A method for constructing an animal model of pressure sores and application thereof

Abstract

The application provides a pressure sore animal model construction method, which comprises the following steps: step 1, feeding the modeling rats according to a standard feeding mode; step 2, constructing an acute spinal cord injury model; step 3, constructing a pressure sore model; and step 4, confirming that the animal model construction is successful. The pressure sore animal model obtained by the construction method can be used in the fields of evaluating the risk of pressure sore occurrence, the molecular mechanism of pressure sore occurrence and development, or preparing and / or screening drugs for preventing and / or treating skin pressure sores.

Description

Technical Field

[0001] This invention relates to an animal model, specifically, to a method for constructing a pressure ulcer animal model and its application. Background Technology

[0002] Pressure ulcers (PUs) are a global public health problem in the context of an aging population. Early-stage PUs are characterized by high incidence, superficial lesions, difficulty in early detection, and challenges in differential diagnosis. However, early detection, early diagnosis, early prevention, and early treatment are key to PU management. Constructing animal models plays a crucial role in pathological research on pressure ulcers, evaluation of pressure ulcer drugs, and optimization of pressure ulcer treatment regimens. Currently, there are many methods for constructing pressure ulcer animal models. For example, Chinese patent CN122478658A discloses a method for constructing a mouse composite pressure ulcer model and its application, and Chinese patent CN115812675A discloses a method for constructing an animal model of skin infectious pressure ulcers and its application. However, these pressure ulcer animal models all involve lifting the skin on both sides of the mouse's back into a strip, placing magnets symmetrically on both sides to clamp the strip of skin, maintaining this position for a period of time, and then removing the magnets. The pressure ulcer model is constructed by compression. However, a recent review study on the incidence of pressure injuries in patients with spinal cord injury (title: Incidence of pressure injuries in patients with spinal cord injury: A systematic review and meta-analysis, [J]. Journal of Tissue Viability, 2025, 34(2): 100881.) shows that the incidence of pressure ulcers in patients with spinal cord injury can be as high as 28.8%. Therefore, there is a need to develop a new animal model of pressure ulcers that can be used to study spinal cord injury. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for constructing an animal model of pressure ulcers and its application.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for constructing an animal model of pressure ulcers, comprising the following steps: Step 1: Feed the modeling rats according to standard feeding methods; Step 2: Perform anesthesia, hair removal, and exposure of spinal cord segments in sequence, and then construct an acute spinal cord injury model using the gravity fall method; Step 3: After acclimatizing the model mice, a pressure ulcer model was constructed using a G-clamp fixator and a standardized ischemia-reperfusion (I / R) circulation model. Step 4: Confirm the successful establishment of the animal model by visually observing the morphology and color changes of the skin at the pressure site of the rat, as well as the integrity of the skin and histological observation.

[0005] In step 1, healthy SPF-grade adult female rats weighing 240-350 g were used for modeling.

[0006] In step 1, rats were numbered with unique identifiers and randomly assigned to 7 groups using a random number table. The rats were randomly divided into a control group and an experimental group. The experimental group was further divided into: acute spinal cord injury group, erythema group, non-pale erythema / stage 1 pressure ulcer group, stage 2 group, stage 3 group, and stage 4 group. After grouping, the weight, activity level, etc. of each group were compared to ensure that there were no significant differences.

[0007] In step 2, after anesthetizing the modeling rats, the hair around the T7 spinous process was removed. The rats were then fixed in a standard prone position on the surgical platform. The area from the occipital protuberance to the tip of the sacrum was disinfected, and a sterile surgical drape was placed. Using a sterile scalpel, a longitudinal incision was made along the midline of the back, centered on the T7 spinous process. The subcutaneous connective tissue was cut, and the muscles on both sides of the spinous process were dissected until the lamina was exposed. A spinal retractor was used to fully expose the T6-T8 spinous processes and the bilateral lamina structures. The interspinous ligaments of T6 / 7 and T7 / 8 were cut. The T7 spinous process was completely removed with bone forceps, and the bilateral lamina were excised to expose the spinal cord segments.

[0008] In step 2, an orthopedic Kirschner wire is dropped vertically along the transparent tube and inserted vertically into the spinal canal, ensuring that the dura mater is not compressed or torn. The needle tip reaches the bottom of the spinal canal and hits the spinal cord, causing edema and congestion. The local swelling height is ≥ 1 mm, and the parenchymal congestion changes to dark red or purplish red. After observing for 1 minute to confirm no cerebrospinal fluid leakage, apply strict pressure for 3 minutes until no active bleeding occurs. Rinse the incision with sterile saline, suture the muscle layer intermittently first, then suture the skin layer continuously, apply povidone-iodine to the wound and cover with sterile gauze, and wait for the rat to awaken. Record the operation time, blood loss and incision length throughout the process. After the operation, administer intraperitoneal fluid resuscitation with saline, place the rat in a constant temperature individual incubator for recovery, and then house it in a single cage. Observe the animal's walking patterns daily, including the movement of the hip, knee and ankle joints, trunk movements and coordination; turn the animal over regularly, massage its abdomen each time it is turned over, and massage its bladder regularly to promote urination and functional recovery and prevent urinary retention; Using a temporal neurological function assessment protocol, the successful establishment of an acute spinal cord injury model was confirmed.

[0009] In step 3, after the skin of the buttocks of rats in the acute spinal cord injury group was prepared, they were placed on an electronic scale and a G-type clamp fixator in sequence according to the tail number. The same pressure was applied to the posterior thigh area of ​​the paralyzed hind limb to cause local ischemia. After 2 hours, the fixators were removed in sequence to allow the ischemic skin to be reperfused. The rats were then put back into the cage and allowed to move freely for 0.5 hours. In a standardized ischemia-reperfusion (I / R) cycle model, each cycle contains two precisely controlled time phases: an ischemic phase (I phase) lasting 2.0 hours, followed by a reperfusion phase (R phase) lasting 0.5 hours.

[0010] The anesthetic is at least one of pentobarbital, isoflurane, tribromoethanol, ketamine, toluenethiazide, and acepromazine.

[0011] The application of an animal model of pressure ulcers constructed by the above method in assessing the risk of pressure ulcer occurrence, the molecular mechanisms of pressure ulcer development, or in the preparation and / or screening of drugs for the prevention and / or treatment of skin pressure ulcers, or in optimizing surgical treatment plans.

