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Traumatic shock, X-irradiation and avoidance behavior
Physiology and Behavior. Vol. 1, pp. 93-95. Pergamon Press Ltd., 1966 Printed in Great Britain
Traumatic Shock, X-Irradiation and Avoidance Behavior' DONALD
L. W H A L E Y , B A R R O N B. S C A R B O R O U G H
AND SHERWOOD
M. R E I C H A R D ~
Department of Psychology, Florida State University, Tallahassee, Florida (Received 18 June 1965) D. L., B. B. SCARBOROUGHAND S. M. REICHARD. Traumatic shock, X-irradiation, and avoidance behavior. PHYSIOL. BEHAV. 1 (1) 93--95, 1966--Spragnc-Dawlcy rats received training in a modified version of the Noble-Collip tumbling apparatus under a regime designed to produce resistance to this form of trauma. These animals were then subjected to a procedure in which X-irradiation was paired with saccharin solution. This procedure typically results in a subsequent conditioned avoidance response of saccharin solution. Trauma resistant rats showed a significantly lessened avoidance response when compared to the appropriate controls. It is suggested that resistance to tumbling trauma served to mitigate the effects of X-irradiation, thus accounting for the lessened saccharin avoidance responses. WHALEY,
Trauma X-irradiation avoidance
Irradiation
Saccharin
Trauma resistance
Avoidance behavior
Conditioned
The present research was concerned with the prospect that animals made resistant to drum-trauma in an apparatus similar to that used by Noble and Collip [5] might also exhibit attenuated reactions to low-dosage ionizing irradiation. The saccharin avoidance phenomenon provided the dependent measures. While saccharin solutions are usually preferred to the exclusion of tap water [12], ff the presentation of saccharin is contiguous with the administration of relatively low doses of ionizing irradiation, the previously preferred solution is subsequently avoided. Further, the degree of avoidance has been shown to b e dependent on such variables as irradiation dosage [2], ordinal presentation of saccharin solution and irradiation, and temporal relationships obtaining between the introduction of irradiation and saccharin solution [2, 9]. In this study the possibility that prior tumbling experience would tend to mitigate saccharin avoidance responses instigated by X-irradiation was investigated.
AN APPARATUSor technique developed for specific or limited scientific purpose, frequently proves later to have broader application. A n example is the technique developed by Noble and Collip [5] which originally devised to administer a graded, well-controlled trauma, subsequent to which typical aspects of shock developed, but without the complicaring factors of infection, hemorrhage or anesthesia. The percentage of animals surviving this type of trauma was directly proportional to the number of revolutions received in the tumbling apparatus. Perhaps of even greater interest, Noble [5] showed that animals could be made resistant to this trauma by gradually increasing the number of revolutions every other day. Such animals survived a degree of trauma far in excess of normal animals. Shorr and his colleagues [10] later found that traumaresistant rats were able to survive a degree of hemorrhage that was fatal to control animals. Zahl et al. [13] exposed rats to sub-lethal degrees of violent shaking and observed an increased tolerance to drum-tranma. Zweifach and Thomas [14] produced an increased tolerance in rats to bacterial endotoxin and found that these animals were also resistant to lethal degrees of drum-tranma or hemorrhagic shock. Griswald and Gray [3] subjected animals to a series of electroconvulsive shocks and observed a greatly enhanced resistance to drum-trauma. Reichard et al. [7] demonstrated that acidified boiled extracts of spleen or plasma taken from drum-trauma conditioned animals resulted in a significantly greater resistance when injected in animals which had not previously been exposed to drum-trauma. The author attributed this transfer of resistance to a humoral principle which may be produced and/or stored in the reticuloendothelial system. The same extract has subsequently been shown to protect against lethal doses of X-irradiation [6].
