Haematology and blood chemistry changes in mice treated with terbuthylazine and its formulation Radazin TZ-50.

TBA is an herbicide in general low acute toxicity and placed into a third category of toxicity. The aim of this study was to determine the effect of TBA and its formulation Radazin TZ-50 in doses of ADI values and 1/100 LD 50 on haematological and biochemical blood parameters in mice. The number of leukocytes was significantly decreased (p < 0.05) in all treated groups compared to non-treated mice (8.81 ± 3.23 × 10(9)/L). The lowest value 3.90 ± 0.74 × 10(9)/L was observed in group treated with TBA (1/100 LD 50) followed by TBA (ADI) 4.49 ± 0.98 × 10(9)/L, Radazin TZ-50 (1/100 LD 50) 4.67 ± 1.24 × 10(9)/L and Radazin TZ-50 (ADI) 4.73 ± 1.15 × 10(9)/L. The values of the enzyme AST was increased from 190.00 ± 26.46-270.00 ± 147.30 U/L in serum of all treated groups as compared to non-treated mice (110.00 ± 20.00). LDH values showed significant increase (3236.67 ± 56.86-4054.33.5 ± 837.16 U/L) as compared to non-treated mice (1010.00 ± 222.71 U/L). Total protein value was significantly (p < 0.05) increased in TBA 1/100 LD50 (63.00 ± 7.48 g/L) and Radazin TZ-50 1/100 LD50 (60.00 ± 2.00 g/L) compared to non-treated mice 52.00 ± 4.00 g/L. Increased serum concentrations of urea and creatinine obtained in mice treated with TBA and Radazin TZ-50 indicates a greater degree of dysfunction of the nephron. TBA and its formulation of Radazin TZ-50 in applied doses demonstrate changes in the number of leukocytes and limited hepatotoxic effects.

Evaluation of cytotoxic and genotoxic effects of two resin-based root-canal sealers and their components on human leucocytes in vitro.

AIM: To evaluate the in vitro genotoxicity and cytotoxicity of two resin-based root canal sealers and to determine the type of cell death they induce. METHODOLOGY: The sealers tested were Epiphany and RealSeal. Each component of the material (Epiphany Primer, Epiphany Thinning Resin, Epiphany Sealant, RealSeal Primer, RealSeal Thinning Resin and RealSeal Root Canal Sealant), components in permutual combinations and all components mixed together were tested on human peripheral blood leucocytes using ethidium bromide/acridine orange viability staining and comet assay. Simultaneously, untreated negative control cultures were analysed in the same manner. DNA damage was evaluated following 4 h of treatment and after 24 h in the absence of the components of the materials. RESULTS: After 4 h of treatment, except thinning resin, each individual component and the different combinations of components induced a significant increase in DNA migration ability (P < 0.05). After 24 h, combination of primer, thinning resin and sealant of both materials caused cell death inducing intense apoptosis. After 24 h, cells exposed to Epiphany Sealant and RealSeal Root Canal Sealant, both in polymerized and unpolymerized form, exhibited a level of DNA damage that was similar to the control. CONCLUSIONS: Primer and thinning resin of both resin-based root canal sealers and their combinations were cytotoxic and induced apoptosis. Both sealants had no significant effect on the viability of the human leucocytes.

Biochemical and epigenetic changes in phytoplasma-recovered periwinkle after indole-3-butyric acid treatment.

