Brain irradiation improves focal cerebral ischemia recovery in aged rats

Journal of the Neurological Sciences 306 (2011) 143–153

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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s

Brain irradiation improves focal cerebral ischemia recovery in aged rats Elena Titova a, Robert P. Ostrowski b, Arash Adami a, Jerome Badaut b,d, Serafin Lalas a, Nirmalya Ghosh d, Roman Vlkolinsky a, John H. Zhang b,c, Andre Obenaus a,d,e,f,⁎ a

Department of Radiation Medicine, Loma Linda University School of Medicine, CA, USA Department of Physiology and Pharmacology, Loma Linda University School of Medicine, CA, USA Departments of Neurosurgery, Anesthesiology, Physiology and Pharmacology, Pathology and Human Anatomy, Loma Linda University School of Medicine, CA, USA d Department of Pediatrics, Loma Linda University School of Medicine, CA, USA e Departments of Radiation Medicine and Radiology, Loma Linda University School of Medicine, CA, USA f Department of Biophysics and Bioengineering, School of Science and Technology, CA, USA b c

a r t i c l e

i n f o

Article history: Received 27 August 2010 Received in revised form 21 January 2011 Accepted 28 February 2011 Available online 8 April 2011 Keywords: Apoptosis Brain irradiation Cerebral ischemia Experimental stroke Middle cerebral artery occlusion Rats

a b s t r a c t Background: Studies have shown that aging is a significant factor in worsening stroke outcomes. While many mechanisms may aggravate brain injury in the elderly, one such potential system may involve increased glial proliferation in the aged stroke patient that could result in increased scar formation. We hypothesized that in aged rats a single brain-only exposure to a low radiation dose prior to focal brain ischemia would reduce glial proliferation and confer a long-term neuroprotective effect. Methods: Brain-only proton irradiation (8 Gy) was performed ten days prior to middle cerebral artery occlusion (MCAO) in aged male rats. Magnetic resonance imaging (MRI) was undertaken in naive, radiationonly (Rad), MCAO, and MCAO + Rad groups at 2, 14 and 28 days post-stroke followed by immunohistochemistry (day 28). Results: Ischemic lesion volume in MCAO + Rad group was decreased by 50.7% with an accelerated temporal reduction in peri-lesional brain edema and increased water mobility within the ischemic core (39.8%) compared to MCAO-only rats. In the peri-lesional brain region of MCAO + Rad rats there was a decreased scar formation (49%, glial fibrillary acidic protein), brain tissue sclerosis (30%, aquaporin-4) and necrosis/ apoptosis (58%, TUNEL positive cells) compared to those in MCAO animals. Conclusion: In aged animals a single exposure to brain-only radiation prior to focal cerebral ischemia is neuroprotective as it prevents glial hyperproliferation, progressive brain tissue sclerosis and reduces the apoptosis/necrosis in the peri-lesional region. Decreased lesion volume is in agreement with accelerated reduction of brain edema in these animals. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Senescence increases the incidence of stroke and results in poorer outcomes in stroke survivors [1]. In experimental settings, although infarct volume does not appear to be different between young and aged animals, critical age-dependent differences were found in the cytological responses to stroke. Compared to young adult animals, aged animals demonstrate increased glial proliferation resulting in accelerated scar development (reviewed in [2,3]). The glial scar, which is composed primarily from reactive astrocytes, produces a range of biologically active molecules that exacerbate brain damage and is associated with delayed infarct expansion [4,5]. The mature glial scar impedes revascularization and axonal restoration in the injured brain tissue and correlates with ⁎ Corresponding author at: Department of Radiation Medicine, Loma Linda University, 11175 Campus St. Chan Shun Pavilion, A-1010 Loma Linda, CA 92350 USA. Tel.: +1 909 558 7108; fax: +1 909 558 0320. E-mail address: [email protected] (A. Obenaus). 0022-510X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2011.02.034

worsened neurological deficits [5–7]. Since the astrocytic and microglial reactivity increases significantly with aging in both normal and injured brains, the modulation of glial proliferation might improve stroke prognosis in the elderly [8–11]. In response to brain injury, astrocyte proliferation is mediated by activated innate immunity mechanisms [12–14]. Control of these mechanisms has proven to be effective in numerous experimental studies, but clinical trials have been unsuccessful [15–17]. In part, the lack of clinical success relates to systemic application of agents proposed to control the immune system response [15–17]. Also, modulation of an acute immunological response to brain injury can result in an imbalance at later phases of the immune response, thus affecting post-stroke neurorepair (reviewed in [18]; see also [19]). In this light, the ability to locally control glial activation is likely to be an attractive therapeutic target. Currently little is known whether local suppression of the immune system post-stroke would reduce glial proliferation and benefit longterm stroke outcomes. To explore this concept, we used brain-only proton irradiation in a focal ischemic stroke model in aged rats. We

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found this model relevant as low dose radiation modulates scar formation and immune system responses [20–24]. Exposure to low radiation doses modulates the synthesis and release the specific growth factors regulating the glial proliferation [20,21]. Whole body radiation pre-conditioning is neuroprotective as it prevents inflammation in stroke models [22–24]. In models of axonal injury, a single low dose of total body or lymphoid organ irradiation reduces secondary neuronal degeneration and stimulates T-cell mediated neuroprotection [25,26]. In our recent study, brain-only radiation preconditioning prior to focal cerebral ischemia decreased lesion volume and apoptotic cell death in rats [27]. However, whether brain-only irradiation affects secondary brain injury progression through control of scar development remains unexplored. In our study we addressed two questions: 1) whether brain-only irradiation in advanced age animals reduces a post-stroke scar formation after focal cerebral ischemia; and 2) whether reduced scar thickness is associated with improvement in long-term outcomes in these animals.

