3 Materials and Methods
3.3 Methods
3.3.9 Biocompatibility tests
3.3.9.3 Histochemistry analysis…
To evaluate SDH activity by histochemistry (SELIGMAN and RUTENBURG, 1951;
BROUILLET et al., 1998) frozen teat sections were cryosectioned at 8 µm at a temperature of -20°C. The udder tissue sections were transferred to glass slides and air-dried for 60 min before histochemical staining. Each tissue section was then incubated for 1 h at 37°C in a dark and humidified chamber in SDH phosphate buffer containing succinic acid (as a substrate) and nitroblue tetrazolium (SELIGMAN and RUTENBURG, 1951; TANJI and BONILLA, 2001; KIYOMOTO et al., 2008). After this procedure, the slices were rinsed with Aqua bidest. to stop the chemical reaction.
The slides were then cover slipped. Endogenous SDH activity resulted in dark blue diformazan deposits from the NBT reduction through succinate oxidation. No blue deposits are formed in the absence of succinate substrate or in the presence of 3NP in the incubation. To quantify SDH, an image of each section was captured using camera connected to an optical microscope. The protocol was applied on cell culture samples by culturing and treating in the same way as mentioned before in 3.3.7.
The medium was removed and cells were washed twice with PBS. 100 µl of histochemistry buffer was added and incubated for 4 h at 37°C and 5 % CO2. The measurements were performed using a spectrophotometer with a wavelength of 570 nm.
Materials and methods
3.3.10 Measurement of biomarkers of inflammatory reactions 3.3.10.1 Preparation of the udder tissue for PGE2 measurements
The teats were cut in small pieces of approximately 1 cm3 (25-30 mg) using a biopsy punch and were transferred in polypropylene tubes over crushed ice. A mixture of 5 mg indometacin + 5 ml DMSO (dimethylsufoxide) + 1 ml PBS was added and the samples were mixed by an ULTRA–TURRAX for 30 min, followed by centrifugation at 3000 x g for 10 min. The supernatants were collected for the PGE2 measurement.
3.3.10.2 Measurement of PGE2 in the udder tissue supernatant
The PGE2 concentration in the culture medium supernatant was measured by a competitive enzyme immunoassay. This assay is based on the forward sequential competitive binding technique, in which PGE2 competes with horseradish peroxidase (HRP) - labelled PGE2 for a limited number of binding sites on a mouse monoclonal antibody. PGE2 in the sample is allowed to bind to the antibody in the first incubation.
During the second incubation, HRP- labelled PGE2 binds to the remaining antibody sites. The amount of PGE2 tracer that is able to bind to the monoclonal antibody is inversely proportional to the concentration of PGE2 in the sample. This antibody-PGE2 complex binds to goat polyclonal anti-mouse IgG that has been previously attached to the well of the assay kit. The plate is washed to remove any unbound material, and a substrate solution is added to the wells to determine the bound enzyme activity. The product of this enzymatic reaction has a distinct yellow colour and absorbs strongly at 412 nm, when the colour development has been stopped.
The intensity of this colour is determined spectrophotometrically and is proportional to the amount of PGE2 tracer bound to the well, which is inversely proportional to the amount of free PGE2 present in the sample during the incubation. A standard curve was performed in each ELISA assay. The percentage of binding was used to establish a calibration curve. The measurements for the extinction of each sample were put in relation to these curves and the PGE2 concentration was calculated. The samples were usually measured in duplicates and the following two controls were included in each. One of the samples was stimulated by the addition of 5 mL lipopolysaccharide (LPS) working solution (500 ng/ml LPS, final concentration) while
Materials and methods
the other control was a pure medium. The supernatant was collected 24 h later and stored at -20°C until determination.
3.3.10.3 Measurement of bovine and mouse TNF-alpha in the culture medium supernatant
The TNF-alpha concentration in the culture medium supernatant was measured.
Briefly, the wells of the assay plates were preincubated with a monoclonal goat anti-mouse or bovine TNF-alpha antibody which binds any TNF-alpha of the sample (bovine or mouse). The bound TNF-alpha is detected using a biotinylated goat anti-mouse or bovine TNF-alpha antibody, and addition of TMB-substrate (tetramethylbenzidine) leads to a color reaction which stopped by using stop solution H2SO4 (2N). TNF-alpha was measured spectrophotometrically at 450 nm. The intensity of the colour is proportional to the concentration of TNF-alpha in the sample.
