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A 7-day study was designed to detect gingipain-induced hippocampal neurodegeneration with Fluoro-Jade C (FJC), a fluorescent stain that has been shown to exhibit maximum staining of degenerating neurons 1 week after a neurotoxic insult (107). Fifteen 8-week-old male BALB/c mice (Envigo) were used in the study. Animals were group-housed (n = 2 to 4 per cage) in plastic cages. Animals were maintained on a 12/12-hour light/dark cycle, with the RT maintained at 22° + 2°C and approximately 50% humidity, and received standard rodent chow and tap water ad libitum.

Mice were anesthetized using isoflurane (2%, 800 ml/min O2). Bupivacaine/epinephrine was used for local analgesia, and carprofen was used for perioperative/postoperative analgesia. A solution of RgpB (5 μg/ml) + Kgp (5 μg/ml) + 5 mM l-cysteine was prepared in sterile saline. Bilateral injections of 0.5 μl were made into coordinates from bregma: anteroposterior −2.0, lateral ±1.5, and ventral −1.4 mm from dura at a rate of 0.1 μl/min with a 5-min rest period using a Hamilton syringe (10-μl syringe with corresponding 30-gauge blunt tip needle; model no. 80308) and the stereotactic micromanipulator (Ultra Micro Pump III with Micro4 Controller, World Precision Instruments). When compound delivery was complete, the needle was left in place for 5 min and then withdrawn such that it took approximately 1 min to fully withdraw the needle.

Mice received a single administration of vehicle or drug 1.5 hours before stereotactic gingipain injection. Inhibitor-treated mice received COR271 (100 mg/kg) in PBS by oral gavage and COR286 (20 mg/kg) in 25% pluronic F127 subcutaneously at a dose volume of 5 and 10 ml/kg, respectively. Vehicle-treated mice received either PBS or pluronic.

Seven days later, mice were anesthetized with isoflurane (2%, 800 ml/min O2) and perfused with PBS. Brains were harvested, fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm.

Serial sections 200 μm apart through the entire hippocampus were stained with the FJC Ready-to-Dilute Staining Kit (Biosensis) according to the manufacturer’s protocol, and FJC-positive cells in the CA1 area were counted on an Olympus BX61 motorized microscope.

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