RU2475251C1 - Method of combination treatment of abscesses in experiment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely surgery, and may be used for treating abscesses, festered residual cavities and ganglions. The cavity contents are daily aspirated through a catheter in laboratory animals (male rabbits) suffering simulated hepatic abscesses of the diameter of 1.5 cm. Then the abscess cavity is filled with copper nanoparticle suspension in 0.9% physiologic saline of the concentration of 1 mcg/ml. A laser light guide provided with a diffuser is placed in the cavity centre to expose to laser light at wave length 630 nm in a continuous mode, output power 35 mWt for 3 minutes.

EFFECT: method enables intensifying antimicrobial action of copper nanoparticles under laser light, starting treating without specifying an agent, suppressing pathogenic microogranisms over a relatively short time, stimulating repair processes successfully, reducing toxic action of nanoparticles on macroorganism, considerably reducing a length of treating festered cavities.

2 dwg, 1 tbl, 1 ex

Description

The invention relates to medicine, in particular to surgery, and can be used to treat abscesses, festering residual cavities and cystic formations.

There are many ways to treat liver abscesses: sanitation of the purulent focus, antibiotic therapy, anti-inflammatory, desensitizing therapy (Akhaladze G.G., Tsereteli I.Yu. Cholangiogenic abscesses in the liver // Consilium medicum. - 2005. - T.7, No. 2. Surgery (adj.). - C.1-9. Parkhisenko Yu.A., Glukhov AA et al. Diagnosis and treatment of liver abscesses // Surgery. - 2000. - No. 8. - S. - 35-37 Shoikhet Ya. N., Fokeev SD et al. Influence of local continuous intra-arterial antimicrobial therapy on the postoperative period in patients with peptic abscesses nor // Annals of Surgery. - 2008. - No. 1. - S. - 50-53), as well as the method of puncture-drainage treatment of abscesses under ultrasound control (A.F. Chernousov, A.D. Timoshin, V.Yu. Mishin, Yu.I. Yashkov. "Puncture-drainage treatment methods in abdominal surgery", Annals of surgery. 1997, No. 2, p. 42-47), which consists in puncture and drainage of the purulent cavity with one or more drains, depending on its volume, with washing with antibiotics and antiseptics. The presence of a large number of various means and methods of sanitizing purulent foci indicates their low efficiency.

The presented methods do not allow high-quality treatment of the walls of the cavity, since dense fibrin deposits that cover the abscess cavity from the inside inhibit the action of drugs, and at the same time, the dosage and group of drugs must be changed due to the formation of antibiotic-resistant forms of bacteria.

In the complex treatment of purulent surgical infections, phage therapy has been widely used recently. A method for the treatment of postoperative intraabdominal abscesses with local administration of a bacteriophage (Kurbangaleev S.M. Purulent infection in surgery. - M .: Medicine, 1985, p.106-109). "Means for the treatment of purulent-inflammatory diseases" (RF patent No. 2105544), "Drug for the treatment of purulent-inflammatory diseases" (RF patent No. 2144368), "Method for the treatment of postoperative abdominal abscesses" (RF patent No. 2243780).

However, despite a slight increase in the effectiveness of drug therapy, the field of application of these drugs in the treatment of abscesses is limited, and treatment is long, and the use of a bacteriophage is possible only after determining the type of pathogen - the minimum study time is 5-7 days.

Techniques and tools used to combat intracavitary infections are not as effective as required. Existing drugs are not able to stimulate sufficiently the regeneration processes, since the structure of the pathogens of purulent complications has changed and the commonly used drugs are ineffective, also because of the formation of polyantibiotic-resistant strains of bacteria, the drug has to be constantly changed.

The bactericidal properties of copper and its ions have been known to man since ancient times. In 2008, the US Federal Environmental Protection Agency (US EPA) officially granted copper the status of a substance with a bactericidal surface. The bactericidal and bacteriostatic effect of copper on the most toxic types of bacteria, fungi and viruses was proved: Akinetobacterium baumann, Black mold, Campylobacter, Aerobacter, Helicobacter pylori, Legionella, MRSA (including E-MRSA), Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus aureus vancomycin resistant, Adenovirus, Candida fungus, Clostridium difficile, Escherichia coli, Type A influenza virus (H1N1), Monocytogenic listeria, Poliovirus, Salmonella (Hartner's bacillus), Tuberculosis bacillus.