AIM: To elucidate the possible mechanism of phytoplasma elimination from periwinkle shoots caused by indole-3-butyric acid (IBA) treatment. METHODS AND RESULTS: It has been shown that a transfer of in vitro-grown phytoplasma-infected Catharanthus roseus (periwinkle) plantlets from medium supplemented with 6-benzylaminopurine (BA) to one supplemented with IBA can induce remission of symptoms and even permanent elimination of 'Candidatus Phytoplasma asteris' reference strain HYDB. Endogenous auxin levels and general methylation levels in noninfected periwinkles, periwinkles infected with two 'Candidatus Phytoplasma' species and phytoplasma-recovered periwinkles were measured and compared. After the transfer from cytokinin- to auxin-containing media, healthy shoots maintained their phenotype, methylation levels and hormone concentrations. Phytoplasma infection caused a change in the endogenous indole-3-acetic acid to IBA ratio in periwinkle shoots infected with two 'Candidatus Phytoplasma' species, but general methylation was significantly changed only in shoots infected with 'Ca. P. asteris', which resulted in the only phytoplasma species eliminated from shoots after transfer to IBA-containing medium. Both phytoplasma infection and treatment with plant growth regulators influenced callose deposition in phloem tissue, concentrations of photosynthetic pigments and soluble proteins, H(2) O(2) levels and activities of catalase (CAT) and ascorbate peroxidase (APX). CONCLUSION: Lower level of host genome methylation in 'Ca. P. asteris'-infected periwinkles on medium supplemented with BA was significantly elevated after IBA treatment, while IBA treatment had no effect on cytosine methylation in periwinkles infected with 'Candidatus Phytoplasma ulmi' strain EY-C. SIGNIFICANCE AND IMPACT OF THE STUDY: Hormone-dependent recovery is a distinct phenomenon from natural recovery. As opposed to spontaneously recovered plants in which elevated peroxide levels and differential expression of peroxide-related enzymes were observed, in hormone-dependent recovery changes in global host genome, methylation coincide with the presence/absence of phytoplasma.

Central nervous system regeneration: from leech to opossum.

A major problem of neurobiology concerns the failure of injured mammalian spinal cord to repair itself. This review summarizes work done on two preparations in which regeneration can occur: the central nervous system of an invertebrate, the leech, and the spinal cord of an immature mammal, the opossum. The aim is to understand cellular and molecular mechanisms that promote and prevent regeneration. In the leech, an individual axon regrows successfully to re-establish connections with its synaptic target, while avoiding other neurons. Functions that were lost are thereby restored. Moreover, pairs of identified neurons become re-connected with appropriate synapses in culture. It has been shown that microglial cells and nitric oxide play key roles in leech CNS regeneration. In the opossum, the neonatal brain and spinal cord are so tiny that they survive well in culture. Fibres grow across spinal cord lesions in neonatal animals and in vitro, but axon regeneration stops abruptly between postnatal days 9 and 12. A comprehensive search has been made in spinal cords that can and cannot regenerate to identify genes and establish their locations. At 9 days, growth-promoting genes, their receptors and key transcription molecules are up-regulated. By contrast at 12 days, growth-inhibitory molecules associated with myelin are prominent. The complete sequence of the opossum genome and new methods for transfecting genes offer ways to determine which molecules promote and which inhibit spinal cord regeneration. These results lead to questions about how basic research on mechanisms of regeneration could be 'translated' into effective therapies for patients with spinal cord injuries.

In vitro genotoxicity of root canal sealers.

AIM: To evaluate the effect of leakage on differences in genotoxicity of root canal sealers ex vivo according to their main components using two different cytogenetic assays. METHODOLOGY: Six materials of different composition (GuttaFlow, Epiphany, Diaket, IRM, SuperEBA and Hermetic) were tested on human peripheral blood lymphocytes using the comet assay and chromosomal aberration analysis. Prepared materials were eluted in physiological solution for 1 h, 1 day, 5 and 30 days. Thereafter cultures were treated with 8 microg, 4 microg and 2 microg of each sealer. Frequencies of chromatide and chromosome breaks and accentric fragments were determined. Comet assay was used to evaluate primary DNA damage by measuring tail length and tail intensity. Chi-square, Fisher's PLSD (Protected Least Significant Difference) and Kruskall-Wallis non parametric tests were used for statistical analysis. RESULTS: After 1-h elution only the highest dose of Diaket, Hermetic and SuperEBA significantly (P = 0.035, P = 0.048, P = 0.037 respectively) affected the measured cytogenetic parameters. The migration ability of DNA was more strongly affected than induction of chromosomal aberrations. After elutions longer than 24 h none of the tested sealers exhibited a genotoxic effect. CONCLUSION: Under the conditions used in the study all sealers had acceptable biocompatibility in terms of genotoxicity.

