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Underdeveloped Extraocular Muscles in the Naked Mole-Rat (Heterocephalus glaber).

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THE ANATOMICAL RECORD 293:918–923 (2010)
Underdeveloped Extraocular Muscles in
the Naked Mole-Rat (Heterocephalus
Departments of Physiology, University of Kentucky, Lexington, Kentucky
Departments of Ophthalmology, Vanderbilt University, Nashville, Tennessee
The extraocular muscles (EOM), the effector arm of the ocular motor
system, have a unique embryological origin and phenotype. The naked
mole-rat (NMR) is a subterranean rodent with an underdeveloped visual
system. It has not been established if their ocular motor system is also
less developed. The NMR is an ideal model to examine the potential codependence of oculomotor and visual system development and evolution.
Our goal was to compare the structural features of NMR EOMs to those
of the mouse, a similar sized rodent with a fully developed visual system.
Perfusion-fixed whole orbits and EOMs were dissected from adult NMR
and C57BL mice and examined by light and electron microscopy. NMR orbital anatomy showed smaller EOMs in roughly the same distribution
around the eye as in mouse and surrounded by a very small Harderian
gland. The NMR EOMs did not appear to have the two-layer fiber distribution seen in mouse EOMs; fibers were also significantly smaller (112.3
46.2 vs. 550.7 226 sq lm in mouse EOMs, *P < 0.05). Myofibrillar
density was less in NMR EOMs, and triad and other membranous structures were rudimentary. Finally, mitochondrial volume density was significantly less in NMR EOMs than in mouse EOM (4.5% 1.9 vs. 21.2% 11.6, respectively, *P < 0.05). These results demonstrate that NMR
EOMs are smaller and less organized than those in the mouse. The ‘‘simpler’’ EOM organization and structure in NMR may be explained by the
poor visual ability of these rodents, initially demonstrated by their primiC 2010 Wiley-Liss, Inc.
tive visual system. Anat Rec, 293:918–923, 2010. V
Key words: extraocular muscle; Heterocephalus glaber; naked
mole-rat; mitochondria
The naked mole-rat (Heterocephalus glaber) is a subterranean rodent that rarely experiences natural environmental light. Its visual system is underdeveloped,
the naked mole-rat retina contains most of the cell types
of visually guided mammals but the structural organization is rudimentary (Mills and Catania, 2004). The neural structures, which arbitrate vision are also atrophied
(Crish, SD et al., 2006).Why nonvisually guided mammals retain a visual system at all remains uncertain.
Potential reasons include that circadian rhythms and
other metabolic activities could still rely on subtle environmental visual cues. Light avoidance, without fine visual details, could also form the sensory arm of an escape
behavior in the event of a tunnel breech. In any event, it
is unclear whether the regression of the visual system is
accompanied by a corresponding change in the ocular
motor system.
Grant sponsor: NEI Francisco H. Andrade; Contract grant
number: EY12998.
*Correspondence to: Colleen A. MCMullen, Department of
Physiology MS508, University of Kentucky, 800 Rose St.,
Lexington, KY 40536-0298, E-mail:
Received 18 September 2009; Accepted 7 December 2009
DOI 10.1002/ar.21107
Published online 23 February 2010 in Wiley InterScience (www.
Fig. 1. Representative light micrographs of cross-sections of
mouse and naked mole rat orbits (H&E stain). (A) Anatomy of the
mouse orbit showing optic nerve (arrowhead), Harderian gland (black
arrow), retractor bulbi (RB), superior oblique, o, orbital layer; g, global
layer; \, rectus muscle. (B) Mouse orbital layer, global layer, retina, re-
tractor bulbi, levator palpebral superioris (lps), and rectus muscles.
(C) Naked mole rat orbit showing optic nerve, muscle, Harderian
gland. (D) Naked mole rat orbit with optic nerve, EOMs, Harderian
gland. (E and F) Naked mole rat retina, EOMs. (scale bars ¼ 100 lm
Figures A, 50 lm Figures B–D, and 25 lm Figures E and F).
