Aim of the study
Various scientific approaches were already been used to investigate the physiology and pathophysiology of inflammatory processes caused by infections as well as pharmacological interventions, ranging from cell culture experiments to in-vivo-studies [12-14]. A benefit of in-vitro-studies is the possibility to use larger numbers of replicates. This is rarely possible in ex-vivo- or in-vivo-experiments due to higher costs, lack of tissue availability and ethical issues [15]. It should be noted that there are also significant individual differences between genetically similar animals, requiring research on a large number of animals and consequently long and expensive experimental set-ups, having a greater impact on animal welfare [16]. Therefore, precision-cut tissue slices may be helpful to perform experimental studies before animal studies are performed. It was the aim of the present study to establish PCBUS as a possible replacement and complement to in-vivo-infection experiments.
Tissue sources and preparation
In the described study, bovine udders of slaughtered dairy cows were used. In the past, it was demonstrated in several studies that slaughter material can be used to investigate different issues with reproducible results [17-19]. Although the udders were already adspectively and palpatorically examined on site and only the udders found to be healthy were selected for transport to the laboratory, in the laboratory additionally, the so-called California Mastitis Test (CMT) was performed with the milk of the supposedly healthy udder. Only udders whose cell count in the milk at the CMT was below 100.000 were included in the experiments. However, these examinations did not reveal totally whether there was a subclinical inflammation in the present organ. It should be helpful to get information about the stage of lactation of the slaughtered cows. Unfortunately, this was not possible in the present study. Because no preliminary report was available for the dissected udders, this is a limitation of the model.
When cutting the udder tissue, it became apparent that the udder quarters have a variable tissue structure and consistency. This interesting discovery mostly correlated with the size of the udder. In consequence, it was always important to adapt the method of obtaining PCBUS; the different lactation stages and the differences for milk in the udder required an adjustment of the number of washing steps to remove the remaining milk from the tissue. Based on the results of this feasibility study, it is therefore recommended that udder health (adspection, palpation, CMT result) and udder size (cow age, lactation stage) must to be taken into account when selecting the sample material. In future studies it is now possible, for example, to investigate differences between the individual lactation or age stages.
The measurement of the tissue slice thickness (Fig. 4) confirmed a good reproducibility with regard to the method of sample preparation. This shows the success of the cutting technique using the dermatome, which is important because tissue thickness can influence its cultivability and nutrient supply. Previous studies showed that precision-cut lung slices (PCLS) are better supplied with oxygen and nutrients at a section thickness of 250 µm [20] than the precision-cut lung slices generated with a thickness of 500–1000 µm [21].
An advantage of the described method is the generation of large number of PCBUS.
If the cutting and punching technique is correctly applied, more than 200 precision-cut udder slices can be obtained from one udder quarter. This allows simultaneous experimental approaches to the material and thereby originate from only one animal. In comparison to other studies, the technique used here has the advantage that the thickness of the samples [9] and thus the diffusion distance for nutrients and metabolites is smaller, resulting in a better exchange between tissue and environment, as already described for PCLS [20].
Morphology
The histological examination of HE stained PCBUS confirms the typical morphology of bovine udder tissue (Fig. 3). This is consistent with results of previous studies [9], also identifying the morphological structure of bovine udder tissue from their udder explants. Our method not only allows the natural cell structure to be preserved, sections can also be studied in histological examinations, for example to check cell integrity after external stimuli. In addition, maintaining a certain slice thickness shows the high reproducibility of the model and ensures that all PCBUS are equally supplied with culture media.
Viability
The viability of the PCBUS were determined by the MTT assay (Fig. 1 and 2). This test has already been used in numerous studies and is one of the standard tests for determining tissue or cell viability [22-24]. The ISO 10993-5:2009 standard [11] describes, that MTT assay results below 70 % compared to 0 indicate reduced viability. Consequently, this limit was used in this work to exclude unsuited tissue slices. No PCBUS with viability of less than 70 % were used in the experiments. However, it should be considered from which region of the udder the samples are generated, as the tissue composition is variable (ratio of gland tissue and connective tissue). Therefore, a sufficient number of PCBUS is necessary to minimize deviations.
The metabolic conversion of the dye during the MTT assay is done by the mitochondrial dehydrogenases of the gland cells, in the connective tissue of PCBUS the dye cannot be produced by corresponding cells. So, if the ratio of glandular to connective tissue in the individual PCBUS is different, the result of this test could fluctuate. However, the PCBUS obtained from one udder showed for the most part a similar tissue structure, so that a similar ratio of glandular to connective tissue is assumed.
Nevertheless, the PCBUS could be reliably cultivated and maintained in a viable condition for more than 96 h, regardless of the tissue composition.
Early inflammatory reaction
LPS and PGN were used as stimulants for the experiment, as these are cell wall components of Gram-negative and Gram-positive bacteria, respectively, which have an immunostimulating effect, thereby recruiting immune cells and inducing the release of cytokines [14, 25, 26]. The feasibility of the experimental setup was confirmed by the fact that stimulation with LPS or PGN was possible. The results of a stimulation by LPS and PGN (1 µg/mL each) showed an enhanced secretion of IL-1ß, TNF‑α and PGE2 after stimulation. After stimulation of PCBUS with LPS, a significant release of IL-1ß as well as PGE2 can be seen after only one hour and is still visible after four and six hours. In contrast to the control, the release of PGE2 is even significant after four and six hours. A combination of the two stimulants (LPS:PGN) even led to a significant release of PGE2 after only one hour, until the end of the study period. In comparison, the release of IL-1ß was significant only after one hour. We also expected a significant increase in IL-1ß release at the other measurement points in contrast to the control. However, this could not be observed, which we explained after the first investigations by the fact that also the cells of the control PCBUS start to become apoptotic and increase the IL-1ß concentration. This requires further attention in future studies and needs to be observed more closely. In contrast, a significant release of TNF-α could only be shown after stimulation with LPS 24 hours. The stimulation of PCBUS with PGN did not show a significant increase in any of the mediators, investigated at any time period considered. It must of course be considered that cell types are confronted with the stimulants that would not encounter the stimuli in vivo. In order to study cells that are only present in the lumen or milk, a different model should be chosen, e.g. the isolated perfused udder [18]. Therefore, the PCBUS model focuses on the large time slot, which allows longer-term examinations than the isolated perfused udder, where only studies of 8 hours maximum are possible.
It can be concluded, that the sections are useful to study the effect of stimulating agents. Further investigations can provide information on how PCBUS react to a bacterial infection.