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The reconstruction of nerve continuity after traumatic nerve injury is the gold standard in hand surgery. Immediate, tension-free, end-to-end nerve suture ensures the best prognosis. The recovery is mostly promising; however, in a few cases, insufficient outcomes in motor or sensory function are observed. Intra- and extra-fascicular scarring accompanies the nerve regeneration process and limits final outcomes. Secondary nerve release in those cases is recommended. Unfortunately, scarring recurrence cannot be eliminated after secondary revision and neurolysis. The supportive influences of mesenchymal stem cells in the process of nerve regeneration were observed in many preclinical studies. However, a limited number of studies in humans have analyzed the clinical usage of mesenchymal stem cells in peripheral nerve reconstruction and revisions. The objective of this study was to evaluate the effects of undifferentiated adipose-derived stromal/stem cell injection during a last-chance surgery (neurolysis, nerve release) on a previously reconstructed nerve. Three patients (one female, two males; mean age 59 ± 4.5 years at the time of injury), who experienced failure of reconstructions of median and ulnar nerves, were included in this study. During the revision surgery, nerve fascicles were released, and adipose-derived stromal/stem cells were administered through microinjections along the fascicles and around the adjacent tissues after external neurolysis. During 36 months of follow-up, patients noticed gradual signs of sensory and in consequence functional recovery. No adverse effects were observed. Simultaneous nerve release with adipose-derived stromal/stem cells support is a promising method in patients who need secondary nerve release after nerve reconstruction. This method can constitute an alternative procedure in patients experiencing recovery failure and allow improvement in cases of limited nerve regeneration. The study protocol was approved by the Institutional Review Board (IRB) at the Centre of Postgraduate Medical Education (No. 62/PB/2016) on September 14, 2016.

The application of mesenchymal stem/stromal cells (MSC) in regenerative medicine offers hope for the effective treatment of incurable or difficult-to-heal diseases. However, it requires the development of unified protocols for both safe and efficient cell acquisition and clinical usage. The therapeutic effect of fat grafts (containing stem cells) in non-healing wounds has been discussed in previous studies, although the application requires local or general anaesthesia. The treatment of MSC derived from adipose tissue (ASC) could be a less invasive method, and efficient delivery could lead to more favourable outcomes, which should encourage clinicians to use such therapeutic approaches more frequently. Therefore, the aim of this study was to optimise the methods of ASC isolation, culture and administration while maintaining their high survival, proliferation and colonisation potential. The ASC were isolated by an enzymatic method and were characterised according to International Society for Cellular Therapy and International Federation for Adipose Therapeutics and Science guidelines. To assess the opportunity to obtain a sufficient number of cells for transplantation, long-term cell cultures in two oxygen concentrations (5% vs. 21%) were conducted. For these cultures, the population doubling time, the cumulative time for cell population doublings and the rate of cell senescence were estimated. In a developed and pre-defined protocol, ASC can be efficiently cultured at physiological oxygen concentrations (5%), which leads to faster proliferation and slower cell senescence. Subsequently, to select the optimal and minimally invasive methods of ASC transplantation, direct cell application with an irrigator or with skin dressings was analysed. Our results confirmed that both the presented methods of cell application allow for the safe delivery of isolated ASC into wounds without losing their vitality. Cells propagated in the described conditions and applied in non-invasive cell application (with an irrigation system and dressings) to treat chronic wounds can be a potential alternative or supplement to more invasive clinical approaches.

Objective/Purpose. Evaluation of efficacy and safety of autologous adipose-derived regenerative cells (ADRCs) treatment in autoimmune refractory epilepsy. Patients. Six patients with proven or probable autoimmune refractory epilepsy (2 with Rasmussen encephalitis, 2 with antineuronal autoantibodies in serum, and 2 with possible FIRES) were included in the project with approval of the Bioethics Committee. Method. Intrathecal injection of autologous ADRC acquired through liposuction followed by enzymatic isolation was performed. The procedure was repeated 3 times every 3 months with each patient. Neurological status, brain MRI, cognitive function, and antiepileptic effect were monitored during 12 months. Results. Immediately after the procedure, all patients were in good condition. In some cases, transient mildly elevated body temperature, pain in regions of liposuction, and slight increasing number of seizures during 24 hours were observed. During the next months, some improvements in school, social functioning, and manual performance were observed in all patients. One patient has been seizure free up to the end of trial. In other patients, frequency of seizures was different: from reduced number to the lack of improvement (3-year follow-up). Conclusion. Autologous ADRC therapy may emerge as a promising option for some patients with autoimmune refractory epilepsy. Based on our trial and other clinical data, the therapy appears to be safe and feasible. Antiepileptic efficacy proved to be various; however, some abilities improved in all children. No signs of psychomotor regression were observed during the first year following the treatment.

To optimise the culture conditions for human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O2 has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs, however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.

Tuning stem cells microenvironment in vitro may influence their regenerative properties. In this study Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) were encapsulated in 3D hydrogels derived from human fibrin (FB) or platelet lysate (PL) and the oxygen level was adjusted to physiological normoxia (5% O2). The influence of the type of the scaffold and physiological normoxia conditions was tested on the WJ-MSCs' survivability, proliferation, migratory potential, the level of expression of selected trophic factors, cytokines, and neural markers. Encapsulated WJ-MSCs revealed high survivability, stable proliferation rate, and ability to migrate out of the hydrogel and the up-regulated expression of all tested factors, as well as the increased expression of neural differentiation markers. Physiological normoxia stimulated proliferation of encapsulated WJ-MSCs and significantly enhanced their neuronal, but not glial, differentiation. Ex vivo studies with indirect co-culture of organotypic hippocampal slices and cell-hydrogel bio-constructs revealed strong neuroprotective effect of WJ-MSCs against neuronal death in the CA1 region of the rat hippocampus. This effect was potentiated further by FB scaffolds under 5% O2 conditions. Our results indicating significant effect of oxygen and 3D cytoarchitecture suggest the urgent need for further optimization of the microenvironmental conditions to improve therapeutical competence of the WJ-MSCs population.

