 |
 |
 |
|
| home | CME | help | feedback | subscriptions | archive |
|
© 1999 The Journal of Bone and Joint Surgery, Inc. The Effects of Melatonin Therapy on the Development of Scoliosis After Pinealectomy in the Chicken*  , EDMONTON, ALBERTA, CANADAInvestigation performed at the Department of Cell Biology and Anatomy and the Department of Surgery, University of Alberta, Edmonton
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.
Although some investigators have successfully produced scoliosis in animals10,12,14, 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 humans6,15. 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 studies2,13. 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.
Pilot Studies 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 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 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.
Pilot Studies
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 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.
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 chickens9,15,16, 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 thirty5,6). 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.
*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.
  CiteULike  Connotea  Del.icio.us  Facebook  Technorati  Twitter What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
| home | CME | help | feedback | subscriptions | archive | site index |
|
Stanford University Libraries' HighWire Press (TM) assists in the publication of JBJS Online. |