A method for constructing a radiation recall dermatitis rat model

Abstract

This invention belongs to the field of biomedical experimental animal model construction technology, specifically relating to a method for constructing a rat model of radiation-induced recurrent dermatitis (RRD). This invention successfully induces recurrent skin inflammation localized to the original irradiated area in rats that have undergone initial radiation injury by inducing drug therapy at a specific induction starting point, thus obtaining a rat model of RRD. The model constructed by this method successfully reproduces the recurrent characteristics of RRD, with inflammation only occurring in the previously irradiated area, while the unirradiated skin remains intact, exhibiting good clinical fit and stability. This model provides a reliable in vivo experimental platform for in-depth research into the pathogenesis of RRD, screening intervention targets, and evaluating treatment regimens.

Description

Technical Field

[0001] This invention belongs to the field of biomedical experimental animal model construction technology, specifically relating to a method for constructing a rat model of radiation recall dermatitis. Background Technology

[0002] Radiation-induced skin injury is a common radiotherapy-related adverse reaction in clinical practice, significantly impacting patient treatment adherence and quality of life. Radiation recall dermatitis (RRD) is a relatively rare and specific type, referring to the recurrence of acute inflammatory skin reactions localized to the original irradiated area after a recovery period, following the use of certain precipitating agents (such as chemotherapy drugs, targeted therapies, or immune checkpoint inhibitors). Chemotherapy drugs are currently one of the most commonly reported precipitating factors, and the occurrence of this adverse reaction poses a significant challenge to the continuity of subsequent systemic antitumor therapy in patients with intermediate-to-advanced stage cancer.

[0003] Typical clinical manifestations of recurrent disease (RD) include erythema, edema, desquamation, and pain in the previously irradiated skin area. In severe cases, it can further develop into blisters, erosion, or even focal or large-area tissue necrosis, while the unirradiated skin area usually remains intact. These reactions not only affect the integrity of the local skin but may also force patients to interrupt or delay their established anti-tumor treatment regimen, thus adversely affecting the overall efficacy.

[0004] Currently, several international expert consensuses and clinical management guidelines have been established for acute radiation-induced skin injuries. However, there is a lack of standardized protocols based on evidence-based medicine for the prevention, prediction, and treatment of chemotherapy-related radiation-induced skin injuries. Current clinical management mainly relies on stopping or adjusting the inducing drugs, supplemented by empirical interventions such as corticosteroids, anti-inflammatory drugs, and local symptomatic supportive treatment. However, the efficacy is unstable, individual differences are significant, and a unified, widely applicable technical approach is lacking.

[0005] Based on current research, the mechanism of chemotherapy-related recurrent skin damage (RRD) is generally considered to be closely related to long-term subclinical damage caused by ionizing radiation to keratinocytes, basal cells, and skin appendages. Radiotherapy can induce DNA damage, increased oxidative stress, and cellular homeostasis imbalance in skin cells, leaving the local skin in a state of low-grade inflammation and immune hypersensitivity for a long time. On this basis, subsequent stimulation by chemotherapy drugs may reactivate the inflammatory response pathways in the previously damaged area, forming a so-called "memory amplification effect," ultimately manifesting as acute inflammatory skin damage limited to the original irradiated area.

[0006] However, current research mainly focuses on hypotheses such as the "memory effect" of radiation damage, changes in local vascular permeability, or drug hypersensitivity reactions. There is a lack of a stable animal model that can simulate the process of radiation-induced memory skin inflammation, enabling systematic evaluation of the pathological processes and potential molecular mechanisms of chemotherapy-related recurrent radiation-induced skin damage (RRD). This lack of animal models limits in-depth research on key cell subsets, inflammatory signaling pathways, and their temporal changes, and objectively restricts the development of predictive methods and intervention targets for chemotherapy-related radiation-induced memory skin damage. Summary of the Invention

[0007] The technical problem to be solved by the present invention is that there is a lack of stable, reliable animal models of RRD that can simulate clinical pathological features in the prior art.

