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1
Post-stroke rapamycin treatment improves post-recanalization cerebral blood flow and 1
outcome in rats 2
Anna M Schneider, MD, PhD, MPH1; Yvonne Couch, PhD1; James Larkin, PhD2; Alastair M 3
Buchan DSc FMedSci 1*; Daniel J Beard, PhD1,3* 4
5
1 Acute Stroke Programme, Radcliffe Department of Medicine, University of Oxford, Oxford, 6
UK 7
2 Department of Oncology, University of Oxford, Oxford, UK 8
3 School of Biomedical Sciences and Pharmacy, University of Newcastle, Australia 9
*These authors contributed equally to this work 10
11
Corresponding author: 12
Professor Alastair M. Buchan, DSc, FMedSci 13
Acute Stroke Programme, Room 7501, Level 7A/B 14
John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom 15
Email: alastair.buchan@medsci.ox.ac.uk 16
Phone: +44 (0)1865 220346 17
18
Running title: Rapamycin improves reperfusion and stroke outcome 19
Abstract word count: 185, Main text word count: 5040 20
References: 70 21
Figures: 7 22
Supplement: 1 Tables 23
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Abstract 24
Ischaemic stroke treatment is limited to recanalizing the occluded vessel, while there is no 25
approved adjunctive cerebroprotective therapy to protect either the neurons and parenchyma or the 26
neurovascular unit. Pharmacological inhibition of mammalian target of rapamycin-1 (mTORC1) 27
with rapamycin has shown promise in reducing infarct volume and improving functional 28
outcomes. However, previous studies that investigated the effects of rapamycin on the vasculature 29
and cerebral blood flow (CBF), administered rapamycin prior to or during stroke induction, thus 30
limiting the potential for clinical translation. Therefore we investigated whether rapamycin 31
maintains its cerebrovascular protective effect when administered immediately after recanalization 32
following 90 minutes stroke in Wistar rats. We show, that rapamycin significantly improved post-33
recanalization cerebral blood flow (CBF), suggesting a beneficial neurovascular effect of 34
rapamycin. Rats treated with rapamycin had smaller infarct volumes and improved functional 35
outcomes compared to the control animals at three days post-stroke. The mechanisms of the overall 36
positive effects seen in this study are likely due to rapamycin’s hyperacute effects on the 37
neurovasculature, as shown with increased CBF during this phase. This paper shows that 38
rapamycin treatment is a promising adjunct cerebroprotective therapy option for ischemic stroke. 39
40
5 keywords: focal ischemia, mTOR, rapamycin, neuroprotection, MRI 41
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Introduction 42
Stroke treatment has recently been revolutionized by recanalization therapy and better patient 43
selection thanks to improved imaging modalities. Despite high recanalization rates (60-70%), half 44
of patients are still left with poor functional outcomes, and only about 10% are symptom-free at 45
three months (1). Therefore, an adjunct pharmacological treatment that has neuronal and vascular 46
protective effects may help improve outcomes post-recanalization. 47
Pharmacological inhibition of mTORC1 with the FDA-approved anti-rejection medication 48
rapamycin has been shown to protect brain tissue from dying in experimental models of stroke (2, 49
3). However, the mechanism by which rapamycin exerts its cytoprotective effects and which 50
cerebral cells and anatomical structures it targets are still being explored. A large body of literature 51
reports that rapamycin can reduce infarct volume and improve neurological function in stroke 52
models (4-19). However, only a few papers have investigated the effects of rapamycin treatment 53
on structural changes of the blood-brain barrier (BBB) and cerebral blood flow (CBF) after 54
transient middle cerebral artery occlusion (tMCAo) (2, 8, 9, 13, 14, 18, 19), of which five reported 55
decreased BBB breakdown due to rapamycin treatment (9, 13, 14, 18, 19). Our recent work has 56
highlighted that rapamycin can also enhance collateral and post-recanalization CBF (Beard et al. 57
2020. However, many of these studies either treated animals before the onset of stroke or prior to 58
recanalisation. Therefore it is not clear if rapamycin maintains its cerebrovascular protective effect 59
if administration is delayed to immediately after recanalization, thus better matching the clinical 60
scenario of administration of an adjunct cerebroprotectant with vessel recanalization. 61
There has been a concerted effort from the preclinical stroke field to improve the quality 62
and thus reduce the bias in clinical studies (20). While our recent meta-analysis of rapamycin 63
showed that no studies have completely adhered to these guidelines, we have designed the present 64
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study in such a way that it avoids these shortcomings (2). In addition to the clinically translatable 65
study design of administering rapamycin in the post-reperfusion phase, the methods used to assess 66
the drug’s effect have also been chosen to be comparable to clinical stroke practice. 67
MRI has proven to be an optimal tool for the longitudinal assessment of cerebral blood 68
flow (CBF), lesion progression and for evaluating translational therapeutic studies (21, 22). 69
