Bacteria Escherichia is the basis of human and animal intestinal microflora. The group of entheropathogenic
E. coli causative colibacillosis has biomedical implication. The virulence of these bacteria associated, at first, by toxins production. The affect of E. coli toxins on physiological processes of microorganism particularly investigated.1 At the same time response of intestine blood vessels on the action of toxins ex tempore in vivo is not describe practically. Changes in blood microcirculation system can be important diagnostic sign, reflecting the interaction of microorganism with surrounding internals´ and tissues´ cells. One of the perspective methods of evaluation of these changes in biomedical researches is speckle-microscopy2-5. Thereby, we carried on an investigation with this method to study the effect of E. coli toxins on blood microcirculation in the course of short intervals of time.
2. Methods and materials
We studied exotoxin producing by strain Escherichia coli A5 and endotoxin producing by strain Escherichia coli B6. Each strain cultured in meat infusion bouillon at 37o C separately. Daily strain cultures centrifuged at 600 g to get supernatants, which used for experiments.
2.2 Laboratory animals
An experimental animals (white rats) sedated by intramuscular injection of 5-Ethyl-5-(1-methylbutyl)-
2,4,6-pyrimidinetrione (Nembutal). Then we abducted the abdominal cavity and eviscerated the ventral mesentery. After the abduction we placed rat on the thermostabilizing stage of speckle-microscope. So, the loop of ventral mesentery was placed directly under the microobjective.
2.3 Speckle-microscope and its optical schemes
Optical scheme of speckle-microscope for investigations of random bioflow is shown in Fig. 12. Beam of He-Ne laser (λ=633 nm) is focused into the spot of small radius ( =1.5 mm) on the investigated microvessel. Conventional optical microscope supplied by monochrome Mutech 1280-USB digital CMOS camera enables observation of blood flow in a vessel visually. Computer image analyzer processes consequence of the video images (frame-by-frame analysis).
As blood or lymph flows through the vessel or probing beam scans the investigated surface, the strongly focused laser beam is modulated in the waist plane. This leads to the formation of dynamic speckle pattern in the far zone of diffraction. Speckles of large size are formed in the case of small number of scatterers, so diameter of aperture of photodetector is essentially smaller then average speckle size. The temporal fluctuations of scattered intensity are detected by photoreceiver. Time-varying signal is amplified, recorded on the tape and processed further by computer6.
Figure 1. Optical scheme of speckle-microscope
1 - laser, 2 - microobjective with 10xmagnification, 3 - beamsplitter, 4 - microobjective with 8x magnification, 5 - stage, 6 - biological object (mesentery of white rat), 7 - mirror, 8 - lamp, 9 - photoreceiver with pinhole, 10 - TV camera, 11 - computer.
2.4 Original research
We brought a quantity one of the daily culture supernatant (~1ml) on the ventral mesentery loop and registered output signal of speckle-microscope for 10 second immediately. Repeated superimposing accomplished after 1, 2, 3, 4 and 5 minutes for dynamic process was studying. Blood flow characteristics before supernatant superimposing were used as control. The speckle-signal is amplified, recorded as avi-file and processed further by the computer with the original algorithm for MathCad 2001 program.
Obtained data are presented as changes of signal spectrum bandwidth (BDW) for each second during the time registration.
We compared the effect of E. coli exotoxin and endotoxin on blood microcirculation in white rats ventral mesentery. Obtained results are presented in Fig. 2 and Fig. 3.
Fig. 2 shows the affect of E. coli A5 on blood microcirculation in capillaries. It is noted immediate increasing of blood flow velocity after superimposing supernatants.
Superimposing of E. coli B6 endotoxin shown lower fluctuations of blood flow velocity (Fig. 3).
Thereby superimposing of E. coli toxins caused immediate change of blood flow velocity in the course of short intervals of time. Thereat superimposing of E. coli A5 exotoxin offered the more intensive reaction, than E. coli B6 endotoxin. There was nearly twice increase of signal spectrum bandwidth in this case, while superimposing of E. coli B6 endotoxin resulted in increase of ~20 Hz maximum.
This work has been funded by Russian Foundation of Basic Researchers (Grant No. 06-04-39016), National Science Foundation of China (Grant No. 30711120171), and US Civil Research and Development Foundation (AWARD REC~006).
Figure 2. The effect of Escherichia coli A5 exotoxin on blood microcirculation in white rats ventral mesentery
Figure 3. The effect of Esherichia coli B6 endotoxin on blood microcirculation in white rats ventral mesentery
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