The pleiotropic molecule NGF regulates the in vitro properties of fibroblasts, keratinocytes, and endothelial cells: implications for wound healing.

Nerve growth factor (NGF) is recognized as a pleiotropic molecule, exerting a variety of biological effects on different cell types and pathophysiological conditions, and its role in tissue wound healing has been recently highlighted. However, the preferential cellular target of NGF is still elusive in the complex cellular and molecular cross talk that accompanies wound healing. Thus, to explore possible NGF cellular targets in skin wound healing, we investigated the in vitro NGF responsiveness of keratinocytes (cell line HEKa), fibroblasts (cell line BJ), and endothelial cells (cell line HUVEC), also in the presence of adverse microenvironmental conditions, e.g., hyperglycemia. The main results are summarized as follows: 1) NGF stimulates keratinocyte proliferation and HUVEC proliferation and angiogenesis in a dose-dependent manner although it has no effect on fibroblast proliferation; 2) NGF stimulates keratinocyte but not fibroblast migration in the wound healing assay; and 3) NGF completely reverts the proliferation impairment of keratinocytes and the angiogenesis impairment of HUVECs induced by high d-glucose concentration in the culture medium. These results contribute to better understanding possible targets for the therapeutic use of NGF in skin repair.

Effects of Topical Application of CHF6467, a Mutated Form of Human Nerve Growth Factor, on Skin Wound Healing in Diabetic Mice.

Nerve growth factor (NGF) is the protein responsible for the development and maintenance of sensory skin innervation. Given the role of appropriate innervation in skin healing, NGF has been indicated as a possible prohealing treatment in pathologic conditions characterized by nerve-ending loss, such as chronic ulcers in diabetes; however, its use as a therapeutic agent is limited by its hyperalgesic effect. We tested the effect of topical application of the nonalgogenic NGF derivative hNGFP61S/R100E in two models of skin ulcer induced in dbdb diabetic mice, investigating healing time, skin histology, reinnervation, and angiogenesis using morphologic and molecular approaches. We showed that the topical administration of CHF6467, a recombinant human NGF in which an amino acid substitution (R100E) abolished the hyperalgesic effect usually associated with NGF, accelerated skin repair in experimental wounds (full-excision and pressure-ulcer) induced in diabetic mice (dbdb). CHF6467-induced acceleration of wound healing was accompanied by increased re-epithelization, reinnervation, and revascularization as assessed by histology, immunohistochemistry, and image analysis. Bioinformatic analysis of differentially expressed genes and signaling pathways in the wound tissues showed that protein kinase B-mammalian target of rapamycin was the most regulated pathway. In spite of the transdermal absorption leading to measurable, dose-dependent increases in CHF6467 plasma levels, no systemic thermal or local mechanical hyperalgesia was observed in treated mice. When tested in vitro in human cell lines, CHF6467 stimulated keratinocyte and fibroblast proliferation and tube formation by endothelial cells. Collectively, these results support a possible use of CHF6467 as a prohealing agent in skin lesions in diabetes. SIGNIFICANCE STATEMENT: Topical application of CHF6467 accelerates reinnervation, neoangiogenesis, and wound healing in diabetic mice in both full-thickness skin-excision and pressure-ulcer models through the protein kinase B/mammalian target of rapamycin pathway and does not induce hyperalgesia.

Antimicrobial activity, toxicity and accumulated hard-tissue debris (AHTD) removal efficacy of several chelating agents.

