JBJS | The Effects of Melatonin Therapy on the Development of Scoliosis After Pinealectomy in the Chicken*

The Journal of Bone & Joint Surgery, Volume 81, Issue 2

Articles | February 01, 1999

The Effects of Melatonin Therapy on the Development of Scoliosis After Pinealectomy in the Chicken*

KEITH BAGNALL, PH.D.†; V. JAMES RASO, M.SC.‡; MARC MOREAU, M.D.†; JAMES MAHOOD, M.D.†; XIAOPING WANG, M.D.†; JIE ZHAO, M.D.†, EDMONTON, ALBERTA, CANADA

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Investigation performed at the Department of Cell Biology and Anatomy and the Department of Surgery, University of Alberta, Edmonton

The Journal of Bone & Joint Surgery. 1999; 81:191-199

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Abstract

The mechanism underlying the development of scoliosis after pinealectomy in young chickens is unknown. However, since the main product of the pineal gland is melatonin, melatonin remains an obvious focus in studies designed to discover this mechanism. One confounding factor is that serum melatonin levels are close to zero after pinealectomy but scoliosis does not develop in all chickens that have had this procedure. Therefore, the role of melatonin in the development of scoliosis in chickens after pinealectomy remains controversial.In the current investigation, two pilot studies demonstrated that a physiological therapeutic dose of melatonin (2.5 milligrams per kilogram of body weight) restored the circadian rhythm of melatonin, as measured by serum assay. In the main study, this dose was administered daily starting either immediately after the pinealectomy or two weeks after it, when scoliosis had developed. Scoliosis was assessed on weekly radiographs, and the Cobb angle was determined for all chickens in which scoliosis developed.Overall, scoliosis developed in only 56 percent (fifty) of the eighty-nine chickens that had had a pinealectomy; this rate was consistent throughout all experimental groups. Scoliosis did not develop in any of the control chickens, which did not have a pinealectomy. On the basis of the average Cobb angles in the chickens in which scoliosis had developed, it was determined that neither the prevalence nor the pattern of the scoliosis was affected by the therapy in any of the experimental groups.It was thus concluded that melatonin therapy after pinealectomy in young chickens has no effect on the development or progression of scoliosis. These results raise doubts regarding the role of melatonin in the development of scoliosis after pinealectomy in the young chicken.CLINICAL RELEVANCE: Although scoliosis has been produced in some animal studies, none of these models has proved to be entirely satisfactory. Consequently, research regarding adolescent idiopathic scoliosis has been hampered. Recently, it was shown that scoliosis with many characteristics similar to those seen in patients who have adolescent idiopathic scoliosis can be produced consistently in chickens after pinealectomy. This finding encourages the development of this model. An understanding of the mechanism involved in the development of scoliosis after pinealectomy in chickens might provide new insights into adolescent idiopathic scoliosis and aid in the development of novel treatment methods.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

Although some investigators have successfully produced scoliosis in animals^10,12,14, none of these models has been completely satisfactory¹¹. Recent studies have shown that pinealectomy in young chickens consistently leads to scoliosis that has characteristics similar to those in adolescent idiopathic scoliosis in humans^6,15. Thus, the animal model of scoliosis after pinealectomy is potentially useful for studying the pathogenesis and mechanism underlying the development of adolescent idiopathic scoliosis.

The hormone melatonin is the principal product of the pineal gland and is a focus of studies of the mechanism underlying the development of scoliosis in chickens. In support of the hypothesis that reduced melatonin levels are the primary cause of scoliosis after pinealectomy, Machida et al.⁶ reported that melatonin therapy suppressed the development of scoliosis in young chickens treated with that procedure. In another study, Machida et al.⁸ found that serum melatonin levels in humans were a useful predictor of the progression of adolescent idiopathic scoliosis. In contrast, other authors found no relationship between serum melatonin levels and the development of scoliosis in either chickens or humans. For example, Wang et al.¹⁶ reported that scoliosis developed in only seventeen (52 percent) of thirty-three chickens that had had a pinealectomy, even though the serum melatonin level in all chickens was zero. This prevalence of scoliosis was consistent with data from other studies^2,13. Similarly, we¹, as well as Hilibrand et al.³, found no difference between melatonin levels in patients who had adolescent idiopathic scoliosis and those in age and gender-matched controls. Such disparity concerning the involvement of melatonin in scoliosis is confusing and requires additional study. Accordingly, the present study was designed to investigate the effects of melatonin therapy on young chickens after pinealectomy. A series of pilot studies was performed to determine an appropriate melatonin therapy regimen with which the normal melatonin secretion patterns of chickens could be reproduced after pinealectomy.

*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding source was Edmonton Civic Employees.

†Department of Cell Biology and Anatomy (K. B., X. W., and J. Z.) and Department of Surgery (M. M. and J. M.), University of Alberta, Edmonton, Alberta T6G 2H7, Canada. E-mail address for Dr. Bagnall: kbagnall@anat.med.ualberta.ca.

‡Glenrose Rehabilitation Hospital, Edmonton, Alberta T5G 0B7, Canada.

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FIG1:: Fig. 1 Graph showing the circadian rhythm of serum melatonin levels in five-day-old chickens. SD = standard deviation.

