Method for establishing small animal dilatation pattern model
By establishing an expansion pattern model on small animals, using methods such as simplified operation, precise incision and sterile dilators, the problem of lack of safe and effective animal models in the existing technology is solved, and efficient and low-cost expansion pattern verification is achieved.
Patent Information
- Application Number
- CN202510453379.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-07-04
AI Technical Summary
The prior art lacks safe and effective animal models for verifying the feasibility of expanded streak therapy instruments and drugs, and there are ethical and high cost issues for verification directly on volunteers.
Methods to establish expansion streak modeling on small animals include simplifying the operation process, precise incision design, using sterile tissue expanders, hormone intervention and quantitative injection of normal saline, combined with multi-dimensional observation and pathological analysis to simulate the occurrence conditions of expansion streaks.
The expansion pattern model with simple operation and high success rate is established, which reduces experimental risks and costs, and improves the safety and effectiveness of verification.
Smart Images
Figure CN120241319A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of pathological models, and particularly to a method for establishing a striae distensae model in small animals. Background Art
[0002] Striae distensae are common lesions characterized by the appearance of linear, smooth bands of atrophic skin that are initially red and finally white, which are two clinically and histopathologically distinguishable forms of striae distensae: red striae and white striae. Pregnancy, sudden weight gain, rapid growth, and endogenous or steroid-induced Cushing's disease are the most common causes of this disease. Other causes also include inadequate skin development (especially the lack of elastic fibers and collagen in the skin), mechanical stretching of the skin, and endocrine imbalance. The lesions usually follow the cleavage lines transverse to the direction of maximum tension and are most commonly found on the abdomen, buttocks, thighs, and groin area. Histologically, striae distensae show varying degrees of thin and flat epidermis, worn and separated collagen bundles, accompanied by vasodilation, and / or separated or completely absent elastic fibers.
[0003] Skin biopsy analysis shows that the expression of collagen, elastin, and fibronectin fibers in striae distensae (SD) is significantly reduced compared to normal skin. Therefore, it is hypothesized that continuous mechanical forces may cause local damage to the skin structure, but permanent damage only occurs in patients in whom dermal fibroblasts cannot rapidly repair these stretch-dependent injuries. When observing the lesional tissue of striae distensae using the standard H&E staining method, early inflammatory changes can be seen, manifested as dermal edema and perivascular lymphocyte infiltration in the lesional skin. The tearing of the extracellular matrix caused by excessive skin stretching leads to the rupture of collagen fibers and simultaneously changes the arrangement structure of collagen and elastic fibers. This also explains why stretch marks are common on the abdomen, which is the area most stretched during pregnancy.
[0004] The pathophysiological mechanism of striae distensae involves changes similar to the scar formation process. In the initial stage, inflammation and edema occur in the dermis, and then collagen in the dermis is deposited along the direction of mechanical stress to form a flat reticular structure. Excessive mechanical stretching of the skin leads to the rupture of elastic fibers, and at the same time, fibroblasts cannot effectively repair the components of the extracellular matrix. In addition, the quality of fibroblasts changes in striae distensae, the content of fibronectin decreases, and the expression of type I and type III procollagen also decreases. As striae distensae atrophy, the collagen at the dermal-epidermal junction decreases, the elastin in the papillary dermis decreases, and at the same time, the elastin and elastic fibers in the deep dermis are also rearranged.
[0005] Currently, there is a lack of an ideal animal model for pre-verifying newly developed striae distensae treatment instruments / drugs. Currently, it is usually directly applied to volunteers, which has certain safety and ethical risks and high costs.
[0006] There is an urgent need to design a method for constructing stretch marks on small animals to overcome the bottleneck of the lack of existing animal models and facilitate a safer and more effective verification of the feasibility of instruments and drugs for treating stretch marks. Summary of the invention
[0007] The present invention proposes a method for establishing a small animal stretch mark model, which has the characteristics of simple operation and high success rate. The innovation of this method is mainly reflected in the following aspects:
[0008] 1. Simplified operation process: The operation steps of this method (such as anesthesia, disinfection, incision, implantation of expander, etc.) make the entire operation process simple and easy, reducing the technical requirements and operation difficulty of the experimenter.
[0009] 2. Precise incision design: A straight incision (1-2 cm in length, preferably 1.5 cm) is made at the tail end of the rat's back skin to minimize the impact on the skin at the expanded site, thereby improving the effectiveness and reliability of the model.