The literature reports on the use of copper nanoparticles in vitro are closest to this work (Babushkina I.V. Study of the antibacterial effect of copper and iron nanoparticles on clinical strains of Staphylococcus aureus / I.V.Babushkina, V.B. Borodulin, G.V. Korshunov, D.M. Puchinyan // Saratov Journal of Medical Scientific Research. - 2010. - T.6. - No. 1. - S.011-014.). When studying the antimicrobial effect of copper nanoparticles on gram-negative microorganisms, it was found that a suspension of copper nanoparticles at a concentration of 1 mg / ml has the greatest properties (V.A. Martyanova et al., 2010). A number of experimental studies have demonstrated that with the introduction of copper nanoparticles into the wound, the mechanisms of regulation of the microelement composition and the activity of antioxidant enzymes are stimulated, a wound healing effect is noted (N.N. Glushchenko et al., 2010), which is the prospect of creating a new class of antibacterial drugs. Unlike antibiotics, copper nanoparticles, due to their multidimensional effect on the bacterial cell, do not cause the selection of resistant strains, which makes it possible to recommend them for use in the treatment of purulent diseases caused by strains resistant to the antibiotics currently used.

There are known negative changes in the biochemical parameters of blood serum upon oral administration of copper nanoparticles. When assessing the biological effect of copper nanopowder on the activity of transaminases (AcAT and AlAT), used as a test for changes in membrane permeability and the severity of hepatocyte cytolytic syndrome, an increase in their activity was found to increase in proportion to an increase in concentration (0.05-5 mg / kg). An increase in the concentration of copper nanoparticles causes an increase in the activity of LDH, the most important biochemical indicator of carbohydrate metabolism, reflecting the severity of interstitial hypoxia, and CPK, which is a mitochondrial-cytoplasmic enzyme (Study of the biological activity of copper nanopowder./ Yu.S. Dudakova, T.A. Budunova. // Materials of the 70th scientific and practical conference of students and young scientists of SSMU: “Young scientists - to the health care of the region.” - 2009. - P.76-77).

There is information in the literature on a series of toxicological studies of copper nanoparticles. In most animals treated with copper nanoparticles, pronounced symptoms of damage to the gastrointestinal tract were observed - loss of appetite, diarrhea, and vomiting. In animals treated with copper nanoparticles, lethargy, oligopnea, tremor, and opistotonus were observed. In experimental animals with necropsy, a discoloration of the renal tissue to bronze was noted, as well as death of proximal tubule cells, glomerulonephrosis, massive necrobiosis, atrophy and color changes in the spleen. In addition to the above shifts, changes in blood biochemical parameters were noted - urea nitrogen, creatinine, total bile acids and alkaline phosphatase, indicating renal and hepatic dysfunction (Chen Z., Meng H., Xing G. et al. Acute toxicological affects of copper nanoparticles in vivo // The journal of physical chemistry. Toxicology letters, 2006 .-- 163. - 109-120).

The target organs of the toxic effects of copper nanoparticles were the liver, spleen, and kidneys (G. G. Onishchenko, B. G. Bikotko, V. I. Pokrovsky, A. I. Potapov. “The concept of toxicological studies, risk assessment methodologies, identification methods and quantification of nanomaterials, 2007).

By the value of the maximum tolerated dose (MTD) (Studying the safety of introducing copper nanoparticles with various physicochemical characteristics into the animal organism) / O.A. Bogoslovskaya, E.A. Sizova, V.S. Polyakova et al. // Vestnik ogu. - 2009. - No. 2. - S.124-127) the differences are in the range of 2.5-10 mg / kg of animal weight, according to LD50 - 7-15 mg / kg of animal weight and according to LD100 - 20-30 mg / kg of body weight animal.

