THE EFFECT OF DIMETHYL SULFOXIDE ON HY POTHALAMIC-PITUITARY-ADRENAL FUNCTIONS 1N THE RAT* John P. Allen Department ojthe Air Force United States Air Force School of Aerospace Medicine Brooks Air Force Base. Texas 78235 Catherine F. Allen Southwest Foundation for Research and Education Son Antonio. Texas 78284
The unusual chemical properties and clinical applications of dimethyl sulfoxide (DMSO) have been the subject of many scientific investigations. These studies have included exploration of the mechanism of action of its anti-inflammatory effect in mammals, We considered it possible that one mode of action of DMSO was to stimulate increased glucocorticoid secretion. Some evidence from a study of the rat supports this hypothesis.' Furthermore, other chemicals, including diethyl ether,2 f ~ r m a l d e h y d e , ethan01,~ ~ and urethane: which resemble DMSO both in configuration and size, stimulate the release of ACTH in the rat; but the administration of DMSO to rats does not apparently cause the same physiological effects (such as irritation and pain, anesthesia, or impaired conciousness) as the others. These previously reported findings prompted us to explore the mechanism through which glucocorticoids are released. This was accomplished by measuring plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations in DMSO-treated rats.
MATERIALS AND METHODS Eflect of DMSO in Vitro In order to test for the presence of interference or cross-reactivity of DMSO in the ACTH and corticosterone radioimmunoassays and the corticosterone acidfluorescence tests, 0.5 ml 100% DMSO was added to 5.0 rnl buffer, reagents, or hormone-free plasma, and the assays were performed. Each sample was assayed in triplicate. Effect of DMSO in the Rat in Vivo Intact Animals. Adult male Sprague-Dawley rats weighing 250 f 50 g were used in all experiments. All animals were given Purina Lab Chow and water ab lib. Four *This study was done in part while Dr. J. P. Allen was affiliated with the Division of Endocrinology, University of Oregon Medical School, Portland, Oregon. It was supported in part by grants from the National Institute of Health.
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were housed in each cage, and they were kept in an environment with constant temperature (24 f 1°C) and controlled lighting (12 hours of light beginning at 6 A.M., and 12 hours of darkness). All animals were maintained under these conditions for 7 days before the administration of DMSO. All studies were performed between 9 A.M. and 10 A.M. Unstressed, unanesthetized intact animals were intraperitoneally (i.p.) injected with 2.0 ml 2.5% o r 25% DMSO in normal saline in the experimental groups, or 2.0 ml normal saline alone in the control group. Another group of controls included untreated animals. After the injection of DMSO or saline, the animals were returned to their respective cages. The saline-treated and the untreated control groups were kept in a separate room from the DMSO-treated group. All rats were alert, awake, and active following the injection of DMSO or saline. At various times after injection, the animals were sacrificed by decapitation. Trunk blood was then collected in heparinized plastic tubes, chilled on ice, centrifuged, and the plasma separated. The plasma samples were frozen and stored at -20°C until assayed for ACTH by radioimmunoassay,6+8or for corticosterone either by acid-fluorescence tests8 or by a modification of a radioimmunoassay technique reported elsewhere.IO All samples from the same experiment were assayed at one time, in order to avoid possible interassay variability. The pattern of ACTH and corticosterone secretion after administration of DMSO was compared t o those observed after administration of other ACTHreleasing stimuli that have the capacity to act directly on the medial basal hypothalamus or through a complex neural pathway. Stimulants used for comparison included diethyl ether inhalation,* endotoxin," or a hind-leg tourniquet.'* Ether stimulation was accomplished by allowing an unstressed rat to inhale an atmosphere that contained diethyl ether for 2.5 min. E. coli endotoxin was given to intact rats in an i.p. dosage of 100 pg/lOO g body weight. Ether and endotoxin were administered to unanesthetized rats, whereas the tourniquet was applied to anesthetized rats (3.5 mg i.p. pentobarbitaI/lOO g body weight). A single tourniquet was applied to a hind leg by a technique described elsewhere,I3 left in place for 2.5 min, and then removed. (The tourniquet was left in place in those animals that were sacrificed 2.5 min after its application.) Following the application of these stresses, the animals were decapitated at various times after the onset of the stress, and blood samples were obtained in the manner described above. Animals with Lesions. In these experiments, 2.0 ml of either 25% DMSO in normal saline or normal saline alone was injected i.p. into adult male rats that weighed 200 f 10 g. The animals were killed by decapitation 40 min after the injetion, and plasma samples were obtained for ACTH and corticosterone analysis. In some animals, adrenalectomies were performed and DMSO or saline was injected 2 hours after application of the lesion. In other animals, hypophysectomies were performed by the parapharyngeal approach," and DMSO or saline was injected 1 hour and 20 min after surgery. In the remaining animals, hypothalamic deafferentiation was performed by a modification of the Halasz techniquei5 that has been reported elsewhere." With this technique the afferent neural input to the ventral hypothalamus concerned with the ACTH release that arises from nociceptors, limbic system nuclei, or olfactory centers, is severed, In rats with this type of lesion, ACTH release following application of a neurogenic stimulant is lost,12 the circadian rhythmicity of corticosterone is abolished,Leand hypersecretion of ACTH following adrenalectomy is abolished," whereas the capacity to release ACTH following application of a systemic stimulant such as hypoglycemia, endotoxin, or ether inhalation is retained.".18 The release of ACTH due to neurogenic stimulants requires the integrity of an ascending contralateral pathway in the central nervous system to
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TABLE1 HUMANSUBJECTS TESTEDWITH ORALDMSO Case I
2 3 4 5
Am
Sex
Diagnosis
DMSO before
years 30 21 53 17 31
male female male male female
normal normal Peyronie’s disease Marie-Strumpell periartenitis
none none none none 5 ml/day, 4 years
the h y p o t h a l a m ~ s . In ~ ~ contrast, systemic stimulants are transported to the hypothalamic-pituitary complex through the circulatory system, and directly stimulate the release of ACTH. Seven days after the complete surgical isolation of the hypothalamus, DMSO or saline was injected and the animals were sacrificed 40 min later. After sacrifice the animals were examined to ensure that the surgical isolation had been complete. Moreover, in those rats with hypothalamic lesions, histological examination of the brain and pituitary was also performed. Effects of DMSO on Plasma ACTH and Cortisol Concentrations in Man
Five adult subjects (2 females and 3 males) between the ages of 17 and 53 years were studied between 9 and 11 A.M. (TABLE1). Eight ml70% DMSO was added to an 8 oz glass of liquid, and the mixture was taken by mouth. Plasma samples were obtained prior to the ingestion of DMSO and at intervals of 30 min after the administration of DMSO, for 120 min; they were later analyzed for ACTH and cortisol by competitive protein-binding.lg The pattern of ACTH and cortisol release was compared to that observed in a standard insulin-tolerance test of 24 normal adult subjects (0.1 unit regular insulin/kg, injected intravenously). The resulting hypoglycemia from the insulin is a known stimulus for ACTH release in man.2o The results of all experiments were pooled and analyzed for significance, by means of an analysis of variance, Student’s t Test, and the Duncan test.2’ RESULTS Effect of DMSO in Vitro
The effect of DMSO added to ACTH-free plasma, as compared to a standard curve and that of plasma obtained from a DMSO-treated intact rat, is shown in FIGURE1. In contrast to the parallel curve generated by plasma obtained from the DMSO-treated rat, DMSO added to ACTH-free plasma was undetectable as ACTH. Similarly, DMSO added to corticosterone-free plasma, buffer, or assay reagents was undetectable as corticosterone by either the radioimmunoassay or the acid-fluorescence technique. Effect of DMSO in the Rat in Vivo Intact Animals. The effect of DMSO on ACTH concentrations in plasma is shown in FIGURE2. There was no significant change in the mean ACTH concen-
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Annals New York Academy of Sciences 50
40
STANDARD CURVES
DMSO IN BUFFER DMSO - HVPOX RAT DMSO . INTACT RAT
+ 16
O'
sio
1:s 250 HUMAN . ACTH pg/TUBE
1:
lob0 2600
FIGURE I . Effect of DMSO on the radioimmunoassay technique for ACTH. The curve obtained from a plasma sample of an intact rat injected with 25% DMSO (0-0) demonstrates parallel immunoreactivity to the standard curves (++). When DMSO is added to the buffer (A-A), or injected into a hypophysectomized rat and a plasma sample obtained (0o), no immunoreactive ACTH is present.
