This study presents the results of a qualitative and quantitative
investigation of the entire complement of encapsulated proprioceptors
in pig EOMs. For the first time, the distribution of all encapsulated
proprioceptors in a certain species is precisely shown over the whole
muscle length. In addition to a high number of muscle spindles, GTOs
are consistently present in the recti and the oblique EOMs in pigs. In
all EOMs of three pigs the quantity of these two proprioceptors is
rather constant. All GTOs are distributed in aponeurotic expansions of
the distal and proximal tendons. GTOs are more numerous in the recti
EOMs than in the oblique EOMs. In each EOM, their number was higher in
the distal aponeurosis than in the proximal aponeurosis. In the EOMs
muscle spindles outnumber GTOs. The ratio of muscle spindles to GTOs is
different in the recti and oblique muscles. In particular, values are
very similar in the recti muscles, whereas they are remarkably high in
the oblique ones. In pig levator palpebrae superioris muscles GTOs are
extremely rare or absent, whereas more GTOs were found in the levator
palpebrae superioris muscles of sheep
21 and
camels.
8 In the current study GTOs are described for the
first time in pig EOMs and the morphology of these receptors is
presented in detail.
Two previous studies supplied data on muscle spindles in pig EOMs. In a
quantitative analysis of EOM spindles in several mammals, Maier et
al.
4 counted the muscle spindles in four EOMs (medial
rectus, lateral rectus, inferior rectus, and inferior oblique) of one
neonatal pig. In the present study, we investigated all EOMs of three
pigs. We found a comparable number of muscle spindles in the recti
EOMs, whereas the number of muscle spindles in the inferior oblique is
higher than previously reported.
4 The superior oblique
muscles are unique in containing a surprisingly high number of muscles
spindles. Kubota
7 described the ultrastructure of pig EOM
spindles and found most of them to contain a single nuclear bag fiber
and three to four nuclear chain fibers. These observations are
congruent with the results of the present study.
As sheep
6 and camel
8 EOMs, we found the
muscle spindles to be distributed over the whole muscle lengths in pig
EOMs. In contrast, the midbelly region of human EOMs contained no
spindles.
9 10 These spindle-free zones coincided with the
zones of en plaque motor endplates.
9 10
This study is in line with previous investigations that described the
presence of GTOs and muscle spindles in EOMs of artiodactyls (i.e.,
cattle,
4 camels,
8 and
sheep.
6 20 21 22 ) Although the presence of spindles in pig
EOMs has been reported
4 7 the occurrence of GTOs was
established in the current study. Evidence at hand suggests that a high
number of both proprioceptors in the EOMs of artiodactyls is a unique
feature among mammals.
Each GTO in pig EOMs has a perineurial capsule and shows an
intracapsular bidirectional arborification of nerve fibers. Nerve
terminals establish contacts with collagen fibrils. These features are
consistent with GTOs of mammalian limb and trunk
muscles
24 25 26 and provide sufficient evidence to classify
these encapsulated organs of this study as GTOs. However, fluid-filled
spaces separating collagen bundles from the GTO capsule and
intracapsular muscle fibers observed in most GTOs of pig EOMs are
absent in GTOs of mammalian limb and trunk muscles. The structural
particularities of GTOs in mammalian EOMs
19 21 22 described in previous reports are also present in numerous GTOs of pig
EOMs. In accordance with findings in GTOs of rhesus
monkey
19 and sheep EOMs,
21 22 this
investigation demonstrates the occurrence of intracapsular muscle
fibers in one third of pig GTOs. As in the distal peripheral patch
layer of sheep EOMs,
22 we found GTOs with traversing
muscle fibers also in pig EOMs. Another similarity between GTOs of both
species is the presence of a fluid-filled space
21 22 which, however, appears less pronounced in pigs. Consequently,
equatorial diameters of pig GTOs (67 ± 25 μm) are smaller than
their counterparts in sheep EOMs (101 ± 18 μm).
22
Recently, we were able to distinguish four morphologic types of GTOs in
the distal peripheral patch layer of sheep EOMs.
22 These
four types correspond to the GTO types presented herein. However,
differences in the numerical proportion have to be emphasized. In the
distal peripheral patch layer of sheep EOMs, GTOs containing
exclusively collagen bundles (GTO type 1) are few, and the majority of
the GTOs contains traversing muscle fibers (GTO type 3).
