An Experimental Inquiry into the Central Source of Preganglionic Fibers to the Chick Ciliary Ganglion C. H. NARAYANAN AND Y. NARAYANAN Depurtment of A n a t o m y , Louzszana State University School of Medtcine, N e w Orleans, Louisiana 70119
ABSTRACT Degenerative changes i n the entire third nerve nucleus were studied following selective removal of the ciliary ganglion, usually of the right side, in day-old chicks. The operated animals were allowed to survive three, six, nine, and twelve days after ciliary ganglionectomy. Retrograde degener ative changes proceed rapidly between three and nine days post-operatively, and are strictly limited to the ipsilateral accessory oculomotor nucleus. The changes are at first observed in the medial division of the accessory oculomotor nucleus, followed by the lateral divisions of the accessory oculomotor nucleus. By nine days following the operation, both the divisions of the accessory oculomotor nucleus are virtually depleted of almost all neurons. The other components of the oculomotor complex were not affected at any stage by ciliary ganglionectomy. These observations provide conclusive evidence that the accessory oculomotor nucleus is indeed the avian homologue of the EdingerWestphal nucleus. In birds, the origin of ocular post-ganglionic fibers from the ciliary ganglion has been well established. There exists no direct evidence, however, on the precise central source of the autonomic pre-ganglionic fibers to the avian ciliary ganglion. The limited data available suggest that the accessory oculomotor nucleus (Cajal, '09; Kappers et al., '36; Yoshida, '53; Nimmi et al., '59), or the urciform nucleus (Kusama, '43), might well be the avian homologue of the Edinger-Westphal nucleus. Removal of the optic vesicle in early stages of development (Cowan and Wenger, '68), or enucleation (Yoshida, '53), has been used to investigate changes affecting the cells of origin of the preganglionic fibers to the ciliary ganglion. In the above experiment in which the optic vesicle was removed the ciliary ganglion was not directly involved in the operative procedure. The degenerative changes in the accessory oculomotor nucleus "follow the time-course of degenerative changes in the ciliary ganglion, although lagging behind by about two or three days" (Cowan and Wenger, '68: p. 113). Although this observation provides indirect evidence on the source of preganglionic fibers to the ciliary ganglion, it has been difficult to exclude cell loss in some of the somatic motor components of J.
COMP.
NEUR., 166: 101-110
the oculomotor nuclear complex, as for instance, in the dorsomedial nucleus where the cell loss has been almost as marked as in the accessory oculomotor nucleus (Cowan and Wenger, '68: p. 113). The following experiment of selective removal of the ciliary ganglion without involving the extrinsic eye muscle was undertaken in an attempt to avoid cell loss in the somatic motor components of the oculomotor nuclear complex observed in previous investigations, and to provide conclusive evidence on the central source of preganglionic fibers of this pathway. MATERIALS A N D METHODS
Subjects Forty-five, day old DeKalb strain of White leghorn chicks raised from fertile eggs were used in this investigation.
Surgical procedure The chick was anesthetized with chloroform and unilateral ciliary ganglionectomy was performed, usually on the right side, under strict aseptic conditions. Repeated doses of chloroform had to be administered, however, with the aid of an etherizer in order to maintain the required level of anesthesia over the period of almost 15101
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20 minutes required for the surgery. First, an incision was made below the lower eye lid extending along the posterior edge of the orbit to about the level of the external auditory opening. The superficial blood vessels were cauterized using a concept “C” size unit as described by Narayanan and Malloy (‘74a,b), in order to control the extent of hemorrhage. Next, the eyeball was freed in this region by cutting very carefully through the fascial lining of the orbit without damaging the extrinsic eye muscles. The inferior rectus along with its nerve was identified. Using a blunt probe to gently retract the inferior rectus, the nerve to the inferior rectus was followed back under the eye towards the apex of the orbit. Additionally, the use of gelatin sponge (Gelfoam-Upjohn) soaked in 0.001% adrenalin hydrochloride helped greatly to keep the operation field dry as described by Warwick (’54), in order to be able to trace the nerve back to the ciliary ganglion more easily. Holding the eyeball away from the apex of the orbit with the aid of a Meyerhoeffer chalazion curette, the ganglion was carefully exposed with the probe. The ganglion was picked away from its connection to the superior branch of the oculomotor nerve very carefully with a pair of needle sharp watchmakers foceps. The wound was closed by drawing the cut edges of the skin together using “000” silk suture. To ensure that ciliary ganglionectomy had been accomplished, the excised nervous tissue was stained with methylene blue and examined under a microscope. Successful removal was also indicated by dilatation of the pupil on the operated side. The pupil remained dilated in all the operated animals regardless of length of the post-operative survival period. As a further check on the effectiveness of the operation and preservation of extrinsic eye muscles, post-mortem dissection of both orbits was carried out routinely. The unoperated side was used as control in all cases. The experimental chicks were divided into three groups of approximately ten animals in each group, on the basis of total survival time: Group I, included chicks with three days total survival time; Group 11, chicks of six days post-operative survival time; Group 111, nine days post-operative survival time, and a last group, of chicks