METHODS
Animals. Sixty male albino rats of the Sprague-Dawley strain were obtained from Dublin Laboratories of Dublin, Virginia. Animals were maintained on ad lib water and Purina rat pellets in home cages until they reached an approximate mean weight of 250 g. Procedures. Glass water bottles and uniform metal nipples were used to administer all oral solutions. The irradiation source employed was a Phillips deep therapy X-ray unit. This unit has a 250 kV, 50 m A capacity, is mounted in a vertical position, and employs a 2.0 m m inherent aluminium filter. The tumbling apparatus was a modified version of the apparatus introduced by Noble and Collip [5]. Exact
1This research was supported in part by U.S.P.H.S. Grant HE 08982--01, National Heart Institute, and by U.S.P.H.S. Research Fellowship Award 1-F1-MH-22, 956-01. 2Division of Radiobiology, Medical College of Georgia, Augusta, Georgia. G
93
94
WHALEY, SCARBOROUGH AND RETCHARD
specifications of the apparatus in this study are given by Reichard et aL [7]. Essentially each compartment consists of a revolving circular drum having a diameter of 15 in. and a depth of 8 in. Two opposing triangular projections 2 in. high and 3 in. at the base extend inward toward the center of the drum and are continuous with the inner surface. As the drum revolves at 40 rpm, the animal is carried up the side on one projection and is then dropped and picked up by the following projection. To prevent animals from jumping over the projections their hind paws are taped together. The tumbling apparatus had 18 such drum units and accommodated as many as 18 animals. The 60 subjects were randomly assigned to 4 groups of 15 each. Groups I and II were made trauma-resistant (TR) by the procedure outlined by Reichard et aL [7] which was designed to establish an increased resistance to drum-shock. Each of the 15 animals in Group I received 200 rev. G r o u p II received identical treatment. On the following day, Groups I and H experienced another 200 rev. On the third and fourth days, revolutions were increased to 300. Groups III and IV were placed in the tumbling apparatus for a comparable period of time (5 rain on days 1 and 2; 7.5 rain on days 3 and 4) required for 200 and 300 rev respectively. During this time, however, the drive mechanism was not activated and tumbling was not experienced. Irradiation procedures began 24 hr after termination of drum-trauma manipulations. Twelve hours prior to irradiation all animals were deprived of all fluids. Groups I and III received irradiation;' Groups II and IV received shamirradiation. The X-ray unit was energized at a 250 kV, 15 mA, and had in supplement to the inherent 2.0 m m aluminium filter, an additional 1.0 mm aluminum and 0.5 m m copper filtration. The distance from the machine head to target was approximately 200 cm. The animals were irradiated with a dose rate of 6 r/min and the total dosage was 60 r. The sham-irradiation groups received the same procedure as the radiation groups with the exception that the power tube of the X-ray machine was not activated. Directly after termination of irradiation, animals were returned to home cages where they were presented with a choice of tap water and saccharin solutions. At the time of the presentation, no more than 20 rain had elapsed since irradiation. Presentation of the choice situation consisted in the introduction of two bottles which differed only in content, one bottle containing tap water and the other 0.1 ~o saccharin solution. All bottles and contents were weighed before presentation and re-weighed 24 hr later and thereafter for each following 24 hr period until the experiment was terminated. The right-left positioning of the two bottles on the cages was changed after each weighing. The amount of each fluid consumed was determined by the differences between weight at time of presentation and weight at the end o f the 24 hr period. A saccharin index [8] was calculated for each animal for each 24 hr period over 5 days, according to the following equation: Saccharin index (S.I.) = Amount saccharin consumed (g) Total fluid consumed (g) × 100 Approximately one month after the termination of the above study (Experiment I), the experiment was repeated (Experiment II) on different animals.
RESULTS AND DISCUSSION Saccharin indices for each animal for each 24 hr period were subjected to a 2 × 2 × 5 analysis of variance after the Winer Case II design [11]. Each two levels of tumbling and irradiation treatments were analyzed across five days of repeated measures. Pair by pair comparisons were made of the cell totals of each group. Figure 1 illustrates the mean saccharin index for each group plotted across the five days of both experiments. Saccharin consumption was significantly depreSSed in both trauma-resistant irradiated and normal irradiated groups from unirradiated groups (p > 0.01). However, while TR-irradiated rats exhibit an avoidance behavior, the degree of avoidance is significantly less than the normal irradiated group (p > 0.05).
I00' 80' 60' o X
Experiment I jo~o. o "~..(~...-o Normal-Sham Irr. o0'.., ~ 0,, "
o"
•
/
••0 TR-Shom Irh
40' 20 0
X---X o --X- --X - ° °X TR - Irr. X~T~X--'-X~.X
Norm01- Irr.