The extraocular muscles (EOMs), responsible for voluntary and reflexive movements of the eyes, are arguably the fastest and most active skeletal muscles (Porter
et al., 1995). These small muscles express mostly fast
myosins, including an ultrafast tissue-specific isoform,
and contain abundant mitochondria and sarcoplasmic
reticulum (SR) (Mayr, 1971). The visual system is anatomically and functionally immature at birth; key prop-
erties, such as binocularity and depth perception develop
postnatally during a species-specific window called the
‘‘critical period’’ (Berardi et al., 2000). Previously, we
demonstrated in mice that dark rearing impairs EOM
function and the neural mechanisms underlying compensatory eye movements (McMullen et al., 2004). This indicates that visual experience early in life is necessary for
the normal development of EOMs (Cheng et al., 2004,
McMullen et al., 2004). The study of the ocular motor
system of a fossorial mammal such as the naked molerat provides the opportunity to assess the role of visual
experience on the development of the extraocular
muscles and corresponding central motor pathways.
Therefore, this project was designed to compare the orbital anatomy and EOM morphology of the naked molerat and the C57BL mouse.
This study was approved by the Institutional Animal
Care and Use Committees at the University of Kentucky
and Vanderbilt University. We used six naked mole-rats
and 13 C57BL mice for histology and immunocytochemistry. Upon arrival, the mice were kept in microisolator
cages with Harlan Teklad rodent food and water provided ad libitum. Naked mole-rats came from a colony
maintained at Vanderbilt University. Naked mole-rats
were kept in a temperature -controlled room housed in
chambers connected with plastic tubing.
Before the collection of tissues, C57BL mice and
naked-mole rats mice were anesthetized with ketamine
hydrochloride/xylazine hydrochloride (100 mg/8 mg per
kg body weight injected i.p.) exsanguinated and perfused
with physiological saline, followed by 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer,
pH 7.4. Whole orbits were dissected and embedded in
paraffin, 10 lm thick sections were stained with hematoxylin and eosin (H&E) to examine morphology. Sections were imaged with a Nikon E600 microscope
equipped with a Spot RT Slider camera and Spot RT
software (v 4.0). Fiber size was measured using Image J
software from NIH ( Quantitative analyses were done by personnel blinded to the experimental conditions.
Electron Microscopy
Mice and naked mole-rats were perfusion-fixed as
described earlier. Individual EOMs were dissected and
postfixed in 1% osmium tetroxide, stained en bloc in uranyl acetate, dehydrated in a methanol series and propylene oxide, and embedded in epoxy resin. Thin (70 nm)
sections were examined and photographed with a Philips
Tecnai 12 transmission electron microscope (UK Imaging
Core). Mitochondria volume density (% of muscle fiber
volume occupied by mitochondria) was determined from
104 extraocular muscle fibers (sampled from both global
and orbital layers) from digital pictures obtained from
six naked mole-rats and 43 extraocular fibers obtained
from 13 C57BL mice using a standard point-counting
method (144-point grid) with systematic sampling
(Weibel, 1979).
Fig. 2. Naked mole-rat fiber size is smaller. Comparison of mouse
(A) and naked mole rat (B) EOM fiber size (H&E stain) (scale bars ¼
50 lm). (C) Mean fiber size in EOMs from mouse and naked mole rat.
*, significant at P < 0.05.
Data Analysis
All results are presented as the mean SE of n observations. Mitochondrial volume density and fiber area
were compared with Student’s t-tests. The significance
level for rejection of the null hypothesis was set at P 0.05 for all comparisons.
The orbits of naked mole-rats were analyzed for overall anatomical features. The basic pattern of six EOMs;
superior, lateral, medial, and inferior rectus muscles;
superior and inferior oblique muscles (Fig. 1a) are
shared among all vertebrate classes (Porter et al., 2003).
