Effect of Yi-Nao-Jie-Yu Prescription on Post-Stroke Depression in Rats using Middle Cerebral Artery Occlusion Combined with Behavioral Restraint

Post-stroke depression (PSD) is a common and treatable complication of stroke, which significantly affects the rehabilitation process and quality of life of patients. PSD patients not only suffer from physical functional impairments but also face mental disturbances such as low mood and loss of interest. This greatly hinders the recovery of neurological functions and even increases the mortality rate. Currently, the pathogenesis of PSD is complex and involves multiple aspects such as neurobiology, neurotransmitter imbalance, and inflammatory response. There is an urgent need to deeply explore effective intervention methods.

The overall goal of this method is to establish a reliable and reproducible rat model of PSD that closely mimics the human condition by combining middle cerebral artery occlusion (MCAO) with chronic mild stress (CMS, primarily behavioral restraint) and isolation housing, and to utilize this model for evaluating potential therapeutics like Yi-nao-jie-yu Prescription (YNJYP). The rationale for developing this technique stems from the need to integrate both the initial ischemic insult and the subsequent chronic psychological stress, which are recognized as key components in PSD pathogenesis, into a single standardized model^1,2,3. Compared to alternative methods, such as those relying solely on extensive CUMS paradigms, this protocol offers a more focused and replicable approach by employing a specific form of behavioral restraint designed to simulate the movement restriction often experienced by stroke patients^3,4,5.

This model contributes to the wider PSD literature by providing a detailed, practical framework for inducing and assessing PSD in rodents, addressing a need for standardization in preclinical research. Researchers interested in studying the combined pathophysiological effects of cerebral ischemia and post-stroke stress, or in screening novel interventions for PSD, may find this protocol appropriate, as it delineates clear procedures for model creation, stress application, and behavioral evaluation.

The animal study protocol was approved by the Animal Care and Use Committee of Beijing University of Chinese Medicine. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation

1. Obtain 216 specific-pathogen-free (SPF) male Sprague-Dawley (SD) rats, weighing 300-320 g.
2. Acclimate the rats to the housing environment for 1 week before the experiment.
   1. Maintain housing conditions at 23 ± 3 °C, 45% ± 5% humidity, and a 12 h light/dark cycle (lights on at 8:00 a.m., off at 8:00 p.m.).
   2. House five rats per cage with ad libitum access to sterile standard food and water.
      NOTE: Minimize the number of animals used and reduce suffering through gentle handling and optimized procedures (e.g., adjustable restraints to prevent respiratory suppression).

2. Middle cerebral artery occlusion (MCAO) surgery

NOTE: This step describes the establishment of the MCAO/reperfusion (MCAO/R) model, modified from Bederson et al.⁶.

1. Prepare the rats for surgery.
   1. Fast the rats overnight (12 h) before surgery (no water restriction).
   2. Anesthetize the rats via intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg).
      NOTE: No additional analgesics were administered, as sodium pentobarbital was used solely as the anesthetic agent.
      CAUTION: Sodium pentobarbital is a controlled anesthetic. Handle in a fume hood, wear gloves and goggles, and store in a locked cabinet according to institutional regulations.
2. Secure and prepare the surgical site.
   1. Place anesthetized rats in a supine position and fix their limbs.
   2. Shave the neck fur and disinfect the skin with an appropriate disinfectant (e.g., 75% ethanol).
3. Expose and prepare the neck vessels.
   1. Make a 2-3 cm midline incision in the neck skin, then bluntly dissect subcutaneous glands and fascia. Separate the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA), which are formed by the branches of the CCA.
   2. Locate the CCA, place two sutures under it as a reserve, ligate one near the heart end, and tie a live knot near the bifurcation of the CCA with the other one.
   3. Use an arterial clamp to close the distal end of the CCA near the bifurcation.
   4. Locate the ECA and ligate it with a suture.
4. Insert the suture to occlude the MCA.
   1. Carefully cut an incision on the wall between the ligation site of the proximal end of the CCA and the clamping site of the artery clip, then insert a special thread plug (monofilament suture) into this incision.
   2. Open the arterial clip and insert the thread plug into the ICA from the fork until slight resistance is felt, then stop (Figure 1).
      NOTE: The suture length (18 mm ± 2 mm) is critical for consistent MCA occlusion; adjust slightly based on rat weight (300-320 g).
5. Induce ischemia and reperfusion.
   1. Maintain ischemia for 2 h (strictly timed with a stopwatch).
   2. After 2 h, gently pull out the suture to restore MCA blood flow, cut off the excess suture, and remove the arterial clip.
6. Close the surgical site and monitor recovery.
   1. Irrigate the incision with sterile saline, then suture the subcutaneous tissue and skin.
   2. Place the rats in a recovery cage (maintained at 25 ± 1 °C) until they regain consciousness; monitor for bleeding or distress for 24 h.
7. Establish the sham operation group.
   1. Perform the same neck dissection and vessel exposure as in steps 2.1-2.3.
   2. Insert the suture into the CCA for only 5 mm, then immediately remove it; suture the incision without MCA occlusion.