Regardless of enormous translational progress in stem cell clinical application, our knowledge about biological determinants of transplantation-related protection is still limited. In addition to adequate selection of the cell source well dedicated to a specific disease and optimal standardization of all other pre-transplant procedures, we have decided to focus more attention to the impact of culture time and environment itself on molecular properties and regenerative capacity of cell cultured in vitro. The aim of this investigation was to determine neuroprotection-linked cell phenotypic and functional changes that could spontaneously take place when freshly isolated Wharton's jelly mesenchymal stem cell (WJ-MSC) undergo standard selection, growth, and spontaneous differentiation throughout passaging in vitro. For determining their neuroprotective potential, we used experimental model of human WJ-MSC co-culture with intact or oxygen-glucose-deprived (OGD) rat organotypic hippocampal culture (OHC). It has been shown that putative molecular mechanisms mediating regenerative interactions between WJ-MSC and OHC slices relies mainly on mesenchymal cell paracrine activity. Interestingly, it has been also found that the strongest protective effect is exerted by the co-culture with freshly isolated umbilical cord tissue fragments and by the first cohort of human mesenchymal stem cells (hMSCs) migrating out of these fragments (passage 0). Culturing of WJ-derived hMSC in well-controlled standard conditions under air atmosphere up to fourth passage caused unexpected decline of neuroprotective cell effectiveness toward OGD-OHC in the co-culture model. This further correlated with substantial changes in the WJ-MSC phenotype, profile of their paracrine activities as well as with the recipient tissue reaction evaluated by changes in the rat-specific neuroprotection-linked gene expression.

Objective. Bone defects or atrophy may arise as a consequence of injury, inflammation of various etiologies, and neoplastic or traumatic processes or as a result of surgical procedures. Sometimes the regeneration process of bone loss is impaired, significantly slowed down, or does not occur, e.g., in congenital defects. For the bone defect reconstruction, a piece of the removed bone from ala of ilium or bone transplantation from a decedent is used. Replacement of the autologous or allogenic source of the bone-by-bone substitute could reduce the number of surgeries and time in the pharmacological coma during the reconstruction of the bone defect. Application of mesenchymal stem cells in the reconstruction surgery may have positive influence on tissue regeneration by secretion of angiogenic factors, recruitment of other MSCs, or differentiation into osteoblasts.Materials and Methods. Mesenchymal stem cells derived from the umbilical cord (Wharton’s jelly (WJ-MSC)) were cultured in GMP-grade DMEM low glucose supplemented with heparin, 10% platelet lysate, glucose, and antibiotics.In vitroWJ-MSCs were seeded on the bone substitute Bio-Oss Collagen® and cultured in the StemPro® Osteogenesis Differentiation Kit. During the culture on the 1st, 7th, 14th, and 21st day (day in vitro (DIV)), we analyzed viability (confocal microscopy) and adhesion capability (electron microscopy) of WJ-MSC on Bio-Oss scaffolds, gene expression (qPCR), and secretion of proteins (Luminex).In vivoBio-Oss® scaffolds with WJ-MSC were transplanted to trepanation holes in the cranium to obtain their overgrowth. The computed tomography was performed 7, 14, and 21 days after surgery to assess the regeneration.Results. The Bio-Oss® scaffold provides a favourable environment for WJ-MSC survival. WJ-MSCs in osteodifferentiation medium are able to attach and proliferate on Bio-Oss® scaffolds. Results obtained from qPCR and Luminex® indicate that WJ-MSCs possess the ability to differentiate into osteoblast-like cells and may induce osteoclastogenesis, angiogenesis, and mobilization of host MSCs. In animal studies, WJ-MSCs seeded on Bio-Oss® increased the scaffold integration with host bone and changed their morphology to osteoblast-like cells.Conclusions. The presented construct consisted of Bio-Oss®, the scaffold with high flexibility and plasticity, approved for clinical use with seeded immunologically privileged WJ-MSC which may be considered reconstructive therapy in bone defects.

Human somatic stem cells can be identified and isolated from different types of tissues and are grouped here based on their developmental maturation and ability to undergo neural differentiation. The first group will represent afterbirth somatic tissues, which are perinatal stem cells including placental blood and tissue, amniotic fluid and tissue, and umbilical cord blood- and umbilical cord tissue-derived cells. The second group of cells discussed in this chapter is the adult stem cells, generally those in a transient period of development, thus placing them in the special position of transitioning from the perinatal to young somatic tissue, and they include the menstrual blood-, the peripheral blood-, and the bone marrow-derived stem cells.

Stem cells (SCs) may constitute a perspective alternative to pharmacological treatment in neurodegenerative diseases. Although the safety of SC transplantation has been widely shown, their clinical efficiency in amyotrophic lateral sclerosis (ALS) is still to be proved. It is not only due to a limited number of studies, small treatment groups, and fast but nonlinear disease progression but also due to lack of objective methods able to show subtle clinical changes. Preliminary guidelines for cell therapy have recently been proposed by a group of ALS experts. They combine clinical, neurophysiological, and functional assessment together with monitoring of the cytokine level. Here, we describe a pilot study on transplantation of autologous adipose-derived regenerative cells (ADRC) into the spinal cord of the patients with ALS and monitoring of the results in accordance with the current recommendations. To show early and/or subtle changes within the muscles of interest, a wide range of clinical and functional tests were used and compared in order to choose the most sensitive and optimal set. Additionally, an analysis of transplanted ADRC was provided to develop standards ensuring the derivation and verification of adequate quality of transplanted cells and to correlate ADRC properties with clinical outcome.

