Download Innervation of the superior tarsal (Müller`s)

Innervation of the Superior Tarsal (Miiller's) Muscle
in the Cynomolgous Monkey: A Retrograde Tracing Study
Frans van der Werf* Bob Baljet,-f Maarlen Prins,* Arie Timmerman,X and Jan A. Otto*
Purpose. The retrograde transport of wheat germ agglutinin-horseradish peroxidase (WGA/
HRP) was used to study the localization of the neurons that innervate the superior tarsal
muscle in the cynomolgous monkey.
Methods. A 5-10% WGA-HRP solution was applied to the medial part of the superior tarsal
muscle. Seventy-two hours later, the animals were killed and perfused with fixative.
Results. The WGA-HRP-labeled neurons were localized in the ipsilateral trigeminal ganglion,
the ipsilateral pterygopalatine ganglion, and the ipsilateral superior cervical ganglion. The
labeled somata of the sensory neurons were all found in the ophthalmic ganglionic part and the
ophthalmic nerve part of the ipsilateral trigeminal ganglion. Most of the labeled somata were
distributed somatotopically in the dorsal region of the ophthalmic part of the ganglion. The
labeled somata of the sympathetic neurons were all located in the ipsilateral superior cervical
ganglion. Most of these labeled somata, which were distributed somatolopically, were found in
the cranial part of the ganglion. The labeled somata of the parasympalhetic neurons were all
located in the ipsilateral pterygopalatine ganglion, particularly in the central ovoid part of the
ganglion. No labeled neurons were detected in the ipsilateral parasympathetic ciliary ganglion,
the ipsilateral nodose ganglion, or the first three ipsilateral sensory spinal ganglia; labeled
somata could not be detected in any of the contralateral ganglia examined.
Conclusions. A complete documentation of all projections of the superior tarsal muscle in the
monkey has been presented, and convincing data has been given about the parasympathetic
innervation of Miiller's muscle. Invest Ophthalmol Vis Sci. 1993; 34:2333-2340.
A he superior tarsal (Miiller's) muscle,1 3 the conjunctiva,4"6 the meibomian glands,7 and the blood vessels8
are autonomically innervated structures of the upper
and lower eyelids in mammals. The role of the superior
From the •Department of Experimental Ophthalmology. The Netherlands
Ophthalmic Research Institute., and the ^Department of Anatomy ami Embryology,
University of Amsterdam, Amsterdam; ami the *Central Animal laboratory of The
National Institute of Public Health ami Environmental Protection, Bilthoven, The
Netherlands.
Submitted for publication: February 5. 1992; accepted February 5, 1992.
Proprietary interest category: N.
Reprint requests: Frans van der Werf, The Netherlands Ophthalmic Research
Institute, P.O. Box 12HI, 1100 AC Amsterdam, the Netherlands.
tarsal muscle is tonic retraction of the upper eyelid;
impulses from the sympathetic nerve fibers cause this
muscle to contract.9
Although neuroanatomic studies of the rat,1
mouse,10 rabbit,11 guinea pig,11 cat,7 monkey,12"14 and
humans12 have been performed, knowledge about the
perikarya of the neurons that take part in the innervation of different components of the eyelid is limited.
Acute ganglionectomy of the superior cervical ganglion (SCG) in rats1 produced complete denervation of
the superior tarsal muscle. Ganglionectomy of the
SCG in the mouse10 caused the disappearance of
Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
adrenergic nerve fibers in the superior tarsal muscle.
