The Last Half Century of Fish Explant and Organ Culture.

Explants are three-dimensional tissue fragments maintained outside the organism. The goals of this article are to review the history of fish explant culture and discuss applications of this technique that may assist the modern zebrafish laboratory. Because most zebrafish workers do not have a background in tissue culture, the key variables of this method are deliberately explained in a general way. This is followed by a review of fish-specific explantation approaches, including presurgical husbandry, aseptic dissection technique, choice of media and additives, incubation conditions, viability assays, and imaging studies. Relevant articles since 1970 are organized in a table grouped by organ system. From these, I highlight several recent studies using explant culture to study physiological and embryological processes in teleosts, including circadian rhythms, hormonal regulation, and cardiac development.

[Correlation between microbial growth in conjunctival swabs of corneal donors and contamination of organ culture media]. / Korrelation zwischen mikrobiellem Wachstum in Bindehautabstrichen von Hornhautspendern und Kontamination des Organkulturmediums.

PURPOSE: The aim of the study was to determine the rate of contamination in conjunctival swabs from corneal donors by microbiological investigations and to correlate this with microbial contamination of the culture medium. PATIENTS AND METHODS: Contamination of conjunctival swabs and culture media was analyzed retrospectively for the years 2009, 2010 and 2011 at the LIONS corneal bank of Saar-Lor-Lux Trier/Westpfalz at the Saarland University Medical Center. The total annual number of conjunctival swabs was 316 in 2009, 341 in 2010 and 381 in 2011. Conjunctival swabs were taken prior to 1.25% povidone-iodine application. After disinfection donor corneas were harvested by in situ corneoscleral disc excision in all cases. The correlation between positive conjunctival swabs and microbial contamination of the culture medium was analyzed. RESULTS: In every year examined the contamination rate of the culture medium was significantly higher in cases of contaminated conjunctival swabs (p < 0.05 in 2009, p < 0.001 in 2010 and p = 0.004 in 2011). Of the conjunctival swabs 38.3% (2009), 53.7% (2010) and 55.6% (2011), respectively exhibited microbial growth. The principal microorganisms detected in the conjunctival swabs were coagulase negative staphylococci, gram negative rods and Staphylococcus aureus. Extending the exposure time to povidone-iodine prior to removal of the corneoscleral disc from 3 min in the year 2009 to 5 min since the year 2010 resulted in a highly statistically significant (p < 0.001) reduction in contamination frequency of the medium from 10.8% (2009) to 7.0% (2010) and 4.5% (2011) was observed. In 2009, 2010 and 2011 the culture medium was contaminated in 16.5%, 11.5% and 7.6% of the donated corneas with positive conjunctival swabs and in 7.2%, 1.9% and 0.6% in donated corneas with negative conjunctival swabs, respectively. CONCLUSIONS: A positive correlation was found between contamination of the culture medium and microbial colonization of the conjunctival swabs, Nevertheless, microbial colonization of the conjunctiva was high and contamination of the culture medium was relatively low. For the microbial contamination rate of the donated corneas in the medium, conjunctival disinfection time with iodine solution before explantation of the corneoscleral disc and the addition of antibiotics to the culture medium seem to play a protective role.

The required sample size in vaccination-challenge experiments with infectious bronchitis virus, a meta-analysis.

For statistical, animal welfare and financial reasons the choice of the number of chickens per group in experiments is important. This estimation, together with the number of tracheal organ cultures (TOCs) that need to be examined from each chicken in order to assess protection, should be based on the difference in level of protection that one would like to be able to detect (effect size), the expected variability of the results between and within the chickens, the desired confidence level and the power of the study. To obtain data that would facilitate this estimation, a meta-analysis was performed on the data from 18 infectious bronchitis virus (IBV) vaccination-challenge experiments performed at the Dutch Animal Health Service Deventer, the Netherlands (GD) in order to determine and quantify the source of variation in the mean level of protection of different groups. For the calculations, 137 groups of chickens were subdivided into 10 clusters based on age (young or adult), vaccination (none, homologous or heterologous), challenge (IBV or mock infected) and location of vaccination (isolator at GD or in the field). The results were used to estimate the required number of chickens per group for the different clusters using 2, 5 or 10 TOCs per chicken to be able to detect effect sizes of 6.25%, 12.5%, 25% and 50% between groups of chickens with 95% confidence (P<0.05) and 80% power. The number of chickens that was required for the mentioned effect sizes varied greatly from 2 to 650. This meta-analysis provided data that allow research workers to estimate the number of chickens that should be included in each group in order to obtain reliable results based on particular combinations of infectious bronchitis vaccination and challenge strains as defined by the presented clusters.

Acamprosate has no effect on NMDA-induced toxicity but reduces toxicity induced by spermidine or by changing the medium in organotypic hippocampal slice cultures from rat.

BACKGROUND: It has been suggested that the antirelapse drug acamprosate can inhibit or potentiate glutamate/NMDA receptor-mediated responses via a polyamine site. Additionally, subchronic exposure to acamprosate increases expression of some NMDA receptor subunits. These effects on NMDA receptors imply that the drug may have neurotoxic or neuroprotective actions under different conditions, and these studies were undertaken to evaluate this possibility in hippocampal neuronal cultures. METHODS: Organotypic hippocampal cultures from 8-day-old neonatal rats were maintained in medium for 28 days. The effects of acamprosate (100 microM) alone or on neurotoxic challenges induced by either 50 microM NMDA or 100 microM spermidine were studied. Neurotoxicity was assessed by uptake of propidium iodide 24 hr after challenge. Calcium entry was measured by uptake of 45Ca2+ into the culture during the challenge. RESULTS: Acamprosate produced no neurotoxicity in these cultures after acute or subchronic exposure. In contrast, the presence of acamprosate significantly reduced "basal" propidium iodide uptake caused by the medium change procedure; similar effects were obtained with dizocilpine (MK-801; 30 microM) and, to a lesser extent, with ifenprodil (50 microM). Acamprosate did not significantly potentiate or inhibit NMDA-induced neurotoxicity, but the presence of acamprosate significantly reduced spermidine-induced neurotoxicity. CONCLUSION: No evidence was obtained that the putative agonist or coagonist effects of acamprosate on the NMDA receptor are able to cause neurotoxicity. Similarly, no evidence for inhibitory effects of acamprosate on NMDA-induced toxicity was observed under any of these conditions. However, acamprosate significantly inhibited the toxicity associated with changing medium and the toxicity induced by spermidine in these hippocampal cultures. The mechanism is unknown but is compatible with previously reported inhibition of polyamine-mediated effects.

Recent developments in cornea transplantation (1997-1999).

Between 1997 and 1999 a steady increase in cornea donations was achieved, but the number of transplantations remained stable because many grafts did not pass quality control. Intermediate organ culture of entire bulbi was examined as a possible solution to reduce post-mortem times and increase suitability for transplantation.