3.3.10.4 Measurement of IL- 6 in the culture medium supernatant
The IL-6 concentration in the culture medium supernatant was measured similarly to the TNF-alpha concentration with an enzyme immunoassay. Briefly, the wells of the assay plates were preincubated with rat anti-mouse IL-6 antibody, which binds any IL-6 of the sample. The bound IL-6 is detected utilizing a biotinylated goat anti-mouse IL-6 (detection antibody) and TMB-substrate. The colour reaction was stopped using stop solution H2SO4 (2N) andwas measured spectrophotometrically at 450 nm. The intensity of the colour is proportional to the concentration of IL- 6 in the sample.
3.3.10.5 Measurement of IL-1 beta in the culture medium
The IL-1 beta concentration in the culture medium supernatant was measured similarly to the IL-6 concentration with an enzyme immunoassay using the same capture and detection antibody.
All those assays are established methods at the Institute of Pharmacology, Toxicology and Pharmacy, Hannover, Germany.
Materials and methods
3.3.11 Detection of antibacterial activity
3.3.11.1 Bouillion dilution test and cultivation of bacteria in petri dishes
Bouillion dilution test and cultivation of bacteria in petri dishes were done by the Milchtierherden-Betreuungs- und Forschungsgesellschaft mbH (MBFG). The aim of the tests is to determine the effect of Mg-Ag-NaCl solutions on the growth of bacteria (E. coli and S. aureus).
Briefly, MgAg1% sticks were incubated in 5 ml NaCl solution in a water bath for 15 days and the degrading supernatants (MgAg1% sticks incubated in NaCl) were used for the bouillion dilution test and the cultivation of bacteria (E. coli and S. aureus).
Firstly, bouillion dilution test was done by preparing a bacterial suspension by incubating about 2 E. coli and S. aureus colonies (GK) in 100 ml buffered peptone water at 37°C for 24 hours on the shaker. The bacterial dilution series was770 X 105, 910 X 105, 100 X106 and 210 X106 KbE/ml. Then 2 test tubes were filled by 5 ml of bacterial suspension, then degrading supernatants (MgAg1% sticks incubated in NaCl) were added to one of the tubes, and the other one serves as a negative control. Later, the number of bacterial colonies was determined.
Secondly, E. coli and S. aureus colonies were cultivated in petri dishes. Afterwards, the bacterial colonies were incubated with the degrading supernatants (MgAg1%
sticks incubated in NaCl), and the number of bacterial colonies was determined.
3.3.11.2 Brilliant black reduction (BRT- MRL Screening test)
The BRT was first described by KRAACK and TOLLE (1967). The test medium is a mixture of nutrients, test bacteria Geobac. Stearothermophilus var. calidolactis C953 (B. stearothermophilus), brilliant black and other supplements which help to improve detection sensitivity towards chosen inhibitors (Fig. 13). Penicillin G serves as a positive control and milk as a negative control.
Materials and methods
Fig.13: The BRT- MRL screening test cavities
MgAg1% sticks were incubated with different volumes of DMEM medium (10, 3 and 1 ml). One tube of medium serves as a negative control, they were incubated in a water bath for 5 days. In addition, different concentrations of AgNO3 solution (control, 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mmol/l) were incubated with DMEM medium in test tubes without adding any supplements in a water bath for 5 days. 100 µl of each sample were added per well. Adhesive tapes were chosen to cover the wells during incubation for 5-6 hours at 65°C in a special incubator (Fig.
14).
Fig.14: Incubator of BRT- MRL Screening test
During the incubation time, the growing test bacteria shift the redox indicator (brilliant black) to its yellow or colourless reduction stage through the division of double azocompounds. Thus test medium changes from blue to yellow or colourless, if inhibitors are not present in the sample, therefore, the sample has no antibacterial effect. While the growth of bacteria will be minimal or non-existent, when there will be no reduction of the colouring agent or to a very small degree and the test medium will
Cavities containing test medium + other supplements
Materials and methods
remain blue, if the inhibitors are present in the medium, therefore, the sample has antibacterial effect.
3.3.12 Statistical analysis
Values are means ± SD. A randomized block design was used in all of the experiments. Samples were usually measured at least in duplicates. The experiments were performed at least 4-6 times.To enable better comparison of the experiments, results are expressed in optical density and percent in comparison to the negative control.
For statistical calculation of differences in the viability of isolated perfused udder (glucose consumption, lactate production and LDH activity), Two-way ANOVA test was performed. For the biocompatibility tests (MTS, neutral red, SDH, PK, IL-1 beta, IL-6 and TNF-alpha), One-way ANOVA test was performed followed by a Dunn’s Multiple comparison test. For the degradation process of MgAg1% sticks in dry off period secretion and in the teats of isolated bovine udder, t-test was performed. For silver and magnesium concentrations in degradation medium, Two-way ANOVA test was performed.
