Figure 1.
Image processing and ROI selection.
T2-WI (A) and T2*-maps (C) were processed into thresholded intensity (B) and T2* maps (D) to calculate areas of affected relaxation times. A control (ROI1) was selected in the unprocessed image and the mean intensity value was used as a reference to select affected pixels (B, D). Only voxels within ROI2 were included in the analysis. Also included are the non-smoothed results of the fit, presented as T2* parametric maps (E) and its standard deviation (F) (scale bars in ms) and intensity (G) and its standard deviation (H) (scale bars in arbitrary units). Note that although figs. A–D are smoothed (Paravision default for image presentation), the ROI statistics are calculated on the non-smoothed data. High resolution T2*-WI of the same animal are represented in Fig. 7.
Figure 2.
High resolution T2*- and T2 WI at different time points after striatal injections.
Upper panels represent 6-OHDA injected animals and lower panels control animals. At two days a strong hyperintensity caused by the presence of edema is visible in T2 and T2*-WI in 6-OHDA- but not in control animals. T2*-WI from 1–4 weeks demonstrated a stronger and more widespread hypointense signal in 6-OHDA injected animals comparing with control animals.
Figure 3.
Hyperintense evaluations from T2-WI after striatal injections.
A) Hyperintense distributions at 2 days postlesion in the striatum expressed as distances anterior (positive) and posterior (negative) to the injection site. Each point represents the summation of hyperintensities from two 0.3 mm-thick contiguous images. There are significant differences between control and 6-OHDA treated animals. B) Time course evolution of total hyperintense areas also differs from control animals. Continuous line represents 6-OHDA injected animals and broken line represents control animals. (*p<0.05, **p<0.01 compared between 6-OHDA and control animals, #p<0.05 compared between time points within 6-OHDA group).
Figure 4.
Hypointense evaluations from T2*- and T2 WI after striatal injections.
A) Time course evolution of total T2*-hypointensities showed no significant differences between 1, 3 and 4 weeks, but showed differences between the two groups. B) T2*-hypointense distribution at 4 weeks expressed as distance anterior (positive) and posterior (negative) to the injection site. The results show a significant difference between 6-OHDA and control animals at different distances. C) Time course evolution of total T2-hypointensities D) T2-hypointense distribution at 4 weeks at different distances to the injection site. Continuous line represents 6-OHDA injected animals and broken line represents control animals. *p<0.05, **p<0.01, ***p<0.001 compared between control and 6-OHDA animals. #p<0.05, ##p<0.01, ###p<0.001 compared between different distances or time points within each group.
Figure 5.
Correlations between edema and iron accumulation.
A) Regression line showing the correlation between hyperintensities at day 2 and corresponding hypointensities at 4 weeks for different positions in the striatum (data points = 1.2 0.6, 0, −0.6, −1.2 mm from injection) of 6-OHDA injected animals. B) Regression line showing the correlation between total hyperintense areas at day 2 and corresponding hypointense areas at 4 weeks, calculated from control, and 6-OHDA groups (represented by blue and white dots respectively).
Figure 6.
Histological analysis of hypointense areas.
Prussian blue staining combined with ED1-inmunoreativity at 2 days postlesion (A, B, E, F) shows that in 6-OHDA injected animals (A, B) and control animals (E, F) iron (A and E) and microglia (B and F) are almost absent. At 4 weeks in 6-OHDA animals (C, D) the iron deposits are located to the same areas as active microglia (D). In control animals accumulation of iron (G) and activation of microglia (H) was less pronounced. Arrows show ED1-negative cells that were iron positive. Scale bar 50 µm.
Figure 7.
Correlations between T2*-hypointensities and iron accumulation.
High resolution T2*-weighted images at four weeks after 6-OHDA injection showed a strong hypointense area at different positions in the striatum A) 0, B) −0.53 and C) −1.06 mm from the injection site which coincided with the presence of iron in Prussian blue histology. The hypointense area and the accumulation of iron was much smaller and more localized in control animals at 0 or −0.53 mm from the vehicle injection (D and E respectively). Scale bar 200 µm.
Figure 8.
Immunoglobulin G (IgG) histochemistry.
A) control- and B) 6-OHDA injected striatum at 2 days postlesion incubated with anti-rat IgG. Control animals showed almost not reactivity to IgG comparing with lesioned animals indicating that 6-OHDA provokes a disturbed BBB. Scale bar 100 µm.