[0008] The technical solution of the present invention:

[0009] In one aspect, the present invention provides a method for constructing a rat model of radiation-induced dermatitis, comprising the following steps:

[0010] (1) Initial radiation damage induction: Rats were selected and the skin of the target area was irradiated with ionizing radiation once to induce acute radiation skin damage in the irradiated area;

[0011] (2) Feeding during the recovery period: After the irradiation, the rats were fed in a routine manner. When the dry desquamation, wet desquamation and scabs on the irradiated area of ​​the skin completely disappeared and there were no open wounds on the skin, it was taken as the induction starting point.

[0012] (3) Drug-induced memory response: Starting from the induction initiation point, the inducing drug was administered to the rats. The dosage of the inducing drug was set such that when the inducing drug was applied to the normal skin of unirradiated rats, it did not produce erythema, desquamation or ulceration, but was sufficient to induce desquamation or ulceration at the irradiated site (this dosage is referred to as the subtoxic dose in this document).

[0013] This can induce recurrent skin inflammation localized to the original irradiated area.

[0014] In the specific implementation plan, the acute radiation skin injury mentioned in step (1) refers to an acute inflammatory skin reaction, including clinical manifestations such as erythema, desquamation, crusting, and ulceration as described in Table 1 below.

[0015] In the specific implementation plan, the rats mentioned in step (1) are males aged 5-6 weeks.

[0016] In a specific implementation plan, the target skin area mentioned in step (1) is the skin of the right hind leg and / or foot of the rat.

[0017] In a specific implementation scheme, the dose of a single ionizing radiation irradiation in step (1) is 20-40 Gy, preferably 30 Gy.

[0018] In a specific implementation, the ionizing radiation irradiation in step (1) is performed using an electron linear accelerator, with an irradiation energy of 4-6 MeV, a source-to-skin distance of 80-120 cm, and a dose rate of 250-350 cGy / min. In a preferred implementation, the irradiation energy is 5 MeV, the source-to-skin distance is 100 cm, the dose rate is 300 cGy / min, and the total dose of a single ionizing radiation irradiation is 30 Gy.

[0019] In a specific implementation plan, in step (1), a tissue compensation membrane of 0.5-1.5 cm thickness is covered on the skin surface of the target area during irradiation to increase the absorbed dose of the epidermis.

[0020] In a specific implementation plan, in step (2), the skin damage score corresponding to the skin condition at the induction starting point is ≤2.0 points.

[0021] In a specific implementation plan, in step (2), the induction start point is 45-47 days after irradiation.

[0022] In a preferred embodiment, the induction initiation point is 45 days after irradiation.

[0023] In a specific implementation plan, the rat skin damage score is quantitatively assessed using the scoring system shown in Table 1 below.

[0024] Table 1. Radiation-induced skin injury score

[0025] score Description of skin damage 1.0 normal skin 1.5 Mild erythema and dryness 2.0 Moderate erythema and dryness 2.5 Obvious erythema and dry desquamation 3.0 Dry scaling and mild dry crusting 3.5 Dry scaling, crusting, and mild epidermal scarring 4.0 Focal wet desquamation with moderate focal ulceration 4.5 Confluent wet desquamation, ulceration, and large areas of deep crusting 5.0 Full-thickness skin loss open wound 5.5 Skin necrosis

[0026] In the construction method of this invention, the selection of the induction initiation point in step (2) is crucial for successful modeling. This invention utilizes a recovery period of 40-50 days to allow the skin of the irradiated rats to recover from the acute phase of wet desquamation (above 4.0 points) to a state of ≤2.0 points (only mild to moderate erythema without desquamation). Unbound by theoretical constraints, at this point, although local microvascular dilation (erythema) exists, the physical structure of the skin (epithelial layer) has been restored to its complete state, and the skin is in a latent state of sensitization but normal appearance. Based on this, a subtoxic dose of the drug is administered. Since this dose does not damage normal skin, the desquamation or crusting reaction induced on the irradiated side can be clearly identified as a recall reaction caused by radiation memory, rather than a drug toxicity reaction.