Sequences used in MRI can detect different aspects of the ischemic lesion (23), and because brain 70
damage following cerebral ischemia is a dynamic process, assessment with MRI requires varied 71
imaging modalities to suit the particular pathophysiologic state of the lesion. For example, T2w-72
MRI provides anatomical information, whereas more dynamic sequences such as diffusion- and 73
perfusion-weighted imaging (DWI and PWI, respectively) can detect tissue microstructure, 74
metabolism, and hemodynamics, and in stroke, inform about potentially salvageable brain tissue 75
using the DWI/PWI mismatch coefficient (23). In this study, we used different MRI sequences at 76
the three days post-stroke onset to study rapamycin’s effect on perfusion and lesion volume, while 77
functional testing assessed the impact on behavioral outcomes and its correlation to the MRI data. 78
The aims of this study are threefold. First, to understand how mTORC1 inhibition with 79
rapamycin affects immediate post-recanalisation CBF. Second, to examine if the effects of 80
rapamycin on perfusion changes persist out to 3 days post-stroke, and to determine any effects on 81
lesion volume and functional outcomes. Third, to study the effect of mTORC1 inhibition on the 82
integrity of the BBB using contrast agent MRI. 83
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Materials and methods 84
Ethics and animal care 85
All experimental procedures were approved by the UK Home Office (1986 Animal Act, Scientific 86
Procedures), conducted in accordance with the Clinical Medicine Ethical Review Guidelines of 87
the University of Oxford, and conformed to the ARRIVE and IMPROVE guidelines for animal 88
and pre-clinical stroke work (24, 25). Male Wistar Han rats (250g - 320g, 8 – 11 weeks old, Envigo 89
Research Model Services in Blackthorn, England) were housed in individually ventilated cages 90
under a 12-hour light/12-hour dark cycle with ad libitum access to food (standard food pellets) and 91
water. The principal investigator began daily animal handling, weighting, and training the rats for 92
the adhesive removal test (see below) 3 days before the surgery. During the 3 days following 93
surgery, the rats underwent daily welfare scoring. 94
95
Study design 96
Controls and exclusion criteria: Appropriate control groups were included in all experiments. 97
For the comparison with rapamycin, vehicle groups were used. Since naïve groups were included 98
in similar animal studies undergoing the same intervention, and the investigators tried to minimize 99
the number of animals used, no sham groups were included in this study (9). Where possible, 100
within-subject controls were used, for example, when comparing the extent of stroke expansion 101
on the ipsilateral versus contralateral hemisphere. Pre-defined exclusion criteria were a laser 102
Doppler flow (LDF) signal decrease of less than 70% from baseline and a confirmed subarachnoid 103
haemorrhage (SAH), a rare but known complication of the focal ischemic stroke model used. 104
Randomization and blinding: Animals were randomized with the sealed envelope method to 105
receive either rapamycin (Sigma Aldrich, 250 μg/kg, n=9) or vehicle (<5% ethanol in saline, n=9), 106
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which was administered by a surgeon blind to treatment allocation (AMS). Blinding was continued 107
throughout the whole experiment and until data acquisition was complete. 108
109
Anesthesia and Monitoring 110
Rats were initially anaesthetized by inhaling 5% isoflurane in 70% N2O and 30% O2 and 111
maintained at 1-2% isoflurane in 70% N2O and 30% O2. During the surgery, the core body 112
temperature of all animals was maintained at 37.0 ± 0.5°C using a rectal thermometer connected 113
to a feedback-controlled heating pad (Harvard Apparatus, Cambourne, UK). Physical parameters, 114
including body temperature and respiratory rate, were checked and recorded every 15 minutes 115
throughout the surgical intervention. Respiration was kept between 50 and 60 breaths per minute 116
by adjusting the isoflurane concentration. 117
118
Cerebral blood flow measurements 119
A fibreoptic probe was firmly positioned on the intact skull surface based on locations described 120
previously (26). An LDF apparatus (Oxyflo 2000 Optronix, Oxford, UK) was used to continuously 121
monitor cerebral perfusion of the lateral MCA territory, corresponding to the core territory after 122
MCA occlusion. LDF traces were recorded onto a Windows XP workstation running with WinDaq 123
Data Acquisition Software (Dataq Instruments, Akron, Ohio, USA). MCAo was confirmed by a 124
>70% decrease in LDF signal from baseline. Selection of a 70% decrease as the threshold was 125
chosen based on previous studies reporting an infarct threshold of 20-30% from baseline (27, 28). 126
127
128
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Focal cerebral ischemia 129
This study used 22 rats. All surgical procedures were conducted under sterile conditions. Focal 130
brain ischemia was induced by transient occlusion of the MCA for 90 minutes, according to a 131
previously described method (29-31). Reperfusion was confirmed by visual reperfusion of the 132
proximal ICA and a sudden increase of the recorded cerebral blood flow in the MCA territory. 133