Cell therapy has been shown to be a key clinical therapeutic option for central nervous system diseases or damage. Standardization of clinical cell therapy procedures is an important task for professional associations devoted to cell therapy. The Chinese Branch of the International Association of Neurorestoratology (IANR) completed the first set of guidelines governing the clinical application of neurorestoration in 2011. The IANR and the Chinese Association of Neurorestoratology (CANR) collaborated to propose the current version “Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017)”. The IANR council board members and CANR committee members approved this proposal on September 1, 2016, and recommend it to clinical practitioners of cellular therapy. These guidelines include items of cell type nomenclature, cell quality control, minimal suggested cell doses, patient-informed consent, indications for undergoing cell therapy, contraindications for undergoing cell therapy, documentation of procedure and therapy, safety evaluation, efficacy evaluation, policy of repeated treatments, do not charge patients for unproven therapies, basic principles of cell therapy, and publishing responsibility. Refereed/Peer-reviewed

Human somatic stem cells can be identified and isolated from different types of tissues and are grouped here based on their developmental maturation and ability to undergo neural differentiation. The first group will represent afterbirth somatic tissues, which are perinatal stem cells including placental blood and tissue, amniotic fluid and tissue, and umbilical cord blood- and umbilical cord tissue-derived cells. The second group of cells discussed in this chapter is the adult stem cells, generally those in a transient period of development, thus placing them in the special position of transitioning from the perinatal to young somatic tissue, and they include the menstrual blood-, the peripheral blood-, and the bone marrow-derived stem cells.

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

Currently, there are no effective therapies to restore lost brain neurons, although rapid progress in stem cell biology and biomaterials development provides new tools for regeneration of central nervous system. Here we describe neurogenic properties of bioactive scaffolds generated by cryogelation of dextran or gelatin linked to laminin - the main component of brain extracellular matrix. We showed that such scaffolds promoted differentiation of human cord blood-derived stem cells into artificial neural tissue in vitro. Our experiments revealed that optimal range of scaffolds' pore size for neural tissue engineering was 80-100 microns. We found that scaffold seeded with undifferentiated, but not neutrally committed stem cells, gave optimal cell adhesion and proliferation in "niche"-like structures. Subsequent differentiation resulted in generation of mature 3D networks of neurons (MAP2+) and glia (S100beta+) cells. We showed that cryogel scaffolds could be transplanted into the brain tissue or organotypic hippocampal slices in a rat models. The scaffolds did not induced inflammation mediated by microglial cells (ED1-, Ox43-, Iba1-) and prevented formation of glial scar (GFAP-). Contrary, laminin-rich scaffolds attracted infiltration of host's neuroblasts (NF200+, Nestin+) indicating high neuroregeneration properties.

The umbilical cord blood-derived neural stem/progenitor cells (HUCB-NSCs) potentially represent a rich source of transplantable material for treatment of a wide range of neurological diseases. Although, recently reported effects of their implementation in animal models of brain pathology are still controversial. As a simplified alternative to in vivo transplantation in this work we have applied a long-term organotypic rat hippocampal slice culture (OHC) as a recipient tissue to study bilateral graft/host cells interactions ex vivo. This type of culture can be considered as a kind of reductionistic model of brain transplantation where direct influence of systemic immunological responses to transplanted human cells would be excluded. The transplantation material derived from a HUCB-NSC line developed and characterized in our laboratory and delivered to the slices either as a single-cell suspension or after formation of typical neurospheres in serum-free medium in vitro (N-HUCBs). Experiments were focused on space-temporal context of cell transplantation in relation to their ability to ingrown, migrate, and differentiate within the slice cytoarchitecture. We gain evidences that these responses are strictly dependent on the engraftment site and that cell movement reflects typical routes used for migratory neuroblasts in vivo. The cells implanted at the second week of slice cultivation ingrown readily and deeply into host cytoarchitecture then matured to the level never observed in our transplantation animal models in vivo. Importantly, transplanted neurospheres, in addition to yield exogenous migratory cells to the host tissue can locally inhibit astrocytosis and promote outgrow of DCX-reactive neuroblasts in the surrounding OHC tissue.

Deep-brain lacunar infarct represents a significant clinical problem as it produces severe symptoms highly resistant to rehabilitation. The limited area of necrosis may facilitate neurorepair via the action of various novel neuroprotective strategies including cell-based therapies. The lesion was induced by stereotactic injection of ouabain into adult rat brains. Subsequent behavioral testing involved beam walking task, rotarod, visual discrimination task and apomorphine rotation. For morphological and topographical analysis brain slices were stained with H-E and evaluated under light microscopy. Lesion size was measured in absolute terms and in relation to the whole brain volume. Immunohistochemical analysis for the co-localization of BrdU with specific cell-type markers (PSA-NCAM, NG2, beta-tubulin III, GFAP, ED1) have has been performed, to determine the fate of newly generated cells with emphasis on evidence of neurogenesis. The lesion involved the basal ganglia, basal forebrain nuclei, internal capsule and striatum (just 1-2% of total brain volume). Significant and relatively stable behavioral deficits were observed up to 30 days. Furthermore, large numbers of cells are seen to be newly generated in response to injury with a significant proportion of these being present on account of neurogenesis.

The access of transplanted cells to large areas of the CNS is of critical value for cell therapy of chronic diseases associated with widespread neurodegeneration. Intrathecal cell application can match this requirement. Here we describe an efficient method for cell injection into the cisterna magna and the assessment of the cell distribution within subarachnoidal space in mice. In order to maximize cell distribution we applied a "concord-like" position, where the cisterna magna is nearly the highest point of the animal's body. A drop of saline on the needle insertion site avoided the outflow of transplanted cells from subarachnoidal space with CSF during surgery. Twenty-four hours later the preparation of the CNS with an intact dura mater by a suitable dissection technique (described in detail) revealed approx. 80% of the injected cells (100,000 cells per animal) within the subarachnoidal space ranging from the skull base (olfactory nerve to premedullary cisterns) to the IV ventricle, and to both the ventral and dorsal surfaces of the spinal cord. Thus the "concorde-like" position proved to be very useful for intrathecal cell application leading to a widespread cell distribution within the subarachnoidal space.

