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

Summary

This protocol describes the establishment of a rat model of poststroke depression (PSD) by combining middle cerebral artery occlusion (MCAO) with chronic mild stress, behavioral restraint, and isolation housing, and the evaluation of the therapeutic effects of the Yi-nao-jie-yu prescription (YNJYP) using behavioral tests.

Abstract

Post-stroke depression (PSD) is a common, treatable complication of stroke, characterized by depressive and somatic symptoms that impair patient recovery and quality of life. This study describes a protocol combining middle cerebral artery occlusion (MCAO) with behavioral restraint and isolation housing to establish a rat PSD model, and evaluates the therapeutic effect of Yi-nao-jie-yu Prescription (YNJYP) on adult neurogenesis in PSD rats. For model establishment, rats in the stroke, PSD, fluoxetine hydrochloride (FXT), and YNJYP groups underwent MCAO: a monofilament suture was advanced from the internal carotid artery to the middle cerebral artery (MCA) for 2 h of ischemia, followed by reperfusion. From post-MCAO day 7, rats in the PSD, FXT, and YNJYP groups were single-housed and restrained in a custom T-shaped platform for 2 h daily for 7 days. Behavioral assessments included the forced swim test (FST, for despair), sucrose consumption test (SCT, for anhedonia), and open-field test (OFT, for exploratory behavior). At 4 and 8 weeks post-stroke, PSD rats showed longer immobility time in FST and lower sucrose preference in SCT than stroke rats (P < 0.01). YNJYP reversed these depressive-like behaviors (P < 0.01), with efficacy comparable to FXT. This protocol confirms the validity of the PSD model and YNJYP's therapeutic potential, supported by rigorous experiments and data analysis.

Introduction

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.

Protocol

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 rats overnight (12 h) before surgery (no water restriction).
      NOTE: All surgical procedures are performed using aseptic technique: surgical instruments are autoclaved before use, the operator wears sterile gloves, and the surgical area is covered with a sterile drape.
   2. Anesthetize rats via intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg). Confirm anesthesia depth by toe pinch reflex and by monitoring respiratory rate. Maintain body temperature during surgery by placing rats on a heating pad (37 ± 0.5 °C) until full recovery. During surgery, apply erythromycin ophthalmic ointment or artificial tears to both eyes of the rat to prevent corneal drying.
      NOTE: No pre‑ or postoperative analgesics were used in this study because NSAIDs and opioids may interfere with post‑ischemic inflammation and infarct volume assessment⁸.
      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. During surgery, place the rat on a heating pad (37 ± 0.5 °C) and monitor body temperature.
   2. Shave the neck fur. Apply a povidone‑iodine scrub in concentric circles starting from the incision site, followed by 75% ethanol in the same circular manner from inside out and repeat this sequence 2 more times. Cover the surgical area with a sterile drape (changed for each rat).
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 rats in a recovery cage (maintained at 25 ± 1 °C) until they regain consciousness; monitor for bleeding or distress for 24 h.<
      NOTE: For the scientific purposes of this study, no systemic analgesics were administered postoperatively (NOTE in 2.1.2 for rationale). However, animals received thermal support during recovery (25 ± 1 °C) and were observed every 30 min for signs of pain. No overt pain behaviors were observed.
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.

Results

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 strok...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adult male Sprague-Dawley rats	Vital River Laboratory Animal Technology, Beijing, China	No. SCXK2012-0001	300–320 g
Fluoxetine Hydrochloride Capsules	Patheon France, Jiangsu, China	0943A
Granules of YNJYP	the Department of Pharmacy, Third Affiliated Hospital of Beijing University of Chinese Medicine	Homemade
Open box for open field test	Homemade	-
Sodium pentophenobarbital	Sigma Corporation of America	P3761-5
the occlusion thread	Beijing Shadong Biotechnology Co., Ltd	2838-A4
Water tank for forced swimming experiment	Homemade	-