3. Grouping of animals

1. Randomly divide the rats into six groups (n = 36 per group initially) for neurological scoring.
   1. Stroke group: MCAO only, no behavioral restraint or drug treatment.
   2. PSD group: MCAO + behavioral restraint + isolation housing, no drug treatment.
   3. FXT group: MCAO + behavioral restraint + isolation housing + fluoxetine hydrochloride gavage.
   4. YNJYP group: MCAO + behavioral restraint + isolation housing + Yi-nao-jie-yu prescription (YNJYP) gavage.
   5. Blank group: No operation performed.
   6. Sham operation group: No blockage of the middle cerebral artery.
2. Exclude rats with scores of 0 or 4 or those that died post-surgery.
   1. Perform Longa⁷ neurological scoring 24 h after MCAO to select qualified rats.
   2. Use the 5-point Longa scale: 0 = no deficit; 1 = forelimb flexion when lifted by the tail; 2 = circular walking toward the paretic side; 3 = falling toward the paretic side; 4 = unconsciousness or inability to walk.

4. Administration of fluoxetine hydrochloride (FXT) and YNJYP

1. Prepare YNJYP and FXT.
   NOTE: YNJYP contains six Chinese medicinal materials: Ciwujia (Acanthopanax senticosus roots) 30 g, Yujin (Curcuma longa rhizomes) 10 g, Wuweizi (Schisandra chinensis fruits) 15 g, Zhizi (Gardenia jasminoides fruits) 10 g, Danshen (Salvia miltiorrhiza roots) 15 g, and Chuanxiong (Ligusticum chuanxiong rhizomes) 15 g (provided by the Department of Pharmacy, Third Affiliated Hospital of Beijing University of Chinese Medicine). Based on previous studies⁸, the intragastric administration dose was determined to be 9.92 g/(kg·d).
   1. Dissolve the fluoxetine hydrochloride capsules in distilled water to achieve a concentration of 0.233 g/L when used. Based on the adult dosage multiplied by seven, set the dosage for rats at 2.33 mg/(kg·d).
2. Administer drugs via gavage.
   1. Start gavage on post-MCAO day 1 and continue until the end of the experiment, with the procedure being consistently performed in the morning across all groups.
   2. Administer FXT to the FXT group at 2.33 mg/(kg·d), YNJYP to the YNJYP group at 9.92 g/(kg·d), and 0.9% normal saline (10 mL/kg) to the stroke and PSD groups.
      NOTE: Use a 1 mL gavage needle; ensure the needle is inserted into the stomach (not the trachea) to avoid aspiration.