Ischemic stroke results in violent impairment of tissue homeostasis leading to severe perturbation within the neurovascular unit (NVU) during the recovery period. The aim of this study was to assess the potential of mesenchymal stem cells (MSCs) originating from Wharton's jelly (WJ) to differentiate into functionally competent cells of endothelial lineage (WJ-EPCs). The protective effect(s) of either primary WJ-MSCs or induced WJ-EPCs was investigated and compared after oxygen–glucose deprivation (OGD) of hippocampal organotypic slices (OHC) in the indirect coculture model. WJ-MSCs, primed in EGM-2 (Lonza commercial medium) under 5% O2, acquired cobblestone endothelial-like morphology, formed capillary-like structures and actively took up DiI-Ac-LDL. Both cell types (WJ-MSCs and WJ-EPCs) were positive for CD73, CD90, CD105, VEGFR-2, and VEGF, but only endothelial-like culture expressed vWF and PECAM-1 markers at significant levels. In the presence of either WJ-MSCs or WJ-EPCs in the compartment below OGD-injured slices, cell death and vascular atrophy in the hypoxia-sensitive CA1 region were substantially decreased. This suggests that a paracrine mechanism may mediate WJ-MSC- and WJ-EPC-dependent protection. Thus, finally, we estimated secretion of the neuro/angio/immunomodulatory molecules IL-6, TGF-β1, and VEGF by these cell cultures. We have found that release of TGF-β1 and IL-6 was TLR ligand [LPS and Poly(I:C)] concentration dependent and stronger in WJ-EPC than WJ-MSC cultures. Simultaneously, the uneven pattern of TLR receptors and modulatory cytokine gene expression was confirmed also on qRT-PCR level, but no significant differences were noticed between WJ-EPC and primary WJ-MSC cultures.

Substantial inconsistencies in mesenchymal stem (stromal) cell (MSC) therapy reported in early translational and clinical studies may indicate need for selection of the proper cell population for any particular therapeutic purpose. In the present study we have examined stromal stem cells derived either from umbilical cord Wharton's Jelly (WJ-MSC) or bone marrow (BM-MSC) of adult, healthy donors. The cells characterized in accordance with the International Society for Cellular Therapy (ISCT) indications as well as other phenotypic and functional parameters have been compared under strictly controlled culture conditions. WJ-MSC, in comparison with BM-MSC, exhibited a higher proliferation rate, a greater expansion capability being additionally stimulated under low-oxygen atmosphere, enhanced neurotrophic factors gene expression and spontaneous tendency toward a neural lineage differentiation commitment confirmed by protein and gene marker induction. Our data suggest that WJ-MSC may represent an example of immature-type "pre-MSC," where a substantial cellular component is embryonic-like, pluripotent derivatives with the default neural-like differentiation. These cells may contribute in different extents to nearly all classical MSC populations adversely correlated with the age of cell donors. Our data suggest that neuro-epithelial markers, like nestin, stage specific embryonic antigens-4 or α-smooth muscle actin expressions, may serve as useful indicators of MSC culture neuro-regeneration-associated potency.

Objective The exposure of human umbilical cord blood mononuclear cells devoid of hematopoietic stem cells (HUCB-MNCs CD34- ) to defined culture condition promotes their conversion into neural lineage. We have asked the question if observed fate change of HUCB-MNCs CD34- results from direct conversion of hematopoietic precursors into neural-like phenotypes due to expression of overlapping genetic program or, alternatively, these neural phenotypes arise from sequential differentiation of more primitive progenitors (embryonic-like cells) preexisting in HUCB-MNCs CD34- fraction. Materials and Methods HUCB-MNCs negatively selected for CD34 antigens were cultured in vitro up to 14 days. Changes in stem/neural cell genes and proteins were successively evaluated during this period and after evoked neuronal differentiation of cells in the presence of RA or BDNF or cocultured with neonatal rat brain astrocytes. Results Freshly isolated HUCB-MNCs CD34- expressed pluripotent cell markers: Oct3/4 , Sox2 , and Rex1 genes. During 24 hours of culture the frequency of Oct3/4 immunopositive cells increased markedly with parallel enlargement of "side population" and CD133 + cell appearance. Concomitantly, cultured cells start to form aggregates and express pro-neural genes, i.e., enhanced Sox2 , OTX1 , Nestin , GFAP , and NF-200 . During the next days of culture immunoreactions for β-tubulin III, MAP2, GFAP, S100β, Doublecortin, and GalC were induced with reciprocal lowering of stem cell gene and protein markers. At this stage cells successively adhered to the bottom, dispersed, and decreased proliferation rate (Ki67 expression). Additional treatments with neuromorphogenes or coculturing with rat brain primary culture induced further differentiation of these neural precursors toward more advanced neuronal phenotypes. Conclusions HUCB-MNCs CD34- fraction contains embryonic-like stem/progenitor cells which increase rapidly but transiently in culture, then differentiate spontaneously after cell aggregate adhesion toward neural lineage. Neurally promoted cells from 10-14 DIV culture acquire three main neural-like phenotypes, i.e., neurons, astrocytes, and oligodendrocytes. In this respect they are promising candidates for experimental treatment of neuronal injury; however, the final proof for conversion of HUCB cells to neural cells can be obtained through transplantation experiments.

The ability of stem and progenitor cells to proliferate and differentiate into other lineages is widely viewed as a characteristic of stem cells. Previously, we have reported that cells from a CD34(-) (nonhematopoietic) adherent subpopulation of human cord blood can acquire a feature of multipotential neural progenitors in vitro. In the present study, using these cord blood-derived stem cells, we have established a clonal cell line termed HUCB-NSCs (human umbilical cord blood-neural stem cells) that expresses several neural antigens and has been grown in culture for more than 60 passages. During this time, HUCB-NSCs retained their growth rate, the ability to differentiate into neuronal-, astrocyte-, and oligodendrocyte-like cells and displayed a stable karyotype. DNA microarray analysis of HUCB-NSCs revealed enhanced expression of selected genes encoding putative stem and progenitor cell markers when compared to other mononuclear cells. dBcAMP-induced HUCBNSCs were further differentiated into more advanced neuronal cells. This is the first report of the establishment and characterization of a nontransformed HUCB-NSC line that can be grown continuously in a monolayer culture and induced to terminal differentiation. These cells should further our understanding of the regulatory mechanisms involved in NSC self-renewal and differentiation.

