Method of modulating post-transplantation changing in kidneys

FIELD: medicine.

SUBSTANCE: to a laboratory animal - rat - carried out is a unilateral nephrectomy, denervation and delymphatisation of the second kidney. After that the renal artery and vein are clamped for 40-60 minutes. After carrying out the surgical operation the triple immunisation of the laboratory animal with a kidney antigen, obtained from the removed kidney is carried out by an adjuvant method. 5 weeks before the surgical operation sampling of bone marrow cells from the laboratory animal is carried out, a culture of multipotent mesenchymal stromal cells is obtained from them in vitro. The denervation and delymphatisation of the remaining kidney is carried out by the removal of the adventitia and cellular tissue, wrapping the ureter, renal artery and vein, in the area of the hiluses renalis at a distance of 7-10 mm and decapsulation of the kidney in the area of its medial edge 7-10 mm wide, extension from one pole of the kidney to the other with the capture of the area of its hiluses. To obtain the kidney antigen the safe kidney is applied and the content of protein in it is brought to 70 mg/ml. On 35-40 day after carrying out the surgical operation the obtained culture of the multipotent mesenchymal stromal cells is introduced to the same animal intravenously in a dose of 1.5-3.0 mln cells in 1 ml of physiological solution.

EFFECT: method makes it possible to create an adequate, available for execution model of chronic rejection in autologous kidneys in small laboratory animals with an accelerated development of visualised destructive post-transplantation processes.

1 ex, 1 tbl, 9 dwg

 

The invention relates to medicine, in particular to experimental pathophysiology and transplantation.

It is known that the hardening of the kidneys and their death (rejection) is the most frequent complication of allogeneic transplantation of donor kidneys. As a result, the number of recipients with long-term functioning grafts is still extremely low.

It is now recognized that chronic rejection of the donor kidney occurs always, regardless of their degree of immune compatibility with the recipient; however, it is noted that in renal transplantation from a related donor, the percentage of long-term functioning grafts was significantly higher than from an unrelated (cadaveric) donor (Balakirev, E. M. prediction of the development of chronic renal failure and transplant organization of care of Nephrology patients. Extended abstract of doctoral. honey. Sciences. M., 1990, 56 p.).

Noted that all of the kidneys as when allograft (blumkin V. N., Ivanov A. E. Cytopathology allotransplantation a person's kidneys: normal and pathological proliferation of the tubular epithelium of the allograft. The Bulletin of the AMS of the USSR. 1983, 11:29-36) and autotransplantation (Kirpatovski I. D., Bykova N. And. Kidney transplantation (experimental and biological bases). M., 1969, 231 S.; Lebedev, A. A. Autot�splantzia kidneys. Leningrad: Nauka, 1971, 121 S.) is their gradual damage, hardening and loss; long-term immunosuppressive therapy used to prevent rejection of kidney, the efficiency is gradually reduced, especially in later crises of rejection, after a few months or years (Ratner M. J., and others. Renal dysfunction. M.: Medicine, 1977, 296 p.). Early rejection crises develop regardless of the degree of immune compatibility of donor and recipient (M Cochran et al. Prognosis of cadaver kidney grafts with a rejection episode occurring within 2 weeks of transplantation. Transplantation. 1979, 27(1):67-8.), the biggest number of crises accounts for 3-10 days (67%) and in the first 2-3 months (86%) after transplantation (Shumakov V. I. Transplantation: a guide for physicians. M: MIA, 2006, 540 S.), that is, for that period of time appear in the blood and circulate renal and renal autoantigens antibodies resulting from ischemic and surgical trauma to the kidneys.

In 3-6 months after transplantation the number of rejection episodes is substantially reduced (approximately 50%), but gradually, starting with 3-4 years in the renal grafts begin to progress, signs of structural damage and sclerosing (tubulo-interstitial nephropathy), the occurrence of which, however, is associated with a toxic effect on long-term graft used immunosuppressive �ERPII.

