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Effect of cold plasmic coagulator on biological tissues

Avramenko K. C., Stupin I. V., Kopenkin E. P.

While treating wounds using Cold Plasmic Coagulator (CPC), coagulation is made with cold plasmic beam. At that the depth of thermal injure of treated wounds comes to 0.1-0.2 mm.

The purpose of research is to study coagulation of liver tissue after resection by means of CPC using gases of helium and argon or by means of plasma flow without carrier gas.

Perfection of coagulation device (Patent of Russian Federation 22100013) were carried out in 45 tests on rats of Vistar line and body mass equal to 200g. Coagulating and keying properties were

appreciated with the following graded frequency variation of current oscillations: 50kHz, 100kHz and 150kHz. While 50 kHz frequency in the moment of contact of tissues with plasma flow shaking of device and animal body was observed, and surgeon-s hands sensed current discharge. The like effect was observed while approaching plasma flow to 2 sm. To the object. Increasing frequency of current oscillations to 100 kHz observed effects were weakening, and disappeared tootally when frequency of current oscillations increased to 150 kHz. Coagulating effect depended on power and volumetric gas flow rate. With speed of 2 litters/min. flow jet didn-t deviate, and dispersion didn-t occur. Increasing this to 6 liters/min. favored the stability of plasma flow, but the effectiveness of coagulation decreased greatly. The latest was higher with low gas flow and high power of electric discharge.

Thus the effectiveness of coagulation is influenced by volumetric gas flow rate and power of electric constituent.

While testing of biological effect of cold plasma 12 unracy dogs of both genders with body mass equal to 15-20 kg., skin sections, hypodermic cellular tissue and muscle were treated with CPC.

The checkups were animals, underwent like operations using standard methods of stanching of bleeding by means of ligating blood vessels with silk thread. General state of animals, body temperature, appetite, wounds- healing rate and presence of complications were considered.

In blood samples taken in 1, 3, 7 and 14 days after operation morphological blood make-up was determined with using of common methods. Also the activity of lysosomal and cytoplasmic ferments was determined with using and analyzer "Impact-400" of "Corning" company (Great Britain).

Animals and control dogs, underwent plasmic effect were diagnosed and flabbiness, drowse, high body temperature and lack of appetite were not found. Cutaneous injures closed up in common dates, without suppurative inflammation.

While biochemical research, directed at research of possibility of damaging hepatic cells, was determined that using of plasmic beam in volume no more thanoperational intervention didn't cause cell alteration. Ferment activity (gamma-glutamiltransferase, alkkaline phosphotase, asparate, alanineminotransferas and lactatedehydrogenase) varied within the limits of physiological norm.

Levels of bilirubin, sugar and overall proteins in blood also were also evidences of integrity of hepatic functions. The activity of creatinephosphokkiase, lactatdehydrogenase, aspartatataminetransferase and butiretehydrogenase in pereferial blood were evidences of cardiomiocites damage; butiratedehydrogenase was the evidence of muscle injury. The activity of the last didn't change in all the diagnosed animals. The increase of lactatedehydrogenase level in 5 daays after operation was noticed, which means that may be referred to the damaging of skeletal muscles. Plasma flow didn't also affect renal functions, because creatinine and residual nitrogen concentration in blood plasma remained unchanged.

Lack of hyperglickemia or reliable change of of activity of amilaze of blood was evidence of correct functioning of pancreas.

Thus the using of cold plasma for tissue coagulation doesn't have toxic effect on vitals and overall organism condition.

Pigs, dogs and rabbits were resected 10% of hepatic mass with scalpel, electrocauter, beam of hot plasma and CPC using argon for plasma-creating gas under hexenaline anesthesia. Resection of liver, temporary excluded from circulation of the blood, occured without bleeding. Injure of liver was treated with CPC of new construction. In different periods after operation organ pieces were placed into neutral 10% formalin, then after dehydretionin spirts of increasing concentration were put into paraffin, and microscopic cuts 5 micron-thick were colored with hematoxiline and eosine.

Liver morphology was studied in 3 series of experiments after resection and treating wounds with CPC.