In vitro studies conducted by our research group documented that neural progenitor cells can be selected from human umbilical cord blood (HUCB-NPs). Due to further expansion of these cells we have established the first human umbilical cord blood-derived neural-like stem cell line (HUCB-NSC) growing in serum-free (SF) or low-serum (LS) medium for over 3 years. The purpose of the study was to evaluate the neurogenic potential of HUCB-NSCs cultured in SF and LS condition in different in vitro settings before transplantation. We have shown that the number of cells attaining neuronal features was significantly higher for cultures expanded in LS than in SF condition. Moreover, the presence of neuromorphogens, cultured rat astrocytes or hippocampal slices promoted further differentiation of HUCB-NSCs into neural lineage much more effectively when the cells had derived from LS cultures. The highest response was observed in the case of co-cultures with rat primary astrocytes as well as hippocampal organotypic slices. However, the LS cells co-cultured with hippocampal slices expressed exclusively a set of early and late neuronal markers whereas no detection of cells with glial-specific markers was possible. In conclusion, certain level of stem/progenitor cell commitment is important for optimal response of HUCB-NSC on the neurogenic signals provided by surrounding environment in vitro.

Neural stem cell line developed from human umbilical cord blood (HUCB-NSC) [Buzańska et al., 2003. Journal of Neurochemistry 85, 33] is an ethically uncontroversial source of stem cells, able to differentiate into neuronal, astrocytic and oligodendroglial lineages. Developmental fate decisions of HUCB-NSC can be experimentally manipulated in vitro by the presence of trophic factors, mitogenes and neuromorphogenes, but can also be influenced by neurotoxins. In this report two-dimensional (2-D) and three-dimensional (3-D) HUCB-NSC cultures are introduced as useful models for testing developmental neurotoxicity. For 2-D culture models we established a standardized method for the assessment of the growth rate and cell differentiation in 96-well plates. The proliferative capacity of the HUCB-NSC was monitored by the MTT test while their ability to differentiate into neural-like cells by immunocytochemistry of beta-tubulin III and MAP-2 for neurons, GFAP and S-100beta for astrocytes and GalC for oligodendrocytes. The 3-D culture of HUCB-NSC is represented by neurospheres. Proliferation and migration of the intermediate precursors from attached neurospheres are shown to be controlled and altered by various growth factors and further modulated by the extracellular matrix component-fibronectin. Thus, neurospheres derived from the HUCB-NSC line can represent a suitable model of the activation of dormant stem cells residing in their niche, and can be used for neurotoxic studies.

Fetal cells with the characteristics of neural stem cells (NSCs) can be derived from the nonhematopoietic fraction of human umbilical cord blood (HUCB), expanded as a nonimmortalized cell line (HUCB-NSC), and further differentiated into neuron-like cells (HUCB-NSCD); however, the functional and neuronal properties of these cells are poorly understood. To address this issue, we used whole-cell patch-clamp recordings, gene microarrays, and immunocytochemistry to identify voltage-gated channels and ligand-gated receptors on HUCB-NSCs and HUCB-NSCDs. Gene microarray analysis identified genes for voltage-dependent potassium and sodium channels and the neurotransmitter receptors acetylcholine (ACh), gamma-aminobutyric acid (GABA), glutamate, glycine, 5-hydroxytryptamine (5-HT), and dopamine (DA). Several of these genes (GABA-A, glycine and glutamate receptors, voltage-gated potassium channels, and voltage-gated sodium type XII alpha channels) were not expressed in the HUCB mono-nuclear fraction (HUCB-MC), which served as a starting cell population for HUCB-NSC. HUCB-NSCD acquired neuronal phenotypes and displayed an inward rectifying potassium current (Kir) and an outward rectifying potassium current (I(K+)). Kir was present on most HUCB-NSCs and HUCB-NSCDs, whereas I(K+) was present only on HUCB-NSCDs. Many HUCB-NSCDs were immunopositive for glutamate, glycine, nicotinic ACh, DA, 5-HT, and GABA receptors. Kainic acid (KA), a non-N-methyl-D-asparate (NMDA) glutamate-receptor agonist, induced an inward current in some HUCB-NSCDs. KA, glycine, DA, ACh, GABA, and 5-HT partially blocked Kir through their respective receptors. These results suggest that HUCB-NSCs differentiate toward neuron-like cells, with functional voltage- and ligand-gated channels identified in other neuronal systems.

Two different models of brain ischemia were used to examine the evoked changes in the tyrosine phosphorylation of NMDA receptor subunits 2A and 2B (NR2A and NR2B), as well as their interactions with non-receptor tyrosine kinases (NRTKs: FAK, PYK2 Src), and PSD-95 protein. Only short-term 5 min ischemia followed by 3 h reperfusion resulted in the elevated tyrosine phosphorylation of both investigated NMDA receptor subunits, but in contrast to previously published data, more pronounced in the case of NR2B. Concomitantly, an increased association of NR2B with FAK, PYK2, Src and PSD-95 has been observed. This sharp early reaction to brief ischemia was markedly attenuated during prolonged recovery (72 h) with almost complete return to control values. The initial recruitment of tyrosine kinases to NMDA receptor during the first 3 h of reperfusion is generally consistent with an active postischemic remodeling of PSD and may participate in the induction of the postischemic signal transduction pathway in gerbil hippocampus. In contrast, ischemia of longer duration (up to 30 min) caused an immediate decrease in the protein levels as well as tyrosine phosphorylation of both NR2A and NR2B subunits which was accompanied by the marked attenuation of the association with their investigated molecular partners--PSD-95 and NRTKs. This effect may be mimicked in vitro by Ca2+-dependent activation of endogenous calpains in purified PSD preparation suggesting irreversible deterioration of the synaptic signaling machinery during irreversible long-term ischemia.

