Increasing Severity of Spinal Cord Injury Results in Microglia/Macrophages With Annular-Shaped Morphology and No Change in Expression of CD40 and Tumor Growth Factor-ß During the Chronic Post-injury Stage.

Determination of the quantitative composition of phenotypically and morphologically different populations of resident microglia and infiltrating macrophages in spinal cord injury (SCI) of various degrees of severity could lead to much needed novel therapeutic interventions in neurotrauma. In this regard, we investigated the CD40 and TGF-ß expressing populations of microglia/macrophages and their morphological states in a rat model of SCI of varying severity. We are the first to describe the annular-shaped microglia/macrophages, the morphology of which was formed due to the spatial orientation of the processes that form round or oval micro-territories, which include disintegrating myelin fibers. This type of cell morphology was found only in the injured spinal cord and mainly in the white matter. At the same time, an assessment of the number of annular-shaped microglia/macrophages and the diameter of micro-territories formed by their processes showed an elevation in these indicators as the severity of SCI increased. While we did not find significant quantitative changes in the populations of Iba1+/CD40+ and Iba1+/TGF-ß+ microglia/macrophages with increased severity of SCI in the chronic period (60 dpi), we did determine changes in the expression of cytokines and mRNAs of genes-encoding microglial marker proteins, finding the greatest changes on days 7 and 14 after SCI between experimental groups with varying severity.

Influence of Genetically Modified Human Umbilical Cord Blood Mononuclear Cells on the Expression of Schwann Cell Molecular Determinants in Spinal Cord Injury.

Spinal cord injury (SCI) unavoidably results in death of not only neurons but also glial cells. In particular, the death of oligodendrocytes leads to impaired nerve impulse conduction in intact axons. However, after SCI, the Schwann cells (SCs) are capable of migrating towards an area of injury and participating in the formation of functional myelin. In addition to SCI, cell-based therapy can influence the migration of SCs and the expression of their molecular determinants. In a number of cases, it can be explained by the ability of implanted cells to secrete neurotrophic factors (NTFs). Genetically modified stem and progenitor cells overexpressing NTFs have recently attracted special attention of researchers and are most promising for the purposes of regenerative medicine. Therefore, we have studied the effect of genetically modified human umbilical cord blood mononuclear cells on the expression of SC molecular determinants in SCI.

Electrophysiological, Morphological, and Ultrastructural Features of the Injured Spinal Cord Tissue after Transplantation of Human Umbilical Cord Blood Mononuclear Cells Genetically Modified with the VEGF and GDNF Genes.

In this study, we examined the efficacy of human umbilical cord blood mononuclear cells (hUCB-MCs), genetically modified with the VEGF and GDNF genes using adenoviral vectors, on posttraumatic regeneration after transplantation into the site of spinal cord injury (SCI) in rats. Thirty days after SCI, followed by transplantation of nontransduced hUCB-MCs, we observed an improvement in H (latency period, LP) and M(Amax) waves, compared to the group without therapy after SCI. For genetically modified hUCB-MCs, there was improvement in Amax of M wave and LP of both the M and H waves. The ratio between Amax of the H and M waves (Hmax/Mmax) demonstrated that transplantation into the area of SCI of genetically modified hUCB-MCs was more effective than nontransduced hUCB-MCs. Spared tissue and myelinated fibers were increased at day 30 after SCI and transplantation of hUCB-MCs in the lateral and ventral funiculi 2.5 mm from the lesion epicenter. Transplantation of hUCB-MCs genetically modified with the VEGF and GNDF genes significantly increased the number of spared myelinated fibers (22-fold, P > 0.01) in the main corticospinal tract compared to the nontransduced ones. HNA+ cells with the morphology of phagocytes and microglia-like cells were found as compact clusters or cell bridges within the traumatic cavities that were lined by GFAP+ host astrocytes. Our results show that hUCB-MCs transplanted into the site of SCI improved regeneration and that hUCB-MCs genetically modified with the VEGF and GNDF genes were more effective than nontransduced hUCB-MCs.

Immunocytochemical demonstration of M(1) muscarinic acetylcholine receptors at the presynaptic and postsynaptic membranes of rat diaphragm endplates.

M(1)-muscarinic acetylcholine (ACh) receptors (M(1)R) were directly demonstrated immunocytochemically in electronmicroscopic images of rat diaphragm neuromuscular junctions (NMJ). Specific electron-dense granules were located at presynaptic nerve ending membranes and in the sarcolemma in the depths of postsynaptic folds. This first visualization of M(1)R on both sides of the NMJ is in agreement with previous pharmacological data on the regulatory role of M(1)R in quantal and non-quantal ACh release.