[0010] 3. Tissue expander used: This method preferably uses a sterile disposable tissue expander, which can effectively control the volume and speed of expansion, simulate the conditions for the occurrence of stretch marks, and reduce the risk of cross-infection that may occur in traditional methods.
[0011] 4. Hormone intervention: During the model establishment process, high hormone secretion levels were simulated by evenly applying hydrocortisone ointment, which further enhanced the biological relevance of the model and improved the formation effect of stretch marks.
[0012] 5. Quantitative injection of saline: The amount of saline injected (e.g. 3 mL on the first day, 0.1-1 mL thereafter, preferably 0.15 mL) is precisely calculated to ensure effective filling and continuous expansion of the expander, avoiding skin damage caused by improper injection.
[0013] 6. Observation and analysis methods: By observing the appearance changes of the rat's back (such as the appearance of red lines) and analyzing the pathological sections using HE staining and Masson staining, a multi-dimensional evaluation standard is provided, so that the success of the model establishment can be based on evidence.
[0014] To achieve the above object, the present invention provides a method for establishing a small animal stretch mark model, comprising the following steps:
[0015] S1. Anesthetize the rat and disinfect the back skin;
[0016] S2. Make an incision at the most caudal part of the rat's back skin, i.e., at the end of the spine, below the dermis;
[0017] S3. Use forceps to create a subcutaneous pocket under the dermis at the junction of the end of the rat's tail and the back skin at the incision site, and place a sterile tissue expander near the neck and above the vertebral column in the incision. Close the subcutaneous pocket with sutures and suture the incision to limit the movement of the tissue expander and close the incision.
[0018] S4. Cover the skin above the tissue expander on the rat's back with 8 ± 1 cm of skin every day 2 , and evenly apply hydrocortisone ointment to evenly cover the skin at the modeling site, and continuously intervene for 3 days; the application site should be as far away from the above incision site as possible.
[0019] S5. Fill the tissue expander with normal saline on the first day, and then inject 0.1 - 1 mL / rat of normal saline into the tissue expander every day, with the limit that there are no obvious wounds on the rat's back skin.
[0020] S6. After 21 days of modeling, remove the tissue expander and let the rats rest for 7 days. Observe the condition of the rats' backs, and based on whether there are red streaks on the appearance and the results of Masson / HE staining analysis, whether the collagen fibers are broken; when it is observed that the characteristics of early red streaks of striae distensae appear, it indicates that the modeling is successful.
[0021] Furthermore, in step S2, the incision is a linear opening with a length of 1 - 2 cm.
[0022] Furthermore, in step S2, the length of the incision is 1.5 cm.
[0023] Furthermore, in step S3, the tissue expander is a disposable tissue expander.
[0024] Furthermore, in step S5, every day after the first day, inject 0.15 mL / rat of normal saline.
[0025] By implanting a tissue expander in small animals and increasing the volume of the tissue expander over time, the purpose of stretching the skin is achieved, simulating the occurrence conditions of striae distensae. And finally, the results of model establishment are determined by methods such as skin section staining. The present invention also provides an experimental scheme for applying it to create a striae distensae model on the back of small animals. Description of the Drawings
[0026] Figure 1 is a picture of a disposable expander implanted under the skin of a rat's back.
[0027] Figure 2 is a picture of a skin sample taken from the back of a control group rat.
[0028] Figure 3 is a picture of a skin sample taken from the back of a model group rat.
[0029] Figure 4 It is a sampling diagram of the back skin of the rats in the model group.
[0030] Figure 5 It is a sampling diagram of the back skin of the rats in the model group.
[0031] Figure 6 It is a diagram of the pathological section staining (HE staining & Masson staining) of the rat back.
[0032] Figure 7 It is a diagram of the pathological section staining (HE staining & Masson staining) of the rat back. Specific implementation mode
[0033] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention. For those specific technologies or conditions not specified in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.
[0034] Example 1: Modeling of striae distensae on the back of small animals
[0035] There are 2 rats in the control group and 4 rats in the experimental group. All 6 rats are SD rats, male, 6 - 8 weeks old, and the body weight is 180 - 250 g.
[0036] 1. Anesthetize the rats in the control group and the experimental group (5% xylazine and 10% ketamine dissolved in PBS), and disinfect the back skin with 10% isopropyl - Betadine (Meda Pharma).
