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Cell death is an essential process occurring during the development of the central nervous system. Despite the availability of wide range of commercially produced antibodies against various apoptotic markers, data regarding the apoptosis in intact spinal cord during postnatal development and adulthood are mostly missing. This study aimed to investigate the apoptosis in the rat spinal cord at different stages of ontogenesis (8, 29 and 90 postnatal days). For this purpose, we used immunofluorescent detection of two widely used apoptotic markers, activated caspase-3 (aC3) and cleaved PARP (cPARP). Surprisingly, we found significant discrepancy between the amounts of aC3+ cells and PARP+ cells, varying with ratio around 500:1–5,000:1 in the rat spinal cord in all postnatal time points. Majority of aC3+ cells were glial cells and did not exhibit apoptotic phenotype. In contrast with the results of in vivo study, in vitro analysis of primary cell culture derived from neonatal rat spinal cord, treated with apoptotic inductor staurosporine, revealed similar onset of occurrence of both markers in cells subjected to apoptosis. Gene expression analysis of spinal cord tissue revealed elevated expression of Birc4 (XIAP), Birc2 and Birc5 (Survivin) genes, which are known as potent inhibitors of apoptosis. Our data indicates that the activated caspase-3 is not an exclusive marker of apoptosis, especially in glial cells, due its possible presence in inhibited forms and/or its participation in other, non-apoptotic roles. Therefore, in the light of our recent results, cPARP appears to be more appropriate marker for detection of apoptosis.

Cerebrospinal fluid contacting neurons (CSF-cNs) represent a specific class of neurons located in close vicinity of brain ventricles and central canal. In contrast with knowledge gained from other vertebrate species, we found that vast majority of CSF-cNs in the spinal cord of C57Bl/6N mice is located in ectopic distal ventral position. However, we found that small number of ectopic CSF-cNs is present also in spinal cord of other investigated experimental mice strains (C57Bl/6J, Balb/C) and mammalian species (Wistar rats, New Zealand White rabbits). Similarly, as the proximal populations, ectopic CSF-cNs retain PKD2L1-immunoreactivity and synaptic contacts with other neurons. On the other side, they show rather multipolar morphology lacking thick dendrite contacting central canal lumen. Ectopic CSF-cNs in the spinal cord of C57Bl/6N mice emerge during whole period devoted to production of CSF-cNs and reach their ventral destinations during first postnatal weeks. In order to identify major gene, whose impairment could trigger translocation of CSF-cNs outside the central canal area, we took advantage of close consanguinity of C57Bl/6J substrain with normal CSF-cN distribution and C57Bl/6N substrain with majority of CSF-cNs in ectopic position. Employing in silico analyses, we ranked polymorphisms in C57Bl/6N substrain and selected genes Crb1, Cyfip2, Adamts12, Plk1, and Herpud2 as the most probable candidates, whose product dysfunction might be responsible for the ectopic distribution of CSF-cNs. Furthermore, segregation analysis of F2 progeny of parental C57Bl/6N and Balb/C mice revealed that polymorphic loci of Crb1 and Cyfip2 underlie the ectopic position of CSF-cNs in the spinal cord of C57Bl/6N mice.

In order to obtain unbiased results of target gene expression, selection of the most appropriate reference gene (RG) remains a key precondition. However, an experimental study focused on the validation of stably expressed RGs in the rat spinal cord (SC) during development or after spinal cord injury (SCI) is missing. In our study, we tested the stability of the expression of nine selected RGs in rat SC tissue during normal development (postnatal days 1&ndash, 43, adulthood) and after minimal (mSCI) and contusion (cSCI) spinal cord injury. The following RGs were tested: common housekeeping genes of basal cell metabolism (Gapdh, Hprt1, Mapk6) and protein translation (Rpl29, Eef1a1, Eif2b2), as well as newly designed RGs (Gpatch1, Gorasp1, Cds2) selected according to the RefGenes tool of GeneVestigator. The stability of RGs was assessed by geNorm, NormFinder, and BestKeeper. All three applets favored Gapdh and Eef1a1 as the most stable genes in SC during development. In both models of SCI, Eif2b2 displayed the highest stability of expression, followed by Gapdh and Gorasp1/Hprt1 in cSCI, and Gapdh and Eef1a1 in the mSCI experiments. To verify our results, selected RGs were employed for normalization of the expression of genes with a clear biological context in the SC&mdash, Gfap and Slc1a3/Glast during postnatal development and Aif1/Iba1 and Cd68/Ed1 after SCI.

