Patrick R. Olson and Dr. Reuben Ward Rhees, Zoology

We have been investigating how stress affects physiological development. My interest dealt with the effects of stress on the immune system. Lymphocyte proliferation is a common measurement of immune function.

A new paradigm for stress is being used dealing with maternal separation, performed under the ethical standards set forth by the animal research and care organizations. This relatively new paradigm is being used in numerous research facilities including: Duke University Medical Center (Pihoker, C. et al., 1993), Stanford University School of Medicine (Bayart, F. et al., 1990), and University of Colorado Health Sciences Center (Laudenslager, M., et al., 1990). We take newborn rat pups and separate them from their mothers for an allotted period of time every day. This maternal separation acts as a stressor on the rats.

This stress was found to affect the development of the rats in several aspects. In an earlier experiment in our laboratory it was found that the body weight was significantly lower in separated rats compared to the control groups. In this experiment, some physiological changes dealing with glucocorticoids were found.

My portion of the experiment was an attempt to replicate the results of an earlier experiment done by Ackerman, S.H. et al., (1988) (1), in which it was found that premature maternal separation of new born rat pups resulted in a highly significant decrease in peripheral blood lymphocyte proliferation in response to the mitogen PHA (F= 18.57, df= 1,62, p<.0001). These authors suggested that “undernutrition associated with premature weaning may be one factor” related to their results. In this experiment we attempted to control for this nutrition factor by only separating the pups for 6 hours a day in three groups and for a half-hour a day in two groups, instead of permanent premature separation. We also attempted to control for the stress hyporesponsive period (SHRP) which usually occurs from day 4-14, by having groups stressed during and continuing after that time period and observing any noticeable differences. There were five groups studied as listed in Table 1.

At day 45, the animals were randomly removed from their cages and exsanguinated by heart puncture under anesthesia within 5-10 minutes. Aliquots of blood from each animal were used for the lymphocyte proliferation studies. Lymphocyte stimulation in response to the mitogen PHA was assessed according to the technique outlined by Keller, Schleifer, McKegney, Sherman, Camerino, and Stein (1982) (2).

In Ackerman, S.H. et al., (1988) (3), lymphocyte activity was determined by the log transformed counts per minute (cpm) of PHA-stimulated lymphocytes minus the log transformed counts per minute (cpm) of PHA-unstimulated lymphocytes. Performing an ANOVA on these numbers, it was found that there was no significant difference between the groups. Further ANOVA calculations showed no significant difference of the average counts per minute as well. Although it is apparent from the above results that there is an increase in PHA-stimulated lymphocytes of the stressed animals in comparison to the control, no significance was found. One probable explanation may be due to the small control sample size (3 rats) in comparison to the total number in the stressed groups (12 rats). Another reason, on more of a humorous note, may be due to the lack of experience this undergraduate researcher had on determining lymphocyte activity.

This project could be a pilot study for future research, with a larger sample size suggested. The literature indeed shows a stress-induced effect on the immune system (see Givalois, L. et al., 1994; Fleshner, M. et al., 1995; Esterling, B. & Rabin, B.S., 1987; Michaut R.J., et al., 1981). Neuroscientist Karen Bulloch said: “Careful, systematic research to decipher the connection between psychological factors and immune function may take years. But the potential benefits will be tremendous” (4).

I am forever indebted to Dr. Ward Rhees of the Zoology Department for his great patience, understanding, and encouragement for such an undertaking. I would also like to thank all those who helped me carry out the tedious and extremely time consuming protocol, especially David Eliason. Also deserving special recognition are the professors and their research undergraduate and graduate students for giving me so much advice, assistance and access to their labs and facilities, including: Dr. Burton, Dr. Judd, Dr. O’neill, Dr. Lephart and Dr. Jensen. Lastly I must acknowledge and thank Tyler Thacker of Dr. Burton’s lab for his tremendous assistance.

References

1. Ackerman, S.H., et. al. (1988). Brief communication: premature maternal separation and lymphocyte function. Brain, Behavior, and Immunity, 2, 161-165.
2.  Keller, Schleifer, McKegney, Sherman, Camerino, and Stein (1982). A simplified method for assessing PHA induced stimulation of rat peripheral blood lymphocytes. Journal of Immunological Methods, 51, 287-291.
3.  Ackerman, S.H., et. al. (1988). Brief communication: premature maternal separation and lymphocyte function. Brain, Behavior, and Immunity, 2, 161-165.
4.  Reader’s Digest (Sep. 1989). Your mind’s healing powers: A compilation.