There are, of course a few difference, for example, in
Fig. 3. EOMs of naked mole-rats are not as compact as mouse EOMs. Representative electron micrographs of EOM fibers from mouse (left) and naked mole rat (right). Ultrastructure profiles of mitochondria
(m), sarcoplasmic reticulum (sr), myofibrillar organization in the A-band (A) and arrows denote large
spaces. Scale bars ¼ 2 mm for top and middle rows, and 500 nm for bottom row.
some rainbow trout, the lateral rectus is oriented vertically instead of horizontally as in other species (Noden
and Fraancecis-West, 2006). Also, in marlins, swordfish
and billfish, the superior rectus is exaggerated and is
used as a heat-generator to warm the brain and eyes
above low water temperatures (Fritsches et al., 2005).
Naked mole-rats have the same number of EOMs in the
same basic arrangement around the optic nerve and
globe, such as the superior oblique, retractor bulbi, and
rectus muscles as the mouse (Fig. 1a–d). The eye is protected by an oily substance from the Harderian glands
that coats the cornea and prevents dryness (Buzzell,
Fig. 4. EOMs of naked mole-rats contain m-lines. Electron micrograph illustrating arrangement of bands
in sarcomere in NMR (A) and mouse (B). Ultrastructure profiles of the M-lines, z-lines (z), mitochondria (m),
triads (t), and arrows denote large spaces in naked mole-rats. Scale ¼ 500 nm for both images.
1996). The Harderian gland from the naked mole-rat is
noticeably smaller compared to mouse (Fig. 1e,f).
In most mammals, EOMs exhibit a distinctive layered
organization known as the orbital and global layers.
These two layers are characterized by their fiber content: the orbital layer consists of smaller fibers and is
typically c-shaped (Fig. 1b). These two layers maintain
rotational stability of the eyes (Demer et al., 2000). The
EOMs from naked mole-rats do not have the two-layer
distribution of fibers (orbital and global layers) typically
seen in mice, cats, dogs, and primates (Fig. 1e).
Muscle Fiber Comparisons
EOMs in other mammals have smaller fibers with
greater mitochondrial content and unique metabolic and
fiber type compositions which are distinct from typical
skeletal muscles (Porter et al., 2001, Cheng et al., 2004,
Cheng and Porter 2002). The EOMs from naked molerats were noticeably smaller compared to mouse (Fig.
2a,b). EOM fiber size was significantly smaller in naked
mole-rat than mouse (112.3 46.2 vs. 550.7 226 lm2,
respectively) (Fig. 2c). A characteristic feature of EOMs
is a small myofibril size compared to other skeletal
muscles. Myofibril density was greatly reduced in the
EOMs from naked mole rats, leading to more space
between myofibrils (Fig. 3,4a).
Sarcomeres produce the typical longitudinal binding
pattern of striated muscles. They are formed by an ordered arrangement of thick and thin filaments. M-lines
contain proteins that interconnect and stabilize adjacent
myosin filaments. These structures, especially prominent
in fast skeletal muscles, are missing in mouse extraocular muscle (Fig. 4b) (Andrade et al., 2003). They are,
however, present in naked mole-rat EOMs (Fig. 4a).
Triad and other membranous structures were rudimentary in naked mole- rat EOMs (Fig. 4a).
Fig. 5. Naked mole-rat EOMs contain less mitochondria. Mitochondrial volume density (% of fiber volume occupied by mitochondria) in
EOMs from mouse and naked mole rat. *, significant at P < 0.05.
The EOMs are reported to have one of the highest mitochondrial contents of mammalian skeletal muscles
(Mayr, 1971). This high mitochondrial content has been
considered to reflect the metabolic demands imposed by
their fast and constant activity. The mitochondrial volume density of naked mole rat EOMs was significantly
less than in mouse (4.5 1.9 vs. 21.2 11.6% of total
fiber volume) (Fig. 5). Mitochondria were also more pleomorphic in the EOM fibers from naked mole rat (Fig. 3,
4a). Naked mole rat mitochondria are more variable in
shape than those of the mouse.