Kainate and metabolic perturbation mimicking spinal injury differentially contribute to early damage of locomotor networks in the in vitro neonatal rat spinal cord.

Acute spinal cord injury evolves rapidly to produce secondary damage even to initially spared areas. The result is loss of locomotion, rarely reversible in man. It is, therefore, important to understand the early pathophysiological processes which affect spinal locomotor networks. Regardless of their etiology, spinal lesions are believed to include combinatorial effects of excitotoxicity and severe stroke-like metabolic perturbations. To clarify the relative contribution by excitotoxicity and toxic metabolites to dysfunction of locomotor networks, spinal reflexes and intrinsic network rhythmicity, we used, as a model, the in vitro thoraco-lumbar spinal cord of the neonatal rat treated (1 h) with either kainate or a pathological medium (containing free radicals and hypoxic/aglycemic conditions), or their combination. After washout, electrophysiological responses were monitored for 24 h and cell damage analyzed histologically. Kainate suppressed fictive locomotion irreversibly, while it reversibly blocked neuronal excitability and intrinsic bursting induced by synaptic inhibition block. This result was associated with significant neuronal loss around the central canal. Combining kainate with the pathological medium evoked extensive, irreversible damage to the spinal cord. The pathological medium alone slowed down fictive locomotion and intrinsic bursting: these oscillatory patterns remained throughout without regaining their control properties. This phenomenon was associated with polysynaptic reflex depression and preferential damage to glial cells, while neurons were comparatively spared. Our model suggests distinct roles of excitotoxicity and metabolic dysfunction in the acute damage of locomotor networks, indicating that different strategies might be necessary to treat the various early components of acute spinal cord lesion.

Low expression of the ClC-2 chloride channel during postnatal development: a mechanism for the paradoxical depolarizing action of GABA and glycine in the hippocampus.

In early postnatal development, during the period of synapse formation, gamma-aminobutyric acid (GABA) and glycine, the main inhibitory transmitters in the adult brain, paradoxically excite and depolarize neuronal membranes by an outward flux of chloride. The mechanisms of chloride homeostasis are not fully understood. It is known that in adult neurons intracellular chloride accumulation is prevented by a particular type of chloride channel, the ClC-2. This channel strongly rectifies in the inward direction at potentials negative to ECl thus ensuring chloride efflux. We have tested the hypothesis that in the developing hippocampus, a differential expression or regulation of ClC-2 channels may contribute to the depolarizing action of GABA and glycine. We have cloned a truncated form of ClC-2 (ClC-2nh) from the neonatal hippocampus which lacks the 157 bp corresponding to exon 2. In situ hybridization experiments show that ClC-2nh is the predominant form of ClC-2 mRNA in the neonatal brain. ClC-2nh mRNA is unable to encode a full-length protein due to a frameshift, consequently it does not induce any currents upon injection into Xenopus oocytes. Low expression of the full-length ClC-2 channel, could alter chloride homeostasis, lead to accumulation of [Cl-]i and thereby contribute to the depolarizing action of GABA and glycine during early development.

'Specific' oligonucleotides often recognize more than one gene: the limits of in situ hybridization applied to GABA receptors.

As exquisite probes for gene sequences, oligonucleotides are one of the most powerful tools of recombinant molecular biology. In studying the GABA receptor subunits in the neonatal hippocampus we have used oligonucleotide probes in in situ hybridization and cloning techniques. The oligonucleotides used and assumed to be specific for the target gene, actually recognized more than one gene, leading to surprising and contradictory results. In particular, we found that a GABA(A)-rho specific oligonucleotide recognized an abundant, previously unknown, transcription factor in both in situ and library screening, while oligos 'specific' for GABA(A) subunits were able to recognize 30 additional unrelated genes in library screening. This suggests that positive results obtained with oligonucleotides should be interpreted with caution unless confirmed by identical results with oligonucleotides from different parts of the same gene, or cDNA library screening excludes the presence of other hybridizing species.

