Научная статья на тему 'Improved results of treatment of purulent wounds with complex use of photodynamic therapy and CO2 laser in the experiment'

Improved results of treatment of purulent wounds with complex use of photodynamic therapy and CO2 laser in the experiment Текст научной статьи по специальности «Фундаментальная медицина»

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Ключевые слова
PHOTODYNAMIC THERAPY / PHOTOSENSITIZER / EXPERIMENT / METHYLENE BLUE / PURULENT WOUND / MICROBE / CO2 LASER / LASER SURGERY

Аннотация научной статьи по фундаментальной медицине, автор научной работы — Teshaev Oktyabr Ruhullaevich, Murodov Alijon Salimovich, Sadykov Rаsul Rustamovich, Hamdamov Bakhtiyor Zaripovich

The aim of research was the evaluation of the effectiveness of photodynamic therapy and CO2 laser destructive purulent wounds of soft tissues in the experiment. Photodynamic therapy is a very effective non-invasive and gentle treatment of purulent wounds and serve as justification for the use of the method of photodynamic therapy in clinical practice for the treatment of acute local inflammatory processes combined with CO2 laser and traditional treatments. These laboratory, thermometry, morphological, microbiological and planimetric data suggest that photodynamic therapy and CO2 laser is very effective non-invasive treatment of purulent wounds and serve as justification for the use of the method in clinical practice for the treatment of local acute purulent-destructive diseases of soft tissues.

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Текст научной работы на тему «Improved results of treatment of purulent wounds with complex use of photodynamic therapy and CO2 laser in the experiment»

I II II

GCh TG LPLD LPHD

I at the moment of discharge in the remote phase

Fig. 3. Indexes of blood lipid profile in MI patients in stationary condition and in the remote monitoring phase (mg/dl)

In the remote monitoring phase the most negative tendency related to absence of reaching a target level oflipids among majority of patients, although it was observed veracious decreasing of GCh (22 %), TG (16 %), LPLD (21 %).

How can we explain insufficient efficacy of ongoing drug therapy in patients with MI in the remote phase?

Results of investigation showed that in 11.7 %cases (n = 39) patients had forgotten to take drug preparations and violated the multiplicity of their taking. In 47.9 % cases (n = 161) violations was not administrated treatment in scheme. But we have to emphasize,

that in 40.4 % cases (n = 135) patients independently changed doses of drug preparations and even multiplicity of their taking.

Conclusions:

1. Results had demonstrated insufficient efficacy of drug therapy in scheme of secondary prophylactic in patients, who suffered from MI as in stationary condition, so in the remote monitoring phase.

2. It was established, that drug preparations is administrated in remote phase in relatively low doses at the remaining nontarget levels of SAP, DAP, HR, GCh, LPLD, LPHD, and TG.

1. 2.

3.

4.

References:

Кардиология. Национальное руководство. Под редакцией Беленкова Ю. Н., Оганова Р. Г. - М.: ГЕОТАР-Медиа, 2007. - 1232 с. Adherence to evidence-based therapies after acute coronary syndrome: a retrospective 1. population-based cohort study linking hospital, outpatient and pharmacy health information systems in Valencia, Spain/G. Sanfelix-Gimeno et al.//J. Manag. Care Pharm. - 2013. -Vol. 19, № 3. - P. 247-257.

Effect of evidence-based drug therapy on long- term outcomes in patients discharged after 2. myocardial infarction: a nested case-control study in Italy/U. Kirchmayer et al.//Pharmacoepidemiol. Drug Saf. - 2013//[Electronic resource]. - Available from: http://www. ncbi.nlm.nih.gov/pubmed/23529919

Kumbhani D. J. Predictors of adherence to performance measures in patients with acute 3. myocardial infarction/D. J. Kumb-hani//Am. J. Med. -2013. - Vol. 126, № 1. - P. 741-749.

