LU504679B1 - Novel Evaluation Method for Rat Hind Limb Venous Ulcers Model - Google Patents
Novel Evaluation Method for Rat Hind Limb Venous Ulcers Model Download PDFInfo
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- LU504679B1 LU504679B1 LU504679A LU504679A LU504679B1 LU 504679 B1 LU504679 B1 LU 504679B1 LU 504679 A LU504679 A LU 504679A LU 504679 A LU504679 A LU 504679A LU 504679 B1 LU504679 B1 LU 504679B1
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- 208000000558 Varicose Ulcer Diseases 0.000 title claims abstract description 15
- 238000011156 evaluation Methods 0.000 title claims abstract description 15
- 208000027418 Wounds and injury Diseases 0.000 claims abstract description 28
- 206010052428 Wound Diseases 0.000 claims abstract description 27
- 210000003141 lower extremity Anatomy 0.000 claims abstract description 15
- 230000029663 wound healing Effects 0.000 claims abstract description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims abstract description 6
- 108090001005 Interleukin-6 Proteins 0.000 claims abstract description 5
- 238000011552 rat model Methods 0.000 claims abstract description 5
- 102000015271 Intercellular Adhesion Molecule-1 Human genes 0.000 claims abstract description 4
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 claims abstract description 4
- 102000008212 P-Selectin Human genes 0.000 claims abstract description 4
- 108010035766 P-Selectin Proteins 0.000 claims abstract description 4
- 238000002604 ultrasonography Methods 0.000 claims abstract description 4
- 241000700159 Rattus Species 0.000 claims description 36
- 238000001514 detection method Methods 0.000 claims description 14
- 210000004204 blood vessel Anatomy 0.000 claims description 9
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- 206010061218 Inflammation Diseases 0.000 claims description 7
- 230000004054 inflammatory process Effects 0.000 claims description 7
- 230000001575 pathological effect Effects 0.000 claims description 6
- 230000035876 healing Effects 0.000 claims description 5
- 208000007536 Thrombosis Diseases 0.000 claims description 4
- 230000017531 blood circulation Effects 0.000 claims description 4
- 210000000265 leukocyte Anatomy 0.000 claims description 4
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- 230000004913 activation Effects 0.000 claims description 3
- 206010047249 Venous thrombosis Diseases 0.000 abstract description 10
- 230000002792 vascular Effects 0.000 abstract description 4
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 abstract 1
- 238000002965 ELISA Methods 0.000 abstract 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 abstract 1
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- 206010051055 Deep vein thrombosis Diseases 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 6
- 206010002091 Anaesthesia Diseases 0.000 description 4
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- 206010072170 Skin wound Diseases 0.000 description 3
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- 230000015271 coagulation Effects 0.000 description 2
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- 102000003390 tumor necrosis factor Human genes 0.000 description 2
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- 239000003154 D dimer Substances 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010034272 Pelvic venous thrombosis Diseases 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 206010040943 Skin Ulcer Diseases 0.000 description 1
- 208000002847 Surgical Wound Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 206010046996 Varicose vein Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
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- 210000003195 fascia Anatomy 0.000 description 1
- 108010052295 fibrin fragment D Proteins 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 210000004013 groin Anatomy 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 231100000019 skin ulcer Toxicity 0.000 description 1
- 210000000273 spinal nerve root Anatomy 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 208000027185 varicose disease Diseases 0.000 description 1
- 201000002282 venous insufficiency Diseases 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
- A61B8/08—Detecting organic movements or changes, e.g. tumours, cysts, swellings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention belongs to the field of bioengineering, and particularly relates to an evaluation method for a hind limb venous ulcers rat model. According to the invention, the presence or absence of venous thrombosis is observed by color Doppler ultrasound, the wound area and wound healing rate, wound tissue HE staining are measured, the morphological changes and immunohistochemistry of wound tissue cells are observed, the expression levels of vascular P-selectin and ICAM-1 are detected, and the levels of plasma IL-6 and TNF-α are detected by ELISA, so that the model is comprehensively evaluated, and the success or failure of model establishment can be reasonably judged.
Description
LU5046 79
Novel Evaluation Method for Rat Hind Limb Venous Ulcers Model
The invention belongs to the field of bioengineering, and particularly relates to a novel evaluation method for a rat hind limb venous ulcers model.
Venous leg ulcers (VLUs) are open skin ulcer lesions in areas affected by venous hypertension of lower limbs, which are often secondary to the the later stages of deep venous thrombosis and varicose veins of lower limbs, accounting for 80% of the ulcer lesions of lower limbs. The disease has a long course, is difficult to heal and is easy to recur. With the progress of the disease, VLUs patients will have serious complications such as infection and canceration, which greatly affects their normal work and life. Therefore, VLUs has become a difficult and hot spot in the research of surgical wound healing. Constructing a stable, feasible and repeatable animal model 1s the basis and premise for the study of pathogenesis and innovative treatment of VLUs.
