Effects of adrenalectomy and hypophysectomy on postictal seizure protection

Brain Research, 402 (1987) 155-159 Elsevier

155

BRE 21980

Effects of adrenalectomy and hypophysectomy on postictal seizure protection Joseph B. Long and Frank C. Tortella Neuropharmacology Branch, Department of Medical Neurosciences, Division of Neuropsychiatry, Walter Reed Army Institute of Research, Washington, DC 20307-5100 (U.S.A.)

(Accepted 2 September 1986) Key words: Pituitary; Adrenal; Endogenous opioid; Anticonvulsant; Maximal electroshock seizure

Endogenous opioid systems activated by seizures appear to contribute to the postictal inhibition of subsequent seizure activity. In consideration of the possible postictal anticonvulsant actions of endogenous opioids of peripheral origin, we examined whether intact adrenal or pituitary sources of these opioids are necessary for the progressive decline in convulsion intensity and duration normally recorded during a series of 6 intermittent maximal electroshocks (MES). Adrenalectomy did not alter the progressive seizure protection associated with repeated MES. Hypophysectomy, in contrast, increased convulsion duration and abolished progressive reductions in convulsion severity. These data indicate that: (1) adrenal secretions do not substantially contribute to postictal protective mechanisms, and (2) endogenous opioids of pituitary origin may be involved in postictal protective mechanisms.

Following a seizure there is a relative resistance to the induction of further seizures 4'6'17"2s and a reduction in the severity of subsequent convulsions 31. While the specific neuronal mechanisms underlying this refractory state are as yet unknown, several observations point to an involvement of endogenous opioids. For example, seizures appear to activate opioid systems as evidenced by a variety of postictal opioid-like effects 8. Since centrally administered opioids are potent anticonvulsants in a variety of experimentally induced seizures 5'26'32, as proposed by Tortella et al. 27"28, the activation of endogenous opioids by seizures may produce similar anticonvulsant actions during the postictal period. In support of this proposal, it has been demonstrated that maximal electroshock (MES)-induced seizures produce significant postictal increase in flurothy128 and kindled 3'24 seizure thresholds which are markedly attenuated by the opioid antagonist naloxone. Additionally, both naloxone and morphine tolerance abolish the progressive decrease in seizure severity that is normally

associated with intermittent MES 31. A n anticonvulsant role for endogenous opioids is further supported by the recent observation that cerebrospinal fluid (CSF) taken from rats following an electrically induced generalized seizure significantly increases flurothyl seizure thresholds in naive recipient rats following its central administration. This anticonvulsant activity was antagonized by opioid antagonists and enhanced by peptidase inhibitors, suggesting an opioid peptide-like nature of this seizure-activated endogenous anticonvulsant substance 3°. The source of the opioid(s) mediating the postictal refractory state has not as yet been determined. It is possible that endogenous opioids released at various locations within the brain play an important role. Alternatively, both the adrenal and pituitary glands are rich sources of endogenous opioid peptides 7. Previous studies have identified apparent contributions of peripherally derived opioid peptides to electroconvulsive shock (ECS)- and stress-induced central opioid effects 12,29. In the present experiments, the

Correspondence: J.B. Long, Neuropharmacology Branch, Department of Medical Neurosciences, Division of Neuropsychiatry, Walter Reed Army Institute of Research, Washington, DC 20307-5100, U.S.A.