5. Chronic mild stress (behavioral restraint) combined with isolation housing

1. Start behavioral restraint and isolation housing on post-MCAO day 7 (to avoid the acute stroke phase).
2. Prepare the custom T-shaped restraint platform.
   1. The platform consists of a base (20 cm × 10 cm × 2.8 cm) and a restraint area (22 cm × 6.6 cm).
   2. The restraint area contains slots for fixing the rat's head and limbs and three adjustable straps to fit different rat sizes (Figure 2, Figure 3, and Figure 4).
      NOTE: The platform is modified from Chen³ and Tian⁴, with added forelimb slots to simulate the paretic posture of stroke patients.
3. Perform daily restraint.
   1. Randomly select a 2 h window each day (e.g., 9:00 a.m.-11:00 a.m. or 2:00 p.m.-4:00 p.m.) to avoid circadian bias.
   2. Secure each rat to the platform using the slots and straps; adjust tightness to prevent struggling but not respiratory suppression.
   3. Maintain restraint for 2 h continuously for 7 consecutive days.
4. House rats in isolation.
   1. Transfer rats in the PSD, FXT, and YNJYP groups from group housing (five rats per cage) to single cages at the start of behavioral restraint.
   2. Maintain single housing until behavioral restraint is completed (post-MCAO day 14).

6. Sucrose consumption test (SCT)

NOTE: Randomly select six rats from each group at 2, 4, and 8 weeks post-MCAO. Test all rats in the order of SCT, OFT, and FST.

1. Prepare rats for the test.
   1. Fast and deprive the rats of water for 24 h before the test.
   2. Ensure the testing room temperature (23 ± 2 °C) and lighting match the housing environment.
2. Conduct the test.
   1. Place two bottles (150 mL each) in each cage: one containing tap water and the other 1% sucrose solution.
   2. After 1 h of free drinking, record the volumes of sucrose solution and water consumed.
3. Calculate sucrose preference.
   1. Use the formula: Sucrose preference (%) = (Volume of sucrose solution consumed / Total volume consumed) × 100%.
   2. Swap the positions of the two bottles halfway through the drinking period to eliminate position bias.

7. Open-field test (OFT)

NOTE: Select the same batch of rats from each group used for the FST at 2, 4, and 8 weeks. Fast and deprive the rats of water for 24 h before the test to maintain consistent physiological conditions.

1. Prepare the open-field apparatus.
   1. Use a black square box with the floor divided into 25 equal grids.
   2. Keep the testing room quiet and dimly lit.
2. Conduct the test.
   1. Place each rat in the center grid of the box and start a 5 min timer.
   2. Record horizontal activity (number of grid crossings with three-fourths of the paws in a new grid) and vertical activity (number of rears with two-fourths of the paws off the floor by manual observation using handheld counters.
   3. Record the time spent in the central grids and grooming frequency (optional, for additional behavioral insights).

8. Forced swim test (FST)

NOTE: Select the same batch of rats from each group used for the FST at 2, 4, and 8 weeks. Fast and deprive the rats of water for 24 h before the test to maintain consistent physiological conditions.

1. Prepare the test apparatus.
   1. Fill a glass cylinder with an appropriate amount of water, ensuring that the water level is high enough so that the rat cannot support itself by touching the bottom with its feet.
   2. Adjust the water temperature to 25 ± 1 °C.
2. Conduct the test.
   1. Place each rat in the cylinder and let it swim for 5 min.
   2. Record the total immobility time during the 5 min.
   3. Define 'immobility' as passive floating (only minimal movements to keep the nose above the water) and 'activity' as limb paddling or wall-scratching.
3. Care for rats post-test.
   1. Remove the rat from the cylinder after 5 min, dry it with a towel, and place it under a heating lamp (30 ± 2 °C) for 30 min to prevent hypothermia (Figure 5).

9. Statistical analysis

1. Organize data as mean ± standard deviation (mean ± SD).
2. Analyze data using SPSS version 20.0 software.
3. Before conducting parametric tests, test the data for normality (using the Shapiro-Wilk test) and homogeneity of variances (using Levene's test). The data of each group conform to a normal distribution and satisfy homogeneity of variance.
   1. Use one-way analysis of variance (one-way ANOVA) for comparisons of sample means between groups, combined with least significant difference (LSD) and Student-Newman-Keuls (SNK) tests. Use Welch's test when variances are unequal.
4. Set statistical significance at p < 0.05.

A total of 5 experimental groups were set up in this study for relevant detection. After statistical analysis of the behavioral data of each group of rats, it was found that at the 4th week of the experiment, the immobility time of PSD group rats in the FST was significantly increased compared with the stroke group rats, and the difference between the two groups was statistically significant (P < 0.01); at the same time, the sucrose preference of PSD group rats in the SCT was significantly lower than that of the stroke group rats, and the differences between the two groups at the 4th and 8th weeks of the experiment were statistically significant (P < 0.01). Based on the changes in these behavioral indicators, it can be determined that the PSD rat modeling in this study was successful (Specific data analysis is shown in Figure 6).

