Abstract
purpose. Orbital and global layers of rectus extraocular muscles (EOMs) are believed to serve different functions. This study sought anatomic and functional evidence of differing blood flow in the two layers of rectus
EOMs.
methods. Four human orbits ranging in age from 17 months to 93 years were
serially sectioned and stained for muscle fibers with Masson’s trichrome and for vascular smooth muscle with monoclonal antibody to smooth muscle α-actin. Digitally assisted microscopy was used to
obtain measurements of luminal cross sections and counts of muscular blood vessels, as well as measurements of muscle fiber number and cross-sectional areas of the two layers. Findings were correlated with
first-pass gadodiamide contrast magnetic resonance imaging (MRI) in two
living humans to demonstrate relative perfusion of EOMs.
results. In all rectus EOMs, the orbital layer had significantly more vessels
per unit area, more vessels per fiber, and more total vascular luminal
area, than the global layer (P < 0.05). Vascularity of
EOMs was greatest in the youngest specimen. First-pass contrast MRI was
consistent with perfusion of the orbital layer earlier than the global
layer of living human rectus EOMs.
conclusions. Orbital layers of human rectus EOMs have significantly more muscular
vessels than the global layers and stain earlier after intravenous
bolus injection of paramagnetic MRI contrast. These findings suggest
higher and even more rapid blood flow in the orbital layers that may
correlate with greater metabolic activity. Greater blood flow is
consistent with more sustained mechanical loading of the orbital than
the global layer.
Mammalian extraocular muscles (EOMs) have been the subjects of
morphologic,
1 2 3 electrophysiological,
4 and
pharmacologic studies.
3 Light and electron microscopy
demonstrate complex fiber composition of EOMs, highly specialized
striated muscles exhibiting a higher innervation ratio, and more
variation in fiber size and types than skeletal
muscles.
1 2 3 The EOMs are among the fastest muscles in
mammals. Yet, in addition to twitch muscle fibers typical of mammalian
skeletal muscle, EOMs possess slow fibers that are more characteristic
of avian and amphibian muscles.
2 3 In the face of this
paradoxical complexity, a fundamental enigma remains regarding the
function of this diversity of EOM fiber types.
The blood supply of EOMs differs from that of other skeletal muscles.
Perfusion of EOMs is luxuriant. In the cat, Wooten and
Reis
5 measured the average blood flow in the six EOMs and
found that it exceeded that of all other skeletal muscles examined and
was surpassed only by myocardium. Wilcox et al.
6 demonstrated a 10-fold greater blood flow per gram of tissue in EOMs
than in the gastrocnemius and soleus muscles of primates and sheep.
The EOMs are classically divided into two distinct
layers.
1 2 3 The peripheral orbital layer lies along the
EOM surface facing the orbital wall. This layer encloses a second
portion, the global layer, closer to the globe. The laminae are
sometimes separated by an internal perimysium. The orbital layer
contains small-diameter fibers with numerous mitochondria and abundant
vessels. The global layer contains relatively large-diameter fibers
with variable mitochondrial content and fewer vessels. The distinction
between the orbital and global layers in EOM is discernible by
histochemistry, particularly in regard to aerobic versus anaerobic
metabolism. Fibers in the orbital layer stain intensely for oxidative
enzymes, whereas the intensity and proportion of stained fibers
gradually decrease through the global layer. By contrast, the activity
associated with glycolytic enzymes is more intense in the global layer
and is weak in the orbital layer.
1 2 3
The classic studies of Koornneef
7 8 indicated stereotypic
organization of connective tissues around the EOMs. More recent
anatomic studies have clarified that each rectus EOM passes through a
pulley consisting of an encircling ring or sleeve of collagen located
near the globe equator in Tenon’s fascia.
9 10 11 Pulleys
are coupled to the orbital wall, adjacent EOMs, and equatorial Tenon’s
fascia by bands containing collagen, elastin, and smooth muscle (SM).
Abundant elastic fibers in and around pulleys provide reversible
extensibility to these resilient tissues.
9 10 Pulleys have
important implications for EOM action, because the functional origin of
an EOM is at its pulley,
9 10 11 and in secondary gaze
positions the EOM path is discretely inflected at the
pulley.
12 Several lines of evidence, including magnetic
resonance imaging (MRI), gross examinations, surgical exposures, and
histologic studies in humans and monkeys indicate that the orbital
layer of each rectus EOM inserts on its corresponding pulley, rather
than on the globe. It appears that only the global layer of the EOM
inserts on the sclera.
11 13 These anatomic differences in
the two EOM layers suggest differences in their functions: the orbital
layer probably acts against the continuous elastic load of the pulley
suspension, whereas the global layer acts against the intermittent,
viscous load of the antagonist EOM.