Thus, the development of post-transplant changes in kidneys (chronic rejection) is always available regardless of the degree of immune compatibility of donor and recipient (immunogenetic differences of donor and recipient increase the severity of the rejection process), and this fact suggests the presence of some additional pathophysiological mechanisms of development of chronic rejection. As a result it becomes evident that for the study of the factors inducing the development of post-transplant changes in the kidneys, and to search for ways to prevent chronic rejection should be created available and adequate model of post-transplant changes in kidneys (chronic rejection) in small laboratory animals, recreating the full range of factors that affect the kidney.

As the most simple models that mimic chronic rejection of the kidney in rats, using a model of its toxic damage, when, after a course of immunosuppressive drug toxic dose (cyclosporine A at a dose of 15 mg/kg for 2-4 weeks) in the kidney develops tubulointerstitial fibrosis (G. Capasso et al. In vivo effect of the natural antioxidant hydroxytyrosol on cyclosporine nephrotoxicity in rats. Nephrol Dial Transplant. 2008, 23:1186-1195; Sanchez-Pozos K. et al. Polymerized type I collagen reduces chronic cyclosporine nephrotoxicity. Nephrol Dial Transplant. 2010, 25:2150-2158). Model emitir�em damage and destruction of the kidneys in the postoperative period under the influence of immunosuppressants, used to prevent rejection of the allograft.

The disadvantage of this model is the use of the notoriously toxic doses of immunosuppressive drug to cause kidney damage; but most importantly an immunosuppressant thus affects anatomically whole kidneys, preserving the connection with the body, whereas transplantation of a kidney becomes decentralized (denervation, delimitate of kidney and ureter) and so sensitized as to toxic damage and the development of immune inflammation, the severity of which varies as you increase the antigenicity of the graft and date of transplantation (decentralization) (Volkova O. V. Neuro degenerative process (morphological aspects). M.: Medicine, 1978, p. 256.).

Known experimental model of chronic rejection of the kidney in rats at their allograft in heterophily when isecheno a kidney transplanted into the abdominal cavity in the descending aorta and inferior Vena cava, the ureter connects with the bladder (Kirpatovski I. D., Smirnova E. D. Fundamentals surgical techniques of organ transplantation. M.: Medicine, 1972, 175 p.). The advantages of the study of chronic rejection of the kidney in this model include the possibility of a reconstruction of the whole complex of factors related to the transplantation of this organ in the clinic.

Untill�taccom model of chronic rejection of the kidney in rats is the technical complexity of its implementation, since the operation is performed on the vessels of small caliber and leads to a large rejection in experimental animals. Performing such operations on mice that allow immunological monitoring is even more problematic.

In addition, on this model it is not possible to study separately the role of the main and permanent factor of transplants factor of decentralization on the development of post-transplant changes in kidneys, as the kidney allograft requires the constant use of immunosuppressants, which with prolonged use (even at therapeutic doses) by themselves have a damaging effect on the transplant. This model of chronic rejection allotransplantation kidney in rats is analogous to the claimed model of chronic rejection histocompatible autologous kidney.

Model of chronic rejection of autologous kidney in small laboratory animals during transplantation into heterophily missing due to technical problems arising in the implementation of double surgery: first - excision of kidney with blood vessels with a wall section of the abdominal aorta and inferior Vena cava, and then their transplantation into the abdominal cavity that is incompatible with life.

Thus, de�ü adequate and available to perform model of chronic rejection of autologous kidney in small laboratory animals (rats, mouse) is missing.

Known method of surgical simulation post-transplant changes in autologous (histocompatible) kidney large animals (dogs), which includes surgical neuro-lymphatic decentralization and electrocoagulation lymphatic outflow pathways in the gate area and the capsule of one kidney, delete a paired organ, the preparation of brain homogenate layer them and immunization of the animal adjuvant method (patent for invention RU №2012928, author of the cat A. G. title of the invention "Method of modeling post-transplant changes in the kidney, published 15.05.1994).

This model of chronic post-transplant changes in autologous kidney was chosen as a prototype.

Known prototype method allows to reproduce the functional and morphological features only the beginning and not pronounced rejection (tubulo-interstitial nephropathy), as they are developing and progressing very slowly (visualisierung peritubular and interstitial sclerosis), manifests itself in dogs only 2-3 years after surgery, which requires large expenditures for long-term keeping of animals, and therefore this model is not suitable for laboratory (screening) studies.