Excitotoxic cell death induces delayed proliferation of endogenous neuroprogenitor cells in organotypic slice cultures of the rat spinal cord.

The aim of the present report was to investigate whether, in the mammalian spinal cord, cell death induced by transient excitotoxic stress could trigger activation and proliferation of endogenous neuroprogenitor cells as a potential source of a lesion repair process and the underlying time course. Because it is difficult to address these issues in vivo, we used a validated model of spinal injury based on rat organotypic slice cultures that retain the fundamental tissue cytoarchitecture and replicate the main characteristics of experimental damage to the whole spinal cord. Excitotoxicity evoked by 1 h kainate application produced delayed neuronal death (40%) peaking after 1 day without further losses or destruction of white matter cells for up to 2 weeks. After 10 days, cultures released a significantly larger concentration of endogenous glutamate, suggesting functional network plasticity. Indeed, after 1 week the total number of cells had returned to untreated control level, indicating substantial cell proliferation. Activation of progenitor cells started early as they spread outside the central area, and persisted for 2 weeks. Although expression of the neuronal progenitor phenotype was observed at day 3, peaked at 1 week and tapered off at 2 weeks, very few cells matured to neurons. Astroglia precursors started proliferating later and matured at 2 weeks. These data show insult-related proliferation of endogenous spinal neuroprogenitors over a relatively brief time course, and delineate a narrow temporal window for future experimental attempts to drive neuronal maturation and for identifying the factors regulating this process.

Mechanisms underlying cell death in ischemia-like damage to the rat spinal cord in vitro.

New spinal cord injury (SCI) cases are frequently due to non-traumatic causes, including vascular disorders. To develop mechanism-based neuroprotective strategies for acute SCI requires full understanding of the early pathophysiological changes to prevent disability and paralysis. The aim of our study was to identify the molecular and cellular mechanisms of cell death triggered by a pathological medium (PM) mimicking ischemia in the rat spinal cord in vitro. We previously showed that extracellular Mg(2+) (1 mM) worsened PM-induced damage and inhibited locomotor function. The present study indicated that 1 h of PM+Mg(2+) application induced delayed pyknosis chiefly in the spinal white matter via overactivation of poly (ADP-ribose) polymerase 1 (PARP1), suggesting cell death mediated by the process of parthanatos that was largely suppressed by pharmacological block of PARP-1. Gray matter damage was less intense and concentrated in dorsal horn neurons and motoneurons that became immunoreactive for the mitochondrial apoptosis-inducing factor (the intracellular effector of parthanatos) translocated into the nucleus to induce chromatin condensation and DNA fragmentation. Immunoreactivity to TRPM ion channels believed to be involved in ischemic brain damage was also investigated. TRPM2 channel expression was enhanced 24 h later in dorsal horn and motoneurons, whereas TRPM7 channel expression concomitantly decreased. Conversely, TRPM7 expression was found earlier (3 h) in white matter cells, whereas TRPM2 remained undetectable. Simulating acute ischemic-like damage in vitro in the presence of Mg(2+) showed how, during the first 24 h, this divalent cation unveiled differential vulnerability of white matter cells and motoneurons, with distinct changes in their TRPM expression.

Neuroprotection of locomotor networks after experimental injury to the neonatal rat spinal cord in vitro.