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FIG2:: Fig. 2 Graph showing the mean serum melatonin levels in five-day-old chickens after a pinealectomy and an intraperitoneal injection of 0.1 milliliter of a solution of phosphate-buffered saline solution and melatonin at a dose of 2.5 milligrams per kilogram of body weight. SD = standard deviation.

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FIG3-A:: Fig. 3-A Graph showing the changes in the mean Cobb angle⁴, during the course of the experiment, in the chickens with scoliosis in the four experimental groups. The data for the normal controls (group E) are shown for comparison. The degree of curvature and the rate of progression were similar among the four experimental groups. SD = standard deviation.

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FIG3-B:: Fig. 3-B Graph showing the changes in the mean Cobb angle⁴, during the course of the experiment, in the chickens in which scoliosis (a curve of more than 9 degrees) did not develop in the five groups. SD = standard deviation.

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FIG4:: Fig. 4 Graph showing the changes in the mean weight, during the course of the experiment, in the five groups. The pattern of growth was very similar among the groups, and no significant differences could be detected at any stage, with the numbers available. SD = standard deviation.

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FIG5:: Fig. 5 Graph showing the changes in the mean length of the spine, during the course of the experiment, in the five groups. The growth patterns were similar, and no significant differences could be detected at any stage, with the numbers available. SD = standard deviation.

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FIG6:: Fig. 6 Graphs showing the changes in the mean weight, in each of the four groups in which a pinealectomy was done, for the chickens in which scoliosis developed compared with those in which it did not. When there is only one symbol, the values overlap and the second symbol is obscured. The mean weight in group E for each week is shown for comparison. No significant differences could be detected, with the numbers available, between the scoliotic and nonscoliotic chickens in any group. SD = standard deviation.

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FIG7:: Fig. 7 Graphs showing the changes in the mean length of the spine, in each of the four groups in which a pinealectomy was done, for the chickens in which scoliosis developed compared with those in which it did not. When there is only one symbol, the values overlap and the second symbol is obscured. The mean length in group E for each week is shown for comparison. No significant differences could be detected, with the numbers available, between the scoliotic and nonscoliotic chickens in any group. SD = standard deviation.

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TABLE I DATA REGARDING THE SCOLIOSIS IN THE FIVE GROUPS OF CHICKENS*

*R = convexity of the curve on the right side, and L = convexity of the curve on the left side. †p = primary curve and s = secondary curve in the chickens that had a double curve.

Group A†	Cobb Angles in Group A (degrees)					Group B†	Cobb Angles in Group B (degrees)					Group C†	Cobb Angles in Group C (degrees)					Group D	Cobb Angles in Group D (degrees)					Group E	Cobb Angles in Group E (Controls) (degrees)
Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy
1	2	3	4	5	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5
1	0	0	0	0	0	24	0	15R	20R	28R	37R	47	0	0	0	17R	20R	69	0	0	8R	11R	12R	1	0	0	0	0	0
2	0	0	0	0	0	25	0	0	0	31L	10L	48	0	0	0	0	0	70	0	15R	25R	26R	29R	2	0	0	0	0	0
3	0	0	8R	10R	15R	26	0	0	0	0	0	49	0	0	0	0	0	71	8R	19L	0	0	0	3	0	0	0	0	0
4	0	12R	16R	20R	25R	27	0	0	0	0	0	50	0	0	0	0	0	72	0	0	0	0	0	4	0	0	0	0	0
5	0	0	0	0	0	28	10R	10R	18R	26R	22R	51	0	0	0	0	0	73	0	0	15R	18R	15R	5	0	0	0	0	0
6	0	0	0	0	0	29	0	0	0	0	0	52	0	0	0	0	0	74	0	0	0	0	0	6	0	0	0	0	0
7p	14R	27R	35R	45R	42R	30	0	10L	18L	10L	10L	53	0	14R	20R	17R	29R	75	10L	22L	23L	28L	24L	7	0	0	0	0	0
7s	0	26L	43L	40L	50R	31	0	20L	25L	30L	32L	54	10R	10R	20R	23R	24R	76	0	10R	11R	14R	15R	8	0	0	0	0	0
8	0	0	0	0	0	32	0	0	0	0	0	55	6L	16L	25L	16L	21L	77	12L	20L	30L	35L	34L	9	0	0	0	0	0
9	0	0	15R	17R	15R	33	11R	25R	32R	36R	—	56p	29L	33R	40R	45R	48R	78	20L	8L	0	0	0	10	0	0	0	0	0
10	9L	15L	20L	23L	28L	34	0	0	10L	10L	14L	56s	0	32L	38L	43L	38L	79	0	0	0	0	0	11	0	0	0	0	0
11	0	10L	10L	10L	12L	35	15R	25R	35R	31R	29R	57	0	10R	14R	17R	12R	80	0	0	0	0	0	12	0	0	0	0	0
12	0	0	0	0	0	36	0	0	0	0	0	58	0	17L	23L	22L	30L	81	0	0	0	0	0	13	0	0	0	0	0
13	0	0	0	0	12L	37	0	0	0	0	0	59	0	0	0	0	0	82	0	19R	25R	23R	28R	14	0	0	0	0	0
14	12L	23L	24L	28L	30L	38	0	0	0	0	0	60	12L	14L	23R	24R	—	83	0	0	20L	14L	15L	15	0	0	0	0	0
15	0	15L	24L	18L	12L	39	0	0	0	0	0	61p	0	12R	25R	29R	32R	84	0	5L	0	0	0	16	0	0	0	0	0
16	0	0	0	0	0	40	0	0	11L	20L	15L	61s	0	0	24L	25L	26L	85	0	0	0	0	0
17	0	0	0	0	0	41	0	10R	22R	17R	22R	62	0	0	15R	20R	15R	86	0	0	19R	20R	20R
18	0	0	0	0	0	42	0	12R	13R	13R	10R	63	10R	18R	31R	34R	40R	87	0	0	0	0	0
19	0	0	0	0	0	43p	0	21L	36L	43L	52L	64	0	18R	23R	28R	27R	88	0	0	0	0	0
20	0	0	0	0	0	43s	0	10R	30R	35R	40R	65	0	0	0	0	0	89	0	20L	25L	25L	22L
21	0	9R	10R	10R	20R	44	0	7R	25L	8R	10R	66	0	0	0	0	0
22	0	0	10L	8L	10L	45	0	12R	16R	25R	28R	67	0	0	0	0	0
23	11R	24R	25R	24R	25R	46	0	0	0	0	0	68	0	0	0	0	0