[0012] This invention represents a significant advancement and substantial improvement over existing technologies. Specifically, it provides a method for constructing an animal model of pressure ulcers. Compared to traditional methods that directly compress pressure ulcers, this invention first constructs an acute spinal cord injury model, and then constructs a pressure ulcer model, thus establishing an animal model of pressure ulcers combined with spinal cord injury. The pressure ulcer animal model obtained through this method can be used to assess the risk of pressure ulcer occurrence, the molecular mechanisms of pressure ulcer development, or to prepare and / or screen drugs for the prevention and / or treatment of skin pressure ulcers. Attached Figure Description

[0013] Figure 1 Images depicting the process of establishing the PUs animal model. A) Establishment of the acute spinal cord injury (SCI) rat model using the gravity fall method; B) Intraoperative positioning of the SCI rat model; C) Postoperative anesthesia and recovery of the SCI rat model; D) Schematic diagram of the PUs animal model establishment; E) PUs animal model establishment; F) Motor gait and local clinical manifestations after successful PUs animal model establishment.

[0014] Figure 2 Images showing the skin and muscle tissue and morphological characteristics of PUs.

[0015] Figure 3 This is a line graph showing the changes in body weight (g) of rats in each group before and after modeling.

[0016] Figure 4Distribution trends of blood biomarkers in different groups of PUs animal models. From left to right in each figure: Group 1: blank group, Group 2: SCI group, Group 3: pre-PUs (BE) stage, Group 4: PUs stage 1, Group 5: PUs stage 2, Group 6: PUs stage 3, Group 7: PUs stage 4.

[0017] Figure 5 ROC curves for predicting PUs using blood biomarkers in animal models.

[0018] Figure 6 Diagram illustrating the diagnostic staging of PUs. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below through specific implementation methods. The experimental animals used in the following experiments were provided by the Experimental Animal Center of Gansu University of Traditional Chinese Medicine [Experimental Animal Use License No.: SYXK (Gan) 2015-0005] and all animal experimental operations were completed. The experimental process strictly followed the following standards: (1) Ethical Guidelines of the International Committee for Assessment and Accreditation of Laboratory Animals (AAALAC); (2) China's "Guidelines for Ethical Review of Laboratory Animal Welfare"; (3) Operating procedures developed by the Institutional Animal Care and Use Committee (IACUC). Example 1

[0020] This embodiment provides a method for constructing an animal model of pressure ulcers, which includes the following steps: Step 1 Fifty-six healthy SPF-grade adult female rats, weighing between 240-350 g, were routinely housed in quiet environments in separate cages. This strain is docile and easy to handle, has a highly stable genetic background, and its spinal cord anatomy (e.g., gray and white matter distribution) and nerve conduction pathways share functional homology with the human spinal cord. A 12-hour light-to-dark cycle (8:00-20:00) was maintained, avoiding direct sunlight. The room temperature was maintained at (25.0 ± 2.0)℃, with temperature fluctuations not exceeding ± 1℃. Humidity was controlled at (50 ± 10)%, and a humidity sensor was used for real-time monitoring. Animals had free access to water (sterilized through a 0.22 μm filter) and were provided with SPF and rodent-specific feed (protein content ≥ 20%). Wood shavings were changed three times a week. Animals were allowed one week to acclimatize to the new environment before the experiment began.

[0021] Rats were numbered with unique identifiers (tail tags) and randomly assigned to 7 groups using a random number table. The rats were randomly divided into a control group and an experimental group. The experimental group was further divided into four groups: acute spinal cord injury (SCI), erythema (BE), non-pale erythema / stage 1 pressure ulcer (NBE), stage 2, stage 3, and stage 4, with 8 rats in each group. After grouping, the weight, activity level, etc. of each group were compared to ensure that there were no significant differences.

[0022] Step 2: Construction of an acute spinal cord injury (SCI) model 1) After one week of acclimatization feeding, rats in both the control and experimental groups were anesthetized by intraperitoneal injection of 3% pentobarbital (30 mg / kg intraperitoneally, injection rate ≤ 0.5 ml / min). Depth of anesthesia was determined by the disappearance of corneal reflex (no blinking response when lightly touching the eyeball with a cotton swab) and relaxation of hind limb muscles (no resistance when pulled) as the signs of successful anesthesia.

[0023] 2) After the experimental animals are anesthetized, surgical preparation is performed according to the following standardized operating procedures: Use an electric shaver to precisely remove hair around the spinous process of the T7 vertebra (5×4cm). 2 The area's hair fully exposes the surgical field; Rats were secured in a standard prone position on a customized surgical platform, with limb straps and a head immobilizer ensuring stability. This procedure strictly adhered to aseptic surgical protocols, with particular attention paid to maintaining visualization of spinal anatomical landmarks and adequate exposure of the surgical area (e.g., ...). Figure 1 (As shown in B).

[0024] 3) All surgical instruments were provided by the Gansu Provincial People's Hospital and the Animal Experiment Center Laboratory of Gansu University of Traditional Chinese Medicine. The scalpel used was a No. 11 pointed blade, the micro forceps had a tip diameter of 0.1 mm, the hemostat was both straight and curved, and the micro scissors had a blade length of 1.5 mm.

[0025] 4) Before starting the surgery, use toe or tail pressure to confirm the appropriate level of anesthesia. Disinfect the surgical area with povidone-iodine in a spiral motion three times (2 minutes apart), from the occipital protuberance to the tip of the sacrum, then place a sterile surgical drape. Using a sterile scalpel, make a longitudinal incision approximately 3 cm long along the midline of the back, centered on the T7 spinous process. Gently incise the subcutaneous connective tissue using small ophthalmic surgical instruments, dissecting the muscles on both sides of the spinous process until the lamina is exposed. If bleeding occurs, apply pressure with gauze pads or ligate the blood vessels before continuing the procedure. The intraoperative anesthesia maintenance protocol involves administering 1 / 3 of the initial dose every 40 minutes, maintaining a respiratory rate of (16-20) breaths / minute.