Acute damage of the adrenergic nerve fibers was
found in the superior tarsal muscle 24 hr after superior cervical ganglionectomy in the cynomolgous monkey.15 Denervation of the branches of the ophthalmic
part of the trigeminal afferent nerve in the rat 1617 resulted in a decrease in substance-P (SP) immunoreactivity and elimination of the calcitonin gene-related
peptide immunoreactivity of sensory nerve fibers in
the conjunctiva and the superior tarsal muscle. Partial
elimination of the pterygopalatine ganglion (PPG) in
the monkey5'6 also caused a decrease in the number of
nerve fibers in the conjunctiva. Electron-microscopic
studies of the superior tarsal muscle in the mouse10
revealed the existence of Type I nerve endings (adrenergic) and Type II nerve endings (cholinergic). In this
study of the mouse, ganglionectomy of the PPG provided support for the hypothesis that cholinergic
nerve fibers originate in the PPG. In the developing
rat,3 it was found that excision of the ipsilateral SCG
caused a 39% reduction in the density of acetylcholinesterase (AChE)-positive nerves 7 days later, indicating
that sympathetic nerves contribute to cholinesterasepositive tarsal muscle innervation. Excision of the PPG
concurrent with the SCG caused a virtually complete
disappearance of AChE-positive innervation within 7
days, indicating that nonsympathetic cholinesterasepositive fibers derive from the PPG and are presumed
to be parasympathetic. Partial diathermic damage of
the PPG in the rabbit reduced the number of SP-positive nerve fibers in the upper eyelid.18 These authors
did not incorporate the superior tarsal muscle in their
study. Intersectioning of the superior levator muscle
in the monkey, according to the Fasanella Servat procedure, did not result in total denervation of the upper eyelid.15 By means of histochemical19 and fluorescent histochemical analysis,U14'20'21 differences in the
number of AChE-positive and adrenergic nerve fibers
in the superior tarsal muscles of the guinea pig, rat,
monkey, and humans have been demonstrated. Fluorescent retrograde tracing with true blue or diamidino
yellow of the superior tarsal muscle in the rat1'2
showed labeled perikarya in the rostral part of the
SCG. AChE in toto12 and AChE thick-section studies13
of the rhesus monkey and humans revealed the presence of fine-meshed intrinsic nerve plexuses in the inferior and superior tarsal muscles. In these investigations, the origins of the AChE-positive nerve fibers
could not be detected.
Therefore, in the monkey, the localization of neurons that innervate the superior tarsal muscle and the
possible somatotopic localization of perikarya in
various ganglia were investigated by means of the
wheat germ agglutinin-horseradish peroxidase
(WGA-HRP) retrograde tracing technique.
MATERIALS AND METHODS
Five young adult cynomolgous monkeys (Macacafascicularis) of both sexes, weighing between 2.3-8.1 kg,
served as experimental subjects. All experiments in
this study were performed in accordance with the
ARVO Resolution on the Use of Animals in Research.
Each animal was anesthetized with a mixture of
ketamine, xylazine, and atropine in the proportion
10:1:0.1 mg/kg, respectively. Two monkeys received
an injection of a 10 /jl of 5% WGA-HRP solution
(Sigma, St. Louis, MO) into the superior tarsal muscle
of the right orbit. For this purpose, a 20-gauge needle
connected by a short length of polyethylene tubing to
a Hamilton syringe was used. In the other three animals, a solidified piece of WGA-HRP (Sigma) in Willospon (Willpharma, Zwanenburg, The Netherlands),
prepared by soaking a small piece of Willospon in 10%
WGA-HRP, was applied to the medial part of the superior tarsal muscle of the right orbit.
All animals were anesthetized 72 hr after surgery
with ketamine (0.4 ml/kg) followed by 5000 units/kg
of thromboliquine. Next, the animals were killed by an
overdose of pentobarbital and perfused directly
through the internal carotid artery with 2000 ml of
phosphate-buffered saline, pH 7.4, at 37°C, followed
by 2-3 1 of fixative containing 0.1 mol/1 phosphatebuffered 2% paraformaldehyde and 2.5% glutaraldehyde, pH 7.4, and finally, with 0.1 mol/1 phosphate
buffer containing 10% sucrose at 4°C.