AIM: To evaluate the antimicrobial, toxicity and cleaning effectiveness of ethylenediaminetetraacetic acid (EDTA) and maleic acid (MA) alone and combined with cetrimide (CTR). METHODOLOGY: Cytotoxic and genotoxic effects were assessed on Chinese hamster cells V79 using the MTT, clonogenic and micronucleus assays, respectively. The bacterial inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined on a strain of Enterococcus faecalis. Antimicrobial tests were performed on a biofilm model after treatment with the chelating agents by using a biofilm eradication concentration (MBEC) and confocal laser scanning microscope (CLSM) assays. Quantification of cell biomass and percentage of live and dead cells in the biomass were assessed for each group. The percentage reduction of accumulated hard-tissue debris (AHTD) after root canal preparation and final irrigation protocols was evaluated by micro-CT. Statistical tests of one-way analysis of variance (anova), Bonferroni test, Kruskal-Wallis test, Dunn's multiple comparison test and Wilcoxon matched-pairs signed-rank tests were used. RESULTS: Cetrimide alone as well as in combination with EDTA and MA at dilutions of 1/10 and 1/100 was significantly more toxic as compared to untreated controls (P < 0.001). All tested mixtures were nontoxic at a dilution of 1/1000. EDTA retained a weak inhibitory and bactericidal effect against planktonic cells, whilst MA inhibited cells growth and killed 99.9% of the cells when diluted. CTR revealed the most prominent effect, being inhibitory and bactericidal, also when diluted. Cetrimide alone or combined with EDTA was able to remove, respectively, 40% (P < 0.01) and 60% (P < 0.001) of the entire biomass after 1 min. Conversely, MA alone and in combination with CTR did not have a significant effect on biomass reduction. After final irrigation, the AHTD volume was significantly decreased in all groups (P < 0.05). EDTA + CTR and MA + CTR were associated with a significant reduction in the percentage of AHTD on the entire root canal compared to the same solutions without surfactant. CONCLUSIONS: 7% MA was less cytotoxic in comparison with 17% EDTA. The addition of cetrimide to EDTA and MA removed accumulated hard-tissue debris effectively from the canal walls and increased their antimicrobial activity when compared to the same solutions without detergents.

Antimicrobial effectiveness of combinations of oxidant and chelating agents in infected dentine: an ex vivo confocal laser scanning microscopy study.

AIM: To evaluate the intratubular antimicrobial activity of several oxidant and chelating agents associated with or without surfactants in experimentally infected root canals, using confocal laser scanning microscopy. METHODOLOGY: Twenty-four dentine blocks from bovine incisors were contaminated for 5 days with Enterococcus faecalis (ATCC- 29212). Ten contaminated dentine specimens were irrigated for 5 min with 5.25% NaOCl followed by 17% EDTA for 2 min, and the other 10 with Hypoclean for 5 min followed by Tetraclean NA for 2 min. The remaining four specimens were used as positive and negative controls (two samples each). Then, dentine blocks were stained with Live/Dead BacLight for analysis of the remaining live or dead bacteria using confocal laser scanning microscopy. Comparison between and within groups was performed using the Mann-Whitney test for independent samples and the Wilcoxon signed-rank test, respectively. RESULTS: After exposure to irrigants, the positive control group had a median of 67.41% of viable bacteria (95% CI: 48.15, 78.9), whilst the NaOCl+EDTA group and Hypoclean+Tetraclean NA group had 3.77% (1.28, 15.92) and 0.87% (-0.42, 4.30) of viable bacteria, respectively. These results were significantly different from each other, both overall and distinct by region (cervical and medium third), or depth (superficial and deep layer) (P < 0.01 in all cases). CONCLUSIONS: The use of adjunctive agents reducing the surface tension associated with oxidant and chelating agents improved the antimicrobial activity of irrigating solutions and intratubular decontamination against Enterococcus faecalis, possibly due to better removal of the smear layer and deeper penetration into dentinal tubules.

Sodium hypochlorite solution penetration into human dentine: a histochemical evaluation.

