Experimental modeling of preclinical and clinical stages of Parkinson's disease.

Degeneration of dopaminergic (DAergic) neurons of the nigrostriatal system is the key stage in the pathogenesis of Parkinson's disease. The first symptoms of this disease are observed after degeneration of 70-80% neurons, which occurs over 20-30 years. The clinical stage of Parkinson's disease begins after this period. Late diagnostics of Parkinson's disease contributes to low efficiency of therapy for this disorder. Detailed study of the pathogenesis and development of preclinical diagnostic methods for Parkinson's disease are the urgent problems. This work was designed to develop a new experimental model of the preclinical and clinical stages of the disease. Experimental modeling was performed on C57Bl/6 mice using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). This agent is converted into the MPP(+)-neurotoxin in brain DAergic neurons. We showed that MPTP in a dose of 4 mg/kg has no effect on the nigrostriatal DAergic system. MPTP in a dose of 8-16 mg/kg produced the toxic effect only on DAergic axons, which simulates the preclinical stage of Parkinson's disease. MPTP in a dose of 20-40 mg/kg had the toxic effect on neuronal axons and bodies, which simulates the clinical stage of Parkinson's disease. The data suggest that progressive degeneration of DAergic neurons is accompanied by activation of compensatory mechanisms for functional deficiency of these cells.

[Optimization of counting process of dopaminergic neurons in substantia nigra of parkinsonian mice].

Parkinson's disease (PD) results from degeneration of dopaminergic (DA-ergic) neurons of sunstantia nigra pars compacta (SNc). In experimental studies, this condition is modelled by administration of neurotoxin's precursor 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). The followed degeneration of DA-ergic neurons is estimated by cell counting. Cell counting with serial sections is exceedingly hard and expensive. Therefore this method is not used extensively in spite of its high precision. Widely known Konigsmark's formula (KF) allows not to count cells in all serial section, but only in sections selected at regular interval. However, its precision decreases with increasing the interval and other parameters. In given work we have developed mathematical method of approximation (MA) by improving of KF. MA allows reducing the time and chemical products expenses for processing and analysis of the material with maintenance of necessary counting precision. Two groups of C56/BL mice were used in this study. The first group received MPTP in a dose of 8 mg/kg with 1 week interval and showed 20% decrease in DA-ergic neurons in SNc. The second group received MPTP in a dose of 12 mg/kg four times with 2-h interval and showed 40 % decrease in DA-ergic neurons in SNc. Degeneration took place mostly in the middle part of SNc within rostra-caudal direction and led to rise of sharp differences in the number of neurons from section to section. These differences substantially decreased precision of FK (6 % error with counting in every 5th section), whereas precision of MA was sufficiently good (4% error with counting in every 7th section). Thus, we have developed MA, that allows us to decrease expenses of the time and chemicals for estimation of DA-ergic cells number in SNc of parkinsonian mice.

[Compensatory reaction during degeneration of arcuate nucleus dopaminergic neurons in rats].

The study has been carried out to verify the authors' hypothesis that degeneration of dopaminergic (DA-ergic) neurons of the hypothalamic tuberoinfundibular system and concomitant development of hyperprolactinemia are accompanied by involvement of compensatory synthesis of dopamine (DA) by non-dopaminergic neurons expressing single complementary enzymes of synthesis of this neurotransmitter. Degeneration of DA-ergic neurons was produced by a stereotaxic injection into the brain lateral ventricles of 6-hydroxydopamine (6-OHDA) - a specific neurotoxin of DA-ergic neurons. 14 and 45 days after the toxin administration there were determined concentration of prolactine in peripheral blood by methods of immunoenzyme and radioimmunological analyses as well as the DA amount in the arcuate nucleus by the method of highly efficient liquid chromatography with electrochemical detection. In a part of the animals, slices were prepared from the mediobasal hypothalamus (arcuate nucleus and medial eminence) and perfused with Krebs-Ringer medium; then the DA concentration was determined in the slices and in the incubation medium. 14 days after the neurotoxin administration there were revealed an increase of blood prolactine concentration and a decrease of DA concentration in the arcuate nucleus in vivo as well a decrease of the total DA amount in the slices and incubation medium in experiments in vitro. 45 days after the neurotoxin administration, all the above parameters returned to the normal level. This, the obtained data indicate that the hyperlactinemia and DA deficit appearing during degeneration of the arcuate nucleus DA-ergic neurons seem to be compensated due to an enhancement of DA synthesis by non-dopaminergic monoenzyme neurons of arctuate nucleus.

[Migration and differentiation of neurons producing gonadotropin-releasing hormone under conditions of serotonin excess in the brain of mouse embryos].

