Current methods for the microglia isolation: Overview and comparative analysis of approaches.

Microglia represent a distinct population of neuroglia, constituting ~ 10% of all CNS cells and exhibit high plasticity. Proper functioning of microglia is critical in the event of CNS damage due to the rapid modulation of their functions. Microglia are not only the first stage of immune defense against injury and infection, contributing to both the innate and adaptive local immune response, but also play a vital role in maintaining homeostasis of the brain and spinal cord. For this reason, microglia deserve special attention in the study of neuropathological responses. Studying microglia behavior in various in vivo models of neuropathologies is certainly a priority, as it allows us to evaluate the behavior in the context of the changing microenvironment of nervous tissue. However, sometimes there are some technological problems that hinder the identification of the features of intercellular interactions, ensured cooperation between microglia and other cell types. In this regard, the use of in vitro models remains relevant today, contributing to a more in-depth understanding of the mechanisms of microglial involvement in neuropathology. The methods considered in this review for obtaining an isolated culture of microglia, along with their advantages and disadvantages, can help researchers in selecting the appropriate source and method for obtaining these cells, thereby opening up opportunities for gaining new neurobiological knowledge.

Health care providers' awareness on medical management of children with autism spectrum disorder: cross-sectional study in Russia.

BACKGROUND: Autism spectrum disorder (ASD) is a complex developmental range of conditions that involves difficulties with social interaction and restricted/repetitive behaviors. Unfortunately, health care providers often experience difficulties in diagnosis and management of individuals with ASD, and may have no knowledge about possible ways to overcome barriers in ASD patient interactions in healthcare settings. At the same time, the provision of appropriate medical services can have positive effects on habilitative progress, functional outcome, life expectancy and quality of life for individuals with ASD. METHODS: This online survey research study evaluated the awareness and experience of students/residents (n = 247) and physicians (n = 100) in the medical management of children with ASD. It also gathered the views and experiences of caregivers to children with ASD (n = 158), all based in Russia. RESULTS: We have established that the Russian medical community has limited ASD knowledge among providers, and have suggested possible reasons for this. Based on results from online surveys completed by students/residents, non-psychiatric physicians, and caregivers of children diagnosed with ASD, the main problems pertaining to medical management of individuals with ASD were identified. Possible problem solving solutions within medical practice were proposed. CONCLUSIONS: The results from this study should be considered when implementing measures to improve healthcare practices, and when developing models for effective medical management, due to start not only in Russia but also in a number of other countries.

Upregulation of proteoglycans in the perilesion perimeter in ventral horns after spinal cord injury.

Spinal cord injury (SCI) results in pronounced focal tissue damage with subsequent formation of a glial scar that blocks axon regeneration and regrowth. Cellular changes and the composition of the extracellular matrix in regions distal from the injured area remain poorly characterized. In the present study, in the spinal cord distal to the damaged area (perilesion perimeter) there were minimal gross histological changes, but there were pronounced alterations in the extracellular proteoglycans even at 30 days after SCI. These abnormalities coincided with the appearance of reactive astrocytes and a reduction in main astrocytic glutamate transporter 1. Proteoglycan levels exhibited different kinetics and changes after SCI in areas near neuronal cell bodies and in areas distal from them. The results of the study suggest that SCI induces widespread changes in the spinal cord that may be responsible for neuronal dysfunction far from the damaged area and further aggravation of the SCI.

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.

PTEN expression in astrocytic processes after spinal cord injury.

The role of the Rho/ROCK/PTEN signaling pathway in the regulation of astrocyte function for consolidation/stabilization of the synapse has not been thoroughly studied. In this study, the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in GFAP-positive astrocytic processes in the ventral horns (VH) of the rat spinal cord has been evaluated in the normal condition and in a delayed period (30 days) after dosed contusion spinal cord injury (SCI) in caudal thoracic segments. In intact rats and at 30 days post-injury (dpi), semi-quantitative immunohistochemical analysis showed that there is approximately 2 folds less synaptophysin reactivity in the motoneuron perikarya than outside the perikarya, i.e., on dendritic spines, in the VH area. At 30 dpi, the square occupied by synaptophysin reactivity on the motoneuron perikarya and dendritic spines decreased ~2.4 and ~2.1 folds, respectively. Western blotting of the postsynaptic density protein 95 (PSD95) showed a decreased amount in the area of injury of ~3 folds at 30 dpi. Expression of GFAP in the astrocytic processes around the synaptophysin spots (APAS) was less than in the astrocytic processes that were located at distance from the synapses (APFS) both in the intact and SCI groups. In the APAS, the expression level of PTEN increased significantly after SCI. In these astrocytic processes, the PTEN expression level was significantly higher than in the APFS for both the intact and SCI rats. In the intact spinal cord, different PTEN expression levels were detected both in APAS and APFS. This may be due to the varying degree of integration of PTEN in the membrane compartment of astrocyte stem processes and possibly the increased delivery of PTEN from the GFAP-positive stem into fine GFAP-negative peripheral processes. The observed shifts after SCI reflect the imbalance in the mechanisms of synaptic plasticity after injury. Thus, strategies that have been developed for the deletion or knockdown of the PTEN gene are quite promising.

