M .A. Yenari et al. Brain Research 885 2000 208 –219
209
MRI PWI can be used to evaluate cerebral perfusion. within cerebral cortex and striata. Brain temperature was
Although PWI does not precisely reflect regional cerebral reduced to 35
8C, 338C, 308C and 288C by placing the blood flow rCBF, it is a non-invasive technique which
animal on a cooling blanket and spraying ethanol onto the can provide information regarding microvascular patency
body, then applying cool air from a hair dryer hose. [41,62]. As these techniques [3,58,60] and hypothermic
Temperature was maintained for 15–20 min prior to therapies [35,51] are beginning to gain interest at the
cooling to the next level. To determine the effects of clinical level, we utilize DWI and PWI to study the
temperature changes during ischemia, temperature probes temporal and regional evolution of mild hypothermia in an
were stereotaxically placed into each striata and a third experimental model of transient focal cerebral ischemia.
probe was placed in the rectum. Temperatures were DWI can also be used to detect transient declines in
monitored during conditions of ischemic normothermia ADC which correspond electrophysiologically with the
n 51 and mild hypothermia n51 as described sub-
transient depolarizations or spreading depressions SD sequently.
observed following ischemia [5,6,17,20,31,48–50,54]. SDs which occur during cerebral ischemia are thought to
2.2. Effects of temperature on ADC contribute to infarct growth by altering ion gradients or
increasing extracellular glutamate [20]. The extent and As micromolecular diffusion is altered with temperature
distribution of SDs also appear to be correlated with changes, the relationship between brain ADC and tempera-
expression of various genes, such as immediate early genes ture was established in our model. Using the correlation
[19], cyclooxygenase [37] and protein kinase C PKC data from the above experiment, we monitored rectal
[29]. While the SDs may result in worsening of ischemic temperature during scanning in this experiment and all
injury, pre-insult induction of SDs by KCl application may subsequent experiments. Three anesthetized non-ischemic
actually protect the brain from subsequent ischemic events animals underwent DWI imaging with brain temperatures
ischemic tolerance [26,27,36]. An earlier report showed varied from 30
8C to 408C. Rectal temperatures were varied that mild hypothermia slows the propagation of the SDs
as described above and DWI images were performed at within cortex, as detected by DC potential measurements
each temperature. Trace DWI images were generated as [59]. We now show complementary results using DWI,
described subsequently in the MRI protocol section. Re- which has the advantage of being non-invasive and offers
gions of interest ROIs were identified within regions of anatomical resolution. To our knowledge, this is the first
cortex, striatum and thalamus, and ADCs from fitted maps report characterizing the temperature dependence of SD-
were measured within these ROIs. Correction curves were like ADC changes in ischemia and KCl models using
determined for each of the structures as a function of DWI.
temperature. 2.3. Ischemia model
2. Materials and methods
Twenty-two male Sprague–Dawley rats weighing be- 2.1. Measurement of brain temperature
tween 290 and 320 g. were anesthetized with 3 halothane by face mask. Oxygen and air were supplied in a ratio of
We first established the relationship between rectal 0.2 l oxygen:0.8 l air. Once surgical planes of anesthesia
temperature and brain temperature in our model, as it is not were attained assessed by absence of hind leg withdrawal
yet feasible to measure simultaneous brain and body to pinch, halothane was decreased to 1–1.5 throughout
temperature during MRI scanning given the susceptibility the remainder of the surgery. A femoral artery catheter was
artifacts caused by the brain temperature probe. Rats were placed to monitor mean arterial blood pressure MAP and
placed in the MRI probe then into the scanning unit to a rectal probe was placed to monitor body temperature.
maintain the
same thermodynamic
environment ex-
The animal was placed on a heating cooling blanket to perienced during scanning. Because we found no differ-
maintain body temperature between 37 8C and 388C. Arteri-
ences in temperature gradients with animals in or out of al blood gases, serum glucose and hematocrit are measured
the MRI unit, subsequent temperature correlations were and recorded. Ischemia was induced using an occluding
made with the animal in the MRI probe. We applied whole intraluminal suture used previously by our group [61]. A
body cooling and warming in the range of 30–40 8C rectal
cervical midline incision was made and the left carotid temperature to halothane-anesthetized rats n
53 and artery and branches were isolated. The common carotid
monitored for heart rate, respirations and rectal tempera- artery CCA, external carotid ECA, and pterygopalatine
ture. Temperature was controlled by placing a bonnet from PPA were identified. The ECA was ligated and bisected.