The work has been carried out on mice of the Tg8 line with knockout of gene of monoamineoxidase A with an increase of serotonin and noradrenaline content in the brain, and on mice of the C3H line with unchanged genome and normal concentration of monoamines. An immunocytochemical study has been performed of development of neurons producing gonadotropin-releasing hormone (GnRH) under conditions of excess of serotonin and noradrenaline in the mice in embryogenesis. The GnRH-neurons were revealed at the 18th day of embryonic development in telencephalon along trajectory of their migration from olfactory bulbs to the retrochiasmatic area. In telencephalon of mouse embryos of the Tg8 line, a redistribution of the GnRH-neurons along their migration trajectory was observed as compared with embryos of the C3H line mice. The percent of the GnRH-neurons in the Tg8 mouse embryos in caudal parts of the migration trajectory was lower than in rostral parts, the opposite distribution of the neurons being observed in the C3H line mouse embryos; at the excess of serotonin and noradrenaline in the Tg8 line mouse embryos, the total amount of GnRH-neurons in the brain was lower than in the C3H mice. In males of the Tg8 line mice under conditions of excess of serotonin and noradrenaline the optical density of neurons, which correlated with the GnRH concentration in the cell, was higher than in control mice. Thus, in the Tg8 mice under conditions of the serotonin and noradrenaline excess, migration of the GnRH-neurons to their final anlage in hypothalamus is accelerated as well as the total number of the GnRH-neurons decreases, which indicates a decrease of proliferation of cells-precursors and the earlier differentiation of neurons.

Pregnancy-induced de-differentiation of media smooth muscle cells in uteroplacental arteries of the guinea pig is reversible after delivery.

The majority media of smooth muscle cells of uteroplacental arteries of the guinea pig is not destroyed during trophoblast invasion. Rather, most of these cells de-differentiate during pregnancy-induced arterial dilatation, forming a population of mesenchyme-like myoblasts ready to reconstitute the media after birth. We have studied the re-differentiation of these cells after delivery by means of transmission electron microscopy and immunohistochemistry using antibodies against a panel of cytoskeletal proteins. The data reveal that post partum re-differentiation of the media myoblasts starts immediately after birth where some of the invasive trophoblast cells are still present. The process of re-differentiation is completed at day 8 after parturition. Post partum re-differentiation can be subdivided into two steps: until day 5 after parturition, the central parts of the media are reconstituted out of the reservoir of vimentin-positive myoblasts by stepwise acquisition of desmin, alpha-smooth muscle actin, gamma-smooth muscle actin and smooth muscle myosin. Only thereafter the same re-differentiation takes place in the peripheral parts of the media. On the ultrastructural level immunohistochemical re-expression of cytoskeletal proteins is accompanied by reconstitution of the intracellular contractile apparatus. The data support our earlier notion that the majority of media smooth muscle cells in the guinea pig uterus does not degenerate during trophoblast-invasion but rather de-differentiate temporarily.

The human placenta is encircled by a ring of smooth muscle cells.

The marginal zone of the human term placenta was studied by transmission electron microscopy and immunohistochemistry using antibodies against cytoskeletal filaments, extracellular matrix molecules and endothelial markers. The marginal sinus of the intervillous space is separated from the chorionic and basal plates by a layer of cells expressing vimentin, desmin, alpha- and gamma-smooth muscle actins, and smooth muscle myosin. Also ultrastructurally, these cells share all features with smooth muscle cells. This muscular ring is continuous with the media of uteroplacental veins entering the marginal sinus. In the basal plate the muscle cells may extend far into the central parts of the placenta. The muscular ring is separated from the intervillous space by a layer of endothelial cells. They are continuous with the maternal endothelium of the marginal uteroplacental veins. Moreover this endothelium covers neighbouring parts of the chorionic and basal plates, locally extending to the surfaces of large stem villi. The data suggest (1) that the marginal zone of the intervillous space ('marginal sinus') represents the dilated and merged parts of uteroplacental veins and (2) that lateral growth of the human placenta partly takes place by expansion into the uteroplacental veins. The functional importance of this muscular ring remains unknown.

Stromal differentiation and architecture of the human umbilical cord.

In order to assess the characteristics of its stromal cells and the distribution of extracellular matrix proteins, we investigated, immunohistochemically and ultrastructurally, term, first and second trimester human umbilical cords. A differential distribution pattern of the various cytoskeletal proteins of stromal cells and extracellular matrix proteins was observed in different zones of the stroma, the subamniotic stroma, Wharton's jelly, and the vessels' adventitia. All three zones showed immunoreactivities for collagen types I, III and VI and for basement membrane molecules such as collagen type IV, laminin and heparan sulphate proteoglycan. Immunoreactivities for these extracellular matrix molecules were observed around cleft-like territories (stromal clefts) in the Wharton's jelly which were occupied by homogeneous ground substance but void of collagen fibrils and basal lamina molecules. Moreover, between the stromal clefts, slender cells were found which immunohistochemically and ultrastructurally corresponded to various stages of myofibroblastic differentiation. In earlier stages of gestation, stromal cells with a less complex expression pattern prevailed. The stromal clefts and the contractile cells together might serve as a system regulating the turgor of the cord.