Laser technologies are widely used in the treatment of purulent diseases. The basis of the mechanism of interaction of low-intensity laser radiation (LLLT) with a biological object is photophysical and photochemical reactions associated with the resonant absorption of light by tissues and the violation of weak intermolecular bonds, as well as the perception and transfer of the effect of laser irradiation with body fluids (Illarionov V.E. Fundamentals of laser therapy. - M .: Publishing house "RESPECT" Association INOTECH-Progress, 1992, p. 42-43).

The objective of the invention is to develop an effective combined method for the treatment of abscesses, which allows to reduce treatment time, reduce the toxic effect of nanoparticles.

The technical result is the complete suppression of pathogenic microorganisms and the successful stimulation of reparative processes. The technical result is achieved by combining the treatment of the abscess cavity with a suspension of copper nanoparticles and irradiation of the LLLT.

The severity of the antimicrobial action of copper nanoparticles and laser radiation in relation to the clinical strain of Staphylococcus aureus was evaluated in vitro by bacteriological methods in several stages:

1) assessment of the effect of laser radiation on the growth of colonies of microorganisms;

2) assessment of the effect of various concentrations of copper nanoparticles on the growth of colonies of microorganisms;

3) the study of the possibility of enhancing the antimicrobial action of copper nanoparticles in combination with the effects of LLLT.

A MacFarland standardized optical turbidity standard turbidity suspension of Staphylococcus aureus was used. The microbial load was 3 × 10 ⁴ microbial bodies / ml. In the first series of experiments, the microorganism culture was irradiated for three minutes with the Matrix VLOK laser therapeutic apparatus, type MLO1KR, wavelength - 630 nm, radiation power of 35 mW in constant mode with a scattering nozzle (spherical scattering diagram). In the second series, 0.2 ml of a suspension of copper nanopowders (average copper particle size of 60-80 nm) with final concentrations of 1000, 100, 10, 1 μg / ml was introduced into the microorganism culture. In the third series, laser irradiation (wavelength - 630 nm, radiation power of 35 mW in constant mode with a scattering nozzle) and the introduction of copper nanoparticles were combined. The control was a culture of microorganisms without laser exposure and the addition of nanoparticles. Immediately and after 1, 2, 3 hours of cultivation, measured sowing (0.1 ml each) was carried out on plates with meat-peptone agar and after 24 hours of incubation at 37 ° C under constant stirring at a speed of 50 rpm, the number of grown colonies was counted . During the statistical processing, they were guided by the method of determining the mean square deviation of the values found in the experiment, proposed by I.P. Ashmarin and A.A. Vorobyov.

The number of colonies of S. aureus strain depending on the time of seeding and cultivation conditions. Stages
work Conditions
cultivating Seeding time, h 0 one 2 3 TO* 3153 ± 5.2 lawn lawn lawn I Laser irradiation 3086 ± 2.0 99 ± 2,3 1212 ± 4.2 5416 ± 8.2 II Copper nanoparticles 1 μg / ml 3123 ± 2.2 lawn lawn lawn Copper nanoparticles 10 μg / ml 3163 ± 3.1 3550 ± 4.6 1094 ± 5.2 0 Copper Nanoparticles 100 μg / ml 3140 ± 4.3 42 ± 1,1 0 0 Copper Nanoparticles 1000 μg / ml 3128 ± 3.7 0 0 0 III Laser + Nano-copper 1 mcg / ml 152 ± 3.6 0 0 0 Laser + Nano-copper 10 mcg / ml 141 ± 4.5 0 0 0 Laser + Nano-copper 100 mcg / ml 103 ± 5.0 0 0 0 Laser + Nano-copper 1000 mcg / ml 98 ± 6.8 0 0 0 * K - control variant of the strain (physiological solution of sodium chloride without ultrafine copper powder and laser irradiation)

The study found that in the first and second stages, the obtained number of grown colonies immediately after sowing did not significantly differ from the control (see table).