Immediately after reperfusion, rapamycin or vehicle was administered through intravenous tail 134
vein injection. The CBF was recorded for another 90 minutes from the time of reperfusion. 135
136
Behavioral assessment 137
Neurological assessments to measure neurological impairments post-stroke were performed on all 138
three days following surgery. Tests involved Bederson (32), Garcia (33), and the adhesive removal 139
test (34). The Bederson scale studies post-stroke behavioral deficits by assessing forelimb flexion, 140
resistance to lateral push, and circling behavior, and using a grading scale of 0 to 3, with 0 being 141
not affected and 3 being severely affected (32). The Garcia testing battery consists of 6 tests to 142
evaluate sensorimotor deficits, where performance is graded on a scale of 0-3, with 0 being not 143
affected and 3 being severely affected by the stroke, representing a minimum score of 0 and a 144
maximum score of 18 points (33). The adhesive removal test involves placing a plaster on the 145
forepaw and measuring the “time-to-contact” (somatosensory deficit in the contralesional 146
forepaw) and “time-to-remove” (contralesional forepaw dexterity). To decrease inter-individual 147
differences within this test, training sessions from 3 days before the surgical procedure (2 trials 148
per day) were performed, and the time used for noticing and removing the sticky tape on the 149
contralateral forepaw was analyzed and compared among the rapamycin and vehicle groups. 150
151
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Magnetic resonance imaging 152
All MRI acquisitions were carried out on a 9.4T horizontal-bore scanner (Agilent Technologies 153
Inc., Santa Clara, USA) with a 72 mm volume transmit coil and a 4-channel surface receive array 154
(Rapid Biomedical, Rimpar, Germany). The animal’s anesthesia was induced with 5% isoflurane 155
in 70% N2 and 30% O2 and maintained at 1-2% isoflurane in 70% N2 and 30% O2. The rats were 156
placed in a cradle equipped with a stereotaxic holder, a rectal thermometer, and a pressure probe 157
to monitor respiration. The rectal thermometer was connected to a feedback-controlled heating pad 158
(Harvard Apparatus, Holliston, United States) to maintain core body temperature at 37.0 ± 0.5°C, 159
and respiration was kept between 50 and 60 breaths per minute by adjusting isoflurane 160
concentration. Physical parameters were recorded and checked regularly throughout the imaging 161
session. 162
Acquisition of baseline maps for T1 and T2 relaxation times (seconds), arterial spin 163
labeling (ASL) for measuring cerebral blood flow (CBF) (mL/100g/min), and apparent diffusion 164
coefficient (ADC) for delineating diffusion (μm²/ms) were performed. T1 and T2 data were 165
acquired using a spin-echo echo-planar imaging (EPI) readout with FOV = 32 x 32 mm², matrix = 166
256 x 256 mm², thickness = 1 mm, 10 slices. T1w images were taken with a scan repetition time 167
(TR) and echo time (TE) of 500 ms and 20 ms, respectively. The parameters were identical for 168
imaging acquisition, both before and after administration of 150 μL gadodiamide (Omniscan, 169
Germany) via an indwelling tail vein cannula. T1-map scans (repetition time/echo time [TR/TE] 170
= 1000 ms/27.16 ms) were obtained using an inversion recovery sequence (TI=13.14, 29.3, 65.3, 171
145, 324, 723, 1610, 8000 ms). T2w anatomical imaging scans (TR = 3000 ms, TEeff = 51.26 ms) 172
were obtained with an echo spacing (ESP) of 8.54. ASL and ADC data were acquired using a spin-173
echo echo-planar imaging (EPI) readout with a field of view (FOV) = 32 x 32 mm², matrix = 64 x 174
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64 mm², thickness = 1 mm, 10 slices. Fast spin-echo multi-slice (FSEMS) scan sequences (TR = 175
1000 ms, TEeff = 40, ESP = 10 ms) were acquired using a midline and axial orientation with FOV 176
= 50 x 50 mm², matrix = 256 x 256 mm², thickness = 2 mm, single slice. Based on the acquired 177
FSMES-images, the labeling plane for ASL imaging (6.2 mm thickness) was placed in the rat’s 178
neck at a 45° angle to the animal’s rostrocaudal axis. CBF maps were generated by multiphase 179
pseudo-continuous arterial spin labeling (35). ADC maps were generated from diffusion-weighted 180
images acquired in 3 orthogonal directions for b=0/mm² and b=1000 s/mm². DW imaging scans 181
were obtained using a two-dimensional spin-echo echo-planar imaging (SE-EPI) sequence. For 182
contrast-enhanced T1w spin echo, multi-slice images were taken before and immediately after 183
0.15 ml injection of gadolinium-based contrast agent (GBCA). 184
MRI analysis: Imaging segmentation and image analysis were performed using itk SNAP 185
(Version 3.8.0, 2019) (36). On T2w maps, areas of ischemia were identified as hyperintense 186
regions (37). On T1w maps, the image before Gd injection was subtracted from the image taken 187
after, and areas of BBB breakdown were identified as hyperintense regions (38). Infarct volume 188
was corrected for edema formation using the formula by Kaplan et al. (39): Corrected infarct size 189
= infarct volume x (volume of contralateral hemisphere/volume of the ipsilateral hemisphere). On 190
ADC maps, areas of ischemia were identified as regions of reduced diffusion of at least 23% 191
relative to the same area on the contralateral hemisphere. Similarly, on ASL maps, areas of 192