AIM: The aim of this study was to evaluate ex vivo the penetration depth of sodium hypochlorite solutions, with and without surfactants, into human dentine using a histochemical stain and the bleaching ability of NaOCl. METHODOLOGY: Extracted maxillary central incisors were decoronated and their root canals were shaped to a size 30, .07 taper. Dentinal tubules were stained with 10% copper sulphate solution followed by a 1% rubeanic acid alcohol solution under vacuum. Specimens were irrigated with 5 mL of Niclor 5 (5.25% NaOCl solution) or 5 mL of Hypoclean (5.25% NaOCl solution + surfactants) according to the corresponding treatment group, for 2 min. A transverse section was obtained in the middle portion of the apical, middle and coronal thirds of each tooth. Under microscopic examination, irrigant penetration was evaluated by measuring the following: the bleached circumference of the root canal with respect to the stained circumference; bleached areas, mean and maximum penetration depth. For statistical analysis, Pearson's chi-squared test and one-way anova were used. RESULTS: Only 30% of dentine tubules in the apical portions were stained; therefore, these were excluded from further comparisons. In the middle and coronal thirds, the average bleached circumference of the root canal was 80-99% of the stained circumference and the bleached area was 73757-135107 µm2 . The average penetration depth ranged from 39 to 62 µm and maximum penetration from 74 to 131 µm. For all reported results, no differences within and between groups were observed (P = 1). CONCLUSION: No differences in penetration of irrigants with and without surfactants into dentinal tubules were observed in the middle and coronal thirds of incisor teeth.

SIK1 localizes with nephrin in glomerular podocytes and its polymorphism predicts kidney injury.

Mutant α-adducin and endogenous ouabain levels exert a causal role in hypertension by affecting renal Na-K ATPase. In addition, mutant ß-adducin is involved in glomerular damage through nephrin down-regulation. Recently, the salt-inducible kinase 1 (SIK1) has been shown to exert a permissive role on mutant α-adducin effects on renal Na-K ATPase activity involved in blood pressure (BP) regulation and a SIK1 rs3746951 polymorphism has been associated with changes in vascular Na-K ATPase activity and BP. Here, we addressed the role of SIK1 on nephrin and glomerular functional modifications induced by mutant ß-adducin and ouabain, by using congenic substrains of the Milan rats expressing either mutant α- or ß-adducin, alone or in combination, ouabain hypertensive rats (OHR) and hypertensive patients. SIK1 co-localized and co-immunoprecipitated with nephrin from glomerular podocytes and associated with caveolar nephrin signaling. In cultured podocytes, nephrin-gene silencing decreased SIK1 expression. In mutant ß-adducin congenic rats and in OHR, the podocyte damage was associated with decreased nephrin and SIK1 expression. Conversely, when the effects of ß-adducin on podocytes were blocked by the presence of mutant α-adducin, nephrin and SIK1 expressions were restored. Ouabain effects were also reproduced in cultured podocytes. In hypertensive patients, nephrinuria, but not albuminuria, was higher in carriers of mutant SIK1 rs3746951 than in wild-type, implying a more direct effect of SIK1 on glomerular damage. These results demonstrate that, through nephrin, SIK1 is involved in the glomerular effects of mutant adducin and ouabain and a direct effect of SIK1 is also likely to occur in humans.

Human mesenchymal stem cells produce bioactive neurotrophic factors: source, individual variability and differentiation issues.

The possible use of cell therapies for neurological lesions and disorders is regarded as a very promising strategy. However, many issues related to cell type, tissue donor, expected biological action etc., are still open. In this study human mesenchymal stem cells derived from different fetal and adult tissues were examined in order to explore growth and neurotrophic factor synthesis and biological action, also considering the individual variability of the donors. Cells were derived from different human tissues and characterized according to the guidelines of the International Society for Cellular Therapy. Growth and neurotrophic factor synthesis was evaluated by real time PCR, biological assays and ELISA. It was found that human mesenchymal stem cells produce vascular endothelial-, nerve-growth factor (VEGF, NGF), brain-derived-, ciliary- and glial-derived neurotrophic factors (BDNF, CDGF, GDNF), which are neuroprotective molecules, but the source and the donor influence the synthesis rate. Accordingly, it is suggested that the source and the individual variability are key issues to be considered in the perspective of the clinical use of mesenchymal stem cells in neurological disorders.