DAY X W a Z Z n,.
I00.
Experiment 2
X''-X~.,.~,X"/,". ' 3
U
"
60"
"O
I'
4(>
TR" Irr" T R - Sham Irr.
"o"
/ It
20' X~X~.x~X.---X
o
Normal- Irr.
i DAY
FIG. 1. Experiment I. Mean saccharin indices for four groups of rats (N=15); irradiated trauma resistant (TR-Irr.), normal irradiated (Normal-lrr.), trauma resistant sham irradiated (TR-Sham Irr.), normal sham irradiated (Normal-Sham Irr), plotted as a function of days. Experiment 2. The experiment was repeated on different animals one month later.
Figure 1 reveals ordinal consistency of comparable groups, in both experiments. Likewise, the analysis of variance for both experiments are oritically similar. In either study both the main effects of irradiation (p > 0.01) and tumbling (p > 0.05) were significant. Comparisons of the trauma-
SHOCK, IRRADIATION AND AVOIDANCE BEHAVIOR resistant (TR) irradiated and normal irradiated groups reveal that these groups were significantly different in both experiments (p > 0.05). The latter differences are of particular interest since they indicate that prior experience in the tumbling apparatus lessens the saccharin avoidance response relative to other groups. If the degree of saccharin avoidance exhibited is a reliable and valid index of tissue damage
95 resulting from irradiation, then it appears that tumbling experience may mitigate these tissue reactions. The reason for this mitigation of the radiation response in animals made resistant to drum-trauma remains largely speculative. The existence of a basic component common to many forms of trauma, such as tumbling, hemorrhage, electric shock, ionizing irradiation, or other stimuli, may be indicated.
REFERENCES 1. Garcia, J. and Kimeldorf, D. J. Some factors which influence radiation conditioned behavior of rats. Rad. Res. 12: 719-727, 1960. 2. Garcia, J., Kimeldorf, D. J. and Hunt, E. L. The use of ionizing radiation as a motivating stimulus. Psychol. Reo. 68: 383-395, 1961. 3. Griswald, R. L. and Gray, I. Conditioning of rats to tumbling trauma by electroconvulsive shock. Am. J. Physiol. 189: 504, 1957. 4. Noble, R. L. The development of resistance by rats and guinea pigs to amounts of trauma usually fatal. Am. J. Physiol. 138: 346, 1942. 5. Noble, R. L. and Collip, J. B. A quantitative method for the production of experimental traumatic shock without hemorrhage in unanesthetized animals. Q. J. Exp. Physiol. 31: 187, 1942. 6. Reichard, S. M. Humoral protection against radiation by the RES. In press. 7. Reichard, S. M., Gordon, A. S. and Tessmer, C. F. Humoral mechanisms in protection against traumatic shock: Role of the RES. In Reticulo-endothelial Structure and Function. Edited by J. H. Heller. New York: Ronald Press, 1960.
8. Scarborough, B. B. and McLaurin, W. A. Saccharin avoidance conditioning instigated immediately after the exposure period. Rad. Res. 21: 299-307, 1964. 9. Scarborough, B. B., Whaley, D. L. and Rogers, J. Postirradiation introduction of saccharin solution and subsequent saccharin solution avoidance: A case of backward conditioning? Psychol. Rep. 14: 475-481, 1964. 10. Shorr, E., Baez, S., Metz, D. B. and Zweifach, B. W. Crossresistance to hemorrhagic shock in rats made resistant to drum-tranma: Relation to hepatic VDM inactivation mechanisms. Fed. Proc. Fedn. Am. Socs. Exp, Biol., 11: 147, 1952. 11. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962. 12. Young, P. T. and Greene, J. T. Relative acceptability of saccharin solutions as revealed by different methods. J. Comp. Physiol. Psychol. 46: 295, 1953. 13. Zahl, P. A., Hutner, S. H. and Cooper, F. S. Noble-Collip shock: Therapeutic effects with autonomic depressants: Motion factors. J. Pharmac. Exp. Ther. 77: 143, 1943. 14. Zweifach, B. W. and Thomas, L. Influence of reticuloendothelial system on response to hemorrhage and trauma. Res. Bull. 3: 31, 1957.
