Numbers of PET scans at each time point.

<p>After status epilepticus induced by pilocarpine administration, four rats were scanned in acute period. Three rats were scanned in subacute period without PET acquisition in acute period and four rats were scanned in chronic period after status epilepticus. Among the four rats scanned in acute period, a rat was scanned in subacute period and among the three rats scanned in subacute period, a rat was scanned in chronic period, repetitively (dotted line).</p

Spatial distribution of changes of mGluR5 BP_ND in chronic epilepsy model rats on [¹¹C]ABP688 PET.

<p>Compared with controls, BP_ND decreased in the bilateral amygdala and dorsal hippocampi (A: p<0.001, uncorrected). (B) Nonparametric post-hoc VOI-based analysis performed on two clusters of decreased BP_ND on voxel-based analysis. BP_ND of cluster 1 (C1) on dorsal hippocampus in chronic epilepsy models decreased significantly compared to controls (p = 0.048). There was a trend for decrease in BP_ND of cluster 2 (C2) on amygdala in chronic epilepsy models compared to controls (p = 0.06).</p

Representative time activity curves (TACs) of experimental epilepsy models.

<p>TACs in the caudate, hippocampus, frontal cortex, amygdala and cerebellum for 60[¹¹C]ABP688 PET in the control (A), and epilepsy model rats in acute period (B), subacute period (C), and chronic period (D) after pilocarpine-induced status epilepticus. (E) Predefined VOIs for TACs and quantitative analyses. CP: Caudate-putamen; H: Hippocampus; F: Frontal cortex; Cb: Cerebellum, and A: Amygdala. SUV: Standardized uptake unit, defined as activity (MBq/mL) within the volume of interest divided by the injected dose per body weight (MBq/g).</p

mGluR5 binding potential (BP_ND) decreased in epilepsy models according to time periods after status epilepticus.

<p>(A–C) BP_ND decreased globally in acute period after status epilepticus. In subacute period, mGluR5 BP_ND recovered in caudate-putamen, while decreased yet in hippocampus and amygdala, and in chronic period, there was no significant difference in BP_ND between model rats and controls using VOI-based analysis. (D) Representative BP_ND parametric maps for model rats and controls were consistent with temporal patterns of VOI-based analysis. Note that BP_ND was visually normalized except in hippocampus and amygdala in chronic period, which corresponded to the voxel-based analysis (arrowheads: hippocampus, arrows: amygdala). Error bars represent standard errors of the mean (SEM). * p<0.05; ** p<0.01.</p

<i>In Vivo</i> Bioluminescence Imaging for Prolonged Survival of Transplanted Human Neural Stem Cells Using 3D Biocompatible Scaffold in Corticectomized Rat Model

<div><p>Stem cell-based treatment of traumatic brain injury has been limited in its capacity to bring about complete functional recovery, because of the poor survival rate of the implanted stem cells. It is known that biocompatible biomaterials play a critical role in enhancing survival and proliferation of transplanted stem cells via provision of mechanical support. In this study, we noninvasively monitored <i>in vivo</i> behavior of implanted neural stem cells embedded within poly-l-lactic acid (PLLA) scaffold, and showed that they survived over prolonged periods in corticectomized rat model. Corticectomized rat models were established by motor-cortex ablation of the rat. F3 cells expressing enhanced firefly luciferase (F3-effLuc) were established through retroviral infection. The F3-effLuc within PLLA was monitored using IVIS-100 imaging system 7 days after corticectomized surgery. F3-effLuc within PLLA robustly adhered, and gradually increased luciferase signals of F3-effLuc within PLLA were detected in a day dependent manner. The implantation of F3-effLuc cells/PLLA complex into corticectomized rats showed longer-lasting luciferase activity than F3-effLuc cells alone. The bioluminescence signals from the PLLA-encapsulated cells were maintained for 14 days, compared with 8 days for the non-encapsulated cells. Immunostaining results revealed expression of the early neuronal marker, Tuj-1, in PLLA-F3-effLuc cells in the motor-cortex-ablated area. We observed noninvasively that the mechanical support by PLLA scaffold increased the survival of implanted neural stem cells in the corticectomized rat. The image-guided approach easily proved that scaffolds could provide supportive effect to implanted cells, increasing their viability in terms of enhancing therapeutic efficacy of stem-cell therapy.</p></div

Schematic representation of the procedure for <i>in vivo</i> optical imaging.

<p>(A) The protocol is for <i>in vivo</i> monitoring of F3-effLuc cells implanted in a corticectomized rat model. The motor cortex region of the Sprague-Dawley rat brain was surgically removed at the given coordinates and after 7 days, the rats were transplanted with F3-effLuc cells alone or F3-effLuc/PLLA scaffold complexes, and then administered cyclosporine A everyday. The grafted cells were monitored at 0, 1, 3, 5, 8, 11, and 14 days using a bioluminescence-imaging device. At the end of the implant period, histological analyses were performed using hematoxylin and eosin (H&E) staining and immunohistochemistry. (B) Behavior tests were performed 7 days after motor cortex ablation. The traumatic brain injury (TBI) models were evaluated by forelimb placing tests and whisker tactile tests in normal and corticectomized rats (n = 10).</p

<i>In vivo</i> bioluminescence imaging of the implanted F3-effLuc/PLLA scaffold in a corticectomized rat model.

<p>(A) After F3-effLuc cells were incubated within the PLLA scaffold for 2 hr, the cell/scaffold complex was implanted into the ablated motor cortex area of the rat brain. Firefly luciferase bioluminescence imaging was performed over 14 days. The prolonged luminescence signals in F3-effLuc cells within the PLLA scaffold were clearly visualized in the ablated area. (B) Quantitative ROI analysis showed significantly enhanced survival duration for F3-effLuc cells within the PLLA scaffold (n = 6). P value, * <0.005.</p

<i>In vitro</i> proliferative effect in F3-effLuc cells within the PLLA scaffold.

<p>(A) Scanning electron microscope (SEM) analysis was conducted to confirm adhesion of F3-effLuc cells to the PLLA scaffold. SEM images showed that F3-effLuc cells were stably attached onto the microfibers of the PLLA scaffold. (B) The luciferase intensity was quantified after F3-effLuc cells were incubated with the sterile PLLA scaffold. F3-effLuc cells incorporated within the PLLA scaffold were stably proliferated at 10 days.</p

Validation of stem cell characteristics in F3 cells and F3-effLuc cells.

<p>Flow cytometric analysis showed F3 and F3-effLuc cells are positive for the stem cell surface marker, (a) CD44, and the intracellular marker s, (b) Nestin, (c) Ki67, (d) Sox1, and (e) Sox2.</p

<i>In vitro</i> luciferase reporter activity in F3-effLuc cells.

<p>(A) Retroviral construct that contains the <i>effLuc</i> gene and Thy1.1 (CD90.1), linked with an IRES (internal ribosomal entry site). (B) Magnetic-activated cell sorting (MACS) was performed to collect the F3-effLuc cells. Flow activated cell sorting (FACS) analysis showed that more than 90% of cells were successively transfected with the <i>effLuc</i> vector. (C) The luciferase activity (n = 3) of F3-effLuc cells cultured in a 96-well plate were measured using an IVIS-100 optical imaging device. Firefly luciferase activity continuously increased in F3-effLuc cells in proportion to cell number, and (D) quantitative analysis showed a linear relationship between the cell number and bioluminescence signals.</p