The brainstem, trigeminal ganglia, SCG, PPG, ciliary ganglia, nodose ganglia, and the first three spinal
ganglia on both sides were dissected. We cut 50-^tm
transverse sections of the brainstem with a cryostat
microtome from the level of the superior colliculus to
the spinal cord. In addition, 20-40-jtm serial cryostat
sections of the ganglia from both sides were mounted
directly on chrome-alum-gelatin-coated slides. The
sections on the slides were then tested for the presence
of WGA-HRP activity using either the tetramethylbenzidine (TMB) procedure22 or the gold-substituted
silver peroxidase procedure.23 All samples were examined by bright- and dark-field microscopy for the presence of WGA-HRP-labeled neurons.
Camera lucida drawings of the sections of the ipsilateral trigeminal ganglia and SCG were made to determine the exact localization of the labeled perikarya.
The labeled neurons in the ipsilateral trigeminal ganglia, SCG, and PPG were counted to estimate the number of labeled somata in each ganglion. All values are
presented as the mean ± the standard error of the
mean.
Immediately after perfusion of the animals, the
upper eyelid, the lacrimal gland, and the cornea of the
right orbit were dissected and processed in the same
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Innervation of the Superior Tarsal Muscle
OPHT
way as the brainstems and ganglia to determine
whether the WGA-HRP had leaked into these tissues.
RESULTS
After retrograde tracing with WGA-HRP of the superior tarsal muscle, labeled somata of the neurons only
appeared in the ipsilateral trigeminal ganglia, SCG,
and PPG (Table 1). The ipsilateral ciliary, nodose, and
first three spinal ganglia were devoid of labeled somata. Labeled somata were never observed in the contralateral ganglia in any animal. The brainstem in four
of five animals did not contain labeled neurons. In one
monkey, 20 labeled motoneurons were located in the
ipsilateral facial motor nucleus. In control sections of
the upper eyelid (including the palpebral conjunctiva),
lacrimal gland, and whole mount of the cornea, leakage of the tracer could not be detected. The control
sections from the monkey with labeled neurons in the
ipsilateral facial motor nucleus revealed small
amounts of WGA-HRP in the orbicularis oculi muscle
of the upper eyelid.
MAX
MAN
FIGURE 1. Schematic drawing of a cryostat-cut section containing the trigeminal ganglion with its three nerves: ophthalmic nerve, opht; maxillary nerve, max; mandibular nerve,
man; ganglionic part, gp; nerve part, np; and transitional
zone, arrow.
Trigeminal Ganglion
The trigeminal ganglion (Gasser) of the cynomolgous
monkey is semilunar in shape. Bundles of nerve fibers
originating in the somata of neurons divide the ganglion into three parts, named for the three nerves (ophthalmic, maxillary, and mandibular nerves) that leave
the ganglion dislally (Fig. 1). Therefore, the terms ophthalmic, maxillary, and mandibular parts of the trigeminal ganglion refer to the topographic distribution of
somata of these neurons. The cluster of neurons located proximally in the ophthalmic nerve itself is
called the ophthalmic nerve part of the trigeminal ganglion (Fig. 1). An average of 465 labeled somata (Table
1 and 2) was restricted to the ipsilateral ophthalmic
part of the ganglion (Fig. 2) and the proximal part of
TABLE l. Presence of Labeled Somata (72 h)
after Injection or Application of WGA/HRP
into the Superior Tarsal Muscle of a Right
Cynomolgous Orbit
Ganglia
Trigeminal
Superior cervical
Pterygopalatine
Nodose
Ciliary
Spinal 1
Spinal 2
Spinal 3
Labeled
Somata
Ipsilateral
Contralatral
+
+
+
_
—
465 ± 62
61 ±32
_
_
_
_
-
-
_
-
38 + 41
the ophthalmic nerve (Figs. 3, 4). An average of 315
WGA-HRP-positive somata was present in the ophthalmic part of the ganglion. An average of 150
WGA-HRP-positive somata was found in the ophthalmic nerve part of the ganglion; in this part, a somatotopic distribution of the perikarya was detected (Fig.
