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UDC:617.559-007.271:591.4-57.081.4
Bengus L.M., Dedukh N.V., Chernyshov A.G., Levshin A.A., Fedotova I.F.
MORPHOLOGICAL ASPECTS OF EXPERIMENTAL LUMBAR SPINAL STENOSIS
MODELING
Sytenko Institute of Spine and Joint Pathology National Ukrainian Academy of Medical
Sciences, Laboratory of connective tissue morphology, chief – professor N.V. Dedukh
80 Pushkinskaya St., Kharkiv 61024, Ukraine
e-mail – L.Bengus@i.ua
This work was completed within the planned research work “Вивчити механізми
розвитку дегенеративного стенозу поперекового відділу хребетного каналу” /2010-2012/
code ЦФ.2010.1.АМНУ. № state registration 01.10.U.0020.88
Introduction
Lumbar spinal stenosis is classified as vertebral diseases in which a reduction of the
spinal canal volume results in compression of neural elements [1]. According to NASS for about
20% of the adult population suffers from this disease [2].
Degenerative lumbar spinal stenosis, being late and severe consequence of degenerative
changes in the spinal motion segment, is the most common form of stenosis. The frequency of
degenerative stenosis increases with the age [3, 4]. Degenerative stenosis may be associated with
ossification of a herniated disc (hard disc herniatio), hypertrophy and ossification of the
ligamentum longitudinale posterius and ligamentum flava, instability, hypertrophic changes and
dislocation of facet joints, the presence of osteophytes, vertebrae antelisthesis, retrolisthesis and
laterolisthesis, systemic diseases, etc [5]. Degenerative stenosis most commonly affects the L3L4, L4-L5 and L5-S1 vertebral motion segments. [6].
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In the initial stages the disease may be asymptomatic one. In the sequel it is possible to be
attended by neurological disorders and leads to significant disability, inability to self-care and as
a consequence – to reduce of life patient quality. Neurological symptoms in spinal stenosis is
caused by compression intracanal formations, but it is pathogenetically linked with many other
factors: the disturbance of the nerve roots trophy due to discirculatory influences, primarily
venous stasis, endo-perineural inflammation, etc. Clinical symptoms of lumbar stenosis is more
pronounced in the upright position by reducing the spinal canal volume due to protrusion. In the
position of flexion the diameter of the spinal canal increases, and the clinical symptoms of
stenosis decreases by reducing the compression of intracanal formations [2].
Given the currently available the phenomenon of "rejuvenation" of degenerative diseases
of the spine and, therefore, reducing the age of the clinical spinal canal stenosis symptom
appearance the profit in-depth study of its pathogenesis becomes clear. In connection with it
experimental models of this state made on animals is of interest, allowing to follow the stages of
spinal stenosis formation, including morphological in dynamics.
The purpose of the investigation is to study morphological features of the spinal canal
and nerve tissue structure in white rats in experimental model of lumbar spinal stenosis.
Materials and methods
Experimental modeling of lumbar spinal stenosis was performed on 15 adult white male
rats. For this purpose, an arc L5 was ripped to the left and right of the spinous processes. Then
the middle part of the arc (with spinous process) was separated from other parts of the arc and
the articular processes. The separated part of the arc with attached ligamentum flava were moved
in the ventral direction of the spinal canal cavity. Contact bone edges were fixed with bone
cement.
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Radiographic studies performed 1 week, 1 month and 3 months later after surgery
showed that the location and degree of stenosis in the animals remained unchanged. One week, 1
month and 3 months later after modeling stenosis animals were killed by an overdose of ether
guiding by international standards on bioethics [7, 8].
For histological study fragments of the lumbar spine of 15 white rats were used.
Fragments of the spine were taken at the level of L4-L6 segments. The material was fixed in
10% neutral formalin. After dehydration in a series of alcohols of increasing concentration the
material was enclosed to celloidin. [9]. Histological sections being 5-7 microns thick, were
stained with hematoxylin and eosin.
Microscopic examination of the material was carried out using a light microscope
«Primostar». Photoprints of histologic specimens were made with a digital camera Canon EOS300D.
Results and its discussion
Modeling stenosis 7 days later
Morphological analysis of bone tissue that forms of the spinal cord canal of rats with
experimental stenosis, showed that it had expressed disturbances of structural organization.
Modeling stenosis 7 days later in the bone at a considerable distance karyopycnosis osteocytes
and cells-shade were presented. Numerous osteocyte lacunes contained the remains of osteocytes
like cellular debris, frequent empty lacunae without osteocytes occurred. A significant part of
osteocytes was located in dilated resorption lacunes, that indicates the activation of periosteocyte
osteolysis.
In the bone matrix of spinal canal walls there were various intensity destructive changes.
Focal basophilia matrix, bundle bundles and unmasking the collagen fibers, the formation of
large-size and variable localization destructive cracks were determined.
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In the spinal canal the terminal filament surrounded by nerve roots of cauda equina and
spinal cord membranes at the L5 vertebra were microscopically revealed. In composition of the
terminal filament the interior part, which has a small of nerve tissue, and outside part, which
does not contain neural tissue and is the extension of the spinal tunics were distinguished. In the
terminal filament the signs of nerve fiber and peri-endoneural edema destruction were marked.