[0027] As used herein, the administration of the induction drug to rats starting from the induction initiation point means administering the induction drug to rats on the day of or the day after the induction initiation point. Preferably, the administration of the induction drug to rats starting from the induction initiation point means administering the induction drug to rats on the day after the induction initiation point.

[0028] In a specific implementation scheme, in step (3), the inducing drug is selected from one or more anthracycline antitumor drugs and antimetabolite antitumor drugs, including but not limited to epirubicin and 5-fluorouracil.

[0029] In a specific implementation, step (3) includes the absence of obvious damage, including the absence of erythema, desquamation, or ulceration.

[0030] In a specific implementation plan, in step (3), the skin damage at the irradiated site includes clinical manifestations such as erythema, desquamation, and crusting.

[0031] In a specific implementation plan, in step (3), the induction drug is administered via local subcutaneous injection, intraperitoneal injection, or intravenous injection at the irradiated site.

[0032] In a preferred embodiment, the application method is local subcutaneous injection at the irradiated site.

[0033] In a preferred embodiment, the subcutaneous injection is administered once daily for 5-10 consecutive days, with each injection volume being 80-120 μL. In a preferred embodiment, when the inducing drug is epirubicin, the subcutaneous injection concentration is 0.01-0.1 μg / μL; when the inducing drug is 5-fluorouracil, the subcutaneous injection concentration is 5-15 μg / μL.

[0034] In a specific implementation plan, the construction method involves administering the drug simultaneously to both the irradiated and unirradiated sides of the same rat, using a self-control to eliminate the toxic interference of the drug itself.

[0035] In a specific implementation plan, the construction method further includes confirming the induction of radiation-induced dermatitis by observing whether erythema reappears on the irradiated skin after the administration of the inducing drug, accompanied by dry desquamation, crusting, or epidermal scarring, while the skin on the unirradiated side of the same body remains normal.

[0036] In the specific implementation plan, the skin damage score of the irradiated area is ≥2.5 points when the model is successfully constructed.

[0037] In another aspect, the present invention provides a rat model of radiation-induced dermatitis obtained by the construction method described herein.

[0038] In another aspect, the present invention provides the use of a rat model of radiation memory dermatitis obtained by the construction method described herein in screening drugs for the prevention or treatment of radiation memory dermatitis.

[0039] The beneficial effects of this invention are:

[0040] 1. High clinical fit. This model successfully reproduces the recall characteristics of RRD, namely, under drug stimulation, inflammation occurs only in previously irradiated areas, while the skin in unirradiated areas remains intact. This self-controlled design effectively eliminates interference from simple drug toxicity or allergic reactions, and is highly consistent with the clinical diagnostic criteria for RRD.

[0041] 2. The model exhibits good stability and accurate parameters. Through extensive experimental screening, this invention has determined that 45 days after 30 Gy irradiation is the optimal induction window, and has identified the sub-threshold concentrations of the inducing drugs (EPI 0.05 μg / μL and 5-Fu 10 μg / μL). These concentrations ensure a significant inflammatory response in sensitized skin without causing non-specific damage to normal skin, thus guaranteeing a high success rate and an extremely low false positive rate.

[0042] 3. Comprehensive evaluation system. This model has been validated in multiple dimensions through macroscopic scoring, histopathological HE staining, and transmission electron microscopy ultrastructure, showing typical pathological changes such as epidermal thickening, parakeratosis, dermal lymphocyte infiltration, and widening of intercellular spaces, providing a reliable in vivo experimental platform for in-depth research on the molecular mechanisms of RRD. Attached Figure Description

[0043] Figure 1 The skin damage in SD rats after 30 Gy electron beam irradiation is shown.

[0044] Figure 2 The skin damage score of SD rats after 30 Gy electron beam irradiation is shown.

[0045] Figure 3 The flowchart for constructing the RRD rat model is shown.

