C57 black mice

C57/Black mice (from Charles River Labs, Margate, UK) were housed in group cages, with unrestricted access to food and water. The mice were anaesthetised with isoflurane, placed in a stereotaxic frame and were unilaterally injected in the striatum (co-ordinates: anterior–posterior: + 0.5; medial–lateral: + 0.21; dorsal–ventral: 3.0). A small hole was drilled in the exposed skull and a Hamilton syringe was used for the delivery of the AAV- α-syn (1 µL in total, at a rate of 0.5 µL/min). The mice were then sutured and allowed to recover in a heated recovery chamber. Eight weeks later, the mice were again anaesthetised with isoflurane, placed in a stereotaxic frame and unilaterally injected in the same striatum with 5 M NaCl (2 µL in total, at a rate of 0.5 µL/min). Control mice were injected with normal saline. The syringe was left in place for 5 min before being slowly removed. The mice were then maintained under isoflurane anaesthetisia until they were sacrificed (5, 10 or 30 minutes post injection; n = 3 at each time point) and their brains were removed for western blot analysis.

Six- to eight-week-old C57 black mice were obtained from Charles River and housed under standard conditions at 22 ± 1 °C, 30% relative humidity and a 12-h light cycle as per Institutional Animal Care and Use Committee (IACUC) protocol. Mice were randomly assigned into two different groups (Control, TWEAK). An acute intrastriatal TWEAK-induced neuroinflammation model was used for this study [45 (link)]. Mice were given intrastriatal injections of 2 µL TWEAK (1 µg/µL) using stereotaxic surgery. Mice were sacrificed 24 h posttreatment to collect the striatum and SN, which were stored at −80 °C until tissue lysates could be prepared for immunoblotting the protein of interest. The use of animals and all animal-related procedures in this study (Protocol ID IACUC-19-259) were approved and supervised by the IACUC at Iowa State University, Ames, IA, USA.