Neuropathy in eosinophilic granulomatosis with polyangiitis: a comparison study of 24 cases with or without prior leukotriene antagonist exposure.

Eosinophilic granulomatosis with polyangiitis (EGPA), also known as Churg-Strauss syndrome (CSS), is a systemic vasculitis affecting almost exclusively patients with asthma. Neuropathy is the presenting feature in 55-75 % of cases. An increased incidence of the syndrome has been reported in asthmatics treated with leukotriene antagonists (LTAs). The causal relation is still debated. We retrospectively examined clinical, biochemical, histological features, and outcome of patients referred between 1990 and 2006 for sural nerve biopsy affected by neuropathy related to EGPA. We identified 24 patients, 6 treated with LTA montelukast (T-group) and 18 not treated (NT-Group). All had chronic asthma; in T-group neuropathy developed from 1 to 150 days after starting montelukast. Demographic features as well as asthma duration and pre-onset treatment were remarkably similar, with the only exception of a statistically nonsignificant larger involvement of the nasal mucosa in T group. Nerve biopsy revealed in both group an axonal neuropathy. At follow-up, all within the T-group and most within the NT-group improved clinically; neurophysiological parameters remained stable, improved or worsened in the same proportion within the two groups. Only 2 NT and no T-patient had stopped steroid treatment before the appearance of the peripheral neuropathy, making withdrawal overall unlikely as a causative factor of the onset of neuropathy. In summary, the temporal relationship between montelukast administration and the onset of neuropathy, would make the latter more likely as an "adverse drug reaction". Despite this, no significant clinical neither neurophysiological differences were noted between the two groups.

Determining the minimum inhibitory concentration of Tetraclean against Candida albicans.

The purpose of the present study was to determine the minimum inhibitory concentrations of Tetraclean, chlorhexidine, hydrogen peroxide, and sodium hypochlorite against Candida albicans.Amphotericin B was used as positive control and RPMI plus 1 ml Candida suspension was used as negative control. Serial dilution method was used to determine MIC of the irrigants. Findings showed that all positive controls demonstrated complete inhibition of C. albicans at concentration of 0.78 microg mL(-1). On the other hand, all negative controls were positive for fungal growth which confirms the methodology of the study. Findings showed that the MIC of CHX was significantly lesser than other tested irrigants (p < 0.05). The MICs of other groups in an ascending order were as follows: Tetraclean, NaOCl, and H2O2. However, the difference betweenTetraclean and NaOCI was not significant (p > 0.05). It can be concluded that MIC of CHX was significantly lower than other irrigations solutions which confirms its strong antifungal activity.

Functional and molecular evidence of myelin- and neuroprotection by thyroid hormone administration in experimental allergic encephalomyelitis.

AIMS: Recent data in mouse and rat demyelination models indicate that administration of thyroid hormone (TH) has a positive effect on the demyelination/remyelination balance. As axonal pathology has been recognized as an early neuropathological event in multiple sclerosis, and remyelination is considered a pre-eminent neuroprotective strategy, in this study we investigated whether TH administration improves nerve impulse propagation and protects axons. METHODS: We followed up the somatosensory evoked potentials (SEPs) in triiodothyronine (T3)-treated and untreated experimental allergic encephalomyelitis (EAE) Dark-Agouti female rats during the electrical stimulation of the tail nerve. T3 treatment started on the 10th day post immunization (DPI) and a pulse administration was continued until the end of the study (33 DPI). SEPs were recorded at baseline (8 DPI) and the day after each hormone/ vehicle administration. RESULTS: T3 treatment was associated with better outcome of clinical and neurophysiological parameters. SEPs latencies of the two groups behaved differently, being briefer and closer to control values (=faster impulse propagation) in T3-treated animals. The effect was evident on 24 DPI. In the same groups of animals, we also investigated axonal proteins, showing that T3 administration normalizes neurofilament immunoreactivity in the fasciculus gracilis and tau hyperphosphorylation in the lumbar spinal cord of EAE animals. No sign of plasma hyperthyroidism was found; moreover, the dysregulation of TH nuclear receptor expression observed in the spinal cord of EAE animals was corrected by T3 treatment. CONCLUSIONS: T3 supplementation results in myelin sheath protection, nerve conduction preservation and axon protection in this animal model of multiple sclerosis.

