Method of treating chronic limb ischemia experimentally

FIELD: medicine.

SUBSTANCE: limb ischemia is simulated in a male Wistar rat anaesthetised with chloral hydrate in a dose of 250-300 mg/kg by the surgical removal of femoral, popliteal arteries and the front shin artery. That is followed by introducing a prepared mononuclear fraction of the autologous bone marrow in a dose of 4×106 cells in an amount of 200 mcl. The above is introduced into the ischemic limb from two points in an amount of 100 mcl each. One point is found directly under the femoral arch paravasally within the anatomical position of collaterals of the inner iliac artery and its branches. The other point is located in the calf muscle on an anteriolateral surface of the midleg.

EFFECT: more effective experimental treatment ensured by stimulating the collateral blood flow in the ischemic limb and improving the arterial blood flow from the proximal to distal parts of the limb.

1 ex, 1 tbl

 

The invention relates to medicine, namely to vascular surgery, and can be used in the treatment of chronic obliterating diseases of lower limb arteries.

The closest to the claimed solution is a method of implantation of autologous bone marrow cells, comprising introducing into the points located on the ischemic thigh muscle of the affected limb (Takafumi Ueno, Toyoaki Murohara, Keith Allen Robinson et all. "Therapeutic angiogenesis by bone marrow-derived cell transplantation in myocardial ischemic tissue and skeletal muscle ischemic tissue", US 7799349 B2). In known methods of bone marrow cells prepared with the removal of red blood cells. The results of the study after the procedure of implantation of bone marrow cells showed that angiogenesis was induced in the ischemic thigh muscle. Laser Doppler showed an increase in blood flow. However, this method still does not provide sufficient treatment efficiency, since bone marrow cells are implanted into the affected thigh muscle infiltration from six different points that without sufficient collateral blood flow at the level of the femoral and popliteal arteries not undermine the experimental limb ischemia, but even exacerbates it by increasing interstitial pressure.

The technical result of the invention is to improve the treatment of eksperimentalnoye limbs by stimulating the development of collateral blood flow in ischemic limbs and thus improving arterial blood flow from the proximal extremity to the distal.

The technical result is achieved by the fact that the background modeling of limb ischemia in the experiment, the rats male Wistar breed by excision of the main vessel, including the femoral artery, popliteal artery and initial parts of the crural arteries under anesthesia with chloral hydrate at a dose of 250-300 mg/kg, the correction of the pathology of the introduction of the mononuclear fraction of autologous bone marrow 4×106cells in a volume of 200 μl in the ischemic limbs of two points, each 100 µl: 1) retroperitoneal immediately below the inguinal ligament in its middle third paravazal in the area of the anatomical location of the collaterals of the internal iliac artery and its branches; 2) intramuscular in gastrocnemius muscle of the anterolateral surface of the middle third of the tibia. This leads to a pronounced correction of disorders of microcirculation in the ischemic limb, as confirmed by laser Doppler flowmetry.

The method is as follows.

Experiments carried out on 100 male rats of Wistar line weighing 300-350 g. the Animals were divided into two series: control and experimental. In the control series included 40 rats, which were divided into four groups, each group included 10 animals, including intact group consisted of 10 rats that have estimated normal level of� microcirculation. The trial series was divided into 2 groups of 30 animals each. 1 group consisted of rats which bone marrow mononuclear cells implanted in infected thigh muscle infiltration from six different points, as shown in the known method. 2 group consisted of animals that bone marrow mononuclear cells implanted in the affected ischemic limb in two points: the retroperitoneal immediately below the inguinal ligament in its middle third paravazal in the area of the anatomical location of the collaterals of the internal iliac artery and its branches and intramuscular in gastrocnemius muscle of the anterolateral surface of the middle third of the Shin Each of the groups experienced the series was divided into 3 subgroups of 10 rats each.

In animals of both series studied the dynamics of the level of microcirculation in the ischemic limb and without the treatment of experimental ischemia with bone marrow mononuclear cells on the 10th, 21st and 28th day. Chronic hind limb ischemia was simulated by the method of M. Sugano et al. (2004), S. Takeshita et al. (1998) by excision of the main vessel, including the femoral artery, popliteal artery and initial parts of the crural arteries.

