Teste Ischemie de Reperfuzie-Inducere de Moarte Celulara Blocare de Apoptoza pe Nanosof

RESEARCH REPORT CONCERNING CERTAIN BIOLOGICAL EFFECTS OF THE POWDERED PRIMARY PRODUCT NANOSOF

  1. Experiments on rat isolated heart – Langendorff system

The study was aimed to identify in a model of global ischemia arrhythmogenic or antiarrhythmic effects, effects on ventricular myocardium contractility, coronary flow, and capacity of recovery following ischemia of the powdered primary product Nanosof at various dilutions applied in an isolated heart preparation. The superfusion solution was of Krebs-Henseleit type (in mM: CaCl2 1.75, KCl 4.70, KH2PO4 1.2, MgSO4 1.2, NaCl 118, NaHCO3 25, D-glucose 11), sterile filtered via vacuum absorption through cellulose acetate filters with 0.45 m pore diameter, saturated with carbogen (95% O2, 5% CO2) and stored in sealed polyethylene plastic bags for perfusion. The Langerdorff apparatus contains 3 ways for superfusion of solutions, an upper vent to eliminate gas bubbles, and a double-walled lower vessel made of heat-resistant glass, forming a thermostated wet chamber for keeping the isolated heart. The heating circuit was assured by a Haake K15 temperature control bath with cooling and external circulation pump. The solution temperature at the level of the isolated heart was measured with an IT-18 T type thermocouple (Physitemp Instruments, Clifton, NJ) and was set to 37 °C. The superfusion pressure was set by adjusting the height of the liquid column (~100 cm H2O). In most of the global ischemia-reperfusion experiments the heart was completely immersed in Krebs-Henseleit solution degassed by boiling throughout the period of ischemia.

The experiments were achieved on adult male Wistar rat (300-350 g) isolated heart. Under deep ether anesthesia, the thorax was largely opened. The heart was removed, cannulated on ice and placed in the Langendorff superfusion system. Within a set of experiments a double monitoring was achieved: of the left ventricular pressure with a home-made balloon catheter coupled to a conductimetric wedge pressure transducer (SensoNor840), and of an electrocardiography lead with a set of two electrodes: the metal cannula inserted in the ascending aorta, and a 0.1 mm in diameter Pt-Ir wire inserted deep in the ventricular wall. Both electrical signals were amplified and filtered with AC/DC Grass CP122 instrumentation amplifiers and sampled with a Digidata 1440A AD/DA unit (Axon Instruments, part of Molecular Devices, Sunnyvale, CA) controlled by the pClamp 10 software. The pharmacological compound to be tested was applied directly in the superfusion system, the moments of solution exchanges being marked with tags on the recordings.

The preparation of the Nanosof primary powdered product suspension was performed by mixing 5 mg of product (the oxygenated variety, of a rust-like color, with paramagnetic properties, sonicated) in 25 ml Krebs-Henseleit solution filtered and treated with carbogen, shaking followed by centrifugation at 2000 rcf for 2 min without activated brake on a Heraeus Labofuge 400R centrifuge with swing-out rotor, and collection of the supernatant only. The primary dilution, used in most of the experiments, was obtained by a 1:100 dilution of the supernatant (e.g. 5 ml added in a perfusion bag containing 500 ml Krebs-Henseleit solution). In a separated experiment the effects of the supernatant were tested directly in successive decimal dilutions up to 10-7 on an isolated heart preparation without global ischemia.

Table 1. Summary of experiments aimed to test the effects of the suspension of the NANOSOF powdered primary product on the rat isolated heart model in Langendorff superfusion system

Exp. no.

Identification code of recording

(xx-x-xx=YY-M-DD)

Experiment description

#7

11 1 03 000

30 min global ischemia (CONTROL)

#8

a1 1 03 000

? (regional ischemia)

#9

11 1 10 000

? (control)

#10

11 1 14 000

global ischemia (CONTROL)

#11

a1 1 14 000

NANOSOF progressive dilutions

#12

11 1 20 000

001

002

NANOSOF progressive concentrations (without TAGs)

#13

11 1 22 000

NANOSOF 1:100 without ischemia (temporary bradycardia, reduced coronary flow; mechanical stimulation)

#14

a1 1 22 000

30 min ischemia-reperfusion with NANOSOF 1:100

(spectacular recovery of electrical and mechanical activity after ischemia – artifact given by temporary obstruction of the LVP monitoring catheter ?)