Moreover, at the present time the use of dogs in Moscow for e�sperimentali research is prohibited (decree of the government of Moscow №819 PP dated 02.10.2002 On the formation system of management and financing, measures to improve the content, use and protection of animals in Moscow").

In addition, this model does not play a damaging effect on the kidney factor of ischemia, which is a constant contributing factor transplantation and is believed, makes the most significant contribution to the development of post-transplant changes in the kidney (Ratner, M. J. "Clinical pathophysiology of diffuse lesions of native kidneys and renal allograft", in the book under the editorship of V. I. Shumakova "Essays on pathophysiological problems of Transplantology and artificial organs, Ed. Repronex 1998 pp. 211-240").

It should also be emphasized that in the prior art there are no activities to address severe surgical stress in the animal (removal of one kidney, decentralization remaining kidney and the antigen-adjuvant effects on the body healthy animal source), which inhibits normal rate recovery (regenerative) processes in the organism and thus increases the time of onset of signs of post-transplant disorders in the kidney (tubulo-interstitial nephropathy, sclerosis).

It is pronounced stress impact on the body inhibits the reparative processes in it and in the kidney and serves as a factor of prolonged detention in animal�x in the experiment in the simulation of post-transplant disorders in the kidney by the method prototype.

In the clinic when the kidney transplantation, in the outer periods (1-2 years) after transplant when danger of development of acute rejection, the patient begins to receive support (i.e. low) doses of immunosuppressive drugs. As a result his body begins to recover the function of bone marrow cells and other organs of immunogenesis, normal immunological and begin to recover the physiological processes of regeneration of organs under the regulatory control of the immune system (Babaev A. G. Reparative processes and the immune system. Izv. An. Ser Biol. 1999, Vol. 128 N11, pp. 484-490). As a result, at remote periods after transplantation in a decentralized body of the activation of regenerative processes is manifested by the formation of distinct signs of recovery, and its damage (the development of interstitial and peritubular nephropathy outcome in tubulo-interstitial fibrosis).

Thus, the prototype method was not included physiological role of enhancing immunity and regeneration in the outer timing as a factor in the induction of accelerated graft damage in the conditions of decentralization.

From the above it follows that for laboratory study of factors of chronic rejection of the kidney (tubulo-interstitial nephropathy, sclerosis) � find ways to prevent its development must be created accessible and adequate model of accelerated development of chronic post-transplant changes of kidneys from small laboratory animals.

The objective of the claimed method is the creation of adequate, affordable to run model of chronic post-transplant changes (chronic rejection) in the autologous kidneys in small laboratory animals (rats, mice) with the rapid development rendered destructive post-transplant processes.

The technical result of the claimed method is:

- the creation of adequate and easily reproducible model of post-transplant changes in autologous kidney small laboratory animals with accelerated visualization of the development of destructive processes, similarly occur in the remote terms after transplantation in the clinic;

- exclusion of animal trauma due to the exclusion of continuous use of immunosuppressive drugs, leading to toxic damage to the kidneys;

- the maintenance of a physiological level of regenerative processes in the kidney;

- as well as the provision of opportunities for therapeutic regulation of the rate and severity of chronic damage (rejection).

The proposed method allows to simplify the method by eliminating the stage of time burning tissue and lymphatics in the gate area of the kidney and on its surface, as proposed by the method prototype.

Summary of the invention the enclosed�is as follows.

For the simulation of post-transplant changes in the kidneys of laboratory animal - the rat perform unilateral nephrectomy, denervation and delimititation second kidney. Then occluded renal artery and vein for 40-60 minutes. After the surgical intervention is carried out three times immunization of laboratory animal renal antigen, obtained from a remote kidneys, adjuvant method. At the same time for 5 weeks before surgery in laboratory animal produce fence of bone marrow cells, obtained from them in vitro culture of multipotent mesenchymal stromal cells. Denervation and delimititation remaining kidney perform by removing the adventitia and tissue surrounding the ureter, renal artery and vein in the gate area of the kidney during 7-10 mm and decapsulation kidneys in the area of the medial margin of a width of 7-10 mm and a length of from one pole of the kidney to the other with a capture area of her gate. To obtain a renal antigen using whole kidney and bring the protein content up to 70 mg/ml For 35-40 days after surgical operation to impose the culture of multipotent mesenchymal stromal cells of the same animal intravenously at a dose of 1.5-3.0 million cells in 1 ml of physiological solution. The method is as follows.

