Cleaved caspase-3 is present in the majority of glial cells in the intact rat spinal cord during postnatal life.

Cell death is an essential process that occurs during the development of the central nervous system. Despite the availability of a wide range of commercially produced antibodies against various apoptotic markers, data regarding apoptosis in intact spinal cord during postnatal development and adulthood are mostly missing. We investigated apoptosis in rat spinal cord at different stages of ontogenesis (postnatal days 8, 29, and 90). For this purpose, we applied immunofluorescent detection of two widely used apoptotic markers, cleaved caspase-3 (cC3) and cleaved poly(ADP-ribose) polymerase (cPARP). Surprisingly, we found significant discrepancy between the number of cC3+ cells and PARP+ cells, with a ratio between 500:1 and 5000:1 in rat spinal cord at all postnatal time points. The majority of cC3+ cells were glial cells and did not exhibit an apoptotic phenotype. In contrast with in vivo results, in vitro analysis of primary cell cultures derived from neonatal rat spinal cord and treated with the apoptotic inductor staurosporine revealed a similar onset of occurrence of both cC3 and cPARP in cells subjected to apoptosis. Gene expression analysis of spinal cord revealed elevated expression of the Birc4 (XIAP), Birc2, and Birc5 (Survivin) genes, which are known potent inhibitors of apoptosis. Our data indicate that cC3 is not an exclusive marker of apoptosis, especially in glial cells, owing its possible presence in inhibited forms and/or its participation in other non-apoptotic roles. Therefore, cPARP appears to be a more appropriate marker to detect apoptosis.

"Neural stem cells puzzle" missing piece: telomerase reverse transcriptase expression patterns during nervous system ontogenesis in rats.

For almost three decades, neural stem cells remain still up-to-date and enigmatic topic. The main problem for their studying is the non-existence of an exclusive neural stem cell marker and the heterogeneity of them across the nervous system. As one of the novel markers of neural stem/progenitor cells may serve telomerase reverse transcriptase (TERT), a catalytic subunit of the telomerase enzyme, responsible for retaining the cell immortality. Thus, the aim of our study was to reveal if TERT, as an enzyme for ensuring the immortality of proliferating cells, could be used as a potential marker of neural stem/progenitor cells during the ontogenesis of the rat central nervous system. In this study, we used various markers related to neural stem or progenitor cells character and examined their co-localization with TERT expression. Our experiments were performed on the tissue of the brain and spinal cord during several stages of postnatal development and the neural tube during the 14th embryonal day. Cytoplasmatic TERT expression was found in alar plate progenitors and ventral horn neuroblasts of E14 rats. In the postnatal stages of spinal cord ontogenesis, a cytoplasmatic expression in neurons and nuclear expression in astrocytes was defined. In the brain, nuclear TERT expression was found in neural progenitor cells of neurogenic areas. This study provides the first comparative study of TERT expression across the central nervous system ontogenesis. The nuclear presence of TERT may be used as a potential marker of neural stem/progenitor cells, however, further studies are required to confirm these assumptions.

Delivery of Alginate Scaffold Releasing Two Trophic Factors for Spinal Cord Injury Repair.

Spinal cord injury (SCI) has been implicated in neural cell loss and consequently functional motor and sensory impairment. In this study, we propose an alginate-based neurobridge enriched with/without trophic growth factors (GFs) that can be utilized as a therapeutic approach for spinal cord repair. The bioavailability of key GFs, such as Epidermal Growth factor (EGF) and basic Fibroblast Growth Factor (bFGF) released from injected alginate biomaterial to the central lesion site significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons (choline acetyltransferase positive motoneurons) and sensory fibres. In addition, we document enhanced outgrowth of corticospinal tract axons and presence of blood vessels at the central lesion. Tissue proteomics was performed at 3, 7 and 10 days after SCI in rats indicated the presence of anti-inflammatory factors in segments above the central lesion site, whereas in segments below, neurite outgrowth factors, inflammatory cytokines and chondroitin sulfate proteoglycan of the lectican protein family were overexpressed. Collectively, based on our data, we confirm that functional recovery was significantly improved in SCI groups receiving alginate scaffold with affinity-bound growth factors (ALG+GFs), compared to SCI animals without biomaterial treatment.

IT delivery of ChABC modulates NG2 and promotes GAP-43 axonal regrowth after spinal cord injury.

Chondroitin sulphate proteoglycans (CSPGs) with the major component NG2 have an inhibitory effect on regeneration of damaged axons after spinal cord injury. In this study, we investigate whether the digestion of CSPGs by chondroitinase ABC (ChABC) may decrease the NG2 expression and promote axon regrowth through the lesion site. Rats underwent spinal cord compression injury and were treated with ChABC or vehicle through an intrathecal catheter delivery at 2, 3, and 4 days after injury. In addition, animals were behaviorally scored using BBB test in weekly intervals after SCI. Based on immunocytochemical analyses, we have quantified distribution of NG2 glycoprotein and GAP-43 in spinal cord tissue in both experimental groups. Multiple injections of ChABC caused decrease of NG2 expression at lesion site at 5 and 7 days, but not at 14 and 28 days in comparison with vehicle-treated rats and significantly enhanced GAP-43 expression during the entire survival. The densitometry analysis showed significantly higher GAP-43 immunoreactivity (1.8-2.2-fold) in the regrowing axons and cell bodies within the central lesion cavity when compared with vehicle group. Longitudinally oriented and disorganized GAP-43-labeled axons were able to infiltrate and penetrate damaged tissue. The outgrowth of GAP-43 axons after CHABC delivery was significantly longer (≤0.457 mm) when compared with the length of axons in vehicle-treated rats (≤0.046 mm). Present findings suggest that degradation of NG2 with acute IT ChABC treatment may promote ongoing (long-lasting) axonal regenerative processes at late survival (14 and 28 days), but with no significant impact on the improvement of motor function.