[Posttraumatic changes of rat spinal cord after transplantation of human umbilical cord blood mononuclear cells transfected with VEGF and FGF2 genes].

Using the model of the rat spinal cord dosed contusion injury at T8 level, cross sectional area of the pathological cavities was measured and the number of myelinated nerve fibers was calculated in the outer zones of white matter after immediate single injection in the damaged area of human umbilical cord blood mononuclear cells (UCB-MC) transfected with plasmid with vegf and fgf2 genes. UCB-MC transfected with pEGFP-N2 plasmid with egfp gene of enhanced green fluorescent protein were injected into the rats of control group under similar conditions. By Day 30 after the injection of UCB-MC transfected with vegf and fgf2 genes, total cross-sectional area of the cavities in outer zones of white matter at a distance of 3 mm caudally from the epicenter of the injury was reduced more than twice as compared with that found in control group. Number of myelinated nerve fibers in the same zones of white matter at the same distance from the epicentre in rostral and caudal directions, was increased by 20% on the average as compared with control, and at a distance of 5 mm in rostral direction--by 40 to 70%. Thus, the delivery to the injury region of the therapeutic genes vegf and fgf2 reduced cavitation, restrained the processes of secondary degeneration and supported the number of myelinated fibers in the injured spinal cord.

Satellite cells of sensory neurons after various types of sciatic nerve trauma in the rat.

Sciatic nerve crushing, transection, and ligation models were used in rats to study the reactions of and changes in the numbers of satellite cells (SC) in spinal dorsal root ganglia in the lumbar segment. Nerve transection was followed by the appearance of neurons surrounded by two layers of SC. The thickness of SC processes and the areas of contacts with neurons increased as a result of invaginations into neuron perikarya. After nerve ligation, SC and their processes were located around parts of large and intermediate neurons in several tightly appressed layers; the area of contact between SC and neuron perikarya showed increased development of invaginations such that lamellar structures appeared in the SC cytoplasm, along with contacts with SC processes surrounding neighboring neurons. The greatest increases in SC numbers were seen after ligation of the nerve. Transection was followed by increases in the numbers of small and intermediate neurons surrounded by vimentin-positive SC. The number of large neurons surrounded by these cells decreased. At all time points following ligation of the nerve, all neurons in the study ganglia were surrounded by vimentin-positive SC. Post-traumatic changes in structure and numbers differed in SC associated with sensory neurons of individual size populations and depended on the type of trauma applied to efferent conductors.

[Satellite cells of the sensory neurons after different types of sciatic nerve trauma in rat].

The reaction of satellite cells (SC) and the changes in their numbers were studied in rat lumbar dorsal root ganglia using the models of sciatic nerve crush, transection and ligation. After the nerve transection, the neurons surrounded by two layers of SC were found. This was accompanied by the increased SC branch thickness and contact area due to invaginations into neuronal perikarya. After the nerve ligation, both SC and their branches were found to form several closely adjacent layers around the part of the large and medium neurons, the area of SC contact with the perikarya of neurons of these populations was increased due to more developed invaginations, there appeared the multilamellar structures in SC cytoplasm and the contacts with the branches of SC, which surrounded the neighboring neurons. The most pronounced increase in SC numbers was demonstrated after the nerve ligation. After the nerve transection, the numbers of small and medium neurons, surrounded by vimentin-positive SC, was increased. At the same time, the number of large neurons surrounded by these cells, was decreased. At all time intervals after the nerve ligation, all the neurons in the ganglia studied were surrounded by vimentin-positive SC. Post-traumatic changes in structure and numbers were different in SC, associated with specific populations of sensory neurons and depended on type of afferent conductor injury.

Comparative studies of the effects of chlorpromazine and 5,6-dihydroxytryptamine on locomotion, defensive reactions in the snail Helix lucorum, and command neuron excitability in long-term sensitization.