156 potential contributions of opioids of peripheral origin to postictal anticonvulsant mechanisms were examined. To this end, we sought to determine whether intact adrenal or pituitary glands are necessary for the progressive decrease in seizure severity normally associated with intermittent MES 31. Hypophysectomized, adrenalectomized, and appropriately sham-operated control rats were obtained from Zivic Miller Labs. (Allison Park, PA) and housed singly in a temperature-controlled room (25 °C) on a 12 h light-12 h dark cycle. In addition to normal drinking water (tap water) and standard laboratory chow, 0.9% saline was provided ad libitum to adrenalectomized and sham-adrenalectomized rats, while hypophysectomized and sham-hypophysectomized rats were supplemented with free access to a 5.0% sucrose solution and 0.9% saline. Experiments were conducted approximately 12 days following surgery, at which time all rats weighed 200-250 g. Rats were subjected to a series of 6 supramaximal seizures 33 induced at 10-rain intervals by intermittent MES (I-MES). MES (2.0 s at 60 Hz, 50 m A ) was delivered transauricularly by alternating current using a constant current Wahlquist shock apparatus (Wahlquist Instruments, Salt Lake City, UT) resulting in a generalized seizure characterized by an initial tonic extension of the fore- and hindlimbs, followed by a clonic jerking of the musculature. Two features of each MES-induced convulsion were analyzed as measures of seizure severity: (1) the duration of tonic forelimb extension, and (2) the motor convulsion pattern. The convulsion patterns were assigned scores which were based upon the extent of the spread of the tonic extension 31. Specifically, using a 0 - 3 grading scale, absence of forelimb extension was assigned a score of 0, complete forelimb extension with absence of hindlimb extension was scored 1, complete forelimb with partial hindlimb extension was scored 2, and complete forelimb and hindlimb extension (with hindlimbs fully extended parallel to the tail) was assigned a maximal score of 3. The durations of tonic forelimb extension were evaluated by analysis of variance with repeated measures 34. Significant differences between means were distinguished by the least significant difference test. Motor seizure pattern scores were evaluated using the M a n n - W h i t n e y U-test 25. Differences were considered significant at the level P < 0.05.

As previously reported 31, I-MES produced a progressive decrease in seizure severity, with both the duration of tonic forelimb extension and m o t o r pattern scores decreasing with successive seizures (Figs. 1 and 2, sham groups). Responses of adrenalectomized rats to I-MES did not differ from the responses recorded in sham-operated controls and, following the sixth MES, tonic forelimb extension durations were significantly shorter than those recorded following the first MES (Fig. 1). Similarly, motor seizure pattern scores progressively declined over the course of 6 I-MES in both adrenalectomized and sham-adrenalectomized rats, resulting in significantly reduced scores for both groups following the sixth MES (Fig. 2). In contrast, hypophysectomy produced significantly increased forelimb extension durations which persisted through all 6 convulsions (Fig. 1). Unlike the sham-hypophysectomized controls, these animals failed to show the characteristic progressive, significant decrease in forelimb extension duration. Hypophysectomized rats also demonstrated higher motor pattern scores (reflecting more severe seizures) throughout all 6 MES-induced con-

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vulsions, which in contrast to the scores from control animals, did not significantly decrease by the sixth MES (Fig. 2). Although not significantly different from controls following the first MES, motor pattern scores of hypophysectomized rats did appear to differ from those of control rats following the sixth MES (P < 0.07, Fig. 2). Prominent among the postictal actions of seizureactivated endogenous opioid systems are their anticonvulsant effects during the acute postictal period 3'9'10'24'28'31. The present observation that hypophysectomy elevated seizure severity (Fig. 1) and abolished the progressive seizure protection associated with I-MES is consistent with the possibility that endogenous opioids of pituitary origin contribute to seizure-activated postictal protective mechanisms. However, since hypophysectomy also results in an array of neuroendocrine and metabolic derangements 16, it is obvious that on the basis of these results alone these effects cannot be exclusively attributed to a loss of pituitary opioids. Adrenalectomy has been reported to increase circulating levels of fl-endorphin 23 and to increase the passage of blood-borne macromolecules into the CNS 13. Thus, one might expect adrenalectomy to