After intervention with YNJYP on PSD rats, the behavioral test results showed that this compound had a significant improvement effect on the depressive-like behavior of rats. Specifically, YNJYP could reduce the immobility time of PSD rats in the FST at the 4th and 8th weeks of the experiment, and after statistical testing, the differences between the intervened rats and the PSD rats without intervention were statistically significant (P < 0.01); in addition, YNJYP could also increase the sucrose preference of PSD rats in the SCT, and at the 4th and 8th weeks of the experiment, the differences between the intervened rats and the PSD rats without intervention were also statistically significant (P < 0.01). Based on the above detection results, it can be considered that in this study, YNJYP could significantly reverse the depressive behavior shown by PSD rats in the FST and SCT.

From the results of the Open Field Test, in the Rearing Frequency aspect, compared with the Sham-operated group, the vertical movement times of PSD group rats at 8 weeks were significantly reduced (P < 0.05); in the Locomotor Activity Counts aspect, the horizontal movement times of PSD group rats at 2 weeks were also significantly lower than those of the stroke group (P < 0.05), which further indicates that the construction of the PSD model was effective. For the YNJYP group (TCM group) and the fluoxetine hydrochloride group (Conventional medicine group), in the vertical movement aspect, the vertical movement times of the TCM group and the conventional medicine group at 8 weeks were increased to a certain extent compared with the PSD group, and the differences were statistically significant (P < 0.05); in the horizontal movement aspect, the horizontal movement times of the TCM group and the conventional medicine group at 2 weeks and 8 weeks were also higher than those of the PSD group, and the differences were statistically significant (P < 0.05). This indicates that both YNJYP and fluoxetine hydrochloride improve the movement of PSD rats and have certain intervention effects on PSD.

The existing methods for establishing cerebral ischemia models mainly include the ligation method, the line occlusion method, the craniotomy method, and the asphyxia method. Compared with the ligation method, the craniotomy method, and the asphyxia method, the line occlusion method demonstrates significant and unique advantages in the construction of rat cerebral ischemia models. The core lies in the relatively constant infarction site (mainly concentrated in the cerebral middle artery supply area), and by strictly controlling technical points such as the insertion depth of the line occlusion, the model success rate can be significantly improved, far higher than the high technical threshold and potential complications of craniotomy method, the low standard compliance rate of the four-vessel ligation method (less than 70 %) of the ligation method (especially the four-vessel method), and the generally lower recovery success rate and survival rate of asphyxia method. Therefore, the line occlusion method, due to its minimally invasive nature, strong controllability, high success rate, and good repeatability, has become the preferred model for studying focal cerebral ischemia and reperfusion pathophysiology and intervention measures.

According to the papers and multiple articles published by Wu Zhangfu et al.9 and Hou Zhitao et al.10, the success rate of ligation method is as follows: the postoperative 48-h survival rate of the traditional bilateral common carotid artery ligation method (2-VO) is 50%; the success rate of the D-gal-induced aging combined with double vessel ligation model group is 40%; the proportion of rats meeting the criteria in the four-vessel ligation method is less than 70%. According to the research of Xu Jia et al.11, "the line occlusion method" with an insertion depth greater than 1.60 cm has a modeling success rate of 83.33%, which is consistent with the 18 mm ±2 mm summarized in this experiment. Comparing the data of the ligation method and line occlusion method, it is found that the modeling success rate of the latter (mainly based on the postoperative survival rate of rats as the main reference) is higher than that of the former. In this experiment, the modified line occlusion method based on the scheme of Tian12 was applied for MCAO surgery, and the postoperative 24 h survival rate of stroke rats was 95%, 75% at 1 week, and 70 % at 2 weeks, showing significant superiority.

