This protocol is significant in that it provides both a comprehensive and low cost time effective technique. It answers key elements of translational research and categorizing component data on both individual and group levels. This technique is advantageous due to the total amount of information that can be extracted regarding the overall profile of the animal.
This includes the categorizing components and the subsequent conclusions. This technique could provide insight into a broad range of research areas, including cardiology, neurology, and pharmacology. This technique is not solely applicable to reproductive studies.
Begin by handling the rat with an age appropriate hold, such as the compression style hold for adolescent aged rats, by placing the subject against the upper chest region with its nose pointed down at the ground. Expose the vaginal canal of the subject by gently flexing the tail before inserting the syringe. While holding the syringe steady with the fore finger and middle finger insert the tip of the syringe into the vaginal canal at an angle parallel to it, slowly expel the sodium chloride into the canal by pushing the plunger inwards with the thumb.
Extract the sodium chloride from the vaginal canal by pulling the syringe's plunger away from the epithelial lining. Use the criteria for each cell type to characterize the cells present in the sample and record the findings. A nucleated keratinized epithelial or quantified epithelial cells will appear jagged or angular edged.
Despite the lack of nuclei, it may show light round areas within the cell representing where a nucleus was once present called ghost nuclei. The large nucleated epithelial, or LNE cells, are typically round to polygonal shaped cells encased by irregular jagged or angular borders. Their nuclei can range from intact to degenerate or pyknotic relating to the irreversible condensation of chromatin in the nucleus of a cell undergoing death or deterioration.
The nuclei take up less space than the cytoplasm within the cell, with a lower nuclear to cytoplasmic ratio than the small epithelial cells. Leukocytes are compact spherical cells. The smallest when compared to all other described cell types.
SNE cells are non-keratinized cells with round nuclei that take up a larger space than the cytoplasm inside the cell, creating a higher nucleus to cytoplasm ratio relative to large epithelial cells. Use the lower objectification, such as four X to get a representative view of the overall cell arrangement. Record whether the cells are clumped together, evenly dispersed, or randomly dispersed, and note the specific organization of each cell type.
Then visually estimate and record the total cell quantity, as a smidge, moderate, or numerous, and the individual cell quantities as a percentage of each cell type present. With these categorizing components combined, determine which estrous cycle stage is being presented. Within the diestrus stage, it can be seen that leukocytes are the dominant or the only cell type.
These are typically arranged in a clumped manner at the beginning of, but more dispersed in late stages. The total quantity of cells may be comparatively low, most often in the later stages of the diestrus period on the second or third day. Concentrated strands of leukocytes may be observed due to the high amount of mucus that may be present in this stage.
Small clumps of cellular strands of SNE cells accompanying the leukocytes may be seen during late phases in the transition to proestrus. The vaginal fluid, when transitioning into, fully transitioned into, and transitioning out of diestrus, appears viscous and opaque. During the proestrus stage, SNE cells are the dominant cells while leukocytes, LNE, and AKE cells may be found in low numbers.
SNE cells are typically arranged in clusters, sheets, or strands during this stage and have a granular appearance that can be observed at higher magnification. The vaginal fluid becomes non viscous and transparent once the subject fully transitions into the proestrus stage. The AKE cells are dominant during the estrous stage, with a decrease in the SNE cells, and an overall increase in the number and size of cells as estrous continues.
The AKE cells are often clustered in the form of keratin bars. They may also contain ghost nuclei that can become more dispersed in the transition from proestrus to metestrus. The vaginal fluid is non viscous and transparent as the rats are transitioning into, fully transition into, and transition out of estrous.
As the rats transition into metestrus, look for all cell types, though there is often a higher amount of SNE and LNE cells compared to the LEUs. A greater amount of debris is observed during metestrus and the transitions due to the epithelial cell decay following estrous. During this stage, there is a lack of consistent arrangement as all cell types are seen and in various amounts.
However, the leukocytes that are packed in proximity to the epithelial cells in the beginning stages may return to the clumped arrangement when transitioning into diestrus. The vaginal fluid appears non viscous and transparent in the stage. And changes to a more viscous and opaque appearance while moving into diestrus.
Within each stage, many variations can be seen, for example within the diestrus stage, both epithelial cells and leukocytes were observed. In the arrangements, clumped mucus strands, composed of leukocytes, and initial clumping of leukocytes, and a later random or even disbursement were also seen. When collecting the vaginal fluid sample, there were non-represented collections that warranted repeat lavages, which included the extraction of squamous cells from the vaginal canal wall due to improper syringe insertion.
A collection of solely debris, a cell count that was too low to categorize, and those that were out of focus. It is common to graph the stage progressions on bar graphs, which allows researchers to examine the overall cycle pattern and identify a-cyclisity. And the samples can then be analyzed by the cycle length and stage progression pattern.
Estrous cycle profiles can be created using a stacked bar graph, either tracking the stage progression per rat or per cohort. Where the categorizing components for each day are graphed either per day or per stage. The categorizing component data can also be organized numerically to create standardized ranges and collect statistics.
Practice the technique so it is minimally invasive and scan the microscope slide for representative samples to decrease selection bias. Overall, remember, there is no such thing as a normal cycle. Following this procedure, experimental variables could be introduced, such as exercise and behavioral paradigms.
This would allow research to establish baseline cycling of animals beforehand and to detect hormonal responses to specific interventions afterwards. This method allows researchers to explore the connection between sex steroid hormones and other bodily systems. An example of this is how cardiac receptors for sex steroid hormones relates to system functionality.