At present, there are few studies on the construction of VLUs animal model. Wang Xi and others made venous ulcer model by combining the lower limbs deep venous thrombosis (DVT) model with the local skin defect model, and made iliac vein thrombosis model by the method of clamping the injury with the mosquito clamp+clamping the iliac vein with the vascular clamp for 24 h. Through hemorheology and D-dimer detection, it was confirmed that rats were in high coagulation and high fibrinolysis state; the increase of concentrations of serum interleukin (IL) 1P, IL-6 and tumor necrosis factor (TNF)-a confirmed the existence of inflammatory reaction in this model, which proved the successful establishment of the venous leg ulcers rat model.
However, the existing evaluation technology has obvious defects. The pathology of venous ulcer changed into distal venous hypertension caused by venous reflux disorder, and white blood cells adhered to the blood vessel wall, thus inducing local inflammatory reaction and causing venous ulcers. The above model evaluation method only confirms the existence of high coagulation and inflammation states in rats, but can't directly confirm the similarity between the modeling method and the pathological process of venous ulcers. The experimental rats have vascular clamps in vivo, and the vascular clamps are partially exposed in vitro, which
? LU5046 79 aggravates the inflammatory reaction of animals in model, and it is impossible to distinguish whether the increase of inflammatory indexes is caused by thrombosis or infecting wound. In a word, the construction system of VLUs animal model is not mature, and new modeling methods need to be explored continuously.
The present application provides an evaluation method for a hind limb venous ulcers rat model, and the ultrasound images, histopathological detection, blood detection, wound micro- morphology observation and macroscopic healing situation are used for evaluation.
Further, the ultrasonic image is to directly observe that thrombosis and the obstruction situation of blood flow backflow in the iliac vein in the model rats.
Further, the histopathological detection is to judge the existence of the adhesion and activation situation of leukocytes in blood vessels through the levels of P-selectin and ICAM- 1 in blood vessels.
Further, the blood detection is to judge the existence of body inflammatory reaction situation in the model rats through the IL-6 and TNF-a levels detection in blood.
Further, the wound micro-morphology observation is to judge the existence of microscopic pathological tissue changes situation in the model rats through pathological observation under the wound microscope.
Further, and the macroscopic healing situation is to judge the existence of the venous ulcer outcome in the model rats through the wound healing situation.
The invention has the following beneficial effects:
According to the invention, it is directly confirmed that the present animal model can simulate the whole pathological process of human venous ulcer through the ultrasound images, histopathological detection, blood detection, wound micro-morphology observation and macroscopic healing situation. Through the ultrasonic observation, it is directly confirmed the existence of thrombosis and the obstruction situation of blood flow backflow in the iliac vein in the model rats; through the levels of P-selectin and ICAM-1 in blood vessels, it is proved the existence of the adhesion and activation situation of leukocytes in blood vessels; through the blood IL-6 and TNF-a levels detection, it is confirmed the existence of body inflammatory reaction in the model rats; through the pathological observation under the wound microscope, it is confirmed the existence of microscopic pathological tissue changes situation in the model rats; through the wound healing situation, it is confirmed the existence of venous ulcer outcome in the model rats.
’ LU5046 79
In order to explain the embodiments of the present invention or the technical scheme in the current technology more clearly, the figures needed in the embodiments will be briefly introduced below. Obviously, the figures described below are only some embodiments of the present invention, and other figures can be obtained according to these figures without creative work for ordinary people in the field.
Fig. 1 shows the process of separating the common iliac vein;
Fig. 2 shows the local skin wound with a diameter of 2 cm;
Fig. 3 shows that the establishment of rat model of venous ulcer of hind limbs is completed;
Fig. 4 shows the limb circumference 2 h after the DVT model is prepared; A: the limb circumference of normal side; and B: the limb circumference of the operation side;
Fig. 5 shows the skin color 2 h after the DVT model is prepared; A: skin color contrast of bilateral hind limbs; B: comparison of skin color of feet.
Now, various exemplary embodiments of the present invention will be described in detail.
Unless otherwise specified, the methods in the embodiments are all conventional methods, and the reagents used are all conventional commercially available reagents or reagents prepared by conventional methods. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.
It should be understood that the terminology described in the present invention is only for describing specific embodiments and is not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed.
Intermediate values within any stated value or stated range, as well as each smaller range between any other stated value or intermediate values within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the field to which this invention relates. Although the present invention only describes the preferred methods and materials, any
* LU504679 methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated document, the contents of this specification shall prevail.
It is obvious to those skilled in the field that many improvements and changes can be made to the specific embodiments of the present invention without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the invention. The description and embodiment of that present invention are exemplary only.
The terms "include", "comprise", "have" and "contain" used in this article are all open terms, which means including but not limited to.
Embodiment 1 Preparation of DVT model
Forty rats after adaptive feeding for one week were randomly divided into model group (n=15), control group (n=15) and blank group (n=10).
The experimental rats were anesthetized by abdominal cavity by using the pentobarbital sodium solution with a concentration of 0.3% and at a dose of 1mL/100g, and the depth of anesthesia was checked every 10 min, when necessary, the rats were given additional anesthesia.