250
r
1 - OMSO (25%1
50
SALINE I
O
d
4 INJECTION
I
20
40 MINUTES
i0
FIGURE2. Plasma ACTH concentrations at various times after the i.p. injection of 2.0 ml 2.5% or 25% DMSO or normal saline. There was no significant change in the plasma ACTH concentrations (mean + SE) after the injection of either 2.5% DMSO or saline. A significant ( P < 0.001) rise occurred 40 min after injection of 25% DMSO.
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50 OMSO 125%1
SILINE
There was no change in COrticostcrone levels in the saline-treated controls.
O
Q
I
I
I
20
40
60
4
MINUTES
INJECTION
tration in plasma following administration of 2.0 ml 2.5% DMSO. Similarly, there was no significant change in ACTH concentrations in the plasma of the salinetreated control groups. When 25% DMSO was injected, ACTH concentrations in plasma reached a peak 40 min later, and then decreased. In contrast to the pattern of ACTH release, corticosterone concentrations in plasma were significantly ( P < 0.00.) elevated 20 rnin after injection of 25% DMSO, and reached a peak at 40 rnin (FIGURE 3). Similarly, a significant ( P < 0.05) increase in corticosterone concentrations in plasma occurred 40 rnin after injection of 2.5% DMSO. It was of interest that there was no correlation between ACTH and corticosterone concentrations in 1000
-
500 400 -
300 -
200 -
-. E 100 E
I-
y
80
--
60 -
i t
= ETHER
4
A-
--4
= ENDOTOXIN TOURWI9UET
OMSO
6-41
I
I
I
2.5
5
10
I
20 MINUTES
I
40
I
I
60 80
I
I
I
I
180220 300 420
STIMULUS
FIGURE 4. The temporal effect of four stimuli on ACTH concentrations in plasma. They reached a peak 2.5 min after the onset of either 2.5 min ether inhalation or application of a 2.5 min hind-leg tourniquet. The peak was delayed after an i.p. injection of either 2.0 ml 25% DMSO (40 min) or 100pg E. coliendotoxin/lOO g body weight (180 rnin).
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Annals New York Academy of Sciences
plasma at either 20 or 40 min. These data suggest that a large dose of DMSO has the capacity to stimulate release of both ACTH and corticosterone. Smaller doses of DMSO appear to stimulate corticosterone release without a significant release of ACTH. The temporal effect of four ACTH-releasing stimuli on ACTH concentrations in 4. Whereas ACTH concentrations reached a peak 2.5 plasma is shown in FIGURE min after the onset of the ether or tourniquet stress, the peak was delayed after application of DMSO or endotoxin. Although the pattern of ACTH release and the mean peak concentration of ACTH varied considerably in these four groups, there was no significant difference between the peak corticosterone concentrations after application of the various stimuli. In the ether-, tourniquet-, and endotoxin-stimulated groups the mean rise in corticosterone concentrations in plasma followed a significant release of ACTH; however, in the DMSO-treated group the mean corticosterone concentration rose significantly before ACTH concentrations that could be measured by radioimmunoassay significantly increased. Animals wirh Lesions. The effect of DMSO in hypophysectomized, adrenalectomized or hypothalamic-deafferentated rats on ACTH and corticosterone concen5 and 6. trations in plasma is compared to the effects of normal saline in FIGURES ACTH concentrations were undetectable in both the DMSO- and the saline-treated hypophysectomized groups. Similarly, cortisol concentrations were undetectable in the plasma of the adrenalectomized groups. These data suggest that DMSO or its metabolites do not cross-react as ACTH or corticosterone. Furthermore, ACTH was significantly ( P < 0.05) released from adrenalectomized rats treated with DMSO, as compared to saline-treated controls. In addition, corticosterone concentrations were low in the plasma of hypophysectomized rats treated with either DMSO or saline. These data indicate that DMSO does not act directly on the
-E 0
J
G.
HYPOX
A DX
DEAFF
FIGURE 5. Plasma concentrations of ACTH in operated rats 40 min after the injection of 2.0 ml25% DMSO or normal saline. ACTH concentrations were undetectable in the DMSO- and saline-treated groups hypophysectomized (HYPOX) 2 hours earlier. DMSO resulted in a significant ( P < 0.05) elevation of ACTH in both the 2-hour adrenalectomized (ADX) and the I-week hypothalamic-deafferentated(DEAFF) groups.
Allen & Allen: Effects on the Hypothalamus I OOO-J
800 600
400
-
5
---
33 1
OMSO
- 0SALINE -
200-
9 0.