According to previous opinion, the derivation of neurons and glia from the central canal (CC) lining of the spinal cord in rodents should occur in the embryonic period. Reports of the mitotic activity observed in the lining during postnatal development have often been contradictory, and proliferation was ascribed to the generation of ependymocytes, which are necessary for the elongation of CC walls. Our study quantifies the intensity of proliferation and determines the cellularity of the CC lining in reference to lumbar spinal segment L4 during the postnatal development of rats. The presence of dividing cells peaks in the CC lining on postnatal day 8 (P8), with division occurring in 19.2% ± 3.2% of cells. In adult rats, 3.6% ± 0.9% of cells still proliferate, whereas, in mice, 10.3% ± 2.3% of cells at P8 and only 0.6% ± 0.2% of cells in the CC lining in adulthood are proliferating. In the rat, the length of the cell cycle increases from 100.3 ± 35.7 hours at P1 to 401.4 ± 80.6 hours at P43, with a sudden extension between P15 and P22. Despite the intensive proliferation, the total cellularity of the CC lining at the L4 spinal segment significantly descended in from P8 to P15. According to our calculations, the estimated cellularity was significantly higher compared with the measured cellularity of the CC lining at P15. Our results indicate that CC lining serves as a source of cells beyond ependymal cells during the first postnatal weeks of the rat. J. Comp. Neurol. 525:693-707, 2017. © 2016 Wiley Periodicals, Inc.

Labelling of DNA in replicating cells using 5-bromo-2′-deoxyuridine (BrdU) is widely used, however the rapid clearance and metabolisation of BrdU in the living organism is a critical issue. Although the pharmacokinetic of BrdU in experimental animals is empirically approximated, the exact time-curve remains unknown. Here we present novel method for estimation of the BrdU content in the blood serum. The application is based on the in vitro cocultivation of tumour cells with the examined serum and the subsequent quantification of the incorporated BrdU in the DNA using flow cytometry analysis. Our results demonstrate that this approach can quantify the BrdU concentration in serum at 1 µmol.dm-3 and might represent an attractive alternative to conventional chromatographic analysis. The employment of tumour cells as “detectors” of the BrdU content in serum provides an advantage over high pressure liquid chromatography (HPLC), as this approach allows us to approximate not only the concentration of BrdU, but also to determine, whether BrdU is present in the blood serum in effective concentration to reliable label all cells undergoing the S-phase of the cell cycle. The presented application might be a helpful tool for studies on pharmacokinetics of BrdU or other thymidine analogues when testing various administration routes or protocols.

Two waves of oligodendrogenesis in the ventricular zone of the spinal cord (SC-VZ) during rat development, which take place between embryonic days 14 and 18 (E14–E18) and E20–E21, have been described. In the VZ of the brain, unlike the SC-VZ, a third wave of oligodendrogenesis occurs during the first weeks of postnatal development. Using immunofluorescence staining of intact rat SC tissue, we noticed the presence of small numbers of Olig2+/Sox-10+ cells inside the lining of the central canal (CC) during postnatal development and adulthood. Olig2+/Sox-10+ cells appeared inside the lining of the CC shortly after birth, and their number reached a maximum of approximately 0.65 ± 0.14 cell/40-μm section during the second postnatal week. After the latter development, the number of Olig2+/Sox-10+ cells decreased to 0.21 ± 0.07 (P36) and 0.18 ± 0.1 cell/section (P120). At P21, Olig2+/Sox-10+ cells inside the CC lining started to express other oligodendroglial markers such as CNPase, RIP, and APC. Olig2+/Sox-10+ cells usually did not proliferate inside the CC lining and were only rarely found to be immunoreactive against oligodendrocyte progenitor markers such as NG2 or PDGFRα. Using 5-bromo-2-deoxyuridine administration at P2, P11, P22, or P120–P125, we revealed that these cells arose in the CC lining during postnatal development and adulthood. Our findings confirmed that the CC lining is the source of a small number of cells with an oligodendroglial phenotype during postnatal development and adulthood in the SC of intact rats. J. Comp. Neurol. 522:3194–3207, 2014. © 2014 Wiley Periodicals, Inc.

Despite extensive investigations of gliogenesis, the time of origin of ependymal cells in the spinal cord has not yet been fully elucidated. Using a single dose of 5-bromo-2-deoxyuridine combined with various survival times we monitored: mitotic activity (short survival time), the presence of newly formed cells in the ventricular zone (intermediate survival time) and the formation of ependymal cells (long survival time) during the late embryonic and early postnatal development in the ventricular zone of the spinal cord of rats. In the period of study it was found that the ependymal cells populated this region in two waves. Most of the ependymal cells originated around embryonic day 18 and then between postnatal days 8 and 15. In addition, it was observed that in the ventricular zone of the spinal cord, proliferation and production of ependymal cells continues at the slower rate at least until day 36 of postnatal development. Elucidation of the relationship between progenitors in the embryonic ventricular zone and the relative quiescent ependymal lining of the central canal in adulthood could be important in the search for the adult neural stem cell niche.

