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LAB 9 CONFINEMENT STRESS AND GLUCOCORTICOID ACTIVITY IN MICE
I. Introduction
This is the first of two labs we will do on cortisol biology. In this lab we will examine the effect of this hormone on blood cells. It is well documented that stress can negatively impact the immune system. One manner in which stress elicits a physiological response is by increasing the levels of glucocorticoids from the adrenal cortex in response to rising levels of ACTH from the pars distalis (anterior pituitary).
In response to a number of stressors including restraint stress, the leukocyte distribution has been shown to change in animals, including mice. After just an hour of restraint, it has been proposed that neutrophils have statistically higher and lymphocytes have statistically lymphocytes lower numbers compared to animals prior to stress application. This is an excellent experimental model to examine the effects of stress on glucocorticoid levels and the resulting distribution of immune system cells in the blood.
In this model system we are proposing that glucocorticoid levels in the blood increase in response to stress resulting from restraint of the mouse. It is thought that the adrenal medullary hormones (epinephrine/norepinephrine) do not play a part in the immune response to restraint in mice. As you know, there are actions actions of cortisol and other glucocorticoids on the immune system. This experiment is designed to examine only leucokyte distribution.
Each lab group will be given 2 female mice. One in each age group will serve as a control. The other will be subjected to stress. Each group will take a blood sample at the beginning of the experiment, another after 60 minutes. Stress will be applied to the experimental animals while the control will be returned to her cage and placed in a quiet place for one hour. After one hour, a blood smear will again be taken from each animal. The smears will be stained and a WBC differential count done on each to look for a change in the percentage distribution of the neutrophils and the lymphocytes .
II. Procedure EXPERIMENTAL DESIGN MATRIX
1. Place each mouse in the restraint device to obtain a blood sample. We will try to use the same technique we used to collect blood in the rats. Use a small needle to nick the lateral vein in the tail of the mouse 2/3 of the distance from the base. Sometimes it is difficult to collect blood from the lateral vein, if so an alternative method involves using a sharp razor to nick the end of its tail. Place a drop of blood on each of two slides.
While one member of the group is applying pressure to the nicked tail to stimulate clotting, a second member of the group is making the blood smears and marking the slides. Be sure to code the slides so you do not get them mixed up.
2. After a blood sample has been taken from the control mouse, it is returned to its cage for one hour.
3. After a blood sample is taken from the ”restraint stressed" mouse, it is left in the restraint chamber to put it under conditions of stress for one hour.
4. During the hour of waiting, you may proceed with the staining of the time=0 slides and examine the commercial prestained blood slides. Stain only one of each pair of slides for each mouse. The WBC differential count of the slides may also be begun.
A. Examine Prepared Blood Smears to Identify WBC’s
Find examples of the following cell types on the prepared slides provides so you will be able to recognize them in the mouse blood slides you will be making. You should be able to see them under high dry magnification. A method of preparing smears of blood and a staining technique is described at the end of this section.
Most of the cells you see in your blood smears will be erythrocytes or RBC's. Leukocytes or WBC's make up 1% or less of all the blood cells on a given slide. There are a number of different leukocytes, for this lab we are interested in identifying and counting the two most common, neutrophils and lymphocytes.
a. Neutrophil are the most numerous of the white cells and make up about 55-65% of the white cells. Neutrophils have nuclei that are characteristically multilobed with 3-5 lobes.
d. Lymphocytes make up about 20-35% of the white cells in adults, more in children. These are smaller cells that contain large, round or kidney shaped nuclei with cytoplasm that often appears as a narrow rim around the large nucleus.
Lymphoctes Neutrophil
B. Preparation of Blood Smears
Slides must be clean for a uniform smear. Handle slides at the edges, keeping fingers off of the clean surface. Touch the next drop of blood to the clean surface of the right end of the slide. Place the narrow edge of another slide at a 20-30 degree angle on the first slide and to the left of the drop of blood. Pull to the right until the slide touches the blood.
As soon as the blood has spread along the line of contact, push the right hand toward the left. Push steadily until all of the blood disappears or the other end of the slide is reached. Move the hand rapidly; if the smear is spread to slowly, the leukocytes concentrate along the edges and in the tail of the smear. This method drags the blood but does not run over it and crush the cells. Dry the slides rapidly in the air by waving them.
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Stock solutions of Wright's stain and buffer will be provided for you. The procedure is as follows:
a. With a wax pencil, draw lines across the slide to delineate the region of the slide to be stained (about 40 mm).
b. Cover this region with 10-12 drops of Wright's stain for 4 minutes.
c. Add an equal amount of buffer for 5 minutes.
d. Rinse in distilled water (1 or 2 dips). You can avoid the formation of precipitate on the slide flushing the slide with water from a pipette.
e. Blot with 2 sheets of filter paper, press to the slide do not rub.
f. Allow the slide to dry before examining under high-dry magnification.
4. After one hour, a second blood sample is taken. The blood sample may be taken while the stressed mouse is still in the commercial restraint chamber. When the stressed mouse has been sampled, it is removed to a cage marked “stressed mouse”.
A blood sample is then taken from the control mouse, after which they are returned to the original home cage. Two slides for each mouse are prepared. Mark the slides.
5. Stain only one of each pair of slides.
6. A WBC differential leukocyte count is performed on each slide. Both members of each group do differentials on each slide and an average is taken of the two observations.
To evaluate the stained blood smears, systematically move the slide across the microscope stage and record each type of WBC until 100 cells have been counted. From these counts, the percentage of each type of WBC can be calculated. Since many students will be evaluating blood smears for the first time, we will find it useful to pool data from the student groups and provide averages to the class.
III. Questions/Data Collection
1. Record the counts from all members of the group. All counts and the averages go into the table directly.
2. Use a t-test or ANOVA to look for significant differences in numbers of WBC’s in the control and stressed mice.
3. Are the data as predicted, why or why not?
IV. Materials
1. Female mice, two per group 2. Mouse restraint for taking blood samples from tail 3. Wrights Stain 4. Microscope slides 5. Slides of stained blood smears 6. Pictures of different white blood cell types 7. Microscopes 8. Dissecting instruments for taking blood samples from mice 9. Small gauge needles and syringe for anesthetizing mice 10. Ketamine solution 11. Confinement containers (examples below)
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