Subarachnoid hemorrhage model

I. Animal-Related Information

1. Animal Strain: SD Rat
2. Animal Gender: Male

3. Animal Age (Weeks) and Body Weight: Body weight ranges from 250 to 300g

   
   

II. Model Establishment Method

1. Preliminary Preparation: First, anesthetize and disinfect the rats, then fix them properly.
2. Blood Collection: Use a heparin-pre-rinsed syringe to draw 0.2 ml of blood.
3. Surgical Operation
   • Firmly fix the rat's head on a stereotaxic instrument equipped with a microinjection pump, and make a 2 cm sagittal incision along the midline of the scalp.
   • Accurately mark the drilling position at 0.1 mm anterior to the bregma and 4 mm to the right, ensuring the hole is located at the coronal suture.
   • Use a drill bit to drill vertically at the marked position, taking care to avoid damaging the dura mater during the operation. Then, draw 0.2 ml of non-heparinized blood into the syringe, place the bevel of the needle in the direction opposite to the midline, and insert the needle tip into the brain tissue until the needle tip disappears completely.
   • Insert the needle vertically into the right caudate nucleus to a depth of 5.5 mm, and slowly infuse 100 μl of autologous whole blood into the right basal ganglia region at a rate of 10 μl/min. After the infusion is completed, leave the needle in place for another 2 minutes.

   • Slowly pull out the needle, seal the drill hole with bone wax, and then suture the skin.

     
     

III. Model Validation

1. General Condition Observation: Closely monitor the daily performance of the experimental animals.
2. Specific Test Detection
   • Placement Test: Evaluate the relevant abilities of the rats through this test.
   • Corner Turn Test: Obtain behavioral data of the rats through this test.
3. Physiological Index Detection
   • Brain Water Content Detection: Since cerebral edema is closely related to the survival rate of patients with cerebral hemorrhage, it is necessary to detect the formation of cerebral edema. The dry-wet weight method is mainly used to determine the brain water content.
   • Blood-Brain Barrier Permeability Detection: Common detection methods include the fluorescent isotope method and Evans blue staining. In addition, IgG staining using the DAB staining method can also be used to detect the permeability of the blood-brain barrier.
   • Brain Genomics Analysis: Aimed at identifying changes in gene expression.

   • Histological Evaluation: Conduct a comprehensive assessment of the amount, location, and range of bleeding, as well as the status of nerve cell death and brain atrophy.

     
     

IV. Suggestions for Subsequent Detection Indicators

1. Brain Water Content: Use the dry-wet weight method to detect the formation of cerebral edema, as cerebral edema has a significant impact on the survival rate of patients with cerebral hemorrhage.
2. Blood-Brain Barrier Permeability: Detect using methods such as fluorescent isotopes, Evans blue staining, and IgG staining via the DAB staining method.
3. Brain Genomics: Identify changes in gene expression.
4. Histology: Evaluate the amount, location, and range of bleeding, the status of nerve cell death, and brain atrophy.

5. Brain Tissue HE Staining: Perform HE staining analysis on brain tissue.

   
   

V. Precautions

1. Housing and Management: Rats after model establishment should be housed in individual cages, and attention should be paid to heat preservation. If a rat dies during or after model establishment, resulting in the inability to collect the required experimental data for that group, a backup rat should be selected as a replacement in accordance with the same randomization principle.
2. Specimen Collection: At the scheduled time points (1 day, 3 days, 5 days) for each group, after the rat is successfully anesthetized, place it in a supine position. Along the abdominal midline, first use tissue scissors to cut the skin in sequence, then bluntly dissect the subcutaneous tissue, followed by cutting the abdominal muscles and peritoneum, and then bluntly dissect the inferior vena cava. Use a disposable venous blood collection needle to draw 3 ml of venous blood. Let the blood stand at room temperature for 2 hours, then place it in a centrifuge and centrifuge at 2 - 8 °C and 1000 r/min for 15 minutes. Collect the upper serum, aliquot it into cryovials, and store it in a -20 °C refrigerator for subsequent detection.
3. Brain Tissue Harvesting: Immediately after blood collection, use hemostats to clamp the inferior vena cava and abdominal aorta. Continue to cut upward along the abdominal midline to the xiphoid process, cut the diaphragm, then cut the ribs on both sides 2 cm away from the sternum respectively. Use tissue forceps to lift the sternum upward and fix it to fully expose the heart. Insert the injection needle connected to the infusion set into the apex of the left ventricle, quickly infuse normal saline, and at the same time cut the right atrial appendage with ophthalmic scissors to continue perfusion. The perfusion speed should be fast first and then slow, and stop perfusion when the rat's eyeballs and upper paws turn white and the perfusate from the right atrial appendage becomes clear. Quickly decapitate the rat to take the brain, completely剥离 (dissect) the brain tissue, and soak it in 4% paraformaldehyde for 48 hours for subsequent pathological detection.
4. Temperature Control: Minor changes in temperature can have an impact in models of ischemic brain injury and cerebral hemorrhage. After cerebral hemorrhage, the rat's own temperature regulation function is affected, and the animal's body temperature may drop sharply in the later stage of the experiment. Such temperature changes can interfere with the experimental results of the cerebral hemorrhage model. Therefore, it is necessary to adopt skull temperature control and anal temperature control measures, and keep the animals in a temperature-controlled incubator within 48 hours after surgery.
5. Gender Factor: Gender has an impact on experimental results in both ischemic stroke models and cerebral hemorrhage models. In ischemic stroke models, the behavioral scores and cerebral infarction volume of female animals are significantly lower than those of male animals; in cerebral hemorrhage models, female animals have a shorter recovery period in terms of behavior and smaller cerebral hematomas. The neuroprotective effect of female animals may be related to circulating estrogen and progesterone, so male animals are usually selected for experiments.
6. Handling of Surgical Bleeding: If bleeding occurs during the operation, hemostatic sponges and bone wax can be used for hemostasis.
7. Consideration of Injection Speed: Different from the rapid injection method used in previous models, this model uses a slow injection method to infuse blood into the brain parenchyma. This slow injection method can not only limit the extravasation of blood into the subarachnoid space to more realistically simulate the natural process but also avoid physiological pressure damage to adjacent tissues.
8. Selection of Blood Source: Cerebral edema is a key part of cerebral hemorrhage research, and hematomas in the brain parenchyma can cause blood-brain barrier damage and edema. Studies have shown that the brain water content in the ipsilateral basal ganglia and cortex of C57BL/6 mice increases significantly 1 - 3 days after autologous blood injection. Moreover, studies have found that 1 day after intracerebral blood injection, donor blood causes more severe cerebral edema compared with autologous blood. Therefore, the donor blood model can be used to further conduct research related to cerebral edema.