I t-
:: loo80: 40
-
20
-
60
10 HYPOX
A DX
DEAFF
FIGURE 6. Plasma concentrations of corticosterone in operated rats 40 min after the injection of 2.0 ml 25% DMSO or normal saline. Corticosterone concentrations were low after DMSO or saline injections in the 2-hour hypophysectomized (HYPOX) and adrenalectomized (ADX) groups. A significant ( P < 0.05) increase in corticosterone concentrations occurred after DMSO (but not saline) injections in the hypothalamic-deafferentated (DEAFF) group. adrenal gland to stimulate corticosterone release, but does directly stimulate ACTH release from the pituitary gland. In the hypothalamic-deafferentated groups, ACTH and corticosterone concentrations in plasma significantly ( P < 0.05) increased 40 min after the injection of DMSO. These data suggest that DMSO acts directly on the hypothalamic-pituitary complex to release ACTH, and does not act primarily through the generation of a neural signal transmitted through the central nervous system to the medial basal hypothalamus.
Effects of DMSO on Plasma ACTH and Cortisol Concentrations in Man FIGURE 7 shows the effect of DMSO on the mean ACTH and cortisol concentrations in the plasma of some subjects, as compared to the effect of insulin-induced hypoglycemia in other normal subjects. There was no significant change in the mean ACTH or cortisol concentrations in plasma following application of DMSO. These data suggest that DMSO at this dosage does not significantly release ACTH and cortisol in man. In contrast, the fall in the mean glucose concentration in plasma to less than 30 mg/100 ml 30 min after the insulin injection resulted in the significant release of ACTH and cortisol. Our data suggest that DMSO administered to the rat has the capacity to stimulate ACTH and corticosterone secretion. The latter observation confirms the findings of Grozdov and colleagues,’ who first reported increased blood levels of glucocorticoids in the rat after administration of DMSO. We have extended these
Annals New York Academy of Sciences
332
500400 -
-
8
L
-
A
300 -
I
L U
200 -
100 1
1
1
I
B 0
M
T
301
3-1
O
v, c
20
oc
0
10
O
3 O 30 60 90 120 4
MINUTES
FIGURE7. The effect of DMSO or insulin-induced hypoglycemia (IIH) on plasma ACTH (A) or cortisol (B) concentrations in human subjects. After administration of 8.0 ml 70% DMSO by mouth, no significant change in the mean ACTH or cortisol concentrations in plasma occurred in the subsequent 120 min. After i.v. injection of 0.1 units regular insulin, a significant increase in the mean plasma ACTH levels occurred at 45 min, followed by a peak in cortisol.
initial observations by showing that a principal mechanism through which DMSO exerts its effect on the hypothalamic-pituitary-adrenal axis is the release of ACTH. The mode of action of DMSO in stimulating the release of ACTH is in part suggested by these studies. Three general possibilities exist. First, and most likely, is that DMSO acts directly on the hypothalamic-pituitary complex to stimulate ACTH release (FIGURE 8). Other substances that are similar in structure to DMSO, such as diethyl ether,2 stimulate ACTH release in the rat by acting on the median eminence of the hypothalamus, not directly upon the adenohypophysis. Hence it is possible that DMSO acts similarly to induce the release of corticotropin-releasing factor (CRF). The second possibility is that DMSO acts as a peritoneal irritant, which results in the generation of an afferent ACTH-releasing signal from nocicep-
Allen & Allen: Effects on the Hypothalamus
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I Anterior Pituitary
CORTICOSTERONE FIGURE 8. Site of action of DMSO on the hypothalamic-pituitary-adrenal axis of the rat. DMSO acts directly on the hypothalamic-anterior pituitary complex (4to ) cause the release of ACTH, presumably through the release of corticotropin-releasing factor (CRF) from the hypothalamus. An additional possibility is that DMSO potentiates the effect of circulating ACTH on the adrenal cortex (----+), which stimulates the release of corticosterone.
tors, and this is carried through pathways of the central nervous system to the ventral hypothalamus. A third possibility is that the unusual odor present in mammals that have been given DMSO may stimulate an ACTH-releasing signal from the olfactory region. Olfactory cues have been shown to result in the release of ACTH in the rat.22 Hypothalamic deafferentation was performed in order to sever the neurogenic stress pathway, hypothalamic input from the olfactory system, and neural input from the limbic system. Because hypothalamic-deafferentated rats release significant amounts of ACTH after the administration of DMSO, the most likely primary mode of action of DMSO in releasing ACTH is direct action upon the hypothalamic-pituitary complex of the rat, and not through the neural pathways named above. Some substances, such as formaldehyde, however, when injected in small doses into the rat, stimulate the release of ACTH through the generation of a neural signal that is carried through ascending neural pathways to the medial basal hypothalamus. In addition, formaldehyde appears to be able to act directly on the medial basal hypothalamus when large doses are ~ s e d . ~ W ~ e. ’did ~ not observe a significant release of ACTH when the small dosage of DMSO was injected i.p.; formaldehyde, however, is very irritating and painful. The rise in corticosterone concentration in the plasma 20 min after the injection of DMSO would be consistent with the hypothesis that a significant release of ACTH occurred at the time of injection (possibly from irritation), with a secondary increase in corticosterone. If this was the explanation, then a persistent elevation of ACTH concentration in plasma would have been expected, because the corticosterone levels in plasma continued to increase. It is possible that there is a bimodal release of ACTH in the rat: the first caused by irritation of the peritoneum, and the second by the substance that acts on
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Annals New York Academy of Sciences
the hypothalamic pituitary complex. The delayed secretion of ACTH following application of DMSO suggests that the agent responsible for ACTH release is either a DMSO metabolite or an endogenous factor that is stimulated by DMSO. Direct observations that support this hypothesis are lacking, however. Some stimuli such as endotoxin stimulate maximal release of ACTH in the rat 2-3 hours after administration, probably through the release of an endogenous humoral factor.I3 The surprising finding was the significant rise in corticosterone concentrations in the intact rat prior to a significant elevation of ACTH concentrations in plasma. Several possibilities exist to explain these data. The first is that DMSO directly stimulates the adrenal cortex to release corticosterone. The failure of DMSO to stimulate corticosterone release in rats hypophysectomized 2 hours earlier makes this possibility most unlikely. That the injection has nonspecific effects capable of stimulating ACTH release in the rat is a second possibility, but it is unlikely, because corticosterone concentrations did not rise in the plasma of saline-treated controls. A third explanation is that the rise in corticosterone concentrations in plasma at 20 min resulted from irritation or an acute metabolic change caused by DMSO at the time of injection, This explanation, however, does not account for the persistent elevation of corticosterone 40 min after injection. The fourth and most intriguing possibility is that DMSO potentiates the effect of ACTH on the adrenal cortex (FIGURE 8). Although conclusive direct evidence that supports this hypothesis is lacking, preliminary studies indicate that DMSO can function in this capacity in an acutely hypophysectomized rat injected with submaximal doses of exogenous ACTH. One question raised at the outset of this study was whether DMSO interferes with the determination of, or cross-reacts with, ACTH or corticosterone when measured by radioimmunoassay, or with corticosterone when measured by acidfluorescence. We found no evidence that DMSO reacted as ACTH or corticosterone when tested by these methods. In man, orally administered DMSO did not stimulate the release of ACTH or cortisol. In this regard a dissociation was observed between the hypothalamicpituitary-adrenal responses to DMSO in man and the rat. Several possibilities exist to account for these differences. ( I ) The dose and the route of administration were different with man, who received a much smaller dose of DMSO. Furthermore, patients who use DMSO for long periods of time do not show clinical signs of excess cortisol. The same dissociation between the responses of man and the rat has been (2) DMSO or its actual metabolite reported when ether was used as the may be interpreted by lower mammals such as the rat as creating stress, and hence the ensuing glucocorticoid response would be protective in nature. The rats injected with the larger dosage of DMSO did not appear ill, hyperexcitable, or lethargic; they did not respond as would be expected if they were significantly stressed. (3) A species-specific physiological adenohypophysial or neurochemical hypothalamic response may occur after the administration of DMSO. ACKNOWLEDGMENTS We are grateful for the opportunity we were given to study patients of Dr. Stanley Jacob. We thank Drs. Monte A. Greer and Joseph W. Goldzieher for their support. REFERENCES I.