Teshaev Oktyabr Ruhullaevich, Murodov Alijon Salimovich, Sadykov Rasul Rustamovich, Hamdamov Bakhtiyor Zaripovich, Tashkent Medical Academy E-mail: saodat.us@mail.ru

Improved results of treatment of purulent wounds with complex use of photodynamic therapy and CO2 laser in the experiment

Abstract: The aim of research was the evaluation of the effectiveness of photodynamic therapy and CO2 laser destructive purulent wounds of soft tissues in the experiment. Photodynamic therapy is a very effective non-invasive and gentle treatment of purulent wounds and serve as justification for the use of the method of photodynamic therapy in clinical practice for the treatment of acute local inflammatory processes combined with CO2 laser and traditional treatments. These laboratory, thermometry, morphological, microbiological and planimetric data suggest that photodynamic therapy and CO2 laser is very effective non-invasive treatment of purulent wounds and serve as justification for the use of the method in clinical practice for the treatment of local acute purulent-destructive diseases of soft tissues.

Keywords: Photodynamic therapy, photosensitizer, experiment, methylene blue, purulent wound, microbe, CO2 laser, laser surgery.

Despite the advances made by medical science, questions of of the wound supports inflammation and slows down the course treatment of patients with suppurative-septic pathology of the skin of reparative processes. According to the literature information, and soft tissues do not lose their actuality. Bacterial contamination the number of patients with acute purulent soft tissue diseases are

among the surgical patients 30-60 % (Svetuhin A. M. et.al, 2002; Goryunov S. V., 2004, Fransuzov V. N., 2006). In this connection continues to develop alternative technologies and methods for the topical treatment of infectious diseases of the skin and soft tissues of different etiology (Baranov Y. V., 2011).

For solving this problem recently successfully uses laser photo-dynamic therapy. Photodynamic therapy is a relatively new method of treatment ofpurulent diseases, and currently PDT has high potential for its use in many clinical directions. In sensitized cells and tissues develops photochemical reaction with the release of singlet oxygen, free radicals and highly active biological objects detrimental effecting, in particular, on tumor cells, microorganisms and etc. (Stranadko Y. F., 2000, Popova N. A., 2007, Ginyuc V. A., 2011, Sibata C. H., 2001).

To study the effects of CO2 laser and photodynamic therapy on current of acute purulent-inflammation and wound healing dynamics, we decided to perform a simulation in laboratory animals in experimental purulent wounds. The experiment was conducted in a vivarium of TMA.

Objective: Improve the treatment of purulent destructive wounds by topical application of the CO2 laser and photodynamic therapy in the experiment.

Materials and methods

For the experiment used: laser-photodynamic apparatus (taken emitters with a wavelength of 670 and 470 nm.), the photosensitiz-er is methylene blue, CO2 laser machine, Wistar male rats weighing from 180 to 250 grams. As an indicator for the purulent surgical infection strains of microorganisms taken: Staphylococcusaureus, Escherichiacoli, Pseudomonasaeruginosa, Bacteroidesfragilis, Clos-tridiumdifficile.

All animals were kept on a standard diet in the vivarium of the Tashkent Medical Academy with free access to food and water. Conditions contents of rats were: air temperature in box 18-20 °C at a relative humidity of 55 %. The animals were kept in a dedicated box for 7 days to adapt to the conditions of the vivarium. Prior to the experiment, all animals were weighed, thoroughly inspected for cash the presence of visible pathology and symptoms of disease. If there is no healthy animals are culled and were not included experiment.

Simulation of purulent wounds, her follow-up treatment, the maintenance of animals, removing them from the experiment and sampling of the material took place in a dedicated control box, corresponding all the rules and regulations when dealing with opportunistic microorganisms, in compliance with all the rules of aseptic and antiseptic [3; 4].

Method of modeling acute local inflammatory processes in the experiment

In the pre-depilated femoral-rump skin of rat with marker using a cardboard template applied outline future wounds: a circle with a diameter of 2.5 cm. Skin treated twice with antiseptic and under general anesthesia excised skin and subcutaneous tissue to the superficial fascia. Next Kocher clamp crushes the edges of the wound and underlying muscles. Then, the bottom and the edges of the wound became infected 24-hour suspension of the mixture of microbes, taken in equal volumes, containing in 1 ml. 10 9 microbial bodies (the concentration was determined by turbidity standard). The volume injected suspension of microbes was 2 ml. Then, to create impermeability, to prevent wound injuring and to contamination by micro-organisms around, the edges of the wound stitched plastic ring with a side height of 1.2 cm. and a diameter of 2.5 cm. The ring is fixed on both sides. In the filed thus ring inserted dry sterile gauze ball. The skin was closed by the imposition of leading individual nodal joints. Thereafter, the

rats were in individual cages to prevent cutting of ligatures and doing additional injury to each other. Access to food and water was free.