The Balkan endemic species, Hypericum rumeliacum, Guttiferae was introduced in vitro for the first time with the aim to study the type of morphogenetic response to plant growth regulators and ability to produce phenolics and flavonoid compounds. The morphoregulatory effect of 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphtaleneacetic acid (NAA), 6-benzyladenine (BA) and combination of BA with NAA in Murashige–Skoog's basal medium on leaf lamina, internode stem segment, stem node and root cuttings was studied. Histological analysis of the structures regenerated from the primary explants proved the presence of both, embryoids and meristemoids. The node explants cultivated on BA-supplemented medium were the most favourable for regeneration through meristemoids. Therefore a double-stage culture approach, allowing an effective multiplication of large quantities of plant shoots in vitro along with maintenance of the biosynthetic capacity of the culture was developed. It comprised one subculture of three-nodal stem explants derived from the stock shoot cultures on MS medium supplemented with 0.2 mg/l BA followed by subculture of the induced multiple shoots on cytokinin-free MS medium. Determination of the total phenolics and flavonoids showed that the decrease of the levels of these secondary metabolites is transitional, as the exclusionof BA from the medium resulted in an increase of their total content.

According to previous opinion, the derivation of neurons and glia from the central canal (CC) lining of the spinal cord in rodents should occur in the embryonic period. Reports of the mitotic activity observed in the lining during postnatal development have often been contradictory, and proliferation was ascribed to the generation of ependymocytes, which are necessary for the elongation of CC walls. Our study quantifies the intensity of proliferation and determines the cellularity of the CC lining in reference to lumbar spinal segment L4 during the postnatal development of rats. The presence of dividing cells peaks in the CC lining on postnatal day 8 (P8), with division occurring in 19.2% ± 3.2% of cells. In adult rats, 3.6% ± 0.9% of cells still proliferate, whereas, in mice, 10.3% ± 2.3% of cells at P8 and only 0.6% ± 0.2% of cells in the CC lining in adulthood are proliferating. In the rat, the length of the cell cycle increases from 100.3 ± 35.7 hours at P1 to 401.4 ± 80.6 hours at P43, with a sudden extension between P15 and P22. Despite the intensive proliferation, the total cellularity of the CC lining at the L4 spinal segment significantly descended in from P8 to P15. According to our calculations, the estimated cellularity was significantly higher compared with the measured cellularity of the CC lining at P15. Our results indicate that CC lining serves as a source of cells beyond ependymal cells during the first postnatal weeks of the rat. J. Comp. Neurol. 525:693-707, 2017. © 2016 Wiley Periodicals, Inc.

1. To test our hypothesis that a transient nonlethal ischemic insult benefits the lumbosacral spinal cord ischemic injury, nestin, the marker of proliferating cells, and Fluoro-Jade B, the marker of degenerating cells, were used in rats. Morphological outcome was evaluated after 12-min ischemia versus 12-min ischemia preconditioned by 3-min ischemic period and 30-min recirculation (IPC), in each group followed by 2, 3, and 4 days of posttreatment survival. 2. Twelve-minute ischemia, inducing nestin-positivity in ependyma and reactive astrocytes at the L(1-3) spinal cord segments, shows this region as the viable region of spinal cord in all postischemic survival periods. On the other hand, abundance of Fluoro-Jade B-positive cells, distributed throughout the dorsal horn and intermediate zone of L4-S2 segments, points out the most injured spinal cord region by ischemia. 3. After the same ischemic insult in IPC rats only a few nestin-positive ependymal cell and reactive astrocytes appeared beside the nestin-positive vessels in the lower lumbar and sacral spinal cord segments of all survival periods. The appearance of nestin-positive cells in the spinal cord segments, which "should have been affected" by ischemia indicates protection of this region by the IPC treatment. 4. The number and density evaluation of Fluoro-Jade B fluorescent cells of L4-S2 segments after ischemia and IPC confirmed that degenerating cells were significantly reduced in the IPC rats in all survival periods. 5. Our results showing the immunohistochemical response of epemdyma, committed to the presence of viable tissue, indicate that the ependymal cells may contribute to the ischemic resistance in the IPC rats.