GROZDOV, S. P..G. 1. BEZIN,1. N. KENDYSH,I . Y.KIR’YANOV& V. V. VASIL’EVSKAYA. 1971. About the mechanism of biological effects and radio-protective effects of dimethyl sulphoxide. Radiobiologiya 11: 522-527.
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2.
MATSUDA, K., C. DUYCK,J. W. KENDALL & M. A. GREER.1964. Pathways by which traumatic stress and ether induce increased ACTH release in the rat. Endocrinology
3.
MANGILI, G., M. MOTTA,W. MUCIACCIA & L. MARTINI.1964. Midbrain stress and ACTH secretion. European REV.Endocrinol. 1: 247-253. FORBES, J. C. & G. M. DUNCAN. 1953. Effect of intraperitoneal administration of alcohol on the adrenal levels of cholesterol and ascorbic acid in rats and guinea pigs. Quart. J. Studies Alc. 14: 19-21. SPRIGGS, T. L. G. & M. A. STOCKHOLM. 1964. Urethane anesthesia and pituitary-adrenal function in the rat. J. Pharm. Pharmacol. 16: 603-610. REES, L. H., D. M. COOK,J. W. KENDALL, C. F. ALLEN,R. M. KRAMER, J. G. RATCLIFFE & R. A. KNIGHT.1971. A radioimmunoassay for rat plasma ACTH. Endocrinology 89: 254261. ALLEN,J. P., D. M. COOK, J. W. KENDALL & R. MCGILVRA. 1973. Maternal-fetal ACTH relationship in man. J. Clin. Endocrinol. 37: 23e-234. COOK,D. M.. J. W. KENDALL, M. A. GREER & R. M. KRAMER. 1973. The effect of acute or chronic ether stress on plasma ACTH concentration in the rat. Endocrinology 93:
74: 98 1-985. 4. 5.
6. 7. 8.
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9. 10. 11.
12. 13.
14.
KENDALL, J. W., M. L. EGANS& A. K. STOTT.1968. Fluorometric determination of corticosteroids: An interfering substance in impure dichloromethane which fluoresces with benzyl alcohol preservative in heparin. J. Clin. Endocrinol. 28: 1373-1376. RUDER,H. J., R. L. GUY& M. 9. LIPSETT.1972. A radioimmunoassay for cortisol in plasma and urine. J. Clin. Endocrinol. Metab. 35: 219-224. MAKARA, G. B., E. STARK & M. PALKOVITS. 1970. Afferent pathways of stressful stimuli: Corticotrophin release after hypothalamic deafferentation. J. Endocrinol. 47: 41 1-416. GREER,M. A., C. F. ALLEN,G. P. GIBBS& C. GULLICKSON. 1970. Pathways at the hypothalamic level through which traumatic stress activates ACTH secretion in the rat. Endocrinology 86: 14041409. ALLEN,J. P., C. F. ALLEN,M. A. GREER& J. J. JACOBS.1973. Stress-induced secretion of ACTH. In Brain-Pituitary-Adrenal Interrelationships. A. Brodish & E. S. Redgate, Eds.: 99-127. Verlag S. Karger, A.-G. Basel, Switzerland. SMITH, P. E. 1930. Hypophysectomy and replacement therapy in the rat. Amer. J. Anat. 45: 205-273.
HALASZ,B. & L. PUPP. 1965. Hormone secretion of the anterior pituitary gland after partial or total deafferentation of all nervous pathways to the hypophysiotrophic area. Endocrinology 77: 553-562. 16. ALLEN,C. F., J. W. KENDALL & M. A. GREER.1972. The effect of surgical isolation of the basal hypothalamus on the nycthemeral rhythm of plasma corticosterone concentration in rats with heterotopic pituitaries. Endocrinology 91: 873-876. C. F., J. P. ALLEN& M. A. GREER.1973. Effect of hypothalamic deafferentation 17. ALLEN, on tonic and stress-induced ACTH secretion in adrenalectomized rats. Progr. 55th Ann. Meeting Endocrinol. SOC.A-79. IY72. Effects of partial hypothalamic S., N. CONFORTI & I. CHOWERS. 18. FELDMAN, deafferentations on adrenocortical responses. Acta. Endocrinol. (Copenhagen) 69: 52615.
530. 9 . E. 1967. Some studies of the protein-binding of steroids and their application 19. MURPHY, to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J. Clin. Endocrinol. 25: 973-990. F. C., J. LANDON & T. C. 9. STAMP.1966. The plasma sugar, free fatty 20. GREENWOOD,
acid, cortisol and growth hormone response to insulin. I . In control subjects. J. Clin. Invest. 45: 429-436. 21. WINER,9 . J. 1962. Statistical Principles in Experimental Design. pp. 101-103. McGrawHill Book Company. New York, N.Y. 1972. Pituitary-adrenal function in photic and 22. DUNN,J., M. BENNETT& R. PEFFLER. olfactory deprived rats. Proc. SOC. Exp. Biol. Med. 140: 755-758. G. B., E. STARK& K. MIHALY. 1967. Site at which formalin and capsaicin act 23. MAKARA, to stimulate corticotrophin secretion. Can. J. Phys. 45: 669-674.
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24. MAKARA,G. B., E. STARK, M . PALKOVITS, T. REVESZ& K. MIHALY.1969. AKerent pathways of stressful stimuli: Corticotrophin release after partial deamerentation of the medial basal hypothalamus. J . Endocrinol. 44: 187-193. 25. HAIBACK, H., R. J. UNDERWOOD, M. A. GREER& W. P. VANDERLAAN. 1970. Failure to stimulate significant cortisol or growth hormone secretion in man by ether infusion. Experientia 26: 1146-1 147.