Histological researches were executed just after operation (1st series v 1 piglet, 3 dogs and 2 rabbits), 3 hours later (2nd series v 1 pigle, 3 dogs and 3 rabbits), and 5 hours after resection (3rd series v 1 piglet, 2 dogs and 3 rabbits).

In the first series cell destruction in the affected area came to 1500-2000 microns, cell outlines could not be found and contents of hepatocites cytoplasm grew more homogeneous, nucleuses acquired elongate, baculiform shapes and were situated perpendicular to affecting factor. Beyond that zone cells with large cytoplasm vacuoles, reduced nucleus sizes and poorly-colored cytoplasm outlines were situated. Leucocyte infiltration was lacking. In the affected zone mesathelium was flaked away. In the other organ sections in structure level traces of affection were not found. The zone of pronounced necrosis with flaking from less damaged organ tissue came to 2000-2500 microns. In this zone strong destruction of hepatocites, lysis of most cell nucleuses, homogenization of cytoplasm contents, and slight infiltration of tissue by leucocytes and signs of seizure of damaged tissue were found. In the outline of the affected zone organ sections lacked of tissues (futility), on the area equal to the one of 10-20 hepatocytes. These were filled with flaky homogeneous contents. Obiviously, these futilities were formed in place of sinusoid blood-circulating capillaries.

Vessel, situated near the staggered zone were filled with erythrocytes and leucocytes. Hepatocites, pertained to the zone were more solid (stronger colored), corpulent cells were not found.

3 hours after the operation (2nd series) the zone of destruction came to 10000 microns and more. In individual organ sections damaged tissue flaked and considerable hemorrhages occurred. After the necrosis zone vast sections of destructive tissue were situated, in which blood-circulating vessels were strongly broadened, filled with blood, mostly with erythrocytes and leucocytes. Hepatocites were changed, vacuolated, feeble-colored compared with undamaged cells. The tissue necrosis zone was made with large vacuoles, filled with tissue liquid. In the outline of necrosis zone considerable number of corpulent cells. On histological cuts the zone came to 2 mm. in the organ's depth. In it destructive hepatocites, vast sections of hemorrhage and homogeneous bar-shaped tissues were found.

Indicated bars of the liver tissue represented merged hepatocites and destroyed liver girders. 5 hours after the operation (3rd series) total necrosis zone came to 2 mm. deep. Destructive hapatocites, hemorrhages and sectors of homogeneous tissue as bars, were detected. They represented merged hepatocites and destroyed liver girders. In the zone, adjacent to the necrotized tissue sector, vessels were strongly widened and fulfilled with blood elements. Liver girders were much apart from each other, and sinusoid capillaries were filled with blood. Among blood cells, infiltrated through tissue, many macrophages were detected. Between the total necrosis zone and tissue, retained its morphology (border zone) mucopolysaccharides were detected in considerable amount (metachromatic colored tissue) in the form of fine fibre and primary matter of connective tissue.

Presence of metachromasis in the tissue was an evidence of mucopolysaccharides synthesis process. Fat cells were not detected.

Liver resection and wound treatment with CPC with use of argon were researched in 4 experimental series.

First series v acute experiment (1 piglet, 3 dogs, 3 rabbits), second series vnext day after the operation (1 piglet, 2 dogs, 3 rabbits), third series v three days after the operation (1 piglet, 2 dogs, 3 rabbits), fourth series v six days after the resection (1 piglet, 3 dogs, 3 rabbits).

In the first series carbonization zone in the spot of coagulator effect was not detected. In the contact spot strong vacuolisation of parenchyma was detected. Vast roundish cavities formed, evidently, through evaporation of in- and between cell liquid with following tissue edema (150 microns deep).

Next to it was the zone of parenchyma carnification with strong effect of coagulation necrosis: deformed, picnotic, unstructured nucleuses lay in weak-oxiphil unstructured homogeneous mass, cell outlines could not be detected, depth of damage came to 100-200 microns. In more distant zones of parenchyma strong dystrophic and necrobiotic shifts were detected. In all parts organ stroma was collabirated, nucleus' structure could not be followed neatly, cytoplasm of parenchymatous cells was large-lumpish. Some of the cells left only picnotic nucleus and withered capsule, and cytoplasm was all but detected (shock cells).

