Download Morphoenzymological aspects of red nucleus cerebral structures

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UDC 591.881:611.815
V.P. Belotserkovsky, E.N. Belotserkovskaya, N.K. Kashrina
Morphoenzymological aspects of red nucleus cerebral structures under the sympathetic denervation
of brain blood vessels
SI "Crimea State Medical University after S.I.Georgivsky ", Simferopol, Ukraine
Key words: nerve system morphology, sympathetic denervation of brain blood vessels, enzymes.
Summary.
Brain blood vessels adrenergic denervation provide morphological and enzyme changes of
neurons cytoplasm and nucleus structures. Among neurons appear different dystrophic changes,
graduate lypofuszin cytoplasm imbibitions, adipose dystrophy, necrosis and also active neuralgia
proliferation in late stage. With TNST designate activity of Kreb’s cycle enzymes, glucose-6phosphate dehydrogenize, lactate dehydrogenize, NAD- and NADPH-diaphoreses in cytoplasm of
red nucleus neurons.
Cardiovascular diseases are the leading place in the overall morbidity, mortality and
disability among other diseases of the population, especially pronounced in developed countries. In
current clinical practice, ganglioblockers and sympatholytics use as a subsidiary, an additional
treatment of arterial hypertension and its complications due to prevent releasing of catecholamines
in adrenergic nerve terminals, including the brain. To date, adequately substantiated information
both on the distribution of the sympathetic innervation in the vessels of the brain, and the question
of the functional capacity of adrenergic fibers by their synaptic endings involved in the change of
cerebral blood flow (1, 2, 3). In particular this applies to cerebral vascular adrenergic fibers
originating from the superior cervical sympathetic ganglion. Undisputed is the fact that any change
in blood flow is of great importance to the nervous tissue of the brain, which has an extremely high
metabolic rate. The purpose of this study was to investigate the influence of partial sympathetic
denervation of brain vessels on the morphological characteristics and intracellular enzimology red
nucleus, as one of the most important structures that integrate the various links of the
extrapyramidal nervous system and has extensive connections with the brain cortex, brain stem
nuclei, spinal cord motor neurons.
Materials and methods. Partial sympathetic denervation of cerebral vessels was achieved by
extirpation of the right superior cervical sympathetic ganglion, sympathetic branches along the
carotid artery under hexenal anesthesia. Investigations were carried out on cats, given the similarity
to the human brain on the structural features of the vascular system and its sympathetic innervation,
the similarity in the structure of the brain stem nuclei. Experiments were carried out in the absence
of a ban on the use of these animals; observe the principle of humane treatment of experimental
animals. From animal experiments 1-series (32) is displayed under hexenal (or using thiopental)
anesthesia by perfusion through the ascending aorta after previous heparinization in time - on the
7th, 15th, 30th day after surgery. Brainstem implemented in Carnoy's fluid, embedded in paraffin
blocks, produced frontal serial sections of the midbrain (10 mkm). Sections were stained by
Einarsson, Nissl, Brachet, sudan black B. Animal of second serie for histoenzymology (24) were
taken out of the experiment by decapitation under hexenal anesthesia, in a cryostat (at -25 ° C)
immediately produced serial sections of standard thickness 10 mkm (I.M. Buykis, 1971). Sections
were incubated in a protective environment at +37 ° C in an oven and identified NAD and NADPdiaphorase method Scarpelli D.J. modified by Reichlin N.T., succinate dehydrogenase (SDH) method Nachlas M.M., lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6FDG), isocitrate dehydrogenase (ITSDG) - the method of A.Rubinstein using quantitative
tetrazolium (TNST, Amersham). The enzymes activity was determined by cytospectrophotometry.
Tackling drugs to probe scanning cytophotometry (probe area S = 0,996 mkm2) conducted
interference method. Among the red nucleus neurons calculates the damage, the extent and severity
of damage. Differentiation of neuronal degenerative changes done with the advice of the
Department of the Institute of Brain morphological AMN Russia. Quantitative data were processed
using the method of variation statistics.
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Results and discussion. In the red nucleus of control animals revealed three types of neurons: large
(50-90 mm diameter soma), medium (20-50 mm) and small (up to 20 microns). Large neurons are
mainly in the caudal and middle third of the nucleus, perikaryin contains abundant and intensely
staining Nissl bodies, centrally located nucleus, and, as a rule, a large nucleolus. Medium-sized
neurons are spread fairly evenly over the stretch of the nucleus, the morphology similar to major. A
small neurons dominate in the ventral and ventrolateral parts of the kernel, are rare in the caudal
part. They are more rounded, contain small Nissl bodies, moderate ectopic nucleolus.