11 Accordingly, the
orbital layer would require a vascular supply adequate for intense,
continuous aerobic metabolism, whereas the global layer would require a
lesser blood supply for its more intermittent and glycolytic function.
Although some aspects of the fine structure of EOM blood vessels and
blood flow within EOMs have been reported,
14 15 16 17 there has
been no quantitative study of the vascularity of human rectus EOM
laminae. Recently, MRI enhanced by the intravenous injection of
paramagnetic contrast has been validated as a means of determining
perfusion in the myocardium.
18 Imaging of the first pass
of contrast through highly perfused muscle maximizes sensitivity to
blood flow changes, and results in similar MRI signal enhancements,
both for contrast agents that remain intravascular and for those that
can diffuse extravascularly.
18 In the present study,
performed on serially sectioned human orbits, the blood vessels were
quantitatively evaluated in the orbital and global layers of EOMs.
Findings were correlated with first-pass contrast perfusion MRI of the
EOMs of living subjects to obtain an indication of physiologic
perfusion.
In conformity with legal requirements, orbital specimens were
obtained from four human cadavers (aged 17 months and 4, 57, and 93
years). The head of a 17-month-old male cadaver was freshly frozen to−
78°C within 24 hours of death by accidental asphyxiation and
obtained by anatomic donation to a tissue bank (IIAM, Scranton, PA).
The head was slowly thawed in 10% neutral buffered formalin for 1
week. Other human orbits were obtained during authorized autopsy from
three cadavers within 12 hours of death. Through an intracranial
approach, the orbits were widely exenterated en bloc with periorbita
intact and fixed in 10% neutral buffered formalin. Orbits were then
lightly decalcified for 24 hours in 0.003 M EDTA and 1.35 N HCl,
embedded in paraffin in a vacuum chamber, serially sectioned in the
coronal plane at 10-μm thickness, and mounted on 50 × 75-mm
gelatin-coated glass slides before staining with Masson’s trichrome to
define EOM fibers and collagen. To detect blood vessels of rectus EOMs,
we used monoclonal mouse antibody to human SM α-actin (Dako,
Copenhagen, Denmark) applied at 4°C overnight at dilutions of 1:100
to 1: 500. Nonspecific peroxidase was blocked using 3%
H2O2 for 5 minutes.
Antigen–antibody reactions were visualized using the ABC kit (Vector,
Burlingame, CA) with diaminobenzidine (Sigma, St. Louis, MO) or blue
chromogen (Alkaline Phosphatase Kit 3; Vector).
Digital light micrographs in 24-bit color were made of each rectus EOM
section using a microscope (BH-2; Olympus, Tokyo, Japan) fitted with a
digital camera (Leaf Lumina; ScyTech, Bedford, MA) at a resolution of
3400 × 2800 pixels. Most EOMs were imaged using a ×2.0
objective, requiring that several fields for each section be combined
into a montage. Using image management software (Photoshop 5.0; Adobe
Systems, San Jose, CA), the images were sharpened on a computer
(Macintosh G-3; Apple Computer, Cupertino, CA) and combined seamlessly
into montages. Sections stained with Masson’s trichrome were used to
distinguish the orbital from the global layer of each EOM by the
smaller, more darkly staining fibers in the former. The border between
layers was digitally outlined and superimposed on the digital montage
of the adjacent section stained with monoclonal antibody to human SMα
-actin, which vividly demonstrated SM in the walls of all muscular
blood vessels. Nonmuscular vessels such as capillaries were not
counted.
Measurements of the cross-sectional areas of the two layers of each EOM
were made using NIH Image (W. Rasband, National Institutes of Health;
available by file transfer protocol from zippy.nimh.nih.gov or on
floppy disc number PB95-500195GEI from NTIS, 5285 Port Royal Road,
Springfield, VA 22161). Blood vessels were counted using a four-digit
hand-held counter in planes selected in the anterior one-third, middle,
and posterior one-third of the length of each EOM. Because complete,
exact counts were made, no sampling approximations were used. For an
estimate of accuracy, duplicate counts were made for all rectus EOMs in
all sections. Counts were repeatable to within less than 3%. In two
orbits, all the fibers were counted in the anterior one-third, middle,
and posterior one-third of each of the four EOMs. In selected sections,
all muscle fibers of the four rectus EOMs were counted using light
microscopy and a hand-held digital counter, again without
approximations.
Measurements of the luminal cross-sectional area were made using NIH
Image software from randomly selected, longitudinally oriented muscular
vessels in the orbital and in the global layers of each EOM at the
level of the midorbit. Effort was made to measure vessels supplying the
EOM itself. Excluded from area measurements were bifurcating vessels,
vessels running tangentially in the plane of section, and the large
ciliary arteries passing through the EOMs to supply the anterior
segment of the globe.