#15

11 1 25 001

30 min ischemia-reperfusion (CONTROL)

#16

a1 1 25 000

30 min ischemia-reperfusion (NANOSOF 1:100 over the entire duration)

#17

b1 1 25 000

30 min ischemia-reperfusion (NANOSOF 1:100 over the entire duration)

#18

failed experiment

#19

11 2 04 000

30 min ischemia-reperfusion (NANOSOF 1:100 late during reperfusion – at 30 min after start of reperfusion – leads to restart of heart beats !)

#20

11 2 04 001

30 min ischemia-reperfusion (NANOSOF 1:100 in reperfusion)

#21

11 2 18 000

45 min ischemia-reperfusion (CONTROL)

#22

a1 2 18 000

45 min ischemia-reperfusion (NANOSOF 1:100 in reperfusion)

Table 2. Coronary flows measured in experiments aimed to test the effects of the suspension of the powdered primary product NANOSOF on the rat isolated heart model in Langendorff superfusion system

Exp. no.

Identification code of recording

(xx-x-xx=YY-M-DD)

Time

Coronary flow

(ml/min)

Remarks

#13

11 1 22 000

2:30

2:37

2:45

2:58

2.3

2.5

1.8

1.4

2:31 NANOSOF 1:100

2:42 mechanical stimulation

2:45 change to normal Krebs

#14

a1 1 22 000

3:10

3:25

3:29

3:31

3:35

3:45

3:47

Reperf

1’

4’

6’

8’

13’

15’

18’

21’

24’

27’

30’

33’

39’

45’

51’

58’

6.8

6.4

6.8

6.2

5.8

4.0

4.0

1.6

2.6

1.8

1.6

1.6

1.6

1.6

1.4

1.4

1.2

1.2

1.2

1.2

1

1

1

37.8 C

*

change to normal Krebs

#15

11 1 25 001

17

26

40

50

60

Reperf 64 (10’)

69 (15’)

76

83

90

6.5

6.8

9.0

7.9

6.2

8

8.5

8.0

6.5

5.6

crash

Record #2 54=12:24

#16

a1 1 25 000

5’

30’

36’

40’

Reperf 5’

10’

20’

25’

30’

4

9.4

12

10

7

6.3

6.0

5.2

6.0

NANOSOF

#17

b1 1 25 000

10

20

30

40

Reperf 5’

10’

15’

20’

25’

30’

35’

40’

45’

50’

55’

60’

11

10, 10.6, 10.4, 10.8

10.4

9.8, 8.8

5

4.3

4.3

4.2

4.2

4.0

4.0

not measured

not measured

3.8

3.6

end-distolic lowered 3 times

#19

11 2 04 000

10

15

20

25

30

Reperf2’

5’

10’

15’

20’

25’

30’

45’

47’

50’

53’

59’

15

13

12

5.5

6

6

5

6.5

6

6

5.5

4.5

NANOSOF

#20

11 2 04 001

10

15

20

25

30

Reperf2’

5’

10’

15’

20’

25’

30’

35’

22

22.5

19

17

17.5

17

12.5

11.5

9

8.5

8

7.5

6.8

Treatment only in reperf.

NANOSOF

Min 12’ – normal Krebs

#21

11 2 18 000

45

50

60

Reperf

2’

5’

10’

15’

20’

25’

30’

10

10

6.5

5.8

4.75

4.25

NBF fixing

#22

a1 2 18 000

30

40

50

Reperf

2’

5’

10’

15’

20’

25’

30’

8.5

7.2

10

5.5

5.7

5.75

5.75

NANOSOF

30 min global ischemia (CONTROL)

? (regional ischemia)

? (control)

global ischemia (CONTROL)

NANOSOF progressive dilutions

NANOSOF progressive concentrations (without TAGs)

NANOSOF progressive concentrations (without TAGs)

NANOSOF progressive concentrations (without TAGs)

NANOSOF 1:100 without ischemia (temporary bradycardia, reduced coronary flow; mechanical stimulation)

(detail – reversibile bradycardia following application of NANOSOF 1:100)

30 min ischemia-reperfusion with NANOSOF 1:100

(spectacular recovery of electrical and mechanical activity after ischemia – artifact given by temporary obstruction of the LVP monitoring catheter ?)