[0046] Figure 4 The skin damage in the RRD rat model is shown.

[0047] Figure 5HE staining of skin tissue at irradiation sites in rats is shown: (A) Representative HE staining images of skin tissue at the right leg of rats in the CON group (unirradiated and injected with saline); (B) Representative HE staining images of skin tissue at the irradiation site of rats in the IR group (irradiated and injected with saline); (C) Representative HE staining images of skin tissue at the lesion site of rats in the RRD (EPI) group (irradiated and injected with EPI); (D) Representative HE staining images of skin tissue at the lesion site of rats in the RRD (5-Fu) group (irradiated and injected with 5-Fu); bar=100.

[0048] Figure 6 Transmission electron micrographs of skin tissue at the irradiation sites in rats are shown: (A) Representative transmission electron micrograph of skin tissue on the right leg of rats in the CON group (unirradiated and injected with saline); (B) Representative transmission electron micrograph of skin tissue on the right leg of rats in the IR group (irradiated and injected with saline); (C) Representative transmission electron micrograph of skin tissue at the lesion site on the right leg of rats in the RRD (EPI) group (irradiated and injected with EPI); (D) Representative transmission electron micrograph of skin tissue at the lesion site on the right leg of rats in the RRD (5-Fu) group (irradiated and injected with 5-Fu); bar = 200 µm. Detailed Implementation

[0049] In a preferred embodiment, the present invention provides a method for constructing a rat model of chemotherapy-associated radiation memory dermatitis, the method comprising the following steps:

[0050] The first step was to establish a basic model of radiation-induced skin injury. Five-week-old male SD rats were selected and subjected to a single high-dose electron beam irradiation of a designated skin area. The irradiation equipment used was an electron linear accelerator, with irradiation conditions of 5 MeV electron beam, source-to-skin distance of 100 cm, dose rate of 300 cGy / min, and a total dose of 30 Gy for a single irradiation. Before irradiation, the rats were anesthetized and shaved, the irradiation field was covered with a tissue compensation membrane, and non-irradiated areas were protected with lead plates.

[0051] The second step is the recovery period. After irradiation, rats were housed in a standard environment and their skin condition was observed. The acute radiation-induced skin damage in the irradiated area was allowed to gradually repair itself after experiencing erythema and moist desquamation. The critical time point was day 45 post-irradiation, when the rat's skin damage score dropped to 2 or below (using the scoring system shown in Table 1 of the instruction manual), no obvious skin lesions were observed visually, and the skin was in a latent state of sensitization but with a normal appearance.

[0052] The third step involved inducing a memory response through medication. Starting 46 days after irradiation, following the end of the injury recovery period, rats were administered chemotherapeutic drugs subcutaneously to induce a memory response. The chemotherapeutic drugs used were epirubicin (EPI) or 5-fluorouracil (5-FU). If epirubicin was used, the concentration was 0.05 μg / μL; if 5-fluorouracil was used, the concentration was 10 μg / μL. Administration was by subcutaneous injection once daily, with each injection consisting of 100 μL, for 7 consecutive days.

[0053] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. The rat skin lesion scores described in the examples and comparative examples were quantitatively assessed using the scoring system shown in Table 1 of the specification. Unless otherwise specified, each treatment group in the examples and comparative examples included 10 rats.

[0054] Example 1: Construction of an epirubicin (EPI)-induced RRD rat model

[0055] The first step was to prepare the experimental animals. Five-week-old male SD rats, weighing 150g±20g, were selected. The rats were housed in an environment with a temperature of 25±2℃ and a humidity of 45% to 60%, with alternating light and dark cycles for 12 hours, and free access to food and water.