[0026] 5) Using a spinal retractor, fully expose the spinous processes of T6-T8 and the bilateral laminae. Carefully sever the interspinous ligaments of T6 / 7 and T7 / 8, and completely remove the T7 spinous process with bone forceps. Use a curette to progressively and precisely remove both laminae. Continuously irrigate the surgical field with saline throughout the procedure to maintain a clear view and reduce the risk of thermal injury. The entire surgical procedure strictly adheres to microsurgical neurosurgical protocols, paying particular attention to protecting the integrity of the dura mater and nerve root structures. Blunt dissect along the surface of the dura mater to expose a spinal cord segment approximately 3 mm in length. Throughout the procedure, ensure the dura mater remains intact while avoiding any compression of the spinal cord.

[0027] 6) When creating the injury model, the gravity drop method was used: An orthopedic Kirschner wire was dropped vertically 10 cm from a height along a transparent tube, inserted vertically into the spinal canal, ensuring that the dura mater was not compressed or torn. The wire tip must reach the bottom of the spinal canal to ensure impact on the spinal cord, causing edema and congestion, with significant local swelling (swelling height ≥ 1 mm), and the parenchymal congestion appearing dark red or purplish-red (see details). Figure 1 A). Observe for 1 minute to confirm no cerebrospinal fluid leakage, then apply strict pressure for 3 minutes until no active bleeding occurs. Rinse the incision with sterile saline, and use 4-0 sutures to intermittently suture the muscle layer first, then continuously suture the skin layer. Apply iodine to the wound and cover with sterile gauze, waiting for the rat to awaken. Record the entire process, including surgical time, blood loss, and incision length. Postoperatively, administer 5 ml of saline intraperitoneally for rehydration, and place the rat in a room-temperature incubator for recovery. Figure 1 C), individual cage rearing, recording respiratory rate and hind limb muscle strength every 15 minutes.

[0028] 7) Observe the animal's walking patterns daily, including the movement of the hip, knee, and ankle joints, trunk movements, and coordination. Turn the animal over every 8 hours, massaging its abdomen each time, and perform a bladder massage every 6 hours to promote urination and functional recovery, and prevent urinary retention.

[0029] 8) Behavioral observation: A time-series neurological function assessment protocol was adopted.

[0030] The specific procedures are as follows: ① Assessment time points: three key time windows: 24h, 48h, and 72h post-surgery; ② Assessment environment: a standardized open testing box with a 5cm thick sterile pad at the bottom; ③ Behavioral observation: the rat was gently placed in the center of the testing box; the box wall was tapped three times at a fixed frequency (1 time / second); hind limb motor function parameters were recorded using a double-blind method, including joint range of motion, weight-bearing capacity, and coordination; ④ Scoring criteria: standardized scoring was performed using the BBB (Basso, Beattie, Bresnahan) scale. The entire assessment process was conducted in a quiet environment with constant lighting (100 lux) to minimize external interference. A score below 4 at 8 hours post-surgery was considered an indicator of successful model establishment. Figure 1 F).

[0031] Animal selection criteria were used to ensure the reliability of experimental data: Individuals with a BBB motor function score below 21 (out of 21) were excluded preoperatively to ensure all enrolled rats had normal baseline motor function; intraoperative monitoring was conducted to exclude individuals experiencing dural injury or accidental death; postoperatively, dual quality control was implemented in the spinal cord injury model group, excluding individuals with a BBB score ≥4 within 8 hours (indicating insufficient injury) and cases of surgical site infection or death; simultaneously, the control group strictly excluded individuals with a BBB score <21 (due to congenital loss of motor function, etc.); during the subsequent observation period, data cleaning was performed using the median absolute deviation method (MAD) to remove outliers in each group whose BBB scores deviated from the median by more than 1.5 times. This standard system implemented comprehensive quality control from preoperative screening and intraoperative monitoring to postoperative follow-up, ensuring the scientific validity and reliability of the experimental results. Step 3: Construction of Pressure Ulcer (PU) Model 1) After the SCI animal model is successfully established, the model mice are acclimatized for 3 days and wait for the anesthetic to be metabolized before PUs modeling is performed. During this period, the surgical incision on the back of the rat is disinfected with povidone-iodine daily to avoid secondary infection of the incision.

[0032] 2) After the skin of the buttocks of the experimental group SCI rats was prepared, they were placed on the electronic scale and G-type clamp fixation device in sequence according to the tail number. The same pressure was applied to the posterior thigh area of ​​the paralyzed hind limb to cause local ischemia. After 2 hours, the fixation device was removed in sequence to allow the ischemic skin to be reperfused. The rats were then put back into the cage and allowed to move freely for 0.5 hours.

[0033] A standardized ischemia-reperfusion (I / R) cycle model was adopted, with each cycle containing two precisely controlled time phases: an ischemic phase (I phase) lasting 2.0 hours, followed by a reperfusion phase (R phase) of 0.5 hours. This cycle design was optimized based on literature and preliminary experimental results and can simulate the pathophysiological process of clinically relevant ischemia-reperfusion injury.

[0034] 3) Place each animal on a digital platform electronic scale (Kaifeng high-precision commercial electronic scale, 10kg / 0.5g) and weigh its tare weight to assess the quantity of load pressure. Calculate the pressure using the following formula:

[0035] Because capillary blood flow may be reduced or stopped when external pressure exceeds 30 mmHg-50 mmHg (approximately 4 kPa-6.7 kPa), the load pressure in the Stadler study was 250 mmHg-1000 mmHg (pressure gradient caused continuous ischemia leading to pressure ulcers of different stages). However, this example is an ischemia-reperfusion injury model, so a pressure of 500 mmHg was selected. The G-type clamp used was in contact with a circular metal plate (2 cm in diameter) on the animal's buttocks, and the load pressure of the electronic scale was approximately 8.52 kg.

[0036] After each cycle, assess the tissues exposed to pressure for pressure sores. Record any skin breaks. After successful modeling, photograph local tissues of each animal model before euthanasia. Specific modeling methods are shown in Table 1. Figure 1 D and Figure 1 E.

[0037] Table 1. Modeling methods for different stages / classes of PUs animal models

[0038] Note: Pressure-related blanchable erythema (BE) is a non-pressure ulcer caused by prolonged external pressure, resulting in localized capillary obstruction. Its clinical presentation on visual assessment is similar to that of a stage 1 pressure ulcer. It is abbreviated as BE stage.