Statistical calculations were performed with GraphPad Prism® 5.03 (GraphPad Software Inc., La Jolla). P values < 0.05 were considered statistically significant.
Results
4. Results
4.1 Establishment of mammary cell culture 4.1.1 Isolation of primary mammary cells
Mammary cells were enzymatically and mechanically isolated and taken into culture.
Both primary mammary fibroblasts and primary mammary epithelial cells were morphologically distinct (Fig. 15 A, B). Each cell type exhibited the typical morphological characters. The fibroblasts showed a spindle shaped morphology and the epithelial cells grew in a cobblestone-like pattern. The purity of each cell population was ensured by the specific isolation method and the typical morphological appearance of the cells, respectively.
Fig. 15: Primary mammary fibroblasts (A) and primary mammary epithelial cells (B) under light microscope showed a spindle shaped morphology and cobblestone-like pattern, respectively cells; magnification = 20x; bar = 100 μm.
4.1.2 Culturing of primary mammary cells
Epithelial cells were first passaged every 7 to 10 days, thereafter cells could be passaged every 3 to 5 days, while fibroblasts were passaged every 12 to 15 days.
Then cells could be passaged every 8 to 10 days with an average number of 3-8 passages without changes in cell morphology.
A B
Results
4.1. 3 Verification of primary mammary cells using immunocytochemistry Immunofluresence was performed to verify the morphological distinction of the two cell types. Fibroblasts were detected using a monoclonal anti-vimentin antibody (Fig.
16, B), while adding anti-cytokeratin to these cells did not result in staining of them.
On the other hand, epithelium cells were detected using a monoclonal anti-pan cytokeratin which resulted in staining (Fig. 16, D), whereas the isotype control with the anti-vimentin antibody was negative.
Fig. 16: Verification of the primary mammary cells: phase contrast microscopy (A, C) and fluorescence microscopy of immunocytological stains (B, D). Mammary epithelial cells (C, D) have a cobblestone-like growth and stain for cytokeratin (D), whereas the fibroblasts (A, B) show a typical spindle-like morphology and are vimentin-positive (B) ; magnification = 40x;
bar = 50 μm.
A B
C D
Results
4.2 Degradation process of MgAg1% sticks 4.2.1 Isolated perfused udder
4.2.1.1 Measuring lactate production in the perfused udder
The udder viability was measured by calculating the amount of lactate production after the perfusation process; the amount ranged from 0.1-0.5 mg/ml which indicates that the udder was viable during the experiment. Lactate production did not show a significant difference on both sides over time (Fig. 17).
Fig. 17: Mean lactate concentration (mg/ml) in the perfusate of perfused udder. There is no significant difference of lactate production of left and right sides of perfused udder, n=6.
4.2.1.2 Measuring glucose consumption in the perfused udder
The glucose consumption during the perfusation process ranged from 45-77 mg/dl on both sides of the udder. Glucose concentration did not show a significant difference on both sides over time (Fig. 18).
Fig. 18: Mean glucose concentration (mg/dl) in the perfusate of perfused udder. There is no significant difference of glucose consumptions lactate production of left and right sides of perfused udder, n=6.
0 2 4 6
0 50 100
150 Left side
Right side
mg/dl
Time (h)
0 2 4 6
0.0 0.2 0.4
0.6 Left side
Right side
mg/ml
Time (h)
Results
4.2.1.3 Measuring lactate dehydrogenase enzyme (LDH) activity in the perfused udder
The lactate dehydrogenase production during the perfusation process ranged from 1000 -1750 mU/l on both sides of the udder. LDH concentration did not show a significant difference on both sides over time (Fig. 19).
Fig. 19: Mean LDH concentration in the perfusate of perfused udder (mU/l). There is no significant difference of (LDH) production of left and right sides of perfused udder, n=6.
4.2.2 Degradation of MgAg1% sticks in bovine udder
The MgAg1% sticks degraded in bovine udder after being incubated for 5-6 hours in the teat of isolated bovine udder (Fig. 20).
Fig. 20:MgAg1% sticks. The stick before incubation in the teat of bovine udder (A), while (B) showed the stick after the incubation.
