Effect of Inducers and Inhibitors of the Keap1/Nrf2/ARE System on the Viability and Functional Activity of Model Neuronal-Like and Glial Cells.

On mouse neuroblastoma (Neuro-2a) and human glioblastoma (U-87 MG) cell lines, we studied the effect of inducers and inhibitors of redox-sensitive signaling system of the antioxidant-responsive element Keap1/Nrf2/ARE on the main processes that determine nerve cell viability and vital activity (proliferative activity, apoptosis, autophagy, and activation of the Keap1/Nrf2/ARE system). Inhibitors of the Keap1/Nrf2/ARE system stimulate apoptosis more pronouncedly than inducers, have a weaker effect on autophagy, and do not change the nuclear to cytoplasmic Nrf2 ratio. In general, the revealed effects testify in favor of the potential effectiveness of stimulating the Keap1/Nrf2/ARE system for the prevention and adjuvant therapy of neurodegenerative diseases.

[Pathological changes in the nervous structures of the aortic wall tissue adjacent to an unstable atherosclerotic plaque]. / Patologicheskie izmeneniya nervnykh struktur v tkanyakh stenki aorty, prilezhashchikh k nestabil'noi ateroskleroticheskoi blyashke.

OBJECTIVE: To study of nerve structures in the aortic wall in atherosclerosis using a complex of immunohistochemical markers. MATERIAL AND METHODS: The objects of the study were excised fragments of the wall of the thoracic and abdominal aorta along with visually determined unstable atherosclerotic plaques. To study nerve structures on paraffin sections, immunohistochemical reactions were performed for the PGP 9.5 protein, tyrosine hydroxylase, and synaptophysin. RESULTS: It has been established that pronounced pathological changes are observed in the nervous structures of the aortic wall near unstable atherosclerotic plaques. Reactive, dystrophic, and severe degenerative changes in neurocytes, nerve fibers, and glial cells are described in the elements of the nervous apparatus of the adventitia (microganglia, nerve trunks, and nerve plexuses). It was found that only sympathetic neurons and their postganglionic fibers remain in the intramural ganglia, while the structures of the parasympathetic nervous apparatus undergo degeneration. Destruction of perivascular nerve plexuses and vasa vasorum in the adventitia, as well as degeneration of varicose axons of the main terminal synaptic plexus at the border of adventitia and superficial smooth muscle layer of the media were demonstrated. CONCLUSION: It is assumed that the presence of inflammatory infiltrates in the adventitia and intima, denervation and death of vasa vasorum can serve as factors determining the development of the atherosclerotic process.

[Features of innervation of epicardial adipose tissue in rats during aging (immunohistochemical study).]

The purpose of the article is to study the structure, innervation and state of the epicardial adipose tissue of the aortic-pulmonary region of the heart of rats at the age of 3-4 months and 18-23 months using neuroimmunohistochemical markers. Using a complex of histological and immunohistochemical methods, various nervous apparatus (ganglia, clusters of chromaffin cells, nerve trunks, nerve fibers, nerve plexuses, synaptic endings) with different mediators were identified in the white and brown adipose tissue of the base of the rat heart. It was found that parasympathetic and sympathetic postganglionic nerve fibers are involved in the innervation of white and brown adipose tissue. They penetrate into adipose tissue as part of Remak's cords of varicose axons along arterial vessels, form terminal synaptic plexuses of the en passant type, and are involved in the innervation of adipocytes of both types of epicardial adipose tissue. It was found that PGP 9.5+ cholinergic terminal nerve fibers predominate over catecholaminergic ones. During aging of rats, neurodegenerative, involutive (desimatization), and destructive pathological changes in white adipocytes were noted in epicardial adipose tissue.

Changes in the Thickness of Rat Nerve Sheaths after Single Subperineural Administration of Rat Bone Marrow Mesenchymal Stem Cells.

The sheaths of the damaged peripheral nerve of Wistar-Kyoto rats were studied after single subperineural administration of bromodeoxyuridine (BrdU)-labeled bone marrow mesenchymal stem cells (MSC) from the same rats. The sciatic nerve was damaged by ligation for 40 sec directly before MSC administration. BrdU+ MSC were identified in the recipient nerve within 1 week after transplantation and were detected not only in the endoneurium, but also in the epineurium and perineurium. It was found that single administration of MSC into the damaged nerve trunk led to an almost 2-fold increase in the thickness of its sheaths (perineurium and epineurium) in comparison with the control group (ligation). It can be hypothesized that MSC induce thickening of nerve sheaths through the production of factors that stimulate angiogenesis and adipogenesis.