30 min ischemia-reperfusion (CONTROL)

30 min ischemia-reperfusion (NANOSOF 1:100 over the entire duration)

30 min ischemia-reperfusion (NANOSOF 1:100 over the entire duration)

30 min ischemia-reperfusion (NANOSOF 1:100 late during reperfusion – at 30 min after start of reperfusion – leads to restart of heart beats !)

30 min ischemia-reperfusion (NANOSOF 1:100 in reperfusion)

45 min ischemia-reperfusion (CONTROL)

45 min ischemia-reperfusion (NANOSOF 1:100 in reperfusion)

Conclusions

  • the suspension of NANOSOF powdered primary product applied in experiments on isolated heart in dilutions of 1 up to 10-7 exerts a series of reversible temporary effects: bradycardia and a coronary flow increase up to 30-50%;

  • the suspension of NANOSOF at 1:100 dilution applied over the entire duration of ischemia-rerperfusion experiments leads to an acceleration in ventricular contracture onset and to an increased degree of contracture (100-120 cm H2O vs. 60-80 cm H2O in control experiments); therefore intravenous application of such a suspension is recommended only during reperfusion, after coronary obstruction removal via different procedures;

  • the suspension of NANOSOF at 1:100 dilution during reperfusion can produce a series of beneficial effects, possibly via neutralization of toxic and arrhythmogenic effects of free oxygen radicals accumulated in afflicted tissues during ischemia: limitation of supraventricular/ventricular fibrillation and acceleration of sinus rhythm recovery, sinus rhythm stabilization and reduction of ventricular premature beats frequency; within an isolated experiment, late application of NANOSOF suspension (30 min after start of reperfusion) resulted in restarting the electrical and contractile activity of the heart.

  • N.B. The conclusions are based on a limited number of experiments; in order to achieve an increased validity these experiments have to be repeated on larger samples.

2. Cell viability and apoptosis experiments on an in vitro model of capsaicin-induced apoptosis

We used a human diploid cell line: HEK293 (human embryo kidney 293) stably transfected with the vanilloid receptor 1 (TRPV1 – transient receptor potential – vanilloid 1), an ion channel acting as a molecular integrator of noxious stimuli, activated by noxious heat (thermal threshold 42 C), extracellular acidity (half-activatory pH 5.2, Hill coefficient 1.8), capsaicin and other vanilloid or non-vanilloid agonists (EC50 for capsaicin: 0.5 M, Hill coefficient 2.0-2.85), and by endovanilloid compounds (e.g. anandamide). This cell line represents a gift from Prof. Makoto Tominaga, Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes for Natural Sciences, Okazaki, Japan. The cells are cultured in DMEM supplemented with 10% fetal calf serum and 1% penicillin-streptomycin, and are passsed weekly via detachment with trypsin-EDTA, centrifugation, resuspension in culture medium and plating in cell culture vials.

For viability and apoptosis experiments we used 12 Petri dishes 35 mm in diameter plated with equal amounts of a homogeneous cell suspension, cultured in identical conditions (humidified incubator at 37 C, 5% CO2). The Petri dishes were divided in four groups, subjected to different treatments; thus, each experiment was repeated in triplicate.

Group 1 (dishes 1,2,3): the control group

Group 2 (dishes 4,5,6): capsaicin 0.1 M applied 2 hours prior to the experiment

Group 3 (dishes 7,8,9): Nanosof suspension 0.5 mg/10 ml culture medium applied 24 hours before the experiment

Group 4 (dishes 10,11,12): Nanosof suspension 0.5 mg/10 ml culture medium applied 24 hours before the experiment and capsaicin 0.1 M applied 2 hours prior to the experiment

At a concentration of 0.1 M we can assume an activatory effect of capsaicin on TRPV1 vanilloid receptors expressed in the cultured cell line, leading to a significant calcium influx into cells and subsequent triggering of the cascade of molecular events known as apoptosis.

For each of the 12 samples of cells within the 4 groups we applied apoptosis and cell viability detection protocols with propidium iodide (PI), based on flow cytometry experiments, using a Beckman-Coulter FC 500 Series instrument. For each sample 30,000 cells were analyzed.