MCAO was performed using an intraluminal thread technique [28]. Briefly, the right carotid complex was exposed through a ventral midline incision, and the external carotid artery and its branches were ligated. Temporary ligatures were placed around the common carotid artery and internal carotid artery to prevent bleeding. A polyethylene tube with an internal diameter of 0.38 mm (TUB3565, Scientific Laboratory Supplies Ltd, UK), coated by liquid silicone rubber (Silastic 732 RTV, Dow Corning GmbH, Merck Ltd, UK) with a rounded tip was used to obstruct the blood flow [29]. Through a transverse incision in the artery, the monofilament was introduced into the external carotid artery lumen, and gently advanced until it met resistance (~23 mm) in the internal carotid artery thus occluding the middle cerebral artery (MCA). After 50 min of occlusion, the thread was removed, the external carotid artery permanently ligated at the level of bifurcation and the common carotid artery and internal carotid artery sutures were removed to allow reperfusion [28]. Non-ischemic (Naive and Rad) animal groups were anesthetized but without surgery.

2. Methods

2.4. MRI data collection

All procedures were approved by the Institutional Animal Care and Use Committee at Loma Linda University and complied with the Guide for the Care and Use of Laboratory Animals. Animals were housed two per cage under 12:12 h light/dark cycle with free access to water and food.

MRI data was acquired using a Bruker 4.7 T with a 57 mm (I.D.) quadrature receiver coil. Images were acquired with 2562 matrix; a 3.5 cm field of view (FOV) producing an in-plane resolution of 0.117 mm/pixel with twenty consecutive 1 mm thick horizontal slices providing full coverage of the brain. The imaging protocol spanned multiple contrast levels including T2-weighted imaging (T2WI) and diffusion-weighted imaging (DWI). A six echo T2 sequence had a TR/ TE = 2850/20 ms with an acquisition time of 24 min. DWI also had similar parameters with a TR/TE = 3000/28 ms for an acquisition time of 51 min. MRI data were collected at 2, 14, and 28 days after MCAO induction. Rad-only animals also underwent an MRI at 2 days postirradiation that provided additional radiation-only control data.

2.1. Animal groups A total of 23 male 18 mo old Sprague Dawley rats (530–580 g; Harlan Laboratories, Inc., Indianapolis, IN, USA), were divided into four groups: naive (Naïve, no irradiation or ischemia), radiation-only (Rad), MCAO-only and MCAO + radiation (MCAO + Rad), n = 5 per group. Three animals were excluded from data analysis because two died (MCAO-only group, 28.6%) within 2 days post transient middle cerebral artery occlusion (MCAO) and one animal was excluded due to a failed MCAO. There were no significant differences in animal survival (Kaplan–Meier survival analysis, LogRank statistics, p N 0.05). 2.2. Irradiation protocol Proton exposure was performed at the Loma Linda University Medical Center Proton Treatment Facility (LLUMC PTF). Briefly, animals were anesthetized with 2% Isoflurane and placed in an animal holder in the beam line. Delivered proton beams were restricted to the brain, using a 1.2 × 0.9 cm rectangular aperture. Brain irradiation was performed 10 days prior to middle cerebral artery occlusion. MCAO + Rad and Rad rats were exposed to 8 Gy 250 MeV/amu protons at a dose rate of 5.3 ± 0.2 Gy/min, while the remaining animal groups (MCAO and Naive) were treated similarly without irradiation. 2.3. MCAO induction Ten days after irradiation rats were anesthetized with 4% Isoflurane, intubated and mechanically ventilated (Harvard Apparatus, Harvard Ventilator 683, Holliston, MA, USA). Anesthesia was maintained with 2–2.5% Isoflurane in a 30%/70% oxygen/air mixture. Ventilation parameters were synchronized to sustain PaCO2 and PaO2 within normal physiological ranges. Rectal temperature was maintained at 37 °C by a thermostatically regulated feedback heating pad (CWE Instruments, Wood Dale, IL, USA). The right femoral artery was cannulated with a PE-50 polyethylene cannula to monitor physiological variables (mean arterial pressure (MAP) and arterial blood parameters) during surgery (1610 pH/Blood Gas Analyzer; Instrumentation Laboratories, Lexington, MA, USA).

2.5. MRI data analysis All MR data sets were quantified using previously published protocols [30]. T2 relaxation rates were determined by varying the echo time (20–120 ms) and coefficients were determined by exponential fits for each pixel to generate T2 maps. Apparent diffusion coefficient (ADC) maps were generated using 2 point linear fit using two diffusion encoding gradients (b = 78 mT/m). Regions of interest (ROIs) were manually drawn on T2 and DW images displaying the largest area of lesion (ischemic core) and perilesion area. The peri-lesion region was defined as a rim of 5 pixels on the outer edge of ischemia surrounding the lesion. Regional statistics, including mean, standard deviation, and number of pixels for each ROI were obtained. All quantitative values from the right and left hemisphere were extracted and summarized. 2.6. Volumetric image analysis Volumetric MR image analysis was undertaken as previously published [30]. Briefly, using Amira software (Mercury Computer Systems, Inc.), T2 and DW images were analyzed for regions of hypoor hyperintensity changes to delineate the spatial development of the infarct volume. Analysis included infarct, non-infarct and total brain volumes. The ischemic lesion volume was calculated as a percentage of the total brain volume. 2.7. Neurological scoring At 2, 14 days and 28 days after MCAO, each rat was graded for neurological deficits in a blinded fashion using an 18-point neurological scoring system [31]. The score consisted of six tests: (i) spontaneous activity; (ii) symmetry in the movement of the four limbs; (iii) forepaw outstretching; (iv) climbing; (v) body proprioception; and (vi) response