The efflux of mitochondrial protein cytochrome C to cytoplasm is one of the key events of mitochondrial dysfunction observed in post-ischemic pathology. We investigated the effect of intra-carotid infusion of 5-10 mg/kg of cyclosporin A (CsA) on the neuronal survival in CA1 sector of hippocampus and on the subcellular localization of cytochrome C in the model of 5 min gerbil brain ischemia. To discriminate between the immunosuppressive and the mitochondria protecting component of CsA action, we compared the effect of CsA with one other immunosuppressant FK506. Almost 75% of neurons in ischemia-affected brain area were saved after CsA but not after FK506 treatment. This protective effect was only observed when the drug was infused immediately upon reperfusion. Early CsA treatment was able to block an initial phase of cytochrome C release, occurring transiently at 30 min post-ischemia, an effect never observed after FK506 administration. We assessed the neuroprotective potency of CsA vs. FK506 in rat cortical primary culture treated with compounds that mimic destructive signals induced by brain ischemia. In all cases, neuronal death and cytochrome C release were evidently suppressed by CsA applied not later than 30 min after the initial insult. Thus, early treatment with CsA in vitro and after bolus intra-carotid injection in vivo can save neurons by inhibition of cytochrome C efflux to cytoplasm.

Paralytic tremor (pt) is a sex-linked mutation in rabbit that affects myelination of the CNS. Myelin in the pt brains represents approximately 30% of the normal levels. Previously we showed that the pt mutation affects primarily proteolipid protein (Plp) gene expression. In the present study we investigated the relative effect of the pt mutation on two distinctive Plp gene products, PLP- and DM-20-specific messenger RNAs. Our results showed that both PLP and DM-20 are affected and that the ratio DM-20/PLP was higher in pt rabbits than in age-matched controls. We sequenced normal rabbit PLP cDNA and characterized pt mutation at the DNA level. Rabbit PLP sequence, deduced from cDNA, differs from the human protein only at Thr198. Sequence analysis of the mutant cDNA revealed a transversion T-->A in exon 2 of the Plp gene. This point mutation, which is placed at the end of the first potential transmembrane domain, results in a substitution of His36 by a glutamine. This transversion abolishes a restriction site that enabled us to screen a large number of animals and observe a perfect correlation between the pt allele and the abnormal phenotype.

Neural stem cells are clonogenic, self-renewing cells with the potential to differentiate into brain-specific cell lines. Our study demonstrates that a neural-stem-cell-like subpopulation can be selected and expanded in vitro by the use of human umbilical cord blood cells, which are a relatively easily available starting material. Through a combination of antigen-driven magnetic cell sorting and subfractionation according to cell surface adhesive properties, we have isolated a clonogenic fraction devoid of hematopoietic or angiogenetic properties but with relatively high self-renewal potency. The resulting clones express nestin, a neurofilament protein that is one of the most specific markers of multipotent neural stem cells. In the presence of selected growth factors or in the rat brain co-culture system, the progeny of these cells can be oriented towards the three main neural phenotypes: neurons,astroglia and oligodendroglia. The cells show high commitment (about 30% and 40% of the population) to neuronal and astrocytic fate, respectively. Interestingly, upon differentiation, the neural-type precursor cells of cord blood origin also give rise to a relatively high proportion of oligodendrocytes — 11% of the total population of differentiating cells.

A stroke in humans may induce focal injury to the brain tissue resulting in various disabilities. Although motor deficits are the most discernible, cognitive impairments seem to be crucial for patients mental well-being. The current lack of effective treatments encourages scientists and clinicians to develop novel approaches. Before applying them in clinic, testing for safety and effectiveness in non-human models is necessary. Such animal model should include significant cognitive impairments resulting from brain lesion. We used ouabain stereotactic injection into the right dorsolateral striatum of male Wistar rats, and enriched environment housing. To confirm the brain injury before cognitive testing, rats were given a beam-walking task to evaluate the level of sensorimotor deficits. To determine the cognitive impairment after focal brain damage, rats underwent a set of selected tasks over an observation period of 30 days. Brain injury induced by ouabain significantly impaired the acquisition of the T-maze habit learning task, where 'win-stay' strategy rules were applied. The injured rats also showed significant deficits in the performance of the T-maze switching task, which involved shifting from multiple clues previously relevant to the only one important clue. Focal brain injury also significantly changed 'what--where' memory, tested in the object exploration task, in which a novel object consecutively appeared in the same place while the location of a familiar item was continuously changed. In conclusion, we developed an animal model of distinct cognitive impairments after focal brain injury that provides a convenient method to test the effectiveness of restorative therapies.

The aim of our study was to investigate whether a human neural cell line could be used as a reliable screening tool to examine the functional conservation, in humans, of transcription factors involved in neuronal or glial specification in other species. Gain-of-function experiments were performed on DEV cells, a cell line derived from a human medulloblastoma. Genes encoding nine different transcription factors were tested for their influence on the process of specification of human DEV cells towards a neuronal or glial fate. In a first series of experiments, DEV cells were transfected with murine genes encoding transcription factors known to be involved in the neuronal differentiation cascade. Neurogenins-1, -2, and -3; Mash-1; and NeuroD increased the differentiation of DEV cells towards a neuronal phenotype by a factor of 2-3.5. In a second series of experiments, we tested transcription factors involved in invertebrate glial specification. In the embryonic Drosophila CNS, the development of most glial cells depends on the master regulatory gene glial cell missing (gcm). Expression of gcm in DEV cells induced a twofold increase of astrocytic and a sixfold increase of oligodendroglial cell types. Interestingly, expression of tramtrack69, which is required in all Drosophila glial cells, resulted in a fourfold increase of only the oligodendrocyte phenotype. Expression of the related tramtrack88 protein, which is not expressed in the fly glia, or the C. elegans lin26 protein showed no effect. These results show that the Drosophila transcription factor genes tested can conserve their function upon transfection into the human DEV cells, qualifying this cell line as a screening tool to analyze the mechanisms of neuronal and glial specification.