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.

Adenoviral vector carrying glial cell-derived neurotrophic factor for direct gene therapy in comparison with human umbilical cord blood cell-mediated therapy of spinal cord injury in rat.

STUDY DESIGN: Experimental study. OBJECTIVE: To evaluate the treatment of spinal cord injury with glial cell-derived neurotrophic factor (GDNF) delivered using an adenoviral vector (AdV-GDNF group) in comparison with treatment performed using human umbilical cord blood mononuclear cells (UCB-MCs)-transduced with an adenoviral vector carrying the GDNF gene (UCB-MCs+AdV-GDNF group) in rat. SETTING: Kazan, Russian Federation. METHODS: We examined the efficacy of AdV-GDNF and UCB-MCs+AdV-GDNF therapy by conducting behavioral tests on the animals and morphometric studies on the spinal cord, performing immunofluorescence analyses on glial cells, investigating the survival and migration potential of UCB-MCs, and evaluating the expression of the recombinant GDNF gene. RESULTS: At the 30th postoperative day, equal positive locomotor recovery was observed after both direct and cell-based GDNF therapy. However, after UCB-MCs-mediated GDNF therapy, the area of preserved tissue and the number of spared myelinated fibers were higher than those measured after direct GDNF gene therapy. Moreover, we observed distinct changes in the populations of glial cells; expression patterns of the specific markers for astrocytes (GFAP, S100B and AQP4), oligodendrocytes (PDGFαR and Cx47) and Schwann cells (P0) differed in various areas of the spinal cord of rats treated with AdV-GDNF and UCB-MCs+AdV-GDNF. CONCLUSION: The differences detected in the AdV-GDNF and UCB-MCs+AdV-GDNF groups could be partially explained by the action of UCB-MCs. We discuss the insufficiency and the advantages of these two methods of GDNF gene delivery into the spinal cord after traumatic injury.

Construction of recombinant adenovirus containing picorna-viral 2A-peptide sequence for the co-expression of neuro-protective growth factors in human umbilical cord blood cells.

STUDY DESIGN: Experimental study. OBJECTIVE: Several neuro-degenerative disorders such as Alzheimer's dementia, Parkinson's disease and amyotrophic lateral sclerosis (ALS) are associated with genetic mutations, and replacing or disrupting defective sequences might offer therapeutic benefits. Single gene delivery has so far failed to achieve significant clinical improvements in humans, leading to the advent of co-expression of multiple therapeutic genes. Co-transfection using two or more individual constructs might inadvertently result in disproportionate delivery of the products into the cells. To prevent this, and in order to rule out interference among the many promoters with varying strength, expressing multiple proteins in equimolar amounts can be achieved by linking open reading frames under the control of only one promoter. SETTING: Kazan, Russian Federation. METHODS: Here we describe a strategy for adeno-viral co-expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) interconnected through picorna-viral 2A-amino-acid sequence in transfected human umbilical cord blood mono-nuclear cells (hUCB-MCs). RESULTS: Presence of both growth factors, as well as absence of immune response to 2A-antigen, was demonstrated after 28-52 days. Following injection of hUCB-MCs into ALS transgenic mice, co-expression of VEGF and FGF2, as well as viable xeno-transplanted cells, were observed in the spinal cord after 1 month. CONCLUSION: These results suggest that recombinant adeno-virus containing 2A-sequences could serve as a promising alternative in regenerative medicine for the delivery of therapeutic molecules to treat neurodegenerative diseases, such as ALS.

Usage of plasmid vector carrying vegf and fgf2 genes after spinal cord injury in rats.

Using rat model of spinal cord contusion injury at TVIII, we compared the effectiveness of immediate single transplantation of human mononuclear umbilical cord blood cells transfected with pBud-VEGF-FGF2 plasmid and immediate direct injection of the same plasmid into the lesion area. The results suggest that the delivery of therapeutic genes vegf and fgf2 in cells is more effective than direct injection of plasmid DNA with the same genes (judging from the number of myelinated fibers). Better tissue preservation and motor function recovery in experiments with direct injection of plasmid pBud-VEGF-FGF2 suggest that direct gene therapy seems to be an effective additional procedure to the method of gene delivery with transfected stem and progenitor cells.