0 2 4 6 8
0 1000 2000
3000 Left side
Right side
Time (h)
mU/l
A B
Results
The initial and the final weight of MgAg1% sticks incubated in the teat of isolated bovine udder for 5-6 hours for 7 separated experiments was listed in (Tab. 8). The mean percentage of weigh loss during incubation of MgAg1% sticks was about 3 % of the initial weight (Fig. 21).
Tab. 8: The initial and final weight of MgAg 1% sticks incubated in the teat of bovine udder for 5-6 hours (n=7)
Trials 1 2 3 4 5 6 7
Initial weight/mg 56.06 53.27 53.97 54.43 54.63 53.3 52.6 Final weight/mg
after 6 h
51.43 52.06 53.56 51.33 53.87 53.23 51.93
Fig. 21: Mean weight of MgAg1% sticks before and after incubation in the teat of bovine udder. There is a significant decrease of weight of MgAg1% sticks incubated 6 h in the teat of bovine udder (* P< 0.05, n= 7).
Initial we ight
Weig ht after loss 45
50 55 60
*
Weight in mg
Results
4.2.3 Degradation of MgAg1% sticks in secretion samples from cows at dry off Period
MgAg 1% stick was cutted into three parts nearly of the same weight.The initial and final weight of MgAg1% sticks after incubation in secretion from dry cows for 21 days was listed in (Tab. 9). The mean loss percentage for 6 separated experiments after incubation was 60 % of the initial weight, regarding that the stick of the highest weight was completely degradable after 21 days.
Tab. 9: The initial and final weight of MgAg1% sticks incubated in dry secretion for 21 days (n=6)
Trials 1 2 3 4 5 6
Initial weight 15.7 mg 14.0 mg 10.3 mg 9.5 mg 10.1 mg 8.6 mg Final weight Completely
degraded
3.8 mg 6.6 mg 4.0 mg 4.7 mg 5.3 mg
The mean final weigh of 6 separated experiments of MgAg1% sticks, after incubation in dry secretion for 21 days, revealed a significant difference compared to the initial weight (Fig. 22).
Fig. 22: Mean weight of MgAg1% sticks before and after incubation in dry off period secretion. There is a significant decrease of weight of MgAg1% sticks incubated 21 days in dry secretion (* P< 0.05, n=6).
initia l we
igh t
Final weight 0
5 10 15 20
weight in mg
*
Results
The MgAg1% sticks degraded after incubation in dry off period secretion for 21 days compared to the initial stick (Fig. 23).
Fig. 23: Cutted MgAg1% sticks. The stick before incubation (A). The stick after the 21 days of incubation in dry off period secretion (B).
A B
Results
4.2.4 Histological parameters
4.2.4.1 Udder tissue incubated with MgAg1% sticks
Both unstained teat tissue (Fig. 24, B) and teat tissues stained with hematoxylin and eosin (H&E) treated with MgAg1% sticks (Fig. 24, D) showed black deposits precipitated on the upper layer, which is more prominent compared to the unstained and stained tissues without treatment (Fig. 24, A & C). This indicates the degradation of MgAg1% sticks during incubation
Fig. 24: The unstained teat tissue treated with MgAg1% sticks (B) and stained teat tissue with hematoxylin and eosin (H&E) treated with MgAg1% sticks (D) showed black deposits precipitated on the upper layer compared to control teat (A, C); magnification = 40x; bar = 50 μm.
C D
A B
Results
4.2.5 Silver concentrations in the degradation medium
The amount of silver ions increased after 25 days of incubation in the degradation medium. The 1 ml volume of degradation medium contains higher concentration of silver than the volume of 3 and 10 ml. The mean concentration of Ag+ ranged from 0.02 -1 mmol/l (Fig. 25)
Fig. 25: Mean silver concentration (mmol/l) in degradatiom medium after incubation of MgAg1% sticks in 1, 3 or 10 ml medium. There is a significant increase of silver concentration in degradation medium after 25 days of incubation (* p< 0.05, n=6).
4.2.6 Magnesium concentrations in the degradation medium
The amount of Mg++ in the degradation medium increased after 10 days up to 25 days of incubation without showing any significant differences between 1, 3 and 10 ml volume of degradation medium. The mean concentration of magnesium ranged from 0.2 - 9 mmol/l (Fig. 26).
Degradation medium
0 0.2 0.4 0.6 0.8 1 1.2 1.4
5 10 15 20 25
Days of incubation
Amount of silver (mmol\l)
1ml 3ml 10ml
*
* *
Results
Fig. 26: Mean Mg++ concentration (mmol/l) in degradation medium after incubation of MgAg 1% sticks in 1, 3 or 10 ml medium. There is an enhanced concentration of magnesium in degradation medium on day 10 up to 25 (* P< 0.05, n=6).