Our findings demonstrate underdeveloped EOMs in
the naked mole rat. Although the naked mole-rat EOMs
retain a somewhat typical organization, EOMs are
remarkably smaller in size and typical sarcomere
arrangement is less well defined. Contrary to adult
mouse EOMS; m-lines are present in the EOMs of naked
mole-rats. M-lines are present in mouse EOMs during
myogenesis, but they disappear soon after birth (Porter
et al., 2003). It is then possible that NMR EOMs may
persist in a state of incomplete development; also m-line
repression may be a characteristic of visually-guided
rodents. The results are consistent with these muscles
being less active and weaker in these nonvisually guided
rodents. It has previously been shown that normal development of the mouse and monkey ocular motor system
and its muscles requires visual experience during the
critical period (McMullen et al., 2004; Cheng et al.,
2004). For example, we have shown that abnormal visual experience postbirth renders mouse EOMs weaker
and more fatigable (McMullen et al., 2004). While this
paradigm is clearly not applicable to NMR, it does serve
to illustrate the connection between the visual pathways
and the motor systems serving the eyes. In the case of
the NMR, vision is basically replaced by somatosensory
inputs, and the visual pathways are correspondingly
diminished compared to visually-guided mammals (Mills
and Catania, 2004; Nikitina, et al., 2004; Hetling et al,
2005). With this in mind, it is not surprising that NMR
EOMs appear underdeveloped.
Mitochondrial content in muscle is dynamic and
reflects the functional demands of the fiber type (Lyons
et al., 2006). Mitochondria content is also a principal determinant of aerobic capacity. Naked mole-rats show
reduced mitochondrial volume density compared to mice.
Reduced mitochondrial density is indicative of a reduction of energy demand in these muscles. (Moyes, 2003).
As mitochondria are the main generators of ATP, it is
possible that this reduction of mitochondria represents a
conservation of energy from the eyes, which seem to be
used only to detect light or to regulate circadian
rhythms (Hetling et al., 2005; Nikitina et al., 2004), so
that other systems receive this metabolic gain. The
reduced mitochondrial volume density also suggests that
naked mole-rat EOMs rely on anaerobic (isolated distribution of small mitochondria) metabolism. Mitochondria
may also be important in regulating [Ca2þ]i kinetics during the activation of extraocular muscle fibers, influencing force production and increasing the dynamic
response range for this muscle group (Andrade et al.,
2005). Reduced mitochondrial content suggests the naked mole-rats move their eyes less than mice; which is
consistent with small fiber size.
These findings correlate with previously shown findings of an overall less well-developed central visual system; a reduced lateral geniculate nucleus, superior
colliculus and visual cortex (Crish et al., 2006; Catania
and Remple, 2002). The lack of architectural specialization and small size of EOMs in naked mole-rats suggests that the development of their ocular motor
system parallels the visual system. The naked mole-rat
provides a novel model to study the coordinated evolution and development of visual and ocular motor
The authors wish to thank Denise Hatala and Gayle
Joseph for technical assistance. This work was sup-
ported by National Health grant EY12998 (to F.H.
Andrade FH, Merriam AP, Guo W, Cheng G, McMullen CA, Hayess
K, van der ven PF, Porter JD. 2003. Paradoxical absence of M
lines and downregulation of creatine kinase in mouse extraocular
muscle. J Appl Physiol 95:692–699.
Andrade FH, McMullen CA, Rumbaut RE. 2005. Mitochondria are
fast Ca2þ sinks in rat extraocular muscles: a novel regulatory
influence on contractile function and metabolism. Invest Ophthalmol Vis Sci 46:4541–4547.