5). In the transitional zone between the ophthalmic
and maxillary parts of the ganglion (Fig. 1) were some
labeled perikarya (maximum number, 30). In the ganglionic part, the labeled sensory neurons differed in
size; small and large perikarya were positive for the
WGA-HRP tracer. In the maxillary part of the ganglion and the principal and mesencephalic nuclei of
the brainstem of cranial nerve V, no labeled somata
could be detected.
TABLE 2. Number of Counted Labeled
Neurons in Each Ganglion
Monkey No.
Tri geminal
Superior
Cervical
Pterygopalatine
396
422
409
502
550
455
502
465
33
38
36
53
112
67
77
61
2
5
3
48
103
33
61
38
A 175
A 519
Mean (n = 2)
710
611
293
Mean (n = 3)
Mean (n = 5)
A = injected animal.
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Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
FIGURE 2. Bright-field lighl micrograph of WGA-HRP-labeled somaia (arrows) in the ophthalmic ganglion part of the
trigeminal ganglion (gold-substituted silver peroxidase procedure), liar = 50 nm.
SCG
Most of the labeled somata were present in the cranial
part of the spindle-shaped ganglion (Figs. 6, 7). In the
upper third of the ganglion, a somatotopic distribution of perikarya was seen (Fig. 8). A few labeled somata (maximum number, ten) were observed in the
medial part of the ganglion; none were detected in the
caudal part of the ganglion. An average of 61 positive
cell bodies was present in this ganglion (Tables 1
and 2).
PPG
WGA-HRP-positive neurons were localized in the ipsilateral PPG. In the three implanted monkeys, labeled
somata were detected in the central ovoid part of the
ipsilateral PPG (Fig. 9). An average of 61 was present
(Table 2). In the two injected animals, a few somata
(two and five) were labeled in the ipsilateral PPG (Ta-
FICURE 4. Dark-field micrograph of WGA-HRP-labeled somata and nerve fibers (arrows) in the ophthalmic nerve part
of the trigeminal ganglion (TMB procedure). Bar = 50 jim.
ble 2). The rami orbitalis of this ganglion did not contain labeled somata. In the ipsilateral PPG of the five
animals, an average of 38 positively WGA-HRP-labeled cell bodies was counted (Table 1); a somatotopic
distribution could not be seen.
DISCUSSION
The results of the current study provide information
on the localization and numbers of perikarya innervating the superior tarsal muscle of the cynomolgous
monkey. Moreover, the sensory (afferent), sympathetic (efferent), and parasympathetic (efferent) innervation of this muscle has been determined. Afferent innervation was restricted to the ophthalmic part
of the trigeminal ganglion; efferent innervation occurs through the SCG and the PPG. There was no
a (0 08)
b (0.28)
c(0.48)
FIGURE 3. Bright-field light micrograph of WGA-HRP-labeled somata (arrows) in the ophthalmic nerve part of the
trigeminal ganglion (TMB procedure). Bar = 50 jtiin.
FIGURE 5. Schematic drawing of serial cryostat-cut sections
illustrates the distribution of labeled somata (solid circles) in
the trigeminal ganglion (shaded areas). Many labeled somaia
are concentrated in the ophthalmic nerve part of the ganglion. The numbers to the left of the figures represent the
distance in millimeters from the dorsal surface of the ganglion.
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Innervation of the Superior Tarsal Muscle
FIGURE 6. Bright-field lighL micrograph of WGA-HRP-labeled somata (arrows) in the cranial part of the SCG (TMB
procedure). Bar = 50 jim.
evidence for direct projections from the superior tarsal muscle toward the brainstem.