E. Titova et al. / Journal of the Neurological Sciences 306 (2011) 143–153

to vibrissae touch. The score (1–3) given to each rat at the completion of the evaluation was the summation of all six individual test scores. Minimum score was 3; maximum 18. 2.8. Euthanasia and sample collection Animals were intracardially perfused at 28 days post-MCAO with ice-cold 0.12 M Millonig's phosphate buffer, pH 7.3 (1 ml/1 g body weight) and decapitated. The brains were cut through the center of ischemic core, as determined from T2WI, and fixed in 4% paraformaldehyde (PFA) (Electron Microscopy Science, Hatfield, PA) for 24 h, then 2 × 5 min wash in Millonig's buffer and stored at 4 °C. 2.9. Histology and immunohistochemistry Fixed brain tissue was cryoprotected in 30% sucrose, and frozen in optimal cutting temperature compound (O.T.C., Tissue Tek). Consecutive coronal sections (30 μm) were cut on a cryostat (Leica CM1850, Leica Microsystems GmbH, Wetzlar, Germany) and stored in cryoprotectant at 4 °C with every tenth coronal section mounted on Superfrost Plus slides for standard Cresyl Violet (CV) staining. The images from five FOVs: two in the motor cortex and three in the peri-lesion in each coronal section (see Fig. 4C, inset) were digitized using Olympus BX-71 microscope (Olympus America Inc., Melville, NY, USA) equipped with a digital camera (Cook, Auburn Hills, MI, USA). To compare the size of the ischemic lesion obtained from MRI to histological data, brain sections with the maximal lesion size were identified and captured at low magnification (Olympus SZ-CTV, Olympus Optical CO, LTD, Tokyo, Japan). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was performed on coronal brain sections from each animal at the level corresponding to the peri-lesion area (approximately 1 mm from the ischemic core) using an In Situ Cell Death Detection Kit (Roche Indianapolis, IN, USA). Briefly, sections were boiled in citric buffer, pH 6.0, for 15 min, washed in 0.01 M PBS and labeled. A mixture of Fluorescein isothiocyanate (FITC)-labeled nucleotides and terminal deoxynucleotidyl transferase was applied onto the sections for 60 min at 37 °C in a dark humidified chamber. Incubation with the labeling solution without the enzyme served as a negative control. Manual counting of TUNEL positive cells was obtained from a visual field of 0.0357 mm2 at ×400 magnification from both hemispheres in two slides per rat (OLYMPUS BX51, Olympus America Inc., Melville, NY). Cell counting was performed independently by two researchers in a blinded fashion and then averaged.

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Burlingame, CA). Immunofluorescent preparations were examined using epifluorescence microscopy (Olympus, BX41, Switzerland). Controls were performed by omitting the primary antibody. All controls gave negative results with no detectable labelling. Depletion of the antibody by an excess of the specific peptide (from Chemicon) was also carried out and also gave negative results.

2.10. Glial scar area assessment The glial scar area was calculated from two histological brain sections per animal from immunolabeled anti-GFAP sections as noted above. GFAP immunofluorescence image ROIs automatically calculated the scar area using customized software. A threshold was applied where the signal intensity higher normal tissues provided a binary mask of the scar. The mask underwent computational morphological closing operations. The area of the binary mask was then calculated.

2.11. Statistical analysis Quantitative data were expressed as the mean ± SEM, where n indicates the number of animals. Statistical significance was tested using repeated measures in one way analysis of variances (one way ANOVA) followed by Holm–Sidak post-hoc tests (Systat Software, Inc., Richmond, CA). A two or three-way ANOVA was used to analyze the interaction effects for factors of time, radiation and ischemia. Survival data was analyzed by Kaplan–Meier survival analysis, LogRank statistics (SyStat Software, Inc., Richmond, CA). Pearson Product Moment Correlation Coefficients were used to measure the dependency between temporal recovery of neurological functions and decreases in peri-lesional brain edema (SyStat Software, Inc., Richmond, CA). P b 0.05 was considered statistically significant. 3. Results 3.1. Physiological variables Arterial blood parameters (pH, PaCO2, PaO2) and MAP were monitored in all animals that underwent MCAO and radiation but no significant differences between the experimental groups were observed (Table 1).

3.2. Neurological outcomes 2.9.1. Immunohistochemistry Double aquaporin-4-glial fibrillary protein (AQP4-GFAP) immunolabeling was evaluated on serial slices as previously published [32]. Infrared immunostaining was carried out in PBS containing 0.25% Triton X-100 and 0.3% bovine serum albumin. For double immunolabeling sections were incubated overnight at 4 °C with rabbit anti-AQP4 (1:300, Chemicon, CA, USA) and mouse anti-GFAP (1:400, Chemicon, CA, USA). Half of the sections were followed by infrared-Alexa-680-nm secondary anti-rabbit (1:1000, Molecular Probes, Invitrogen, CA, USA) and infrared-Dye-800-nm secondary anti-mouse (1:1000, Rockland Immunochemicals, PA, USA) for 2 h at room temperature. After incubation the mounted sections were rinsed in PBS 3 × 10 min. Sections were then scanned using an infra-red scanner and fluorescence from GFAP- and AQP4-immunoreactivity was quantified (Odyssey-system, LI-COR Biotechnology, USA) from the same five FOVs used in our quantitative TUNEL assessment. Fluorescence signal intensity was reported as integrated-intensities (a.u). The second half of the floating sections were incubated for 2 h at room temperature with an Alexa-Fluor568 nm coupled secondary rabbit antibody (Molecular Probes, Invitrogen, 1:500). Sections were mounted and coverslipped with anti-fading medium Vectashield containing DAPI (Vector, Vector laboratories,

No significant change in neurological status was found in irradiatedonly compared to naïve animals demonstrating no radiation-only effects. Significant neurological deficits were observed in all animals that underwent MCAO during the observation period (p b 0.05, MCAO vs. Naïve, MCAO + Rad vs. Rad-only, repeated measures one way ANOVA). No differences in neurological function between irradiated and nonirradiated ischemic animals were significant at any time point. At days 14 and 28 post-stroke all ischemic animals demonstrated significant improvement from the initial neurological deficits (pb 0.001 days 14 and 28 vs. day 2, repeated measures one way ANOVA, Fig. 1A). In both groups of animals that underwent cerebral ischemia, recovery of neurological function was significantly dependent on the time postMCAO. A strong negative correlation over time revealed a reduction in peri-lesional brain edema (ischemia, pb 0.001; time, p =0.044; time x ischemia, p=0.036, two way ANOVA; Pearson Correlation Coefficient, r: MCAO r= −0.601, p =0.01, n =16; MCAO+ Rad, r=−0.595, p=0.02, n=15; Fig. 1B). Temporal analyses demonstrated that reductions in brain edema over 28 days post-stroke exhibited improved neurological function (Table 2).

