Viral Manipulation of Neural Stem/Precursor Cells - Viral Vector Approaches in Neurobiology and Brain Diseases - page 269

2. Load the syringe with the cell suspension containing trans-
duced NPCs;
3. After the vein is located, insert the needle by directing the
needle into the vein with its bevel pointing upward at an angle
of approximately 20°. Slowly insert the needle visualizing it as
it enters the vein and inject the cell suspension ( see Note 13 );
4. To assess in situ proliferation of i.v-injected NPCs, mice can be
i.p.-treated with bromodeoxyuridine (BrdU, 50 mg/kg) for
three consecutive days and sacrificed soon thereafter [ 26 , 86 ].

1. Weigh the animal and calculate the appropriate dose of anes-
thesia. Deeply anesthetize the animal with an appropriate
injectable (e.g., intraperitoneal injection of ketamine 100 mg/
kg and xylazine 10 mg/kg) or breathable (e.g., isoflurane)
anesthesia ( see Note 14 ). Shave the fur on dorsal area of the
mouse in the region of interest and clean the skin with 70 %
ethanol or another disinfectant;
2. Under a surgery microscope, make a midline incision using a
sterile scalpel. Then separate the muscles from either side of
the vertebral column using an iridectomy scissor and perform
a laminectomy at the desired level using mouse laminectomy
forceps, Dumont forceps and tissue forceps ( see Note 15 );
3. Measure the volume of cell suspension to inject (250 nl/injec-
tion site) with 5 μl Hamilton syringe and then place it on a
sterile surface. Draw it up by capillarity using a glass capillary
(diameter 40-50 μm) connected to an insulin syringe. While
visualizing the spinal cord under a surgery microscope, slowly
inject the NPCs at the site(s) of interest;
4. Close the incision by applying two to three staples (depending
on size of incision) to the skin and keep the animal warm in an
individual cage until it fully recovers;
5. Treat mice with the analgesic buprenorphine (0.05-0.1 mg/kg)
following surgery.

3.4.3 Surgical
Transplantation of NPC into
the Spinal Cords

3.5 Organotypic
Slice Cultures

1. Humanely kill the animal(s) in accordance to the laws and
guidelines regarding animal research in your country and
institution. Decapitate the dead animal;
2. Remove the brain from the skull and transversely slice it in
350 μm thick slices using a tissue chopper. Collect the slice cut
at the level of the hippocampus ( see Note 16 );
3. Immediately transfer each slice on a filter in the bottom of a
culture plate with the appropriate culture medium;