The cellular processes with a potential to lead to delayed death of neurons following transient (5 min) ischemia in gerbil hippocampus were evaluated. Neuronal apoptosis, visualized by the terminal transferase dUTP nick-end labelling (TUNEL) reaction, selectively appeared in the CA1 region of the pyramidal cell layer between the third and fourth days after the insult. Concomitantly, an enhanced immunoreactivity to anti-cJun/AP1 (N) antibody as a major component of activator protein 1 (AP1) transcriptional factor was observed in CA1 neurons. In contrast, in the early postischemic phase, the cJun/AP1 reaction was noticed in numerous neurons and glia-like cells of the CA2/CA3 region, hilus of the dentate gyrus, and region of mossy fiber terminals. In parallel, hippocampal protein binding to AP1, measured by the electrophoretic mobility shift assay (EMSA), showed biphasic enhancement at 3 and then 72-120 hours after ischemia. Supershifts, with antibodies against c-Fos and phospho-c-Jun constituencies of the AP1 dimer, revealed an increased amount of phosphorylated c-Jun in the late postischemic phase. Collectively, these results suggest diversity of AP1 complex function, regulated by its dimer composition as well as time and place of expression during postischemic reperfusion. The early, survival-supporting AP1 response, located mainly in ischemia-resistant areas of CA2/3, is followed by the delayed phase, characteristic of massive neuronal apoptosis in CA1 with concomitant increase of phospho-c-Jun in AP1 dimer.

Calpains, Ca(2+)-dependent neutral proteinases (microM and mM Ca(2+)-sensitive), and their endogenous inhibitor calpastatin were examined in rat brain. Specific activity of m-calpain exceeded almost 10 times that of mu-calpain, and the both isoforms of calpain together with calpastatin were mainly located in the soluble fraction of homogenate. Acute postdecapitative ischemia of 15 min duration resulted in a gradual, time-dependent decrease of total mu-calpain activity (to 60% of control values) and in the moderate elevation of calpastatin activity (by 28%). The decrease of total mu-calpain activity coincided with its remarkable increase (above 300% of control values) in particulate fraction. In the case of m-calpain, the only observed effect of ischemia was its redistribution and, as a consequence, the elevation of activity in particulate fraction. The accumulation of breakdown products, resulting from calpain-catalyzed proteolysis of fodrin (as revealed by Western blotting) indicated activation of calpain under ischemia. The findings suggest that this rapid activation involves partial enzyme translocation toward membranes, and is followed (at least in acute phase) by mu-calpain downregulation and increased calpastatin activity.

Cerebral ischemia is known to modify the expression of genetic information in the brain. To complement this knowledge, in the present study we have estimated the expression of calcium- and phospholipid-dependent (classical) protein kinase C (c PKC) isoform mRNAs (alpha, beta1 and gamma) at different time following ischemia. Forebrain cerebral ischemia was performed on Mongolian gerbils by 5 minutes bilateral occlusion of common carotid arteries. At the pointed time the cytoplasmic RNA was extracted from hippocampus and the expression of PKC mRNA quantified by RT PCR technique using GAPDH expression as an internal standard. Results indicate that only one gamma isoform of cPKC mRNA expression becomes significantly modified in postischemic hippocampus. A transient increase up to 145% of control within the first 3 h was followed by its decline to 60-65% at a longer recirculation period. This lowered levels returned back to control at 72 h postischemic recovery. This result indicates that gamma PKC could be particularly sensitive to ischemic insult and would react in accordance with the other early signals determining ischemic outcome.

Background aims As we approach the era of mesenchymal stem cell (MSC) application in the medical clinic, the standarization of their culture conditions are of the particular importance. We re-evaluated the influences of oxygens concentration on proliferation, stemness and differentiation of human umbilical cord Wharton Jelly–derived MSCs (WJ-MSCs). Methods Primary cultures growing in 21% oxygen were either transferred into 5% O 2 or continued to grow under standard 21% oxygen conditions. Cell expansion was estimated by WST1/enzyme-linked immunosorbent assay or cell counting. After 2 or 4 weeks of culture, cell phenotypes were evaluated using microscopic, immunocytochemical, fluorescence-activated cell-sorting and molecular methods. Genes and proteins typical of mesenchymal cells, committed neural cells or more primitive stem/progenitors (Oct4A, Nanog, Rex1, Sox2) and hypoxia inducible factor (HIF)-1α–3α were evaluated. Results Lowering O 2 concentration from 21% to the physiologically relevant 5% level substantially affected cell characteristics, with induction of stemness-related-transcription-factor and stimulation of cell proliferative capacity, with increased colony-forming unit fibroblasts (CFU-F) centers exerting OCT4A, NANOG and HIF-1α and HIF-2α immunoreactivity. Moreover, the spontaneous and time-dependent ability of WJ-MSCs to differentiate into neural lineage under 21% O 2 culture was blocked in the reduced oxygen condition. Importantly, treatment with trichostatin A (TSA, a histone deacetylase inhibitor) suppressed HIF-1α and HIF-2α expression, in addition to blockading the cellular effects of reduced oxygen concentration. Conclusions A physiologically relevant microenvironment of 5% O 2 rejuvenates WJ-MSC culture toward less-differentiated, more primitive and faster-growing phenotypes with involvement of HIF-1α and HIF-2α–mediated and TSA-sensitive chromatin modification mechanisms. These observations add to the understanding of MSC responses to defined culture conditions, which is the most critical issue for adult stem cells translational applications.

Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin–laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Wharton's jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the host's immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Here we provide a comprehensive data on the unique features of mesenchymal stem cells (MSCs) which makes them feasible and preferred candidate for cell-based therapy in neurological clinic. From this point of view the most important features of these cells are: (1) availability from autologous sources independently from age of patient; (2) extensive expansion in vitro; (3) immunomodulatory "bystander" function after transplantation in vivo; (4) potentiality to protect, repair or eventually replace impaired or dysfunctional host cells. For complete this last task of functional regeneration of central nervous system, we have to take advantages of MSCs capability for transient, time-locked proliferation, migration to site of injury and their commitment to neuronal differentiation. However, if we are to make progress in the use of MSCs for therapy in the clinic it will be necessary to establish more unified, advanced standards for cells processing in vitro as well as safer and improved procedures for their delivery in vivo.

The activity of Ca2+/calmodulin-dependent protein kinase II (CaM-KII) during short-term global ischaemia was investigated in the gerbil brain hippocampus and cortex. Ischaemia of 0.5 min duration significantly stimulated Ca(2+)-independent 'autonomous' activity, indicating activation of the first step of intramolecular enzyme phosphorylation just after ischaemia has developed. Prolongation of the ischaemic period up to 5 min inhibited both Ca(2+)-dependent and, to a lesser extent, Ca(2+)-independent activities of CaM-KII. These last events coincide with an extensive translocation of CaM-KII protein from the soluble to the membranous fraction. In effect, in spite of inhibition of total CaM-KII activity, its Ca(2+)-independent, persistently active component can still remain more abundant at specific membrane regions.