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Table 1 Physiological variables. Parameters

pH PaCO2, mm Hg PaO2, mm Hg MAP, mm Hg HR/min Temp, °C

15 min before MCAO

30 min during MCAO

15 min after reperfusion

MCAO

MCAO + Rad

MCAO

MCAO + Rad

MCAO

MCAO + Rad

7.3 ± 0.01 37.6 ± 0.9 136.2 ± 3.8 98.8 ± 1.5 284.7 ± 4.1 36.8 ± 0.1

7.4 ± 0.01 36.4 ± 1.1 140.8 ± 7.1 99.1 ± 1.4 280 ± 3.6 36.6 ± 0.1

7.3 ± 0.01 39.3 ± 0.9 134.3 ± 2.9 99.7 ± 1.4 317.7 ± 4.4 36.7 ± 0.1

7.4 ± 0.01 35 ± 0.7 137 ± 5.3 96.9 ± 1.4 326.9 ± 2.4 36.6 ± 0.1

7.3 ± 0.02 41 ± 1.3 125.5 ± 6.2 110 ± 2.2 316.3 ± 9.7 36.8 ± 0.1

7.4 ± 0.01 37.5 ± 1.2 133.3 ± 10.7 105 ± 0.0 321.3 ± 6.6 37 ± 0.0

3.3. MRI analysis Acute (day 2) ischemic lesion volume, measured from the T2images, was reduced 50.7% in MCAO + Rad animals compared to MCAOonly (pb 0.05, two way ANOVA, Fig. 2A). Although not significant at day 14 or 28, decreased lesion volumes in the MCAO + Rad group were observed at all time periods (Fig. 2). The temporal improvement in lesion volume (i.e. decrease) was evident in both groups of ischemic animals; however, the time dependent reduction in the ischemic lesion volume was significantly affected by brain irradiation (effects of time, p b 0.001; radiation, p = 0.003; tendency to interaction radiation x time, p = 0.07, two way ANOVA). Quantitative analysis of DWI (ADC values) allows in vivo monitoring of water mobility within brain tissues. A significant acute increase in ADC values in the ischemic peri-lesional region was

found in MCAO + Rad but not in MCAO-only animals (day 2, p b 0.05, MCAO + Rad vs. Rad, one way ANOVA, Fig. 3A). There was a chronic temporal increase in ADC values up to 28 days within the ischemic core in MCAO + Rad animals that was not observed in MCAO-only animal group (MCAO + Rad: p b 0.01 vs. Rad, p b 0.01 vs. MCAO, p b 0.01 vs. day 2, one way ANOVA, Fig. 3B). This temporal increase in ADC values in MCAO + Rad animals exhibited a dependence on radiation and ischemia interactions within the ischemic core but not in the ischemic peri-lesion (ischemic core: time, p = 0.031, radiation, p = 0.017, ischemia, p = 0.008, interaction radiation x ischemia, p = 0.028, three way ANOVA). This difference between the MCAO + Rad ischemic peri-lesion and ischemic core ADC values was significantly increased at day 28 (peri-lesion vs. core, p b 0.01, one way ANOVA). Analysis of T2 values allows evaluation of total brain water content following stroke. A dramatic increase in T2 values in the ischemic peri-lesion and core regions was found in all animals that underwent cerebral ischemia (MCAO + Rad vs. Rad; MCAO vs. Naïve, peri-lesion: p b 0.05; core: p b 0.001, one way ANOVA, Fig. 3C, D). At 28 days postMCAO there was an increase in T2 values within the ischemic core compared to the peri-lesion (peri-lesion vs. core, p b 0.01, repeated measures one way ANOVA). Both groups of ischemic animals demonstrated a temporal improvement in T2 values in the ischemic peri-lesion. However, this improvement was significantly accelerated in irradiated ischemic animals (MCAO + Rad, days 14, 28 vs. day 2, p b 0.001, repeated measures one way ANOVA, Fig. 3C, D). We further analyzed the effects of brain irradiation and cerebral ischemia and their interactions on the temporal evolution of T2 values. Cerebral ischemia was found to be the only significant factor that independently increased the T2 values in both, ischemic perilesion and ischemic core at all experimental time points (ischemic core and ischemic peri-lesion: ischemia, p b 0.001, three way ANOVA). 3.4. Histology and immunohistochemistry 3.4.1. Infarct lesion size evaluated by MRI and by morphology At the final time point (28 days post-stroke) the infarction size was found to be similar between the Cresyl Violet (CV) stained sections and lesion size obtained from T2 images. 3.4.2. Cresyl violet staining Our MCAO model resulted in primarily a subcortical infarct without overt direct involvement of the motor cortex (Figs. 2 and 4). However,

Table 2 Temporal improvements in neurological function (Pearson correlation coefficients, r). Fig. 1. Recovery of post-stroke neurological deficits. A. A temporal improvement in neurological function (Neuroscore) was found in both ischemic animal groups without group differences (*p b 0.05 vs. Day 2, repeated measures one way ANOVA). Dotted line indicates the averaged neurological score from naïve and Rad-only animals. B. Summary of the temporal profile and correlations between neurological recovery (Neuroscore) and reduction in brain edema within the ischemic peri-lesion (T2 values). All animals that underwent focal cerebral ischemia demonstrated a significant negative correlation between these two parameters: MCAO, r = −0.601, n = 16; MCAO+ Rad, r = −0.595, n = 15; p b 0.05).