hypoperfusion were defined as areas with reduced blood flow of at least 57% relative to the same 193
area on the contralateral hemisphere (40). Relative diffusion and perfusion of the pre-defined 194
cortical area were calculated as follows: diffusion/perfusion of the ipsilateral 195
ROI/diffusion/perfusion of the mirrored region on the contralateral hemisphere. In all imaging 196
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modalities, ROIs were selected manually by the main investigator blinded to treatment or control 197
(AMS). 198
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Western blotting 199
After imaging, rats were killed by intraperitoneal pentobarbital injection (800 mg/kg) (41). Brains 200
were collected and sliced into 2 mm thick coronal sections with an ice-cold stainless-steel matrix 201
(Kent Scientific). Areas of the striatum and cortex of 1 mm2 from both hemispheres were taken 202
from one section and snap-frozen on dry ice. Proteins were extracted from the cortical ipsilateral 203
lesion side by cellular lysis using RIPA buffer supplemented with a protease inhibitor cocktail. 204
Protein quantification was performed using the BCA assay (Pierce BCA Protein Assay Kit, 23225, 205
Thermo Fisher Scientific), 50 µg of protein was denatured (95°C for 5 minutes) using lysis and 206
Laemmli sample buffer (161-0737, Bio-Rad, with DTT). Samples were separated on a 10 % 207
gradient gel (Criterion TGX Precast Gel, 5671033, Bio-Rad) using an Electrophoresis Unit (Bio-208
Rad, Cressier, Switzerland). Proteins were then transferred onto a polyvinylidene difluoride 209
(PVDF) membrane using the electrophoresis unit. Nonspecific binding sites were blocked with 210
PBS containing 5% BSA and 0.1% Tween-20 for 1 hour at room temperature before being 211
incubated in primary antibody (mTOR and phospho-mTOR (Ser 2448), both from Cell Signaling) 212
in 5% PBS-T solution overnight. After 3 x 5-minute-washes in PBS-T, the membrane was 213
incubated in secondary antibody goat anti-rabbit (IgG H&L (HRP), ab6721, Abcam, dilution 214
1:2000) in 5% BSA PBS-T for 1 hour at RT. After 3 x 5-minute-washes in PBS-T, the membrane 215
was developed using enhanced chemiluminescence (ECL; 12644055, Fisher Scientific) for 5 216
minutes and immediately imaged after that. Western Blot analysis and quantification were 217
performed using densitometry and corrected for loading using b-tubulin which was used as the 218
housekeeping protein. The membrane was probed for antibodies twice, using the following mild 219
antibody-stripping protocol: The membrane strips were incubated in mild stripping solution (7.5 g 220
glycine, 0.5 g SDS, 5 ml Tween-20, pH to 2.2, and diluted up to 500 ml distilled water) for 2 x 7 221
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minutes, following by 2 x 10-minute-washes in PBS, and 2 x 5-minute washes in PBS-T, before 222
blocking again in 5% BSA and 0.1% Tween-20. 223
224
Statistical analysis 225
The experimental design and number of animals have been determined and optimized to test the 226
specific hypotheses of the project and follow the standards for scientific reporting and 227
reproducibility (42). Statistical analyses were performed at the end of the experiment, and no 228
further animals were included at that point. 229
We planned the study with n=9/group, based on the maximum effect size (difference 230
between rapamycin and vehicle) and variability of the change in lesion volume which was defined 231
as the primary outcome (30% with a standard deviation of 20%). We were able to reject the null 232
hypothesis that rapamycin does not change lesion volume with a probability (power) of 0.80. The 233
type I error probability of this null hypothesis (alpha) was 0.05. 234
Statistical tests were performed using GraphPad Prism 8.42 (La Jolla, USA). The 235
D’Agostino and Pearson normality tests were performed on all data. Appropriate statistical tests 236
were chosen based on the normality of the data. To compare differences between the two treatment 237
groups, an unpaired Student’s t-test was used. The correlation of imaging parameters with 238
functional outcomes was analyzed by simple linear correlation calculation. The correlations were 239
classified as tiny or < 0.05, very small (0.05 < = r < 0.1), small (0.1< = r < 0.2), medium (0.2< = r 240
< 0.3), large (0.3< = r < 0.4), or very large (r > = 0.4) according to Funder and Ozer (43). A P 241
<0.05 is accepted as statistically significant and data are presented as mean ± standard deviation 242
(SD). 243
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Results 244
Excluded animals: A total of 4 rats were excluded from the study, 3 of which had an insufficient 245
LDF drop during filament insertion and 1 due to filament-induced SAH, which occurred before 246
either the treatment or the vehicle was administered. 247
248
Rapamycin improves immediate post-recanalization blood flow. 249
There was no difference in blood flow measured at the time points immediately before re-250
canalization/treatment administration in control and rapamycin groups, respectively (rapamycin: -251
56.58 ± 7.64 % versus vehicle: 66.16 ± 12.35 % pre-MCAo baseline, P=0.1412, Figure 1A). 252
Rapamycin significantly increased the average MCA territory blood for the first 90 minutes post-253