Treatment to block the pathophysiological processes triggered by acute spinal injury remains unsatisfactory as the underlying mechanisms are incompletely understood. Using as a model the in vitro spinal cord of the neonatal rat, we investigated the feasibility of neuroprotection of lumbar locomotor networks by the glutamate antagonists 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and aminophosphonovalerate (APV) against acute lesions induced by either a toxic solution (pathological medium (PM) to mimic the spinal injury hypoxic-dysmetabolic perturbation) or excitotoxicity with kainate. The study outcome was presence of fictive locomotion 24 h after the insult and its correlation with network histology. Inhibition of fictive locomotion by PM was contrasted by simultaneous and even delayed (1 h later) co-application of CNQX and APV with increased survival of ventral horn premotoneurons and lateral column white matter. Neither CNQX nor APV alone provided neuroprotection. Kainate-mediated excitotoxicity always led to loss of fictive locomotion and extensive neuronal damage. CNQX and APV co-applied with kainate protected one-third of preparations with improved motoneuron and dorsal horn neuronal counts, although they failed with delayed application. Our data suggest that locomotor network neuroprotection was possible when introduced very early during the pathological process of spinal injury, but also showed how the borderline between presence or loss of locomotor activity was a very narrow one that depended on the survival of a certain number of neurons or white matter elements. The present report provides a model not only for preclinical testing of novel neuroprotective agents, but also for estimating the minimal network membership compatible with functional locomotor output.

Extensive glial apoptosis develops early after hypoxic-dysmetabolic insult to the neonatal rat spinal cord in vitro.

The current etiopathogenesis of spinal cord injury comprises a growing number of nontraumatic causes, including ischemia generating hypoxic-dysmetabolic conditions. To mimic the metabolic disruption accompanying nontraumatic acute spinal cord injury and to characterize the type and dynamics of cell death in relation to locomotor network function, we used, as a model, the rat neonatal spinal cord preparation in vitro transiently (1 h) exposed to a "pathological medium" (PM), i.e. hypoxic/aglycemic solution containing toxic radicals. PM induced, in the ventrolateral spinal region, pyknosis already detectable after 2 h and stabilized 24 h later (affecting 55% of white matter cells). Glial cells were much more vulnerable than neurons. The amplitude of fictive locomotor patterns recorded from lumbar ventral roots was decreased and periodicity delayed by PM, in keeping with substantial preservation of neuronal networks. Repeated application of PM intensified such a functional impairment. White matter astrocytes and oligodendrocytes displayed nucleolytic pyknosis mainly dependent on caspase-mediated death processes as shown by active caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) positivity. Expression of cleaved poly(ADP-ribose) polymerase-1 (PARP-1) (the active caspase-3 executor) also grew with similar time course. The caspase-3 inhibitor II counteracted, in a dose-dependent fashion, white matter pyknosis. Our results suggest the important involvement of apoptotic pathways in early glial cell death during the first 24 h after a hypoxic-dysmetabolic insult, associated with impaired locomotor output. Residual locomotor network activity together with distinctive apoptotic damage to white matter cells suggests that early protection against glial destruction may help to prevent subsequent damage extension responsible for paraplegia.

Kainate-induced delayed onset of excitotoxicity with functional loss unrelated to the extent of neuronal damage in the in vitro spinal cord.

While excitotoxicity is a major contributor to the pathophysiology of acute spinal injury, its time course and the extent of cell damage in relation to locomotor network activity remain unclear. We used two in vitro models, that is, the rat isolated spinal cord and spinal organotypic cultures, to explore the basic characteristics of excitotoxicity caused by transient application of the glutamate analogue kainate followed by washout and analysis 24 h later. Electrophysiological records showed that fictive locomotion was slowed down by 10 microM kainate (with no histological loss) and fully abolished by 50 microM, while disinhibited bursting with unchanged periodicity persisted. Kainate concentrations (> or =50 microM) larger than those necessary to irreversible suppress fictive locomotion could still elicit dose-dependent motoneuron pool depolarization, and dose-dependent neuronal loss in the grey matter, especially evident in central and dorsal areas. Motoneuron numbers were largely decreased. A similar regional pattern was detected in organotypic slices, as extensive cell loss was dose related and affected motoneurons and premotoneurons: the number of dead neurons (already apparent 1 h after kainate) grew faster with the higher kainate concentration. The histological damage was accompanied by decreased MTT formazan production commensurate with the number of surviving cells. Our data suggest locomotor network function was very sensitive to excitotoxicity, even without observing extensive cell death. Excitotoxicity developed gradually leaving a time window in which neuroprotection might be attempted to preserve circuits still capable of expressing basic rhythmogenesis and reconfigure their function in terms of locomotor output.