Time/group

Day 2

Day 14

Day 28

MCAO

r = −0.200 p = 0.704 n=5 r = −0.266 p = 0.666 n=5

r = −0.748 p = 0.146 n=5 r = −0.355 p = 0.558 n=5

r = −0.730 p = 0.161 n=5 r = 0.812 p = 0.095 n=5

MCAO + Rad

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3.4.3. TUNEL staining Cerebral ischemia and radiation each affected the number of TUNEL positive apoptotic/necrotic cells (Fig. 4A, e–h, B, e–h white arrows). In the ischemia-only animals, the TUNEL positive cell numbers were increased by 5.8- and 20.1-fold in the cortex (Fig. 4Ag) and peri-lesion (Fig. 4Bg), respectively, compared to nonischemic naive animals (MCAO vs. Naive, p b 0.001, one way ANOVA, Fig. 4C, D). Compared to naive control animals, in the Rad-only group the 6.4-fold increase of TUNEL positive cells was only observed in the region corresponding to the ischemic peri-lesion (Fig. 4Bf) with no significant differences in the motor cortex (Fig. 4Af) (Rad vs. Naive, p b 0.001, one way ANOVA, see Fig. 4D). In irradiated ischemic animals the number of TUNEL positive cells was reduced by 57.1% in the perilesional (Fig. 4Bh) and 56.6% in the cortical (Fig. 4Ah) regions compared to non-irradiated ischemic (MCAO + Rad vs. MCAO, perilesion; motor cortex, p b 0.001, one way ANOVA, Fig. 4C, D). The reduction in the number of TUNEL positive cells was significantly dependent on a radiation and ischemia interaction (cortex: radiation, p = 0.215, ischemia, p b 0.001, radiation × ischemia, p = 0.003; perilesion: radiation, p = 0.142, ischemia, p b 0.001, radiation × ischemia, p b 0.001, two way ANOVA). 3.5. Glial fibrillary acidic protein (GFAP) In naïve animals, differences in GFAP expression between cortex and peri-lesional regions reflect the normal spatial distribution in accordance with previous published data [33] (p b 0.05, Naive: cortex vs. peri-lesion, ANOVA, Fig. 5G). In irradiated-only animals GFAP expression was significantly increased in both the cortex and the perilesional regions (p b 0.01, cortex, peri-lesion: Rad vs. Naive, one way ANOVA). At 28 days, the post-ischemic up-regulation of GFAP was limited to the peri-lesional area in the MCAO-only with no differences found in the cortex (peri-lesion: p b 0.01 vs. Naive, one way ANOVA, Fig. 5G). In irradiated ischemic animals the increased GFAP expression was significantly reduced within peri-lesion (p b 0.01, MCAO + Rad vs. MCAO, one way ANOVA; Fig. 5A, D, G; effect of factors: cortex: radiation, ischemia and ischemia × radiation, p b 0.001; peri-lesion: radiation, p = 0.389, ischemia and ischemia × radiation, p b 0.001, two way ANOVA). Fig. 2. Radiation reduces lesion volume in ischemia-injured brain tissue. A. Acute (day 2) reduction in lesion volume was significant in the MCAO + Rad compared to the MCAOonly group and remained decreased for 28 days post-injury (*p b 0.05 vs. MCAO, 2 way ANOVA). The temporal decrements in lesion volume were significant in each group of animals that underwent cerebral ischemia († MCAO, # MCAO + Rad, p b 0.05 vs. day 2; two way ANOVA). B, C. T2- and DWI-derived 3D reconstructions clearly illustrate the time course of the reduction in the ischemic lesion volumes.

scattered neurons with fragmented nuclei were observed in the MCAOonly (wide white arrows, Fig. 4Ac) but not in the MCAO + Rad cortex (Fig. 4Ad). In addition, tissue density appeared to be reduced in the Rad-only cortex (Fig. 4Ab) compared to naive animals (Fig. 4Aa). No observable differences between groups were found in the number of shrunken neurons (open black arrows) and pyknotic small sized cells (solid white arrows, Fig. 4A–B). Within the ischemic peri-lesion (Fig. 4B, a–d) a few dark small sized cells (solid white arrows) and shrunken neurons (open black arrows) were found in Naive (Fig. 4Ba) and Rad-only (Fig. 4Bb) animal groups. Pale stained neurons with loss of discernible internal cell structures (degenerating neurons, wide white arrows) were found only in the Rad-only animals (Fig. 4Bb). Scattered clusters of small cells with non-structured backgrounds, dark apoptotic/necrotic cells and apoptotic bodies (Fig. 4Bc,d, white arrows) were observed in both ischemic animal groups: MCAO (Fig. 4Bc) and MCAO + Rad (Fig. 4Bd). Infiltration of small sized cells and the occurrence of dark apoptotic/necrotic cells appeared to be lower in the MCAO+ Rad peri-lesion compared to MCAO.

3.6. Aquaporin 4 (AQP4) In contrast to GFAP expression, brain irradiation had no effect on AQP4 expression in either the cortex or the peri-lesion region (Rad vs. Naïve: cortex vs. peri-lesion, two way ANOVA; Fig. 5H). However, a dramatic increase in AQP4 expression was evident in the peri-lesion of the ischemic-only animals, but not in the motor cortex (p b 0.001, peri-lesion, MCAO vs. Naive, p b 0.05 cortex vs. peri-lesion, one way ANOVA; Fig. 5B, E, H). In irradiated ischemic animals AQP4 expression returned to control levels in peri-lesion (p b 0.01, MCAO + Rad vs. MCAO, one way ANOVA; effect of factors: radiation, p = 0.044, ischemia, p = 0.009, radiation × ischemia, p = 0.011; two way ANOVA, Fig. 5H). 3.7. Post-ischemic scar In all ischemic animals an ischemic scar around the lesion was clearly detectable by increased GFAP immunostaining surrounding the ischemic cavity (Fig. 6). The area of ischemic scar that surrounded the ischemic cavity was found to be reduced 49% in radiation exposed animals (p b 0.05, MCAO + Rad vs.MCAO-only, t-test; Fig. 6B). 4. Discussion We report for the first time that a single low dose of brain-only proton radiation prior to MCAO results in the long-term improvement