which were allowed to survive twelve days after surgery. Experimental animals were sacrificed at periods varying from three to twelve days following surgery. Each animal was anesthetized and perfused through the heart with chick Ringer’s solution (New, ’66), followed by 10% neutral buffered formalin. The brain was dissected out, dehydrated, cleared in amyl acetate, embedded in paraffin and sectioned serially at 12 p. A complete series of sections over the extent of the midbrain was mounted and stained in buffered 1 % thionin. For the normal series of brain sections used in this study, embryos of eleven days incubation age (fig. 1) were prepared by fixing the chloralhydr ate/alcohol/nitric acid formula r ecommended for the Cajal-De Castro silver technique (Levi-Montalcini, ’49). RESULTS
General comments The chicks recovered uneventfully after the ciliary ganglion of the right side was excised according to the method described above. In the most successful cases, the chicks showed characteristic fixed pupillary dilatation after surgery regardless of the post-operative survival period. No detectable changes were observed in their behavior, since the experimental chicks pecked at grain and drank water with as much fervour as the control animals of corresponding age groups. At autopsy, on days three, six, nine, and twelve, ciliary ganglionectomy was found to be complete and successful in the majority of our experimental animals as determined by the total absence of the ciliary ganglion of the right side. Normal cytoarchitechtre of the oculomotor nucleus Although the normal cytoarchitectonic organization of the oculomotor nucleus has been described previously by Niimi et al. (‘59), and Cowan and Wenger (‘68), a brief account is presented here in order to be able to understand the effect of selective ciliary ganglion removal on the entire third nerve nucleus. The terminology proposed by Niimi et al. (‘59) to designate the various components of the oculomotor nucleus, and the abbreviations of Cowan and
PREGANGLIONIC FIBERS TO CILIARY GANGLION
Wenger ('68),will be used in the following description. The present observations, made in silver impregnated material of the 11day chick embryo, confirm the conclusions made in previous studies regarding the various subnuclear components which form the entire third nerve nucleus (fig. 1). The oculomotor nucleus in the chick is confined to the more dorsal region of the tegmentum, lying between the raphe and the Medial longitudinal fasciculus. At a glance, two principal nuclei on each side are evident: (1) A dorsal group, comprised of a dorsolateral (DL) and a dorsomedial (DM) subnuclei. (2) A ventromedial group of cells which in turn is formed of a dorsal division (VMD), and a ventral division (VMV). Between the dorsomedial subnuclei is the relatively ill defined arciform nucleus (AR). The cells of both the dorsal group and the ventromedial group are predominantly large and stand out in comparison with another cell group called the accessory oculomotor nucleus. The latter, is dorsal to the dorsolateral subnucleus of the dorsal group, and is separated from the other cell groups by a cell free zone. Its cells are distinctly smaller and form a more nearly elliptical group of cells with its long axis directed diagonally in a dorsolateral aspect (Niimi et al., '59). It is also subdivided into medial (AM) and lateral (AL) subnuclei.
103
cessory oculomotor nucleus of the operated side, on the other hand, a few intact neurons were still recognizable. The other nuclei of the oculomotor complex were unaffected by the operation. Group I1 The experimental chicks of this group survived the operation for six days. In seven out of a total of 16 experimental chicks belonging to this group, ciliary ganglionectomy was successful and complete. The changes observed in the accessory oculomotor nucleus of the operated side were significantly more severe in comparison to those observed in Group I. The accessory oculomotor nucleus as a whole was reduced to about one-half of its original size compared to the unoperated side. The degenerative changes now affect both the subdivisions of the accessory oculomotor nucleus, except for a few cells of the lateral accessory oculomotor nucleus, which were in various stages of cellular atrophy. A photomicrograph of a section through the oculomotor nucleus in the experimental chick of this group (EW 29) is shown in figure 3.