recanalisation (rapamycin: -9.609 ± 3.374 versus vehicle: -28.32 ± 6.683% of pre-MCAo baseline 254
CBF, P<0.0001, Figure 1B). 255
256
Rapamycin significantly reduces infarct volume. 257
Rapamycin significantly reduced infarct volume at 3 days post-stroke (rapamycin 44.77 ± 30.93 258
mm3 versus vehicle: 113.3.44 ± 60.19 mm3, P=0.0114, Figure 2A). The Waxholm Space Atlas 259
was then overlaid onto the T2w MRI sequences showing that, amongst other areas, the 260
somatosensory cortex was affected by stroke and at least in part salvaged by rapamycin treatment 261
(Figure 2 (B), Supplementary Table 1). 262
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Rapamycin significantly improves functional outcomes. 263
Rapamycin significantly improved performance on the Garcia test (rapamycin: 12.78 ± 1.039 264
points versus vehicle: 11.67 ± 0.8660 points, P=0.0295, Figure 3A). Rapamycin did not improve 265
performance on the Bederson test (rapamycin: 1.556 ± 0.7265 points versus vehicle: 2.111 ± 266
0.6009 points, Figure 3B, P=0.0962). Rapamycin-treated animals required significantly less time 267
to notice the adhesive tape on their forepaw, indicating improved sensory functioning (rapamycin: 268
45.04 ± 11.91 s versus vehicle: 72.33 ± 12.17 s, P=0.0002, Figure 3 C). Rapamycin-treated animals 269
also required less time to remove the adhesive tape on their forepaw (rapamycin: 49.89 ± 23.09 s 270
versus vehicle: 76.56 ± 12.92 s, P=0.0146, Figure 3D). 271
There was a tight correlation between infarct volume and neurological test results, with 272
larger lesions leading to worse functional outcomes (Infarct volume vs. Garcia: r=-0.5502, 273
P=0.0272; Figure 4A; Infarct volume vs. Bederson: r=0.6138, P=0.0114, Figure 4B; Infarct 274
volume vs. time to notice of contralateral forepaw in adhesive removal test: r=0.5486, P=0.0278, 275
Figure 4C). 276
277
Rapamycin does not alter diffusion or perfusion. 278
After evaluating rapamycin’s effect on blood flow in the hyperacute phase, we next wanted to 279
investigate its effects on spatially matched dynamic changes in the subacute phase of disease 280
progression. 281
At 3 days, no rat had a large enough diffusion deficit to define the cortical MCA region as 282
ischemic. Rapamycin did not significantly change relative diffusion (rapamycin 1.122 ± 0.04604 283
versus vehicle 1.109 ± 0.08839, P=0.5765, Figure 5A). Rapamycin did not significantly change 284
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absolute diffusion in the ipsilateral ROI rapamycin 0.00081 ± 0.0001 104 mm2/sec versus vehicle: 285
0.00080 ± 0.000 104 mm2/sec, P=0.6099, Figure 5B). 286
One rat exhibited a stark enough difference with ASL data between the ipsi- and 287
contralateral hemispheres to define a perfusion abnormality. Rapamycin did not significantly 288
change relative perfusion (rapamycin 0.9997 ± 0.3364 versus vehicle: 1.065 ± 0.1856, P=0.6340 289
Figure 5C). Rapamycin did not significantly change absolute perfusion (rapamycin 94.60 ± 290
34.6697 ml/100g/min versus vehicle: 139.1 ± 87.92 ml/100g/min, P=0.4807, Figure 5D). 291
292
The effect of rapamycin on edema formation and BBB integrity. 293
Edema formation of the T2w images was assessed using the edema calculation after Kaplan (39): 294
Extent of edema = (volume of the ipsilateral hemisphere – the volume of the contralateral 295
hemisphere)/volume of the contralateral hemisphere. Rapamycin did not significantly change 296
edema volume (rapamycin 9 ± 5.657 mm3 versus vehicle: 14.25 ± 7.186 mm3, P=0.1129, Figure 297
6A). Further, T1w imaging with gadolinium injection was performed to assess BBB integrity. 4 298
additional animals had to be excluded because the amount of Gd reaching the area of interest was 299
insufficient. Rapamycin did not significantly change BBB breakdown (rapamycin 3.270 ± 2.336 300
mm3 versus vehicle: 7.647 ± 4.500 mm3, P=0.0606, Figure 6B). 301
302
mTOR activity was not reduced at 3 days. 303
For mTOR, the ratio of phosphorylated to total protein can indicate its level of pathway activity, 304
where values over 1 indicate activation and values below 1 suggest an inhibitory effect. The ratio 305
of phosphorylated mTOR (p-mTOR) to total mTOR did not differ significantly in the stroke 306
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animals treated with either vehicle or rapamycin (rapamycin: 0.5502 ± 0.3153 versus vehicle: 307
0.9032 ± 0.4139, P=0.1880, Figure 7). 308
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Discussion 309
This study explored the effects of postischemic mTOR inhibition on cerebral blood flow, BBB 310
integrity, and stroke outcome in rats using MRI. The clinically approved mTORC1 inhibitor 311
rapamycin improved reperfusion CBF immediately after recanalization and decreased infarct 312
volume on day 3. Rapamycin also reduced somatosensory deficits in stroke animals and had no 313
effect on BBB, as shown by MRI 3 days following stroke onset. 314
Rapamycin significantly increased CBF in the MCA territory immediately after 315
recanalizing the occluded artery in rats. This result is in agreement with previous findings from 316
our lab showing increased collateral perfusion and increased post-reperfusion CBF in Wistar and 317
spontaneously hypertensive rats following rapamycin administration at 30 minutes post-stroke 318