5-Bromo-2ʹ-deoxyuridine (BrdU) is a marker that is widely used to label S-phase cells in neurobiological research in most common doses 50 or 100 mg/kg per single intraperitoneal (i.p.) injection. However, the important data regarding its pharmacokinetics in rodents are still missing. The aim of our study was to investigate the BrdU level in serum after a single i.p. injection to adult rats (doses: 50 or 100 mg/kg) and adult mice (50 mg/kg). The animals were killed at selected time-points after the BrdU injection, and proliferating tumour cells (cell lines HCT-116 and HL-60) were co-cultivated with isolated blood sera. BrdU incorporated in the DNA of the S-phase tumour cells was stained with an anti-BrdU antibody and analysed using flow cytometry. In rats, the efficacies of BrdU labelling of S-phase cells in both in vitro and in vivo conditions were compared in the 50 and 100 mg/kg groups. According to our results, BrdU was in saturated concentration to label almost all S-phase cells for 60 min in both doses and was detectable in blood serum until 120 min after the single i.p. injection. However, the 100 mg/kg dose of BrdU did not provide a prolonged staining period to offset the potentially higher toxicity in comparison with the 50 mg/kg dose. In mice, due to their faster metabolism, the concentration of BrdU in blood serum was sufficient to label the whole population of S-phase cells for only 15 min after the i.p. injection, then dropped rapidly.

Two waves of oligodendrogenesis in the ventricular zone of the spinal cord (SC-VZ) during rat development, which take place between embryonic days 14 and 18 (E14-E18) and E20-E21, have been described. In the VZ of the brain, unlike the SC-VZ, a third wave of oligodendrogenesis occurs during the first weeks of postnatal development. Using immunofluorescence staining of intact rat SC tissue, we noticed the presence of small numbers of Olig2(+) /Sox-10(+) cells inside the lining of the central canal (CC) during postnatal development and adulthood. Olig2(+) /Sox-10(+) cells appeared inside the lining of the CC shortly after birth, and their number reached a maximum of approximately 0.65 ± 0.14 cell/40-μm section during the second postnatal week. After the latter development, the number of Olig2(+) /Sox-10(+) cells decreased to 0.21 ± 0.07 (P36) and 0.18 ± 0.1 cell/section (P120). At P21, Olig2(+) /Sox-10(+) cells inside the CC lining started to express other oligodendroglial markers such as CNPase, RIP, and APC. Olig2(+) /Sox-10(+) cells usually did not proliferate inside the CC lining and were only rarely found to be immunoreactive against oligodendrocyte progenitor markers such as NG2 or PDGFRα. Using 5-bromo-2-deoxyuridine administration at P2, P11, P22, or P120-P125, we revealed that these cells arose in the CC lining during postnatal development and adulthood. Our findings confirmed that the CC lining is the source of a small number of cells with an oligodendroglial phenotype during postnatal development and adulthood in the SC of intact rats.

Despite the abundance of cerebrospinal fluid-contacting neurons (CSF-cNs) lining the central canal of the spinal cord of mammals, little information is known regarding the phenotype and fate of these cells during development and in adulthood. Using immunofluorescence of spinal cord tissue of rats from the first postnatal day (P1) until the end of the 5th postnatal week (P36), we observed that these neurons show both immature (doublecortin+, β-III-tubulin+, neurofilament 200 kDa-) and more mature (weak NeuN+, P2X2+, GAD65+) characteristics during the first postnatal weeks. Because of the gradually decreasing number of CSF-cNs in the central canal lining during development, we were also interested in the migration potential of these cells. However, the assessment of the number of CSF-cNs in the lining of the central canal during postnatal development revealed that this decline is most likely associated with the growth of the spinal cord. Lastly, to reveal the birth date of CSF-cNs, we performed 5-bromo-2-deoxyuridine administration and colocalization analyses. We found that production of these cells appears from day 12 of embryonic development (E12) until E22. The vast majority of CSF-contacting neurons arise on E14 and E15. In contrast with other types of spinal neurons, the production of CSF-cNs is not restricted to a particular neuroepithelial region and occurs even after what is thought to be the termination of neurogenesis.

In the last quarter of the embryonic development of rat and shortly after a termination of neurogenesis, the transformation of the spinal cord primitive lumen (pL) to the central canal (CC) occurs. In this work, we show that this phenomenon is not an insignificant event but it is directly associated with the processes of gliogenesis. Using a light microscopy and immunohistochemistry, we monitored the development of the rat embryonic spinal cord from the end of the neurogenesis on the embryonic day 17 until the maturation of the spinal cord during the first postnatal weeks. Our observations demonstrate the importance of the transformation of the pL to the CC and its connection with gliogenesis, and the mechanism of this transformation is proposed. It is found that a segregation of the glutamate transporter (GLAST) immunopositive cells from the alar plates and transformation of the radial glial cells to the fibrous and protoplasmic astrocytes play presumably a key role in the diminution of the ventricular zone. Results indicate that the very transformation and migration of the radial glial cells during gliogenesis could result in a transformation of the pL to the CC.