Group 111 The chicks of this group were sacrificed nine days following ciliary ganglionectomy. Of the ten cases belonging to this group, seven showed a complete absence of the Group 1 ciliary ganglion of the right side. EW 37 The first group of chicks were sacrificed is a typical case. The degenerative changes three days following surgery. Of the 13 on both subdivisions of the accessory ocucases that survived the operation, seven lomotor nucleus of the right side have proexperimental chicks showed a complete ab- ceeded even further amounting to almost sence of the ciliary ganglion of the right a total cell loss (fig. 4). The other comside. Experimental animal EW 25 is a rep- ponents of the oculomotor complex were resentative case sectioned through the ocu- unaffected and indistinguishable from those lomotor complex. A photomicrograph of a of the unoperated side. section through the oculomotor complex With longer post operative survival peis shown in figure 2. Unequivocal signs of riods up to 12 days after ciliary gangliondegeneration were observed in the acces- ectomy, the degenerative changes in the sory oculomotor nucleus of the operated accessory oculomotor nucleus proceed to side. However, the cells belonging to the virtually complete cell loss and show the two subnuclei which form the accessory same severe degeneration as observed in oculomotor nucleus of this side reacted the experimental group of nine days, theredifferently to ciliary ganglionectomy. The fore, will not be described separately. degenerative changes were severe in the The following general conclusions can medial oculomotor nucleus. The few sur- be drawn: viving cells of this group appeared shrunk(1) In all of the experimental cases we en with pycnotic nuclei. In the lateral ac- have examined the reaction to ciliary gan-
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glionectomy was limited to the accessory oculomotor nucleus. (2) The response to ciliary ganglion removal appeared f m t in the medial accessory oculomotor nucleus followed by degenerative changes in the lateral accessory oculomotor nucleus in subsequent stages. ( 3 ) The degenerative changes are considerably marked at every stage and progresses between the third and ninth day after surgery from a partial cell loss to almost a total cell loss in both subdivisions. (4) None of the other components of the oculomotor nuclear complex seem to be affected by the operation. Although no attempts were made to quantify the results by cell counts of the other components of the oculomotor nuclear complex, it is our impression that they were identical in every respect compared to cell groups of the unoperated control side in each case. DISCUSSION
The main objectives of this investigation were: (1) to determine whether or not severe degenerative changes are restricted to the accessory oculomotor nucleus after selective removal of the ciliary ganglion without impairment to any of the extraocular muscles of the eye, and (2) to examine the pattern of degenerative changes, if any, in the entire third nerve nucleus in general, and specifically, in the two subdivisions which form the accessory OCUlomotor nucleus. Our experimental findings point unequivocally to the conclusion that, in the chick, cell loss following ciliary ganglionectomy is strictly limited to the ipsilateral accessory oculomotor nucleus. The other components of the oculomotor complex are unaffected by the operation, and are similar to the cell groups of the unoperated control side in all cases. In this respect, our results of ciliary ganglionectomy are quite different from that of optic vesicle removal in early stages of development (Cowan and Wenger, '68; Amprino, '43). Optic vesicle removal in early stages of development in the chick embryo has been used successfully to investigate neuronal hypoplasia in the ciliary ganglion and various nuclear centers during development (Amprino, '43; Levi-Montalcini and Amprino, '47; Hammond and Yntema, '58; cowan and Wenger, '68). It is noteworthy
that in all of the investigations involving lesions of the optic vesicle, particularly those of Cowan and Wenger ('68), and Amprino ('43), the ciliary ganglion was not directly involved by the operative procedure. This is further confirmed by the fact that at least in the younger embryos, removal of the optic vesicle does not injure the developing ciliary ganglion or affect its early development, until the ganglion has attained numerical completion in the chick embryo, i.e. day 6 or 7 (Cowan and Wenger, '68). In older embryos, the ciliary ganglion was not available for study in the experiments of Cowan and Wenger ('68), since the brains were dissected out before fixation. Hence, their evidence on the nucleus of origin of preganglionic fibers to the ciliary ganglion is indirect, and as stated by Cowan and Wenger ('68), to some degree, circumstantial. Nevertheless, in their experiments, marked degenerative changes were observed in the ipsilateral accessory oculomotor nucleus at every stage between the tenth to sixteenth days of incubation, from a general cell shrinkage to virtually complete cell loss in later stages. The severity of cell loss in the accessory oculomotor nucleus, as compared to the rest of the third nerve nucleus, would seem to indicate that its terminations are quite different from those of the other components of the oculomotor complex. Cowan and Wenger ('68) noted that the other components of the oculomotor complex showed a more variable reaction to optic vesicle removal, which has been accounted for in two ways. One, is that in most of the components other than the accessory oculomotor nucleus and with the exception of the dorsomedial nucleus, the reaction was presumably due to differing degrees of involvement of the extrinsic eye muscle mesoderm. Second, the severity of cell loss in the dorsomedial nucleus in most of their experimental material according to them was probably due to its innervation of a muscle or muscles in proximity to the optic vesicle and almost invariably resulted in the removal of the innervated muscle(s) during the operation. Yet, in other situations such as the effect of eye removal, immediately after hatching, in the chick (Cowan and Wenger, '68), in adult pigeons (Yoshida, '53), the results are inconclusive (see below). No