A novel model of in vitro osteocytogenesis induced by retinoic acid treatment.

Despite recent research which more and more stresses the importance of osteocytes in regulating bone and systemic mineral metabolism, current molecular and functional knowledge of osteocyte properties are still incomplete, mostly due to limited availability of in vitro models. Osteocytes are terminally differentiated dendritic cells, and therefore are not easy to obtain and maintain in primary cultures. As an alternative, osteocyte differentiation can be induced by progressive osteoblast embedding in mineralised extracellular matrix. In this model, which is suitable for reproduction of bone development, the presence of calcified matrix prevents several cell biological methods from being used. Therefore, the osteocyte-like MLO-Y4 cell line continues to be the most widely used cellular system. Here we show that treatment of primary osteoblasts or MC3T3-E1 cells with retinoic acid generates a homogeneous population of ramified cells with osteocyte features, as confirmed by morphological and molecular analyses. The first morphological changes are detectable in primary cells after 2 days of treatment, and in the cell line after 4 days of treatment. Differentiation is complete in 5 and 10 days, respectively, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers, and up-regulation of osteocyte-specific molecules, most notably among them sclerostin. Compared to other published protocols, our method has a number of advantages. It is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in the complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Comparison of the surface tension of 5.25% sodium hypochlorite solution with three new sodium hypochlorite-based endodontic irrigants.

AIM: To investigate the surface tension characteristics of 5.25% sodium hypochlorite and three recently introduced sodium hypochlorite solutions, which had been modified to reduce their surface tension: Chlor-Xtra, Hypoclean A and Hypoclean B. METHODOLOGY: Freshly produced MilliQ water was used as a reference liquid. All measurements of surface tension were taken by the Wilhelmy plate technique, using a Cahn DCA-322 Dynamic Contact Angle Analyzer at the temperature of 22 °C. A glow-discharge cleaned glass slide was immersed in 5 mL of the test liquid in a beaker cleaned with hot chromic acid, rinsed with MilliQ water and finally air plasma-cleaned in a glow-discharge reactor. The force on the glass slide was recorded continuously by the instrument software as the beaker was raised and withdrawn at the constant speed of 40 micron/s, until at least 1 cm of the glass slide was immersed. The typical accuracy was 0.5 mJ m(-2). For each sample, fifteen measurements were taken, and mean values were calculated. A Kruskal-Wallis anova analysis, followed by Mann-Whitney's U rank sum test for pair-wise comparisons, was used to compare surface tension values. Statistical significance was set at α = 0.05. RESULTS: MilliQ water (72.13 mJ m(-2)) and 5.25% sodium hypochlorite (48.90 mJ m(-2) ) had the highest surface tension values (P < 0.01) compared to Chlor-Xtra (33.14 mJ m(-2)), Hypoclean B (30.00 mJ m(-2) ) and Hypoclean A (29.13 mJ m(-2)). Hypoclean A had the lowest surface tension (P < 0.01). CONCLUSIONS: The new 5.25% sodium hypochlorite solutions modified with surfactants, Hypoclean A and Hypoclean B, had surface tension values that were significantly lower (P < 0.01) than Chlor-Xtra and 5.25% NaOCl. Because of their low surface tension and increased contact with dentinal walls, these new irrigants have the potential to penetrate more readily into uninstrumented areas of root canal system as well as allow a more rapid exchange with fresh solution, enabling greater antimicrobial effectiveness and enhanced pulp tissue dissolution ability.

Laser application in tooth bleaching: an update review.

