Muscular spasms and death in thyroparathyroidectomized rats subjected to pinealectomy

Life Sciences Vol. 11, Part I, pp. Printed in Great Britain

123-139, 1972 .

Pergamon Press

MUSCULAR SPASMS AND DEATH IN THYROPARATHYROIDECTOMIZED RATS SUBJECTED TO PINEALECTOMY( 1 ) Russel J. Reiter, Sandy Sorrentino, Jr . and Roger A. Hoffman Department of Anatomy, University of Tezas . Medical School at San Antonio, Department of Aaatamy, University of Rochester, and Department of Biology, Colgate University (Received 8 October

1971 ;

in final form 23 December

1971)

S umma~ If rate were thyroparathyroidectomf$ed when 24 to 25 days of age and were later (10 to 21 days) pinealectomised, all animals ezperienced severe convulsive attacks and most of them died within 14 hours . Sham thyroparathyroidectomized rats that were subjected to pinealectamy exhibited no similar convulsive episodes nor did they die . Thyroparathyroidectomy caused the anticipated decrease in serum calcium levels but had no influence on serum sodium, potassium or magnesium concentrations . Pinealectomy was followed by a slight elevation in serum potassium levels regardless of the status of the thyroparathyroid gland complez. Pineal removal did not influence the serum levels of either sodium, calcium or magnesium . Introduction In an almost serendipitous manner, we recently observed that if previously thyroparathyroidectomiaed rats had their pineal glande removed, the majority of the animals died within 12 hours.

Prior to death the animals

ezperienced severe tonic contractions of the trunk and limb musculature . The cause of death was probably asphy~dation due to spasm of the laryngeal and thoracic muscles . Pineal removal alone has never been reported to precipitate convulsive attacks as was observed under these ezperimental conditions .

The findings seemed of sufficient merest and probable

importance to justify the following preliminary report . (1) Reprint requests to R. J. Reiter, Departme~ of Anatomy, University of Tezas Medical School at San Antonio, San Antonio, Texas T8229 . 123

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Materiale and Methode All animals used in these studies were male Sprague-Dawley rate born in the Vivarium at the University of Rochester, Rochester, New York .

The

rate were weaned when they were 21 days of age and thereafter were provided with Purina rat cl}ow and tap water ad libitum.

They were housed

4 or 5 per clear plastic cage (25 z 45 z 20 cm) in light (12 hours of light per day) and temperature (23 t 2°C) controlled rooms.

Lights were automati-

cally turned on at 6:00 A. M. and off at 6:00 P. M.

Light was provided by

40 watt "cool white" fluorescent bulbs which yielded an intensity at the level of the cages of 50-85 ft . c. In the first experiment, thirteen 24 to 25 day old rats were subjected to thyroparathyroidectomy (TPTX) under sodium pentobarbital anesthesia . Twelve control rate of the same age were sham-operated (SHAM) .

The

latter operation included eaposure and gentle manipulation of the thyroparathyroid gland complez. Skin incisions were closed with wound clips. All animals (TPTX and SHAM) were allowed to recover for 10 days . time all the animals were subjected to pineal gland removal (PX) .

At this The

technique for pinealectomy was that described by Hoffmaa and Reiter (1) and a Neuman Pinealectomy Kit (H . Neuman and Co . , Skokie, Illinois) was used . The pinealectomies were done with the animals aneethesised with ether and each operation required approximately 3 minutes .

All animals were pineal-

ectomised between 0915 and 1100 hours and the specific time of pineal removal was recorded for each rat.

After this operation the rats were

placed 4 or 5 per cage eo that each cage contained animals from each of the two categories ; i. e. , each cage contained ea~me TPTX + PX and some SHAM + PX rate . After pinealectomy the rats were observed continuously

Vol. 11, No. 3

Süect ad Plaealectomy

lab

and the time of appearance ad muscular spasms and death was recorded . In the second study 10 rats were thyroparathyroidecto~naised and 11 were sham-operated when they were 24 days old.

Three weeks later the

rate were anesthesised with ether and 1 ml of blood was drawn from the external jugular vein.

Two days after blood withdrawal, the rats were

pinealectomised, between 0935 and 1050 hours, and they were caged as in the first experiment . In the second experiment, rather than allowing the animals to die, the TPTX + PX rats were decapitated during their first intensive muscular spasm. At the time of decapitation the second blood sample was collected. Within 4 hours after pinealectomy, all the TPTX rats had experienced their first muscular spasm and had been sacrificed. The SHAM + PX rats were decapitated 1 hour after the last TPTX + PX rat had been killed.

The first (taken 2 days before pinealecto~my) and the second

blood samples were centrifuged and the serum was analysed for calcium, potassium and magnesium.

Unfortunately, in this experiment the sera were

inadvertently discarded before the sodium levels were determined .

The ion

levels were measured using a Perkin-Elmer Atomic Absorption Spectrophotometer (Model 290) after the serum was diluted 1:50 with deionised water. To check the serum levels of sodium in convulsing rate a third experiment was conducted. Eight rats were TPTX and 8 were sham-operated when they were 22 days of age.