105
PREGANGLIONIC FIBERS TO CILIARY GANGLION
doubt, cellular degeneration was observed following enucleation in both subdivisions of the accessory oculomotor nucleus in the experiments of Cowan and Wenger (‘68), but, many shrunken neurons persisted at all levels throughout the rostro-caudal extent of the nucleus. As Cowan and Wenger (‘68) have pointed out, a proper assessment of the degenerative changes was not possible since the orbital tissues were not available for study. It would suffice to note here that there is really no clear evidence from these studies as to whether the degenerative changes seen in the accessory oculomotor nucleus were secondary to a neuronal degeneration in the ciliary ganglion or retrograde due to direct injury of the ciliary ganglion at the time of eye removal (Cowan and Wenger, ’68). It is clear from our experiments, that selective ciliary ganglionectomy does have a direct effect on the accessory oculomotor nucleus. It operates as a direct consequence of injury to the ciliary ganglion thus leading to a rapid and total loss of cells in the accessory oculomotor nucleus. In summary, our results indicate that retrograde degenerative changes in the accessory oculomotor nucleus proceed very rapidly between three and nine days following ciliary ganglionectomy. Experiments currently in progress on neural hyperplasia following transplantation of an additional eye (Narayanan and Narayanan, ’75) have shown a significant increase in the number of cells of the ciliary ganglion. Further work is underway which will explore more thoroughly the effects of peripheral overloading by transplantation of an additional eye on other nuclear centers. If a corresponding increase in the number of cells in the accessory oculomotor nucleus should occur, we should have additional evidence in s u p port of the notion that the accessory oculomotor nucleus does in fact represent the avian homologue of the Edinger-Westphal nucleus. ACKNOWLEDGMENTS
The authors wish to express their sincere thanks to Mr. Bill Stallworth of the
Audio-visual Department of the L. S. U . School of Dentistry, for photographic assistance, to Lynn Barrett for technical help and to Mrs. Susan Orazio for typing the manuscript. LITERATURE CITED Amprino, R. 1943 Correlazioni quantitative fra centri nervosi e territori d’innervazione Deriferica durante lo sviluppo. Arch. Anat. Embriol., 49.261-300. ~ . . Cajal, S. Ramon y 1909 Contribucion a1 estudio de 10s ganglios de la substancia reticular del bulbo, con algunos detalles concenientes B 10s focos motores y vias reflejas bulbares y mesocefalicas. Trab. lab. invest. biol. Madrid, 7: 259-284. Cowan, W. M., and E. Wenger 1968 Degeneration in the nucleus of origin of the preganglionic fibers to the chick ciliary ganglion following early removal of the optic vesicle. J. Exp. Zool., 168: 105-124. Hammond, W. S., and C. L. Yntema 1958 Origin of ciliary ganglia in the chick. J. Comp. Neur., 110:3 6 7 4 8 9 . Kappers, C. U. A,, G. C. Huber and E. C. Crosby 1936 The comparative anatomy of the nervous system of vertebrates, including man. Vol 11. McMillan Co.,New York. Kusama, T. 1943 On the comparative anatomy of the Edinger-Westphal nucleus. Nisshin Igaku, 32 (cited by Yoshida, ’53, and Niimi et al., ’59). Levi-Montalcini, R. 1949 The development of the acoustico-vestibular centers in the chick embryo in the absence of the afferent root fibers and of descending tracts. J. Comp. Neur., 91: 20 9-242. Levi-Montalcini, R., and R. Amprino 1947 Recherches experimentales sur l’origine du ganglione ciliare dans l’embryos de poulet. Arch. Biol., 58: 265-288. Narayanan, C. H., and R. B. Malloy 1974a Deafferentation studies on motor activity in the chick. I. Activity patterns of hind limbs. J. Exp. Zool., 189: 163-176. 1974b Deafferentation studies on motor activity in the chick. 11. Activity patterns of wing. J. Exp. Zool., 189: 177-188. Narayanan, C. H., and Y. Narayanan 1975 On neural hyperplasia in the chick ciliary ganglion following peripheral overloading. Anat. Rec., 181: 434 (abstract). Niimi, K., T. Sakai and J. Takasu 1959 The ontogenic development of the oculomotor nucleus in the chick. Tokushima. J. Exp. Med., 5: 3 1 1 4 2 5 . Warwick, R. 1954 The ocular parasympathetic nerve supply and its mesencephalic sources. J. of Anat. (London), 88: 71-92. Yoshida, K. 1953 Comparative anatomical and experimental studies on the oculomotor nucleus and neighboring nuclei. Acta Med. et Biol., I : 14S161.
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PLATE 1 EXPLANATION OF FIGURES
106
1
A photomicrograph of the oculomotor complex i n a normal 11-day old chick embryo. De Castro silver preparation. AL. Lateral accessory nucleus; AM. Medial accessory nucleus; DM. Dorsornedial nucleus; DL. Dorsolateral nucleus; VMD. Dorsal division of ventromedial n u cleus; VMV. Ventral division of ventromedial nucleus. Scale: 0.1 mm.
2
Photomicrograph of a transverse section about the middle of the oculornotor complex of experimental chick EW 25 (three days after ciliary ganglionectomy). The cells of the medial accessory nucleus of the operated side indicated by arrows are severely depleted; i n the lateral accessory some intact neurons are still present. Thionin preparation. Scale: 0.1 mm.
PREGANGLIONIC FIBERS TO CILIARY GANGLION C. H. N a r a y a n a n and Y . N a r a y a n a n
PLATE 1
107
PLATE 2 EXPLANATION OF FIGURES
108
3
Photomicrograph of a transverse section about the middle of the oculomotor complex of experimental chick EW 29 (six days after surgery). The accessory nucleus a s a whole on the operated side (arrows) shows a reduction to about one half its normal volume. The other components of the oculomotor complex are unaffected. Thionin preparation. Scale: 0.1 mm.
4
Photomicrograph of a transverse section through the middle of the oculomotor nucleus of experimental chick EW 37 (nine days after ciliary ganglionectomy). The accessory oculomotor nucleus on the operated side indicated by arrows shows a n almost complete cell loss. T h e other components of the oculomotor complex are unaffected. Thionin preparation. Scale: 0.1 mm.