(26). Similarly, Wang et al. showed that intraperitoneal rapamycin administration immediately 319
after MCAo in Sprague Dawley rats increased the diameter of deep brain collaterals between the 320
terminal branches of the PCA and MCA (44). Although these findings are encouraging, the timing 321
of administration is of central importance, especially given the potential translational capacity of 322
rapamycin, an already FDA-approved clinically used drug. Therefore, administration of rapamycin 323
after recanalization, as in this study, is of greater clinical significance compared to studies in which 324
the drug was administered before or at stroke induction (9, 13, 14, 18, 19). The mechanism by 325
which rapamycin exerts its beneficial vasodilatory effects is not fully elucidated, but studies by 326
our group and others suggest an endothelial nitric oxide synthase (eNOS) dependent mechanism 327
as seen in mouse models and isolated collateral vessels, a RhoA-dependent pathway, or direct 328
reduction in calcium sensitivity in smooth muscle cells (26, 45-47). Better understanding the 329
pathway by which rapamycin exerts its beneficial effects, which cell types are primarily targeted, 330
and which dose exerts optimal effects are essential questions for future studies. 331
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Rapamycin significantly reduced lesion volume at 3 days when compared to the vehicle-332
treated group. Our group's previous meta-analysis on rapamycin treatment in rodent models of 333
stroke included a global estimate of 30 comparisons and suggested that rapamycin significantly 334
reduced infarct volume by 22% (2). The current study exceeded the expected number with a mean 335
reduction of 39%. MRI imaging didn’t show a difference in diffusion or perfusion when 336
rapamycin-treated animals. Further, no diffusion-perfusion mismatch (DPM) was observed in 337
either the treatment or control group, suggesting that there was no “salvageable” brain tissue at 338
this time point, which might also explain why there was no effect in the treatment group. Literature 339
suggests that DPM volume gradually decreases over time, with a stark decrease to a fourth of the 340
initial penumbral region from 45 to 210 minutes after permanent MCA occlusion (27, 48). There 341
have been far fewer studies on the evolution of DPM after recanalization in temporary MCAo. We 342
found one study by Meng et al. that found the DPM was greatest within the first 2 hours following 343
reperfusion (49). Therefore, a two-hour window post reperfusion is thought to have the most 344
salvageable tissue and therefore be the optimal time for the administration of adjunct 345
cytoprotective therapies. It is likely that by administering rapamycin at the time of recanalisation 346
we were most likely to have an effect as there is still brain left to save. This is evidenced by a 347
reduced infarct volume at 3 days. 348
BBB integrity was not improved in rapamycin-treated animals versus control. Subtle 349
impairment of BBB function has been reported as early as 30 minutes to 6 hours after ischemia-350
reperfusion (I/R) in a mouse model of t-MCAo, resulting in extravasation of small macromolecules 351
(≤3 kDa) from blood to brain parenchyma (50-52). After more than 3 hours, larger macromolecules 352
(≥ 40kDa) leak through the barrier, contributing to high BBB leakage during this phase. Tight 353
junction maintain their integrity during the hyperacute phase following the insult but lose their 354
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19
integrity at around 48 hours post-I/R (51, 53, 54) due to damage caused by cytokine-activated 355
matrix-metalloproteinases MMP-3, MMP-9, and cyclooxygenase-2. The loss of tight-junction 356
integrity during the acute phase of BBB breakdown is thought to be the leading cause of the 357
development of vasogenic edema (50, 55). Previous studies have reported that rapamycin 358
treatment significantly reduced BBB breakdown after stroke (8, 9, 13, 14, 19, 56). A potential 359
explanation for the lack of benefit of rapamycin treatment is that assessment of BBB breakdown 360
in our study was a secondary analysis and it is possible that we were underpowered to detect an 361
effect. Alternatively, the lack of effect may have been due to the concentration of rapamycin 362
dropping at 3 days (as evidenced by our Western blotting data), the timepoint when BBB 363
breakdown is known to peak following ischaemic stroke. Our findings of a lack of effect of 364
rapamycin on the BBB is also consistent with our T2W brain edema analysis, which also showed 365
that rapamycin did not significantly reduce edema volume at 3 days. 366
Rapamycin significantly improved somatosensory function after stroke. Studying behavior 367
is very important for evaluating the effectiveness of neuroprotective therapy to understand its 368
impact on functional recovery. Three days is an optimal time point to assess potential long-lasting 369
functional deficits and improvements with treatment, as it is not masked by acute deficits and 370
weaknesses due to the surgery. The testing battery chosen in this study aimed to include both 371
functional and neurological deficit scores that test broad motor and somatosensory control and 372
overall functional outcome. We found that rapamycin significantly improved the time to notice 373