[Morphological signs of neurogenic inflammation in the heart of rats during aging.]

Using histological methods of staining with toluidine blue, hematoxylin-eosin and immunohistochemical reactions for the PGP 9,5 protein, tyrosine hydroxylase (TH), Iba-1 protein, cellular changes in different parts of the heart of Wistar rats at the age of 18-23 months were studied. In the connective tissue of the heart base, focal inflammatory infiltrates were found, near which PGP 9.5+ and TH+ plexuses, consisting of parasympathetic and sympathetic nerve fibers, were detected. In the area of the valvular heart apparatus, at the border of the anneau fibreux and the myocardium of the right atrium, pathological changes in nerve structures were found: degeneration of nerve fibers and granular destruction varicose axons of the terminal plexus. A close relationship has been established between axons of the terminal nervous network and cells of inflammatory infiltrates and blood capillaries. The features of the localization of neurocellular inflammatory complexes consisting of nerve fibers, blood capillaries and cells participating in the local inflammatory process (mast cells, histiocytes, monocytes, fibroblasts, plasma cells) in various parts of the myocardium in old animals are described. The chronic nature of neurogenic inflammation in the heart during aging has been established.

[Structural and functional changes of endothelium of heart's vessels of old rats according to immunistochemical studies.]

The aim of the work is to investigate the morphofunctional changes of the vascular endothelium in different parts of the heart of aging rats using immunohistochemical detection of Willebrand factor. Reactive, dystrophic and pathological changes of endotheliocytes in different parts of the heart of old (18-23 months) rats were first identified using an immunohistochemical marker vWF. They are most expressed in the endothelium of the arterial vessels of the aortic root, coronary arteries of the epicardium, and myocardial capillaries. In this work, desquamation of endothelial cells into the blood, death of capillaries in the myocardium, an increase in the number of sinusoidal vessels, their mummification, the formation of lacunae or cisterns filled with blood were found. It has been established that the secretory activity of endotheliocytes increases with aging. It is established that the products of protein metabolism, pigment, Weibel-Palade body and various sizes of blood clots accumulate in the cavity subaroundal cone of heart in old rats. Pigment structures were also found in the valves and the fibrous ring. The data obtained are of great diagnostic value.

Changes in the Distribution of Cell Contacts and Mitotic Cycle Disturbances in Cells of the Allograft of Rat Embryonic Neocortex.

Morphological changes in the allograft of rat anterior cerebral vesicle at the early stages after transplantation into the peripheral nerve of an adult rat were studied by immunohistochemical methods. Immunohistochemical reaction to bromodeoxyuridine showed that the delay of mitotic division in neural stem/progenitor cells in the grafts occurred during S/G2 stage. In transplants of rat embryonic neocortex (E13), changes in the cell cycle of neural stem/progenitor cells in 3 h after transplantation into the nerve correlated with abnormal distribution of adherens junctions and interkinetic nuclear migration.

Vascularization of the Damaged Nerve under the Effect of Experimental Cell Therapy.

Quantitative analysis of blood vessels in the distal segment of rat sciatic nerve after its ligation for 40 sec and subperineurial administration of mesenchymal stem cells or dissociated cells of rat embryonic spinal cord was carried our by immunohistochemical tracing of von Willebrand factor, a marker of endothelial cells of blood vessels. It was found that the number of blood vessels per unit area of the nerve trunk in 21 days after injury and administration of mesenchymal stem cells increased by more than 1.5 times in comparison with the control (damaged nerve). After administration of dissociated cells of the embryonic spinal cord, this effect was not observed. It is assumed that mesenchymal stem cells stimulate the growth of vessels of the damaged nerve via production of angiogenic factors.

[Neurons with Different Neurotransmitters in Embryonic Neocortical Allografts in the Rat Sciatic Nerve].

Different subsets of interneurons in the Wistar rat neocortex and in neocortical transplants developing in a damaged nerve were identified by the following immunohistochemical markers: glutamate decarboxylase (GAD 67) for GABAergic nerve cells, NO-synthase (NOS) for NO-ergic neurons, choline acetyltransferase (ChAT) for cholinergic cells, and tyrosine hydroxylase for catecholaminergic structures. Twenty-eight days after surgery, individual GAD 67-ir, NO-ir, ChAT-ir, and very rarely TH-ir cells were detected in the graft. It was shown that the number of GAD 67-ir neurons per unit area in the grafts was less than in the rat neocortex P20.

