All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Materials
- Preparation of lipopolysaccharide (LPS) injection
- Add 25 mL of distilled water to 25 mg of LPS powder derived from Salmonella typhimurium to a final concentration of 1 mg/mL. Store the injection in a sterile tube at 4 °C.
CAUTION: LPS is toxic.
- Prepare a 2% Evans blue (EB) solution in normal 0.9% saline solution to keep EB injection at working concentration.
2. Animals
Administer LPS to male Sprague-Dawley (SD) rats, aged 10 weeks (average weight: 280 ± 20 g) by intraperitoneal injection.
CAUTION: If rats are purchased from another organization, then the adaptive phase should not be less than 7 days.
3. LPS Injections
- Inject LPS intraperitoneally into each rat at a dose of 1 mg/kg and return the rats to their home cage.
NOTE: Anesthesia is not necessary.
- Six hours later, inject the same dose of LPS injection into the rats.
- Sixteen hours later inject the same dose of LPS into the rats.
CAUTION: Injecting SD rats with LPS at a dose of 1 mg/kg may result in a 5% mortality rate. The mortality rate could further increase in younger or older rats or pregnant female rats.
- Return the rats to their cages after LPS injection and provide ad libitum access to food and drink.
NOTE: Rats will exhibit a distinct systemic inflammatory response, and thus it is essential that the cages remain clean throughout the experiment. Rats should be frequently (2x day) monitoring for weight, general appearance and attitude after the first LPS injection. If the rats start to exhibit a hunched posture, unwillingness to move, significant weight loss (>20%), porphyrin staining that they be humanely euthanized prior to the 7-day EB study end point.
4. Sample Collection
- EB injection
- Seven days after the first injection, anesthetize the rat intraperitoneally according to your approved IACUC protocol.
- Wait 1–2 min until the rats do not show corneal reflex responses. Perform a 5–8 mm-deep cardiac puncture on each rat; the puncturing point should be 5 mm to the left margin of sternum at the 3rd and 4th intercostals space.
- Wait until blood recovery is observed, and then inject EB at a dose of 0.2 mL/100 g body weight into the left heart ventricle.
CAUTION: Cardiac puncture and EB injection should be carefully performed. EB might be injected into thoracic cavity or pericardium instead of the left heart ventricle.
- Suck back with an empty tube and replace this volume with an EB tube before injection to help reduce the failure rate (e.g., injection to rib cage by error).
- Keep the rat in a supine position for 10 min.
NOTE: If sample is not used to detect EB leakage in immunofluorescence imaging, then this step may be omitted.
- Gross observation
- Perform cardiac perfusions using ice-cold 1 M phosphate buffer saline (PBS) to clear the cerebral vasculature and brains.
- Using a scalpel, make an abdominal incision across the entire length of the diaphragm.
- Cut through ribs just left of the rib cage midline.
- Open up the thoracic cavity. Use clamps to expose the heart and to facilitate drainage of blood and other fluids.
- While steadily holding the heart with forceps (the heart should still be beating), directly insert a needle into the protrusion of the left ventricle to extend this to about 5 mm. Secure the needle at that position by clamping near the point of entry.
CAUTION: Do not excessively extend the needle, as it can pierce the interior wall and compromise the circulation of solutions.
- Release the valve to allow the flow ice-cold PBS solution (200–300 mL) at a slow and steady rate at around 20 mL/min using a perfusion pump. If the animals require fixation instead of gross observation, then use the same dose of ice-cold 0.9% saline solution.
- Using sharp scissors, make an incision in the atrium, ensuring that the solution continuously flows. If the fluid does not flow freely or is coming from the animal's nostrils or mouth, reposition the needle.
- Screen for CMHs by gross observation.
NOTE: If the sample is not used in calculating the number of CMHs by gross observation, then this step may be omitted.
- Fixation
- Perform cardiac perfusions using ice-cold 0.9% saline solution (200–300 mL) to clear the cerebral vasculature and brains.
- Perform cardiac perfusions using ice-cold 4% paraformaldehyde for fixation.
- Decapitate the rat, isolate the brain, and immerse the brain in 20% sucrose solution for at least 6 h.
- Change the solution into 30% sucrose and fix for another 6 h.
- Prepare 10 mm-thick brain tissues sections using a cryostat.
5. Hematoxylin and eosin (HE) Staining
NOTE: This procedure is performed using a HE Staining Kit.
- Wash the slides in distilled water.
- Stain in hematoxylin solution for 8 min. Wash in running tap water for 5 min.
- Differentiate in 1% acid alcohol for 30 s. Wash in running tap water for 1 min.
- Stain in 0.2% ammonia water (Bluing) or saturated lithium carbonate solution for 30 s to 1 min. Wash in running tap water for 5 min.
- Rinse in 95% alcohol (10 dips).
- Counterstain with eosin solution for 30 s to 1 min.
- Dehydrate through 95% alcohol, two changes of absolute alcohol, for 5 min each.
- Clear in xylene for 30 s.
- Mount using Liu's method.
- Analyze using a brightfield fluorescence microscope.
NOTE: Red blood cells released from blood vessels, which are the components of CMHs, appear in red-orange under HE staining.