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Fig. 3. Analysis of ADC and T2 values. There were no significant differences between the two control animal groups (naïve and Rad-only) in ADC or T2 values at any time point. A. Increased ADC values within the ischemic peri-lesion were found significant in the MCAO+ Rad but not in the MCAO-only animal group (day 2, *pb 0.05, MCAO + Rad vs. Rad, one way ANOVA). B. At days 14 and 28 ADC values were remarkably increased within the ischemic core in MCAO+ Rad group whereas no ADC values differences were found in MCAO-only animals (* p b 0.01 vs. Rad, ‡ p b 0.01 vs. MCAO, ‡‡ p b 0.01 vs. Day 2, one way ANOVA). C. There was a significant increase in T2 values within the peri-lesion in both MCAO and MCAO + Rad animal groups compared to non-MCAO groups (# MCAO vs. Naïve, * MCAO + Rad vs. Rad-only, p b 0.05, one way ANOVA). While no T2 differences between irradiated and non-irradiated ischemic animals were found at any time point, the temporal improvement in T2 was accelerated in the MCAO+ Rad peri-lesion (MCAO + Rad: Days 14 and 28 vs. Day 2, † p b 0.05, one way ANOVA). D. There were elevated T2 values within the ischemic core in all MCAO animals at 28 days post-stroke (* MCAO+ Rad vs. Rad, # MCAO vs. Naive, p b 0.001; one way ANOVA).

of several brain injury measures in aged rats. The key findings between irradiated and non-irradiated ischemic animal groups were: at the acute post-stroke stage—(i) reduction of the ischemic lesion volume, at the chronic stage—(ii) accelerated temporal reduction of brain edema in the ischemic peri-lesion accompanied by increased water mobility in the ischemic core, (iii) decreased necrotic/apoptotic cell death and reactive astrogliosis in the ischemic peri-lesion with reduced glial scaring surrounding the ischemic cavity. Based on quantitative MRI analysis and brain histopathology we concluded that brain-only radiation pre-conditioning was neuroprotective in aged animals. The neuroprotection was effected by accelerating decrements in post-stroke brain edema in the potentially salvable ischemic peri-lesion and clearing of necrotic tissue from the ischemic core with reduced tissue scarring. Using two established MR imaging techniques, relaxometric (T2WI, T2-mapping) and diffusion-weighted imaging (DWI, ADCmapping), we monitored the effects of local radiation pre-conditioning on stroke progression and recovery. T2WI and DWI are recognized as highly valid diagnostic and prognostic tools for noninvasive monitoring and evaluation of therapeutic interventions in stroke and study of stroke pathophysiology (reviewed in [34,35]). While T2 values reflect total brain water content, ADC values characterize water mobility in extracellular and intracellular compartments indicative both histological structure and cellularity [36,37]. The dynamics of MRI changes in stroke and corresponding histopathophysiological consequences are well documented in both, experimental and human studies [38– 40]. In experimental stroke an acute biphasic reduction of ADC values correlates with an initial drop in cerebral blood flow, progressing to

cytotoxic brain edema and inflammation [41]. At this early post-stroke phase, brain histology accompanying MRI studies reveal reactive gliosis, astrocytic swelling, shrunken neuronal perikarya and nuclei surrounded by swollen cellular processes and narrowing of the microvascular lumen [42,43]. Declining ADC values are associated with elevated T2 values delineates irreversible ischemic damage and correlates with neurological deficits and a final infarct volumes [39,44]. Further ischemic injury development: loss of cell membrane integrity, structural tissue disintegration and cellular necrosis coexisting with a massive influx of hematogenous inflammatory cells and advanced glial activation is reflected by an ADC rebound (“pseudonormalization”) [44–46]. ADC pseudo-normalization occurs around 2 days post-experimental stroke and about 10 days in human stroke [47]. Co-occurrence “pseudo-normal” ADCs with elevated T2 values indicates existence of free, unbound water, i.e. progressive vasogenic brain edema [48]. Finally, unbound water replaces the necrotic cells in the core of ischemia; in surrounding tissue, it accumulates in extracellular space shrunken as a result of advancing brain tissue sclerosis and glial activation [49]. The chronic increase in ADC values reflects progressive necrotic cell loss followed by wound cleaning by actively recruited peripheral blood phagocytes [37,49,50]. In the ischemic cavity the co-occurrence of highly increased T2 values and elevated ADC values is linked to removal necrotic cell debris by phagocytes [46,49,51]. In the older individuals, both in animals and humans, the ADC values pseudo-normalization occurs earlier and the chronic ADC increase is found to be lower [45,52]. In the present study, we evaluated MRI data beginning from day 2 with focus on the late phase: necrosis and wound cleaning in ischemic

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Fig. 4. Radiation reduces apoptosis at 28 days after MCAO induction. A. Motor cortex. Cresyl Violet (CV) (a–d) staining demonstrated no differences in the number of shrunken neurons (open black arrows), pyknotic smaller cells and apoptotic bodies (thin white arrows) in the motor cortex between all four experimental animal groups. There was an apparent reduced tissue density in the Rad-only group (b) when compared to Naive (a). Neurons containing fragmented nuclei were only found in the MCAO-only animals (wide white arrows, c) but not in the MCAO + Rad cortex (d). Bar 20 μm. The number of TUNEL (e–h) positive cells (white arrows) appeared to be higher in the MCAO (g) compared to the MCAO + Rad group (h). No differences were found in naive (e) and Rad-only (f) animals. Bar 30 μm. B. Ischemic peri-lesion. Cresyl violet (a–d) shrunken neurons (open black arrows) and dark apoptotic/necrotic cells (thin white arrows) were found in naive (a) and Rad-only animal groups (b). Pale stained degenerating neurons with no nuclei (wide white arrows) were observed in Rad-only animals (b). Clusters of small pyknotic cells and apoptotic bodies (thin white arrows) were observed in each of ischemic animal groups. These numbers appeared to be reduced in MCAO + Rad (d) compared to MCAO-only (c) animals. Bar 20 μm. No TUNEL (e–h) positive cells (white arrows) were observed in the area corresponding to the ischemic peri-lesion in Naive (e) in contrast to Rad-only animals (f). There was a reduced number of TUNEL positive cells in the MCAO + Rad rats (h) compared to the MCAO-only group (g). Bar 30 μm. C, D. In irradiated-only animals the number of TUNEL positive cells were increased in area corresponding to the ischemic peri-lesion (D) but not in the motor cortex (C) (*p b 0.001, Rad vs. Naive, one way ANOVA). In the ischemic-only animals, TUNEL positive cell numbers were significantly increased in the motor cortex (C) and the peri-lesion (D) (**p b 0.001, MCAO vs. Naive, one way ANOVA). In irradiated ischemic animals TUNEL positive cell numbers were significantly decreased in both the motor cortex (C) and ischemic peri-lesion (D) (# p b 0.001 MCAO + Rad vs. MCAO, one way ANOVA). Inset in (C) are the fields from which the cell counts were performed. (Contr— contralateral, Ipsi—ipsilateral hemispheres). Bar 1000 μm.