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TABLE I DATA REGARDING THE SCOLIOSIS IN THE FIVE GROUPS OF CHICKENS*

*R = convexity of the curve on the right side, and L = convexity of the curve on the left side. †p = primary curve and s = secondary curve in the chickens that had a double curve.

Group A†	Cobb Angles in Group A (degrees)					Group B†	Cobb Angles in Group B (degrees)					Group C†	Cobb Angles in Group C (degrees)					Group D	Cobb Angles in Group D (degrees)					Group E	Cobb Angles in Group E (Controls) (degrees)
Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy
1	2	3	4	5	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5
1	0	0	0	0	0	24	0	15R	20R	28R	37R	47	0	0	0	17R	20R	69	0	0	8R	11R	12R	1	0	0	0	0	0
2	0	0	0	0	0	25	0	0	0	31L	10L	48	0	0	0	0	0	70	0	15R	25R	26R	29R	2	0	0	0	0	0
3	0	0	8R	10R	15R	26	0	0	0	0	0	49	0	0	0	0	0	71	8R	19L	0	0	0	3	0	0	0	0	0
4	0	12R	16R	20R	25R	27	0	0	0	0	0	50	0	0	0	0	0	72	0	0	0	0	0	4	0	0	0	0	0
5	0	0	0	0	0	28	10R	10R	18R	26R	22R	51	0	0	0	0	0	73	0	0	15R	18R	15R	5	0	0	0	0	0
6	0	0	0	0	0	29	0	0	0	0	0	52	0	0	0	0	0	74	0	0	0	0	0	6	0	0	0	0	0
7p	14R	27R	35R	45R	42R	30	0	10L	18L	10L	10L	53	0	14R	20R	17R	29R	75	10L	22L	23L	28L	24L	7	0	0	0	0	0
7s	0	26L	43L	40L	50R	31	0	20L	25L	30L	32L	54	10R	10R	20R	23R	24R	76	0	10R	11R	14R	15R	8	0	0	0	0	0
8	0	0	0	0	0	32	0	0	0	0	0	55	6L	16L	25L	16L	21L	77	12L	20L	30L	35L	34L	9	0	0	0	0	0
9	0	0	15R	17R	15R	33	11R	25R	32R	36R	—	56p	29L	33R	40R	45R	48R	78	20L	8L	0	0	0	10	0	0	0	0	0
10	9L	15L	20L	23L	28L	34	0	0	10L	10L	14L	56s	0	32L	38L	43L	38L	79	0	0	0	0	0	11	0	0	0	0	0
11	0	10L	10L	10L	12L	35	15R	25R	35R	31R	29R	57	0	10R	14R	17R	12R	80	0	0	0	0	0	12	0	0	0	0	0
12	0	0	0	0	0	36	0	0	0	0	0	58	0	17L	23L	22L	30L	81	0	0	0	0	0	13	0	0	0	0	0
13	0	0	0	0	12L	37	0	0	0	0	0	59	0	0	0	0	0	82	0	19R	25R	23R	28R	14	0	0	0	0	0
14	12L	23L	24L	28L	30L	38	0	0	0	0	0	60	12L	14L	23R	24R	—	83	0	0	20L	14L	15L	15	0	0	0	0	0
15	0	15L	24L	18L	12L	39	0	0	0	0	0	61p	0	12R	25R	29R	32R	84	0	5L	0	0	0	16	0	0	0	0	0
16	0	0	0	0	0	40	0	0	11L	20L	15L	61s	0	0	24L	25L	26L	85	0	0	0	0	0
17	0	0	0	0	0	41	0	10R	22R	17R	22R	62	0	0	15R	20R	15R	86	0	0	19R	20R	20R
18	0	0	0	0	0	42	0	12R	13R	13R	10R	63	10R	18R	31R	34R	40R	87	0	0	0	0	0
19	0	0	0	0	0	43p	0	21L	36L	43L	52L	64	0	18R	23R	28R	27R	88	0	0	0	0	0
20	0	0	0	0	0	43s	0	10R	30R	35R	40R	65	0	0	0	0	0	89	0	20L	25L	25L	22L
21	0	9R	10R	10R	20R	44	0	7R	25L	8R	10R	66	0	0	0	0	0
22	0	0	10L	8L	10L	45	0	12R	16R	25R	28R	67	0	0	0	0	0
23	11R	24R	25R	24R	25R	46	0	0	0	0	0	68	0	0	0	0	0