Degradation medium
0 2 4 6 8 10 12 14
5 10 15 20 25
Days of incubation
Amount of Mg (mmol/l)
1 ml 3 ml 10 ml
*
*
*
* *
*
**
*
* *
*
Results
4.3 Biocompatibility tests 4.3.1 Cell viability and proliferation 4.3.1.1 MTS assay
1. Murine cells
a. Incubated with silver ions
Murine fibroblasts (L929 cells) were treated with different concentrations of AgNO3
solution. A significant reduction in the viability of murine fibroblasts of 50 % was observed, when incubated with concentrations equal or above 0.1 mmol/l and more than 75 %, when incubated with concentrations above or equal to 0.3 mmol/l compared to the negative control cells (Fig. 27).
Fig. 27: Cell viability (MTS) of murine fibroblasts as indicated by the mean optical density of formazan after incubation in various concentration of AgNo3. A significant reduction in the viability of murine fibroblasts at 0.1 mmol/l of AgNO3 solution compared to control was observed (* p< 0.05, n= 6).
Raw murine macrophages and primary murine macrophages were treated with different concentration of AgNO3 solution. A significant reduction in the viability of raw macrophages was observed by 30 %, when incubated with concentrations equal or higher than 0.3 mmol/l, while primary macrophages showed a significant reduction in viability by 35 % when incubated with concentrations equal or below 1 mmol/l (Fig.
28).
control
0.00010.0003 0.001 0.003 0.01 0.03 0.1
Concentration of AgNO3 (mmol/l)
L929 cells
Results
Fig. 28: Cell viability (MTS) of raw macrophages and primary macrophages as indicated by the mean optical density of formazan after incubation in various concentration of AgNo3. A significant reduction in the viability in raw macrophages at 0.3 mmol/l AgNo3 solution compared to control, while primary macrophages showed a significant reduction in the viability at 1 mmol/l AgNo3 solution compared to control (* p< 0.05, n= 5).
Raw macrophages
Concentration of AgNO3 (mmol/l)
Optical density
Concentration of AgNO3 (mmol/l)
Optical density
Primary macrophages
Results
b. Incubated with MgAg1% sticks
Murine fibroblasts were treated with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). A significant reduction in viability of about 75 % was observed, when murine fibroblasts were incubated with volumes equal or below 1 ml of degradation medium (1 ml = 0.5 mmol/l Mg++,0.2 mmol/l Ag+) (Fig. 29).
Fig. 29: Viability test (MTS assay) of murine fibroblasts as indicated by the mean optical density of formazan after treatment with degradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). A significant reduction in the viability of murine fibroblasts at 1ml compared to control regarding that, 10 ml ≈ 0.2 mmol/l Mg++, 0.02 mmol/l Ag+, 3 ml ≈ 0.3 mmol/l Mg++,0.04 mmol/l Ag+ and 1 ml ≈ 0.5 mmol/l Mg++,0.2 mmol/l Ag+ (* p< 0.05, n= 5).
control 10 ml 3 ml 1 ml 0
1 2 3
*
Different volumes of degradation medium (ml)
Optical density
L929
Results
Both raw murine macrophages and primary murine macrophages did not show a significant reduction in the viability, when treated with degradation medium (MgAg1%
sticks incubated in 1, 3, 10 ml medium) (Fig. 30).
Fig. 30: Cell viability (MTS asaay) of raw macrophages and primary macrophages as indicated by the mean optical density of formazan after treatment withdegradation medium (MgAg1% sticks incubated in 1, 3, 10 ml medium). No significant reduction in the viability of raw macrophages and primary macrophages compared to control regarding that, 10 ml ≈ 0.2 mmol/l Mg++, 0.02 mmol/l Ag+, 3 ml ≈ 0.3 mmol/l Mg++,0.04 mmol/l Ag+ and 1 ml ≈ 0.5 mmol/l
Different volumes of degradation medium (ml)
Raw macrophages
Different volumes of degradation medium (ml)
Primary macrophages
Results
2. Bovine cells
a. Incubated with silver ions
Primary mammary epithelial cells were treated with different concentrations of AgNO3 solution and showed a slight tendency toward reduction, when incubated with concentrations equal or below 1 mmol/l (Fig. 31).
Fig. 31: Cell viability (MTS assay) of primary mammary epithelial cells as indicated by the
Fig. 31: Cell viability (MTS assay) of primary mammary epithelial cells as indicated by the