Introduction
Tendinopathy of the superficial digital flexor
tendon (SDFT) is a
common, debilitating and expensive disorder of equine athletes.
It is characterised by gradual degeneration (tendinosis) of the
central portion of the mid-metacarpal region of the tendon. The
processes that underlie tendinosis are largely undescribed, but
it is surmised that tendinosis results from physical fatigue
causing the accumulation of microtrauma within extracellular
matrix components. Tendinosis represents a localised, progressive
weakening of the tendon, which predisposes the tendon to partial
or complete rupture. Partial tendon rupture and its inflammatory
sequelae are referred to as ‘bowed tendon’, or
tendinitis.
Bowed tendon is usually sustained during intense activity such as
during training or racing. The condition is so named because the
normal straight lateral profile of the tendon is replaced by a
bulge in the mid-metacarpal region. Bowed tendon is a serious
injury that prohibits training and race activity for several
months. No treatment is known to significantly enhance the
healing of a bowed tendon, and once bowed it is likely to be re-bowed
on resumption of athletic activity.
It is generally assumed that tendinosis represents a failure of
tenocytes to repair the microtrauma caused by repetitive strain
on tendon. However, the cellular strategies of tenocytes to
repair microtrauma are not understood. Current investigations
into the tenocyte’s role in tendinosis include studies of
cell type, gene expression and cytokine action. Recent
unpublished work from this institute has confirmed a bimodal
distribution of tenocyte nuclear lengths, described qualitatively
by Holmes in 1971. This information suggests the presence of two
types of tenocyte, which may represent two distinct activity
states. The relative proportions of these cells may provide an
indicator for the metabolic state of tendon.
Electron microscope studies have illustrated an association
between nuclear morphology and synthetic organelles. Those cells
with short nuclei were described as having abundant rough
endoplasmic reticulum (RER), while cells with long nuclei had
less RER.
Workers first identified alpha-smooth muscle actin (aSMA) in
normal tenocytes in the 1970’s. No attempt was made to
quantify the relative proportions of aSMA-positive and aSMA-negative
tenocytes, nor has that proportion been related to tenocyte
nuclear morphology. Qualitatative statements include
" most, if not all tenocytes from foal
DDFT stain positively for aSMA"
Hartzel et al. 2001
Thus the relationship of aSMA to tenocyte
typing by nuclear morphology is unknown.
The function of aSMA in tendon is unknown. aSMA is generally
associated with cell processes that involve contractility, for
example wound closure, cell migration and ECM remodelling. aSMA
is associated with skin fibroblasts in wound environments; it has
also been shown to be prevalent in the acute tendon wound
environment. No attempt has been made to visualise aSMA in
tendinotic lesions, therefore the potential of aSMA to play a
part in tendinosis is unknown.
Hypotheses
Tenocytes from normal equine SDFT taken from
the mid-metacarpal segment stain positively for aSMA
Images can be generated using confocal microscopy and 3D
visualisation software that reveal the 3D structure of tenocyte
nuceli and alpha-SMA
Aims
The present study is involved in investigating
the 3D
intracellular distribution of aSMA in normal tenocytes. The
confocal microscope generates very thin (<0.2 micron) optical
sections that can be taken serially and reconstructed with
computer software to visualise 3D structures. This technique in
concert with fluorescent immunohistochemistry will be used to
visualise aSMA within the tenocyte cytoplasm in 3D. In addition,
a nucleic acid stain (propidium iodide, PI) will be used to label
tenocyte nuclei. Two-channel detection will be used to colocalise
aSMA with tenocyte nuclei.
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