PREGANGLIONIC FIBERS TO CILIARY GANGLION C. H. N a r a y a n a n a n d Y. N a r a y a n a n
PLATE 2
109
ABSTRACT Degenerative changes i n the entire third nerve nucleus were studied following selective removal of the ciliary ganglion, usually of the right side, in day-old chicks. The operated animals were allowed to survive three, six, nine, and twelve days after ciliary ganglionectomy. Retrograde degener ative changes proceed rapidly between three and nine days post-operatively, and are strictly limited to the ipsilateral accessory oculomotor nucleus. The changes are at first observed in the medial division of the accessory oculomotor nucleus, followed by the lateral divisions of the accessory oculomotor nucleus. By nine days following the operation, both the divisions of the accessory oculomotor nucleus are virtually depleted of almost all neurons. The other components of the oculomotor complex were not affected at any stage by ciliary ganglionectomy. These observations provide conclusive evidence that the accessory oculomotor nucleus is indeed the avian homologue of the EdingerWestphal nucleus. In birds, the origin of ocular post-ganglionic fibers from the ciliary ganglion has been well established. There exists no direct evidence, however, on the precise central source of the autonomic pre-ganglionic fibers to the avian ciliary ganglion. The limited data available suggest that the accessory oculomotor nucleus (Cajal, '09; Kappers et al., '36; Yoshida, '53; Nimmi et al., '59), or the urciform nucleus (Kusama, '43), might well be the avian homologue of the Edinger-Westphal nucleus. Removal of the optic vesicle in early stages of development (Cowan and Wenger, '68), or enucleation (Yoshida, '53), has been used to investigate changes affecting the cells of origin of the preganglionic fibers to the ciliary ganglion. In the above experiment in which the optic vesicle was removed the ciliary ganglion was not directly involved in the operative procedure. The degenerative changes in the accessory oculomotor nucleus "follow the time-course of degenerative changes in the ciliary ganglion, although lagging behind by about two or three days" (Cowan and Wenger, '68: p. 113). Although this observation provides indirect evidence on the source of preganglionic fibers to the ciliary ganglion, it has been difficult to exclude cell loss in some of the somatic motor components of J.
COMP.
NEUR., 166: 101-110
the oculomotor nuclear complex, as for instance, in the dorsomedial nucleus where the cell loss has been almost as marked as in the accessory oculomotor nucleus (Cowan and Wenger, '68: p. 113). The following experiment of selective removal of the ciliary ganglion without involving the extrinsic eye muscle was undertaken in an attempt to avoid cell loss in the somatic motor components of the oculomotor nuclear complex observed in previous investigations, and to provide conclusive evidence on the central source of preganglionic fibers of this pathway. MATERIALS A N D METHODS
Subjects Forty-five, day old DeKalb strain of White leghorn chicks raised from fertile eggs were used in this investigation.
Surgical procedure The chick was anesthetized with chloroform and unilateral ciliary ganglionectomy was performed, usually on the right side, under strict aseptic conditions. Repeated doses of chloroform had to be administered, however, with the aid of an etherizer in order to maintain the required level of anesthesia over the period of almost 15101
102
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20 minutes required for the surgery. First, an incision was made below the lower eye lid extending along the posterior edge of the orbit to about the level of the external auditory opening. The superficial blood vessels were cauterized using a concept “C” size unit as described by Narayanan and Malloy (‘74a,b), in order to control the extent of hemorrhage. Next, the eyeball was freed in this region by cutting very carefully through the fascial lining of the orbit without damaging the extrinsic eye muscles. The inferior rectus along with its nerve was identified. Using a blunt probe to gently retract the inferior rectus, the nerve to the inferior rectus was followed back under the eye towards the apex of the orbit. Additionally, the use of gelatin sponge (Gelfoam-Upjohn) soaked in 0.001% adrenalin hydrochloride helped greatly to keep the operation field dry as described by Warwick (’54), in order to be able to trace the nerve back to the ciliary ganglion more easily. Holding the eyeball away from the apex of the orbit with the aid of a Meyerhoeffer chalazion curette, the ganglion was carefully exposed with the probe. The ganglion was picked away from its connection to the superior branch of the oculomotor nerve very carefully with a pair of needle sharp watchmakers foceps. The wound was closed by drawing the cut edges of the skin together using “000” silk suture. To ensure that ciliary ganglionectomy had been accomplished, the excised nervous tissue was stained with methylene blue and examined under a microscope. Successful removal was also indicated by dilatation of the pupil on the operated side. The pupil remained dilated in all the operated animals regardless of length of the post-operative survival period. As a further check on the effectiveness of the operation and preservation of extrinsic eye muscles, post-mortem dissection of both orbits was carried out routinely. The unoperated side was used as control in all cases. The experimental chicks were divided into three groups of approximately ten animals in each group, on the basis of total survival time: Group I, included chicks with three days total survival time; Group 11, chicks of six days post-operative survival time; Group 111, nine days post-operative survival time, and a last group, of chicks