Berardi N, Pizzorusso T, Maffei L. 2000. Critical periods during sensory development. Curr Opin Neurobiol 10:138–145.
Buzzell GR. 1996. The Harderian gland: perspectives. Microsc Res
Tech 34:2–5.
Catania KC, Remple MS. 2002. Somatosensory cortex dominated by
the representation of teeth in the naked mole-rat brain. Proc Natl
Acad Sci 99:5692–5697.
Cheng G, Merriam AP, Gong B, Leahy P, Khanna S, Porter JD.
2004. Conserved and muscle group-specific gene expression patterns shape postnatal development of the novel extraocular muscle phenotype. Physiol Genomics 18:184–195.
Cheng G, Porter JD. 2002. Transcriptional profile of rat extraocular
muscle by serial analysis of gene expression. Invest Ophthalmol
Vis Sci 43:1048–1058.
Crish SD, Dengler-Crish CM, Catania KC. 2006. Central visual system of the naked mole-rat (Heterocephalus glaber). Anat Rec A
Discov Mol Cell Evol Biol 288:205–212.
Demer JL, Oh SY, Poukens V. 2000. Evidence for active control of
rectus extraocular muscle pulleys. Invest Ophthalmol Vis Sci
Fritsches KA, Brill RW, Warrant EJ. 2005. Warm eyes provide superior vision in swordfishes. Curr Biol 15:55–58.
Hetling JR, Baig-Silva MS, Comer CM, Pardue MT, Samaan DY,
Qtaishat NM, Pepperberg DR, Park TJ. 2005. Features of visual
function in the naked mole-rat. J Comp Physiol 191:317.
Lyons CN, Mathieu-Costello O, Moyes CD. 2006. Regulation of skeletal muscle mitochondrial content during aging. J Gerontol A Biol
Sci Med Sci 61:3–13.
McMullen CA, Andrade FH, Stahl JS. 2004. Functional and
genomic changes in the mouse ocular motor system in response to
light deprivation from birth. J Neurosci 24:161.
Mayr R. 1971. Structure and distribution of fibre types in the external eye muscles of the rat. Tissue Cell 3:433–462.
Mills SL, Catania KC. 2004. Identification of retinal neurons in a
regressive rodent eye (the naked mole-rat). Vis Neurosci 21; 107.
Moyes CD. 2003. Controlling muscle mitochondria content. J Exp
Biol 206:4385–4392.
Nikitina NV, Maughan-Brown B, O’Rian MJ, Kidson SH. 2004.
Postnatal development of the eye in the naked mole rat (Heterocephalus glaber). Anat Rec 227A:317.
Noden DM, Francis-West P. 2006. The differentiation and
morphogenesis of craniofacial muscles. Dev Dyn 235:1194–
Porter JD, Andrade FH, Baker RS. 2003. The extraocular muscles.
In: Kaufman PL, Alm A, Editors. Adler’s physiology of the eye
clinical application. St. Louis, Misssouri: Mosby Inc. p 787–817.
Porter JD, Baker RS, Ragusa RJ, Brueckner JK. 1995. Extraocular
muscles: basic and clinical aspects of structure and function. Surv
Ophthalmol 39:451–484.
Porter JD, Khanna S, Kaminski HJ, Rao JS, Merriam AP, Richmonds CR, Leahy P, Li J, Andrade FH. 2001a. Extraocular muscle is defined by a fundamentally distinct gene expression profile.
Proc Natl Acad Sci USA 98:12062–12067.
Porter JD, Merriam AP, Gong B, Kasturi S, Zhou X, Hauser KF,
Andrade, FH, Cheng G. 2003. Postnatal suppression of myomesin,
muscle creatine kinase and the M-line in rat extraocular muscle.
J Exp Biol 206:3101–3112.
Weibel ER. 1979. Stereological methods. London: Academic Press.
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underdeveloped, muscle, molek, heterocephalus, naked, rat, glaber, extraocular
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