cavity and brain tissue repair in the surrounding peri-lesional area. Consistent with published data, T2 values were notably increased in the ischemic core without between ischemic animal group differences [38]. However, the dynamic pattern of ADC values differed. In ischemic-only animals, no anticipated ADC increases were observed; instead, ADC values remained unchanged across our experimental timelines. Several possible explanations exist. One feature of stroke in aged animals in our study was that the stroke induced a relatively

small and predominantly subcortical infarct. This primary subcortical infarct may not alter ADC values on the same scale as reported when longer occlusion times in young animals were used [53,54]. Also, accelerated and increased temporal activation of post-stroke inflammatory and glial responses in aged animals likely increases tissue cellularity, thus contributing to our observed lower ADC values compared to those reported in younger animals [6,45,52,55]. Longterm MRI monitoring data in aged stroke animals is limited with

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Fig. 5. Radiation reduces post-ischemic glial proliferation. Confocal microscopy illustrates immunohistochemical localization of glial fibrillary acidic protein (GFAP, A, D) and aquaporin-4 (AQP4, B, E) in the ischemic peri-lesion in MCAO (A–C) and MCAO+ Rad (D–F) animal groups. GFAP (A, D) was up-regulated in the peri-lesion of MCAO-only animals (A) compared to the MCAO+ Rad animals (D). Bar 40 μm. We also observed an increased expression of AQP4 within the peri-lesion of MCAO animals (B) but not in the MCAO + Rad animal group (E). The merged GFAP (green) and AQP4 (red) images (C, F) in MCAO and MCAO+ Rad animals are shown. Nuclei are counterstained with DAPI (blue). G. Differences in GFAP expression between cortical and peri-lesional regions were found in both Rad-only and naïve animals (** p b 0.05 cortex vs. peri-lesion, two way ANOVA). In irradiated-only animals a dramatic increase in GFAP signal intensity within the motor cortex and peri-lesion was observed reflecting reactive astrogliosis († p b 0.01 Rad vs. Naive, one way ANOVA). The ischemic-only animals increased GFAP signal intensity in the ischemic peri-lesion was observed but not in the motor cortex (* p b 0.01 MCAO vs. Naïve, one way ANOVA). GFAP expression was significantly reduced in irradiated ischemic brains (# p b 0.01 MCAO-Rad vs. MCAO, one way ANOVA). H. Increased AQP4 expression was found in the MCAO-only ischemic peri-lesion (MCAO: perilesion, * p b 0.001 vs. Naive; ** p b 0.05 peri-lesion vs. cortex, two way ANOVA). In the peri-lesion of MCAO + Rad animals the AQP4 staining recovered to control levels compared to MCAO-only animals (# p = 0.006 vs. MCAO, two way ANOVA).

significantly different experimental paradigms being utilized [43,54,56,57]. We compared MRI data in non-irradiated and irradiated ischemic animal groups. In irradiated ischemic rats ADC values were significantly elevated across all time points. The increase in ADC values at day 2 in the ischemic peri-lesion continued to increase within the ischemic core until day 28. ADC elevations suggest reduced tissue density in irradiated animals [39,58]. The dynamics in apparent tissue density and ADC values are related to ongoing inflammation and inflammatory cell infiltration [39,58–60]. We previously showed that

massive glial infiltration results in a dramatic reduction in ADC [61]. Extensive blood-brain barrier breakdown and acute neutrophil recruitment after a single intrastriatal injection of interleukin 1-beta leads to a long (up to 5 days) suppression of ADC values [62]. Also, massive macrophage infiltration in the infarct border zone is linked to post-ischemic ADC pseudo-normalization [39,58]. In contrast, increases in ADC values were found in similar brain areas with reduced cresyl violet staining, thus mirroring both apparent tissue density and cellularity [59]. Decreased tissue density and ADC increases co-existing with elevated T2 values and reflects local

Fig. 6. Radiation reduces glial scaring around the ischemic cavity. GFAP immunofluorescence images from ischemic-only (A) and irradiated ischemic (B) brain sections. The area of the binary mask was then obtained (Ab, Bb). The area of the scar (C) was significantly reduced in irradiated ischemic animals compared to non-irradiated (* p b 0.05, t-test).