After full anesthesia, the hair on the ventral root of the left thigh of the rat was removed with an electric shaver, and the rat was taken to a sterile operating table at the supine position, the limbs were abduction-fixed, medical iodophor was used for disinfection, and a sterile hole towel was laid to expose the inner visual field of the left groin of the rat. An incision with a length of 2.0 cm~2.5 cm was made in the middle of the visual field with an ophthalmic scissors, and then the subcutaneous tissue was bluntedly separated, and common iliac vein was exposed and separated. See Fig. 1. The proximal ends, the branches and the distal ends of the left common iliac vein in the model group were ligated with 3-0 surgical silk thread in turn. After ligation, the blood vessels at the ligation site became congested and thickened. Common iliac vein of rats in the control group was separated without ligation, and the blank group was not treated.
After the surrounding tissues were reset, the muscularis and skin were sutured continuously with silk thread, and the incision was disinfected with iodophor. Rats were raised in a single cage, and the abdominal incision was disinfected with iodophor once a day.
Embodiment 2 Preparation of local skin wound model
Seven days after the successful preparation of DVT model, the above anesthesia steps
) LU504679 were repeated, and the rats were placed in a sterile operation table at the prone position, and the long hair on the back of the left hind limb of the rats was removed, and the short fluff was removed with depilatory cream to form a hairless area with a diameter of about 4 cm, so as to ensure that the skin of the rats was intact and undamaged. A circular mark was printed with a circular stamp with a diameter of 2 cm, routine disinfection was performed, and a sterile hole towel was laid; on the basis of keeping the skin tight, the full-thickness skin was removed to the deep fascia according to the mark range, resulting in a local skin wound. See Fig. 2 and Fig. 3. After hemostasis and disinfection, the wound was wrapped with sterile gauze and fixed with medical paper tape. The wound was disinfected routinely every day and observed for 10 days.
Embodiment 3 General state
During the experiment, the hair, weight and mental state of rats in each group were observed in a quiet environment, and the swelling degree of left hind limbs of rats were observed after the preparation of DVT model. The changes of blood vessels and blood flow in the left hind limbs were observed by color Doppler ultrasonic diagnostic instrument before, immediately after and on the 7 th day after DVT model preparation.
Embodiment 4 Wound index
The changes of rat wounds were observed every day, and the wounds situation of rats were recorded on the Sth and 10th day after the preparation of VLUs model. Calculation method of wound area: the wound is shot by camera, ensuring the vertical distance between the camera and the wound and the focal length of the camera to be constant, and at the same time a ruler is placed next to the wound as a reference. The clearest picture was selected and the wound area was calculated with Image J medical image analysis software. Each wound was measured three times, and the average value was taken as the final wound area and the wound healing rate was calculated. Wound healing rate (%)=[(initial wound area-observation time point wound area)/initial wound area ]x 100%.
Embodiment 5 Observation on general state of rats in each group
No rats died during the experiment, and the success rate of estabilishment of model was 100%. The mental state of 40 rats was good, their activities were normal, and their weight increased steadily. There was no significant difference among the groups. All the rats in the model group showed swelling of the left hind limb and dark purple skin at 2 h after operation, but there was no significant difference between the two hind limbs in the control group. See
Fig. 4 and Fig. 5.
° LU5046 79
The above-mentioned embodiments only describe the preferred mode of the invention, and do not limit the scope of the invention. Under the premise of not departing from the design spirit of the invention, various modifications and improvements made by ordinary technicians in the field to the technical scheme of the invention shall fall within the protection scope determined by the claims of the invention.
Claims (6)
1. An evaluation method for a hind limb venous ulcers rat model, wherein, the ultrasound images, histopathological detection, blood detection, wound micro-morphology observation and macroscopic healing situation are used for evaluation.
2. The evaluation method as claimed in claim 1, wherein, the ultrasonic image is to directly observe that thrombosis and the obstruction situation of blood flow backflow in the iliac vein in the model rats.
3. The evaluation method as claimed in claim 1, wherein, the histopathological detection is to judge the existence of the adhesion and activation situation of leukocytes in blood vessels through the levels of P-selectin and ICAM-1 in blood vessels.
4. The evaluation method as claimed in claim 1, wherein, the blood detection is to judge the existence of body inflammatory reaction situation in the model rats through the IL-6 and TNF-a levels detection in blood.
5. The evaluation method as claimed in claim 1, wherein, the wound micro-morphology observation is to judge the existence of microscopic pathological tissue changes situation in the model rats through pathological observation under the wound microscope.
6. The evaluation method as claimed in claim 1, wherein, and the macroscopic healing situation is to judge the existence of the venous ulcer outcome in the model rats through the wound healing situation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU504679A LU504679B1 (en) | 2023-07-06 | 2023-07-06 | Novel Evaluation Method for Rat Hind Limb Venous Ulcers Model |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU504679A LU504679B1 (en) | 2023-07-06 | 2023-07-06 | Novel Evaluation Method for Rat Hind Limb Venous Ulcers Model |
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| LU504679B1 true LU504679B1 (en) | 2024-01-11 |
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| LU504679A LU504679B1 (en) | 2023-07-06 | 2023-07-06 | Novel Evaluation Method for Rat Hind Limb Venous Ulcers Model |
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