1.5 weeks before surgery on the kidney in rats harvest autologous bone marrow by M. B. Askarov (M. B. Askarov. Transplantation of autologous bone marrow cells for the treatment of long-term non-healing ulcers of the stomach. Author. Diss.Dr. med. of Sciences, Moscow, 2009, 46 p.). To do this, under ether anesthesia from the medullary canal of the two femurs of the rats receiving cells in the bone marrow (km) by washing the cavity of the bone with phosphate-buffered saline containing 50 U/ml of heparin and 0.25 mg/l of gentamicin, with a needle 18 G, mounted on a syringe. The cell suspension km centrifuged at 1500 rpm for 3-5 min, the precipitate of cells was resuspended in solution for lysis of red blood cells (114 mm NH4Cl, 7.5 mm JISC3, 100 μm EDTA) for 5 min and again centrifuged.

Demolitionary supernatant is removed by aspiration and the cell pellet resuspended in medium JMEM (Gibco, USA) containing an additional 10% bovine fetal serum (Well, Clone, USA), 0.4 µm insulin and 025 mg/l gentamicin. Then, the resulting cells are plated onto culture plastic in an amount of 2.0-2.5 million cells/ml of growth medium JMEM, on Petri dishes (D=100 mm) at 37°C in CO2the incubator, in an atmosphere of air with 5% CO2and 95% humidity. 3-4 days of cell cultivation km environment completely replaced by DMEM containing 10% bovine serum and insulin 0.4 µm/l also dexamethason 10 nm/l and basic fibroblast growth factor (FGFb) 20 ng/ml (Sigma, USA) to stimulate proliferative�activity of multipotent mesenchymal stromal cells (MMSC), contained in the total cell fraction from km. A change of environment is carried out every 3 days to eliminate reprecipitate cells. At 12-14 days of culturing MSCS form 80-85% of the monolayer (1.5 million cells) with fibroblast-like morphology, they are removed from the plastic using a standard solution of Trypsin-Versene, centrifuged, a precipitate of cells, the supernatant removed and the cell pellet resuspended in the medium for cryopreservation (90% fetal bovine serum and 10% DMCO), the cell concentration was 1.5 million per ml. the cell Suspension is transferred into cryoprobe and freeze at a speed of 0.8-1.2°C in 1 min. At the temperature in the test tube -70°C it is transferred into liquid nitrogen for storage. 2 weeks before using cryoprobes with Aspasia frozen cells were placed in a water thermostat at 37°C for 2 min hold defrosting. Next, the tubes were centrifuged, the supernatant removed, the precipitate cells - MSCS was resuspended warm growth medium and sown on 3 culture Petri dishes (D=100 mm). After 12-14 days of cultivation in each plate the number of cells reached up to 1.5 million; they are removed by a standard solution of trypsin-versene, centrifuged, supernatant udalyayte, and in the amount of 3 million cells (2 cups) or 1.5 million cells (1 Cup) was resuspended in 1 ml of physiological solution direct� before using (after surgery on the kidney and the antigen-adjuvant immunization of rats).

2. 1 week after cell collection bone marrow are training rats to surgery. Using rats that underwent sanitary and epidemiological control. 5 weeks after cell harvesting under ether anesthesia in the supine position perform median laparotomy, the left kidney was excised, then the right kidney surgically decentralize - deservered and delimatsis. To do this the right kidney is isolated from retroperitoneal tissue by blunt and sharp perform precise dissection of the nerve and lymphatics in the gate area of the kidney and adjacent to the gate of the tissues of the kidney. Dissection of the nerve and lymphatics when this is achieved not only by surgical destruction and removal of the adventitia and tissue surrounding the renal artery, vein and ureter in the gate area of the kidney, where the nerve trunks and lymphatic vessels (skeletization renal artery, vein and ureter in juxta-junction Department, with a length of 7-10 mm), but also by partial surgical decapsulate (partial removal of the capsule) of the kidney: decapsulation kidneys in the area of the medial margin of a width of 7-10 mm and a length of from one pole of the kidney to the other with a capture area of her gate. Partial decapsulate kidney increases the reliability of surgical denervation and delimitacii kidney, because the additional closes Mac�oscopies visible and non-visible nervous and lymphatic path.