Damage of blood vessel cells was detected: endothelium pavement in many of them was lacking, edema, segmental and total plasmarrhages were detected. Part of blood vessels was spasmated, endotheliocydes' nucleuses stood in opening fence-shaped in the meanwhile. Perivascular edema and widening of perisinusoid Disse areas were detected. Transgression of rheological blood properties was detected: separation of form elements from plasma. Part of vessels was strongly plethoric. Along with disseminated coagulation hepatocytes' changes parenchymatous cells in colliquation necrosis with cytolisis of nucleuses were detected. From the side of bile-ducts transgressions were not detected. It is necessary to note, that damage found in liver parenchyma spread deep from the place of coagulator effect to all thickness of researched organ sector (0,5 sm).

In the second series strong leucocyte reaction was observed (polymorph-nucleus leucocytes infiltrated affected parenchyma 500 microns deep from coagulator-treated surface). The zone of strong necro-biotic transgressions of liver tissue 800-900 microns wide then followed. Parenchyma had a view of oxyphil denuclearized mass (karyolysis), organ cytoarchetonics was utterly transgressed, roundish disconnected oxyphil formations were all what was left from the cells. Needed to say, that one day after the liver resection with coagulator "intact" parenchyma zone was neatly delimited from irreversibly damaged tissue, boundary line was situated 1300-1400 microns deep from the place of contact. At the border with unchanged parenchyma demarcation billow was formed. It was represented with polymorph-nucleus leucocytes, macrophage reaction was also shown. Directly after the billow dystrophic changes developed, in some parenchymatous cells grainy dystrophy was found, in others - vacuole dystrophy. Parenc

hyma-disseminated hepatocytes in condition of coagulation and colliquacious necrosis with plasmo- and cariolysis were observed. But joisted formation of organism was observed in all fields of vision. Some veil of nucleus structure of parenchymatous cells, nidal reactive hyperemia in-segment sinusoid capillaries and central veins were noted. Peri-sinusoid spaces of Disse were enlarged. Blood vessels' walls were hydropic, endothelius pavement was damaged. Diapadese and nidal hemorrhages were observed.

In the third series part of irreversibly damaged parenchyma was represented by necrotic masses with nidi of purulent fusion. At the border with "intact" zone formed demarcation billow, development of grainy tissue with large macrophage amount, not numerous phiroblasts, and structuring blood capillaries were observed. In "intact" parenchyma organ cytoarchetonics could be neatly observed in all fields of vision. Hepatocyes were polygonal-shaped, with small-grained cytoplasm and neat nucleus structure. Only in parenchyma sections, belonging to developing granulation tissue vacuole dystrophy was observed (200-250 microns). Nucleuses of star-shaped reticular-endotheliocytes in irritated condition, swollen, were directed into opening of in-segmental sinusoid capillaries. Nidal parenchyma hyperemia was observed.

In the fourth series granulation tissue formed neat border between necrobiotic changed and intact parenchyma. In granulation tissue large number of macrophages, phiroblasts and capillaries were present. In adjoining sections of "undamaged" parenchyma (150-200 microns) vacuole hepatocytes dystrophy was observed, in-segmental sinusoid capillaries were enlarged and fulfilled with blood. In parenchyma lymphoid infiltrations were seldom met. Cellular infiltration was found around bile-ducts, and also around triads, portal fields were enlarged and infiltrated mainly by the cells of lymphoid row. Walls of some blood vessels were damaged. Organ cytoarchetonics remained unchanged in all fields of vision. Reticular-endotheliocytes system heightened activity was observed.

Comparative study of liver morphology after resection and wound treatment with CPC without using carrier gas was carried out in 3 experiment series accordingly 1, 3 and seven days after the operation (in each 1 piglet, 2 dogs and 3 rabbits took part).