The unusual chemical properties and clinical applications of dimethyl sulfoxide (DMSO) have been the subject of many scientific investigations. These studies have included exploration of the mechanism of action of its anti-inflammatory effect in mammals, We considered it possible that one mode of action of DMSO was to stimulate increased glucocorticoid secretion. Some evidence from a study of the rat supports this hypothesis.' Furthermore, other chemicals, including diethyl ether,2 f ~ r m a l d e h y d e , ethan01,~ ~ and urethane: which resemble DMSO both in configuration and size, stimulate the release of ACTH in the rat; but the administration of DMSO to rats does not apparently cause the same physiological effects (such as irritation and pain, anesthesia, or impaired conciousness) as the others. These previously reported findings prompted us to explore the mechanism through which glucocorticoids are released. This was accomplished by measuring plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations in DMSO-treated rats.
MATERIALS AND METHODS Eflect of DMSO in Vitro In order to test for the presence of interference or cross-reactivity of DMSO in the ACTH and corticosterone radioimmunoassays and the corticosterone acidfluorescence tests, 0.5 ml 100% DMSO was added to 5.0 rnl buffer, reagents, or hormone-free plasma, and the assays were performed. Each sample was assayed in triplicate. Effect of DMSO in the Rat in Vivo Intact Animals. Adult male Sprague-Dawley rats weighing 250 f 50 g were used in all experiments. All animals were given Purina Lab Chow and water ab lib. Four *This study was done in part while Dr. J. P. Allen was affiliated with the Division of Endocrinology, University of Oregon Medical School, Portland, Oregon. It was supported in part by grants from the National Institute of Health.
325
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Annals New York Academy of Sciences
were housed in each cage, and they were kept in an environment with constant temperature (24 f 1°C) and controlled lighting (12 hours of light beginning at 6 A.M., and 12 hours of darkness). All animals were maintained under these conditions for 7 days before the administration of DMSO. All studies were performed between 9 A.M. and 10 A.M. Unstressed, unanesthetized intact animals were intraperitoneally (i.p.) injected with 2.0 ml 2.5% o r 25% DMSO in normal saline in the experimental groups, or 2.0 ml normal saline alone in the control group. Another group of controls included untreated animals. After the injection of DMSO or saline, the animals were returned to their respective cages. The saline-treated and the untreated control groups were kept in a separate room from the DMSO-treated group. All rats were alert, awake, and active following the injection of DMSO or saline. At various times after injection, the animals were sacrificed by decapitation. Trunk blood was then collected in heparinized plastic tubes, chilled on ice, centrifuged, and the plasma separated. The plasma samples were frozen and stored at -20°C until assayed for ACTH by radioimmunoassay,6+8or for corticosterone either by acid-fluorescence tests8 or by a modification of a radioimmunoassay technique reported elsewhere.IO All samples from the same experiment were assayed at one time, in order to avoid possible interassay variability. The pattern of ACTH and corticosterone secretion after administration of DMSO was compared t o those observed after administration of other ACTHreleasing stimuli that have the capacity to act directly on the medial basal hypothalamus or through a complex neural pathway. Stimulants used for comparison included diethyl ether inhalation,* endotoxin," or a hind-leg tourniquet.'* Ether stimulation was accomplished by allowing an unstressed rat to inhale an atmosphere that contained diethyl ether for 2.5 min. E. coli endotoxin was given to intact rats in an i.p. dosage of 100 pg/lOO g body weight. Ether and endotoxin were administered to unanesthetized rats, whereas the tourniquet was applied to anesthetized rats (3.5 mg i.p. pentobarbitaI/lOO g body weight). A single tourniquet was applied to a hind leg by a technique described elsewhere,I3 left in place for 2.5 min, and then removed. (The tourniquet was left in place in those animals that were sacrificed 2.5 min after its application.) Following the application of these stresses, the animals were decapitated at various times after the onset of the stress, and blood samples were obtained in the manner described above. Animals with Lesions. In these experiments, 2.0 ml of either 25% DMSO in normal saline or normal saline alone was injected i.p. into adult male rats that weighed 200 f 10 g. The animals were killed by decapitation 40 min after the injetion, and plasma samples were obtained for ACTH and corticosterone analysis. In some animals, adrenalectomies were performed and DMSO or saline was injected 2 hours after application of the lesion. In other animals, hypophysectomies were performed by the parapharyngeal approach," and DMSO or saline was injected 1 hour and 20 min after surgery. In the remaining animals, hypothalamic deafferentiation was performed by a modification of the Halasz techniquei5 that has been reported elsewhere." With this technique the afferent neural input to the ventral hypothalamus concerned with the ACTH release that arises from nociceptors, limbic system nuclei, or olfactory centers, is severed, In rats with this type of lesion, ACTH release following application of a neurogenic stimulant is lost,12 the circadian rhythmicity of corticosterone is abolished,Leand hypersecretion of ACTH following adrenalectomy is abolished," whereas the capacity to release ACTH following application of a systemic stimulant such as hypoglycemia, endotoxin, or ether inhalation is retained.".18 The release of ACTH due to neurogenic stimulants requires the integrity of an ascending contralateral pathway in the central nervous system to
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TABLE1 HUMANSUBJECTS TESTEDWITH ORALDMSO Case I
2 3 4 5
Am
Sex
Diagnosis
DMSO before
years 30 21 53 17 31
male female male male female
normal normal Peyronie’s disease Marie-Strumpell periartenitis
none none none none 5 ml/day, 4 years
the h y p o t h a l a m ~ s . In ~ ~ contrast, systemic stimulants are transported to the hypothalamic-pituitary complex through the circulatory system, and directly stimulate the release of ACTH. Seven days after the complete surgical isolation of the hypothalamus, DMSO or saline was injected and the animals were sacrificed 40 min later. After sacrifice the animals were examined to ensure that the surgical isolation had been complete. Moreover, in those rats with hypothalamic lesions, histological examination of the brain and pituitary was also performed. Effects of DMSO on Plasma ACTH and Cortisol Concentrations in Man
Five adult subjects (2 females and 3 males) between the ages of 17 and 53 years were studied between 9 and 11 A.M. (TABLE1). Eight ml70% DMSO was added to an 8 oz glass of liquid, and the mixture was taken by mouth. Plasma samples were obtained prior to the ingestion of DMSO and at intervals of 30 min after the administration of DMSO, for 120 min; they were later analyzed for ACTH and cortisol by competitive protein-binding.lg The pattern of ACTH and cortisol release was compared to that observed in a standard insulin-tolerance test of 24 normal adult subjects (0.1 unit regular insulin/kg, injected intravenously). The resulting hypoglycemia from the insulin is a known stimulus for ACTH release in man.2o The results of all experiments were pooled and analyzed for significance, by means of an analysis of variance, Student’s t Test, and the Duncan test.2’ RESULTS Effect of DMSO in Vitro
The effect of DMSO added to ACTH-free plasma, as compared to a standard curve and that of plasma obtained from a DMSO-treated intact rat, is shown in FIGURE1. In contrast to the parallel curve generated by plasma obtained from the DMSO-treated rat, DMSO added to ACTH-free plasma was undetectable as ACTH. Similarly, DMSO added to corticosterone-free plasma, buffer, or assay reagents was undetectable as corticosterone by either the radioimmunoassay or the acid-fluorescence technique. Effect of DMSO in the Rat in Vivo Intact Animals. The effect of DMSO on ACTH concentrations in plasma is shown in FIGURE2. There was no significant change in the mean ACTH concen-
328
Annals New York Academy of Sciences 50
40
STANDARD CURVES
DMSO IN BUFFER DMSO - HVPOX RAT DMSO . INTACT RAT
+ 16
O'
sio
1:s 250 HUMAN . ACTH pg/TUBE
1:
lob0 2600
FIGURE I . Effect of DMSO on the radioimmunoassay technique for ACTH. The curve obtained from a plasma sample of an intact rat injected with 25% DMSO (0-0) demonstrates parallel immunoreactivity to the standard curves (++). When DMSO is added to the buffer (A-A), or injected into a hypophysectomized rat and a plasma sample obtained (0o), no immunoreactive ACTH is present.