[0039] Step 4: Criteria for Judging Model Validity (1) Visual observation The morphological and color changes of the skin at the pressure site in rats, as well as the integrity of the skin, are detailed in [the document / reference needed]. Figure 2 A. Currently, there is no unified international naming convention for the grading / classification of PUs. The most widely used classifications are roughly the following three categories, as detailed below. Figure 6 This study used the diagnosis and staging of pressure ulcers from the Rapid Reference Guide for the Prevention and Treatment of Pressure Ulcers, a system first proposed in 1989 and last revised in 2019.

[0040] (2) Histological observation - HE staining After successful modeling, rats were anesthetized and euthanized. Skin tissue from the wound and surrounding 1 cm³ was collected, fixed in 4% paraformaldehyde, prepared into paraffin sections, stained with hematoxylin and eosin (HE), and observed under a light microscope. Panoramic slide scanner: 3DHISTECH (Hungary), PANNORAMIC MIDI / SCAN 150; upright white light microscope: Nikon (Japan), ECLIPSE CI-L. It was observed that as the PUs stage progressed, rat muscle fibers, myocytes, and fat exhibited regular changes, as detailed in the following section. Figure 2 Among them, the PUs4 stage rats and humans showed similar morphology, indicating that the animal model was successful: the overall muscle tissue structure was slightly abnormal, with a small amount of muscle fiber degeneration and necrosis, and a small amount of inflammatory cell infiltration in the necrotic areas (see details). Figure 2 B).

[0041] from Figure 2 B. Analysis of organizational morphology: 1) Control group (normal animals): The overall muscle tissue structure is basically normal. The muscle fibers are arranged relatively regularly and are of relatively uniform size. The cross striations of the muscle fibers are clear, and the cell nuclei are located at the edge of the muscle fibers. The yellow arrows in the figure indicate the muscle fibers. No obvious inflammatory cell infiltration is observed in the tissue. 2) SCI animals: The overall muscle tissue structure is basically normal. The muscle fibers are arranged relatively regularly and are of relatively uniform size. The cross striations of the muscle fibers are clear, and the cell nuclei are located at the edge of the muscle fibers. The yellow arrows in the figure indicate the muscle fibers. No obvious inflammatory cell infiltration is observed in the tissue. 3) BE stage (pre-PUs stage): The overall muscle fiber structure is slightly abnormal. Some muscle fibers are twisted and wavy. The muscle fibers are loosely arranged with significantly increased gaps, as shown by the green arrows in the figure. No obvious inflammatory cell infiltration is observed in the tissue. 4) PUs1 stage animals: The overall muscle tissue structure is slightly abnormal. The muscle fibers are arranged disorderedly, and the outlines of the muscle fibers are unclear. Some muscle fibers are swollen and broken, as shown by the yellow arrows in the figure. A small amount of inflammatory cell infiltration is observed in the tissue, as shown by the black arrows in the figure. 5) PUs2 stage animals: The overall muscle tissue structure is abnormal, with disordered arrangement of muscle fibers. Numerous muscle fibers show swelling and vacuolar degeneration, and large areas of muscle fiber dissolution and disappearance, as shown by the yellow arrows in the figure. Myofibroblast cell nuclei are largely absent, and no obvious inflammatory cell infiltration is observed. 6) PUs3 stage animals: The overall muscle tissue structure is abnormal, with disordered arrangement of muscle fibers. Numerous muscle fibers show swelling and vacuolar degeneration, and large areas of muscle fiber dissolution and disappearance, as shown by the yellow arrows in the figure. Myofibroblast cell nuclei are largely absent, and no obvious inflammatory cell infiltration is observed. 7) PUs4 stage animals: The overall muscle tissue structure is slightly abnormal. The muscle fibers are regularly arranged and relatively uniform in size, with clear muscle fiber striations. A small number of muscle fibers show degeneration and necrosis, as shown by the green arrows in the figure. A small number of inflammatory cells are visible in the necrotic areas, as shown by the black arrows in the figure. 8) PUs4 stage human body: The overall structure of muscle tissue is abnormal, the arrangement of muscle fibers is disordered, a large number of muscle fibers are swollen and vacuolated, and a large area of ​​muscle fibers are dissolved and disappeared, as shown by the yellow arrow in the figure. The tissue basically does not have muscle fiber cell nuclei, and a small number of inflammatory cells are infiltrated in the tissue, as shown by the black arrow in the figure.

[0042] In both animals and humans at stage PUs4, muscle tissue exhibited significant structural abnormalities and muscle fiber damage. Common features included disordered muscle fiber arrangement (mild in animals, significant in humans), varying degrees of muscle fiber degeneration and necrosis (few focal cases in animals, extensive dissolution in humans), inflammatory cell infiltration (visible in both but to a lesser extent), and disruption of the muscle fiber striations (striations were still clear in animals, severe vacuolation in humans). Inflammatory response and degenerative changes in muscle fibers were core pathological features shared by both, but the damage in human tissue was more extensive and severe, manifested as the disappearance of muscle fiber cell nuclei and extensive dissolution.

[0043] Example 2 This embodiment provides a method for applying the pressure ulcer animal model obtained in Example 1, which includes the following steps: 1. Specimen Collection and Processing (1) Arterial blood sample collection and storage: Blood samples were collected after successful model establishment. The specific collection method was as follows: After anesthetizing the rats with 3% sodium pentobarbital via intraperitoneal injection, they were fixed in a supine position on the operating table. Strict aseptic operation procedures were followed: the surgical area was shaved and disinfected three times with 5% povidone-iodine solution; a modified abdominal aortic puncture technique was used: the operator accurately pressed the proximal end of the abdominal aorta (1 cm below the xiphoid process) with the index finger of the left hand, and punctured the bifurcation of the abdominal aorta at a 45° angle with the right hand with a 22 G blood collection needle. After blood return was observed, a 5 ml vacuum blood collection tube (without anticoagulant) was connected; if the blood collection was insufficient, the assistant performed a Valsalva-like operation (raising the upper body 30° and pressing the chest cavity), which could increase the blood collection volume by 15%-20%. The entire operation should be completed within 2 minutes to ensure blood quality. After collection, avoid direct sunlight and let stand at room temperature for two hours. Then, centrifuge the venous blood at 3000 rpm for 15-20 min at low temperature. Aliquot the supernatant serum into 0.5 ml EDTA anticoagulant EP tubes, label them, and freeze them in liquid nitrogen. Place them in a -80 ℃ freezer within 2 hours for testing.