a commercially available hair dryer directly onto the An uncoated 30-cm long segment of 3-0 nylon monofila-
animal, and the air temperature could be adjusted by the ment suture Ethicon, Somerville, NJ with the tip rounded
unit’s controls. A small burr hole was drilled over the by a flame is inserted into the stump of the ECA and
fronto-parietal cortex and temperature probes were placed advanced under direct visualization into the ICA approxi-
210 M
mately 19–20 mm from the bifurcation in order to occlude mic n
53, or were cooled to 308C rectal temperature, the ostium of the MCA. A temporary aneurysm clip was
n 53 immediately prior to suture insertion.
also placed on the CCA. Animals were either kept normothermic normo, rectal
5378C, n511 or rendered mildly hypothermic hypo, rectal
5308C, n511 within 2.6. MRI scanning
15–20 min after MCA occlusion, and maintained for 2 h. After 2 h of ischemia, the suture was withdrawn with the
DWI and PWI images were obtained intra-ischemically animal remaining in the MRI scanner. The animal was
30 min following MCAO, post-reperfusion within 5–10 rewarmed within 15–20 min using a warming blanket and
min of reperfusion and 24 h later. Imaging was performed by blowing warm air into the scanner bore. Following the
on a Bruker CSI 2.0 Tesla system. The complete protocol final scan, the animal was returned to the operating table.
for each DWI and PWI scan set took approximately 30 The aneurysm clip was removed and the surgical incisions
min to complete. Because of the prolonged time under closed. The animal was allowed to recover, then trans-
halothane anesthesia, many animals were incapable of ported to the intensive care unit at the animal facility for
surviving the full 24 h. Only four normothermic and three post-operative monitoring. At the completion of the experi-
mildly hypothermic animals were able to complete imag- ment, the animal was euthanized with a barbiturate over-
ing at all three time points. Therefore, subsequent animals dose.
were randomized into two groups. In the first group three normothermics, four mildly hypothermics, animals were
imaged intra-ischemically and early into reperfusion. In the 2.4. KCl-induced SD experiments
second group four normothermics and four mildly hypo- thermics, the animal was removed from the scanner after
In separate, non-ischemic, halothane anesthetized ani- obtaining the intra-ischemic images and the mild hypo-
mals n 54, a KCl chamber was placed 2 mm anterior to
thermia was completed. The suture was removed and the the lambdoidal suture and 2 mm lateral to the sagittal
animal was allowed to recover. The following day, a suture over the right hemisphere. A DC electrode was
second set of scans at 24 h was performed. For all groups, placed 2 mm anterior to the coronal suture and 2 mm
the total duration under anesthesia was similar 4.5 h. A lateral to the sagittal suture. KCl 3 M was applied during
total of 22 animals, 11 normothermic and 11 mildly imaging acquisition and a DC shift was confirmed by
hypothermic, were imaged for this part of the study. simultaneous recordings from the electrode. Rectal tem-
perature was varied and images were obtained at 30 8C,
33 8C and 378C, corresponding to brain temperatures of
2.7. DWI protocols 33
8C, 358C and 388C, respectively. DWI imaging de- scribed below commenced immediately before and up to
Spin echo echo planar images SE EPI was used with 20 min after KCl application.
2
the following imaging parameters: FOV 540340 mm and
resolution 564364. For the ischemia and correlative ex-
2.5. Ischemia-induced SD experiments periments, a 5-cm diameter bird-cage radio frequency coil
was used. For the SD experiments, a surface coil was used. In six separate animals, MCA occlusion was induced
Both isotropic diffusion-weighted IDW and DWI trace remotely, with the animal inside of the MRI scanner. This
images were obtained. Imaging parameters were: TE TR 5
method has been published previously [49], but some 66 ms 4 s, no. of slices
54, coronal plane, slice thickness5 modifications have been made. PE-50 tubing 10 mm in
2.0 mm. For IDW scans, 16 images with varying gradient
2
length was tapered on one end. The opposite end of the amplitudes 16 b-values between 100 and 1200 s mm in
tubing was inserted approximately 5 mm into PE-90 tubing random order were acquired for each slice. For the trace
which extended the length of the MR cradle |30 cm. The DWI images, eight images were acquired from each slice
2
tapered tip of the PE-50 tubing was inserted 5 mm into the eight b-values between 0 and 1400 s mm , and repeated
ECA stump and secured. A 30-mm monofilament suture with the gradients applied in the x, y and z directions.