After the stage of denervation and delimitacii her right kidney is exposed to ischemia for a period of 40-60 minutes. To do this, allocate the vessels of the kidney (artery and vein) distal to the zones of the carcass, bring them under the ligature, subcutaneously administered heparin, and after 15 minutes with a tourniquet is occluded vessels of the kidney for a period of 40-60 min.

3. After the first 7-10 days after surgery immunizations animal by 3-fold introduction of water-salt kidney antigen. To obtain a renal antigen using whole kidney and bring the protein content up to 70 mg/ml For immunization of laboratory animal extract prepared from a single homogenized whole autologous kidneys in 3-4 ml of saline, mixed with incomplete adjuvant of frand in the ratio of 1:1, divided into three portions and injected into the subcutaneous tissue of the 4-xlegs three times every 7-10 days (1.8 to 2.0 ml of a mixture of water-salt solution of the antigen with the adjuvant of freind in a 1:1 ratio with the total protein content of 35 mg/ml). After subcutaneous injection of a solution of a kidney antigen and adjuvant of frand the injection site for 30 seconds moderately pressed aseptic ball with alcohol to the exclusion of the reverse current of the liquid, then treated with a solution of povidone - iodine.

4. After 35-40 days after surgery in�of Estella the animal under ether anesthesia intravenous (tail vein) injected pre-allocated MMSC cultured autologous bone marrow in an amount of 1.5-3.0 million cells in 1 ml of physiological solution (for restore the original level of activity of the regenerative processes in the body).

Provide proof of implementation of the stated purpose and achievement of the specified technical result.

The table presents some indicators of renal function after unilateral nephrectomy and modeling of chronic rejection of the remaining kidney in the control group (method prototype) and in the experimental group (modeling the claimed method).

An example of the method.

5 weeks before surgery under ether anesthesia in the supine position, after the treatment the skin of the thigh antiseptics with a needle of size 18 G has carried out harvesting of bone marrow cells sequentially from two femurs in an amount up to 0.5 ml (the contents of the bone marrow cells reaches 100×103 cells). Further, bone marrow cells were treated and cultured in standard conditions according to the method described by M. B. Askarov, 2009, and received MMSC in an amount up to 3 million cells per animal.

Under ether anesthesia in the supine position was treated with antiseptics surface of the anterior abdominal wall of the rat on Piloncillo-Grossau. Then the middle section cut through the skin and subcutaneous tissue over the white line of the abdomen and penetrated into the abdominal cavity. Legero�Ali artery and vein left kidney, deleted it (hereinafter this kidney were used for the preparation of renal antigen); then the right kidney was surgically genererally and delimitative (decentralization kidney) by releasing from the surrounding tissue and precision of dissection of the nerve and lymphatics. Performed skeletization renal artery, vein and ureter in juxta-junction Department, with a length of 7 mm and partial surgical decapsulation (partial removal of the capsule) kidneys: decapsulate kidneys in the area of the medial margin width of 10 mm, a length from one pole of the kidney to the other with a capture area of its gates.

Performed surgical destruction and removal of the adventitia and tissue surrounding the renal artery, vein and ureter, in the gate area of the kidney with a length of 7 mm, while maintaining blood flow through the renal artery and vein (Fig. 1).

In Fig. 1 is a diagram of a surgical decentralization (denervation, deemphasize) kidney, where

1 - renal artery, 2 - renal vein, 3 - ureter.

After the stage of denervation and delimitacii her right kidney was subjected to ischemia for a period of 60 min.

To do this, allocate the vessels of the kidney (artery and vein) distal to the zones of the carcass, conducted under the ligature, were injected subcutaneously heparin in a dose of 0.2 ml per rat, and after 15 minutes with a turnstile p�resimli vessels of the kidney for a period of one hour. After removal of the tourniquet, the blood flow in the kidney of spontaneously recovered. Surgery the animal was accompanied by irrigation of the abdominal cavity with saline solution with antibiotics. Ended operation layer-by-layer suturing of the wound.

