Effect of exosomes derived from multipluripotent mesenchymal stromal cells on functional recovery and neurovascular plasticity in rats after traumatic brain injury methods
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Methods
Wistar rats were subjected to TBI followed by tail vein injection of 100 µg protein of exosomes derived from MSCs or an equal volume of vehicle phosphate-buffered saline (n = 8/group) 24 hours later. To evaluate cognitive and sensorimotor functional recovery, the modified Morris water maze, neurological severity score and footfault tests were performed. Animals were sacrificed at 35 days after TBI. Histopathological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation.
Methods
MSC expansion was performed according to previously described methods. Briefly, bone marrow from adult male rats was mechanically harvested by flushing the cavity of the femurs with PBS, and the cells were washed and suspended in culture medium. Three days later, cells that tightly adhered to the plastic flasks were considered as P0 MSCs. MSCs were conventionally cultured with a modified MEM medium (Hyclone, Logan, UT, USA) containing 20% fetal bovine serum (Gibson Laboratory, Grand Island, NY, USA) and penicillin-streptomycin on 75cm tissue culture flasks (Corning, St Louis, MO, USA). For the exosome isolation, conventional culture medium was replaced with an exosome-depleted fetal bovine serum-contained (EXO-FBS-250 A-1, System Biosciences, Mountain View, CA, USA) medium when the cells reached 60% to 80% confluence, and the MSCs were cultured for an additional 48 hours. The me...
Morris Water Maze (MWM) Test
To measure spatial learning impairments, an updated version of the MWM test was used. The procedure was modified from previous versions, and has been used for spatial memory assessment in rodents with brain injury. The MWM test was performed monthly postinjury. At each testing interval, animals were tested with 4 trials per day for 5 consecutive days on Day 31-35 after mTBI. A blue swimming pool (1.8 m in diameter) was located in a large room, where there were many clues external to the maze (e.g., pictures on the walls, lamps and a camera on the ceiling); these were visible from the pool and presumably used by the rats for spatial orientation. The position of the cues remained unchanged throughout the experiment. Data collection was automated using the HVS Image 2020 Plus Tracking System (US HVS Image, San Diego, CA), as described previously. For data collection, the swimming pool wa...
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Wistar rats were subjected to TBI followed by tail vein injection of 100 µg protein of exosomes derived from MSCs or an equal volume of vehicle phosphate-buffered saline (n = 8/group) 24 hours later. To evaluate cognitive and sensorimotor functional recovery, the modified Morris water maze, neurological severity score and footfault tests were performed. Animals were sacrificed at 35 days after TBI. Histopathological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation.
MSC expansion was performed according to previously described methods. Briefly, bone marrow from adult male rats was mechanically harvested by flushing the cavity of the femurs with PBS, and the cells were washed and suspended in culture medium. Three days later, cells that tightly adhered to the plastic flasks were considered as P0 MSCs. MSCs were conventionally cultured with a modified MEM medium (Hyclone, Logan, UT, USA) containing 20% fetal bovine serum (Gibson Laboratory, Grand Island, NY, USA) and penicillin-streptomycin on 75cm tissue culture flasks (Corning, St Louis, MO, USA). For the exosome isolation, conventional culture medium was replaced with an exosome-depleted fetal bovine serum-contained (EXO-FBS-250 A-1, System Biosciences, Mountain View, CA, USA) medium when the cells reached 60% to 80% confluence, and the MSCs were cultured for an additional 48 hours. The media were then collected and exosomes were isolated by ExoQuick exosome isolation method according to the manufacture's instruction. Briefly, ExoQuick-TC (2.5 ml) was added to 10 ml of media, incubated 12 hours at 4°C, and centrifuged at 1500 × g for 30 min to obtain pelleted exosomes. The...
To measure spatial learning impairments, an updated version of the MWM test was used. The procedure was modified from previous versions, and has been used for spatial memory assessment in rodents with brain injury. The MWM test was performed monthly postinjury. At each testing interval, animals were tested with 4 trials per day for 5 consecutive days on Day 31-35 after mTBI. A blue swimming pool (1.8 m in diameter) was located in a large room, where there were many clues external to the maze (e.g., pictures on the walls, lamps and a camera on the ceiling); these were visible from the pool and presumably used by the rats for spatial orientation. The position of the cues remained unchanged throughout the experiment. Data collection was automated using the HVS Image 2020 Plus Tracking System (US HVS Image, San Diego, CA), as described previously. For data collection, the swimming pool was subdivided into four equal quadrants formed by imaging lines. At the start of each trial, the rat was placed at one of four fixed starting points, randomly facing toward a wall (designated North, South, East and West) and allowed to swim for 90 seconds or until it found the platform which was tran...
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