The highest level of neuron dystrophic changes (82%) appeared on the 7th day after the
operation. There were dominated by reversible changes in the nerve cells. Most neurons (31,87%)
were observed swelling and rounding of the soma and nuclei of cells, ectopic nuclei, reducing the
size of nucleoli, enlightenment karyoplasm and mainly perikaryon Nissl bodies lysis. A number of
medium and small neurons size take round shape, there nuclei are surrounded by a thin belt of
cytoplasm with single small Nissl bodis clumps. In 21.43% of the neurons revealed highly dark
homogeny staining cytoplasm and karyoplasm, accompanied by the formation of angular and
inverted loops bodies of neurons, their nuclei and decreasing of cell sizes.
Pronounced degenerative changes according to the type of homogenizing changes and
intensive Nissl bodies lysis are detected in 10.99% of neurons are more common among large and
medium-sized neurons. In a few neurons (2.1%) showed evidence of primary irritation. Irreversible
changes (in 2.3% of neurons), usually present by type neuronophagia with the phagocytosis of
neurons and active proliferation of neuroglia. Morphometry designated a significant increasing in
the neurons body area and volume – respectively by 17.38% (229,14 mkm2) and 22.14% (1770,13
mkm3). More significantly increased the area and volume of the nuclei of neurons - respectively by
25.24% (53,37 mkm2) and 35.78% (184.83 mkm3). Area and volume of neuroglia bodies and nuclei
increased more significantly - by 23.24% (11.03 mkm2) and 48.05% (2,88 mkm3). A marked
tendency to increase the number and size of neuroglia satellites.
On the 15th day after the operation, reducing the number dystrophic neurons (up to 74.21%)
accompanied by an increase in the maximum severity of the neurons (51%). Degenerative changes
in the nerve cells are amplified along with the active proliferation of neuroglia. The number of
neurons with symptoms of acute swelling is reduced (to 24%), but they increases lysis and
homogenization Nissl bodies, enlightenment and ectopic changes of nuclei. Smaller number of the
neurons undergo more intensive edematous changes, often marked perinuclear swelling. Increases
the number of nerve cells (to 16%) with evidence of homogenizing changes, the extent of which is
also growing. At the same time a decrease in the number of nerve cells with signs of highly dark
homogeny staining cytoplasm and karyoplasm. On the 15th day reaching maximum expression
degenerative processes, neuron degeneration, significant inclusions of lipofuscin, lipids, karyolysis,
neuron phagocytosis, necrosis. Often identified cells with "glassy" cytoplasm and nucleus
destruction. In 47.62% of the neurons appear visible deposits of lipofuscin. Localization of
lipofuscin varies - it is found in a discharge of one of the processes, compact clumps in the
cytoplasm or fine diffusely located granules. Fatty degeneration of neurons is less common (12%).
Lipofuscin and fatty degeneration are usually combined with other dystrophic neuron changes.
Neuroglia proliferation provided the significant increasing of the satellites and the total glial
number. Due to reduction of fluid overload of nervous tissue remains only a tendency to edematous
changes.
On the 30th days after the operation of the red nucleus neurons showed a significant number
of neurons with signs of recovery of the structure and development of morphological intracellular
repair processes - increasing content of Nissl bodies, the number of ribosomes on the outer nuclear
membrane, nucleolar hypertrophy, appearance of the paranucleolar bodies, indicating that increased
activity of protein synthesis. Reversible dystrophic neuron changes were found in 25% of cells. In
most of the neurons it was founded lipofuscin (42,87%) and fatty degeneration (11%). Around
neurons with inclusions of lipofuscin was an increase satellites in the cytoplasm often identified
lipofuscin granules. Expression of glia proliferation to form chains, greater number of glial cells
increases around the degenerative neurons as well as around the nerve cells with signs of recovery
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structures. Interstitial change ratio of the cellular elements of the nervous tissue in the red nucleus,
which is manifested by significant increase of the total number of glial cells, the number of
satellites. The above changes reflect improvement of trophic neurons through the activation of the
trophic function, detoxification and other functions of the neuroglia. Morphometric study revealed
no significant changes in the profile of the field area and the volume of bodies of nerve cells and
their nuclei and cytoplasm. Development of signs of repair processes in many neurons of the red
nucleus show high plasticity of the nervous tissue, its significant opportunities for the development
of compensative, adaptive and repairing processes.
