A glutamate model of stroke was analyzed from the standpoint of the development of a typical pathological process that is thought to occur when major regulatory mechanisms are violated. The analysis made it possible to isolate the main mechanisms that underlie a transition from normal physiological processes to common pathological changes. This review considers a generalizing concept of how the pathological process develops. Following the concept, the typical pathological process is based on nonspecific distortion of cyclic regulatory processes and arises when reactive nitrogen species (RNS) and reactive oxygen species (ROS) increase simultaneously. Once RNS and ROS concentrations are beyond the regulatory capabilities of biochemical antioxidant systems, nitric oxide and superoxide anion radical cycles are disrupted. In the context of the concept, damage to cell membranes and subcellular structures in glutamate toxicity arises because the above alterations lead to the generation of nitrogen dioxide, which is a highly reactive compound, is involved in free radical chain reactions, and oxidizes the main biochemical components of living organisms: DNA/RNA (guanines primarily), fatty acids (unsaturated fatty acids that are components of phospholipid membranes), and proteins (the SH groups of sulfur-containing amino acids and the OH groups of tyrosine residues to produce nitrotyrosine). The concept agrees well with the ideas that every disease starts with a failure of regulatory mechanisms (R. Virkhov) and that dysregulatory pathology forms its basis (G.N. Kryzhanovsky). The mechanisms of the toxic effects of glutamate- and NO-generating compounds as a model of stroke made it possible to suggest methods to reduce their damaging effects. The methods have already been used as part of therapy for ischemic and hemorrhagic strokes, hemorrhages, and head injuries.

This review summarizes published data and original findings on the morphology of neurons and glial cells in normal conditions and model conditions that simulate those in the human brain during stroke. Ultrastructural changes that occur in the presence of high concentrations of glutamate (Glu, 0.1–5.0 M) and the NO-generating compound NaNO2 (0.1–5.0 mM) were studied in the cerebella (frog cerebellum), which is one of the simplest circuitries structurally. Such studies and data analyses are important because hyperstimulation of Glu receptors is a leading pathogenetic factor of neuronal damage during a stroke. High Glu concentrations exert a toxic effect and damage cerebellar neurons and glial cells. Mitochondria are de-energized, ionic homeostasis is distorted, the intracellular Ca2+ concentration increases, and constitutive NO synthases are activated in the process. The changes result in an increase in the contents of NO and its transformation products, which are involved in a negative feedback mechanism from postsynaptic neurons to presynaptic cells. Biochemical processes are consequently affected, and morphological changes are induced in neurons and glial cells, leading to their swelling. At the same time, ultrastructural compensatory adaptive mechanisms develop to reduce the damaging effect of high concentrations of Glu and NO-generating compounds.

1 Институт вышей нервной деятельности и нейрофизиологии РАН (117485, г. Москва, ул. Бутлерова, 5а), 2 Национальный научно-практический центр здоровья детей (119991, г. Москва, Ломоносовский пр-т, 2/1), 3 Институт проблем передачи информации им. А.А. Харкевича РАН (101447, Москва, Большой Каретный пер., 19), 4 Тихоокеанский государственный медицинский университет (690002, г. Владивосток, пр-т Острякова, 2)

Ultrastructural changes in synapses between parallel fibers (PF) and the spines of Purkinje cell dendrites (PCD) in frog cerebellum were studied after exposure to high concentrations (1 mM) of glutamate (Glu) and NO-generating compound in experimental model. It was shown that exposure to Glu resulted in the envelopment of the terminal bouton by the spine, while under the influence of NO-generating compound, on the contrary, the spine was surrounded by the bouton. Morphological study has shown that in Glu solution there was the predominance of synapses in which the glial cells surrounded the spines, while in the presence of NO they covered the boutons. After the electrical stimulation of PF, the relative number of synapses, containing the boutons surrounded by glial cells, was 10 times higher as compared to those in which the glial cells surrounded the spines. The observed morphological changes reflect the functional state of synapses between PF and PCD in response to the damaging effects of excess Glu and NO, that is expressed in different forms of synaptic contacts and neuronglial structures.

