Tribromoethanol

The mouse SCI model was established as previously described [51 (link)]. Briefly, male C57BL/6J mice of 12 weeks of age were anesthetized with 330 mg/kg of 2-2-2 Tribromoethanol (Sigma) intraperitoneally and undertook a laminectomy at the T12 vertebrae. Then a contusion SCI (hemitransection) was induced on the exposed dorsal surface of dura mater through using a SCI micro-scissor devise (Infinite Horizon impactor at 60 kDyn, Precision Systems and Instrumentation, USA). Sham group includes the complete procedures without hemitransection of the spine. During recovery, mice were maintained in an isothermic cage. For animal welfare, all animal experimental procedures were approved by the Ethics Committee of the third hospital, Hebei Medical University.

Eight-week-old male C57BL/6 mice were used for measurement of body temperature and locomotor activity using biotelemetry transmitters (E-mitter, STARR Life Science Corp., Oakmont, PA, USA) as previously described [34 (link)]. Briefly, mice were anesthetized with tribromoethanol (250 mg/kg, Sigma-Aldrich) and a biotelemetry transmitter was implanted into the abdominal cavity. Output was monitored by a receiver (ER-4000, STARR Life Science Corp.) laid under each cage. A data acquisition system (Vital View, STARR Life Science Corp.) was used for automatic data collection and analysis. Mice were adapted to the vital view system for 24 h prior to SPX injection.

Eight-week-old male MPTP-induced Parkinson model mice (on a C57BL/6J background, MPTP-PD) and recommended control (C57BL/6J, CN) were purchased from the Shanghai Model Organisms Center, Inc, Shanghai, China. The animals were anesthetized with 500 mg/kg tribromoethanol (Sigma, Saint Louis, MO, USA) and were killed by cervical dislocation. After the animals were sacrificed, brain tissues (cerebral cortex, hippocampus, striatum and cerebellum) were isolated, quickly frozen in liquid nitrogen and stored in liquid nitrogen (n = 1). Four brain regions were pooled for nuclei isolated according to the ‘Nuclear Isolation by Single-cell RNA Sequencing’ protocol of 10X Genomics^®. In brief, the tissue was lysed in a chilled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40). Then, the suspension was filtered and nuclei were pelleted by centrifugation. Nuclei pellets were then washed in ‘nuclei wash and resuspension buffer’ (1× PBS, 1% BSA, 0.2 U/μL RNase inhibitor, 2 mM DTT), filtered and pelleted again. Cell count was then performed to calculate the concentration of nuclear suspension.

A linear accelerator (Varian Clinac 1600 C-2 (250 Mu/min, 2.4 Gy/min) and True Beam STX (600 Mu/min, 5.6 Gy/min), Radiation Oncology Systems, LLC, San Diego, CA, USA) with 6 MV nominal photon energy were used to irradiate mice on P14. The mice were anesthetized with an intraperitoneal (i.p.) injection of tribromoethanol (Sigma, Stockholm, Sweden), placed on a polystyrene bed in prone position (head to gantry) and irradiated with a symmetrical 2 × 2 cm radiation field. A tissue equivalent material covered the head to obtain an even radiation dose distribution in the underlying tissue. The source to skin distance was approximately 99.5 cm and the irradiated tissue received a single absorbed dose of 8 Gy with a dose variation of ±5%. Using the LQ model^{59 (link)} and an alpha/beta ratio of 3 for late effects in the normal brain tissue, the acute exposure of 8 Gy is equivalent to approximately 18 Gy when delivered in repeated 2 Gy fractions. Animals were kept on a warm bed (36 °C) both before and after CIR to maintain body temperature. Control animals were anesthetized but did not receive any CIR.