[0044] (2) Collection and storage of muscle tissue specimens: After the experimental group rats were modeled, a circular skin tissue with a diameter of about 12 mm was cut from one side of the symmetrical wound, including the wound edge. If there was blood or foreign matter on the tissue, it was rinsed with pre-cooled PBS buffer or physiological saline, and the water was dried with clean, dust-free absorbent paper. About 100 mg / tube was dispensed into centrifuge tubes, weighed, and then dispensed into cryovials and labeled. The tubes were then quickly frozen in liquid nitrogen and stored at -80℃ for testing.

[0045] 2. Experimental conditions and quality control Complete blood count and biochemical assays: Standardized venous blood collection procedures were followed, using a vacuum blood collection tube system (red additive-free tubes and purple EDTA anticoagulant tubes) to collect whole blood samples. The collected blood samples were immediately placed in a 4°C centrifuge at 3500 r / min for 10 minutes. After centrifugation, clear three-phase separation was observed. The upper pale yellow serum layer was carefully aspirated using a sterile pipette and aliquoted into 1.5 ml EP tubes, then stored at -80°C for later use. This procedure strictly followed clinical laboratory specimen handling standards to ensure that the serum sample quality met the requirements for subsequent testing. Analytical blood tests were performed using a fully automated blood analysis system.

[0046] 3. Statistical Analysis Repeated measures ANOVA was used to analyze the changes in body weight of rats in each group using SPSS 26.0. One-way ANOVA was used to compare the differences in biochemical / blood routine indicators among the seven groups of rats. Graphs were generated using Origin Pro 2021.

[0047] 4. Results of blood biomarker studies in PUs animal models 1) Changes in body weight in PUs animal models Repeated measures ANOVA and Mauchly's test of sphericity were used (W = 0.036, P < 0.05). There was no significant difference in body weight among the groups (F = 2.222, P > 0.05). However, the body weight of rats in each group gradually decreased over time (F = 42.588, P < 0.001, partial variance). 2 = 0.838), see details Figure 3 .

[0048] 2) Differential analysis of blood biomarkers One-way ANOVA was used to compare differences in continuous biochemical / blood routine indicators in animal models. Data are shown in Tables 2-3. If the data did not meet the requirements of normality or homogeneity of variance (Shapiro-Wilk test, P<0.05 or Levene test, P<0.05), the Kruskal-Wallis test was used instead. After ANOVA or Kruskal-Wallis was significant, pairwise comparisons were performed using the Tukey HSD or Dunn-Bonferroni post-hoc test. Data are presented as mean ± standard deviation (normal distribution) or median (interquartile range) [non-normal distribution]. The significance level was set at α = 0.05, and the effect size was expressed as partial η. 2 (ANOVA) or Cohen's d (pairwise comparison) is used.

[0049] Table 2. Comparison of differences in blood biomarkers among seven groups of rats (normal distribution)

[0050]

[0051]

[0052]

[0053] Note: *P<0.05, **P<0.01; Group 1: Blank group, Group 2: SCI group, Group 3: PUs pre-phase (BE phase), Group 4: PUs phase 1, Group 5: PUs phase 2, Group 6: PUs phase 3, Group 7: PUs phase 4.

[0054] Some indicators do not meet the normality test, and the data are presented as median (interquartile range) [non-normal], as shown in Table 3.

[0055] Table 3. Comparison of differences in blood biomarkers among seven groups of rats (non-normal distribution)

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] Note: *P<0.05, **P<0.01; Group 1: Blank group, Group 2: SCI group, Group 3: Pre-PUs (BE period), Group 4: PUs1 period, Group 5: PUs2 period, Group 6: PUs3 period, Group 7: PUs4 period.

[0062] Blood routine and biochemical tests of different groups of PUs animal models revealed significant differences among the groups in 11 biomarkers, including lymphocyte percentage, neutrophil percentage, monocyte count and percentage, platelet volume, proportion of large platelets, blood urea nitrogen to creatinine ratio, glucose, aspartate aminotransferase, total protein, and globulin. Figure 4 Box plots are used to show the distribution trends of each indicator in different groups of animal models.

[0063] Differences in blood biomarkers in animal models were analyzed, and the distribution trends were analyzed using multiple comparisons and box plots. See Tables 2-3 for details. Figure 4 ,Discover: (1) Percentage of lymphocytes: This marker decreases after PUs occur, but increases again as the damage deepens (P<0.01). (2) Neutrophil percentage: This marker increased in rats after the onset of stage 1 PUs (P<0.05). (3) Monocyte count and percentage: This marker increases after PUs occur, but decreases as the damage worsens (P<0.01). (4) Platelet volume and the proportion of large platelets: After PUs occur, these two markers will decrease, but as the degree of damage increases, the levels will increase again (P<0.01). (5) Blood urea nitrogen to creatinine ratio: This marker increases after PUs occur and further increases from the original level as the degree of damage deepens (P<0.01). (6) Glucose: This marker will rise after PUs occur, but the level will decrease slightly as the damage deepens (P<0.05). (7) Aspartate aminotransferase: The level of this marker increased in the stage 4 PUs animal model compared with that in stage 1 (P<0.01). (8) Total protein: The levels of this marker in BE, 1 and 4 stage PUs animal models were lower than those in normal rats (P<0.01). (9) Globulin: This marker increases after PUs occur and increases further as the degree of damage deepens (P<0.01).

[0064] 3) Predictive value of blood biomarkers The dependent variable was whether PUs occurred (yes = 1, no = 0), where PUs occurred in stages 1-4, and did not occur in the blank group, SCI group, and BE group. The variable selection method was forward. Based on the univariate analysis results of the factors related to the occurrence of PUs in the animal model, factors with P < 0.05 were included. All of them were continuous variables. The original values ​​of each variable were used to perform binary logistic regression analysis.

[0065] The results showed that among the 11 risk factors that showed differences in univariate analysis, NEU%, MON%, GLU, and BUN / Cr were independent risk factors for PUs in the animal model. A PUs prediction model in the animal model was constructed.