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Pilot Studies

Determination of the daily melatonin rhythm in five-day-old chickens: One hundred and ten newly hatched Mountain Hubbard chickens were obtained from a local hatchery (Lilydale, Edmonton, Alberta, Canada) and were immediately introduced to a 12:12-hour dark:light cycle (twelve hours of dark [6:00 A.M. to 6:00 P.M.] and twelve hours of light) with food and water provided ad libitum. Five days after hatching, beginning at the start of the light cycle (at 6:00 P.M.), ten chickens were killed at each of eleven times during the next twenty-four hours (at the start of the light cycle, at six hours, at twelve hours, at hourly intervals from fifteen to twenty-one hours, and at twenty-four hours), and one milliliter of blood was collected from the carotid artery. The blood samples were analyzed for serum melatonin levels with use of radioimmunoassay techniques, as described previously¹. Previous experiments had shown that consistently low levels of serum melatonin could be expected during the light cycle; therefore, only three samples were collected during this time. Higher values and greater fluctuation were expected during the dark cycle; therefore, eight samples were collected during this time. Samples were collected at both the start of the light cycle and twenty-four hours after the start to provide continuity in the cycle.

Determination of an appropriate therapeutic dose of melatonin to restore the normal daily rhythm: Eighty-five newly hatched Mountain Hubbard chickens were obtained from a local hatchery (Lilydale) and were immediately introduced to a 12:12-hour dark:light cycle, with the dark cycle from 6:00 A.M. to 6:00 P.M.; food and water were provided ad libitum. Three days after hatching, a pinealectomy was performed with previously described methods¹⁶. Five days after hatching, seventy chickens were given an intraperitoneal injection of 0.1 milliliter of a solution of phosphate-buffered saline solution and melatonin at a dose of 2.5 milligrams per kilogram of body weight. Fifteen chickens were not given an injection. Ten of these fifteen chickens were killed immediately, and one milliliter of blood was collected from the carotid artery and was analyzed as described previously. Starting thirty minutes after the injection, ten of the seventy chickens were killed at hourly intervals and blood samples were collected and analyzed for serum melatonin levels. The remaining five chickens that had not been given an injection were killed 210 minutes after the injections were performed in the experimental group; these five chickens served as the normal controls. Blood samples were collected and analyzed for serum melatonin levels in the same manner as described for the experimental chickens.

Main Experiment

One hundred and five newly hatched Mountain Hubbard chickens were obtained from a local hatchery (Lilydale) and were immediately introduced to a 12:12-hour dark:light cycle, as described for the pilot studies, with food and water provided ad libitum. Three days after hatching, eighty-nine chickens had a pinealectomy. All of the chickens were then assigned to one of five groups. Group A (twenty-three chickens) received a daily intraperitoneal injection of 0.1 milliliter of a solution of phosphate-buffered saline solution and melatonin at a dose of 2.5 milligrams per kilogram of body weight, starting the day after the pinealectomy. Group B (twenty-three chickens) received a daily intraperitoneal injection of phosphate-buffered saline solution starting the day after the pinealectomy; these chickens served as a control for group A. Group C (twenty-two chickens) received a daily intraperitoneal injection of 0.1 milliliter of a solution of phosphate-buffered saline solution and melatonin at a dose of 2.5 milligrams per kilogram of body weight, starting fourteen days after the pinealectomy, by which time scoliosis had been given an opportunity to develop. Group D (twenty-one chickens) received no other treatment after the pinealectomy; this group served as a general control for the chickens that had had a pinealectomy. Finally, group E (sixteen chickens) did not receive any treatment at all and served as normal controls.

On the basis of the results of the pilot studies, the injections were given approximately four hours after the beginning of the dark cycle and, when appropriate, the dose of melatonin of 2.5 milligrams per kilogram of body weight was adjusted weekly when each chicken was weighed during radiography.

At the end of the experiment, the chickens that had had a pinealectomy were killed and the brain was removed. The area posterior to the third ventricle was examined with a dissecting microscope to confirm that the pineal gland and its stalk had been removed at the time of the operation and that no extraneous tissue had been left behind or had regenerated.

Radiographic Evaluation

Once a week, beginning one week after the pinealectomy and continuing until five weeks after it, each chicken (including the normal controls) was anesthetized with halothane and was evaluated radiographically in a standardized supine position with the neck and legs fully extended by light weights attached to strings. Care was taken to align the sternum and the vertebral column and to ensure that the pelvic and pectoral girdles were parallel to each other and level. Each radiograph was evaluated for the presence of scoliosis and the degree of curvature (the Cobb angle⁴). The cephalad and caudad end vertebrae were identified, and lines were drawn parallel to their cephalad and caudad borders, respectively. Additional lines were drawn perpendicular to these lines, and the intersecting angle of the perpendicular lines was measured as the Cobb angle. Scoliosis was defined as a lateral curve of more than 9 degrees. The length of the spine was determined on each radiograph by measuring the straight-line distance, regardless of lateral curve, from the cephalad border of the first thoracic vertebra to the tip of the most caudad vertebra in the tail.