Twelve days later a 1 ml blood sample was drawn

from the external jugular vein.

Three days following the blood collection all

the animals were pinealectaazised while snestheeised with ether. 8 TPTX + PX rats exhibited

Each oaf the

a convulsive episode within 5 hours after PX .

During their Hret convulsion they were decapitated and the trunk blood was

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Eitect of Pinealectomy

collected.

The SHAM + PX rate were killed 6 hours after pinealectamy and

the blood samples were also retained .

The sera from these blood samples

were analysed for sodium using the epectrophotametric method noted above. Results Eaperimeat one. None of the rate died from the thyroparathyroidectomy procedure or from the,sham operation.

Within several days after these

operations all animals were eating and drinking normally .

Likewise, all of

the rate tolerated pineal removal well with the animals recovering from the ether anesthesia within about 10 minutes . About 2 hours after pinealectomy, however, it became apparent that the TPTX + PX rate were reacting differently to this procedure than were control animals (SHAM + PX).

At

1311 hours (Table 1, animal no . 11) the first TPTX+ PX rat ezhibited an intensive muscular spasm. Within a short time other TPTX + PX rate experienced similar convulsive attacks (Table 1) .

The sequence of events

for these episodes was usually as follows : hypermobility, clonic contractions of the hind limbs and eventually sustained hyperextensioa of all the limbs accompanied by pronounced rigidity of the trunk musculature.

The

time interval from the onset of hypermobility to the conclusion of the spasm was 30 seconds to several minutes. An attack, if death did not ensue, was followed by a period (1 to several minutes) of inactivity and increased respiratory rate . Ezamination of Table 1 reveals that of the 13 TPTX rate that were pinealectomised in the morning, all had experienced at least one generalised convulsive attack by late that afternoon.

Characteristically, these animals

sustained 2 or 3 such spasms before death ensued .

When they did succumb,

they usually died during a convulsion or shortly thereafter .

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rat (Table 1, no . 7) experienced a single convulsion but survived until the experiment was terminated (3 weeks after piaealectomy). On several occasions a spastic attack is one rat seemed to precipitate a convulsive reaction is a similarly-treated animal in the same cage . Note, for example, the first attack sustained by rat no . 9 was accampasied by the second convulsion in rat no . 8 (Table 1) .

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episodes of rate numbers 10 and 11, respectively, coincided.

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did not seem to be coincidentally related, although they may have been . The 10 SHAM + PX rats tolerated the experimental procedures well . None of these animals ezhibited a convulsion and all survived for 3 weeks after pinealecto~my, at which point they were sacrificed. Fsperiment two. The experimental protocol for the second study was only slightly different from that of experiment one. Again, the animals that were pinealectomized after having been thyroparathyroidectamized 3 weeks earlier sustained convulsive attacks several hours subsequent to removal of the pineal .

The average length of time between pia+ealectomy and the first

convulsion was 208 minutes (Table 2) . As in the first study, none of the SHAM + PX rats Buffered any untoward consequences of the operations . As ezpected, at the tei`mination of the experiment the TPTX + PX rate weighed significantly lees than the animals with intact thyroparathyroid glands (Table 2) .

Also, the relative weights of anterior pituitary glands

ezhibited the characteristic hypertrophic response . TPTX caused the anticipated decrease in serum calcium levels but these values were not significantly influenced by pineal removal (Table 3) . On the other hand, serum magnesium concentrations remained unchanged

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130

$üect ad Piae~.lectomy

after either TPTX or PX .

Vol . 11, No. 3

Although excision of the thyroparathyroid gland

complex did not affect serum potassium levels, pineal removal caused a modest increase (P<0 . 02) in this serum constituent in both TPTX and shamoperated rate . Experiment three.

The purpose of the final study was to determine whether

the sodium levels in the serum of convulsing male rate was altered. Again, the TPTX + PX rats eaperieaced their first convulsive episode within several hours after pineal gland removal while SHAM + PX rats exhibited no similar attacks.

TPTX did not significantly change plasma sodium levels

when compared to SHAM rate (144 t 5 . 9 versus 156 t 5 . 3 mEq/1) (mean f S. E. ) . Similarly, the sodium levels of convulsing rate (141 f 9. 1 mEq/1) were not statistically significantly different from those of non-convulsing animals (143 t 7. 7 mEq / 1) . Discussion Although the present report deals with only two experiments in which a total of 64 rate were used, the study has been repeated on several occasions using 150-200 rate and the results were similar.

Pineal removal

from rats that are already lacking their thyroid and parathyroid glands leads to convulsions and somel:imes death. Furthermore, this condition is not restricted to rate .

We (M. K. Vaughan and R . J. Reiter, unpublished

observations) recently found that pinealectomy (even without prior removal of the thyroparathyroid gland complex) occasionally initiates convulsions and death in voles Microtue montanus ).

The sequence of events and the

time requirement for the onset ie about the same is both species .

It should

be noted that subjecting TPTX rate to sham pinealectomy (removal of the bone flap and rupture of the venous einueea in the vicinity of the pineal) is

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not followed by convulsions .