Traumatic Shock, X-Irradiation and Avoidance Behavior' DONALD
L. W H A L E Y , B A R R O N B. S C A R B O R O U G H
AND SHERWOOD
M. R E I C H A R D ~
Department of Psychology, Florida State University, Tallahassee, Florida (Received 18 June 1965) D. L., B. B. SCARBOROUGHAND S. M. REICHARD. Traumatic shock, X-irradiation, and avoidance behavior. PHYSIOL. BEHAV. 1 (1) 93--95, 1966--Spragnc-Dawlcy rats received training in a modified version of the Noble-Collip tumbling apparatus under a regime designed to produce resistance to this form of trauma. These animals were then subjected to a procedure in which X-irradiation was paired with saccharin solution. This procedure typically results in a subsequent conditioned avoidance response of saccharin solution. Trauma resistant rats showed a significantly lessened avoidance response when compared to the appropriate controls. It is suggested that resistance to tumbling trauma served to mitigate the effects of X-irradiation, thus accounting for the lessened saccharin avoidance responses. WHALEY,
Trauma X-irradiation avoidance
Irradiation
Saccharin
Trauma resistance
Avoidance behavior
Conditioned
The present research was concerned with the prospect that animals made resistant to drum-trauma in an apparatus similar to that used by Noble and Collip [5] might also exhibit attenuated reactions to low-dosage ionizing irradiation. The saccharin avoidance phenomenon provided the dependent measures. While saccharin solutions are usually preferred to the exclusion of tap water [12], ff the presentation of saccharin is contiguous with the administration of relatively low doses of ionizing irradiation, the previously preferred solution is subsequently avoided. Further, the degree of avoidance has been shown to b e dependent on such variables as irradiation dosage [2], ordinal presentation of saccharin solution and irradiation, and temporal relationships obtaining between the introduction of irradiation and saccharin solution [2, 9]. In this study the possibility that prior tumbling experience would tend to mitigate saccharin avoidance responses instigated by X-irradiation was investigated.
AN APPARATUSor technique developed for specific or limited scientific purpose, frequently proves later to have broader application. A n example is the technique developed by Noble and Collip [5] which originally devised to administer a graded, well-controlled trauma, subsequent to which typical aspects of shock developed, but without the complicaring factors of infection, hemorrhage or anesthesia. The percentage of animals surviving this type of trauma was directly proportional to the number of revolutions received in the tumbling apparatus. Perhaps of even greater interest, Noble [5] showed that animals could be made resistant to this trauma by gradually increasing the number of revolutions every other day. Such animals survived a degree of trauma far in excess of normal animals. Shorr and his colleagues [10] later found that traumaresistant rats were able to survive a degree of hemorrhage that was fatal to control animals. Zahl et al. [13] exposed rats to sub-lethal degrees of violent shaking and observed an increased tolerance to drum-tranma. Zweifach and Thomas [14] produced an increased tolerance in rats to bacterial endotoxin and found that these animals were also resistant to lethal degrees of drum-tranma or hemorrhagic shock. Griswald and Gray [3] subjected animals to a series of electroconvulsive shocks and observed a greatly enhanced resistance to drum-trauma. Reichard et al. [7] demonstrated that acidified boiled extracts of spleen or plasma taken from drum-trauma conditioned animals resulted in a significantly greater resistance when injected in animals which had not previously been exposed to drum-trauma. The author attributed this transfer of resistance to a humoral principle which may be produced and/or stored in the reticuloendothelial system. The same extract has subsequently been shown to protect against lethal doses of X-irradiation [6].