was attached to another segment of PE-50 tubing 45 cm Trace images were derived by averaging the images
in length and fed through the P-90 tubing and into the obtained from each direction. T2-Weighted T2W images
ECA approximately 15 mm from the CCA bifurcation. were obtained using b-values
50. This design permitted the suture to be advanced no more
In order to detect SDs, continuous, two slice EPI images than 20 mm from the CCA bifurcation. Two minutes after
were acquired. Imaging parameters were TE TR 550 ms 2
the beginning of image acquisition, the suture was ad- s, slice thickness
51.65 mm, no. of slices52, axial plane. vanced another 2–5 mm from the CCA bifurcation. Two
Three images with varying gradient amplitude gradients slice DWI imaging to detect SDs continued for a total of
applied in x, y and z simultaneously, total b-values 56,
30 min, followed by multislice protocols for DWI and PWI 1347, and 1347 were obtained. This was repeated 600
to confirm ischemia. Animals were maintained normother- times with a time resolution of 2 s.
M .A. Yenari et al. Brain Research 885 2000 208 –219
211
2.8. PWI protocols Maps of bolus arrival delays were fitted from the PWI
scans using a fitting routine previously reported by our PWI scans were performed using a T2-weighted gra-
group MRVision, Menlo Park, CA [62]. We and others dient echo EPI to track the contrast bolus [48,49,62]. Three
have found the delay maps to be the most sensitive coronal slices were repeatedly acquired during rapid
perfusion index compared to relative cerebral blood vol- intravenous injection of a contrast agent 0.2 mmol kg
ume rCBV and bolus peak effect [41,62]. Regions of gadopentitate dimeglumine. Imaging parameters were the
significant delay were mapped out using a thresholding same as for DWI imaging. All three slices were acquired
routine. Only areas where bolus arrival delays were greater every 2 s prior to and during contrast injection, and
than 2 s compared to the contralateral hemisphere were continued for a total of 32 s.
included and expressed as a percentage of the total ipsilateral hemisphere area [62].
2.9. MRI analysis Because the scans could only be continued for a finite
period of time 20 min for each SD trial, the total number All analyses were performed by an investigator blinded
of SDs could not be reliably determined; therefore, we to treatment groups, although information regarding the
chose to only analyze parameters from the initial SD wave time point of the scan sets was provided. Data sets for all
from each KCl application or ischemia experiment. From MRI scans from a specific time point were pooled,
the KCl-induced SD experiments, ADC maps were gener- regardless of when the animal was euthanized. ROIs
ated and ROIs were defined in the ipsilateral hemisphere delineating the DWI hyperintensity from four adjacent
near the region of KCl application. ADC changes within slices were traced by hand and expressed as the percent
the same ROI were computed from the sequential images area relative to the ipsilateral hemisphere. We have previ-
and plotted as a function of time. The maximum ADC ously shown that these DWI hyperintensities at 24 h
decrease, the time from initial ADC decline to maximal strongly correlate with infarction as seen by triphenyl
ADC decrease ADC decline time, and the time from tetrazolium chloride TTC staining [61]. ROIs were also
maximal ADC decrease to ADC recovery ADC recovery delineated within the striata Fig. 1, Si to determine the
time were measured from the first transient depolarization. percent change in ADC value between the ipsilateral
For the SD experiments conducted in ischemic animals, ischemic Si and corresponding contralateral non-is-
ROIs were identified within the cortex corresponding to a chemic Sc sides from the ADC maps. For comparison
peri-infarct region P, see Fig. 1. Another ROI within the between temperature groups, all ADCs from images ac-
striatum was also defined Si, Fig. 1 in order to measure quired under mild hypothermic conditions were corrected
the time to terminal ADC decrease, defined as the time to to a brain temperature in a normothermic animal using the
maximum ADC decline in regions where no ADC re- previous correlative data.
covery was observed during the 20-min scanning period.
Fig. 1. Diffusion-weighted MRI scan showing a large middle cerebral artery territory lesion. Regions of interest ROI which were used to measure changes in apparent diffusion coefficients ADCs following ischemia are indicated P, peri-infarct region in cortex; Si, ipsilateral ischemic striatum; Sc,
contralateral non-ischemic striatum.
212 M
Similar parameters from an ROI within the cortex adja- cent to the area of KCl application were measured from
the KCl experiments.
2.10. Statistical analysis Standard statistical methods were used to analyze data.
Differences between groups and the various parameters was determined using one-way analysis of variance
ANOVA and ANOVA with repeated measures to detect overall significance, followed by a post hoc multiple
comparison procedure Scheffe’s. Correlation was de- termined by Pearson’s correlation coefficient, followed by
a linear regression. Statistical significance was determined at
the P
,0.05 level. All data are presented as mean
6S.E.M.
Fig. 2. Striatal temperature in ischemic and non-ischemic brain under normothermic and mildly hypothermic conditions. Under normothermic
3. Results
