Construction scheme of sham operation control model of ischemic stroke in SD rats

Experimental Animals: 6-8 week-old SPF-grade male SD rats (200-220g, reared in a barrier environment)

I. Standardized Sham Operation Procedure (Modified Control Method)

1. Anesthesia and Body Position
   • 10% chloral hydrate (3.5ml/kg, intraperitoneal injection) combined with 2% lidocaine (0.1ml for incision infiltration)
   • Fixed in supine position, depilated the anterior cervical region, and disinfected with iodophor-alcohol 3 times
   • Maintained rectal temperature at 36.8±0.5℃ during the operation (controlled by a body temperature maintenance instrument)
2. Vascular Dissection Operation
   • Made a 2cm longitudinal incision in the midline of the anterior neck, and separated the sternohyoid muscle with micro forceps (precision 0.1mm)
   • Exposed the bifurcation of the left common carotid artery (CCA), and dissected 5mm each of the external carotid artery (ECA) and internal carotid artery (ICA) (preserving the vagus nerve)
   • Key Control Step: Do not ligate the ECA, only use 8-0 silk thread to loosely circle and mark (simulate thread embolization operation but do not insert the thread)
   • Rinsed the surgical field with normal saline, confirmed no bleeding, and sutured layer by layer (intradermal suture of the skin with 6-0 absorbable thread)
3. Postoperative Resuscitation
   • Monitored with a constant temperature blanket (37℃) until the rats opened their eyes voluntarily, and reared them in individual cages (to avoid interference from the model group)
   • Recorded respiratory rate and corneal reflex every hour within 6 hours after operation, and administered subcutaneous injection of 0.9% NaCl (5ml/kg) for fluid replacement

II. Validation of Sham Operation Efficacy (Double-Blind Evaluation)

Additional Validation: Transcranial Doppler (TCD) was used to detect the middle cerebral artery (MCA) blood flow velocity at 48h postoperation (Peak Systolic Velocity, PSV ＞50cm/s, difference from preoperative baseline ＜10%)

III. Control Cycle Management (Synchronized with the Model Group)

• Acute Phase (0-24h): Observed incision exudation every 2 hours, and administered ampicillin (20mg/kg, intramuscular injection) for infection prevention
• Subacute Phase (2-3d): Weighed the rats daily (weight loss ＜5%), and recorded food intake (≥15g/d)
• Special Records: Operation duration (accurate to minutes, difference from the model group ＜2min), blood loss (cotton ball weighing method ＜0.1g)

IV. Histological Control Detection (Parallel Operation with the Model Group)

1. TTC Staining (72h Postoperation)
   • Before brain extraction, perfused 200ml of normal saline through the ascending aorta, and made 2mm coronal sections (6 sections in total)
   • Stained with 2% TTC staining solution (pH7.4, containing 0.1% NaN3) at 37℃ in the dark for 25 minutes
   • Image Processing: Measured the infarct volume using ImageJ (expected to be ＜1% of brain volume in the sham operation group)
2. Blood-Brain Barrier Detection
   • Injected 2% Evans Blue (4ml/kg) via the tail vein at 72h postoperation, and extracted the brain 2 hours later
   • Homogenized the brain tissue and centrifuged (3000rpm, 10min), then measured the OD620 value of the supernatant with a spectrophotometer
   • Evans Blue content in the sham operation group ＜5μg/g (＞20μg/g in the model group)

V. Quality Control Key Points

1. Operation Standardization: Performed by the same surgeon (with experience in over 200 MCAO operations), and the sham operation includes an additional "vascular dissection-repositioning" step (to simulate thread embolization insertion)
2. Blinding Implementation: Neurological scoring and specimen processing were completed by researchers unaware of the grouping (double-blind coding system)
3. Statistical Preset: Independent samples t-test was used to compare the sham operation group and the model group, with P＜0.01 considered a statistically significant difference