Histochemistry study of control animals was founded the low activity of SDH and LDH, in
the neuron cytoplasm is dominated by small and dust-like granules of formazan deposits are
significant around the cells nuclei. Often there are neurons in the soma that the enzyme activity is
lower than in the surrounding structures. Activity ITSDG high, in the cytoplasm of cells revealed a
dense cluster of small intensely colored formazan granules. In the surrounding soma neuronal
structures of the enzyme activity is low. Active D-6FDG high, small granules intensely colored,
tightly arranged, dominated in the perinuclear cytoplasm region. The high activity of NADPHdiaphorase and NAD-diaphorase activity is detected in the cytoplasm of neurons and the lowest - in
the structures surrounding soma.
Partial sympathetic denervation of the brain vessels leads to a sharp increase in the activity
of SDH in the cytoplasm of neurons (respectively 2,75, 1.98 and 1.84 times - on the 7th, 15th and
30th day), whereas in the surrounding structures amount of formazan granules increases
insignificantly. Dynamics of changes in the activity of another enzyme of the Krebs cycle - ITSDGwas quite different. The enzyme activity was significantly reduced (by 18,51-17,62%). Reduced
activity ITSDG indicates a decrease in the rate of the reactions in the Krebs cycle, which are
characteristic of hypoxic disorders in the brain (5), as the NAD-dependent allosteric enzyme is
ITSDG limits the speed of the processes in the Krebs cycle. Increased activity of LDH is probably
the result of reactions anaplerotic restocking succinate. Under hypoxic disorders in sympathectomy
eliminate the deficit in the energy-rich compounds is carried out by the pre-emptive use of succinate
to go oxidative transformations at the level of the Krebs cycle of NAD-dependent substrates to
succinate. Thus, these enzymological changes developing in the neuron cytoplasm, should be
considered as a compensatory metabolic reconstruction.
General activity of G-6FDG in the cytoplasm of neurons was significantly reduced on the
15th day (at 23.97%) and tended to decrease on day 30th. The main value of reducing activity of this
enzyme is not the influence of a violation of the energy balance, as pentose phosphate path splits
only 5-10% of the consumed glucose, and decreased synthesis of pentose conjugate to form nucleic
acids, essential to life of the nerve cells. This is confirmed by the maximum expression of
degenerative changes in the nerve cells of the red nucleus on the 15th day of the study.
The sharp increase in the activity of LDH (2.07, 2.32 and 2.3 times, respectively, on the 7th,
15th and 30th day) creates favorable conditions for the flow of glycolysis and lactic acid, which in
turn contributes to the development acidosis and irreversible changes in the appearance of neurons.
Since high capacity for anaerobic glycolysis is characteristic of early stages of embryogenesis,
increased glycolysis is a result of adaptation to the conditions of sympathetic denervation may be a
kind of return of adaptive metabolic exchange. Activity of NAD-and NADP-diaphorase, which are
part of electron transport system, significantly reduced on the 15th day in the cytoplasm of the red
nucleus of nerve cells, when found most pronounced degenerative changes in neurons. Recovery of
activity diaphoreses on the 30th day after the operation shows the development of compensatory and
adaptive responses that contribute to the normalization of metabolism of the nerve cells and is
accompanied by activation of reparative processes.
CONCLUSIONS
1. Early stage of partial sympathetic brain vessels denervation revealed morphological changes
according to the type of tissue hyper hydration and hypoxic disorders dominate the reversible
neuron changes of red nucleus.
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2. By the 15th day reach maximum development degenerative neuron changes, widely spread
lipofuscin and fatty degeneration, neuron necrosis. Proliferation of the neuroglia is accompanied by
an increase in common glia and especially –satellites.
3. By the 30th day significantly reduced the reversible neuron changes, mainly reduced dehydration
components of nervous tissue in the red nucleus. However, the number of lipofuscin and fatty
neuron degeneration not significantly altered.
4. Quantitative methods histochemistry set multidirectional changes activity of the main enzymes of
the Krebs cycle, pentose phosphate shunt, glycolysis and electron transport system in nerve cells,
indicating the development of hypoxia in these disorders, moderate some compensatory adaptive
reactions and repair.