[0056] The second step involved constructing a radiation-induced skin injury model. SD rats were anesthetized before irradiation, and the skin on the right leg was physically shaved and fixed to a foam board. The irradiation field was set as the right thigh and foot, covered with a 1 cm thick tissue compensation membrane. Non-irradiated areas were tightly shielded with lead plates. The right leg skin of the SD rats was irradiated using an electron linear accelerator with an energy of 5 MeV electron beams, a source-to-skin distance of 100 cm, a dose rate of 300 cGy / min, and a total single dose of 30 Gy. Post-irradiation observation revealed that erythema appeared on the rat skin on day 9, and the damage peaked on day 15, manifesting as moist desquamation, followed by gradual healing. By day 45 post-irradiation, all rats' skin injury scores had recovered to 2 points or below, with no obvious skin lesions visible to the naked eye. This was used as the induction starting point (…). Figure 1 and Figure 2 ).

[0057] The third step involved determining the inducing drug concentration. To ensure that the drug only induced a memory response without damaging normal skin, epirubicin at different concentration gradients was tested on the skin of normal rats. The results showed that at concentrations of 0.25 μg / μL and above, erythema or ulceration appeared on the rat skin; while at concentrations of 0.05 μg / μL and 0.1 μg / μL, no significant damage was observed to the rat skin. Therefore, in this embodiment, 0.05 μg / μL was selected as the inducing concentration.

[0058] The fourth step was to induce the RRD model. Starting on day 46 post-irradiation, subcutaneous injections were administered to both thighs of the rats (right side irradiated, left side unirradiated). The injected drug was 0.05 μg / μL epirubicin solution, 100 μL once daily for 7 consecutive days. Results showed that in all rats injected with the drug, erythema, dryness, desquamation, and crusting reappeared on the irradiated skin during the injection period. Figure 4 The injury score rose to 2.5 to 3.5 points; while the skin on the unirradiated side remained normal. Pathological examination showed significant thickening of the epidermis on the irradiated side, accompanied by parakeratosis and lymphocytic infiltration, consistent with the pathological features of RRD (Reduced Rye Damage). Figure 5 and Figure 6 ).

[0059] Example 2: Construction of a 5-fluorouracil (5-Fu)-induced rat model of RRD

[0060] The first and second steps are the same as in Example 1: a rat model irradiated with 30 Gy electron beams is constructed and the rats are fed until day 45 after irradiation, when the skin damage has basically recovered.

[0061] The third step involved determining the inducing drug concentration. Different concentration gradients of 5-fluorouracil were used to test the skin of normal rats. The results showed that at concentrations of 20 μg / μL and above, the rat skin exhibited dryness and desquamation; however, at concentrations of 10 μg / μL and 15 μg / μL, no skin damage was observed. Therefore, in this embodiment, 10 μg / μL was selected as the inducing concentration.

[0062] The fourth step was to induce the RRD model. Starting on day 46 post-irradiation, rats underwent subcutaneous injections into both thighs. The injected drug was a 10 μg / μL solution of 5-fluorouracil, administered once daily at a dose of 100 μL for 7 consecutive days. Observations showed that the irradiated skin exhibited typical RRD symptoms, including erythema and desquamation, while the unirradiated skin showed no significant changes. Figure 4 ). Observation was performed using HE staining and transmission electron microscopy ( Figure 5 and Figure 6 The study confirmed the presence of specific inflammatory responses and ultrastructural changes in the skin of the model group, proving the successful construction of the model.

[0063] Comparative Example 1: Construction of an epirubicin (EPI)-induced RRD rat model (by changing the induction initiation point)

[0064] The construction method was the same as in Example 1, except that the starting point was selected as day 48 after irradiation, and starting on day 49 after irradiation, the drug was injected subcutaneously into the bilateral thighs of rats with a solution of 0.05 μg / μL epirubicin.

[0065] The results showed that not all rats injected with the drug developed obvious erythema, dryness, desquamation, and crusting on the irradiated side of the skin during the injection period. A few rats injected with the drug did not develop dryness, desquamation, or crusting on the irradiated side of the skin, and pathological examination showed that the pathological features of RRD on the irradiated side were not obvious.