22 In contrast, most GTOs in pig EOMs exhibit type one characteristics and
few GTOs are consistent with type 3. The remaining GTO types 2 and 4
are present in comparable percentages in both species.
Intracapsular muscle fibers in the GTOs of rhesus monkey
19 and in sheep EOMs
21 22 resembled the multiply innervated
type in their fine structure. This was further confirmed by an
immunohistochemical analysis of the MHC pattern of intracapsular muscle
fibers in sheep EOMs.
22 In the present study,
intracapsular muscle fibers in GTOs of pig EOMs were analyzed by
transmission electron microscope and light microscope, by means of
histochemistry and immunohistochemistry. Intracapsular muscle fibers in
pig GTOs have few and small mitochondria, sparse sarcoplasmic
reticulum, and densely arranged myofibrils. These fine structural
features correlate with multiply innervated muscle fibers in EOMs of
various mammals.
32 33 Further, it was shown that
intracapsular muscle fibers in GTOs of pig EOMs were positive for
mATPase after acid preincubation and positive for slow-twitch MHC
antibody. From studies on other mammalian species
32 and
humans
34 it is well known that muscle fibers exhibiting
this staining pattern for mATPase and slow-twitch MHC belong to the
multiply innervated type. Recently, it was demonstrated in rat EOMs
that multiply innervated muscle fibers of the orbital layer contain
slow-twitch MHC and additionally embryonic MHC.
35 We
cannot exclude that multiply innervated intracapsular muscle fibers in
GTOs of pig contain other MHC isoforms. Based on fine structure and on
histochemical staining for mATPase and SDH, intracapsular GTO muscle
fibers of pigs correspond to the multiply innervated muscle fibers in
the distal peripheral patch layer of sheep EOMs.
23 Further, based on mATPase and SDH activity and MHC expression,
intracapsular muscle fibers in GTOs of pig EOMs resemble the
low-oxidative, multiply innervated muscle fibers in the marginal zone
of human EOMs.
34 In conclusion, these results indicate
that intracapsular GTO muscle fibers of pig EOMs may derive from a
particular muscle layer comparable with the peripheral patch layer in
sheep EOMs
23 and the marginal zone in human
EOMs.
34
GTOs containing intracapsular muscle fibers with sensory myoneural
contacts have been observed in developing limb muscle of
rats.
36 A few days after birth, these muscle fibers
withdraw from the receptors and in mature GTOs sensory nerve terminals
is exclusively found among the collagen bundles.
36 Ruskell
21 argued that the occurrence of intracapsular
muscle fibers observed in numerous GTOs of young sheep
21 (11–14 months old) and young rhesus monkeys EOMs
19 may be
a sign of a prolonged GTO maturation. These intracapsular muscle fibers
may get lost with advancing age.
21 If, however, this
particular morphology were also observed in GTOs of old animals of the
same species, Ruskell
21 stated that this would indicate
the retained development of these receptors. Recently, we compared GTOs
in the EOMs of a 1-year-old and a 5-year-old sheep
22 but
found no differences in the morphology of their GTOs. The results of
the present study are in line with this investigation.
22 The incidence of intracapsular muscle fibers is almost the same in GTOs
of four pigs ranging from 2 months to 5 years of age. Moreover, sensory
neuromuscular contacts observed in developing GTOs of
rat
36 are not found in the GTOs of pig EOMs. We conclude
that the particular morphology of numerous GTOs in pig EOMs indicates
that these receptors are retained in development, as we previously
observed in sheep EOMs.
22
It was proposed by Ruskell
21 that in case of a retained
development in GTOs of mammalian EOMs, the utility of GTOs may be
questionable. However, the following arguments favor the endowment of
pig EOMs with special but functioning GTOs. The number of GTOs is high
in those pig EOMs that enable movements of the globe. Moreover, all
GTOs in pig EOMs are richly supplied with nerve terminals that are
distributed in each collagen bundle. These nerve terminals contain
numerous mitochondria and are partly invested with Schwann cells.
Because large portions of the nerve terminals are covered only with a
basal lamina they establish intimate contacts with the neighboring
collagen fibrils. In some areas, the basal lamina of nerve terminals is
interrupted. These findings are consistent with those of nerve
terminals in GTOs of other mammalian skeletal
muscles.
24 25