[0066] This is named the prediction model. See Table 4 for details. Figure 5 .

[0067] Table 4. Blood-related influencing factors of PU occurrence in animal models

[0068] Depend on Figure 5The AUC of the PUs prediction model in the animal model was 0.928 (95% CI: 0.846-1.000), indicating that the model has a very strong predictive ability for PUs and has significant clinical application value. However, due to the limited sample size, further verification of sample independence and avoidance of overfitting are needed.

[0069] Based on the analysis of differential results of blood biomarkers in animal models, combined with multicenter clinical analysis and the pathogenesis of pressure ulcers, the above 11 biomarkers were divided into 4 categories.

[0070] 1. Biomarkers related to inflammation and immune response (1) Lymphocyte percentage: LYM% is an important indicator for assessing the body's immune status. The blood analysis studies of the above-mentioned animal models found that its decrease may be closely related to the occurrence and development of pressure ulcers. Pressure ulcer patients often have suppressed immune function due to prolonged bed rest, malnutrition, or chronic inflammation, which manifests as a decrease in lymphocytes.

[0071] Studies have shown that low LYM% can reflect impaired T cell and B cell function, affecting the cellular and humoral immune responses required for wound healing. In addition, chronic inflammatory states (such as pressure ulcers complicated by infection) may promote the release of pro-inflammatory cytokines (such as TNF-α and IL-6), further inhibiting lymphocyte proliferation and forming a vicious cycle.

[0072] Clinical observations have revealed that the LYM% (experienced ulcer percentage) in pressure ulcer patients is often lower than that in healthy individuals, and is negatively correlated with the severity of pressure ulcers, consistent with the findings of the aforementioned study. Therefore, monitoring LYM% helps assess a patient's immune status and risk of pressure ulcer progression, and provides a basis for immunomodulatory therapies (such as nutritional support and anti-inflammatory interventions).

[0073] (2) Neutrophil percentage: Elevated NEU% is a typical marker of acute inflammation or infection and has important clinical significance in the development of pressure ulcers. Pressure ulcer patients often experience neutrophil infiltration due to local tissue ischemia, necrosis, and secondary infection, resulting in a significantly increased NEU% percentage. In addition, neutrophils participate in the inflammatory response by releasing reactive oxygen species (ROS) and proteolytic enzymes (such as elastase), but excessive activation may lead to tissue damage and delay wound healing. Studies show that when pressure ulcers are complicated by bacterial infections (such as Staphylococcus aureus and Pseudomonas aeruginosa), NEU% further increases and is positively correlated with inflammatory markers such as C-reactive protein (CRP). A persistently elevated NEU% indicates an increased risk of pressure ulcer infection, requiring timely anti-infective treatment. Therefore, dynamic monitoring of NEU% helps assess the degree of inflammation and infection risk of pressure ulcers and guides clinical intervention.

[0074] (3) Monocyte count and percentage: MON# and MON% play a dual role in the chronic inflammation and tissue repair process of pressure ulcers. Monocytes can migrate to the injury site and differentiate into macrophages, participating in inflammation regulation and wound repair. In the early stages of pressure ulcers, monocyte infiltration helps clear necrotic tissue and promotes angiogenesis. However, long-term chronic inflammation may lead to monocyte phenotypic polarization (e.g., M1 pro-inflammatory macrophages predominate), exacerbating tissue damage. Clinical studies have found that MON% is often slightly elevated in patients with pressure ulcers and is associated with delayed wound healing; in addition, monocyte-derived cytokines (such as IL-1β and TGF-β) may affect collagen deposition and scar formation. Therefore, MON# and MON% can serve as indicators for assessing the chronic inflammatory status and repair potential of pressure ulcers and provide a basis for targeted immunomodulatory therapy.

[0075] 2. Biomarkers related to coagulation and microcirculation disorders (1) Platelet volume: MPV is an indicator of platelet size, and its changes are closely related to the occurrence, development, and prognosis of pressure ulcers. Increased MPV usually reflects enhanced platelet activation and an accelerated rate of young platelet production by bone marrow megakaryocytes. These large platelets have higher procoagulant activity and the ability to release inflammatory mediators. In patients with pressure ulcers, prolonged immobilization, local ischemia-reperfusion injury, and chronic inflammation can lead to platelet activation, manifested as elevated MPV. Activated platelets promote local inflammatory response and microthrombus formation by releasing cytokines such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), further aggravating tissue ischemia and necrosis. In addition, elevated MPV is associated with endothelial dysfunction, which may affect microcirculation perfusion in the pressure ulcer area and delay wound healing. The above studies show that the MPV value of pressure ulcer animals is significantly higher than that of healthy controls, and the MPV level is positively correlated with the severity of pressure ulcers. Elevated MPV may also indicate an increased risk of pressure ulcer infection, as bacterial toxins and inflammatory factors (such as IL-6 and TNF-α) can further stimulate platelet activation. Furthermore, dynamic changes in MPV may serve as a potential biomarker for assessing the effectiveness of pressure ulcer treatment; for example, effective anti-inflammatory and anticoagulant therapy can reduce MPV and improve tissue repair. Therefore, MPV is not only an important participant in the pathophysiological process of pressure ulcers but may also be a useful indicator for predicting pressure ulcer progression and prognosis.

[0076] (2) Proportion of large platelets: P-LCR refers to the percentage of platelets in the blood with a volume greater than 12 fL. Its elevation is usually associated with platelet activation, hypercoagulable state and enhanced inflammatory response. In patients with chronic wounds, an elevation of P-LCR may reflect an accelerated rate of large platelet production by bone marrow megakaryocytes, which have stronger procoagulant and pro-inflammatory properties. Large platelets are rich in dense granules and α-granules, which can release a large number of procoagulant substances (such as thromboxane A2 and P-selectin) and inflammatory mediators (such as CD40 ligands), promoting local microthrombus formation and inflammatory response

[142] . The pathophysiological process of pressure ulcers involves ischemia-reperfusion injury caused by long-term pressure, chronic inflammation and microcirculatory disturbances, and an elevation of P-LCR may exacerbate these pathological changes.

