Fixation of Rat Tail Causes Muscle Fiber Type Transition in Paraspinal Muscles
Koshi N. Kishimoto, Hiroshi Okuno, Masahiko Tanaka, Masayuki Kuwahara, Eiji Itoi.
Tohoku University, Sendai, Japan.
Disclosures:
K.N. Kishimoto: None. H. Okuno: None. M. Tanaka: None. M. Kuwahara: None. E. Itoi: None.
Introduction: Vertebral fracture and disc degeneration lead to local kyphosis and loss of flexibility in the spine. Accumulation of
such segmental degeneration develops deformity in posture. Degenerative postural deformities often accompany back pain,
easy fatigability, fatty degeneration and atrophy of back muscles[1]. Reduction in back muscle strength, furthermore,
deteriorates postural deformities.
There are two types of skeletal muscle fibers according to oxidative activities: slow-twich (Type 1) and fast-twitch (Type 2) fibers.
Type 2 fibers were subdivided into three types: Type 2A, 2B and 2D/X. Each fiber type primarily expresses a specific isoform of
myosin heavy chain (MHC). It has been reported that back muscles contain higher proportion of MHC type 1[2]. The impact of
kyphosis on the proportion of fiber types in the back muscles, however, has not been fully understood.
We have developed a model for kyphotic spine using a rat tail. A rodent tail has vertebrae segmented by intervertebral discs.
Pairs of dorsal coccygeal muscles exist on the both side of spinal process spanning tail vertebrae longitudinally. The anatomical
characteristics of these muscles are similar to the multifidus muscles in human paravertebral muscles. In this model, rat tail was
fixed in straight or kyophosis position by custom made external fixator.
The aim of this study is to analyze the transition of muscle fiber types after kyophotic or straight fixation using a rat tail model.
The muscle fiber diameter of the dorsal coccygeal muscles was assessed histologically. The gene expression profiles of each MHC
fiber type were analyzed by quantitative RT-PCR. Extracted MHC proteins were subjected to fiber type analyses by SDS-PAGE.
Methods: The experimental procedure of this study was approved by the Committee of Animal Experiment in the author’s
institution.
Rat tail fixation
Male rats at 12-week old were used in this study. The rats were randomly divided into three groups; sham, straight, and
kyphosis group. Four 1-mm-diameter Kirschner wires were inserted horizontally through the rat tail. Proximal two wires were
inserted 5 mm apart in the most proximal part of the tail. A distal wire was inserted at 5 cm distal to the most proximal wire in
the straight group and 7.5 cm distal in the kyphosis group. Another distal wire was inserted 5 mm distal to the first distal wire.
Both ends of Kirschner wires were fixed by custom-designed stainless plates (Fig.1). The plates were sandwiched and bolted
together with rubber inserts. The sham group which served as control received only pierced wound in the tail by the Kirschner
wire. In the straight group, the wires were fixed in the original distance to maintain the tail in the straight position. In the
kyphosis group, the interval between the most proximal and the most distal wires were fixed with a distance of 5 cm to maintain
the tail in kyphotic position. The rats were sacrificed at 7 and 28 days after the tail fixation for molecular analysis (n=6), and at 28
days after the tail fixation for histological analysis (n=5).
Fiber diameter measurement
Dorsal coccygeal muscles were harvested under dissection microscope. The axial sections were stained with hematoxylin and
eosin. Images under microscope were captured by digital camera (Olympus). Diameter of muscle fiber was measured using
ImageJ software (NIH: v1.48). Minimum Feret’s diameter of muscle fiber was considered as fiber diameter.
Quantitative RT-PCR
Total RNA was extracted from the harvested dorsal coccygeal muscles by Trizol reagent (Invitrogen) and purified using RNeasy
Mini Kit (QIAGEN). First strand cDNA was synthesized using High capacity cDNA archive kit (Applied Byosystems). Quantitative
RT-PCR reactions were performed using ABI StepOne plus and Power SYBR Green PCR MasterMix reagent (Applied Byosystems)
routinely in duplicate. The primers for each types of myosin heavy chain were synthesized based on the data previously
published elsewhere[3]. Sample values were normalized to the threshold value for beta-actin. The values of sham group were
used as reference. The fold change in mRNA expression was calculated by ∆∆CT method.
Fiber type analysis
MHC isoforms were defined by SDS-PAGE as previously described. Dissected muscles were homogenized in 40 times volume of
extracting buffer containing 5M urea, 2M thiourea, 10mM sodium pyrophosphate and 0.1% 2-mercaptoethanol. Muscle extract
was diluted 50 times in sample buffer was loaded on the gel containing 7.5% Acrylamide and 30% glycerol. Gels were run at a
constant voltage of 150 V for 48 hours at 4°C. Same amount of extracted sample was loaded into each well and gels were silver
stained. MHC bands were quantified using ImageJ software. The overall order of migration by MHC type was known as
1>2b>2x>2a.
Statistics
Differences between control and experimental groups were examined for statistical significance using one-way ANOVA with
Dunnett’s multiple comparison test. The level of statistical significance was set at p < 0.05.
Results: Gene expression profiles of MHCs and protein expression in the dorsal coccygeal muscles were compared with the tibilis
anterior and the gastrocunemius muscles. Higher proportion of MHC type 1 gene and protein expression were confirmed in the
dorsal coccygeal muscles than tibialis anterior and gastrocuneimus muscles. MHC type 2B protein expression was not detected
in dorsal coccygeal muscles (Fig 2).
Histology and histogram of muscle fiber diameter in each group at 28 days were shown Fig 3. Diameter of muscle fiber exhibited
shifted histogram peak toward larger diameter in straight and kyphotic groups (Fig 3).
Muscle fiber types in the coccygeal muscles after straight or kyphotic fixation were analyzed and compared with control. Gene
expression of MHC type 1 was decreased at 7 and 28 days after fixation in straight and kyphosis group. The significant difference
was seen at 28 days in kyphosis group (Fig 3A). The band densities of MHC protein type 1 and 2A plus 2D/X were decreased in
both straight and kyophosis groups at 28 days after fixation while sample volume was adjusted by wet wight of dissected
coccygeal muscles. The mean proportion of MHC protein type 1 separated by SDS-PAGE were decreased in straight and kyphosis
group. The difference was significant in straight group (Fig 3B).
Discussion: Our results demonstrated that the fixation of the rat tail induced transition of muscle fiber types in the coccygeal
muscles characterized by the decrease in the proportion of the MHC type 1. Back muscles are required to contract continuously
to keep posture. Slow-twitch fibers in back muscle contribute for continuous contraction. Slow-twitch fibers utilize energy
efficiently by oxidative process while fast-twitch fibers mainly consume glucose through glycolysis producing lactate acid. Not
only decreased amount of MHC but also decreased proportion of MHC type 1 might be the reason of easy fatigability in patients
with degenerative postural deformity. The limitations of this study are the difference between human paravertebral and rat
coccygeal muscles and short duration of observation.
Significance: Paraspinal muscles contain higher proportion of slow-twich fibers. The fixation of the rat tail induced transition of
muscle fiber types in the paravertebral muscles characterized by the decrease in the proportion of the slow type myosin heavy
chain. This result may explain easy fatigue in the patients with degenerative postural deformity.
Acknowledgments: The authors thank to Ms. Michiko Fukuyama for her technical assistance.
References: [1] Clin Radiol 62(5):479-486, 2007. [2] Spine 35(13):1265-1270, 2010. [3] J Commun Disord 43(4):327-334,
2010.
ORS 2014 Annual Meeting
Poster No: 1399