which were allowed to survive twelve days after surgery. Experimental animals were sacrificed at periods varying from three to twelve days following surgery. Each animal was anesthetized and perfused through the heart with chick Ringer’s solution (New, ’66), followed by 10% neutral buffered formalin. The brain was dissected out, dehydrated, cleared in amyl acetate, embedded in paraffin and sectioned serially at 12 p. A complete series of sections over the extent of the midbrain was mounted and stained in buffered 1 % thionin. For the normal series of brain sections used in this study, embryos of eleven days incubation age (fig. 1) were prepared by fixing the chloralhydr ate/alcohol/nitric acid formula r ecommended for the Cajal-De Castro silver technique (Levi-Montalcini, ’49). RESULTS
General comments The chicks recovered uneventfully after the ciliary ganglion of the right side was excised according to the method described above. In the most successful cases, the chicks showed characteristic fixed pupillary dilatation after surgery regardless of the post-operative survival period. No detectable changes were observed in their behavior, since the experimental chicks pecked at grain and drank water with as much fervour as the control animals of corresponding age groups. At autopsy, on days three, six, nine, and twelve, ciliary ganglionectomy was found to be complete and successful in the majority of our experimental animals as determined by the total absence of the ciliary ganglion of the right side. Normal cytoarchitechtre of the oculomotor nucleus Although the normal cytoarchitectonic organization of the oculomotor nucleus has been described previously by Niimi et al. (‘59), and Cowan and Wenger (‘68), a brief account is presented here in order to be able to understand the effect of selective ciliary ganglion removal on the entire third nerve nucleus. The terminology proposed by Niimi et al. (‘59) to designate the various components of the oculomotor nucleus, and the abbreviations of Cowan and
PREGANGLIONIC FIBERS TO CILIARY GANGLION
Wenger ('68),will be used in the following description. The present observations, made in silver impregnated material of the 11day chick embryo, confirm the conclusions made in previous studies regarding the various subnuclear components which form the entire third nerve nucleus (fig. 1). The oculomotor nucleus in the chick is confined to the more dorsal region of the tegmentum, lying between the raphe and the Medial longitudinal fasciculus. At a glance, two principal nuclei on each side are evident: (1) A dorsal group, comprised of a dorsolateral (DL) and a dorsomedial (DM) subnuclei. (2) A ventromedial group of cells which in turn is formed of a dorsal division (VMD), and a ventral division (VMV). Between the dorsomedial subnuclei is the relatively ill defined arciform nucleus (AR). The cells of both the dorsal group and the ventromedial group are predominantly large and stand out in comparison with another cell group called the accessory oculomotor nucleus. The latter, is dorsal to the dorsolateral subnucleus of the dorsal group, and is separated from the other cell groups by a cell free zone. Its cells are distinctly smaller and form a more nearly elliptical group of cells with its long axis directed diagonally in a dorsolateral aspect (Niimi et al., '59). It is also subdivided into medial (AM) and lateral (AL) subnuclei.
103
cessory oculomotor nucleus of the operated side, on the other hand, a few intact neurons were still recognizable. The other nuclei of the oculomotor complex were unaffected by the operation. Group I1 The experimental chicks of this group survived the operation for six days. In seven out of a total of 16 experimental chicks belonging to this group, ciliary ganglionectomy was successful and complete. The changes observed in the accessory oculomotor nucleus of the operated side were significantly more severe in comparison to those observed in Group I. The accessory oculomotor nucleus as a whole was reduced to about one-half of its original size compared to the unoperated side. The degenerative changes now affect both the subdivisions of the accessory oculomotor nucleus, except for a few cells of the lateral accessory oculomotor nucleus, which were in various stages of cellular atrophy. A photomicrograph of a section through the oculomotor nucleus in the experimental chick of this group (EW 29) is shown in figure 3.