Immunohistochemical localization of extracellular matrix in perivillous fibrinoid of normal human term placenta.

The extracellular matrix of perivillous fibrinoid in normal human term placenta was investigated by means of the indirect immunofluorescent technique. Polyclonal antibodies to collagen types I, III, IV, V, fibronectin, fibrinogen, laminin, entactin and heparan sulphate proteoglycan and monoclonal antibodies BC-1, IST-9 and IST-4 to human fibronectin were used. The antigens can be grouped according to their presence in fibrinoid as abundant (fibrinogen, fibronectin, heparan sulphate proteoglycan, basement membrane collagen types IV and V), absent (laminin) and variable between fibrinoids (interstitial collagen types I and III, entactin). Our results also demonstrate that fibronectin in fibrinoid originates from placental cells (presumably cytotrophoblast). Monoclonal antibodies BC-1 and IST-9 specific to tissue fibronectin do not stain neighbouring placental extracellular matrix but do bind to fibrinoids on the same sections. Work by other authors has presented evidence that fibrin actually originates from maternal blood and even makes an attempt to substitute the term "fibrinoid" for "fibrin deposition". Our data on the composition of perivillous fibrinoids and the abundance of extracellular matrix components do not support this view and suggest that fibrinoid is a more relevant term for this interesting phenomenon, which deserves further investigation.

A kinase-related protein stabilizes unphosphorylated smooth muscle myosin minifilaments in the presence of ATP.

An apparent paradox in smooth muscle biology is the ability of unphosphorylated myosin to maintain a filamentous structure in the presence of ATP in vivo, whereas unphosphorylated myosin filaments are depolymerized in vitro in the presence of ATP. This suggests that additional uncharacterized factors are required for the stabilization of myosin filaments in the presence of ATP. We report here that an abundant smooth muscle protein forms sedimentable complexes with unphosphorylated smooth muscle myosin, partially reverses the depolymerizing effect of ATP on unphosphorylated myosin, and promotes the assembly of minifilaments as revealed by electron microscopy. This protein is called kinase-related protein (KRP) because it is derived from a gene within the gene for myosin light chain kinase (MLCK) and has an amino acid sequence identical to the carboxyl-terminal domain of MLCK. Consistent with the results with purified KRP, deletion of the KRP domain within MLCK results in a diminished ability of MLCK to interact with unphosphorylated myosin. KRP binds to the heavy meromyosin fragment of myosin but not to myosin rod or fragments lacking the hinge region and light chains. Altogether, these results suggest that KRP may play a critical role in stabilizing unphosphorylated myosin filaments and that the KRP domain of MLCK may be important for subcellular targeting to filaments.

Immunofluorescent study of heterogeneity in smooth muscle cells of human fetal vessels using antibodies to myosin, desmin, and vimentin.

Immunofluorescence-microscopy was applied to study the distribution of desmin, vimentin, and smooth muscle myosin in smooth muscle of human fetal vessels. Serial cryostat sections of the vessels examined all reacted positively with myosin and vimentin antibodies. However, heterogeneous staining of the vessels with desmin antibodies was observed. Thus, 2 types of smooth muscle staining were documented--desmin-negative and desmin-positive. Elastic and muscular arteries of the fetus (aorta, femoral and branchial artery) were desmin-negative while femoral and branchial veins were desmin-positive. In umbilical cord arteries and veins, the distribution of desmin-positive cells was largely localized to the outer layer of media, but not to the inner layer. In placenta, both desmin-positive and desmin-negative vessels were also revealed. Thus, differences in desmin expression by human vascular smooth muscle cells already exists during early stages of ontogeny.

Expression of calponin in rabbit and human aortic smooth muscle cells.