At 1 and 2 hours of cultivation after laser irradiation, a decrease in the number of colonies of the experimental strain was noted to 99 ± 2.3 and 1212 ± 4.2, respectively, however, at the third hour, the growth of Staphylococcus aureus resumed. The antibacterial properties of LLLT are not effective enough.

Copper nanopowder (second stage) at a concentration of 1 μg / ml did not affect the growth of the experimental culture. A decrease in the number of colonies of microorganisms was noted with an increase in the concentration of ultrafine copper powder and cultivation time in comparison with the control, where growth was observed in the form of a lawn. The concentration of copper nanopowder 10 μg / ml causes a decrease in the number of colonies of Staphylococcus aureus in the first hours of contact with the culture with complete suppression of growth after 3 hours of exposure. The concentrations of nanomedi 100 and 1000 μg / ml inhibited the growth of the experimental culture at 2 and 1 hour, respectively.

At the third stage of work, immediately after exposure, a sharp inhibition of S. aureus growth is noted. The concentration of copper nanopowder 1, 10, 100 and 1000 μg / ml in combination with LLLT completely suppressed the vital activity of Staphylococcus aureus at 1, 2 and 3 hours of cultivation.

For the first time, the effect of enhancing the antimicrobial effect of copper nanoparticles in combination with the effect of LLLT on the culture of Staphylococcus aureus was revealed, which makes it possible to obtain the necessary antibacterial effect at lower concentrations of copper nanoparticles, thereby reducing the toxic effect of this substance on the body in vivo.

To create a liver abscess, laboratory animals (male rabbits) modeled a fibrous cyst with a diameter of 1.5 cm according to the method described in RF patent 2394278 (performed percutaneous liver puncture with laser support under ultrasound control, a two-channel catheter with a balloon was infused through the needle, which was inflated to the desired size of the simulated cavity in the liver tissue, sclerotherapy solutions were injected into the cyst cavity through the second channel of the catheter, the balloon was emptied for 5 days, x-ray and ultrasound cysts were monitored ), on day 6, 2 × 10 ⁹ CFU in 1.0 ml of a clinical strain of Staphylococcus aureus was injected into the formed cyst through the sanitizing channel of the catheter. Analysis of the obtained data showed that on the 3rd day after infection with S. aureus strain, the average number of bacterial cells in the aspirated animals separated from the cavity of the cyst was 5.12 × 10 ⁵ CFU / ml — a liver abscess was detected.

According to the variant of local treatment, laboratory animals with simulated abscesses were divided into 4 groups: 1 - standard sanitation (furatsilin, dioxidin), 2 - the use of laser therapy, 3 - the use of a suspension of copper nanoparticles, 4 - copper nanoparticles in combination with the effects of LLLT.

In all groups, treatment began with aspiration of the contents of the abscess. In the first group, the cavity was daily sanitized with a solution of furatsilin 1: 5000 with the subsequent administration of 0.5 ml of a 1% solution of dioxidine. In the second group, laser treatment was performed with a Matrix ALT apparatus, type MLO1KR (wavelength - 630 nm, constant mode, radiation power - 35 mW, scattering head with a spherical scattering diagram; power flux density (PPM) for a cavity with a diameter of 1.5 cm was 5 mW / cm ² ). Sessions were carried out daily for 3 minutes for 14 days. In the third group, once a day, the cavity was filled with a suspension of copper nanoparticles in a physiological solution of sodium chloride with a concentration of 100 μg / ml. In the fourth day, an abscess cavity was filled daily for ten days with a suspension of copper nanoparticles in a physiological sodium chloride concentration of 1 μg / ml, a laser fiber equipped with a scatterer was placed in the center of the cavity through the catheter sanitizing channel, and laser irradiation was performed (the exposure mode is similar to the second group of experiment) .

For a comprehensive assessment of the inflammatory process, an ultrasound scan was used (ultrasound), which was carried out on the 3rd, 5th, 7th, 10th, 14th, 30th day, daily microbiological examination of the contents of abscesses was carried out, and the general condition was assessed. animals. Ultrasound determined the diameter of the cavity and the thickness of the capsule. A bacteriological study of the contents of liver abscesses included a study of the qualitative composition and quantification of microorganisms.