Further, from the resected left kidney homogenate was prepared for the 3-fold immunization of rats, which was held surgical denervation and deemphasize right kidney, and its semiseria. For this remote mechanical kidney was crushed on a cold, suspended in 4 ml of DMEM (company PANECO, Russia), conducted filtering the homogenate through a filter system for blood transfusion (sterile PC system 21-01-"Synthesis"), was determined in solution the protein content by Lowry and, if necessary, add a fresh environment, DMEM in water-salt antigen, bringing in it the protein content up to 70 mg/ml.

Was then mixed with 1 ml of the prepared water-salt antigen with 1 ml of incomplete adjuvant of franda (firm Gifco, Usa). The prepared mixture (2 ml) was administered to the operated rat at 7 days in the subcutaneous tissue of the 4-xfeet (0.5 ml each foot). On 17 and 27 days after surgery - the mixture of water-salt antigen and adjuvant of frand in the amount of 2 ml, prepared in volumetric ratio of 1:1, additionally injected in the subcutaneous tissue of the 4 legs of the same rat. On�Le each introducing a mixture of a kidney antigen and adjuvant to the injection site for 30 seconds moderately pressed aseptic alcohol ball to prevent the escape of liquids, then the foot was treated with a solution of povidone - iodine.

At 35 days after surgery and 3-fold injection of antigen with adjuvant rat under ether anesthesia intravenous (tail vein) injected MSCS autologous bone marrow in an amount of 1.5 million cells in 1 ml of physiological solution.

From this point, we carried out a comparative study of function and morphological state only kidneys subjected to nonspecific factors transplantation according to the method prototype and the proposed method for 7 months.

For this purpose, the terms 1, 3, 5 and 7 months after completing the simulation, chronic rejection of the kidney was studied the level of creatinine and urea in the blood of animals, as well as parameters: the reabsorption of sodium, the sodium concentration in the urine and protein in urine daily in the morning conditions intraperitoneal water load (5% of body weight). After functional studies under ether anesthesia the animals were sacrificed and examined the morphological condition of the kidneys when they are coloring hematoxylin-eosin or Mallory.

The results of the functional studies carried out in experimental (the inventive method, n=25) and control (method-prototype (n=20) groups are presented in the table above.

The table shows that the kidneys in the experimental and control�encourages creativity groups during the 7 months retain their basic homeostatic function excretion of nitrogenous toxins from the body (the level of creatinine and urea in the blood is maintained within normal values).

Meanwhile tubular reabsorption function Na, Na excretion and protein is impaired compared to normal (baseline), and to a significantly greater extent in the model of chronic renal failure claimed method than in the method prototype (table).

More severe violations of the excretory renal function (canalave functions) claimed method were confirmed by morphological examination of the kidneys in 2 groups of experiments (Fig. 2 and Fig. 3).

In Fig. 2 shows the morphological condition of the kidney in the development of post-transplant changes in the method prototype. Coloring hematoxylin-eosin.

A - after 3 months of modeling chronic rejection.

B - 5 months after modelling chronic rejection.

In - 7 months after modelling chronic rejection.

From Fig. 2 shows that the glomeruli of the same size, numerous; there is slightly pronounced hyperemia capillary loops, increased cellular infiltration of part of the glomerular inflammatory cells (lymphocytes, polymorphonuclear leukocytes). The epithelium of the convoluted tubules in the state of protein malnutrition; notes ocaho�s and peritubular periglomerular lymphocytic infiltrates.

In Fig. 3 presents the morphological condition of the kidney cortical layer dynamics in the development of post-transplant changes in the claimed method. Coloring hematoxylin-eosin.

A - after 3 months. after simulation.

B - after 5 months. after simulation.

In in 7 months. after modeling chronic rejection.

In Fig. 4 shows the dynamics of post-transplant changes in the medulla of the kidney after 7 months of modeling chronic damage to the claimed method, A and B different fields of view.