METHODS
Animals. Sixty male albino rats of the Sprague-Dawley strain were obtained from Dublin Laboratories of Dublin, Virginia. Animals were maintained on ad lib water and Purina rat pellets in home cages until they reached an approximate mean weight of 250 g. Procedures. Glass water bottles and uniform metal nipples were used to administer all oral solutions. The irradiation source employed was a Phillips deep therapy X-ray unit. This unit has a 250 kV, 50 m A capacity, is mounted in a vertical position, and employs a 2.0 m m inherent aluminium filter. The tumbling apparatus was a modified version of the apparatus introduced by Noble and Collip [5]. Exact
1This research was supported in part by U.S.P.H.S. Grant HE 08982--01, National Heart Institute, and by U.S.P.H.S. Research Fellowship Award 1-F1-MH-22, 956-01. 2Division of Radiobiology, Medical College of Georgia, Augusta, Georgia. G
93
94
WHALEY, SCARBOROUGH AND RETCHARD
specifications of the apparatus in this study are given by Reichard et aL [7]. Essentially each compartment consists of a revolving circular drum having a diameter of 15 in. and a depth of 8 in. Two opposing triangular projections 2 in. high and 3 in. at the base extend inward toward the center of the drum and are continuous with the inner surface. As the drum revolves at 40 rpm, the animal is carried up the side on one projection and is then dropped and picked up by the following projection. To prevent animals from jumping over the projections their hind paws are taped together. The tumbling apparatus had 18 such drum units and accommodated as many as 18 animals. The 60 subjects were randomly assigned to 4 groups of 15 each. Groups I and II were made trauma-resistant (TR) by the procedure outlined by Reichard et aL [7] which was designed to establish an increased resistance to drum-shock. Each of the 15 animals in Group I received 200 rev. G r o u p II received identical treatment. On the following day, Groups I and H experienced another 200 rev. On the third and fourth days, revolutions were increased to 300. Groups III and IV were placed in the tumbling apparatus for a comparable period of time (5 rain on days 1 and 2; 7.5 rain on days 3 and 4) required for 200 and 300 rev respectively. During this time, however, the drive mechanism was not activated and tumbling was not experienced. Irradiation procedures began 24 hr after termination of drum-trauma manipulations. Twelve hours prior to irradiation all animals were deprived of all fluids. Groups I and III received irradiation;' Groups II and IV received shamirradiation. The X-ray unit was energized at a 250 kV, 15 mA, and had in supplement to the inherent 2.0 m m aluminium filter, an additional 1.0 mm aluminum and 0.5 m m copper filtration. The distance from the machine head to target was approximately 200 cm. The animals were irradiated with a dose rate of 6 r/min and the total dosage was 60 r. The sham-irradiation groups received the same procedure as the radiation groups with the exception that the power tube of the X-ray machine was not activated. Directly after termination of irradiation, animals were returned to home cages where they were presented with a choice of tap water and saccharin solutions. At the time of the presentation, no more than 20 rain had elapsed since irradiation. Presentation of the choice situation consisted in the introduction of two bottles which differed only in content, one bottle containing tap water and the other 0.1 ~o saccharin solution. All bottles and contents were weighed before presentation and re-weighed 24 hr later and thereafter for each following 24 hr period until the experiment was terminated. The right-left positioning of the two bottles on the cages was changed after each weighing. The amount of each fluid consumed was determined by the differences between weight at time of presentation and weight at the end o f the 24 hr period. A saccharin index [8] was calculated for each animal for each 24 hr period over 5 days, according to the following equation: Saccharin index (S.I.) = Amount saccharin consumed (g) Total fluid consumed (g) × 100 Approximately one month after the termination of the above study (Experiment I), the experiment was repeated (Experiment II) on different animals.
RESULTS AND DISCUSSION Saccharin indices for each animal for each 24 hr period were subjected to a 2 × 2 × 5 analysis of variance after the Winer Case II design [11]. Each two levels of tumbling and irradiation treatments were analyzed across five days of repeated measures. Pair by pair comparisons were made of the cell totals of each group. Figure 1 illustrates the mean saccharin index for each group plotted across the five days of both experiments. Saccharin consumption was significantly depreSSed in both trauma-resistant irradiated and normal irradiated groups from unirradiated groups (p > 0.01). However, while TR-irradiated rats exhibit an avoidance behavior, the degree of avoidance is significantly less than the normal irradiated group (p > 0.05).
I00' 80' 60' o X
Experiment I jo~o. o "~..(~...-o Normal-Sham Irr. o0'.., ~ 0,, "
o"
•
/
••0 TR-Shom Irh
40' 20 0
X---X o --X- --X - ° °X TR - Irr. X~T~X--'-X~.X
Norm01- Irr.