[0077] The aforementioned studies found that P-LCR levels in patients with pressure ulcers were significantly higher than in healthy individuals, and were positively correlated with the severity of pressure ulcers (such as deep tissue damage or unstageable pressure ulcers). Furthermore, elevated P-LCR may indicate an increased risk of deep vein thrombosis (DVT) associated with pressure ulcers, as large platelets are more likely to adhere to damaged endothelial cells and form thrombi [144-145]. Dynamic monitoring of P-LCR may be helpful in assessing the effectiveness of pressure ulcer treatment. For example, antiplatelet therapy (such as aspirin or clopidogrel) can reduce P-LCR and improve local blood perfusion. In addition, the correlation between P-LCR and inflammatory markers (such as CRP and IL-6) suggests that it may serve as a surrogate indicator of the inflammatory burden of pressure ulcers. Therefore, P-LCR is not only a biomarker of hypercoagulable state and inflammatory response in pressure ulcers, but may also serve as a reference indicator for individualized treatment strategies.

[0078] 3. Nutrition and metabolism-related biomarkers (1) Total protein: Total protein (TP) is an important indicator reflecting the body's nutritional status and protein metabolism. Its decreased level is significantly associated with the occurrence, severity, and poor prognosis of pressure ulcers. Hypoproteinemia (TP < 6.0 g / dL) can lead to a decrease in plasma colloid osmotic pressure, causing tissue edema, which in turn aggravates local microcirculatory disturbances and ischemic damage. Protein is the basic raw material for tissue repair. Its deficiency will hinder collagen synthesis, fibroblast proliferation, and angiogenesis, thus delaying pressure ulcer healing. Clinical studies have shown that TP levels are also positively correlated with Braden scores. Patients with TP < 5.5 g / dL have a 3.2 times higher incidence of pressure ulcers than the normal population. In addition, low TP status is often accompanied by lymphopenia and elevated inflammatory markers, forming a vicious cycle of "malnutrition-inflammation-pressure ulcers." At the same time, enteral / parenteral nutritional support can increase TP levels, increasing the healing rate of stage 3 and 4 pressure ulcers by 40%. Therefore, TP is not only a predictor of pressure ulcer risk but also an important target for nutritional intervention.

[0079] (2) Globulins: As a major component of immune proteins, abnormal levels of GLB are associated with chronic inflammation of pressure ulcers. Pressure ulcer status and infection risk are closely related. Elevated GLB (>3.5 g / dL) is commonly seen in pressure ulcers complicated by chronic inflammation or infection, reflecting increased B cell activation and antibody (IgG / IgM) production. Gamma globulins contain complement components and acute-phase proteins; their overactivation may lead to tissue-damaging inflammatory responses and hinder wound re-epithelialization. Conversely, decreased GLB (<2.0 g / dL) suggests immunodeficiency, increases the risk of bacterial colonization, and raises the pressure ulcer infection rate by 2.4 times. A GLB / ALB ratio >1.2 can predict pressure ulcer progression to infected wounds with a sensitivity of up to 78%. Therefore, dynamic monitoring of GLB is of great value in assessing the immune status and infection risk of pressure ulcers.

[0080] (3) Glucose: Hyperglycemia (GLU>140 mg / dL) promotes pressure ulcers and hinders healing through multiple mechanisms. Persistent hyperglycemia leads to the accumulation of advanced glycation end products (AGEs), resulting in abnormal collagen cross-linking, decreased elasticity, and a 35% reduction in tissue pressure resistance

[155] . Blood glucose fluctuations >50 mg / dL can induce oxidative stress, reducing the activity of superoxide dismutase (SOD) in the pressure ulcer area by 42%. Microvascular complications in diabetic patients reduce the partial pressure of oxygen in subcutaneous tissue by 28%, accelerating pressure necrosis. Clinical data show that for every 1% increase in HbA1c, the risk of pressure ulcers increases by 18%, and intensive blood glucose control (target glucose of 110-140 mg / dL) can increase the healing speed of pressure ulcers in diabetic patients by 2.3 times; in addition, the hyperglycemic environment promotes the formation of Staphylococcus aureus biofilm, increasing the incidence of infectious pressure ulcers. Therefore, blood glucose management is the core link in the prevention and treatment of pressure ulcers.

[0081] (4) Blood urea nitrogen to creatinine ratio: Elevated BUN / Cr reflects dehydration or hypercatabolism and is significantly associated with the risk of pressure ulcers. For every 10 mg / dL increase in blood urea nitrogen, the water content of skin tissue decreases by 15%, increasing the risk of shear injury by 2.1 times

[158] . In patients with renal insufficiency (BUN / Cr>25:1), the accumulation of uremic toxins reduces the proliferative capacity of fibroblasts

[159] . A high BUN / Cr state (>30:1) indicates accelerated protein breakdown, leading to a reduction in wound healing-related growth factors (such as VEGF and PDGF)

[160] . Therefore, BUN / Cr is a key indicator for assessing the hydration status and metabolic environment of patients with pressure ulcers.

[0082] 4. Biomarkers related to tissue damage and liver function Aspartate aminotransferase (AST): AST is an important liver enzyme, mainly found in the liver, myocardium, skeletal muscle, kidneys, and erythrocytes. It plays a crucial role in amino acid metabolism (such as transamination) and energy metabolism (such as the tricarboxylic acid cycle and gluconeogenesis), and elevated serum AST levels may be potentially associated with the occurrence and development of pressure ulcers. Patients with pressure ulcers often experience muscle atrophy and increased catabolism due to prolonged immobilization, prompting the release of AST from damaged muscle cells into the bloodstream. Studies have shown that patients bedridden for more than 14 days have elevated AST levels on average, and these levels are significantly positively correlated with muscle mass reduction. Furthermore, ischemia-reperfusion injury in pressure ulcer tissues can induce oxidative stress, further increasing cell membrane permeability and promoting AST reduction. Notably, in diabetic patients with pressure ulcers, AST levels are correlated with glycated hemoglobin (HbA1c), suggesting that hyperglycemia may exacerbate muscle damage and enzyme release. However, AST lacks specificity as a marker of tissue damage and requires comprehensive evaluation in conjunction with indicators such as creatine kinase (CK) and lactate dehydrogenase (LDH). Therefore, dynamic monitoring of AST may help assess the degree of muscle damage and metabolic status in patients with pressure ulcers, but its value as an independent predictor still needs further research to verify.