Polyclonal antibodies to chicken gizzard calponin were used to localize calponin and determine calponin expression in rabbit and human aortic smooth muscle cells in culture. Calponin was localized on the microfilament bundles of cultured smooth muscle cells. Early in primary culture, calponin staining was accumulated preferentially in the central part of the cell body. With time in culture, the number of calponin-negative smooth muscle cells increased while the distribution of calponin in calponin-positive cells became more even along the stress fibers. Calponin content and the calponin/actin ratio decreased about 5-fold in rabbit aortic smooth muscle cells during the first week in primary culture and remained low in proliferating cells. The same tendency in calponin expression was observed when human vascular smooth muscle was studied. On cryostat sections of human umbilical cord, calponin antibodies mainly stained vessel walls of both the arteries and veins, although less intensive labelling was also observed in non-vascular tissue. When primary isolates of human aortic intimal and medial smooth muscle cells were compared with corresponding passaged cultures, it was found that calponin content was reduced about 9-fold in these cells in culture and was similar to the amount of calponin in endothelial cells and fibroblasts. Thus, high calponin expression may be used as an additional marker of vascular smooth muscle cell contractile phenotype.

Confocal and conventional immunofluorescent and immunogold electron microscopic localization of collagen types III and IV in human placenta.

Confocal and conventional indirect immunofluorescence and immunogold electron microscopic methods were applied to examine the distribution of extracellular matrix constituents (collagens types III and IV) in the villi of immature and term human placentae. The immunofluorescence study revealed that collagen type III is more distinct in the villous stroma of term placenta as compared with that of the first trimester. Collagen type IV was detected mainly in endothelial and epithelial basement membranes and interestingly also to a certain extent in the stroma. Results obtained using immunoelectron microscopy support the proposal that collagen types III and IV are characteristic of stromal and basement membranes, respectively. Stromal collagen type IV is apparently localized in association with the interstitial types of collagen (I and III), in the villous stroma of term placenta.

Immunohistochemical investigation of autoantibodies to nerve fibers in cardiomyopathy.

The immunofluorescence technique was used to trace auto-antibodies to neural structures in the blood serum of 180 patients with various cardiomyopathies and 20 healthy probands (controls). Incubation of cryostat slices of heart, kidney, spinal cord and medulla oblongata of Wistar-rats or of cell cultures of embryonal spinal cord with the blood serum of patients with cardiomyopathies resulted in immunofluorescence of nerve fibres and neuronal perikaryon. The controls were negative. The question remained unanswered for the nature of the involved antigen. Neurohistochemical findings suggested auto-antibodies in sera of patients to be orientated to cholinergic endings. The authors suggest the immunomorphological approach as practicable for revealing auto-antibodies in neurohistological studies.

[Distribution of myosin, desmin and vimentin in smooth muscle cells of human embryonic vessels]. / Raspredelenie miozina, desmina i vimentina v gladkomyshechnykh kletkakh émbrional'nykh sosudov cheloveka.

Immunofluorescent study of embryonal vessels of man using antibodies to myosin, desmin and vimentin showed heterogeneity of smooth muscle cells. It is supposed that the use of desmin as a marker of cell differentiation can increase the role of modified phenotypes in the development of the pathological process in the vascular wall.

Participation of collagen types I, III, IV, V, and fibronectin in the formation of villi fibrosis in human term placenta.

The indirect immunofluorescence method was used to study the human term placenta in pathological pregnancy for the distribution of collagen types I, III, IV, V, and fibronectin in fibrosis stromatis villi. All collagen types and fibronectin were shown to participate in fibrosis villorum formation. Fibronectin was also detected in the fibrinoid that surrounded villi at stroma. The presence of free cytotrophoblast cells in the fibrinoid was accompanied by a noticeable increase in fibronectin fluorescence. A significant amount of collagen types IV and V and a less amount of collagen types I and III were identified.

[Distribution of collagen types III and IV in human placental villi]. / Raspredelenie kollagena III i IV tipov v vorsinakh platsenty cheloveka.

Immunofluorescent examination showed more significant accumulation of interstitial collagen type III in the stroma of mature placenta compared with immature one. Localization of membrane collagen type IV was found neither in basal membranes of epithelium and villous vessels of mature term placenta, nor in their stroma. The described patterns of distribution of collagen types III and IV in human placenta villi were proved by immunoelectronmicroscopic method.

[The localization of the components of the extracellular matrix and cytoskeleton in myocardial biopsies in alcoholic cardiomyopathy]. / Lokalizatsiia nekotorykh komponentov vnekletochnogo matriksa i tsitoskeleta v miokarde bioptatov pri alkogol'noi kardiomiopatii.

Distribution of I, III, IV and V-type collagen, fibronectin, vimentin, filamin, and desmin in myocardial biopsy specimens of 8 patients with alcoholic cardiomyopathy was studied by indirect immunofluorescence. It is found that all types of collagen and fibronectin participate in formation of diffuse cardiosclerosis while small cardiosclerosis is formed by interstitial types of collagen and to a lower extent of fibronectin. Filamin and desmin distribution in cardiomyocytes proves deformation and destruction of Z-lines and intercalated discs.