Chondroitinase ABC treatment and the phenotype of neural progenitor cells isolated from injured rat spinal cord.

The aim of the present study was to investigate whether enzyme chondroitinase ABC (ChABC) treatment influences the phenotype of neural progenitor cells (NPCs) derived from injured rat spinal cord. Adult as well as fetal spinal cords contain a pool of endogenous neural progenitors cells, which play a key role in the neuroregenerative processes following spinal cord injury (SCI) and hold particular promise for therapeutic approaches in CNS injury or neurodegenerative disorders. In our study we used in vitro model to demonstrate the differentiation potential of NPCs isolated from adult rat spinal cord after SCI, treated with ChABC. The intrathecal delivery of ChABC (10 U/ml) was performed at day 1 and 2 after SCI. The present findings indicate that the impact of SCI resulted in a decrease of all NPCs phenotypes and the ChABC treatment, on the contrary, caused an opposite effect.

Enrichment of rat oligodendrocyte progenitor cells by magnetic cell sorting.

The embryonic, neonatal, as well as adult rat spinal cords harbor a pool of neural stem cells (NSCs), which may be easily isolated and used to replace neuronal cell loss or remyelinate damaged axons following various neurodegenerative disorders. In the present study we have used magnetic cell sorting (MACs) technology to generate enriched oligodendroglial cell populations from the embryonic (E16) rat spinal cord. Target cells were separated by positive selection, using specific A2B5 antibody-labeled MicroBeads achieving optimal recovery and high purity of pro-oligodendroglial cells. Based on immunocytochemical analyses for oligodendroglial developmental markers (A2B5, NG2, RIP and MBP) we were able to characterize and quantify oligodendroglial progenitors (OPCs) and mature oligodendroglial cells in: (i) unseparated heterogeneous population of NSCs, or in (ii) antigen-antibody separated NSCs. Our results showed that MACs technology enable us to gain enriched OPCs from heterogeneous population of spinal NSCs, resulting in a 58-61% of mature oligodendrocytes content (MBP+, RIP+) in comparison to 6-12% of oligodendroglial cells acquired from unseparated population. In addition, the enriched OPCs could be cultured in vitro for several >8 passages, giving rise to a high number of newly formed spheres, as well as high expansion potential. These experiments indicate that MACs technology provide a feasible approach for experimental cell enrichment of desired oligodendroglial progeny, which may be used in future trials for cell-based therapies to treat spinal cord injury.

Effect of paternal rat irradiation transmitted to the progeny during prenatal development.

The transgenerational transmission of radiation damage was investigated on the basis of quantitative changes of nucleic acids and histones, the integral index of tissue and organ cellularity. Male Wistar rats, whole-body irradiated with the dose of 3 Gy of gamma rays, were mated with non-irradiated females 25 or 80 days after exposure and their progeny were investigated on the 15th (embryos), 17th (embryos), or 19th (embryonic brain) day of prenatal development (E15, E17, and E19Br, respectively). A significant increase in DNA and RNA concentration and content was found on the 15th day and predominantly on the 17th day of gestation in the progeny of males irradiated 80 days before mating. On the contrary, in the progeny of the same males, concentration of histones was decreased in groups E15 and E19Br. Finally, the radiation alterations in the progeny arisen from irradiated spermatogonia (by paternal exposure 80 days before mating) were more profound in nucleic acids than in histones. Our findings suggest an incidence of radiation-induced genome instability manifested as enhanced proliferating activity of cells in response to DNA damage in the progeny of males, mated at later intervals after exposure.

Effects of combined treatment of rats with cadmium and ionizing radiation on nucleic acids in the kidneys, liver and haemopoietic organs.

The influence of Cd (1 mg/rat CdCl2 i.p.) and/or gamma radiation (6 Gy) on RNA and DNA content and/or concentration in the intact kidney and hypertrophic kidney (on the 44th hour after UN) and in other slowly and quickly proliferating organs was studied. The period between administration of Cd and Ir in the group with combined treatment was 30 min; between treatment (administration of Cd, Ir and combination of both treatments--Cd + Ir) and UN it was 1, 7, 14 and 21 days. The total extent of damage caused by the treatments in the investigated organs was following: intact kidney < liver < hypertrophic kidney and bone marrow < spleen < thymus. In the intact and hypertrophic kidney and liver, the administration of Cd caused more extensive changes in comparison with gamma irradiation, and the effects of combination of the treatments were similar to those of Cd alone. In the bone marrow, spleen and thymus, more profound changes were observed after Ir in comparison with Cd administration, and the effects of combined treatment were similar to the effects of Ir alone. The changes in the hypertrophic kidney after administration of Cd and/or Ir were more extensive than in the intact kidney, which suggests latent injury induction in the rat kidney by these noxa. The higher effectiveness of the treatments in the hypertrophic kidney than in the intact one was manifested mostly by the decrease in the RNA and DNA content, which was mainly due to inhibition of growth induced by UN and not by a real decrease in DNA and RNA contents caused by loss of damaged cells.