This study used the modified Chen et al.4self-made T-shaped restraint platform scheme to screen rats that met the Longa neurological score criteria. Their heads, necks, and thoracolumbar and lumbar dorsal regions were fixed by adjustable soft bands on the wooden restraint table. They were subjected to arbitrary 2 h continuous restraint for 1 week, and were also fasted and dehydrated for 24 h. These stress stimuli made the rats able, and combined with the isolation method, the rat PSD model was successfully replicated.

The research articles of Sun Peiyang et al.1, Sun Meifang et al.2, and Sun Yi et al.3 showed that after a period of behavioral restraint, the consumption ratio of sucrose water of the model group rats compared with the sham operation group and the stroke group was lower, and the difference was statistically significant (P < 0.05 or P < 0.01); there were also studies showing that although the sugar water intake of the simple stroke group decreased, there was no significant difference compared with the blank control group (P > 0.05). The research of Huang Qiaoling et al.13and Sun Yi et al.3 verified that compared with the sham operation group and the stroke group, the PSD group showed an increase in immobility time, and the difference was statistically significant (P < 0.01). The above studies all used chronic unpredictable mild stress (CUMS), and its commonly used stimulation methods include food and water deprivation, foot shock, reversed light-dark cycle, ice water swimming, heat stimulation, bright light stimulation, noise stimulation, horizontal oscillation, cage tilting, damp cage, tail suspension or tail clamping, and behavioral restraint, etc. It emphasizes the diversity of stimulation types and the complete randomness and unpredictability of occurrence time, making it more difficult for animals to adapt.

In contrast, this experiment chose chronic mild stress (CMS, behavioral restraint was mainly used in this study), which mainly consists of mild, single, or fixed stimulation, with low intensity and a fixed pattern that is easy to replicate. The self-made T-shaped platform can restrict the behavior of rats after a stroke. This stimulation specifically simulates the real state of stroke patients in clinical practice. The ones mentioned are the innovative advantages of the T-shaped platform.

Due to technical limitations, this study did not directly quantify the restraint pressure, but attempted to increase the adjustable strap design to ensure animal welfare: referring to the method of Chen et al.4, it fixed and did not compress the rat's chest cavity. The three-point fixation design of the T-shaped restraint platform (head, thorax, abdomen, waist, and back) reduced the animal's struggle and respiratory inhibition, and segmented fixation could also reduce unnecessary stimulation, which may increase the survival rate. Specific data needs further statistical verification. This study chose to initiate restraint 7 days after the surgery primarily based on the avoidance of the acute phase of stroke: Longa et al.7 demonstrated that the peak of brain edema occurred within 0-3 days after MCAO, and premature restraint might aggravate the damage; as Bernhardt et al.14 showed, the blood-brain barrier permeability increased within 0-72 h after stroke, and the expression of IL-6 mRNA in the cortex significantly increased, suggesting that there was a significant inflammation and barrier disruption in the acute phase; as Oppenheimer et al.15 demonstrated, the sympathetic over-activation state in the acute phase is prone to induce arrhythmias due to additional stress, confirming that the sympathetic over-activation in the acute phase of stroke can trigger arrhythmias. Secondly, many clinical studies have shown that PSD often occurs 1-4 weeks after stroke4, and this study conducted behavioral restraint 7 days after the surgery, which is in line with the known depression induction window of neuroplasticity in current research. As Arvidsson et al.16demonstrated, the peak of neural stem cell proliferation occurred 7 days after MCAO, marking the initiation of endogenous repair, and this time was also the optimal timing for determining the restraint stress. As Sapolsky et al.17 demonstrated, the expression of glucocorticoid receptors in the hippocampus increased 7-14 days after surgery, leading to enhanced sensitivity to stress hormones, providing a molecular-level explanation for the basic mechanism of stress sensitivity in rats; as Robinson et al.18 demonstrated, approximately 60% of stroke patients presented initial symptoms of depression at this stage, which was highly consistent with the time window for stroke-induced depression modeling in this experiment. To more accurately simulate the situation of PSD patients, Wang Qian et al.5 pointed out that the specific model of post-stroke depression still needs to simultaneously simulate focal brain injury and movement restriction. This study attempted to simulate the typical limb position or unilateral limb movement disorder of stroke patients by adjusting the fixation angle of the rat's forelimbs on the T-shaped platform and the limb straps (refer to the investigation results of Nakayama et al.19, compared with the traditional tubular restraint method, which cannot achieve customized body position (refer to the method of Luo L.20, the behavioral restraint stimulation in this experiment can more reliably simulate the restrictive body position and hemiplegic posture of humans after stroke.