Differentiation of Cholinergic Neurons in Rat Spinal Cord Under Conditions of Allotransplantation into a Peripheral Nerve and In Situ Development.

The method of ectopic transplantation of embryonic anlages of CNS allows studying histoblastic potencies of progenitor cells developing under conditions of changed microenvironment. Some progenitor cells in the transplants of rat embryonic spinal cord retained their ability to express choline acetyltransferase after transplantation into the sciatic nerve of adult animals. Comparative analysis of cholinergic neurons in the neurotransplants and neurons formed in rat spinal cord during normal ontogeny showed that choline acetyltransferase-positive cells after transplantation into the nerve reached morphological differentiation of motor neurons at later terms than cells developing in situ. They were scattered one by one and did not form nuclear nerve centers. We did not fi nd structures similar to presynaptic cholinergic buds typical of intact spinal cord near these cells throughout the observation period. Solitary cholinergic neurons survived in the transplants for 19 months.

Injured Nerve Regeneration using Cell-Based Therapies: Current Challenges.

This paper reviews the recent research progress in the past several years on promoting peripheral nerve recovery using stem and progenitory cells. The emphasis is placed on studies aimed at assessing various stem cells capable of expressing neurotrophic and growth factors and surviving after implantation in the nerve or a conduit. Approaches to improving nerve conduit design are summarized. The contribution of stem cells to axonal regeneration and neural repair is discussed. The side effects associated with cell-based treatment are highlighted. From the studies reviewed, it is concluded that the fate of transplanted stem cells needs further elucidation in a microenvironment-dependent manner.

Effect of allotransplants containing dissociated cells of rat embryonic spinal cord on nerve fiber regeneration in a recipient.

Regeneration of nerve fibers in rat sciatic nerve was quantitatively assessed after injury (ligation) and injection of dissociated cells derived from embryonic spinal cord. A suspension of dissociated spinal cord cells from rat embryos was transplanted under the perineurium of a nerve trunk. After transplantation, bromodeoxyuridine-labeled precursor cells survived and retained the label for more than 2 months; some of these cells differentiated into NeuNpositive neurons. Analysis of semithin sections of the distal nerve segment from the recipient taken at a distance of 0.5 cm from the site of injury showed that transplantation of dissociated cells of embryonic spinal cord led to an increase in the number of myelinated nerve fibers in the recipient nerve.

Development of rat embryonic spinal ganglion cells in damaged nerve.

The development of dissociated cells from rat embryonic spinal ganglion after transplantation to damaged nerve of adult animals was studied using immunohistochemical differentiation markers of neural and glial cells. The cell suspension obtained after dissociation of rat embryonic spinal ganglia (embryonic day 15) was injected into the proximal segment of crushed sciatic nerve. The nerve was damaged by ligation for 40 sec. Progenitor cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) before transplantation. BrdU-immunopositive cells were detected in the nerve trunks of recipients on days 1, 21, and 28 after transplantation. Dissociated cells of rat embryonic spinal ganglion (embryonic day 15) survived for at least 4 weeks after transplantation to the nerve and differentiate into NeuN-immunopositive neurons with morphological properties of sensory neurons and satellite cells containing S100 protein.

[Effect of embryonic anlage allografts of the rat spinal cord on growth of regenerating fibers of the recipient nerve].

A comparative study of the effect of tissue and suspension allografts of an embryonic spinal cord on regeneration of nerve fibers of impaired (by application of a ligature) sciatic nerve in rats was conducted. It was demonstrated that unlike tissue grafts that reach a large volume 21 and 60 days after transplantation, suspension grafts do not inhibit the growth of axons of the recipient to the periphery. It was established that introduction of a suspension of dissociated cells of the spinal cord embryonic anlages (but not fragments of these anlages) into the impaired sciatic nerve in rats results in an increase in the amount of myelinated regenerating nerve fibers of the recipient 60 days after the operation.

[Features of chronic pancreatitis pathomorphism in the validity of surgical approaches].