Comparing the injection method versus the Willospon implantation method, the best results were obtained with the latter. This is probably the result of the
amount of uptake of WGA-HRP into the nerve endings. The WGA-HRP-soaked small piece of Willospon
localized in the superior tarsal muscle enhanced the
uptake of WGA-HRP, thus increasing the amount of
WGA-HRP available for transport, which in turn, may
increase the neuron labeling.24
Controls
Because of selective application of the tracer to the
medial part of the superior tarsal muscle, no leakage to
the adjacent tissues was detected in four of our five
monkeys. The absence of labeled somata in the ciliary
ganglia of the five animals investigated further suggested that tracer leakage from the superior tarsal
FIGURE 7. Dark-field micrograph of WGA-HRP-labeled somaia with axons (arrows) in the cranial part of the SCG
(TMB procedure). Bar = 50 /xm.
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a (0.1)
b(0.2)
c(0.3)
d (0.4)
FIGURE 8. Schematic drawing of serial cryostat-cut sections
illustrates the distribution of labeled somata (solid circles) in
(he SCG (shaded areas). Labeled somata are concentrated
mainly in the upper third of the ganglion. The numbers at
the bottom of the figures represent the distance in millimeters from the ventral surface of the ganglion.
muscle into adjacent tissues of the eyelid was negligible. In one monkey, with some leakage into the adjacent orbicularis oculi muscle, a few labeled motoneurons were found in the ipsilateral facial motor nucleus.
This was in full agreement with the results of previous
tracer studies on the orbicularis oculi muscle in the
cynomolgous monkey.25 After application of WGAHRP and HRP to the orbicularis oculi muscle, these
authors found labeled motoneurons in the ipsilateral
facial motor nucleus with a few neurons in the locus of
the contralateral facial motor nucleus.
Trigeminal Ganglion
In the cynomolgous monkey, we chose the same nomenclature for the trigeminal ganglion, the trigeminal
nerve, and its branches as is used in humans.26 In ro-
FIGURE 9. Dark-field micrograph of WGA-HRP-labeled somata (arrows) in the central part of the PPG (TMB procedure). Bar = 50 nm.
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Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
dents, 4 ' 2728 a different nomenclature is generally applied because the distribution of the branches of the
trigeminal nerve differs from that in humans. In particular, rodents have a thick infraorbital nerve caused by
innervation of the vibrissae and other structures.27
Moreover, in the rat,28 the trigeminal ganglion has a
common ophthalmic-maxillary nerve. The current
study of primates has demonstrated that afferent neurons are concentrated in the ophthalmic ganglionic
part and the ophthalmic nerve part of the ipsilateral
trigeminal ganglion. A somatotopic distribution was
present in the ophthalmic nerve part, specifically in
the dorsal region. This finding is in accordance with
the results of studies in which transsected branches of
the ophthalmic nerve of the cat were labeled.29 Immunohistochemical studies of the eyelids of the rat15'16
showed calcitonin gene-related peptide- and SP-positive nerve fibers in the superior tarsal muscle. In that
experiment, the calcitonin gene-related peptide-positive nerve fibers disappeared after denervation of the
ophthalmic and maxillary nerves; the SP-positive
nerve fibers were reduced in number but did not disappear. These results are in agreement with the findings of the current investigation.
SCG
The results show that the superior tarsal muscle in the
cynomolgous monkey is sympathetically innervated by
moderate numbers of neurons located in the ipsilateral SCG. Several other studies confirm this observation.1-2 A fluorescent tracer applied to the superior
tarsal muscle1'2 and to both the superior tarsal muscle
and the nictitating membrane2 in rats resulted in the
appearance of labeled somata in the ipsilateral rostral
part of the SCG. In the double-labeling experiments,2
double labeling of the somata of the SCG could not be
detected. It can be concluded that collateral innervation does not exist in the SCG of the rat. In our investigation, a cranial somatotopic distribution of labeled
somata in the SCG was seen in the cynomolgous monkey. This sympathetic connection between SCG and
superior tarsal muscle has also been observed in the
mouse10 and in one cynomolgous monkey15 after
ganglionectomy of the SCG. Histochemical studies
(using the Falck and Hillarp and the AChE methods)
showed both adrenergic11'20 and AChE-positive19
nerve fibers in the superior tarsal muscle.
