All experiments were carried out in accordance with the European communities’ council directive (86/609/EEC) and institutional guidelines for animal care after ethics committee approval. Male Wistar rats (Charles River Laboratories, Sulzfeld, Germany) weighing between 250-300 g, were either subjected to 90 minutes of focal cerebral ischemia followed by 48 hours of reperfusion (Experimental group, N=11) or sham operation (Sham group, N=6). Serial MRI was performed at the following time points: Pre-ischemic (Control), 1 hour after ischemia (BR), following reperfusion (AR), 4 hours post-reperfusion (04PR), 24 hours post-reperfusion (24PR) and 48 hours post-reperfusion (48PR). Experimental animals were scanned at six time points (Control, BR, AR, 04PR, 24PR and 48PR), and sham animals at three time points (04PR, 24PR and 48PR).
Transient middle cerebral artery occlusion (tMCAO) was performed as previously described (Longa et al. 1989
; Spratt et al. 2006
). Briefly, anaesthesia was induced with 5% isoflurane in a mixture of 70% nitrous oxide and 30% oxygen. After endotracheal intubation, anaesthesia was maintained using 1.5% isoflurane. The body temperature was maintained at 37-37.5°C throughout surgery. A cannula was inserted into the left femoral vein for contrast agent administration. The left common carotid artery (CCA) bifurcation was exposed through a midline neck incision and the occipital artery branches of the external carotid artery (ECA) were isolated, ligated and dissected. After careful isolation of the internal carotid artery (ICA), a 3 cm long silicone coated, polyamide 4-0 monofilament (Ethicon) was advanced through the ICA to the MCA until mild resistance was felt. After the intra-luminal suture was placed, the neck incision was closed with a silk suture. Sham operation was performed in the same manner, except for MCA occlusion. The animals were then allowed to recover. One hour post occlusion, the animals were re-anesthetized with 1.5% isoflurane and MRI was performed for the time point BR. Following this, animals were subjected to reperfusion by gently pulling back the filament.
All experiments were conducted on a standard 3T clinical dedicated head MR scanner (Magnetom Allegra, Siemens Healthcare, Erlangen, Germany), where the gradients achieve a 40 mT/m amplitude with a slew rate of 400 T/m/s per axis. The MR scanner was complemented only by a dedicated four-channel phased array rat head coil assembly (RAPID Biomedical GmbH, Rimpar, Germany). All MRI acquisitions throughout this longitudinal study has been conducted at a constant receiver gain, which was initially set during the optimization phase of the standard Siemens product sequences for performing small animal imaging.
To detect ischemic changes especially at the early time points (BR and AR), DW-EPI images were acquired with repetition time (TR) = 3300 ms, echo time (TE) = 105 ms, field of view (FoV) = 6.7 cm, image matrix (IM) = 104 × 104 and at five different ‘b’ values (0, 500, 1000, 1500 & 2000 s/mm2) applied in three orthogonal directions. Trace weighted apparent diffusion co-efficient (ADC) maps were also generated. T2-turbo spin echo (TSE) images were acquired with TR = 3330 ms, TE = 70 ms and Turbo factor = 7.
To assess BBB permeability changes, T1-spin echo (SE) images were acquired before (pre-contrast) and after contrast agent (post-contrast) administration. The images were acquired with TR = 900 ms and TE = 10 ms. After acquiring the pre-contrast images, Gd-DTPA (MW: 590 Da. Magnevist, Shering, Germany) was administered (0.2 mmol/kg) and post-contrast T1-SE images were acquired after 25 minutes. This dose was chosen from preliminary experiments showing that following subtraction and during the calculation of enhanced volumes there was a significant variability in the estimated volumes by two authors of this paper (DRP and DB) when using lower doses of MRI contrast agent (0.1 mmol/kg)
For assessment of brain water content/ edema formation, T2 relaxometry was performed at all time points (Control, BR, AR, 04PR, 24PR & 48PR) using a SE sequence with a TR of 3330 ms and employing 7 TE values (29, 58, 88, 117, 146, 175 & 204 ms).
For TSE and SE acquisitions, a FoV of 2.5 cm, with an IM of 128 × 128 was used. The spin-echo and EPI images had an in-plane resolution of 200 μm and 600 μm respectively with 1 mm slice thickness. Sequences were applied in the following order: DW-EPI, T2-TSE, T2 relaxometry, T1-SE and post-contrast T1-SE.
All the required parameters were acquired from a single coronal slice (1 mm thick) located at 7 mm posterior to the anterior tip of the frontal cortex. A preliminary analysis had demonstrated that this slice revealed the largest volume of infarct at all three relevant time points with good assessment of the midline shift and was best suited for standardized T2 relaxometry in the striatum and cortex. The variation in infarct volume was also found to be lower for this slice region at 24 and 48 hours, compared to adjacent regions with similar infarct volumes (data not shown).
Quantitative T2 relaxometry was performed at three regions of interest (RoIs); the ipsi- and contralateral striatum and on the ipsilateral cortex. In the striatal region, circular RoIs have been defined over the entire striatal region excluding the ventricles ().
Figure 1 Representative images of T2-TSE for edema analysis (upper) and post-contrast T1-SE (lower) for blood-brain barrier permeability at 04, 24 and 48 hours post reperfusion. Highlighted areas (as upper left image) have been considered for T2 relaxometric estimations. (more ...)
Mono-exponential non-linear curve fitting was performed using Graphpad Prism Version 5.00 for Windows (Graphpad Software, San Diego, California, USA) to determine T2 relaxation parameters. Volumetric estimations of the cerebral hemispheres were again performed on T2-weighted images using the available built-in tools of Siemens Syngo 2004A software (Siemens Healthcare, Erlangen, Germany).
Analysis of BBB permeability changes was conducted on subtracted maps from the pre- and post-contrast T1-SE images to highlight regions of Gd-DTPA extravasation. Gd-DTPA permeable BBB volume (PBV) in cubic centimeters (cm3) representing brain tissue with leaky BBB and the average pixel intensity (T1SIdiff) of the hyper-intense enhanced regions derived from the subtraction maps were calculated using the built-in tools. The obtained values of the mean pixel intensity for the subtracted images (T1SIdiff) are software generated (Siemens syngo 2004A) and standard across all the current generation of Siemens scanners. A product of T1SIdiff and PBV (T1SIdiff x PBV) has been considered to account for the observed temporal and spatial changes in the average pixel intensity of the enhanced regions (T1SIdiff) and the brain volume with leaky BBB (PBV). The T1SIdiff x PBV product serves as an indicator to quantify the overall entry of contrast agent into the brain over time. For sham analysis, the average PBV from the experimental group was projected onto the subtracted images of the sham animals, and T1SIdiff was determined within this region.
Throughout the study, values are treated as mean ± standard error (SEM). Repeated measures ANOVA followed by Tukey-Kramer post-hoc tests have been considered for within-group comparisons. For between-group comparisons (experimental v/s sham group) at different time points, unpaired ‘t’ tests with Welch correction were applied. A P value < 0.05 was considered significant.