The chickens were also weighed while the radiographs were made. Differences in the mean weights and in the mean lengths of the spine among the various experimental groups during the course of the experiment were evaluated for significance with use of the Student t test. The level of significance was set at p < 0.05.

Results

Introduction | Materials and Methods | Results | Discussion | References

Pilot Studies

Determination of the daily melatonin rhythm in five-day-old chickens: The results showed that five-day-old chickens have a circadian melatonin cycle (Fig. 1). Serum melatonin levels are very low during the light cycle. As the dark cycle begins, the levels gradually increase until a peak is reached in the middle of the cycle. Levels then decline gradually until very low levels are reached again at the end of the dark cycle and the beginning of the light cycle.

Determination of an appropriate therapeutic dose of melatonin to restore the normal daily rhythm: Intraperitoneal injection of 0.1 milliliter of a solution of phosphate-buffered saline solution and melatonin at a dose of 2.5 milligrams per kilogram of body weight reproduced the normal serum melatonin pattern in the chickens that had had a pinealectomy (Fig. 2). The chickens that had had a pinealectomy but had not been given an injection had very low serum melatonin levels. The injection of melatonin led to a rapid increase in serum melatonin levels, which was maintained for approximately ninety minutes. These high levels then quickly declined and were again close to zero within five hours after the injection.

Main Experiment

Daily injections of melatonin did not cause any adverse effects, and the procedure was tolerated well by the chickens. The chickens that were given the injections were just as active as the chickens that did not have any treatment (group E) and those that had had a pinealectomy with no subsequent injections (group D).

The prevalence of scoliosis was approximately the same in each group: twelve (52 percent) of twenty-three chickens in group A, fourteen (61 percent) of twenty-three in group B, twelve (55 percent) of twenty-two in group C, and twelve (57 percent) of twenty-one in group D (Table I). Scoliosis did not develop in any of the chickens in group E. The scoliosis developed within three weeks after the pinealectomy in eleven of twelve chickens in group A, thirteen of fourteen in group B, eleven of twelve in group C, and all twelve in group D. More than 80 percent of the curves that developed in each group were single curves. The only regressive curve (defined as a curve that was at least 10 degrees at some point during the experiment but then decreased to less than 10 degrees) developed in group D. A progressive curve (an increase in the Cobb angle of 3 degrees or more on each consecutive radiograph) developed in two chickens in group A, five in group B, and four in group C. The curve decreased (the final measurement was not the highest recorded) in some chickens in all of the groups except the normal controls (in which a pinealectomy was not carried out), but the prevalence of such decreases was similar among the groups (three of twelve chickens in group A, seven of fourteen chickens in group B, five of twelve chickens in group C, and eight of twelve chickens in group D).

The pattern of development, the degree of curvature (Figs. 3-A and 3-B), the mean weight (Fig. 4), and the mean length of the spine (Fig. 5) were similar in all groups at all times during the experiment.

The data for groups A, B, C, and D were separated into two subgroups: chickens in which scoliosis developed and those in which it did not. We detected no significant differences in the mean weight of the chickens (Fig. 6) or the mean length of the spine (Fig. 7) between the two subgroups in any of the groups.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

The results of the present study show that when melatonin therapy with an appropriately determined, physiological daily dose is started immediately after pinealectomy it has no effect on the development of scoliosis in young chickens. Furthermore, when such therapy is started fourteen days after pinealectomy it does not affect the continued development of recognized scoliosis in its initial stages. These results are in sharp contrast to those reported by Machida et al.⁵, who conducted a similar study and found that melatonin therapy had positive results when used to suppress the development of scoliosis. The differences between these two studies are difficult to explain.

The major difference between the present study and that by Machida et al.⁵ is the therapy. Machida et al. administered a dose of 2.5 milligrams per 100 milligrams of body weight in the late stages of the light cycle every other day. The method of injection was not specified, but we assume that it was intraperitoneal. There are several concerns regarding that treatment regimen. First, the dose is very high. In our pilot study, we determined that a much lower physiological dose of 2.5 milligrams per kilogram of body weight closely simulated the normal serum melatonin levels in the chicken. Machida et al.⁵ did not measure serum melatonin levels, so a comparison of the effectiveness of the two doses is not possible. In addition, our pilot study showed that melatonin injected intraperitoneally is conveyed quickly to the bloodstream but is depleted within a few hours. Machida et al.⁶ injected melatonin several hours before the end of the light cycle. This would result in peak serum melatonin levels at the end of the light cycle, but serum melatonin levels normally peak in the middle of the dark cycle. Furthermore, melatonin has a circadian rhythm that reaches a maximum value during the dark cycle and a minimum value during the light cycle. Injections every other day would not duplicate this rhythm, especially since melatonin is removed from the bloodstream within a few hours after intraperitoneal injection. However, it is not clear how these differences in therapy might explain the contrasting results. In the present study, in which the melatonin therapy closely approximated normal rhythms, the therapy had no effect on the development of scoliosis. In contrast, Machida et al.⁶, who used a therapy that was somewhat different from a physiological dose and regimen, reported substantial effects. These results cloud the issue of the role of melatonin in the development of scoliosis after pinealectomy in young chickens.