The cells within each sample were detached with EDTA, centrifuged 5 min at 1000 rcf, resuspended in 1 ml PBS, then equal amounts (0.5 ml) were used for the two experimental protocols.

Cell viability protocol

0.5 ml cell suspension in PBS was treated with PI 5 g/ml 15 min at dark.

The samples were centrifuged 5 min at 1000 rcf, the supernatant was removed and the pellet was resuspended in PBS for flow cytometry.

DNA content analysis protocol for detection of apoptosis

0.5 ml cell suspension in PBS was added to 10 ml 70% ethanol kept on ice and cells were fixed for 2 hours.

Cells were centrifuged 2 min at 2000 rfc.

The cell pellets were resuspended in 50 L buffer for DNA extraction (0.2 M phosphate citrate buffer pH 7.8: 683.48 mg Na2HPO4 and 16.81 mg citric acid for 20 ml).

The vials were mechanically shaked 30 min at 37 C.

The cell suspensions were centrifuged 10 min at 1500 rfc.

The pellets were resuspended in 1 ml PBS with PI 5 g/ml and Triton X-100 0.1 % (unfortunately we could not add RNase A) and incubated 30 min at room temperature.

Results of the viability test

Sample no.

Group

Viability

(% PI negative cells)

1

control

75.00

2

65.16

3

66.04

4

Capsaicin 0.1 M 2 hours

56.36

5

54.22

6

46.55

7

Nanosof 0.05 mg/ml 24 hours

57.69

8

67.61

9

61.30

10

Nanosof 0.05 mg/ml 24 hours +

Capsaicin 0.1 M 2 hours

81.82

11

63.29

12

58.40

Results of the DNA contents analysis for detection of apoptosis

Quality of analysis Model fit %G1 %S %G2 G1 µ G2 µ G1 cv G2 cv %Apoptosis %Nonapoptotic Sample no.
OK

0.26

15.8

44.2

27.3

235

486

10.1

16.3

35

65

1

OK

0.47

57

16.4

27.3

321

609

24.8

19.5

5.26

94.74

2

OK

0.83

4.18

82.3

0

266

519

5.05

8.28

6.42

93.58

3

OK

0.28

25.5

60.9

11.3

263

564

12.3

8.82

24.8

75.2

4

OK

0.63

20.1

62.2

11.2

224

562

10.8

7.88

12.9

87.1

5

OK

0.63

13.7

31

52.6

235

460

14

29.4

7.2

92.8

6

Low

0

15.5

54.6

0

227

460

12.5

0

20.5

79.5

7

Low

0

4.2

71.1

0

206

460

2.01

0

33

67

8

OK

0.21

29.2

38.2

28.7

261

492

10.3

17.5

14.7

85.3

9

OK

0.29

23.4

45.4

28.4

262

529

13.3

28.8

15.8

84.2

10

OK

0.19

21.7

67.9

7.05

260

550

8.86

6.83

11

89

11

OK

0.45

24.6

61.9

13.7

258

525

12.7

8.34

8.45

91.55

12

Remarks

Quality of analysis: Low = uncertain results

Model fit: how well does the mathematical model of phases fit the real curve; the smaller the value the better

%G1, S, G2: percentages of non-apoptotic cells in the corresponding phases of the cell cycle

%G1, 2: the fluorescence channel containing the peak corresponding to the listed cell cycle phase

%G1, 2 cv: variation coefficient of data for phases G1, G2

%Apoptosis: percentage of apoptotic cells of total cells identified by size and granularity

%Non-apoptotic: percentage of non-apoptotic cells of total cells identified by size and granularity

Conclusions

The pretreatment of the HEK293 cell line stably transfected with TRPV1 with a 0.05 mg/ml suspension of the Nanosof powdered primary product for 24 hours can prevent induction of apoptosis via intracellular calcium influx triggered by exposure of cells to 0.1 M capsaicin for 2 hours, in an experimental in vitro model of apoptosis.

The results of DNA contents analysis for detection of apoptosis are less reliable because of the reduced sizes of samples subjected to measurement.

Dr. Sorin Muşat Dr. Bogdan Amuzescu