From Fig. 3 and 4 shows that the glomeruli of the same size and numerous; there is slightly pronounced hyperemia capillary loops; in some glomeruli are defined by cells of the inflammatory infiltrate (lymphocytes, polymorphonuclear leukocytes). The epithelium of the convoluted tubules in the cortical layer and collecting tubules in the medulla in a state of protein malnutrition, some dilated tubules, lined by flattened epithelium and filled with homogeneous eosinophilic protein liquid. The number of such tubules and collecting ducts increases as you increase the period of observation; there are also moderately expressed focal peritubular, periglomerular and perivascular lymphocytic infiltrates.

Comparing the results of morphological examination�ing kidney you can upgrade to the conclusion that the modeling of post-transplant disorders in the kidney by the inventive method increases the degenerative processes in the convoluted tubules of the cortical layer, breaks the flow of urine, causes dilation of tubules and the appearance of cysts, increases the protein content in the ultrafiltrate (primary urine) and urine collecting ducts; leads to rapid atrophy of the tubular system, which is responsible for ensuring excretory processes in the kidney. Strengthening degenerative and atrophic processes in a tubular apparatus of the cortex and medulla of the kidney arising in the simulation of the claimed method, usually accompanied by a more severe course of chronic rejection of transplanted kidneys, which is clinically characterized by the appearance (or increase) of protein in the urine (proteinuria).

Thus, the inventive method allows in a shortened time frame (after 3 and 5 months) play heavy tubulo-interstitial nephropathy with severe functional (protein in the urine) and structural manifestations in the bone cortex and medulla of the kidneys. Prototype method does not allow a specified time to play distinctly pronounced proteinuria and protein accumulation in the renal tubule.

The invention is useful in the pathophysiology and transplant surgeons� in the study of pathogenetic mechanisms of post-transplant damage getsomething of neurotransplantation and to develop effective methods of protecting the transplanted kidney from the action of nonspecific factors of damage.

The method of simulation of post-transplant changes in the kidney, including the execution of laboratory animal unilateral nephrectomy, denervation, delimitacii second kidney and triple immunization renal antigen, obtained from a remote kidneys, adjuvant method, characterized in that as a laboratory animal using a rat, which is 5 weeks before surgery produce fence of bone marrow cells, obtained from them in vitro culture of multipotent mesenchymal stromal cells, denervation and delimititation remaining kidney perform by removing the adventitia and tissue surrounding the ureter, renal artery and vein in the gate area of the kidney during 7-10 mm and decapsulation kidneys in the area of the medial margin of a width of 7-10 mm and a length of from one pole of the kidney to the other with a capture area of her gate, then occluded renal artery and vein for 40-60 minutes; to get kidney antigen using whole kidney and bring the protein content up to 70 mg/ml, 35-40 days after surgical operation to impose the culture of multipotent mesenchymal stromal cells of the same animal intravenously at a dose of 1.5-3.0 million cells in 1 ml of physiological solution.



 

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1 tbl

FIELD: medicine.

SUBSTANCE: cecum lymphoma is modelled by the introduction of 2,4,6-trinitrobenzosulfonic acid to a rat in a dose of 0.1-0.15 ml, diluted in 0.1-0.15 ml of a 50% ethanol solution. Introduction is realised into a submucosal layer of the cecum cupola 2-3-times daily for 2-3 weeks.

EFFECT: creation of an adequate model of the cecum lymphoma.

2 ex

FIELD: medicine, experimental abdominal surgery.

SUBSTANCE: as experimental animals one should apply mongrel dogs of 12-17 kg body weight. Under general anesthesia one should conduct superior-median laparotomy, introduce 3.0 ml 70%-ethanol solution under pancreatic capsule and then laparotomic wound should be sutured up. Manipulation should be performed once. The method provides modeling adequate acute pancreatic inflammation at no side effects being very simple in implementation.

EFFECT: higher efficiency.

3 dwg

FIELD: medicine; medical engineering.

SUBSTANCE: method involves studying transverse longitudinal and rotation stiffness characteristics. The studies are carried out step-by-step from the first order units to complete external fixation apparatus structure. The device has frame and is provided with calibration loads, wire rope, displacement indicators, strip for fastening to loading end of bone imitator fragment, beam for fixing displacement indicators, beams having unit for modeling longitudinal and transverse loadings. The frame is manufactured as parallelepiped. The fixing panel has openings for bone imitator, for fixing external fixation apparatus and yoke connection union and is fixed in end face part of the frame. Beam for fixing displacement indicators has longitudinal slit for fixing the indicators and arranging them on lateral slots in frame base. The beams having unit for modeling rotational, longitudinal and transverse loadings are arranged on lateral frame sides on lateral slots in base.