DAY X W a Z Z n,.
I00.
Experiment 2
X''-X~.,.~,X"/,". ' 3
U
"
60"
"O
I'
4(>
TR" Irr" T R - Sham Irr.
"o"
/ It
20' X~X~.x~X.---X
o
Normal- Irr.
i DAY
FIG. 1. Experiment I. Mean saccharin indices for four groups of rats (N=15); irradiated trauma resistant (TR-Irr.), normal irradiated (Normal-lrr.), trauma resistant sham irradiated (TR-Sham Irr.), normal sham irradiated (Normal-Sham Irr), plotted as a function of days. Experiment 2. The experiment was repeated on different animals one month later.
Figure 1 reveals ordinal consistency of comparable groups, in both experiments. Likewise, the analysis of variance for both experiments are oritically similar. In either study both the main effects of irradiation (p > 0.01) and tumbling (p > 0.05) were significant. Comparisons of the trauma-
SHOCK, IRRADIATION AND AVOIDANCE BEHAVIOR resistant (TR) irradiated and normal irradiated groups reveal that these groups were significantly different in both experiments (p > 0.05). The latter differences are of particular interest since they indicate that prior experience in the tumbling apparatus lessens the saccharin avoidance response relative to other groups. If the degree of saccharin avoidance exhibited is a reliable and valid index of tissue damage
95 resulting from irradiation, then it appears that tumbling experience may mitigate these tissue reactions. The reason for this mitigation of the radiation response in animals made resistant to drum-trauma remains largely speculative. The existence of a basic component common to many forms of trauma, such as tumbling, hemorrhage, electric shock, ionizing irradiation, or other stimuli, may be indicated.
REFERENCES 1. Garcia, J. and Kimeldorf, D. J. Some factors which influence radiation conditioned behavior of rats. Rad. Res. 12: 719-727, 1960. 2. Garcia, J., Kimeldorf, D. J. and Hunt, E. L. The use of ionizing radiation as a motivating stimulus. Psychol. Reo. 68: 383-395, 1961. 3. Griswald, R. L. and Gray, I. Conditioning of rats to tumbling trauma by electroconvulsive shock. Am. J. Physiol. 189: 504, 1957. 4. Noble, R. L. The development of resistance by rats and guinea pigs to amounts of trauma usually fatal. Am. J. Physiol. 138: 346, 1942. 5. Noble, R. L. and Collip, J. B. A quantitative method for the production of experimental traumatic shock without hemorrhage in unanesthetized animals. Q. J. Exp. Physiol. 31: 187, 1942. 6. Reichard, S. M. Humoral protection against radiation by the RES. In press. 7. Reichard, S. M., Gordon, A. S. and Tessmer, C. F. Humoral mechanisms in protection against traumatic shock: Role of the RES. In Reticulo-endothelial Structure and Function. Edited by J. H. Heller. New York: Ronald Press, 1960.
8. Scarborough, B. B. and McLaurin, W. A. Saccharin avoidance conditioning instigated immediately after the exposure period. Rad. Res. 21: 299-307, 1964. 9. Scarborough, B. B., Whaley, D. L. and Rogers, J. Postirradiation introduction of saccharin solution and subsequent saccharin solution avoidance: A case of backward conditioning? Psychol. Rep. 14: 475-481, 1964. 10. Shorr, E., Baez, S., Metz, D. B. and Zweifach, B. W. Crossresistance to hemorrhagic shock in rats made resistant to drum-tranma: Relation to hepatic VDM inactivation mechanisms. Fed. Proc. Fedn. Am. Socs. Exp, Biol., 11: 147, 1952. 11. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962. 12. Young, P. T. and Greene, J. T. Relative acceptability of saccharin solutions as revealed by different methods. J. Comp. Physiol. Psychol. 46: 295, 1953. 13. Zahl, P. A., Hutner, S. H. and Cooper, F. S. Noble-Collip shock: Therapeutic effects with autonomic depressants: Motion factors. J. Pharmac. Exp. Ther. 77: 143, 1943. 14. Zweifach, B. W. and Thomas, L. Influence of reticuloendothelial system on response to hemorrhage and trauma. Res. Bull. 3: 31, 1957.