Depending on the procedure conducted by the local treatment ofpurulent wounds the animals were divided into 4 groups, characteristics of which are presented in Table 1.

Table 1. - Distribution of the animals selected for experimental studies

№ Monitoring groups Number of animals

1 Observing the natural course of wound process 20

2 Study of the influence of the traditional method of treatment on the course of wound process 20

3 Study of the effect of photodynamic influence on the course of wound process 20

4 Study the influence of the traditional method of treatment in combination with CO2 laser and photodynamic therapy on the course of wound process 20

Results and their discussion

The purulent wounds in rats were formed after 48 hours and had all the classic signs of inflammation. The edges of the wound were with necrotic areas, slightly hyperemic, edematous. The bottom of the wounds was wet, had a color from yellow-green and purple-bluish to black-necrotic areas with previously injured muscle necrosis and fibrin overlays. Discharge from the wound, which was soaked gauze ball, delivered on the first day of the experiment, there was purulent in moderate amounts from 0.7 to 1.0 ml. of turbid, yellow-green color with hemorrhagic component, with specific smell. All animals have the same shape, area and location of the wound, which is important for further comparison and subsequent analysis of the dynamics of the wound early healing.

Some animals have evolved putrid infection in the wound, the generalization of infection process. These animals are a further experiment, did not take part, derived from the experiment by an overdose of anesthesia (including provisions for the humane treatment of animals).

All the animals were divided into control and experimental groups, depending on the method of treatment. In each group were of 20 rats. In the course of the experiment for evaluating the effectiveness of the treatment was carried out a thorough dynamic monitoring of the general state ofthe animals, the local course ofwound process, the healing progress. Monitor and evaluate the rate of contraction of the skin, the presence and disappearance of redness around the wound, the rate of formation of primary and secondary scab, the nature of discharge from the wound — its color, smell and number. Watch for changes in the peripheral blood, a qualitative and quantitative change in the dynamics of microbial inoculation of the wound. In this regard, at certain intervals a part of rats was deduced from an experiment. Fence material took place at 3, 7, 10, 14, 21, 28th day from the start of the experiment. Usually in each group in terms were withdrawals of these for 3-4 rats. We have estimated the « survival — mortality», for statistical processing of material. It was conducted a dynamic photo archive.

On the third day from the moment of infection, depending on the intended method of treatment is carried out following manipulations.

The control animals (the 1st group), no therapeutic intervention was used. We monitor and evaluate the natural course of the wound healing process.

Animals (2nd group) whose treatment was carried out only with the help of traditional methods of treatment, on the third day after the infection was removed suggestive stitches, plastic ring and a gauze ball, delivered on the first day of the experiment, and the wound treated with a solution of hydrogen peroxide and after draining inserted Levomekol ointment. And also with the aim of antibiotic therapy Ceftriaxone pricked 1.0 grams in a dosage of 0.3 ml. of 1 times a day.

The next group of animals whose treatment was carried out using photodynamic therapy (3rd group), on the third day after infection apposition sutures removed and wound was opened, bead gauze was removed, placed on the first day of the experiment, and the wound treated with a solution of hydrogen peroxide. Subsequent session conducted with the application of photodynamic therapy bead sterile gauze soaked with an aqueous solution of 0.005 % MB at the wound surface layer to 1-2 mm. with fully closing the wound surface. It was applied opaque bandage. After 30 minutes of application held next treatment session, which consisted of installing irradiation FDD-1 with a power density of up to 200 mW/cm2 with duration of 10 minutes in the field. The procedure does not require anesthesia.

The animals of the main group (4th group) whose treatment was carried out using traditional methods of treatment, CO2 laser and photodynamic therapy, on the third day after infection, the wound opened after taking out of leading stitches, removed gauze ball, delivered on the first day of the experiment, and the wound treated with a solution of hydrogen peroxide and had a CO2 laser session for early necrectomy and sterilizing wounds. The session ended with the CO2 laser destruction by applying sterile gauze balls soaked in an aqueous solution of 0.005% MB on the surface of the wound bed to 1-2 mm. with fully closing the wound surface. It was applied opaque bandage. After 30 minutes of application held next treatment session, which consisted of installing irradiation FDD-1 with a power density of up to 200 mW/cm2 with duration of 10 min. in the field. The procedure does not require anesthesia. And also with the aim of antibiotic therapy Ceftriaxone pricked 1.0 grams in a dosage of 0.3 ml. of 1 times a day.