Extracellular magnesium enhances the damage to locomotor networks produced by metabolic perturbation mimicking spinal injury in the neonatal rat spinal cord in vitro.

An acute injury to brain or spinal cord produces profound metabolic perturbation that extends and exacerbates tissue damage. Recent clinical interventions to treat this condition with i.v. Mg(2+) to stabilize its extracellular concentration provided disappointing results. The present study used an in vitro spinal cord model from the neonatal rat to investigate the role of extracellular Mg(2+) in the lesion evoked by a pathological medium mimicking the metabolic perturbation (hypoxia, aglycemia, oxidative stress, and acid pH) occurring in vivo. Damage was measured by taking as outcome locomotor network activity for up to 24 h after the primary insult. Pathological medium in 1 mM Mg(2+) solution (1 h) largely depressed spinal reflexes and suppressed fictive locomotion on the same and the following day. Conversely, pathological medium in either Mg(2+)-free or 5 mM Mg(2+) solution evoked temporary network depression and enabled fictive locomotion the day after. While global cell death was similar regardless of extracellular Mg(2+) solution, white matter was particularly affected. In ventral horn the number of surviving neurons was the highest in Mg(2+) free solution and the lowest in 1 mM Mg(2+), while motoneurons were unaffected. Although the excitotoxic damage elicited by kainate was insensitive to extracellular Mg(2+), 1 mM Mg(2+) potentiated the effect of combining pathological medium with kainate at low concentrations. These results indicate that preserving Mg(2+) homeostasis rendered experimental spinal injury more severe. Furthermore, analyzing ventral horn neuron numbers in relation to fictive locomotion expression might provide a first estimate of the minimal size of the functional locomotor network.

Increase of annexin 1 immunoreactivity in spinal cord of newborn opossum (Monodelphis domestica) at the time when regeneration after injury stops being possible.

Annexins constitute a family of proteins that associate reversibly with cell membranes in a calcium dependent manner. We have studied the distribution of annexin 1, which is known to mediate anti-inflammatory actions of glucocorticoids, and which is upregulated after spinal cord injury, in newborn and adult South American opossum (Monodelphis domestica) spinal cord. We show the increase in the annexin 1 immunoreactivity in spinal cords of neonatal opossums over the critical period when regeneration after injury ceases to be possible. We further show the restricted and specific sites at which it is detected in adult opossum cerebellum and hippocampus. Since the procedures used in immunochemistry of annexin in CNS have in the past yielded conflicting results, different procedures were tested and shown to be reliable. As a control, annexin 1 distribution was surveyed in kidney.

Strategies for identifying genes that play a role in spinal cord regeneration.

A search for genes that promote or block CNS regeneration requires numerous approaches; for example, tests can be made on individual candidate molecules. Here, however, we describe methods for comprehensive identification of genes up- and down-regulated in neurons that can and cannot regenerate after injury. One problem concerns identification of low-abundance genes out of the 30,000 or so genes expressed by neurons. Another difficulty is knowing whether a single gene or multiple genes are necessary. When microchips and subtractive differential display are used to identify genes turned on or off, the numbers are still too great to test which molecules are actually important for regeneration. Candidates are genes coding for trophic, inhibitory, receptor and extracellular matrix molecules, as well as unknown genes. A preparation useful for narrowing the search is the neonatal opossum. The spinal cord and optic nerve can regenerate after injury at 9 days but cannot at 12 days after birth. This narrow window allows genes responsible for the turning off of regeneration to be identified. As a next step, sites at which they are expressed (forebrain, midbrain, spinal cord, neurons or glia, intracellular or extracellular) must be determined. An essential step is to characterize proteins, their levels of expression, and their importance for regeneration. Comprehensive searches for molecular mechanisms represent a lengthy series of experiments that could help in devising strategies for repairing injured spinal cord.

Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus.

The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.