Assessment of the level of microcirculation in the muscles of the femur and tibia of rats were performed by laser Doppler flowmetry (LDF). The study was carried out using the apt�rata - laser Doppler flowmetry "Biopac systems MP-100 and sensor TSD-144". Recording and processing of data was performed using the software AcqKnowledge 38. The study was performed under anesthesia with chloral hydrate at a dose of 250-300 mg/kg, was administered intraperitoneally in an aqueous solution. After the onset narcotic sleep dissected area of the skin on the anterolateral surface of the right femur and tibiae of the hind limbs. The animal was placed on the opposite side. The sensor against put to the muscle of the thigh, Shin and then flowmetry was performed. The level of microcirculation were recorded in five points: 1) the middle of the length of the muscle; the point at 3-4 mm 2) above, 3) lower 4) lateral and (5) medial from the first (patent No. 2353977 from 27.04.2009). The entry level curve of the microcirculation was performed for 30 sec at each point. From the resulting five values deduced average, which contributed to the Protocol and adopted at the level of microcirculation in the muscles of the femur and tibia from this animal. 10 obtained values deduced average, which was hosted at the level of microcirculation in the group of animals at this time.

In animals of the control series of the first group - intact - evaluated the level of microcirculation in the muscles of the femur and tibia of the right hind limb by LDF. Obtained average values of the level of microcirculation in the group animal�x were taken as "normal" and amounted to 588,74±19,28 p. E. (perfusion units) in the thigh muscle and 535,22±17,53 p. E. in the muscle of the calf.

Animals of the control series of treatment is not received. The rats experienced a series of treatment was started on the seventh day after the operation. For that, immediately prior to administration, a suspension of bone marrow was isolated from femurs of animals as follows. In rats undergoing surgery modeling ischemia of the right hind limb, anesthetized with chloral hydrate at a dose of 250-300 mg/kg, on the opposite limb was pullervale femoral-tibial joint is formed by the lateral and medial condyles of the femur and tibia, fixed him with one arm in the flexion position and the other took a 20G needle diameter, connected with a syringe containing 2500 IU of heparin, diluted with 0.9% NaCl in the ratio 1:1 in a volume of 1 ml, and after rehabilitation with 70% ethyl alcohol via percutaneous cartilage menisci under the patella puncture was performed on the bone marrow cavity. Bone marrow was serioulsy in the syringe by pushing the needle in the cavity back and forth in a volume of 1 to 1.5 ml. Then the mononuclear fraction of bone marrow was isolated by the method of Boyum (Boyum A. Separation of leukocytes from blood and bone marrow //Scand. J. Clin. Lab. Investig. - 1968. - Vol. 21 - Suppl. 97. p. 1-9), the principle of which is based on the difference in the buoyant density of formed elements. The mixture of the polysaccharide ficoll � radiopaque substance verografin creates a gradient with a density, which allows for centrifugation to separate cells of a bone-marrow mononuclear fraction (MF), which includes lymphocytes, a subpopulation of monocytes and hematopoietic progenitor cells, and the fraction containing granulocytes and erythrocytes. Mononuclear fraction is less than the gradient, density and placed above the gradient. The density of granulocytes and red blood cells is greater than the density gradient, they pass through the gradient down to the bottom of the tube.

Method for the separation of mononuclear fraction of heparinized bone marrow.

The bone marrow is diluted with saline solution (pH of 7.2 )at a ratio of 1:5; centrifuged in a density gradient ficoll-verografin at 400g for 30 min. the Obtained mononuclear fraction was washed three times in SFR (saline, buffered with phosphate-buffered saline) and was resuspended in RPMI-1640 medium at a concentration of 106 cells per ml. cell Viability, determined according to the method of staining with trypan blue, exceeds 96%.

The obtained mononuclear fraction of autologous bone marrow 4×106cells in a volume of 200 μl in the first group injected into the ischemic limbs hips infiltration from six different points on the previously known methods closest to the claimed method, and in the second group of two points in each�th of which 100 µl: 1) retroperitoneal immediately below the inguinal ligament in its middle third the area of the anatomical location of the collaterals of the internal iliac artery and its branches; 2) intramuscular in gastrocnemius muscle of the anterolateral surface of the middle third of the tibia.

The animals were removed from the experiment by an overdose of anesthesia at 10, 21 and 28 days. Before removal from the experiment in rats of control and experimental series assessed the clinical and flowmetrics indicators in the simulation of limb ischemia in different periods. The data obtained were processed statistically: calculated average shift values (M), the average error of the arithmetic average (±m) and the probability of error (p). Differences were evaluated as significant at p<0,05.

Clinical examination in the majority of animals registered the presence of pain, redness of the skin of leg and foot a symptom of "punctuating the limp", the limited involvement of the limbs in movement.