The results of examination and surgical treatment of 298 patients with chronic pancreatitis and the original morphological investigations of material of the pancreas were studied. The data allowed the detection of additional criteria features of inclusion of the patients with chronic pancreatitis to be made in groups according to foreign Marseilles-Roman classification (1988). It is shown, that the basis of study of morphogenesis of chronic pancreatitis is immunohistochemical method, which let the authors diagnose not only the pathological changes of exo- and endocrine sections of pancreas, but at the same time the structural features of nervous apparatus and vessels of microcircular bed. The revealed morphological features of different forms of chronic pancreatitis vs clinical finding characteristics and the data of instrumental and laboratory methods of research allow the substantiation of surgical treatment version to be made.

Differentiation of dissociated rat embryonic brain after allotransplantation into damaged nerve.

Fragments of the dorsolateral wall of the anterior brain vesicle from rat embryos on embryonic day 15 were dissociated and the resultant suspension containing single cells and cell aggregates was injected into the proximal segment of crushed sciatic nerve of adult animals for evaluation of their engrafting and differentiation under conditions of changed microenvironment. On days 1 and 21 postoperation, Msi-1, GFAP, NeuN, vimentin, and PCNA were detected by immunohistochemical methods. Small clusters of Msi-1-immunopositive cells were detected in the nerve trunks on the next day after transplantation. On day 21 after surgery, these precursors differentiate into nerve cells, astrocytes, and primarily ependymocytes.

[Development of dissociated cells of various rat cns primordia after transplantation into the damaged nerve].

The purpose of this paper was to examine the possibilities of engraftment, and to study the differentiation of the dissociated cells from the embryonic primordia of the spinal cord and the neocortex of Wistar rats, after their transplantation into the sciatic nerve of adult animals. The cell suspension obtained as a result of a dissociation of fragments of the cervical spinal cord and the anterior cerebral vesicle from rat fetuses at day 15 of development, was injected into the proximal segment of a previously damaged sciatic nerve. Using the immunocytochemichal marker of neural stem/progenitor cells (Msi-1) the transplanted cells were identified in the nerve trunks after 1 day after the operation. After 21 day some of these cells underwent differentiation into NeuN-immunopositive neurons, however their number was small. Thus, dissociated precursor cells from embryonic rat spinal cord and neocortex survive for three weeks under conditions of transplantation into the damaged nerve and retain the ability to differentiate into neurons, but the number is small. Most of the cells in the neocortex transplants, unlike those from spinal cord transplants, within 21 days after the operation were represented by the ependymocytes.

[Advantages and disadvantages of zink-ethanol-formaldehyde as a fixative for immunocytochemistry and confocal laser microscopy].

The paper presents an analysis of authors' results obtained using fixation of various tissues in zink-ethanol-formaldehyde (ZEF). It was found that fixation in ZEF, in comparison with other methods of fixation, allowed to achieve higher sensitivity of immunocytochemical reaction for a large number of antigens studied and, in many cases, to avoid heat unmasking of antigens. It also provided high resolution of the images obtained using the fluorescent and confocal laser microscopy. However, the studies of antigens with small molecular mass revealed the antigen diffusion from the site of original localization. The data obtained suggest that fixation of a material in ZEF fixative is promising for both immunocytochemical studies, including those using the fluorescent and confocal laser microscopy, and general histological practice.

[The use of stem cells to stimulate regeneration of damaged nerve].

The purpose of the review was the summation of the results of experimental studies on the use of stem cells to repair damaged nerves. There are two basic strategies to connect the segment of nerve damage: the use of biodegradable scaffolds and conduits and the application of stem cells. Stem cells are source of the growth and trophic factors. Embryonic stem cells, neural stem/progenitor cells, olfactory ensheathing cells, mesenchymal stem cells, adipose-derived stem cells, dental pulp stem cells, skin-derived precursor cells were used in the past ten years. Genetically modified cells with overexpression of growth factors were also used. Analysis of the results of experimental studies have shown that stem cells contribute to improving the recovery of damaged nerves. Further basic research on the mechanisms of the influence of the transplanted stem cells in regenerating axons and Schwann cells of the recipient are required. Investigation of the fate of transplanted cells in the long-term after the operation is necessary to eliminate the risk of any adverse effects of stem cell transplantation. The use of stem cells in experimental modeling of nerve injury allows us to study patterns of development of stem cells, mechanisms of their differentiation and malignant transformation.