EFFECT: high vision acuity without applying spectacle-based correction; accelerated treatment course.

2 cl, 16 dwg, 1 tbl

FIELD: experimental medicine.

SUBSTANCE: the present innovation deals with modeling urinary calculosis in rats due to injecting intraperitoneally 60%-glucose solution at 1 ml/100 g animal body weight twice daily for 2 mo. The method is very simple and enables to achieve lithogenesis in 25% experimental animals.

EFFECT: higher efficiency of experimental modeling.

2 dwg, 2 ex, 4 tbl

FIELD: medicine.

SUBSTANCE: method involves using Hann diode crystal with proper frequencies of pathogenic microorganisms and cells during their death period or during the stimulating factors action period being applied.

EFFECT: enhanced effectiveness of treatment; wider range of biophysical action types.

3 cl, 1 tbl

FIELD: medicine, experimental physiology.

SUBSTANCE: hypoxia with hypercapnia should be modeled due to creating a closed system of inhaled air circulation. Air enters lungs out of hermetically sealed reservoir and at expiration returns back. The process of recirculation is supplied with an apparatus of artificial pulmonary ventilation. The innovation suggested provides steadiness in development of hypoxia with hypercapnia excluding the development of stressor reaction. Conditions should be created to carry out any manipulations with an animal in the course of an experiment.

EFFECT: higher efficiency of modeling.

5 dwg, 1 ex

FIELD: medicine, stomatology.

SUBSTANCE: the present innovation should be carried out for the purpose to study ethiology and pathogenesis of parodontitis. One should affect with emotional stress in experimental animals (mature rats) due to placing 10-11 experimental animals into the cage at area of 0.018 sq. cm/animal. Before placing into the cage one should create artificial dental plaque around the cervix of the upper and lower incisors with the help of stomatological cement for every experimental animal. In the course of modeling all experimental animals should eat paste-like food. The method enables to shorten terms for obtaining the model desired and increase its similarity with pathomorphological manifestations of human parodontitis.

EFFECT: higher efficiency of investigation.

2 ex

FIELD: medical equipment.

SUBSTANCE: device has input first variable resistor, capacitor and permanent resistor. Permanent resistor is connected to arm second and third variable resistors. Second ends of variable resistors are connected with motionless contacts of polarized relay. Movable contact of relay is connected to common bus. Input of device is connected to amplifier which has output connected with control wiring of polarized relay. Second end of wiring is connected with common bus. Device is intended for electrical modeling of balanced and misbalanced conditions of acupuncture point at electropunctural action with unlike-poled signals due to liquidation mutual errors at any circuit of opposite arms of the device. Values of active resistances can be installed independently at any arm of device.

EFFECT: increased precision.

2 dwg

FIELD: experimental medicine.

SUBSTANCE: laboratory animals should be once injected intraperitoneally or intravenously with phenylhydrazine at the dosage of 100-150 mg/kg.

EFFECT: higher efficiency.

1 cl, 4 ex, 2 tbl

FIELD: medicine, experimental biology, ecology, toxicology.

SUBSTANCE: at studying the mechanisms of heavy metals toxic action, in particular, cadmium upon renal function, it is suggested to introduce cadmium sulfate solution into stomach once daily for 2 mo at the dosage of 0.5 mg/kg, on conversion to metal, where cadmium corresponds to 0.5 mg per 1 ml solution. The present innovation enables to study the pathology in dynamics of development and elaborate and searching preparations for treating and preventing chronic toxic nephropathy.

EFFECT: higher efficiency.

1 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: method involves exposing cell or cell group to external power source. At least two electrodes are introduced before treating the cells. One of electrodes is set on cytoplasmatic external cell membrane surface and the other one cell membrane and membrane potential value is measured. External electric voltage source is connected to the introduced electrodes oppositely in polarity with cell membrane potential difference value being not less than cell membrane potential.

EFFECT: enhanced effectiveness in building cell damage model by means of energy burst and death.

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