250
r
1 - OMSO (25%1
50
SALINE I
O
d
4 INJECTION
I
20
40 MINUTES
i0
FIGURE2. Plasma ACTH concentrations at various times after the i.p. injection of 2.0 ml 2.5% or 25% DMSO or normal saline. There was no significant change in the plasma ACTH concentrations (mean + SE) after the injection of either 2.5% DMSO or saline. A significant ( P < 0.001) rise occurred 40 min after injection of 25% DMSO.
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50 OMSO 125%1
SILINE
There was no change in COrticostcrone levels in the saline-treated controls.
O
Q
I
I
I
20
40
60
4
MINUTES
INJECTION
tration in plasma following administration of 2.0 ml 2.5% DMSO. Similarly, there was no significant change in ACTH concentrations in the plasma of the salinetreated control groups. When 25% DMSO was injected, ACTH concentrations in plasma reached a peak 40 min later, and then decreased. In contrast to the pattern of ACTH release, corticosterone concentrations in plasma were significantly ( P < 0.00.) elevated 20 rnin after injection of 25% DMSO, and reached a peak at 40 rnin (FIGURE 3). Similarly, a significant ( P < 0.05) increase in corticosterone concentrations in plasma occurred 40 rnin after injection of 2.5% DMSO. It was of interest that there was no correlation between ACTH and corticosterone concentrations in 1000
-
500 400 -
300 -
200 -
-. E 100 E
I-
y
80
--
60 -
i t
= ETHER
4
A-
--4
= ENDOTOXIN TOURWI9UET
OMSO
6-41
I
I
I
2.5
5
10
I
20 MINUTES
I
40
I
I
60 80
I
I
I
I
180220 300 420
STIMULUS
FIGURE 4. The temporal effect of four stimuli on ACTH concentrations in plasma. They reached a peak 2.5 min after the onset of either 2.5 min ether inhalation or application of a 2.5 min hind-leg tourniquet. The peak was delayed after an i.p. injection of either 2.0 ml 25% DMSO (40 min) or 100pg E. coliendotoxin/lOO g body weight (180 rnin).
3 30
Annals New York Academy of Sciences
plasma at either 20 or 40 min. These data suggest that a large dose of DMSO has the capacity to stimulate release of both ACTH and corticosterone. Smaller doses of DMSO appear to stimulate corticosterone release without a significant release of ACTH. The temporal effect of four ACTH-releasing stimuli on ACTH concentrations in 4. Whereas ACTH concentrations reached a peak 2.5 plasma is shown in FIGURE min after the onset of the ether or tourniquet stress, the peak was delayed after application of DMSO or endotoxin. Although the pattern of ACTH release and the mean peak concentration of ACTH varied considerably in these four groups, there was no significant difference between the peak corticosterone concentrations after application of the various stimuli. In the ether-, tourniquet-, and endotoxin-stimulated groups the mean rise in corticosterone concentrations in plasma followed a significant release of ACTH; however, in the DMSO-treated group the mean corticosterone concentration rose significantly before ACTH concentrations that could be measured by radioimmunoassay significantly increased. Animals wirh Lesions. The effect of DMSO in hypophysectomized, adrenalectomized or hypothalamic-deafferentated rats on ACTH and corticosterone concen5 and 6. trations in plasma is compared to the effects of normal saline in FIGURES ACTH concentrations were undetectable in both the DMSO- and the saline-treated hypophysectomized groups. Similarly, cortisol concentrations were undetectable in the plasma of the adrenalectomized groups. These data suggest that DMSO or its metabolites do not cross-react as ACTH or corticosterone. Furthermore, ACTH was significantly ( P < 0.05) released from adrenalectomized rats treated with DMSO, as compared to saline-treated controls. In addition, corticosterone concentrations were low in the plasma of hypophysectomized rats treated with either DMSO or saline. These data indicate that DMSO does not act directly on the
-E 0
J
G.
HYPOX
A DX
DEAFF
FIGURE 5. Plasma concentrations of ACTH in operated rats 40 min after the injection of 2.0 ml25% DMSO or normal saline. ACTH concentrations were undetectable in the DMSO- and saline-treated groups hypophysectomized (HYPOX) 2 hours earlier. DMSO resulted in a significant ( P < 0.05) elevation of ACTH in both the 2-hour adrenalectomized (ADX) and the I-week hypothalamic-deafferentated(DEAFF) groups.
Allen & Allen: Effects on the Hypothalamus I OOO-J
800 600
400
-
5
---
33 1
OMSO
- 0SALINE -
200-
9 0.