[0037] 2. Make an incision (a linear opening, 1 - 2 cm in length (preferably 1.5 cm), under the dermis layer) at the most caudal part of the back skin (i.e., the most distal end of the spine, so as to minimize the impact of the incision on the skin at the expansion site) to reduce the mechanical expansion force on the wound during the experiment and maximize the mechanical expansion force on the skin at the dilator position.
[0038] 3. Use forceps to separate the back skin tissue and muscle tissue of the rat through the incision to facilitate the implantation of the dilator, and place a sterile disposable tissue expander in the incision near the neck, above the vertebral column (i.e., between the left and right scapulas of the rat, above the spine). Use sutures (nylon thread, model 5 - 0) to close the subcutaneous pocket and sew up the incision to limit the movement of the tissue expander and close the incision.
[0039] In the control group, only subcutaneous pouches were created in the mice without implanting tissue expanders. In the experimental group, subcutaneous pouches were created in the mice and tissue expanders were implanted.
[0040] 4. Every day, cover the skin above the tissue expander on the back of the rats with a length of about 8 cm 2 or so (the purpose is to simulate a high hormone secretion level), evenly apply about 3 - 10 mg of hydrocortisone ointment (subject to evenly covering the skin at the modeling site, preferably 5 mg), and continuously intervene for 3 days; the application site should be as far away from the incision site in step 2 as possible;
[0041] 5. On the first day, it is 3 mL (the injection volume on the first day is determined according to the volume of the disposable tissue expander. The disposable tissue expander is 3 ml, so 3 ml needs to be injected on the first day after implantation to fill the volume of the tissue expander). Thereafter, inject 0.1 - 1 mL / rat of normal saline into the tissue expander every day (preferably 0.15 mL, with the boundary that there are no obvious wounds on the back skin of the rats);
[0042] 6. After 21 days of modeling (normal feeding can be carried out during modeling), remove the disposable tissue expander and then let the rats rest quietly for 7 days (steps 4 - 5 are no longer needed during the rest period, and standard feeding is okay), and observe the back conditions of the rats. All analyses are carried out in the cylindrical top area caused by the skin expander because this area experiences the highest strain;
[0043] 7. Whether there are red streaks in appearance, and the results of HE staining and Masson staining are compared with the existing striae distensae staining results in the literature as the judgment basis;
[0044] 8. When one of the following situations occurs in the experimental animals, immediately terminate the experiment:
[0045] (1) Weight loss: Rapidly (such as within 1 day) losing 15 - 20% of the original weight, or growing animals continuously having no weight gain, not monitoring the weight but the animals showing cachexia and persistent muscle wasting.
[0046] (2) Loss of appetite: Completely losing appetite for 24 hours or having poor appetite (less than 50% of the normal amount) for 3 days.
[0047] (3) Weakness: Unable to eat and drink by itself, unable to stand for 24 hours or extremely reluctantly able to stand. It is necessary to first exclude whether it is the recovery period of the animals after anesthesia, and then evaluate whether the animals are weak due to diseases or experiments and other factors.
[0048] (4) Infection of body organs: Presenting physical indicators and abnormal blood test values, having no good response to drug treatment and continuously evolving into a systemic disease.
[0049] The following lists some abnormal indicators. If any one of them exists, it is determined as abnormal;
[0050] 1. Physical indicators
[0051] Fever: A common symptom of infection, with body temperature rising.
[0052] Pain: Pain or tenderness may occur at the infection site.
[0053] Redness and swelling: Local infections are often accompanied by redness, swelling and fever.
[0054] Discharge: Such as pus, mucus, etc.
[0055] Fatigue: Systemic infections are often accompanied by fatigue and weakness.
[0056] Shortness of breath: Lung infections may cause rapid breathing.
[0057] Frequent urination or urgency: Common symptoms of urinary tract infections.
[0058] 2. Abnormal blood test values
[0059] White blood cell count (WBC):
[0060] Increase: Common in bacterial infections.
[0061] Decrease: May be caused by certain viral infections.
[0062] C-reactive protein (CRP):
[0063] Increase: A sensitive indicator of inflammation or infection.
[0064] Erythrocyte sedimentation rate (ESR):
[0065] Increase: An indicator of inflammation or infection.
[0066] Procalcitonin (PCT):
[0067] Increase: An indicator of severe bacterial infection or sepsis.
[0068] Platelet count:
[0069] Increase: Inflammatory response.
[0070] Decrease: Severe infection or sepsis.