In our series of several experiments involving pinealectomy in a total of approximately 600 young chickens^9,15,16, scoliosis developed in only approximately 55 percent. Machida et al.^5-7, who allowed chickens who had had a pinealectomy to grow until they were twelve weeks old, reported that scoliosis developed in 100 percent (thirty of thirty^5,6). The reasons for this difference in the rate of scoliosis are not clear, but we are convinced that it is not due to the duration of the observation period because scoliosis usually develops in chickens within the first few weeks after pinealectomy, as was observed by us and by Machida et al.⁶. We previously questioned the role of melatonin¹⁶ because serum melatonin levels after pinealectomy are close to zero and yet scoliosis does not develop in all chickens that have had a pinealectomy. The results of the present study, in which melatonin therapy appeared to have no effect on the development of scoliosis, support our contention that serum melatonin levels are a poor indicator of the development of scoliosis in young chickens. However, the production and secretion of melatonin and its involvement with other molecules, particularly growth hormone, are very complex, and thus we are reluctant to dismiss the role of melatonin entirely. Instead, we are continuing to study the myriad relationships of melatonin, particularly the effects of reduced levels of melatonin in complex growth patterns. We are also examining the other products of the pineal gland and their effects on growth.

The differences in the rates of scoliosis among studies of melatonin therapy are disturbing. A pinealectomy is an extensive and delicate operation, and we have conducted several morphological and histological studies to determine if any aspect of the operative technique, rather than removal of the pineal gland, is responsible for the development of scoliosis. Sham operations, removal of the pineal bulb after sectioning of the stalk at various levels, and different methods of actual removal of the pineal gland have all resulted in the development of scoliosis in approximately 55 percent of chickens. We will continue to investigate the different aspects of the operative technique, but the results so far confirm that removal of the pineal gland, and not some aspect of the operative technique, is solely responsible for the development of scoliosis. For this reason, the development of scoliosis after pinealectomy in chickens warrants additional study.

References

Introduction | Materials and Methods | Results | Discussion | References

Bagnall, K. M.; Raso, V. J.; Hill, D. L.; Moreau, M.; Mahood, J. K.; Jiang, H.; Russell, G.; Bering, M.; and and Buzzell, G. R.: Melatonin levels in idiopathic scoliosis. Diurnal and nocturnal serum melatonin levels in girls with adolescent idiopathic scoliosis. Spine,21: 1974-1978, 1996.211974 1996 [PubMed]

Coillard, C.; Badeaux, J.; and Rivard, C.: Vertebral deformation and scoliosis. In Proceedings of the Quebec Scoliosis Society, p. 24. Quebec City, Quebec, Canada, 1994.

Hilibrand, A. S.; Blakemore, L. C.; Loder, R. T.; Greenfield, M. L.; Farley, F. A.; Hensinger, R. N.; and and Hariharan, M.: The role of melatonin in the pathogenesis of adolescent idiopathic scoliosis. Spine,21: 1140-1146, 1996.211140 1996 [PubMed]

Keim, H. A.: Scoliosis. In Clinical Symposia, vol. 31, no. 2. Dorval, Quebec, Ciba, 1979.

Machida, M.; Dubousset, J.; Imamura, Y.; Iwaya, T.; Yamada, T.; and and Kimura, J.: An experimental study in chickens for the pathogenesis of idiopathic scoliosis. Spine,18: 1609-1615, 1993.181609 1993 [PubMed]

Machida, M.; Dubousset, J.; Imamura, Y.; Iwaya, T.; Yamada, T.; and and Kimura, J.: Role of melatonin deficiency in the development of scoliosis in pinealectomised chickens. J. Bone and Joint Surg.,77-B(1): 134-138, 1995.77-B(1)134 1995

Machida, M.; Dubousset, J.; Imamura, Y.; Miyashita, Y.; Yamada, T.; and and Kimura, J.: Melatonin. A possible role in pathogenesis of adolescent idiopathic scoliosis. Spine,21: 1147-1152, 1996.211147 1996 [PubMed]

Machida, M.; Miyashita, Y.; Murai, I.; Dubousset, J.; Yamada, T.; and and Kimura, J.: Role of serotonin for scoliotic deformity in pinealectomized chicken. Spine,22: 1297-1301, 1997.221297 1997 [PubMed]

O'Kelly, C.; Wang, X.; Raso, V. J.; Moreau, M.; Zhao, J.; and Bagnall, K. M.: The production of scoliosis following pinealectomy in young chickens, rats and hamsters. Unpublished data.

Pincott, J. R.; Davies, J. S.; and and Taffs, L. F.: Scoliosis caused by section of dorsal spinal nerve roots. J. Bone and Joint Surg.,66-B(1): 27-29, 1984.66-B(1)27 1984

Robin, G.: The Aetiology of Idiopathic Scoliosis. Boca Raton, Florida, CRC Press, 1990.