At the same time, similar to the PSD modeling methods used in most studies, this experiment additionally provided single cage feeding for the rats, which can psychologically increase the despair of the rats and better match the psychological and social situation of stroke patients with depression, stroke patients usually leave their relatives and friends when hospitalized and are in a state similar to living alone, therefore, the isolated and solitary-raised rats that leave their original social life are closer to the actual state of the patients. Figure 7 illustrates the timeline of the complete experimental protocol.

Figure 1: Schematic of MCAO surgery. (A) After anesthesia, neck muscles are dissected to expose the CCA, ECA, and ICA. (B) The ECA is ligated; a small incision is made in the CCA. (C) A monofilament suture (18 mm ± 2 mm) is inserted into the ICA to occlude the MCA. Please click here to view a larger version of this figure.

Figure 2: Side view of the T-shaped restraint platform. The platform has a lower base (20 cm × 10 cm × 2.8 cm) and an upper restraint area (22 cm × 6.6 cm) with adjustable straps. Please click here to view a larger version of this figure.

Figure 3: Top view of the T-shaped restraint platform. The restraint area includes slots for fixing the rat's head, limbs, and forelimbs (modified to simulate paretic posture). Please click here to view a larger version of this figure.

Figure 4: Photograph of the constructed T-shaped restraint platform. The wooden platform with soft adjustable straps ensures secure fixation without respiratory suppression. Please click here to view a larger version of this figure.

Figure 5: Procedure for warming rats after the forced swim test (FST). Rats are dried with a towel and placed under a heating lamp (30 ± 2 °C) for 30 min to prevent hypothermia. Please click here to view a larger version of this figure.

Figure 6: Behavioral test results. This figure shows OFT horizontal activity counts, OFT vertical rear frequency, SCT sucrose preference, and FST immobility time. Groups: Sham-operated (Sham), Stroke, PSD, Conventional medicine (FXT), TCM (YNJYP). Statistical symbols: *P < 0.05, **P < 0.01 vs. Sham; #P < 0.05, ##P < 0.01 vs. Stroke; △P < 0.05, △△P < 0.01 vs. PSD. Error bars represent SD. Please click here to view a larger version of this figure.

Figure 7: Timeline of the complete experimental protocol. The diagram outlines the study duration, including model preparation, MCAO surgery, post-stroke depression (PSD) model establishment, daily drug administration, and behavioral assessments. Please click here to view a larger version of this figure.

Figure 8: Photograph of the sucrose preference test (SPT) setup. Two bottles (one with water and one with sucrose solution) are simultaneously presented to the rat for consumption. Please click here to view a larger version of this figure.

In the process of constructing the rat brain ischemia model, there are various different modeling methods, including the ligation method, craniotomy method, asphyxia method, and wire occlusion method. Compared with the three modeling methods of ligation, craniotomy, and asphyxia, the wire occlusion method demonstrates significant and unique advantages. These advantages mainly lie in three key aspects: the minimally invasive nature of the operation, the precision of time and space control, and the reliability of the model^1,2,3.

Firstly, from the perspective of the invasive nature of the operation, the suture method differs significantly from other methods. When using the suture method to establish a rat model of cerebral ischemia, no craniotomy is required for the rat, nor extensive vascular dissection and ligation procedures. The specific operation method is to simply make a small incision at the rat's neck, and then insert the suture through the carotid system to achieve the occlusion of the target vessel. This operation method significantly reduces the physical trauma caused by the surgery itself to the rat and also significantly reduces the risk of postoperative infection. In contrast, the craniotomy method is prone to causing cerebrospinal fluid leakage in the rat during the operation, and the probability of postoperative infection is also relatively higher.

