add 200 microliters of cold 75%ethanol and vortex the tube to resuspend the RNA pellet.Centrifuge at 8, 000G for five minutes at 4 degrees Celsius. After removing ethanol and drying the tube for 20 to 30 minutes, add 20 microliters of RNase-free water and resuspend the pellet. Then, measure the optical density in the micro volume spectrophotometer at 260 and 280 nanometers to assess the purity of RNA.To synthesize cDNA from Tear RNA, add 10 to 5, 000 nanograms of RNA. Mix it with one microliter of oligo dT and up to 12 microliters of RNase-free water. Spin at 500G for 30 seconds and incubate at 65 degrees Celsius for five minutes.Then, add four microliters of reaction buffer, one microliter of RNase inhibitor, two microliters of dNTP mix, and one microliter of reverse transcriptase to the mixture. After spinning the tube, incubate at 42 degrees Celsius for 60 minutes. After that, incubate at 70 degrees Celsius for 10 minutes to conclude the reaction.Use the program and primers shown here for the qPCR cycle. Perform a milk curve analysis to check for the presence of primer dimers or unspecific amplicons in the reaction. The qPCR revealed CT values between 20 and 24, a suitable range for the starting amount of target mRNA in the sample.However, the melt curve analysis suggested the low presence of target mRNA. The 2%agarose gel confirmed the presence of amplicons at the expected size.