Methods of evaluating the effectiveness of treatment of purulent wounds

The following parameters were included in the complex of the treatment effectiveness evaluation: laboratory, thermometry, the nature and amount of wound, planimetric (changes of the wound area), and clearance of the wounds, the time of appearance of granulation, morphological, bacteriological parameters.

To perform thermometry in rats it's quite suitable conventional mercury thermometer. For morphological evaluation were taken

during process ofwound tissue samples obtained from a wound and bottom walls at the 1st, 3rd, 7th days after its application. To control for the passage of wound healing in experimental animals immediately before treatment and on the 3rd, 7th and 10th days was used as planimetric method. And also conducted bacteriological research microflora isolated from necrotic wounds and the level of microbial contamination of the wound tissue.

The results of laboratory studies

On the third day, all animals showed signs of intoxication varying degrees of severity, as evidenced by changes in blood leukocyte formula: leukocytosis, increased amounts of immature neutrophils, plasma cells, the appearance, reducing the number of monocytes and lymphocytes, as well as increase in the LII (6-8 conventional units). After 3 hours from the moment of the beginning of the traditional methods of treatment observed decrease in the values of LII — 2.9 conv. units, with the additional effect on wound laser methods (CO2 laser and photodynamic therapy) there is a big reduction in the indicator LII — 1.9 conv. units. After 10 days of the beginning of purulent wounds treatment with using PDT, LII matched normal value of the index, while the control group animals remained elevated — 19.3 ± 0.18 conv. units.

The results of thermometric studies

Immediately prior to the treatment of the initial value of the rectal temperature of the animals of all groups were as follows: 38.7 °C.

In the animals of the 1st group there is a slight decrease in rectal temperature only the 10th day. The animals in group 2 who received conventional standard treatment, there was a decrease in rectal temperature on the 7th day. Similar changes were found in animals of group 3, which received PDT treatment only. The most noticeable drop in temperature, which registered in the animals already on the 3rd day it was 4th group animals, which used for the treatment of a combination of effects of photodynamic therapy and standard methods of treatment.

The results of changes the wound area (planimetry) in dynamics

In experimental animals of all groups on the first day before the start of the treatment the wound area were averaged 745 ± 23.2 mm.

On examination, there was a moderate hyperaemia and swelling of the wound edges, the bottom was covered with a dirty grayish bloom. In the tissues surrounding the wound, there were areas of diapedetic hemorrhage.

The obtained data of the planimetric studies in dynamics are shown in Table 2. It reflects changes in the average index of the area of the wound surface in animals of different groups that have occurred at different times after the start of treatment under the influence of applicable treatments.

Table 2. - Indicators of planimetric studies in the dynamics at different times from the start of treatment

Groups № Treatment methods Planimetric injury indicators (mm)

Before treatment On the 3rd day On the 7th day On the 10th day

1 Observing the natural course of wound healing 745 ± 23.2 723.0 ± 19.0 661.5 ± 15.9 581.4 ± 11.3

2 Traditional method of treatment 745 ± 23.2 619.0 ± 16.1 518.8 ± 17.1 401.3 ± 10.3

3 Study of the effect of photodynamic influence on the course of wound healing 745 ± 23.2 581.6 ± 10.2 495.8 ± 6.8 381.8 ± 8.3

4 The influence of the traditional method of treatment in combination with CO2 laser and photodynamic therapy on the course ofwound process 745 ± 23.2 381.6 ± 8.3 274.6 ± 9.1 121.6 ± 3.6

At evaluating the cleansing of the wound surface in the dynamics of the animals in Group 1, which did not apply the treatment, cleansing the wound surface from necrotic masses observed by day 17 (16.5 ± 0.7), in the 2nd group of animals cleansing of

the wound surface was observed by day 10 (9.4 ± 0.8), and the animals of group 3 the cleansing of the wound surface observed by the 7th day (6.3 ± 0.8), animal group 4 on the 6th day (5.2 ± 0.9) from the start of treatment.