Group 111 The chicks of this group were sacrificed nine days following ciliary ganglionectomy. Of the ten cases belonging to this group, seven showed a complete absence of the Group 1 ciliary ganglion of the right side. EW 37 The first group of chicks were sacrificed is a typical case. The degenerative changes three days following surgery. Of the 13 on both subdivisions of the accessory ocucases that survived the operation, seven lomotor nucleus of the right side have proexperimental chicks showed a complete ab- ceeded even further amounting to almost sence of the ciliary ganglion of the right a total cell loss (fig. 4). The other comside. Experimental animal EW 25 is a rep- ponents of the oculomotor complex were resentative case sectioned through the ocu- unaffected and indistinguishable from those lomotor complex. A photomicrograph of a of the unoperated side. section through the oculomotor complex With longer post operative survival peis shown in figure 2. Unequivocal signs of riods up to 12 days after ciliary gangliondegeneration were observed in the acces- ectomy, the degenerative changes in the sory oculomotor nucleus of the operated accessory oculomotor nucleus proceed to side. However, the cells belonging to the virtually complete cell loss and show the two subnuclei which form the accessory same severe degeneration as observed in oculomotor nucleus of this side reacted the experimental group of nine days, theredifferently to ciliary ganglionectomy. The fore, will not be described separately. degenerative changes were severe in the The following general conclusions can medial oculomotor nucleus. The few sur- be drawn: viving cells of this group appeared shrunk(1) In all of the experimental cases we en with pycnotic nuclei. In the lateral ac- have examined the reaction to ciliary gan-
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C. H. N A R A Y A N A N A N D Y. N A R A Y A N A N
glionectomy was limited to the accessory oculomotor nucleus. (2) The response to ciliary ganglion removal appeared f m t in the medial accessory oculomotor nucleus followed by degenerative changes in the lateral accessory oculomotor nucleus in subsequent stages. ( 3 ) The degenerative changes are considerably marked at every stage and progresses between the third and ninth day after surgery from a partial cell loss to almost a total cell loss in both subdivisions. (4) None of the other components of the oculomotor nuclear complex seem to be affected by the operation. Although no attempts were made to quantify the results by cell counts of the other components of the oculomotor nuclear complex, it is our impression that they were identical in every respect compared to cell groups of the unoperated control side in each case. DISCUSSION
The main objectives of this investigation were: (1) to determine whether or not severe degenerative changes are restricted to the accessory oculomotor nucleus after selective removal of the ciliary ganglion without impairment to any of the extraocular muscles of the eye, and (2) to examine the pattern of degenerative changes, if any, in the entire third nerve nucleus in general, and specifically, in the two subdivisions which form the accessory OCUlomotor nucleus. Our experimental findings point unequivocally to the conclusion that, in the chick, cell loss following ciliary ganglionectomy is strictly limited to the ipsilateral accessory oculomotor nucleus. The other components of the oculomotor complex are unaffected by the operation, and are similar to the cell groups of the unoperated control side in all cases. In this respect, our results of ciliary ganglionectomy are quite different from that of optic vesicle removal in early stages of development (Cowan and Wenger, '68; Amprino, '43). Optic vesicle removal in early stages of development in the chick embryo has been used successfully to investigate neuronal hypoplasia in the ciliary ganglion and various nuclear centers during development (Amprino, '43; Levi-Montalcini and Amprino, '47; Hammond and Yntema, '58; cowan and Wenger, '68). It is noteworthy
that in all of the investigations involving lesions of the optic vesicle, particularly those of Cowan and Wenger ('68), and Amprino ('43), the ciliary ganglion was not directly involved by the operative procedure. This is further confirmed by the fact that at least in the younger embryos, removal of the optic vesicle does not injure the developing ciliary ganglion or affect its early development, until the ganglion has attained numerical completion in the chick embryo, i.e. day 6 or 7 (Cowan and Wenger, '68). In older embryos, the ciliary ganglion was not available for study in the experiments of Cowan and Wenger ('68), since the brains were dissected out before fixation. Hence, their evidence on the nucleus of origin of preganglionic fibers to the ciliary ganglion is indirect, and as stated by Cowan and Wenger ('68), to some degree, circumstantial. Nevertheless, in their experiments, marked degenerative changes were observed in the ipsilateral accessory oculomotor nucleus at every stage between the tenth to sixteenth days of incubation, from a general cell shrinkage to virtually complete cell loss in later stages. The severity of cell loss in the accessory oculomotor nucleus, as compared to the rest of the third nerve nucleus, would seem to indicate that its terminations are quite different from those of the other components of the oculomotor complex. Cowan and Wenger ('68) noted that the other components of the oculomotor complex showed a more variable reaction to optic vesicle removal, which has been accounted for in two ways. One, is that in most of the components other than the accessory oculomotor nucleus and with the exception of the dorsomedial nucleus, the reaction was presumably due to differing degrees of involvement of the extrinsic eye muscle mesoderm. Second, the severity of cell loss in the dorsomedial nucleus in most of their experimental material according to them was probably due to its innervation of a muscle or muscles in proximity to the optic vesicle and almost invariably resulted in the removal of the innervated muscle(s) during the operation. Yet, in other situations such as the effect of eye removal, immediately after hatching, in the chick (Cowan and Wenger, '68), in adult pigeons (Yoshida, '53), the results are inconclusive (see below). No