Paralytic tremor (pt), a hereditary neurological disorder of rabbits is a recessive, X-linked point mutation of the gene for proteolipid protein (PLP) biosynthesis. This mutation results in substitution of histidine by glutamine in the PLP molecule and produces severe hypomyelination. In the present study, we investigated the developmental expression of myelin-oligodendrocyte-specific glycolipid markers by means of ELISA assay. While immunoreactivity with antibodies recognising proligodendroblast (POA) antigen was unchanged, only minute amounts of the other glycolipid markers characteristic for more advanced stages of OLs maturation, such as 04 and 01 antigens, were expressed in pt brain. The degree of down-regulation was similar to that for MBP. Concomitantly, the level of in situ expression of the mutated PLP gene mRNA in glial cells of 14 day old pt brain was found to be as high as in age-matched controls. Northern blot analysis of developmental PLP gene expression showed a significant deficit of this message in pt brain, but only at more advanced developmental stages. However, aside from changes in myelin structure, no changes in glial cell number or morphology were evident by light microscopic examination of pt mutants. In contrast, electron microscopy revealed substantial abnormalities in pt oligodendrocyte cytoarchitecture, indicating functional impairment of intracellular transport and utilisation of myelin constituents. Thus, only POA expression is positively correlated with the unchanged content of OLs in pt brain, whereas decreases of 04 and 01 antigens, together with MBP immunoreactivity, are indicators of the degree of hypomyelination. Furthermore, oligodendrocyte differentiation appears to proceed normally in pt mutant brain up to the stage of PLP gene expression. Then, due to intracellular accumulation of this abnormal gene product, synthesis of PLP as well as the other myelin-specific constituents is inhibited by a "feed-back" control mechanism.

An X-linked, recessive paralytic tremor (pt) mutation is characterized by CNS hypomyelination. In our previous work, we presented developmental studies on the expression of several myelin-specific proteins (PLP/DM-20, MBP, CNP, MAG, and MOG) in the brain homogeates of both pt mutant and age-matched control Chinchilla rabbits aged 1-120 postnatal days. A moderate reduction in all examined proteins and a striking PLP deficiency were observed in the pt mutant rabbits. In the present study, we investigated isolated and purified myelin fractions. A severe (approximately 30% of control values) and approximately constant hypomyelination of pt mutant CNS was observed during the entire investigated development (28-120 postnatal days). Although the neurological symptoms gradually regressed and a partial recovery of the affected animals usually occurred, no tendency toward regression of the hypomyelination was noticed. Whereas the content of CNP, MBP, and MAG in isolated myelin membrane fractions seemed close to normal, a drastic PLP deficiency was observed. A significantly elevated amount of MOG was found in the myelin of pt mutant rabbits. The controversy between the high degree of hypomyelination and the slight reduction in myelin protein marker expression (except for PLP) in mature brain homogenates is discussed with respect to retarded oligodendrocyte maturation and deficient processing of myelin membranes in pt mutant rabbits.

The potential involvement of platelet activating factor (PAF, 1-O-alkyl 2-O-acetyl-sn-glycero-3-phosphocholine) in aggravation of ischemic brain injury has been recently postulated. Reported evidences in support of this thesis include increases of brain PAF concentration during ischemia and the neuroprotective effect exerted by PAF antagonists. In this article, we demonstrate that several PAF-mediated biochemical responses in synaptoneurosomes in vitro resemble these observed previously in ischemic brain and are widely acknowledged as the potentially causal factors in this pathology. In synaptoneurosomes prepared from rat hippocampus, 10 nM PAF caused an observable elevation of intracellular calcium as measured by fluorescence Fura-2A probe. A similar elevation of synaptoneurosomal [Ca2+]i was evoked by 1 mM glutamate treatment. As an effect of calcium entry after PAF application, a translocation of protein kinase C (PKC) toward plasma membranes was demonstrated by 3H-labeled phorbol-binding method. It was followed by an increase of 50 kDa proteolytic fragment of the enzyme (PKM) recognized on Western blots with anti-PKC antibody. Incubation of synaptoneurosomes in the presence of calcium chelators abolished these effects of PAF and significantly decreased the content of PKC in the membranes. Furthermore, PAF treatment markedly attenuated the receptor- and postreceptor-activated cAMP accumulation in synaptoneurosomes. The decrease of cAMP level seems to be secondary to the PAF-induced calcium entry with subsequent activation of cAMP-specific phosphodiesterase, since it was completely blocked by IBMX, a potent inhibitor of this enzyme. Our observations indicate that PAF in a concentration found in ischemic brain can elevate [Ca2+]i and potentiate calcium-dependent intracellular signalling in synaptoneurosomes in vitro, including PKC translocation/activation and proteolysis, followed by IBMX-sensitive inhibition of cAMP production. The relative contribution of these events to ischemic brain injury is currently under extensive investigation.

The effect of the platelet activating factor (PAF) antagonist BN52021 on [3H]D-aspartate (D-Asp) release was investigated in rat hippocampal slices during and after incubation (20 min) in ischaemia-like conditions. Ischaemia did not influence spontaneous D-Asp outflow whereas K(+)-evoked, calcium-dependent release was markedly enhanced in reoxygenated, post-ischaemic slices. These slices also showed a substantial translocation/activation of protein kinase C (PKC). BN52021 blocked both ischaemia-induced effects. Moreover, the PKC inhibitor H7 attenuated post-ischaemic K(+)-evoked D-Asp release when beta-PDBu, a PKC activator, was used to enhance the response of normoxic slices. Assuming that PKC is activated by ischaemia in a PAF-dependent manner and that this activation proceeds to enhanced glutamate exocytosis, we speculate on the involvement of PAF receptor stimulation in the pathology of cerebral ischaemia.