According to the results of our study, the performance of wound granulation tissue was observed in the animals of group 1 to the beginning of the 4th week (23.6 ± 1.1), the animals of the 2nd group to the top of the 3rd week (15.1 ± 0.9), animals of group 3 to the 9th day (8.8 ± 0.9), the animals of group 4 to the 7th day of (7.1 ± 0.3) from the start of treatment.

According to studies conducted in animals of group 4 the wound surface area decreased more intensively. Contractation and granulation ofwounds observed faster than in the control groups. This proves once again that the combined application of traditional methods of treatment sessions with CO2 laser and photodynamic therapy clearly effective when compared to other methods studied individually. The results of histological examination Before starting the experiment, the animals of all groups were identified inflammatory and necrotic changes in tissue structures on the background of an abundant infiltration of polymor-phonuclear leukocytes.

Fig. 1. Home purulent wounds on the 3rd day without treatment. Ulceration of the skin epidermis, edema and leukocyte infiltration of the dermis. Coloring: hematoxylin-eosin.

Incr.: oc. 10, ob. 20

On the border with intact tissues are found demarcation leukocyte shaft and expressed frustration hemodynamics and microcirculation in the form of full-blooded vessels, increased permeability of their walls for plasma proteins and blood cells, stasis, fibrinoid necrosis of the vessel walls with perivascular and focal hemorrhages.

Morphological studies have shown that conventional treatment purulent-destructive soft tissue wounds inefficiently and only affects the decrease of the activity of inflammatory and destructive changes in tissue structures of the skin surface. In the deep layers of the dermis and subcutaneous musculature picture pathological changes is maintained until the end of the observation period.

Fig. 2. Saving inflammatory leukocyte infiltration into the intramuscular connective tissue within 7 days after the traditional treatment of purulent-destructive wounds. Coloring: hematoxylin-eosin. Incr.: oc. 10, ob. 90

Identified dyscirculatory disorders also stored until the end of experimental observation, that is, apparently, cause edema and infiltration conservation deeper layers of the dermis. All this leads to delay ripening of granulation tissue fibroblasts and proliferation of the wound surface epithelization.

Treatment of purulent-destructive wounds only by PDT has a less effective impact on the severity of the recovery processes, leading to a slowdown in terms of filling the wound granulation tissue and its subsequent epithelialization.

Fig. 3. Saving and interstitial infiltrate perivoskulyarnogo lymphohistiocytic 10 days after treatment with PDT.

Coloring: hematoxylin-eosin. Incr.: oc. 10, ob. 90

The use of traditional treatment combined with CO2 laser and photodynamic therapy is the most effective impact of the intensity of regenerative processes in all areas of purulent-destructive wounds. The use of traditional treatment combined with CO2 laser and photodynamic therapy is the most effective impact of the intensity of regenerative processes in all areas of purulent destructive wounds.

Fig. 4. Reducing the volume of edema, destruction and inflammatory infiltration in the affected area of purulent destructive process through 3 days of traditional treatment combined with PDT. Coloring: hematoxylin-eosin.

Incr.: oc. 10, ob. 90

The significant reduction in the microcirculation disorders is responsible for the fast clearance of the wounds from purulent necrotic detritus and enhances the phagocytic activity of neutrophils. All this contributes to a more active and early formation of granulation tissue, activation of cellular proliferation elements macrophage and fibroblastic series, fast maturation and fibrosing granulation tissue, which ultimately leads to a reduction in terms of complete epithelialization of the wound surface.

Fig. 5. Saving proliferation of histiocytic cells intramuscular interstitial after 10 days of traditional treatment destructive purulent wounds combined with PDT. Coloring: hematoxylin-eosin. Incr.: oc. 10, ob. 90

Diagnostics and treatment tactics of non-stable pelvis injuries

The results of microbiological studies

Microbiological studies were carried out to identify the causative agent and determination of CFU, as well as to monitor the effectiveness of the treatment. Gathering pathologic releasing substance from the wound to the qualitative analysis of the microflora was performed using a sterile swab, which was placed in a special tube to transport the coal environment.