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doubt, cellular degeneration was observed following enucleation in both subdivisions of the accessory oculomotor nucleus in the experiments of Cowan and Wenger (‘68), but, many shrunken neurons persisted at all levels throughout the rostro-caudal extent of the nucleus. As Cowan and Wenger (‘68) have pointed out, a proper assessment of the degenerative changes was not possible since the orbital tissues were not available for study. It would suffice to note here that there is really no clear evidence from these studies as to whether the degenerative changes seen in the accessory oculomotor nucleus were secondary to a neuronal degeneration in the ciliary ganglion or retrograde due to direct injury of the ciliary ganglion at the time of eye removal (Cowan and Wenger, ’68). It is clear from our experiments, that selective ciliary ganglionectomy does have a direct effect on the accessory oculomotor nucleus. It operates as a direct consequence of injury to the ciliary ganglion thus leading to a rapid and total loss of cells in the accessory oculomotor nucleus. In summary, our results indicate that retrograde degenerative changes in the accessory oculomotor nucleus proceed very rapidly between three and nine days following ciliary ganglionectomy. Experiments currently in progress on neural hyperplasia following transplantation of an additional eye (Narayanan and Narayanan, ’75) have shown a significant increase in the number of cells of the ciliary ganglion. Further work is underway which will explore more thoroughly the effects of peripheral overloading by transplantation of an additional eye on other nuclear centers. If a corresponding increase in the number of cells in the accessory oculomotor nucleus should occur, we should have additional evidence in s u p port of the notion that the accessory oculomotor nucleus does in fact represent the avian homologue of the Edinger-Westphal nucleus. ACKNOWLEDGMENTS
The authors wish to express their sincere thanks to Mr. Bill Stallworth of the
Audio-visual Department of the L. S. U . School of Dentistry, for photographic assistance, to Lynn Barrett for technical help and to Mrs. Susan Orazio for typing the manuscript. LITERATURE CITED Amprino, R. 1943 Correlazioni quantitative fra centri nervosi e territori d’innervazione Deriferica durante lo sviluppo. Arch. Anat. Embriol., 49.261-300. ~ . . Cajal, S. Ramon y 1909 Contribucion a1 estudio de 10s ganglios de la substancia reticular del bulbo, con algunos detalles concenientes B 10s focos motores y vias reflejas bulbares y mesocefalicas. Trab. lab. invest. biol. Madrid, 7: 259-284. Cowan, W. M., and E. Wenger 1968 Degeneration in the nucleus of origin of the preganglionic fibers to the chick ciliary ganglion following early removal of the optic vesicle. J. Exp. Zool., 168: 105-124. Hammond, W. S., and C. L. Yntema 1958 Origin of ciliary ganglia in the chick. J. Comp. Neur., 110:3 6 7 4 8 9 . Kappers, C. U. A,, G. C. Huber and E. C. Crosby 1936 The comparative anatomy of the nervous system of vertebrates, including man. Vol 11. McMillan Co.,New York. Kusama, T. 1943 On the comparative anatomy of the Edinger-Westphal nucleus. Nisshin Igaku, 32 (cited by Yoshida, ’53, and Niimi et al., ’59). Levi-Montalcini, R. 1949 The development of the acoustico-vestibular centers in the chick embryo in the absence of the afferent root fibers and of descending tracts. J. Comp. Neur., 91: 20 9-242. Levi-Montalcini, R., and R. Amprino 1947 Recherches experimentales sur l’origine du ganglione ciliare dans l’embryos de poulet. Arch. Biol., 58: 265-288. Narayanan, C. H., and R. B. Malloy 1974a Deafferentation studies on motor activity in the chick. I. Activity patterns of hind limbs. J. Exp. Zool., 189: 163-176. 1974b Deafferentation studies on motor activity in the chick. 11. Activity patterns of wing. J. Exp. Zool., 189: 177-188. Narayanan, C. H., and Y. Narayanan 1975 On neural hyperplasia in the chick ciliary ganglion following peripheral overloading. Anat. Rec., 181: 434 (abstract). Niimi, K., T. Sakai and J. Takasu 1959 The ontogenic development of the oculomotor nucleus in the chick. Tokushima. J. Exp. Med., 5: 3 1 1 4 2 5 . Warwick, R. 1954 The ocular parasympathetic nerve supply and its mesencephalic sources. J. of Anat. (London), 88: 71-92. Yoshida, K. 1953 Comparative anatomical and experimental studies on the oculomotor nucleus and neighboring nuclei. Acta Med. et Biol., I : 14S161.
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PLATE 1 EXPLANATION OF FIGURES
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1
A photomicrograph of the oculomotor complex i n a normal 11-day old chick embryo. De Castro silver preparation. AL. Lateral accessory nucleus; AM. Medial accessory nucleus; DM. Dorsornedial nucleus; DL. Dorsolateral nucleus; VMD. Dorsal division of ventromedial n u cleus; VMV. Ventral division of ventromedial nucleus. Scale: 0.1 mm.
2
Photomicrograph of a transverse section about the middle of the oculornotor complex of experimental chick EW 25 (three days after ciliary ganglionectomy). The cells of the medial accessory nucleus of the operated side indicated by arrows are severely depleted; i n the lateral accessory some intact neurons are still present. Thionin preparation. Scale: 0.1 mm.
PREGANGLIONIC FIBERS TO CILIARY GANGLION C. H. N a r a y a n a n and Y . N a r a y a n a n
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
108
3
Photomicrograph of a transverse section about the middle of the oculomotor complex of experimental chick EW 29 (six days after surgery). The accessory nucleus a s a whole on the operated side (arrows) shows a reduction to about one half its normal volume. The other components of the oculomotor complex are unaffected. Thionin preparation. Scale: 0.1 mm.
4
Photomicrograph of a transverse section through the middle of the oculomotor nucleus of experimental chick EW 37 (nine days after ciliary ganglionectomy). The accessory oculomotor nucleus on the operated side indicated by arrows shows a n almost complete cell loss. T h e other components of the oculomotor complex are unaffected. Thionin preparation. Scale: 0.1 mm.
PREGANGLIONIC FIBERS TO CILIARY GANGLION C. H. N a r a y a n a n a n d Y. N a r a y a n a n
PLATE 2
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