Suk, S. I.; Song, H. S.; and and Lee, C. K.: Scoliosis induced by anterior and posterior rhizotomy. Spine,14: 692-697, 1989.14692 1989 [PubMed]

Thillard, M. J.: Déformations de la collone vertébrale consécutives à l'épiphysictomie chez le poussin. Extrait des comptes rendus de l'Association des Anatomistes,46: 22-26, 1959.4622 1959

Thomas, S., and and Dave, P. K.: Experimental scoliosis in monkeys. Acta Orthop. Scandinavica,56: 43-46, 1985.5643 1985

Wang, X.; Jiang, H.; Raso, J.; Moreau, M.; Mahood, J.; Zhao, J.; and and Bagnall, K.: Characterization of the scoliosis that develops after pinealectomy in the chicken and comparison with the adolescent idiopathic scoliosis in humans. Spine,22: 2626-2635, 1997.222626 1997 [PubMed]

Wang, X.; Moreau, M.; Raso, V. J.; Jie, Z.; Jiang, H.; Mahood, J.; and and Bagnall, K.: Changes in serum melatonin levels in response to pinealectomy in the chicken: its relation to the development of scoliosis. Spine,23: 2377-2381, 1998.232377 1998 [PubMed]

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FIG1:: Fig. 1 Graph showing the circadian rhythm of serum melatonin levels in five-day-old chickens. SD = standard deviation.

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TABLE I DATA REGARDING THE SCOLIOSIS IN THE FIVE GROUPS OF CHICKENS*

*R = convexity of the curve on the right side, and L = convexity of the curve on the left side. †p = primary curve and s = secondary curve in the chickens that had a double curve.

Group A†	Cobb Angles in Group A (degrees)					Group B†	Cobb Angles in Group B (degrees)					Group C†	Cobb Angles in Group C (degrees)					Group D	Cobb Angles in Group D (degrees)					Group E	Cobb Angles in Group E (Controls) (degrees)
Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy
1	2	3	4	5	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5
1	0	0	0	0	0	24	0	15R	20R	28R	37R	47	0	0	0	17R	20R	69	0	0	8R	11R	12R	1	0	0	0	0	0
2	0	0	0	0	0	25	0	0	0	31L	10L	48	0	0	0	0	0	70	0	15R	25R	26R	29R	2	0	0	0	0	0
3	0	0	8R	10R	15R	26	0	0	0	0	0	49	0	0	0	0	0	71	8R	19L	0	0	0	3	0	0	0	0	0
4	0	12R	16R	20R	25R	27	0	0	0	0	0	50	0	0	0	0	0	72	0	0	0	0	0	4	0	0	0	0	0
5	0	0	0	0	0	28	10R	10R	18R	26R	22R	51	0	0	0	0	0	73	0	0	15R	18R	15R	5	0	0	0	0	0
6	0	0	0	0	0	29	0	0	0	0	0	52	0	0	0	0	0	74	0	0	0	0	0	6	0	0	0	0	0
7p	14R	27R	35R	45R	42R	30	0	10L	18L	10L	10L	53	0	14R	20R	17R	29R	75	10L	22L	23L	28L	24L	7	0	0	0	0	0
7s	0	26L	43L	40L	50R	31	0	20L	25L	30L	32L	54	10R	10R	20R	23R	24R	76	0	10R	11R	14R	15R	8	0	0	0	0	0
8	0	0	0	0	0	32	0	0	0	0	0	55	6L	16L	25L	16L	21L	77	12L	20L	30L	35L	34L	9	0	0	0	0	0
9	0	0	15R	17R	15R	33	11R	25R	32R	36R	—	56p	29L	33R	40R	45R	48R	78	20L	8L	0	0	0	10	0	0	0	0	0
10	9L	15L	20L	23L	28L	34	0	0	10L	10L	14L	56s	0	32L	38L	43L	38L	79	0	0	0	0	0	11	0	0	0	0	0
11	0	10L	10L	10L	12L	35	15R	25R	35R	31R	29R	57	0	10R	14R	17R	12R	80	0	0	0	0	0	12	0	0	0	0	0
12	0	0	0	0	0	36	0	0	0	0	0	58	0	17L	23L	22L	30L	81	0	0	0	0	0	13	0	0	0	0	0
13	0	0	0	0	12L	37	0	0	0	0	0	59	0	0	0	0	0	82	0	19R	25R	23R	28R	14	0	0	0	0	0
14	12L	23L	24L	28L	30L	38	0	0	0	0	0	60	12L	14L	23R	24R	—	83	0	0	20L	14L	15L	15	0	0	0	0	0
15	0	15L	24L	18L	12L	39	0	0	0	0	0	61p	0	12R	25R	29R	32R	84	0	5L	0	0	0	16	0	0	0	0	0
16	0	0	0	0	0	40	0	0	11L	20L	15L	61s	0	0	24L	25L	26L	85	0	0	0	0	0
17	0	0	0	0	0	41	0	10R	22R	17R	22R	62	0	0	15R	20R	15R	86	0	0	19R	20R	20R
18	0	0	0	0	0	42	0	12R	13R	13R	10R	63	10R	18R	31R	34R	40R	87	0	0	0	0	0
19	0	0	0	0	0	43p	0	21L	36L	43L	52L	64	0	18R	23R	28R	27R	88	0	0	0	0	0
20	0	0	0	0	0	43s	0	10R	30R	35R	40R	65	0	0	0	0	0	89	0	20L	25L	25L	22L
21	0	9R	10R	10R	20R	44	0	7R	25L	8R	10R	66	0	0	0	0	0
22	0	0	10L	8L	10L	45	0	12R	16R	25R	28R	67	0	0	0	0	0
23	11R	24R	25R	24R	25R	46	0	0	0	0	0	68	0	0	0	0	0