The sensitivity of the isolates to antibiotics was determined by the standard technique for disks. In addition, it was conducted quantitative research methods to determine the level of microbial contamination in 1 gram of tissue injury. It should be noted that this method is the most informative.

When determining overall characteristics of the selected agents, it was found, that they mainly represented by staphylococcus and gram-negative flora. The leading role in microbiological study of releasing substance from purulent wounds took St. Aureus (62.5 %); Proteus mirabilis (54.8 %); E. Colli (50.8 %) and a small amount Str. Pyogenus (12.5 %). It should be noted, that the above listed organisms are major pathogens of purulent infection in surgery.

When determining the sensitivity was defined that most of the identified bacteria showed resistance to the most widely used antibiotics and retained sensitivity to cephalosporin, aminoglycoside and fluoroquinolones antibiotics past generations. So, they were the most sensitive to ciprofloxacin, cefazolin and rifampicin and less sensitive to penicillin and erythromycin. Furthermore, they were multi-resistant to several antibiotics simultaneously. These results suggest that the microflora isolated from purulent wounds is often a multi-resistant. Therefore, at the beginning of the complex treatment of purulent wounds it is recommended to appoint ciprofloxacin and rifampicin, ceftriaxone, and after receiving the results of the study of bacterial purulent wounds, antibiotic therapy should be corrected taking into account the sensitivity of microorganisms to antibiotics.

Along with the quality of microbiological studies of quantitative methods of research have been conducted, which today are the

most informative, because allows to define levels microbial content per 1 g. of tissue injury.

Results of the study showed that before treatment in all groups of animals determined by the high level of contamination of tissue wounds, an average of1 x 10 6-9 CFU/g.

On the third day simulation of purulent wounds destructive level of microbial contamination was in the animals of group 1 — 10 5-10 9 CFU/g. The animals in group 2, treated with the standard treatment, on the 3rd day level of microbial contamination was 10 4-10 6 CFU/g. The animals of the 3rd group, which were received treatment PDT only in these terms the level of contamination was on average 10 3-10 6 CFU/g. The lowest level of microbial contamination is observed in the animals of group 4, which was carried out comprehensive treatment with PDT, it was 10 2-10 5 CFU/g.

On the 7th day from the start of treatment ofpurulent wound microbial contamination level ofperformance in the animals of group 1 are without much dynamics. It should be noted that a significant reduction in microbial load of 1 g. of tissue observed in the animals of group 4 (10 2-10 5 CFU/g), compared to the contamination level data tissue wounds of animals in 2nd and 3rd groups (in average 10 3-10 6).

On the 10th day of observation in almost all groups noted a progressive decrease in the level of microbial contamination. However, in the group of animals received a combination treatment of antibiotic therapy with PDT observed sustained reduction in the level of microbial contamination of the wound tissue below the critical. The results suggest greater effectiveness of this type of exposure to destructive purulent wounds as compared to other treatments studied.

Thus conducted studies allow to evaluate the effectiveness of the method of photodynamic therapy of intraoral method of application of the photosensitizer methylene. These laboratory, ther-mometry, morphological, microbiological and planimetric data show, that photodynamic therapy and CO2 laser is very effective non-invasive treatment of purulent wounds and serve as justification for the use of the method in clinical practice for the treatment of local acute purulent- destructive diseases of soft tissues.

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2. Shishkin O. E., Butakova L. Y., Ivanchenko J. O., Antonov S. Microbiological study the effectiveness of the photosensitizers in photodynamic therapy//Laser medicine. - 2013. - 17(1): 35-37.

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Tilyakov Akbar Buriyevich, MD, PhD, Senior researcher of the traumatology department, The Republic Research Centre of Emergency Medicine,

Tashkent city, Uzbekistan Bobur Sobirovich Ubaydullaev, MD, Head of the traumatology department E-mail: akbar-tilak@mail.ru

Diagnostics and treatment tactics of non-stable pelvis injuries

Abstract: Treatment analysis of 415 patients with non-stable and poly-focal fractures of pelvis bones has been described in the article. All patients have been divided to the groups subject to the types of injury, the optimal algorithm of supplying medical aid was worked-out for each group and according to the severity of the combined injuries an appropriate treatment method has

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