[0071] Liver function indicators:
[0072] Elevation of ALT / AST: Liver infection or inflammation.
[0073] Kidney function indicators:
[0074] Elevation of creatinine and blood urea nitrogen: Kidney infection or impaired function.
[0075] By implanting a cylindrical disposable tissue expander (commercially available directly) on the back of rats, the depth of the expander implantation is in the deep layer of the subcutaneous tissue, on the surface of the muscle tissue, and the bottom is supported by the rat spine. After surgery and suturing, as Figure 1 shown, Figure 1 is a picture of the disposable expander implanted under the back skin of rats. Figure 1 In Figure, A is the control group, only creating a subcutaneous pocket without implanting an expander. Figures B - D are the experimental groups, creating a subcutaneous pocket and implanting an expander. Part of the expander is implanted into the rat body, and the other part remains outside the body. After the entire experimental period, the back skin of the mice is taken for observation as Figures 2 - 5 shown, and then the back skin is sectioned and stained for observation (as Figures 6 - 7 ), it can be seen that: after implanting the expander, the back skin of the rats is subjected to a certain degree of mechanical expansion force, simulating the tensile force on the local skin due to individual growth during the formation of striae distensae. The control group is the normal rat skin staining. In the pictures of model groups 1, 2, and 3, it can be seen that the epidermal layer is significantly thickened, with downward epidermal protrusions visible, and part of (the dermal sawtooth ridge) disappears. There are scattered inflammatory cell infiltrations in the dermis, and the collagen fibers are broken, with scar hyperplasia.
[0076] From the external observation and pathological section analysis, it conforms to the characteristics of the early stage of striae distensae - red striae:
[0077] ● The accumulation of degranulated mast cells and macrophages around the elastic fibers in the dermis, leading to elastinolysis;
[0078] ● Interlacing of lymphocytes around blood vessels and an increase in dermal edema and glycosaminoglycans;
[0079] ● Fine dermal elastic fibers are dominant, and there are signs of structural changes in collagen;
[0080] ● Dermal edema;
[0081] ● The increase in microvessels leads to its erythematous color.
[0082] Example 2: Modeling of striae distensae on the back of small animals
[0083] According to the method of Example 1, 2 mice are set as the control group and 4 mice are set as the experimental group, and finally 3 mice in the experimental group are successful. The 2 rats in the control group are the same as the control in Example 1.
[0084] Among them, 1 rat that died in the experimental group died due to factors outside the experimental process, not due to experimental operations. Therefore, the success rate of this experiment is greater than or equal to 75%.
[0085] Therefore, the method of the present invention is feasible and can be repeatedly implemented, with a high success rate.
[0086] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.
Claims
1. A method for establishing a striae distensae model in small animals, characterized in that, It includes the following steps: S1. Anesthetize the rats and disinfect the back skin; S2. Make an incision at the most caudal part of the back skin of the rats, i.e., the most distal end of the spine, and this incision is below the dermis layer; S3. Use forceps to create a subcutaneous pocket under the dermis layer at the junction of the distal end of the rat's tail and the back skin at the incision, and place a sterile tissue expander in the incision near the neck, above the vertebrae. Use sutures to close the subcutaneous pocket and sew up the incision to limit the movement of the tissue expander and close the incision; S4. Cover the skin above the tissue expander on the back of the rats with a size of 8 ± 1 cm every day 2 , apply hydrocortisone ointment to evenly cover the skin at the modeling site, and continuously intervene for 3 days; the application site should be as far away from the above incision site as possible; S5. On the first day, fill the tissue expander with normal saline, and then inject 0.1 - 1 mL of normal saline into the tissue expander per rat every day, with the boundary being that there are no obvious wounds on the back skin of the rats; S6. After 21 days of model establishment, take out the tissue expander and let the rats rest quietly for 7 days. Observe the back conditions of the rats, based on whether there are red streaks in appearance and the results of Masson / HE staining analysis on whether the collagen fibers are broken. When it is observed that the characteristics of early red streaks of striae distensae appear, it indicates that the model establishment is successful.
2. The method according to claim 1, characterized in that, In the step S2, the incision is a linear opening with a length of 1 - 2 cm.
3. The method according to claim 2, wherein In the step S2, the length of the incision is 1.5 cm.
4. The method according to claim 2, wherein In the step S3, the tissue expander is a disposable tissue expander.
5. The method according to claim 1, characterized in that In the step S5, every day after the first day, inject 0.15 mL of normal saline per rat.