In the first series affected zone was represented with narrow line of coagulation of yellow-colored blood (10-20 microns), it was followed by the vacuolization zone - coagulation parenchyma necrosis with evaporation of in- and between-cellar liquid with the following tissue edema. Then deep from contact place parenchyma had the view of unstructured strong-oxyphil denuclearized mass (karyolysis), among which polymorph-nucleus leucocytes appeared. Border of parenchyma intact zone was neatly delimited from damages and was situated at the distance of 1800-1850 microns from the zone of contact with plasma beam. In "intact" parenchyma, adjacent to the damaged one, organ structure remained unchanged. In the depth of 200-250 microns enlargement and plethora of in-segmental sinusoid capillaries were observed, in them besides erythrocytes dead parenchymatous cells and isolated pycnotic nucleuses (cell detritus) were found. In all fields of vision single hepatocytes in condition of coagulation necros

is were seldom met. Nidal microdrop vacuolization of parenchymatous cells was noted. While moving away from the affected zone these transgressions graded. At the same time blood vessels' walls damage (desquamation of endothelium layer, edema and plasmorhages in the wall) were sometimes met in more distant sectors of "intact" parenchyma. In some vessels transgression of rheological blood properties: mixing of form elements from plasma, fibrin sutures were seen.

In the second series at the liver border zone of cold plasma affection was detected as coagulated yellow-colored blood with lesser surface tissue carbonization. Here and there gatherings of fibrin , infiltrated by polymorph-nucleus leucocytes were adjacent to the carbonization area, dystrophic changes of neutrophils were noted. Then vacuolated layer was found (as consequence of evaporations of in- and between-cellar liquid); deeper - unstructured, denuclearized oxyphil mass, infiltrated with polymorph-nucleus leucocytes. On the border with unchanged parenchyma demarcation billow was formed.

It was represented with polymorph-nucleus leucocytes. Initial stages of reparation process in parenchyma were detected - formation of granulation tissue, consisting of young tissue-connecting cells, macrophages, forming blood vessels. In adjacent zones (400-450 microns) of "intact" parenchyma strong vacuole hepatocytes dystrophy was observed. In some parenchymatous cells evidences of coagulation necrosis were observed (nucleuses were pycnotic, cytoplasm was strongly basphilic, unstructured); in others colliquatous transgressions prevailed (vacuolization of cytoplasm, cytolysis of nucleuses). Not large amount of "shock" cells was found. Hepatocytes' dystrophic changes were noted in onsiderable distance in the depth of "intact" parenchyma. Cupfer's cells (star-shaped reticular-endotheliocytes) were in condition of irritation everywhere (nucleuses were swollen, outstood in open space of in-segmental sinusoid capillaries, some cells peeled off from capillary walls and polymorphed into free macrophages). Peri-sinusoid Disse spaces were enlarged. Wall damage of some blood vessels and transgressions of rheological blood properties were detected.

In the third series the damaged parenchyma section was represented by necrotised tissue with nidals of purulent fusion. On the border with "intact" parenchyma goodly formed fresh connecting tissue with numerous newly-formed blood capillaries could be seen. In adjacent sections of "intact" parenchyma (100-150 microns) large-vacuolar dystrophy was detected, in other parenchyma mild dystrophic hepatocytes changes could be seen. Lymphoid infiltrates were seldom noticed. Around bile-ducts leucocyte (eosinophilic) infiltration was observed. Peri-vascular infiltrations were sometimes met. Nidal vacuole dystrophy was detected. Portal fields in triad region were enlarged, lymphoid-histo-cytar infiltration by them with admixture of polymorph-nuclear leucocytes was noted. Organ tissue was moderately fulfilled with blood.

Conclusion. Possibility of coagulation of bleeding tissue with Cold Plasmic Coagulator of new construction with resection using helium argon as plasma-creating gas and without carrier gas when coagulating with plasma beam (or beam of charged particles) was determined. In all cases safe bleed-stop was attained. In place of crossing of bigger vessels second coagulation or more long exposition (10-20 seconds) may be needed. Bleeding stops faster when using plasma beam without carrier gas.