PPG
In the classic concept of the primate, the superior tarsal muscle is innervated exclusively by the sympathetic
In this study, three of the five
nervous system.'
monkeys, implantation with WGA-HRP in the medial
part of the superior tarsal muscle resulted in the detection of labeled somata in the ovoid part of the ipsi-
lateral PPG. In the two injected animals, only a few
somata of the PPG were labeled. Ganglionectomy of
the PPG of the mouse10 caused partial disappearance
of the autonomic Type II nerve endings in the superior tarsal muscle; destruction of the PPG in the rabbit18 led to a decrease in the number of SP-positive
nerve fibers in the upper eyelid, particularly in the
conjunctiva and not in the superior tarsal muscle.
Experiments combined with immunohistochemical studies17'18 revealed that, in the rat, neither the
number nor the distribution of SP-immunoreactive
nerve fibers in the superior tarsal muscle and the (palpebral) conjunctiva were affected by ganglionectomy
of the SCG or denervation of the ophthalmic and maxillary nerve parts of the trigeminal ganglion. It is well
known that innervation of the (palpebral) conjunctiva
is parasympathetic.4~6'18 Some nerve fibers originating
in the PPG pass through the superior tarsal muscle
and are probably destined for the conjunctiva.1617 In
the cynomolgous monkey, electron microscopy revealed parasympathetic nerve fibers and endings in
the palpebral conjunctiva.5 A dense meshwork of sensory and autonomic nerve fibers was found in the palpebral conjunctiva of both humans and the rhesus
monkey.12'13 In the same studies, dense intrinsic nerve
plexuses were identified in the superior and inferior
tarsal muscles. The two plexus are connected with one
another. These AChE studies10'1213 did not give a definite answer about the origin of neurons innervating
the tarsal muscles. Because bundles of nerve fibers
located in the superior tarsal muscle stained positively
for AChE,12'13 this was considered strong evidence for
cholinergic innervation of this muscle. Staining for
AChE is an accepted method for identifying cholinergic parasympathetic nerves if there is a strong staining
of perikarya and/or nerves.3 However, several studies
have documented the presence of this enzyme in nonparasympathetic nerves, such as the sympathetic and
sensory nerves.30'31 Definite demonstration of the origin of cholinergic innervation in the PPG has been
obtained with this retrograde tracing study of the cynomolgous monkey. So, in this study, it has been proved
that, in the primate, the tarsal muscles have parasympathetic (PPG) and sympathetic (SCG) innervation.
These findings suggest a possible mechanism of interactions between sympathetic and parasympathetic
nerves in this smooth muscle, as was postulated in the
rat.3 Sympathetic nerves can modulate parasympathetic nerves indirectly by influencing production
and/or release of neurotrophic substances from
smooth muscle cells. Neuroanatomic studies of the
eyelids of humans21 have shown that pathways of sympathetic nerves accompany the branches of the ophthalmic nerves toward the tarsal muscles. However,
these authors neglected the parasympathetic compo-
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Innervation of the Superior Tarsal Muscle
nent. Dissection of the palpebral levator muscle15 resulted in partial denervation of the upper eyelid,
which could be explained by the fact that the autonomic nerve pathways run alongside the blood vessels
and branches of the ophthalmic nerve. In conclusion,
the parasympathetic nerve fibers located in the superior tarsal muscle in the primate originate in the ipsilateral PPG. To our knowledge, this is the first observation of parasympathetic innervation of Miiller's muscle in the monkey.
11.
12.
13.
Key Words
WGA-HRP, innervation, superior larsal muscle, trigeminal
ganglion, superior cervical ganglion, pterygopalatine ganglion, cynomolgous monkey
14.
15.
Acknowledgments
The authors thank Annelies C. Vinju for typing the manuscript and Nico C. M. Bakker and Marina E. M. Danzmann
for preparing the photographs.
16.
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