The ultrastructure of the retinal pigment epithelium of a diurnal rodent ( Lasiopodomys [Microtus] brandti Radde, 1861) was described considering (1) the functions of the pigment epithelium as a participant in the renewal of a photoreceptor outer segment and (2) digestion of outer segment membranes in phagosomes of the retinal pigment epithelium. The myeloid bodies were searched for after exposure of the pigment epithelium to light (200 lux, 4 h) and darkness (0.1 lux, 1.5 h). No myeloid bodies were observed in the cytoplasm of the pigment epithelium of the vole. Instead, small lamellar bodies, which have a spiral form and a size from ~ 200 to 400 nm were found. The structure of these lamellar bodies is described. Furthermore, structures that are presumably responsible for the transport of the digested material were revealed. The evidence for this is the presence of (1) a dense precipitate in the apical domain of the pigment epithelium and (2) microtubules that participate in the transport of this precipitate.

The study detected common ultrastructure of pigmented epithelium cells of Eolagurus luteus and other mammals and described its features characteristic of this species. Stages of interactions between the pigmented epithelium cells and external segments of retinal rod cells were studied. Migration of the external segments to the apical zone of a pigmented epithelium cell was observed. A pigmented epithelium cell was characterized by the presence of tubular endoplasmic reticulum and electron dense cytoplasm with processes directed to the retina. Multilamellar formations were detected, regarded as the initial stages of the myeloid body formation. These structures were characterized by periodicity of layers of ~4.2 and ~13.3 nm. The periodicity of layers in the formed myeloid body was ~23 nm. Interactions between the external segments and cells of pigmented epithelium, leading to formation of myeloid bodies, and the significance of this process for normal work of retinal elements are discussed.

Методом электронной микроскопии исследована структура глиальных клеток (астроцитов) мозжечка (МЖ) лягушки и их взаимодействие с нейронами в условиях повреждения нейронной сети МЖ токсическими дозами глутамата и NO-генерирующего соединения. Повреждения такого рода можно рассматривать как модель повреждений, имеющих место при инсультах. Показано, что отростки астроцитов, сохранившие свою структуру в этих условиях и имеющие большое количество зерен гликогена, способны защищать нервные клетки. Однако сами отростки при этом претерпевают значительную структурную трансформацию. Отросток вытягивается в жгут, практически лишенный цитоплазмы, а расстояние между его стенками уменьшается до 25–30 нм. Внутри жгута появляются поперечные мостики, скрепляющие его стенки. Эта структура носит название аутотипического септального контакта. Такой жгут может обкручивать либо синапс, либо его элементы (бутон, шипик), образуя плотную капсулу (обкрутку). Чем сильнее повреждение, тем больше рядов в обкрутке, и тем меньше расстояние между стенками жгута (18–20 нм). Предполагается, что аутотипические контакты могут защищать нейроны зернистого слоя МЖ при токсическом воздействии глутамата и NO-генерирующего соединения.

Ultrastructural changes in cerebellar granule cells were studied in a model of stroke after the toxic action of glutamate (Glu) and an NO-generating compound. Toxic doses of glutamate produced two types of change in the nuclear chromatin. In some cases, cells with almost completely decondensed nuclear chromatin appeared; in others, cells with partially decondensed chromatin appeared. Pathological fusion of granule cells was seen in both cases. The toxic action of an NO-generating compound on granule cells also induced the appearance of cells with essentially complete and partial decondensation (flocculent) nuclear chromatin. Thus, Glu and NO, inducing changes in the nuclear chromatin, activate the process leading to the formation of clusters of granule cells able to undergo cytoplasmic fusion to form multinucleate conglomerates. The possible physiological role of the fusion of cerebellar granule cells in the presence of high concentrations of Glu and an NO-generating compound is discussed. This process is regarded as the realization of a compensatory-adaptive reaction in extreme conditions as seen in stroke and oxidative stress.