191

It is posdble, hoavever, that the loss a~f the

pineal was not directly responsible for the changes but rather the pinealectomy procedure disturbed an adjacent neural area which mediated the seisuree . Pineal-thyroid and pineal-parathyroid interactions have both been proposed previously .

With regard to the thyroid gland, the pineal and pineal

substances have been studied relative to their inhibitory effect on pituitary thyroid stimulating hormone and thyroxine metabolism [for review see (2)] . The results obtained thus far have been equivocal .

There have been only a

few studies concerned with the interplay between thyrocalcitonin, an important hypocalcemic and hypophosphatemic factor derived from the parafollicular cells of the thyroid gland (3, 4), and the pineal .

The thyrocalci-

tonin-producing parafollicular cells reportedly undergo a hyperplaetic reaction after pinealectomy (5) .

Whether the absence of thyrocalcitonia was

concerned with the convulsive reactions of the rate observed in the present ezperime~e is unknown, but seems unlikely . The interplay of the pineal and the parathyroid glands is also problematical .

According to Kiss et al. (6), excision of the pineal gland causes

decreased parathyroid activity ae evidenced by the increased plasma levels and reduced urinary output of phosphorus and by the depressed incorporation of radioactive methionine into the parathyroid glands .

Seemingly co~rary

to these results are those of Krstic (7) who noted that the administration of pineal eztracts to rats caused involution of the parathyroid glands .

It' is

obvious fraaa studies such as these that the interaction of this organ with the pineal still requires clarification . It was a~icipated that the analyses of serum cation levels would provide

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a partial explanation for the generalized muscular contractions which were recorded .

The usual drop in serum calcium attendant upon parathyroidec-

tomy was apparent . However, subsequent pinealectomy caused no further detectable depression in the level of this inorganic constituent.

Although

this suggests that the spasms were not due to low-calcium tetany, on the basis ad these preliminary data it seems umvise to disregard depressed eztracellular calcium levels se a causative factor .

The failure of pineal

removal to depress serum calcium is consistent with the observations of Karppaaen et al . (8). .Unexpectedly, the potassium levels were elevated after pinealectomy regardless of whether the thyroparathyroid gland complex was intact or removed.

These findings are contrary to those of other

workers (8) who observed a depression of serum potassium concentrations after ezcisioa of the pineal .

The slightly elevated serum levels of potassium

is the present experiment does not provide us with an adequate explanation oaf the observed muscular hypereacitability.

The serum magnesium levels

were equivalent in all animals. Magnesium deficiency may also induce episodes of tetany . In 1965, Quay (9) reported that pinealectomized rate on a sodium deficient diet were unable to maintain cerebral potassium content.

This

illustrates the possibility that the pineal hormone may have a direct influence on brain hameoetasis and it is conceivable that the ionic effects of pineal ezcisioa are exaggerated in TPTX rate .

Considering the importance of

sodium and potassium balance in neurone and their paramour role in regulacing neural transmission (lp), an explanation for the present results may lie, at least partially, within the CNS.

It may be noteworthy that injected

tritiated-melatonin, the radioactive form of the pineal hormone, is taken up

vol.

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xo.

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Ettect ~ Pinealectomy

lss

by the brain and peripheral nerves (11) . Whether this substance, which would be reduced after pinealectosny, has any influence on neural ionic Hazes, neuromuscular ezcitability or synaptic transmission remains to be seea. Acknowledgment-Supported by U. S. P. H. S. grants HD-02937 and HD-06523 . References 1.

R. A. HOFFMAN and R. J. REITER, Anat . Rec. 153, 19 (1965) .

2.

R. J. REITER and F. FRASCHINI, Neuroendocrinology 5, 219 (1968) .

3.

G. V. FOSTER, J. MacINTYRE and A. G. E. PEARSE, Nature 203, 1029 (1964) .

4.

P. L. MUNSON, In P. P. FOA (ed. ), Action oaf Hormones , pp . 231254. Charles C. Thomas, Springfield (1971) .

5. R. MILINE, M. SCEPOVIC and R. KRSTIC, Bull. l'Assoc. Anat . 5Z, 893 (1967) . 6. .J . KISS, D. BAHEGYI and G. CSABA, Acta Med. Acad . Sci. Hung . 26, 363 (1969) . 7 . R . KRSTIC, Z. Zellfor. ~, 73 (1968) . B. H. KARPPANEN, H. VAPAATALO, S. LAHOVAARA and M. K. PAASONEN, Pharmacology 3, 76 (1970) . 9 . W. B. QUAY, PrOA . Brain Res. 10, 646 (1965) . 10 . H. HOAGLAND, In K. A. C . ELLIOTT, I. H. PAGE and J. H. QUASTEL (eds . ), Neurochemistry . 2nd. Ed . , pp. 954-976. Charles C. Thomas, Springfield (1962) . R . J. WURTMAN, J. AXELROD and L. T. POTTER, J. Pharm. Ezp. Therap . 143, 314 (1964) .