I t-
:: loo80: 40
-
20
-
60
10 HYPOX
A DX
DEAFF
FIGURE 6. Plasma concentrations of corticosterone in operated rats 40 min after the injection of 2.0 ml 25% DMSO or normal saline. Corticosterone concentrations were low after DMSO or saline injections in the 2-hour hypophysectomized (HYPOX) and adrenalectomized (ADX) groups. A significant ( P < 0.05) increase in corticosterone concentrations occurred after DMSO (but not saline) injections in the hypothalamic-deafferentated (DEAFF) group. adrenal gland to stimulate corticosterone release, but does directly stimulate ACTH release from the pituitary gland. In the hypothalamic-deafferentated groups, ACTH and corticosterone concentrations in plasma significantly ( P < 0.05) increased 40 min after the injection of DMSO. These data suggest that DMSO acts directly on the hypothalamic-pituitary complex to release ACTH, and does not act primarily through the generation of a neural signal transmitted through the central nervous system to the medial basal hypothalamus.
Effects of DMSO on Plasma ACTH and Cortisol Concentrations in Man FIGURE 7 shows the effect of DMSO on the mean ACTH and cortisol concentrations in the plasma of some subjects, as compared to the effect of insulin-induced hypoglycemia in other normal subjects. There was no significant change in the mean ACTH or cortisol concentrations in plasma following application of DMSO. These data suggest that DMSO at this dosage does not significantly release ACTH and cortisol in man. In contrast, the fall in the mean glucose concentration in plasma to less than 30 mg/100 ml 30 min after the insulin injection resulted in the significant release of ACTH and cortisol. Our data suggest that DMSO administered to the rat has the capacity to stimulate ACTH and corticosterone secretion. The latter observation confirms the findings of Grozdov and colleagues,’ who first reported increased blood levels of glucocorticoids in the rat after administration of DMSO. We have extended these
Annals New York Academy of Sciences
332
500400 -
-
8
L
-
A
300 -
I
L U
200 -
100 1
1
1
I
B 0
M
T
301
3-1
O
v, c
20
oc
0
10
O
3 O 30 60 90 120 4
MINUTES
FIGURE7. The effect of DMSO or insulin-induced hypoglycemia (IIH) on plasma ACTH (A) or cortisol (B) concentrations in human subjects. After administration of 8.0 ml 70% DMSO by mouth, no significant change in the mean ACTH or cortisol concentrations in plasma occurred in the subsequent 120 min. After i.v. injection of 0.1 units regular insulin, a significant increase in the mean plasma ACTH levels occurred at 45 min, followed by a peak in cortisol.
initial observations by showing that a principal mechanism through which DMSO exerts its effect on the hypothalamic-pituitary-adrenal axis is the release of ACTH. The mode of action of DMSO in stimulating the release of ACTH is in part suggested by these studies. Three general possibilities exist. First, and most likely, is that DMSO acts directly on the hypothalamic-pituitary complex to stimulate ACTH release (FIGURE 8). Other substances that are similar in structure to DMSO, such as diethyl ether,2 stimulate ACTH release in the rat by acting on the median eminence of the hypothalamus, not directly upon the adenohypophysis. Hence it is possible that DMSO acts similarly to induce the release of corticotropin-releasing factor (CRF). The second possibility is that DMSO acts as a peritoneal irritant, which results in the generation of an afferent ACTH-releasing signal from nocicep-
Allen & Allen: Effects on the Hypothalamus
333
I Anterior Pituitary
CORTICOSTERONE FIGURE 8. Site of action of DMSO on the hypothalamic-pituitary-adrenal axis of the rat. DMSO acts directly on the hypothalamic-anterior pituitary complex (4to ) cause the release of ACTH, presumably through the release of corticotropin-releasing factor (CRF) from the hypothalamus. An additional possibility is that DMSO potentiates the effect of circulating ACTH on the adrenal cortex (----+), which stimulates the release of corticosterone.
tors, and this is carried through pathways of the central nervous system to the ventral hypothalamus. A third possibility is that the unusual odor present in mammals that have been given DMSO may stimulate an ACTH-releasing signal from the olfactory region. Olfactory cues have been shown to result in the release of ACTH in the rat.22 Hypothalamic deafferentation was performed in order to sever the neurogenic stress pathway, hypothalamic input from the olfactory system, and neural input from the limbic system. Because hypothalamic-deafferentated rats release significant amounts of ACTH after the administration of DMSO, the most likely primary mode of action of DMSO in releasing ACTH is direct action upon the hypothalamic-pituitary complex of the rat, and not through the neural pathways named above. Some substances, such as formaldehyde, however, when injected in small doses into the rat, stimulate the release of ACTH through the generation of a neural signal that is carried through ascending neural pathways to the medial basal hypothalamus. In addition, formaldehyde appears to be able to act directly on the medial basal hypothalamus when large doses are ~ s e d . ~ W ~ e. ’did ~ not observe a significant release of ACTH when the small dosage of DMSO was injected i.p.; formaldehyde, however, is very irritating and painful. The rise in corticosterone concentration in the plasma 20 min after the injection of DMSO would be consistent with the hypothesis that a significant release of ACTH occurred at the time of injection (possibly from irritation), with a secondary increase in corticosterone. If this was the explanation, then a persistent elevation of ACTH concentration in plasma would have been expected, because the corticosterone levels in plasma continued to increase. It is possible that there is a bimodal release of ACTH in the rat: the first caused by irritation of the peritoneum, and the second by the substance that acts on
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Annals New York Academy of Sciences
the hypothalamic pituitary complex. The delayed secretion of ACTH following application of DMSO suggests that the agent responsible for ACTH release is either a DMSO metabolite or an endogenous factor that is stimulated by DMSO. Direct observations that support this hypothesis are lacking, however. Some stimuli such as endotoxin stimulate maximal release of ACTH in the rat 2-3 hours after administration, probably through the release of an endogenous humoral factor.I3 The surprising finding was the significant rise in corticosterone concentrations in the intact rat prior to a significant elevation of ACTH concentrations in plasma. Several possibilities exist to explain these data. The first is that DMSO directly stimulates the adrenal cortex to release corticosterone. The failure of DMSO to stimulate corticosterone release in rats hypophysectomized 2 hours earlier makes this possibility most unlikely. That the injection has nonspecific effects capable of stimulating ACTH release in the rat is a second possibility, but it is unlikely, because corticosterone concentrations did not rise in the plasma of saline-treated controls. A third explanation is that the rise in corticosterone concentrations in plasma at 20 min resulted from irritation or an acute metabolic change caused by DMSO at the time of injection, This explanation, however, does not account for the persistent elevation of corticosterone 40 min after injection. The fourth and most intriguing possibility is that DMSO potentiates the effect of ACTH on the adrenal cortex (FIGURE 8). Although conclusive direct evidence that supports this hypothesis is lacking, preliminary studies indicate that DMSO can function in this capacity in an acutely hypophysectomized rat injected with submaximal doses of exogenous ACTH. One question raised at the outset of this study was whether DMSO interferes with the determination of, or cross-reacts with, ACTH or corticosterone when measured by radioimmunoassay, or with corticosterone when measured by acidfluorescence. We found no evidence that DMSO reacted as ACTH or corticosterone when tested by these methods. In man, orally administered DMSO did not stimulate the release of ACTH or cortisol. In this regard a dissociation was observed between the hypothalamicpituitary-adrenal responses to DMSO in man and the rat. Several possibilities exist to account for these differences. ( I ) The dose and the route of administration were different with man, who received a much smaller dose of DMSO. Furthermore, patients who use DMSO for long periods of time do not show clinical signs of excess cortisol. The same dissociation between the responses of man and the rat has been (2) DMSO or its actual metabolite reported when ether was used as the may be interpreted by lower mammals such as the rat as creating stress, and hence the ensuing glucocorticoid response would be protective in nature. The rats injected with the larger dosage of DMSO did not appear ill, hyperexcitable, or lethargic; they did not respond as would be expected if they were significantly stressed. (3) A species-specific physiological adenohypophysial or neurochemical hypothalamic response may occur after the administration of DMSO. ACKNOWLEDGMENTS We are grateful for the opportunity we were given to study patients of Dr. Stanley Jacob. We thank Drs. Monte A. Greer and Joseph W. Goldzieher for their support. REFERENCES I.