Dynamics of the assessment of the level of the microcirculation using laser Doppler flowmetry in animals of the experimental series (group 2) compared with the control and intact groups is shown in table 1.

As can be seen from table 1, after the operation the level of the microcirculation sharply and rapidly decreases and then slowly recovering, but up to 28 days inclusive) remains significantly below the normal value, is recorded in the group of intact rats. Previous level microcir�ulali reaches only 3 months after surgery. Compared with the group of intact animals the level of microcirculation in group 1 treated rats by well-known methods, for 28 days at thigh level was higher by 1.2 times, at the level of the tibia 1.1 times; compared with the control at the hip level was above 10 days - in 1,6 times, on day 21 - 1,4 times, in 28 days - 1.7 times; at the level of the tibia the level of microcirculation increases in 1,5 times, 1.3 times and 1.7 times, respectively, on the same terms.

Compared with the group of intact animals the level of microcirculation in the 2nd group of rats treated with the claimed method, was higher by 1.2 times at the hip level and 1.1 times at the level of the tibia already on day 21 of 28 day at thigh level, 1.5 times, and at the level of the tibia - 1.4 times, as compared with the control at the hip for 10 days increased 1.8-fold at 21 days, 2 times and at 28 days - 2.1 times; at the level of the tibia to the level of the microcirculation is increased 1.7 times 1.8 times and 2 times, respectively, on the same terms.

When comparing these indicators between 1 and 2 groups was that the level of the microcirculation at the level of both femur and tibia on the 10th day above 1.1 times, on day 21 - 1.4 times and at 28 days - 1.2 times, which suggests that the essence of the invention is expressed in the totality of significant features, sufficient to achieve provided by the invention a technical result.

EXAMPLE �KONKRETNOGO RUN

Rats experienced series in group No. 1 in the experiment were injected mononuclear fraction of the bone marrow on day 7 after modeling chronic limb ischemia by well-known methods infiltration from six different points in the affected muscle of the thigh at a dose of 4×106cells in a volume of 200 μl, which led to the increase of microcirculation in the ischemic extremities for 10 days at thigh level, to 366,86±17,7 p. E. and at the level of the tibia to 310,31±for 15.39 p. E.; on day 21 at the hip level to 496,92±26,93 p. E. and at the level of the tibia to 423,41±23,42 p. E.; 28 days at thigh level, to 717,46±37,11 p. E. and at the level of the tibia to 615,18±32,27, p. E.

The rats experienced a series in the 2nd group after modeling chronic limb ischemia at day 7 of mononuclear fraction of bone marrow in a dose of 4×106cells in a volume of 200 µl was injected by the proposed method from two points, each 100 µl: retroperitoneal immediately below the inguinal ligament in its middle third the area of the anatomical location of the collaterals of the internal iliac artery and its branches and intramuscular in gastrocnemius muscle of the anterolateral surface of the middle third of the tibia. This resulted in a significant increase in the level of microcirculation in the ischemic extremities for 10 days at thigh level, to 420,28±22,86 p. E. and at the level of the tibia to 350,23±19,05 p. E.; on day 21 at the hip level to 695,47±46,6 p. E. and at the level of g�laziness to 579,56±38.83 p. E.; 28 day at thigh level, to 873,98±43,72 p. E. and at the level of the tibia to 728,32±36,43 p. E., which is higher compared to group 1 at the level of the thigh and the Shin level on the 10th day 1.1 times, on day 21 - 1.4 times and at 28 days 1.2 times.

Thus, the results of the experimental study indicate that the proposed method is more effective in the treatment of limb ischemia compared to known by stimulating the development of collateral blood flow in ischemic limbs and thus improve arterial blood flow from the proximal extremity to the distal.

A method for the treatment of chronic limb ischemia in the experiment, including the modeling of limb ischemia in rats male Wistar breed by excision of the femoral, popliteal arteries and initial segments of the crural arteries under anesthesia with chloral hydrate at a dose of 250-300 mg/kg followed by an introduction of the obtained mononuclear fraction of autologous bone marrow 4×106cells in a volume of 200 μl, characterized in that the correction of pathology conducted the introduction of the mononuclear fraction in the ischemic limbs of two points, each 100 µl: immediately below the inguinal ligament paravazal in the area of the anatomical location of the collaterals of the internal iliac artery and its branches and intramuscular in gastrocnemius muscle p�replacerules surface of the middle third of the tibia.



 

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