2.14 shRNA transfection
The Gal-3 knockdown lentiviruses (Hanheng China) were transiently
transfected into the cells using a transfection reagent (Beyotime China)
following the manufacturer’s instructions. Transfection efficiency was
evaluated by harvesting the cells after 3 days. The sequence of Gal-3
shRNA and control is presented in Table 2. Stable HT22 cell lines with
Gal-3 knockdown were established through lentivirus infection and
subsequent selection with 5ug/ml puromycin (Sigma, China) for
approximately one week. Finally, the stability of the generated cell
lines was confirmed by Western blot analysis and qPCR.
Mice were anesthetized with intraperitoneal injection of chloral hydrate
(400 mg/kg, Sigma) and secured onto a stereotactic frame (RWD, Shenzhen,
China). The rectal temperature was maintained at 37.5°C using a heating
blanket. A midline scalp incision was performed to fully expose the
bregma and lambda. For intracortical injections, a borehole was created
on the left side using a high-speed drill at the following coordinates
relative to bregma (X=1.0 mm, Y=0.80 mm). The syringe was connected to a
microinjection pump and the needle was inserted into the brain through
the burr hole (Z=1.5 mm from the bone surface) to infuse 1μl Gal-3
knockdown adeno-associated virus(AAV) (Hanheng
China)44. The Gal-3 shRNA sequence and control are
presented in Table 2. Following the surgery, the cranial defect was
sealed using bone wax, and the incision was closed with sutures.