Fig 1.
The experiment-timeline design.
The picture of the rat head represents the parameters used for tumor induction: 10µL of C6Luc solution were administered during 10 min, according to the following coordinates: AP 2 mm; LL 2 mm; D 2.5 mm from bregma. The experimental groups were divided as control, sham and tumor. In vivo experiments comprised behavioral tests, BLI, and MRI analysis, using the same animals of each group to perform all timepoints (X) (4 rats per group, totalizing 12 rats). Ex vivo experiments comprised BLI and histological analysis. A new group of 48 rats was used (4 rats per each group and each time point) until the 21st day after the induction (O). 1 rat per group/time was used in the BLI analysis, and the same group of rats was used in the BLI analysis to complete the number of 4 rats per group/ time used for histological analysis. The “X” into the red square represents that the same 12 animals of the in vivo experiments were used in the last time point of the ex vivo experiments (28th day).
Fig 2.
Dynamic parameters for gait analysis by CatWalk test.
(A) Animal preview and automatic analysis of paws classification (RF, right forepaw in blue color; RH, right hindpaw in pink color; LF, left forepaw in yellow color; LH, left hindpaw in green color); (B) footprint spatial view (cm); (C) footprint timing view (s); (D) 2D footprint intensities measured in arbitrary units (range: 0–255 A.U.); (E) footfall patterns; (F) 3D footprint intensities (range: 0–255 A.U.); and (G) footprint dimensions of each paw (cm2). Abbreviations: PP, print positions; BOS, base of support; SL, stride length; SC, step cycle; mCA, maximum contact area; s, seconds; cm, centimeters.
Fig 3.
Tumor growth analysis by image.
(A) Coronal plane of T2-weighted MRI images, at base; (B) C6 glioma cells injection localization according to coordinate (2.0;2;0;2.5 mm); (C–F) Coronal plane of T2-weighted MRI images at days 7, 14, 21, and 28 after tumor brain induction ().; (G–H) Microscopic and macroscopic images at base and (I–J) at the day 28 after tumor induction; (K–N) in vivo 2D-bioluminescence imaging at days 7, 14, 21, and days after tumor induction and corresponding brain (ex vivo) with bioluminescence signal after 10 min of brain extraction; (O) MRI volumetric results at each time point. (P) The mean tumor volume by MRI is plotted against the mean BLI in vivo signal at four points of analysis (n = 4 per group), and the Pearson correlation coefficient (r = 0.96) between both results plotted and (Q) bioluminescence tumor growth results of each time point (n = 4 per group).
Fig 4.
Spontaneous locomotor analysis by Actimeter.
The following parameters were analyzed in slow (S) and fast (F) thresholds: (A–B) Horizontal movement (MOV), (C–D) stereotypic movements (STE), and (E–F) rearing (REA) or vertical movement. Red line: tumor group (n = 4), blue line: sham group (n = 4), and black line: control group (n = 4).
Table 1.
Estimated mean and 95% confidence interval of each group for spontaneous motor activity analysis.
Fig 5.
Temporal parameters of gait analysis by CatWalk test.
Stand (A–C), step cycle (D–F), cadence (steps/s) (G), duration (H), for control, sham, and tumor groups (n = 4 per group) and each of the paws or side paws. Abbreviations: s, seconds; RF, right forepaw; RH, right hindpaw; LF, left forepaw; LH, left hindpaw.
Fig 6.
Spatial parameters of gait analysis by CatWalk test.
(A–C) SL, stride length (cm); (D–F) mAC, maximum contact area (cm²); (G–I) PP, print positions (cm); (J–L) BOS, base of support (cm) for control, sham, and tumor groups (n = 4 per group) and each of the paws or side paws; (M–O) the basal and 28 days after induction spatial parameters are represented. Abbreviations: RF, right forepaw; RH, right hindpaw; LF, left forepaw; LH, left hindpaw; LS, left side; RS, right side; FP, forepaw; HP, hindpaw.
Table 2.
Estimated mean and 95% confidence interval of each group in some temporal parameters of gait analysis.
Table 3.
Estimated mean and 95% confidence interval of each group in some spatial parameters of gait analysis.