and time to remove the adhesive in the adhesive removal test. Interestingly, time to notice (an 374
assessment of somatosensory function) showed greater improvement in rapamycin treatment 375
compared to the time to remove (encompassing both somatosensory and motor components) (57). 376
Furthermore, time to notice showed a stronger correlation with final infarct volume than time to 377
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20
remove. These differences may be due to the increased sensitivity of time to notice to detect 378
sensory disturbances owing to the somatosensory cortex being affected by MCAo in the rat and 379
potentially subsequently salvaged by rapamycin treatment. This was depicted in Figure 2B and 380
Supplementary Table 1, where the Waxholm Space Atlas was overlaid onto the T2w MRI 381
sequences showing that, amongst other areas, the somatosensory cortex was affected by stroke and 382
at least in part salvaged by rapamycin treatment. 383
Alternatively, these findings could simply be due to the stroke surgery itself requiring the 384
ligation of the external carotid artery thus inducing ischaemia of muscle of mastication making it 385
more difficult for the animal to remove the adhesive with its mouth (58). Rapamycin treatment did 386
not significantly improve the outcome of the Bederson or Garcia tests, which assess sensorimotor 387
deficits, independent of the use of the mouth (59). Therefore our results suggest that rapamycin 388
was better at rescuing sensory deficits compared to motor deficits in our model. Our findings are 389
in agreement with previous studies that showed that rapamycin treatment improved neurological 390
outcomes (5, 6, 10, 11, 13, 14, 19, 60). Our previous study reported that rapamycin administered 391
at the time of stroke significantly improved the time to notice on the adhesive removal test (26). 392
Furthermore, our recent systematic review and meta-analysis of rapamycin in stroke also found 393
that rapamycin improved neurological scores by 30% and there was a significant correlation 394
between infarct volume and neurobehavioural scores (2). 395
Western blot of tissue from the ipsilateral cortical lesion showed that mTOR activity (as 396
measured by pmTOR:mTOR ratio) at three days was not downregulated at 3 days post-stroke. 397
suggesting that rapamycin had already exerted the entirety of its effects. To the best of our 398
knowledge, there is no information on mTOR activity after rapamycin treatment in rats after 3 399
days. However, a study in Sprague Dawley rats showed that 72 hours following a 1 mg/kg dose of 400
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21
rapamycin, the blood concentration of rapamycin dropped to ~1 nM (estimated half-life of 25 401
hours), which is below the effective concentration needed for mTOR inhibition in neuronal cell 402
culture (61, 62). Another study by Nalbandian et al reports that rapamycin’s half-life is 58 hours 403
in mice (63). Conversely, Böttiger and colleagues reported rapamycin’s half-life to be 79 hours in 404
healthy males, suggesting a species-dependent difference in the biochemical effect and metabolism 405
of the drug (64). To test this, future studies should repeat the experiments but choose earlier study 406
endpoints and compare the effects of rapamycin treatment on mTOR activity, the 407
microvasculature, and infarct volume with MRI, histology, and functional outcome. Alternatively, 408
to test whether the positive effects of rapamycin treatment are due to its mediating effects on the 409
blood flow in the hyperacute phase (i.e. within the 2-hour window of salvageable tissue post-410
recanalisation), treatment could be started after 2 hours post-recanalisation to test whether the 411
beneficial effect of rapamycin is maintained, diminished or lost. A better understanding of the 412
temporal pattern under which post-ischemic mTOR inhibition exerts its beneficial effects on stroke 413
outcomes might increase our knowledge of the underlying physiological properties of rapamycin 414
in the context of stroke and eventually optimize the treatment approach for a clinical setting. 415
In conclusion, this is the first high-quality preclinical trial of rapamycin taking into account 416
key translational considerations. We found that inhibiting mTOR activity upon reperfusion with 417
the FDA-approved drug rapamycin in an ischemic rat model immediately increases CBF. At 3 418
days, lesion volume was significantly smaller, and functional outcome improved. Thus our study 419
confirms previous findings of rapamycin’s positive impact on stroke outcomes, reinforcing its 420
potential as a cerebroprotective drug for ischemic stroke patients. Confirmation of our positive 421
results in rodents using larger animal models is an important next step for establishing the 422
translatability of our research findings. 423
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Acknolwedgements 424
AMB is currently funded by a Trans-Atlantic network grant from the Leducq Foundation and by
425
an Einstein Visiting Fellowship to the Charite in Berlin, from the Einstein Foundation, Berlin. YC
426
is funded by Alzheimer’s Research UK. DJB was funded by the Medical Research Council UK
427
(MR/M022757/1) and the Australian National Health and Medical Research Council
428
(APP1182153).