Secondly, in the application of the suture method, its advantage in time control is unparalleled by other methods and has irreplaceable accuracy. This method can meet the needs of researchers to freely set the duration of suture occlusion of the target vessel and other requirements. Similarly, when reaching the predetermined time point, researchers can achieve precise reperfusion of the rat's brain vessels by withdrawing the suture. This feature is a crucial prerequisite for the molecular mechanism of ischemia-reperfusion injury and the determination of the time window for related treatments. However, the ligation method usually only achieves permanent occlusion of the vessel and cannot flexibly control the time of vessel occlusion and reperfusion; asphyxia causes global cerebral ischemia in the rat, and the control process of the resuscitation time point is relatively complex, so these two methods are difficult to meet the precise control requirements of the ischemia-reperfusion time in the research.

Furthermore, the focal cerebral ischemia model established by the suture method shows high advantages in reliability and repeatability. The core advantage of this method lies in the relatively constant infarction site of the model, mainly concentrated in the blood supply area of the rat's middle cerebral artery. In addition, by strictly controlling the key technical points such as the depth of suture insertion, the success rate of the model can be significantly improved. In contrast, the craniotomy method has a high technical threshold for operation and is prone to potential complications during the operation, resulting in an increased difficulty in model construction; the ligation method, especially the four-vessel ligation method, has a relatively low proportion of standard rats, usually less than 70%; asphyxia causes a low success rate of resuscitation and a low survival rate of rats. In summary, the model constructed by the suture method is far superior to the craniotomy method, ligation method, and asphyxia method in terms of reliability and repeatability.

In conclusion, precisely because the suture method has the advantages of being minimally invasive, strong controllability, a high success rate, and good repeatability, it has become the preferred method for model construction in the study of focal cerebral ischemia and reperfusion pathophysiological mechanisms, as well as the exploration of related intervention measures.

The current mainstream method for modeling post-stroke depression is to first successfully establish a rat stroke model, then apply chronic unpredictable mild stress (CUMS) combined with the isolation housing method^1,2,3, which has become a depression model of rodents with practical application value.

The behavioral test and evaluation methods for PSD modeling are diverse, and the evaluation time after stress stimulation varies. There is no fixed quantitative standard yet. The following are common behavioral test methods for PSD.

The sucrose water consumption experiment (also known as sugar water preference detection, abbreviated as SCT) is operated by fasting and water deprivation for 24 h, then placing one bottle of drinking water and 1% sucrose water beside each rat, allowing the rats to drink for a period of time, and calculating the sucrose water consumption (Figure 8). The formula for calculating sucrose water consumption is the consumed sucrose water volume / total sucrose water volume × 100%, and the positions of the two bottles of water will be exchanged during the experiment; Sun Meifang et al.² pointed out that the amount of sucrose solution consumed by rats can reflect their pleasure level, a significant decrease in the sucrose water consumption ratio indicates a decline in the desire for pleasant events in depressed rats, which is a specific manifestation of pleasure deficiency after stroke; Wang Shuling et al.²¹ proposed that the sugar water preference index is currently the most suitable and main method for evaluating the core symptoms of depression.

The forced swimming experiment (abbreviated as FST) is operated by preparing a cylindrical container of a certain volume, maintaining an appropriate water depth, and controlling the water temperature at a certain level, then placing the rat alone in the glass cylinder for 5 min of swimming. During this process, record the behavioral patterns of the rat in the water and the cumulative immobile time (total time immobile) of each rat.

The open field experiment (also known as the open field test, abbreviated as Open-Field Test) is operated by placing the rat in the center of a black open box, scoring the horizontal activity based on the number of times the rat crosses the adjacent grid with three or four feet, and the vertical activity based on the number of times the rat stands upright with both feet or both forelimbs reaching a certain distance from the bottom, and measuring the horizontal activity score and vertical activity score of the rat within 5 min; Sun Yi et al.³ also observe the time the rat stays in the middle grid and the number of grooming times, etc.; He Jieying et al.²² pointed out that the open field experiment is an important behavioral research method for rodents, which can reflect the interest level and activity level of rats, and is of great significance for evaluating the movement ability, emotional change, and cognitive ability of rodents.