The actions of the neuroleptic chlorpromazine (CPZ) and the neurotoxin 5,6-dihydroxytryptamine (5,6-DHT) on defensive reactions, locomotion, formation of long-term sensitization, and the electrical characteristics of command neurons in the common snail were compared. Prolonged (chronic) treatment with CPZ led to a significant increase in the pneumostoma closure time, as well as changes in motor behavior, with a decrease in the rate of locomotion. Administration of 5,6-DHT in small daily doses for one week was accompanied by gradual decreases in the rate of locomotion of the snails, which persisted for one week. A similar effect was seen after administration of the same total dose of neurotoxin, 30 mg/kg, as a single injection. Administration of CPZ prevented the formation of long-term sensitization, as did treatment with 5,6-DHT. The procedures of treatment with CPZ, long-term sensitization, long-term sensitization followed by CPZ, and acquisition of long-term sensitization on the background of treatment with CPZ gave a locomotion speed which was directly proportional to the length of the sole. No such relationship was seen during the acquisition of long-term sensitization on the background of treatment with 5,6-DHT. Electrophysiological studies showed that chronic CPZ led to a depolarization shift in the membrane potential and a decreased action potential generation threshold in command neurons, which also occurred on treatment with 5,6-DHT. It is concluded that the actions of the neuroleptic CPZ on defensive behavior and locomotion in the common snail, as well as on the electrical characteristics of identified neurons, were similar to the toxic actions of serotonin.

Locomotor responses and neuron excitability in conditions of haloperidol blockade of dopamine in invertebrates and vertebrates.

Levels of movement activity were used to identify two groups of rats: those with high- and low-activity levels. Blockade of dopamine receptors with haloperidol led to suppression of locomotor activity in both groups of rats; in common snails, haloperidol decreased the rate of locomotion. The excitability of spinal centers in rats decreased 5 min after single i.v. injections, with gradual recovery seen by 30 min. Chronic administration of haloperidol suppressed post-tetanic potentiation of the H response in the gastrocnemius muscle of spinal rats. Prolonged use of haloperidol induced significant hyperpolarization of the membrane potential of command neurons in common snails and increased the action potential generation threshold. Selective pharmacological exclusion of the brain dopamine system was found to lead to decreases in the excitability of defined neurons in snails and the spinal motor centers in rats, also producing impairments in locomotor responses in these animals.

[Comparative investigation of influence of injections of chlorpromazine and 5,6-dihydroxytryptamine on locomotion, defensive reactions of snall helix lucorum and excitability of command neurons in long-term sensitization].

Comparative analysis of the action of chlorpromazine (CPZ) and neurotoxin 5,6-dihydroxytryptamine (5,6-DHT) on defensive reactions and locomotion of grape snail and elaboration of long-term sensitization (LTS), was carried out. Long-term (chronic) injection of chlorpromazine led to significant increasing of a pneumostome closing time and to changing of motor behaviour towards decrease of the velocity of the locomotion. Daily injections of 5,6-DHT in small doses within a week were accompanied by the gradual decrease of the velocity of snails locomotion, which was kept for a week. Similar effect was observed in injection of neurotoxin (30 mgs/kg). Injections of CPZ prevents elaboration of LTS, as well as injections of 5,6-DHT. After the action of CPZ, LTS, LTS followed by CPZ, and also during elaboration of LTS after injection of CPZ, the velocity of locomotion directly depended on the length of leg. During elaboration of LTS after injection of 5,6-DHT, such dependency is not retained. Electrophysiological study revealed that chronic injections of CPZ led to depolarizing shift of membrane potential and decrease of the threshold of action potential generation in command neurons as after injection of neurotoxin 5,6-DHT. Therefore, the action of neuroleptic drug CPZ on the defensive behaviour, locomotion of grape snail and electrical characteristics of identifying neurons is comparable with the action of toxic analogue of serotonin.

[Locomotor reactions and excitability of neurons during the blockade of dopamine by haloperidol in vertebrate and invertebrate animals]. / Lokomotornye reaktsii i vozbudimost' neironov v usloviiakh blokady dofamina galoperidolom u bespozvonochnykh i pozvonochnykh zhivotnykh.

Two groups of rats with different level of motor activities: high- and low-active animals, were distinguished. The blockade of dopamine receptors by haloperidol led to depression of locomotor activity in both groups of rats; in grape snails, haloperidol caused a decrease of the velocity of locomotor responses. In was found that within 5 minutes of intravenous injection of haloperidol the excitability of spinal centers of rats decreased; but in 30 minutes in started restoring. Chronic application of the preparation depressed the effect of posttetanic potentiation of H-response in gastrocnemius muscle of spinal rats. In command neurons of grape snail, chronic injections of haloperidol causes a significant hyperpolarization shift of membrane potential and an increase of threshold of the generation of action potential. It was shown that the selective pharmacological inhibition of dopaminergic system of the brain led to a decrease of excitability in some determined neurons of the snail and spinal motor centers of rats, as well as inhibited the locomotor responses both in vertebrate and in invertebrate animals.