429
430
Author Contributions 431
AMS, YC, JL, AMB, and DJB contributed to the study conception and design. Material 432
preparation, data collection, and analysis were performed by AMS, YC, JL, and DJB . The first 433
draft of the manuscript was written by AMS, and all authors commented on previous versions of 434
the manuscript. All authors read and approved the final manuscript. 435
436
Disclosures/conflicts of interest 437
AMB is senior medical science advisor and co-founder of Brainomix, a company that develops 438
electronic ASPECTS (e-ASPECTS). The other authors declare no competing conflict of interest. 439
440
Supplementary information available on JCBFM Website 441
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23
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chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show 615
promising results in valosin containing protein multisystem proteinopathy. PLoS One. 616
2015;10(4):e0122888. 617
64. Böttiger Y, Säwe J, Brattström C, Tollemar J, Burke JT, Häss G, et al. Pharmacokinetic 618
interaction between single oral doses of diltiazem and sirolimus in healthy volunteers. 619
Clin Pharmacol Ther. 2001;69(1):32-40. 620
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65. Papp EA, Leergaard TB, Calabrese E, Johnson GA, Bjaalie JG. Waxholm Space atlas of 621
the Sprague Dawley rat brain. Neuroimage. 2014;97:374-86. 622
623
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32
Figures 624
625
Figure 1. Rapamycin improves post-recanalization blood flow acutely. (A) Cerebral blood flow 626
(CBF) of the middle cerebral artery (MCA) territory after recanalization and rapamycin treatment. 627
(B) Change in CBF calculated as % change from before middle cerebral artery occlusion (MCAo). 628
Data are mean (SD), ****P<0.0001, compared to vehicle. n=7 for vehicle- and n=9 for rapamycin. 629
.CC-BY-NC-ND 4.0 International licenseavailable under a
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630
631
Figure 2. Rapamycin significantly reduces infarct volume. (A) Infarct volume assessment with 632
MRI (T2w sequence) 3 days post-stroke, with correction for edema. Data are mean (SD), * P<0.05, 633
compared to vehicle. n=7 for vehicle- and n=9 for rapamycin. (B) Coronal view, T2w MRI of 634
representative stroke brains 72 h post tMCAo. Red areas salvaged by rapamycin. 635
636
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637
Figure 3. Rapamycin significantly improves functional outcomes. (A) Neurological assessment 638
with Garcia scale 72 h post-stroke (3-15 points). (B) Neurological assessment with Bederson scale 639
72 h post-stroke (0-3 points). Data are mean (SD), * P<0.05, when compared to vehicle, n=9 for 640
all groups. (C) Rapamycin significantly reduces sensory deficits assessed by the adhesive removal 641
test. Time to notice the left paw (contralateral to stroke side), calculated as the difference between 642
baseline and 72h post-stroke (seconds). (D) Rapamycin significantly reduces motor deficits 643
assessed by the adhesive removal test. Time to remove the sticky tape from the left paw 644
(contralateral to stroke side), calculated as the difference between baseline and 72h post-stroke 645
(seconds). Data are mean (SD), *** P<0.001, when compared to vehicle. n=9 for all groups. 646
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35
647
Figure 4. There is a very strong correlation between infarct volume and neurological test results, 648
with larger lesions leading to worse functional outcomes. (A) Stroke volume vs. Garcia r=-0.5502, 649
* P<0.05; (B) Stroke volume vs. Bederson r=0.6138, * P<0.05; (C) Stroke volume vs. time to 650
notice of contralateral forepaw in adhesive removal test r=0.5486, * P<0.05. n=16 for all groups. 651
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36
652
Figure 5. Rapamycin does not alter diffusion or perfusion. (A) Ipsilateral diffusion of the stroke 653
area relative to the same area on the contralateral hemisphere was assessed with MRI (ADC 654
sequence) 3 days post-stroke. (B) Absolute diffusion values of the ipsilateral hemisphere. (C) 655
Ipsilateral perfusion of the stroke area relative to the same area on the contralateral hemisphere 656
assessed with MRI (ASL sequence) 3 days post-stroke (D) Absolute perfusion values of the 657
ipsilateral hemisphere. Data are mean (SD), n=9 for rapamycin and n=9 for the vehicle group. 658
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659
Figure 6. The effect of rapamycin on edema formation and BBB integrity. (A) Rapamycin did not 660
reduce edema formation. Brain edema expressed as a percentage of whole-brain volume, assessed 661
with MRI (T2w sequence) 3 days post-stroke. Data are mean (SD), n=8 for control and n=9 for 662
the rapamycin-treated group. (B) BBB breakdown assessment with contrast-enhanced T1w spin 663
echo, multi-slice images were taken before and immediately after 0.15 ml injection of gadolinium-664
based contrast agent (GBCA) and corrected for edema formation. Data are mean (SD), n=6/group 665
in all groups. 666
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38
667
Figure 7. Rapamycin does not inhibit mTOR pathway activity at 3 days. At 3 days, brain tissue 668
from the ipsilateral cortical lesion side from animals treated with rapamycin versus control was 669
used to investigate mTOR activity (measured as pmTOR/mTOR ratio). Western Blot did not show 670
a significant difference in mTOR activity in the lesion area 3 days following the rapamycin 671
treatment. Data are mean (SD), n=4 for rapamycin and n=6 for vehicle. 672
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