We studied the effect of locomotor activity on the ultrastructure of cerebellar neurons, neurological disturbances, and survival rate in Krushinsky–Molodkina rats during the development of hemorrhagic induced by acoustic stress. In animals with high spontaneous locomotor activity, severe edema of cerebellar neurons (resulting in the destruction of surrounding structures) and swelling of the synapses (terminals of mossy fi bers on granule cell dendrites) were observed. By contrast, the areas of intracerebral, subdural, and subarachnoid hemorrhages were lower in rats under conditions of forced rest.

Ultrastructure of processes of glial cell, astrocytes of the molecular layer of cerebellar cortex in Rana temporaria frog, under conditions of damage to the cerebellum caused by NO-generating compound sodium nitrite was studied under an electron microscope. It is found that astrocytes have at least two types of processes: the first (fibrillar) primarily contained numerous fibrils and few glycogen granules and the second (granular) primarily containing glycogen granules. In the presence of NO-generating compound in toxic doses, fibrillar processes are damaged or completely degrade more rapidly than granular ones. The processes containing glycogen can protect both damaged synapses and individual synaptic buttons by forming a compact structure, wrapping, around them. We analyzed the possible role of glycogen of cerebellar glial cell processes in neuroglial interactions in the presence of sodium nitrite.

Ultrastructure of synaptic vesicles in axon terminals of granule cells from isolated cerebellum of Rana temporaria frogs under the influence of NO-generating compound NaNO2 in various concentrations and electrical stimulation was evaluated by the method of electron microscopy. NO-generating compound in low concentration induced translocation of synaptic vesicles and formation of small clusters. The size and structure of synaptic vesicles remained unchanged under these conditions. Increasing the concentration of NaNO2 led to swelling of synaptic vesicles, formation of arranged heaps from individual vesicles or fusion of their content. Electrical stimulation of the cerebellum in the presence of NaNO2 increased damage to synaptic vesicles. These experimental data model some stages observed in stroke. The formation of clusters from synaptic vesicles is a compensatory and adaptive response maintaining the structure of synaptic vesicles and protecting neurons from high concentrations of glutamate. Glutamate produces a toxic effect on nerve cells and glial cells of the cerebellum under pathological conditions, which is accompanied by impairment of signal transduction from presynaptic to postsynaptic neurons.

The molecular layer of the cerebellum of the frog Rana temporaria was studied by light and electron microscopy after electrical stimulation in the presence of an NO-generating compound. In these conditions, there was severe swelling of granule cell axon terminals (boutons) and astrocyte processes (AP), with loss of cytoplasmic elements. However, along with damaged structures, there were also undamaged structures: boutons with synaptic vesicles and AP with glycogen granules. It is suggested that these persisting viable AP may form 1) glial “wrappings” around damaged synapses or boutons and 2) neuron-glial contacts, which form when synaptic vesicles cross damaged bouton membranes to AP containing glycogen granules. It is also suggested that the presence of glycogen in AP in conditions of oxygen and glucose deficiency may provide a source of high-energy substrates such as glucose and ATP, thus providing conditions for neuron survival in pathological states (ischemia/hypoxia).

Molecular layer of frog (Rana temporaria) cerebellum was studied using light and electron microscope after electrical stimulation of parallel fibers in presence of NO-generating compound. Under these conditions, significant swelling of axonal terminals (boutons) of granular cells and astrocyte processes (AP) with a loss of cytoplasmic elements. However, along with the damaged structures, intact boutons were found with synaptic vesicles and APs containing glycogen granules. It is suggested that the remaining viable APs are capable of forming 1) protective glial "wrappings" around damaged synapses or boutons, and 2) neuron-glial junctions, that are formed due to transmission of synaptic vesicles through the damaged membrane of bouton into AP containing glycogen granules. It is also proposed that the presence of glycogen in APs under conditions of oxygen and glucose deficit may serve as the source of such energy-containing substrates, such as glucose and ATP, and thus may provide for neuronal survival in pathological states (ischemia/hypoxia).