either eliminate the potential postictal anticonvulsant actions of adrenal-derived opioids or to promote the postictal anticonvulsant actions of circulating opioids of pituitary origin. However, adrenalectomy failed to alter either seizure parameter observed over the course of 6 I-MES seizures, indicating that endogenous opioids of adrenal origin (as well as other adrenal secretions) are of little if any importance in the postictal regulation of seizure activity in this model. The influence of hypophysectomy on endogenous opioid-mediated postictal events has been examined in several previous studies, with the results varying depending upon the specific endpoint under evaluation. For example, hypophysectomized rats demonstrate complete opioid-mediated ECS-induced antinociception 11. Similarly, hypophysectomy did not eliminate the MES-induced postictal rise in flurothyl seizure threshold, nor did it alter the attenuation of this refractory state by naloxone 28. Thus, it would appear that opioids of pituitary origin play little if any role in these particular postictal opioid effects. However, more recent results revealed that hypophysectomy did partially attenuate ECS-induced electrogenesis and postictal depression 29. Furthermore, in contrast to sham-operated controls, in hypophysectomized rats naloxone was ineffective in preventing ECS-induced increases in E E G voltage output and postictal depression. These latter observations are consistent with the present findings in suggesting that opioids originating from the pituitary may contribute in part to certain postictal conditions. Based on differences in these varied observations, it would appear reasonable to conclude that ECS- or MES-induced seizures activate endogenous opioid systems at multiple loci, each of which may contribute to varying degrees to the different postictal opioid effects associated with a seizure. The apparent disparity with the presence and absence of an effect of hypophysectomy on opioid anticonvulsant effects in the flurothyl and intermittent MES models, respectively, most likely stems from the different threshold and suprathreshold natures of these models. The absence of an effect of hypophysectomy in the flurothyl model indicates that pituitary secretions may not greatly affect those conditions associated with the initiation of a seizure, which in turn serve to determine flurothyl seizure thresh-

158 olds. In contrast, the elimination following hypophysectomy of the normally occurring reduction in seizure severity in the suprathreshold I-MES model reveals that pituitary secretions (such as opioids) may normally influence those conditions determining seizure spread. Presumably, endogenous opioid influences on seizure parameters are mediated within the CNS. However, as circulating pituitary products (including opioids) are normally excluded from the CNS by the blood-brain barrier 2'22, the influence of pituitary opioids on CNS function is probably quite limited. For circulating peptides, entry into the CNS is normally limited to circumventricular sites, where the presence of endothelial fenestrations and the absence of tight junctions provide for immediate localized access 2°. Further passage of blood-borne macromolecules through the rest of the CNS is restricted by the ventricular ependymal barriers in these locations 2'2°'22. As the integrity of the blood-brain barrier to macromolecules has been reported to be compromised by seizures el, under these conditions substances normally excluded from the brain (such as circulating endogenous opioids) may pass through the cerebral microvascular endothelium. Alternatively, attention has recently been redirected to the possibility of retrograde transport of pituitary secretions directly to the median eminence, with subsequent distribution of pituitary humors into the CSF and throughout the CNS 15'18'19. While to date no direct evidence of pituitary-brain retrograde flow has been produced under physiological circumstances, backflow has been demonstrated under experimental conditions and remains an attractive possibility based on several lines of investigations 15'18'19. While we failed to observe acute alterations in Met-enkephalin, Leu-enkephalin and /3-endorphin levels in brain tissue samples following I-MES-induced seizures (unpublished observations), we have

reported significant increases in CSF fl-endorphinlike immunoreactivity associated with a single generalized seizure TM. Evaluation of CSF fl-endorphin concentrations in hypophysectomized rats following seizures might shed light on both the origin of the increases in CSFfl-endorphin and on the possible retrograde transport of fl-endorphin under conditions associated with a convulsion. Given the fine dynamics and pressure sensitivities of the pituitary portal system, it is conceivable that the dramatic pressor responses to MES 1 might temporarily alter the direction of blood flow in the portal system and facilitate entry of pituitary secretions. If so, seizure-induced elevations in CSF levels of other pituitary hormones might be expected as well, and might similarly contribute to postictal events. In summary, removal of the pituitary prevents the progressive decrease in seizure severity normally associated with I-MES. When considered with the previously demonstrated involvement of endogenous opioids in this postictal anticonvulsant mechanism, the present findings point to the potential involvement of opioids of pituitary origin in postictal refractory states.

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In conducting the research described in this report, the investigator(s) adhere to the 'Guide for the Care and Use of Laboratory Animals', as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The views of the author(s) do not purport to reflect the position of the Department of the Army or the Department of Defense (para 4-3, A R 360-5). We thank Lydia Robles for technical assistance, Dr. John W. Holaday for thoughtful suggestions and discussions, and Dr. Rochelle M. Long for assistance with statistical evaluations.

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