The hanging tail experiment is operated by applying adhesive tape to the tail tip of the rat 1 centimeter away from the tail tip, attaching the rat head down and about 10 cm away from the bottom surface, placing the hanging tail support in a well-ventilated and quiet box, and recording the behavior of the rat during this process and counting the cumulative immobile time during this period.

The body weight detection is operated by weighing the body weight of all rats in a room with suitable temperature and humidity before the stress begins, and measuring the body weight of each rat in the same environment before feeding at 8:00 in the morning every weekend during the stress period until the end of the experiment; Huang Qiaoling et al.¹³ pointed out that the slow weight gain of rats may be related to the decreased appetite and reduced food intake caused by depression, and the decreased appetite itself is one of the core symptoms of depression patients. Xiao Wei et al.²³ believe that one of the indicators of the successful establishment of the PSD model is that rats exhibit obvious depressive symptoms. Specifically, these include listlessness, dull coat color, reduced various activities, weight loss, decreased food intake, diarrhea, decreased body temperature, poor cold tolerance, low energy metabolism, arched backs, and lack of activity, etc.

Limitations of PSD modeling and the practical development significance
During the application of the commonly used wire-tie method in rat cerebral ischemia models, there is a problem of inconsistent wire-tie insertion lengths. Specifically, the length of the wire-tie insertion lacks standardization, and in some experimental operations, the insertion length of the wire-tie failed to reach the reference standard of 1.65 cm. This operational difference directly affects the models created by the wire-tie method, resulting in a slightly lower success rate. However, even with this issue, the success rate of the wire-tie method is still higher than that of the currently widely used ligation method, and its relative advantage has not been completely lost.

In the current relevant literature, there are significant differences in the research content of PSD models. Most studies focus on the preparation methods of animal models of stroke, and have conducted extensive discussions on the construction process, operation points, and optimization strategies of MCAO. However, in sharp contrast, there is a significant deficiency in the research on chronic mild stress, especially the lack of complete and systematic operation methods.

The chronic stress model, as a classic animal model in the field of depression research, is widely used by many researchers in the study of depression mechanisms and the screening of intervention drugs due to its correlation with the pathological physiological process of depression. However, this model has problems with stability. Due to the significant differences in tolerance to the same stress intensity among different types, different strains of animals, and even different individuals of the same type and strain, there are no unified and effective operation standards for the same stress method in different research teams, which directly leads to the need for improvement in the stability of the chronic stress model and the difficulty in effectively guaranteeing the reproducibility of the model results. Its modeling method still needs to be further standardized to enhance the reliability and applicability of the model.

Currently, the pathogenesis of PSD is not yet fully clear, and related research is still in the exploration stage. The pathogenesis process of PSD involves complex interactions between cerebral stroke pathological changes and abnormal depressive emotions, and its pathogenesis mechanism has uniqueness, which cannot be simply explained by the pathogenesis mechanism of simple depression. Clinical research has found that many antidepressants with clear therapeutic effects in the treatment of simple depression have failed to achieve the expected therapeutic effects when applied to PSD patients²⁴. This clinical phenomenon also indirectly confirms the particularity of the pathogenesis of PSD. The lack of clarity in the pathogenesis directly leads to the inability to provide a precise etiological replication method for the PSD animal model, making the model construction lack clear pathological mechanism guidance, and making it difficult to precisely simulate the pathogenesis process of clinical PSD.

In the evaluation system of experimental stroke animals, the applicability of evaluation methods varies for different types of functional impairments. For neurological deficits symptoms such as hemiplegia and other motor functional impairments that occur in experimental stroke animals, there are already relatively mature objective evaluation methods. This can be achieved through objective assessment scales (such as Zea Longa⁷ score, Berderson⁶ score) for quantifying the animal's motor function, balance ability, etc., or through neuroimaging techniques for intuitive observation and analysis of cerebral ischemic lesions and neural structure changes.

However, for depressive behaviors and emotional changes that occur in stroke animals, researchers face significant challenges. Since animals cannot convey their emotional states through language or clear subjective expressions, their depressive-like behaviors are difficult to evaluate and diagnose accurately through objective means. At the same time, there are essential differences in physiological structure and neural function between experimental animals and humans, which further exacerbate the difficulty in establishing and evaluating PSD animal models.