Since the development of laser, a variety of potential applications for lasers in endodontics such as pulp diagnosis, disinfection of the root canal system, canal shaping, obturation of the root canal, apicoectomy, treatment of dentin hypersensitivity, and tooth bleaching have been proposed. The aim of this paper was to review the benefits and drawbacks of laser tooth bleaching.

A single prenatal exposure to the endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin alters developmental myelination and remyelination potential in the rat brain.

Polychlorinated dibenzo-dioxins, furans and dioxin-like polychlorinated biphenyls are ubiquitous in foodstuffs of animal origin and accumulate in the fatty tissues of animals and humans. The most toxic congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a lipophilic endocrine-disrupting molecule that accumulates in adipose tissue, placenta and milk. polychlorinated biphenyls and TCDD are known to interfere with thyroid hormone metabolism and signaling in the developing brain. As thyroid hormone is critical in the myelination process during development, we investigated the effect of a single dose of TCDD prenatal exposure (gestational day 18) on the myelination process. A semi-quantitative analysis of oligodendrocyte markers at different stages of maturation was performed in the offspring's medulla oblongata, cerebellum, diencephalon and telenchephalon at different postnatal days (2/3, 14, 30 and 135). The most significant alterations observed were: (i) cerebellum and medulla oblongata: altered expression of oligodendroglial lineage and platelet-derived growth factor alpha receptor, myelin basic protein (MBP) mRNAs (P2/3, P135) and MBP protein (P135); (ii) diencephalon: increase in platelet- derived growth factor alpha receptor mRNA level (P2/3); (iii) telenchephalon: decrease in MBP mRNA expression. The oligodendroglial generation capability of adult neural stem/precursor cells obtained ex vivo from TCDD and vehicle-treated dams was then explored. TCDD impairs neurosphere proliferation and retards CNPase-positive cell generation from adult neurospheres.

The galanin receptor 2/3 agonist Gal2-11 protects the SN56 cells against beta-amyloid 25-35 toxicity.

The neuropeptide galanin is a modulator of cholinergic function and may play a role in A beta peptide-induced degeneration of cholinergic forebrain neurons. We have studied the effect of galanin and its galanin receptor subtype 2/3 agonist Gal2-11on toxicity induced by freshly-prepared beta-amyloid(25-35) in the cholinergic cell line SN56. Both nuclear fragmentation and caspase-3 expression were analysed. beta-amyloid(25-35)-exposure induced a significant increase in caspase-3 mRNA expression after 30, 60, 90 or 150 min of beta-amyloid(25-35) exposure. These effects were abolished in the presence of Gal2-11 (10 nM). Similarly, beta-amyloid(25-35)-induced nuclear fragmentation was prevented by the galanin agonist at all time points studied. These findings indicate that the galanin 2/3 agonist Gal2-11 protects SN56 cholinergic cells from beta-amyloid(25-35)-induced cell death and that this action is mediated by an early reduction of caspase-3 expression.

Ex vivo study of dentate gyrus neurogenesis in human pharmacoresistant temporal lobe epilepsy.

AIMS: Neurogenesis in adult humans occurs in at least two areas of the brain, the subventricular zone of the telencephalon and the subgranular layer of the dentate gyrus in the hippocampal formation. We studied dentate gyrus subgranular layer neurogenesis in patients subjected to tailored antero-mesial temporal resection including amygdalohippocampectomy due to pharmacoresistant temporal lobe epilepsy (TLE) using the in vitro neurosphere assay. METHODS: Sixteen patients were enrolled in the study; mesial temporal sclerosis (MTS) was present in eight patients. Neurogenesis was investigated by ex vivo neurosphere expansion in the presence of mitogens (epidermal growth factor + basic fibroblast growth factor) and spontaneous differentiation after mitogen withdrawal. Growth factor synthesis was investigated by qRT-PCR in neurospheres. RESULTS: We demonstrate that in vitro proliferation of cells derived from dentate gyrus of TLE patients is dependent on disease duration. Moreover, the presence of MTS impairs proliferation. As long as in vitro proliferation occurs, neurogenesis is maintained, and cells expressing a mature neurone phenotype (TuJ1, MAP2, GAD) are spontaneously formed after mitogen withdrawal. Finally, formed neurospheres express mRNAs encoding for growth (vascular endothelial growth factor) as well as neurotrophic factors (brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor). CONCLUSION: We demonstrated that residual neurogenesis in the subgranular layer of the dentate gyrus in TLE is dependent on diseases duration and absent in MTS.