[0066] Comparative Example 2: Construction of an epirubicin (EPI)-induced RRD rat model (by changing the induction initiation point)

[0067] The construction method was the same as in Example 1, except that the starting point was selected as day 51 after irradiation, and starting on day 52 after irradiation, the drug was injected subcutaneously into the bilateral thighs of rats with a solution of 0.05 μg / μL epirubicin.

[0068] The results showed that most rats injected with the drug did not show obvious erythema, dryness, desquamation, or crusting on the irradiated skin during the injection period, and the damage score was ≤2.0. Pathological examination showed no obvious RRD pathological features on the irradiated side.

[0069] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for constructing a rat model of radiation-induced dermatitis, characterized in that, Includes the following steps: (1) Initial radiation damage induction: Rats were selected and the skin of the target area was irradiated with ionizing radiation once to induce acute radiation skin damage in the irradiated area; (2) Feeding during the recovery period: After the irradiation, the rats were fed in a routine manner. When the dry desquamation, wet desquamation and scabs on the irradiated area of ​​the skin completely disappeared and there were no open wounds on the skin, it was taken as the induction starting point. (3) Drug-induced memory response: Starting from the induction initiation point, the inducing drug is administered to the rats. The dosage of the inducing drug is set such that when the inducing drug is applied to the normal skin of unirradiated rats, it does not produce erythema, desquamation or ulceration, but is sufficient to induce desquamation or ulceration at the irradiated site. This can induce recurrent skin inflammation localized to the original irradiated area.

2. The construction method according to claim 1, characterized in that, The rats mentioned in step (1) are male SD rats aged 5-6 weeks; Furthermore, the target skin area is the skin of the right hind leg and / or foot of the rat.

3. The construction method according to claim 1, characterized in that, The dose of a single ionizing radiation irradiation in step (1) is 20-40 Gy, preferably 30 Gy; Furthermore, the irradiation is performed using an electron linear accelerator with an irradiation energy of 4-6 MeV, a source-to-skin distance of 80-120 cm, and a dose rate of 250-350 cGy / min.

4. The construction method according to claim 1, characterized in that, In step (1), a tissue compensation membrane of 0.5-1.5 cm thickness is covered on the skin surface of the target area during irradiation.

5. The construction method according to claim 1, characterized in that, In step (2), the skin damage score corresponding to the skin condition at the induction initiation point is ≤2.0 points; Furthermore, the induction initiation point is 45-47 days after irradiation, preferably 45 days after irradiation; Furthermore, the rat skin lesion score was quantitatively assessed using the scoring system shown in the table below:

6. The construction method according to claim 1, characterized in that, In step (3), the inducing drug is selected from one or more anthracycline antitumor drugs and antimetabolite antitumor drugs.

7. The construction method according to claim 6, characterized in that, In step (3), the absence of obvious damage includes the absence of erythema, desquamation, or ulceration.

8. The construction method according to claim 1, characterized in that, In step (3), the induction drug is administered by local subcutaneous injection, intraperitoneal injection, or intravenous injection at the irradiated site. Furthermore, the method of administration is local subcutaneous injection at the irradiated site, once a day for 5-10 consecutive days, with each injection volume being 80-120 μL; Furthermore, when the inducing drug is epirubicin, the subcutaneous injection concentration is 0.01-0.1 μg / μL; when the inducing drug is 5-fluorouracil, the subcutaneous injection concentration is 5-15 μg / μL.

9. The construction method according to claim 1, characterized in that, The construction method involves administering the drug simultaneously to both the irradiated and unirradiated sides of the same rat, using a self-control to eliminate the toxicity interference of the drug itself. Furthermore, the construction method also includes confirming the induction of radiation-induced dermatitis by observing whether erythema reappears on the irradiated skin after the administration of the inducing drug, accompanied by dry desquamation, crusting or epidermal scarring, while the skin on the unirradiated side of the same body remains normal.

10. The use of a rat model of radiation memory dermatitis obtained by the construction method according to any one of claims 1-9 in screening drugs for the prevention or treatment of radiation memory dermatitis.

Images