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activation of immune system and T-cell modulated lysis of cells [60]. In our study, local brain immunity modulated by brain irradiation altered the dynamics of water mobility derived from ADCs. In the acute post-stroke phase peri-lesional ADC increases can indicate both, reduced influx of inflammatory cells and activated necrotic cell lysis [58,60,62]. At the late time points, increases in ADC and T2 values in the ischemic core correspond to extended phagocyte activation, cell lysis and ischemic cavity debridement [58,60,63]. At these time points no group differences were found in peri-lesional ADC values. However, in irradiated ischemic animals brain histology revealed visibly reduced cellular infiltration and tissue density. In these animals, unchanged ADC values suggest that other mechanisms contribute to the balance of inter-compartmental water distribution. For example, Lin et al., proposed that “re-normalized” ADCs can reflect a temporal post-ischemic improvement of cerebral blood flow (CBF) and cerebral blood volume (CBV) and increased vascular density [64]. In their model, parallel increases in CBV and vascular density were interpreted as activated angiogenesis that peaked at 7 days postischemia and return to basal level by day 14 [64]. Reduced brain tissue density in cresyl violet staining agrees with remarkable (by 57%) decrease in TUNEL positive cells in irradiated ischemic brains. In a focal cerebral ischemia model, more than 95% of TUNEL positive cells in the inner boundary zone of the infarct were reported to be neurons [65,66]. TUNEL positive cells are thought to reflect late apoptotic and/or necrotic phases [67]. At 28 days poststroke the decrease in TUNEL positive cells provides evidence for attenuated neuronal death and long-term neuroprotection induced by local radiation pre-conditioning. Specific molecules are expressed on apoptotic cell surfaces allowing their recognition by phagocytes [68]. In irradiated ischemic animals, attenuated apoptotic cells signaling appeared to recruit fewer phagocytes to the injury site as reflected by decreased cell abundance and brain tissue density [69,70]. While neuroprotective in the stroke-injured brain tissue, in healthy, non-ischemic brain tissue (Rad-only animals) radiation activated apoptotic cell death mechanisms [71,72]. Single exposures to low or moderate radiation doses induces apoptotic cell death in brain and spinal cord as increased numbers of TUNEL positive cells [72,73]. The majority of TUNEL positive cells were reported to be oligodendrocytes; post-mitotic neurons are less sensitive to radiation exposure [72–74]. In our study, increased TUNEL positive cells in the Rad-only animal group were significant only in the corresponding ischemic stroke areas (i.e. predominantly white matter regions, oligodendrocytes) but not in the cortical areas. Radiation alone induces astrogliosis (defined as up-regulated GFAP expression), microglial activation and blood-brain barrier damage in numerous experimental and humans studies [71,75,76]. However, ischemic brain irradiation reduces severity of a chronic astrogliosis in the surrounding ischemic cavity areas, indicative of reduced postischemic scar thickness. Scar formation is known to inhibit nerve fiber growth by releasing chondroitin sulfate proteoglycans and physical blockade of axonal and vasculature regeneration [77]. The increased severity of post-stroke scar development in aged animals significantly contributes to worsening of the neurorepair process [7]. In our study, there was a reduction of post-stroke reactive astrogliosis by 28% in the ischemic animals pre-treated with radiation. The decreased gliosis was further evidenced by a 49% reduction of the peri-lesional scar area, thus likely contributing to the post-stroke neurorepair in the MCAO + Rad animals [78]. Brain tissue sclerosis, i.e. stiffening of structures, cell shrinkage, and replacement of normal brain tissue with connective scar tissue can be reflected by AQP4 expression levels [79,80]. AQP4, the primary water channel of the mammalian brain, is normally expressed by astroglia at the border between brain parenchyma and major fluid compartments (cerebrospinal fluid, blood) and has a critical role in acute brain edema formation. In the chronic post-injury state AQP4 facilitates astrocyte migration toward the site of injury and thus

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contributes to scar formation [79,81,82]. Inhibition of AQP4 impairs astroglial mobility thus affecting glial scarring in the injured brain tissue [81,82]. In human studies, chronic AQP4 up-regulation is associated with increased brain tissue sclerosis and atrophy [80]. In our present study, up-regulated AQP4 expression in ischemic-only animals returned to baseline when the brain was irradiated accompanied by significantly reduced glial scar formation. As expected, overall neurological function recovery was found to negatively correlate with the temporal decrease in brain edema [54,83]. Somewhat surprisingly, in every time point evaluated irradiated ischemic animals demonstrated a positive correlation between decreasing T2 values with improved neurological scores at day 28. Considering accelerated brain edema reduction and progressive temporal neurological improvements, an inverse correlation at the delayed time point in the irradiated ischemic animal group reflects a more complicated relationship involved in activated neurorepair mechanisms [64,84]. 4.1. Conclusions and significance We observed significant short- and long-term improvements in measures of stroke injury in irradiated aged animals including decrements in glial scarring. Pre-conditioning-induced ischemic tolerance is an effective experimental paradigm to understand how the brain protects itself [83,85,86]. While the underlying mechanisms remain unclear, there are several possibilities. We found a similar pattern in the reduction of acute brain edema in the brain-only compared to whole body irradiated animals. Strachan et al. reported that 5 Gy of whole body irradiation 7 days prior to MCAO significantly reduced brain edema 24 h post-stroke. They interpreted their results as radiation-induced immunosupression [24]. While no immune competent organs were directly irradiated in our study, the brain irradiation could effect normal post-stroke microglia signaling and following activation of inflammatory cascade mechanisms. Complimentary mobilization of stem cells are known to be neuroprotective in stroke and have been reported to be amplified by brain irradiation [87,88]. Additional studies are required to elucidate the underlying mechanisms of radio-neuroprotection in stroke and evaluate its potential benefits, including older, more aged animals. Conflict of interest The authors declare no conflict of interest. Acknowledgements The authors would like to thank Suzzanne Marcantonio for MCAO induction and Maria Moldovan for cresyl violet image acquisition. This work was supported by research funds from the Non-Invasive Imaging Laboratory, Department of Radiation Medicine. References [1] Marini C, Triggiani L, Cimini N, Ciancarelli I, De Santis F, Russo T, et al. Proportion of older people in the community as a predictor of increasing stroke incidence. Neuroepidemiology 2001;20:91–5. [2] Popa-Wagner A, Carmichael ST, Kokaia Z, Kessler C, Walker LC. The response of the aged brain to stroke: too much, too soon? Curr Neurovasc Res 2007;4:216–27. [3] Buga AM, Dunoiu C, Balseanu A, Popa-Wagner A. Cellular and molecular mechanisms underlying neurorehabilitation after stroke in aged subjects. Rom J Morphol Embryol 2008;49:279–302. [4] Mori T, Tan J, Arendash GW, Koyama N, Nojima Y, Town T. Overexpression of human S100B exacerbates brain damage and periinfarct gliosis after permanent focal ischemia. Stroke 2008;39:2114–21. [5] Wanner IB, Deik A, Torres M, Rosendahl A, Neary JT, Lemmon VP, et al. A new in vitro model of the glial scar inhibits axon growth. Glia 2008;56:1691–709. [6] Petcu EB, Sfredel V, Platt D, Herndon JG, Kessler C, Popa-Wagner A. Cellular and molecular events underlying the dysregulated response of the aged brain to stroke: a mini-review. Gerontology 2008;54:6–17.

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