Treatment results

Figure 1 is a photograph of the control seeding separated from the abscess cavities on the seventh day of treatment, where in the animals of the fourth group the growth of the colonies of the pathogen was absent during microbiological examination, in animals of the third group there was a more pronounced inhibition of the growth of S. aureus (104 ± 2.6) Compared with the first group (1936 ± 4.2), in animals of the second group there was no inhibition of the growth of colonies of microorganisms (lawn).

Purification of abscess cavities from the pathogen in animals of the first group was noted by the fourteenth day. When treating an isolated LILI (second group) from the cavities of animal abscesses, the separation of pus continued throughout the observation. In animals of the third group, the cavities were cleaned by the twelfth day of treatment.

Figure 2 is a photo of an ultrasound examination on the 14th day of treatment, where in the animals of the first group the cavity diameter decreased to 1.1 cm, in the second it remained the same (1.5 cm), in animals of the third group - 0.9 cm, in the fourth - up to 0.5 cm.

As a result of the application of standard rehabilitation of liver abscesses in the experiment (animals of the first group), the cavities were cleared of the pathogen only by the fourteenth day, while the size decreased to 1.1 cm, and by the thirtieth day the cavity size was 0.7 cm. Treatment of LILI (second group ) did not lead to a purification of the abscess cavity even by the thirtieth day of the experiment; with ultrasound, the diameter of the cavity remained the same throughout the entire observation. When treated with copper nanoparticles (third group) by the fourteenth day, the size of the residual cavities decreased to 0.9 cm, the cavity was cleared from the pathogen only by the twelfth day, and by the thirtieth day the diameter of the abscesses was 0.5 cm.

The first identified effect of enhancing the antimicrobial effect of copper nanoparticles under the influence of LLLT allows you to start treatment without determining the type of pathogen, significantly reduce the concentration of nanoparticles, which completely suppresses the growth of pathogenic microorganisms, and reduce its toxic effect on the macroorganism.

With the combination of nanomedi and LLLT, the infected cavities are cleaned of the pathogen as soon as possible, as evidenced by the lack of growth of colonies of the microorganism by the seventh day. According to ultrasound, by the fourteenth day of the combined treatment of liver abscesses, the size of the cavities was reduced by three times, by the thirties they were not determined.

The method is illustrated by the following example.

EXAMPLE

A male rabbit of a 1-year-old chinchilla breed weighing 3.5 kg with a simulated liver abscess 1.5 cm in diameter was aspirated daily from the cavity through a catheter, then the abscess cavity was filled with a suspension of copper nanoparticles in 0.9% physiological saline solution of sodium chloride at a concentration of 1 μg / ml, was placed in the center of the cavity with a laser fiber equipped with a scatterer through the catheter sanitizing channel and laser irradiation with a wavelength of 630 nm was carried out in a constant mode with an output power of 35 mW for 3 minutes. On the seventh day, there was a lack of growth of Staphylococcus aureus colonies during control seeding from the abscess cavity. On the tenth day of treatment, the catheter was removed. According to the ultrasound data by the fourteenth day, the diameter of the residual cavity was 0.5 cm. The control ultrasound was performed on the thirtieth day, while the abscess cavity was not determined.

Thus, the effect of enhancing the antimicrobial effect of copper nanoparticles under the influence of LLLT allows you to start treatment without determining the type of pathogen, completely suppress pathogenic microorganisms as soon as possible, successfully stimulate reparative processes, reduce the toxic effect of nanoparticles on a macroorganism, and significantly reduce the duration of treatment of purulent cavities.

Claims (1)

A method for the combined treatment of abscesses in an experiment, including aspiration of contents from a cavity through a catheter and exposure to a suspension of copper nanoparticles in physiological sodium chloride solution, characterized in that the abscess cavity is filled with a suspension of copper nanoparticles in physiological sodium chloride concentration of 1 μg / ml and at the same time an intracavitary laser is performed irradiation with a wavelength of 630 nm in a constant mode with an output power of 35 mW daily for 3 minutes for ten days.

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