In vitro antibacterial action of Tetraclean, MTAD and five experimental irrigation solutions.

AIM: To investigate the antibacterial effect of Tetraclean, MTAD and five experimental irrigants using both direct exposure test with planktonic cultures and mixed-species in vitro biofilm model. METHODOLOGY: Tetraclean, MTAD and five experimental solutions that were modifications of existing formulae including MTAD + 0.01% cetrimide (CTR), MTAD + 0.1% CTR, MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 (Tween 80 replaced by 0.1% CTR) and MTAD-D (MTAD without the Tween 80 and no CTR added) were used as disinfectants in the experiments. In the direct exposure test, a suspension of Enterococcus faecalis was mixed with each of the solutions. After 0.5, 1, 3 and 10 min, an inactivator was added and the number of surviving bacteria was calculated. A mixed-species biofilm from subgingival plaque bacteria was grown in brain heart infusion broth in anaerobic conditions on synthetic hydroxyapatite discs. Two-week-old biofilms were exposed to the solutions for 0.5, 1 and 3 min. The samples were observed by confocal laser scanning microscopy after bacterial viability staining. The scans were quantitatively analysed, and the volume of killed cells of all cells was calculated for each medicament. RESULTS: Tetraclean and MTAC-2 (0.1% CTR) killed planktonic E. faecalis in <30 s. Complete killing of bacteria required 1 min by MTAC-1, 3 min by MTAD + 0.1% CTR and 10 min by MTAD, MTAD-D and MTAD + 0.01% CTR. In the biofilm test, there were significant differences in microbial killing between the different solutions and times of exposure (P < 0.005). MTAC-2 showed the best performance, killing 71% of the biofilm bacteria in 3 min, followed by MTAC-1 and Tetraclean. MTAD and the three MTAD modifications demonstrated the lowest antibacterial activity. CONCLUSION: Tetraclean was more effective than MTAD against E. faecalis in planktonic culture and in mixed-species in vitro biofilm. CTR improved the antimicrobial properties of the solutions, whereas Tween 80 seemed to have a neutral or negative impact on their antimicrobial effectiveness.

Changes in brain cholinergic markers and spatial learning in old galanin-overexpressing mice.

The cholinergic forebrain system is involved in learning and memory, and its age-dependent decline correlates with a decrease in cognitive performance. Since the neuropeptide galanin participates in cholinergic neuron regulation, we have studied 19- to 23-month-old male mice overexpressing galanin under the platelet-derived growth factor B promoter (GalOE) and wild-type (WT) littermates by monitoring behavioral, neurochemical and morphological/histochemical parameters. In the Morris water maze test, old transgenic animals showed a significant impairment in escape latency in the hidden platform test compared to age-matched WT animals. The morphological/histochemical studies revealed that cholinergic neurons in the basal forebrain display a slight, age- but not genotype-related, alteration in choline acetyltransferase- (ChAT) immunoreactivity. The neurochemical studies showed an age-related decline in ChAT activity in the cerebral cortex of all mice, whereas in the hippocampal formation this effect was seen in GalOE but not WT animals. Expression of BDNF mRNA in the hippocampal formation, as evaluated by RT-PCR, was reduced in old animals; no age- or genotype-induced variations in NGF mRNA expression were observed. These data suggest that galanin overexpression further accentuates the age-related decline of the cholinergic system activity in male mice, resulting in impairment of water maze performance in old animals.