GROZDOV, S. P..G. 1. BEZIN,1. N. KENDYSH,I . Y.KIR’YANOV& V. V. VASIL’EVSKAYA. 1971. About the mechanism of biological effects and radio-protective effects of dimethyl sulphoxide. Radiobiologiya 11: 522-527.
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MATSUDA, K., C. DUYCK,J. W. KENDALL & M. A. GREER.1964. Pathways by which traumatic stress and ether induce increased ACTH release in the rat. Endocrinology
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MANGILI, G., M. MOTTA,W. MUCIACCIA & L. MARTINI.1964. Midbrain stress and ACTH secretion. European REV.Endocrinol. 1: 247-253. FORBES, J. C. & G. M. DUNCAN. 1953. Effect of intraperitoneal administration of alcohol on the adrenal levels of cholesterol and ascorbic acid in rats and guinea pigs. Quart. J. Studies Alc. 14: 19-21. SPRIGGS, T. L. G. & M. A. STOCKHOLM. 1964. Urethane anesthesia and pituitary-adrenal function in the rat. J. Pharm. Pharmacol. 16: 603-610. REES, L. H., D. M. COOK,J. W. KENDALL, C. F. ALLEN,R. M. KRAMER, J. G. RATCLIFFE & R. A. KNIGHT.1971. A radioimmunoassay for rat plasma ACTH. Endocrinology 89: 254261. ALLEN,J. P., D. M. COOK, J. W. KENDALL & R. MCGILVRA. 1973. Maternal-fetal ACTH relationship in man. J. Clin. Endocrinol. 37: 23e-234. COOK,D. M.. J. W. KENDALL, M. A. GREER & R. M. KRAMER. 1973. The effect of acute or chronic ether stress on plasma ACTH concentration in the rat. Endocrinology 93:
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KENDALL, J. W., M. L. EGANS& A. K. STOTT.1968. Fluorometric determination of corticosteroids: An interfering substance in impure dichloromethane which fluoresces with benzyl alcohol preservative in heparin. J. Clin. Endocrinol. 28: 1373-1376. RUDER,H. J., R. L. GUY& M. 9. LIPSETT.1972. A radioimmunoassay for cortisol in plasma and urine. J. Clin. Endocrinol. Metab. 35: 219-224. MAKARA, G. B., E. STARK & M. PALKOVITS. 1970. Afferent pathways of stressful stimuli: Corticotrophin release after hypothalamic deafferentation. J. Endocrinol. 47: 41 1-416. GREER,M. A., C. F. ALLEN,G. P. GIBBS& C. GULLICKSON. 1970. Pathways at the hypothalamic level through which traumatic stress activates ACTH secretion in the rat. Endocrinology 86: 14041409. ALLEN,J. P., C. F. ALLEN,M. A. GREER& J. J. JACOBS.1973. Stress-induced secretion of ACTH. In Brain-Pituitary-Adrenal Interrelationships. A. Brodish & E. S. Redgate, Eds.: 99-127. Verlag S. Karger, A.-G. Basel, Switzerland. SMITH, P. E. 1930. Hypophysectomy and replacement therapy in the rat. Amer. J. Anat. 45: 205-273.
HALASZ,B. & L. PUPP. 1965. Hormone secretion of the anterior pituitary gland after partial or total deafferentation of all nervous pathways to the hypophysiotrophic area. Endocrinology 77: 553-562. 16. ALLEN,C. F., J. W. KENDALL & M. A. GREER.1972. The effect of surgical isolation of the basal hypothalamus on the nycthemeral rhythm of plasma corticosterone concentration in rats with heterotopic pituitaries. Endocrinology 91: 873-876. C. F., J. P. ALLEN& M. A. GREER.1973. Effect of hypothalamic deafferentation 17. ALLEN, on tonic and stress-induced ACTH secretion in adrenalectomized rats. Progr. 55th Ann. Meeting Endocrinol. SOC.A-79. IY72. Effects of partial hypothalamic S., N. CONFORTI & I. CHOWERS. 18. FELDMAN, deafferentations on adrenocortical responses. Acta. Endocrinol. (Copenhagen) 69: 52615.
530. 9 . E. 1967. Some studies of the protein-binding of steroids and their application 19. MURPHY, to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J. Clin. Endocrinol. 25: 973-990. F. C., J. LANDON & T. C. 9. STAMP.1966. The plasma sugar, free fatty 20. GREENWOOD,
acid, cortisol and growth hormone response to insulin. I . In control subjects. J. Clin. Invest. 45: 429-436. 21. WINER,9 . J. 1962. Statistical Principles in Experimental Design. pp. 101-103. McGrawHill Book Company. New York, N.Y. 1972. Pituitary-adrenal function in photic and 22. DUNN,J., M. BENNETT& R. PEFFLER. olfactory deprived rats. Proc. SOC. Exp. Biol. Med. 140: 755-758. G. B., E. STARK& K. MIHALY. 1967. Site at which formalin and capsaicin act 23. MAKARA, to stimulate corticotrophin secretion. Can. J. Phys. 45: 669-674.
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24. MAKARA,G. B., E. STARK, M . PALKOVITS, T. REVESZ& K. MIHALY.1969. AKerent pathways of stressful stimuli: Corticotrophin release after partial deamerentation of the medial basal hypothalamus. J . Endocrinol. 44: 187-193. 25. HAIBACK, H., R. J. UNDERWOOD, M. A. GREER& W. P. VANDERLAAN. 1970. Failure to stimulate significant cortisol or growth hormone secretion in man by ether infusion. Experientia 26: 1146-1 147.