[0083] In summary, based on the ischemia-reperfusion mechanism, seven animal models of acute spinal cord injury (SCI) pressure ulcers were constructed, including pre-PUs, PUs stage 1, PUs stage 2, PUs stage 3, PUs stage 4, the SCI group, and the control group. Through analysis of animal models and clinical data, 11 pressure ulcer-related blood biomarkers were systematically classified into four categories: 1) Inflammation and immune response-related biomarkers (LYM%, NEU%, MON#, and MON%), whose abnormal changes reflect the immunosuppressive state, acute inflammatory response, and chronic tissue repair imbalance in pressure ulcer patients; 2) Coagulation and microcirculation disorder biomarkers (MPV, P-LCR), which exacerbate tissue damage by mediating microthrombus formation and local ischemia through platelet activation; 3) Nutrition and metabolism-related biomarkers (TP, GLB, GLU, BUN / Cr), which together constitute the pathological cycle of "malnutrition-metabolic disorder-pressure ulcer worsening," with hypoproteinemia and hyperglycemia being particularly critical; and 4) Tissue damage biomarkers (AST), reflecting muscle breakdown and oxidative stress damage caused by prolonged immobilization. These biomarkers not only reveal the multi-mechanism pathogenesis of pressure ulcers (inflammation, coagulation, metabolism, and tissue damage) from different dimensions, but also provide objective evidence for clinical risk stratification (such as LYM% predicting immune status and P-LCR warning of thrombosis risk) and precision intervention (such as nutritional support to improve TP and glycemic control to optimize GLU) through dynamic monitoring. However, the specificity of some indicators (such as AST) still needs to be further verified by multi-center studies and further exploration at the molecular level is required.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A method for constructing an animal model of pressure ulcers, comprising the following steps: Step 1: Feed the modeling rats according to standard feeding methods; Step 2: Perform anesthesia, hair removal, and exposure of spinal cord segments in sequence, and then construct an acute spinal cord injury model using the gravity fall method; Step 3: After acclimatizing the model mice, a pressure ulcer model was constructed using a G-clamp fixator and a standardized ischemia-reperfusion circulation model. Step 4: Confirm the successful establishment of the animal model by visually observing the morphology and color changes of the skin at the pressure site of the rat, as well as the integrity of the skin and histological observation.

2. The method for constructing a pressure ulcer animal model according to claim 1, characterized in that: In step 1, healthy SPF-grade adult female rats weighing 240-350 g were used for modeling.

3. The method for constructing an animal model of pressure ulcers according to claim 1 or 2, characterized in that: In step 1, rats were numbered with unique identifiers and randomly assigned to 7 groups using a random number table. The rats were randomly divided into a control group and an experimental group. The experimental group was further divided into: acute spinal cord injury group, erythema group, non-pale erythema / stage 1 pressure ulcer group, stage 2 group, stage 3 group, and stage 4 group. After grouping, the weight, activity level, etc. of each group were compared to ensure that there were no significant differences.

4. The method for constructing a pressure ulcer animal model according to claim 3, characterized in that: In step 2, after anesthetizing the modeling rats, the hair around the T7 spinous process was removed. The rats were then fixed in a standard prone position on the surgical platform. The area from the occipital protuberance to the tip of the sacrum was disinfected, and a sterile surgical drape was placed. Using a sterile scalpel, a longitudinal incision was made along the midline of the back, centered on the T7 spinous process. The subcutaneous connective tissue was cut, and the muscles on both sides of the spinous process were dissected until the lamina was exposed. A spinal retractor was used to fully expose the T6-T8 spinous processes and the bilateral lamina structures. The interspinous ligaments of T6 / 7 and T7 / 8 were cut. The T7 spinous process was completely removed with bone forceps, and the bilateral lamina were excised to expose the spinal cord segments.

5. The method for constructing an animal model of pressure ulcers according to claim 4, characterized in that: In step 2, an orthopedic Kirschner wire is dropped vertically along the transparent tube and inserted vertically into the spinal canal, ensuring that the dura mater is not compressed or torn. The needle tip reaches the bottom of the spinal canal and hits the spinal cord, causing edema and congestion. The local swelling height is ≥ 1 mm, and the parenchymal congestion changes to dark red or purplish red. After confirming there was no cerebrospinal fluid leakage, apply strict pressure for 3 minutes until there was no active bleeding. Rinse the incision with sterile saline, suture the muscle layer intermittently first, and then suture the skin layer continuously. Apply iodine to the wound and cover it with sterile gauze, and wait for the rat to wake up. Record the operation time, amount of bleeding and incision length throughout the process. After the operation, inject saline into the peritoneum for fluid replacement. After the rat is revived in a constant temperature incubator in the room, it is housed in a single cage. Observe the animal's walking patterns daily, including the movement of the hip, knee and ankle joints, trunk movements and coordination; turn the animal over regularly, massage its abdomen each time it is turned over, and massage its bladder regularly to promote urination and functional recovery and prevent urinary retention; Using a temporal neurological function assessment protocol, the successful establishment of an acute spinal cord injury model was confirmed.

6. The method for constructing a pressure ulcer animal model according to claim 1, characterized in that: In step 3, after the skin of the buttocks of rats in the acute spinal cord injury group was prepared, they were placed on an electronic scale and a G-type clamp fixator in sequence according to the tail number. The same pressure was applied to the posterior thigh area of ​​the paralyzed hind limb to cause local ischemia. After 2 hours, the fixators were removed in sequence to allow the ischemic skin to be reperfused. The rats were then put back into the cage and allowed to move freely for 0.5 hours. In the standardized ischemia-reperfusion cycle model, each cycle contains two precisely controlled time phases: an ischemic phase lasting 2.0 h, followed by a reperfusion phase of 0.5 h.

7. The method for constructing an animal model of pressure ulcers according to claim 1, characterized in that: The anesthetic is at least one of pentobarbital, isoflurane, tribromoethanol, ketamine, toluenethiazide, and acepromazine.

8. The use of an animal model of pressure ulcer constructed by the construction method according to any one of claims 1-6 in assessing the risk of pressure ulcer occurrence, the molecular mechanism of pressure ulcer development, or in preparing and / or screening drugs for the prevention and / or treatment of skin pressure ulcers, or in optimizing surgical treatment plans.

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