This method can help answer key questions in cerebral micro-hemorrhage field such as the etiology, distribution, and detection of cerebral micro-hemorrhage and so on. The main advantage of this technique is that LPS injections stably induce inflammation. In addition, LPS injection is quite simple, economical, and cost-effective.
Though this method can provide insight into cerebral hemorrhage induction, it can also be applied to other brain disorders such as depression, schizophrenia, Parkinson's disease, Alzheimer's disease, and prion disease. Administer LPS at a dose of one milligram per kilogram of body weight to 10-week-old male Sprague Dawley rats by intraperitoneal injection, then return each rat to the home cage. Repeat the LPS injection after six hours and 16 hours.
Please note that injecting SD rats with LPS at a dose of one milligram per kilogram may result in a 5%mortality. The mortality 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.
Under terminal anesthesia, perform a five to eight-millimeter deep cardiac puncture on each rat. The puncture point should be five millimeters to the left margin of the sternum at the third and fourth intercostal space. Hold the needle in place and replace the empty tube with a tube containing Evans blue.
Wait until blood recovery is observed, and then inject Evans blue at a dose of 0.2 milliliters per 100 grams of body weight into the left heart ventricle. Keep the rat in a supine position for 10 minutes if assessing Evans blue leakage through the blood/brain barrier by immunofluorescence imaging. Perform cardiac perfusions using ice-cold 200 to 300 milliliters of 0.9%saline solution to clear the cerebral vasculature and brains.
Switch to ice-cold 4%paraformaldehyde for fixation. Then, after isolating the brain, immerse it in 20%sucrose in PBS for at least six hours or until the brain sinks to the bottom of the tube. Change the solution to 30%sucrose and fix it for another six hours.
Finally, prepare 10-millimeter thick brain tissue sections using a cryostat. Wash the slides with PBS three times for five minutes each, then incubate slides with 4, 6'diamidino-2-phenylindole or DAPI solution for 15 minutes at room temperature. After washing the slides in PBS as before, mount the slides with PBS-glycerol solution.
Capture images on a fluorescence microscope. EB deposition is indicated by red fluorescence, nuclei are indicated by blue fluorescence. Begin H&E staining by washing the slides in distilled water.
Then immerse in hematoxylin solution for eight minutes. Alternatively, for Perl Prussian blue staining, stain in reaction solution with equal-parts mixture of ferrocyanide and hydrochloric acid for 10 minutes. Following staining, wash in running tap water for five minutes.
Differentiate in 1%acid alcohol for 30 seconds. After washing in running tap water for one minute, stain in 0.2%ammonia water or saturated lithium carbonate solution for 30 seconds to one minute. Next, wash in running tap water for five minutes.
Counter-stain with eosin solution for 30 seconds to one minute. Dehydrate through 90%alcohol, then 95%alcohol, and absolute alcohol for 0.5 to two minutes each. After dehydration, clear in xylene for 30 seconds.
After mounting, analyze the H&E staining using a brightfield fluorescence microscope. The red blood cells released from blood vessels, which are components of the CMHs, appear in red-orange under H&E staining. Surface CMHs can be detected by gross observation as indicated by the red arrows.
Evans blue staining reveals visible red fluorescence where Evans blue molecules leaked from an injured blood-brain barrier. H&E staining shows red blood cells found outside the capillaries, and Prussian staining reveals ferric ion points derived from lysis of red blood cells. While attempting this procedure, it is important to remember that the dose of LPS to induce cerebral micro-hemorrhage is larger than that used in Parkinson's disease or schizophrenia model.
Therefore, the environment of animals should be kept clean to reduce mortality rate. Following this procedure, other methods like electron microscopy, magnetic resonance imaging, and immunofluorescent imaging could be performed to determine the damage to outer structure of blood-brain barrier in cerebral micro-hemorrhage, distribution of cerebral micro-hemorrhages in brain parenchyma in vivo, as well as glial cell activation.