There were degenerative changes of neural structures of cauda equina manifested by hyperemia
of peri-and endoneural vessels (Fig. 1). In the part of the nerve roots of cauda equina destruction
and vacuolization were marked. In the epidural space of spinal canal foci of hemorrhage were
revealed.
As it is known, one of the main causes of the symptoms of lumbar spinal stenosis is
ischemia causing circulatory disorders of the spinal motion segment and, accordingly, its neural
elements [10]. Observable phenomena of blood vessel hyperaemia in the peri-and endoneurium
of the nerve roots, as well as the foci of hemorrhage in the epidural space testified of deep
damages in the neural elements blood supply that occur in this experimental model.
Modeling stenosis 1 month later
After modeling stenosis 1 month later in nerve roots of cauda equina expressed leukocyte
infiltration was defined. On the background of hyperaemia and vascular endoneural stasis the
progression of the nerve root destruction, accompanied by their pronounced vacuolization was
marked (Fig. 2). In the spinal canal near the destructive changed nerve roots bone sequesters
were present. In the epidural space over a large area there were extensive hemorrhages (Figure 3)
and connective tissue areas. In the fibrous tissue which occupied large areas of the epidural space
perivascular infiltrates and empty blood vessels were identified.
As we know, chronic compression of the nerve roots, which occurs in spinal stenosis,
leading to edema and fibrosis of these structures, causes an increase in their irritability.
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Pathologic studies showed the presence of blood stasis in the vessels of the stenotic spinal canal
and hypoxia, due to the influence of mechanical factors, and led to an increase in functional
disorders of the neural structures [2]. Marked in this study angiogenic disorders increase
(hyperemia and stasis of peri-endoneural nerve roots vessels, extensive hemorrhage in the
epidural space) indicated the progression of ischemia in the area of experimental stenosis
reproduction. The presence of empty blood vessels in the epidural fibrous tissue indicated the
appearance foci without blood supply.
Modeling stenosis 3 months later
At the period of the study in the bone tissue forming the wall of the spinal canal, the
number and size of destructive cracks increased. On the surface of a hard tunic of the spinal cord
canal single local deposition of new bone tissue with a high density of young osteocytes were
observed, it indicates about during the regenerative processes in that region. However, these
weak attempts to the regeneration could not compensate apparent disturbance of the structural
bone tissue organization taking place on the background of this experimental model.
On the surface of the annulus fibrosus of the intervertebral disc and bone tissue of the
vertebral bodies, forming the ventral surface of the spinal canal, new forming connective tissue
foci with a high density of large fibroblasts were identified. In areas this tissue bordered with
been present there spinal canal bone sequesters.
In the cauda equina nerve roots remained destructive changes, marked at 1 month followup. Besides destruction in the spinal canal fragmentation of the nerve roots and bone sequesters
was observed. Fibrotization of individual nerve roots was revealed on the background, that
indicated the presence of chronic inflammation in the spinal canal. The destruction areas of the
nerve roots were filled with fibrous tissue massifs.
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Epidural fibrosis progressed in the spinal cord canal (Fig. 4). Fibrous tissue contained
powerful multidirectional bundles of collagen fibers. Its cellular composition was presented by
fibroblasts and flattened elongated fibrocytes, which were the final stage of fibroblast
differentiation. Massifs of epidural and perineural fibrosis interspersed with hyalinosis areas. In
perineural area massive diapedetic hemorrhages were observed, indicating that ischemic
persisted for some time. Fibrous tissue areas in the epidural space at considerable length had
signs of edema and contained foci of destruction.
Fig. 1. Cauda equina nerve roots. Hyperemia
Fig. 2. Marked vacuolization and destruction of
perineural vessels. Hematoxylin and eosin.
the nerve roots. Hematoxylin and eosin.
Original magnification × 40.
Original magnification × 400.
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Fig. 3. Extensive hemorrhage in the epidural
Fig. 4. Expressed epidural fibrosis spinal canal.
space. Epidural fibrosis. Destruction and
Hematoxylin and eosin.
vacuolization of the nerve roots. Hematoxylin
Original magnification × 100.
and eosin. Original magnification × 100.
Conclusions:
1.
Analysis of the results of morphological study showed that in experimental model the
rats had significant pathologic changes of bone tissue during lumbar spinal stenosis.
There were destruction areas of cells and extracellular matrix for a considerable
distance. There were cells-shade, osteocyte lacunes containing cellular debris and
empty lacunes without osteocytes. A considerable part of osteocyte lacunes was
extended, indicating the activation of periosteocyte osteolysis. Destructive cracks
were presented in the bone matrix, the number and size of which increased with time.
3 months later after the experimental modeling the lumbar spinal stenosis in the bone
tissue forming its walls cracks and large gaps were determined. Weak manifestation of
regenerative processes could not compensate for a destructive changes taking place in
the background of this experimental model.
2.
In the cauda equina nerve roots and terminal filament the leukocyte infiltration,
hyperemia and stasis of the endo- and perineural vessels, vacuolization and destruction
were defined. Over the time, the progression of destruction, the nerve roots
fragmentation as well as their local fibrotization were noted.
3.
In the spinal canal at the level of experimental reproduction of spinal stenosis there
were presented bone sequesters, in the epidural space revealed extensive hemorrhage,
empty blood vessels, the defining features of a chronic inflammation, which
contributed to the progression of epidural fibrosis and hyalinosis.