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TABLE I DATA REGARDING THE SCOLIOSIS IN THE FIVE GROUPS OF CHICKENS*

*R = convexity of the curve on the right side, and L = convexity of the curve on the left side. †p = primary curve and s = secondary curve in the chickens that had a double curve.

Group A†	Cobb Angles in Group A (degrees)					Group B†	Cobb Angles in Group B (degrees)					Group C†	Cobb Angles in Group C (degrees)					Group D	Cobb Angles in Group D (degrees)					Group E	Cobb Angles in Group E (Controls) (degrees)
Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy					Weeks After Pinealectomy
1	2	3	4	5	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5	1	2	3	4	5
1	0	0	0	0	0	24	0	15R	20R	28R	37R	47	0	0	0	17R	20R	69	0	0	8R	11R	12R	1	0	0	0	0	0
2	0	0	0	0	0	25	0	0	0	31L	10L	48	0	0	0	0	0	70	0	15R	25R	26R	29R	2	0	0	0	0	0
3	0	0	8R	10R	15R	26	0	0	0	0	0	49	0	0	0	0	0	71	8R	19L	0	0	0	3	0	0	0	0	0
4	0	12R	16R	20R	25R	27	0	0	0	0	0	50	0	0	0	0	0	72	0	0	0	0	0	4	0	0	0	0	0
5	0	0	0	0	0	28	10R	10R	18R	26R	22R	51	0	0	0	0	0	73	0	0	15R	18R	15R	5	0	0	0	0	0
6	0	0	0	0	0	29	0	0	0	0	0	52	0	0	0	0	0	74	0	0	0	0	0	6	0	0	0	0	0
7p	14R	27R	35R	45R	42R	30	0	10L	18L	10L	10L	53	0	14R	20R	17R	29R	75	10L	22L	23L	28L	24L	7	0	0	0	0	0
7s	0	26L	43L	40L	50R	31	0	20L	25L	30L	32L	54	10R	10R	20R	23R	24R	76	0	10R	11R	14R	15R	8	0	0	0	0	0
8	0	0	0	0	0	32	0	0	0	0	0	55	6L	16L	25L	16L	21L	77	12L	20L	30L	35L	34L	9	0	0	0	0	0
9	0	0	15R	17R	15R	33	11R	25R	32R	36R	—	56p	29L	33R	40R	45R	48R	78	20L	8L	0	0	0	10	0	0	0	0	0
10	9L	15L	20L	23L	28L	34	0	0	10L	10L	14L	56s	0	32L	38L	43L	38L	79	0	0	0	0	0	11	0	0	0	0	0
11	0	10L	10L	10L	12L	35	15R	25R	35R	31R	29R	57	0	10R	14R	17R	12R	80	0	0	0	0	0	12	0	0	0	0	0
12	0	0	0	0	0	36	0	0	0	0	0	58	0	17L	23L	22L	30L	81	0	0	0	0	0	13	0	0	0	0	0
13	0	0	0	0	12L	37	0	0	0	0	0	59	0	0	0	0	0	82	0	19R	25R	23R	28R	14	0	0	0	0	0
14	12L	23L	24L	28L	30L	38	0	0	0	0	0	60	12L	14L	23R	24R	—	83	0	0	20L	14L	15L	15	0	0	0	0	0
15	0	15L	24L	18L	12L	39	0	0	0	0	0	61p	0	12R	25R	29R	32R	84	0	5L	0	0	0	16	0	0	0	0	0
16	0	0	0	0	0	40	0	0	11L	20L	15L	61s	0	0	24L	25L	26L	85	0	0	0	0	0
17	0	0	0	0	0	41	0	10R	22R	17R	22R	62	0	0	15R	20R	15R	86	0	0	19R	20R	20R
18	0	0	0	0	0	42	0	12R	13R	13R	10R	63	10R	18R	31R	34R	40R	87	0	0	0	0	0
19	0	0	0	0	0	43p	0	21L	36L	43L	52L	64	0	18R	23R	28R	27R	88	0	0	0	0	0
20	0	0	0	0	0	43s	0	10R	30R	35R	40R	65	0	0	0	0	0	89	0	20L	25L	25L	22L
21	0	9R	10R	10R	20R	44	0	7R	25L	8R	10R	66	0	0	0	0	0
22	0	0	10L	8L	10L	45	0	12R	16R	25R	28R	67	0	0	0	0	0
23	11R	24R	25R	24R	25R	46	0	0	0	0	0	68	0	0	0	0	0