Figure 1. The experimental schematic diagram and operation procedure.

(a) The schematic diagram for imaging the tracer’s transportation into the ISS or ISF flow process. MRI scans were performed before and after the injection of Gd-DTPA into the ISS of the rat brain under anesthesia (A). The MRI scans were performed at 30 min and each hour post-injection until no enhancement could be demonstrated on the subtracted MR images, which was obtained by a pixel-by-pixel co-registration and subtraction of the post-injection images from the pre-injection images (B). Developed software was used to perform the post-processing of the images. The transportation of the tracer in the ISS on the global, whole-brain scale can be shown three-dimensionally, and the flow or diffusion parameters can be derived from the time profile of the net enhancement on the subtracted images. (b) The procedure in Ts and Tc groups. Ts, Tss and Cs indicate the stimulation groups; Tc and Cc indicate the non-stimulation control groups. The anesthetized rat was scanned to acquire a basic image for further subtraction and was immobilized so that a small trephine hole could be made using a stereotaxic instrument (Lab Standard Stereotaxic-Single, Stoelting Co, Illinois, USA). 2 μl Gd-DTPA solution (10 mmol/L) was automatically infused into the brain ISS at the rate of 0.2 μl/min in all groups. The stereotactic coordinates for T. were set at the bregma: −3.0 mm, lateral: 2.0 mm, vertical: 6.0 mm and for Cn. at bregma: + 1.0 mm, lateral: 3.5 mm, vertical: 5.0 mm. The rats were then placed in the scanner to acquire the post-injection MR images at 0.5 h. When an MRI scan was applied at the 0.5-hour timepoint following injection, the painful stimulation was conducted in the Ts and Tss groups while a positive righting reflex was maintained to ensure the conscious state of the rats (see details in Fig. 4). No stimulation was conducted in the Tc or Cc groups. After the stimulation period, the rats were anesthetized and a series of MRI scans were performed.