Alteration in cerebral MFG-E8 protein level and gene expression after permanent focal cerebral ischemia by middle cerebral artery occlusion (MCAO). (a) Cerebral MFG-E8 protein levels were measured by Western blot at 24 h post-MCAO. Data are presented as mean±SE, and analyzed by Student’s t-test. Compared with Sham, cerebral ischemia (MCAO) decreased MFG-E8 levels (n = 4–5, *p<0.05 vs. (b)Sham). Cerebral MFG-E8 gene expression was measured at 24 h post-MCAO by real-time PCR. Data are presented as mean±SE, and analyzed by Student’s t-test. Cerebral ischemia did not cause a significant alteration in MFG-E8 gene expression (n = 4).

rhMFG-E8 does not alter blood pressure after cerebral ischemia. Mean arterial blood pressure (MABP) was monitored continuously by right femoral artery catheterization, starting from just before MCAO to 90 minutes post-MCAO. Data are presented as mean±SE, and analyzed by Student’s t-test. In both vehicle and rhMFG-E8 treatment groups there was no significant change in MABP throughout the whole period of monitoring. Intravenous administration of rhMFG-E8, commencing at 60 minutes post-MCAO did not cause significant alteration of MABP compared with vehicle (n = 5)

rhMFG-E8 treatment decreases sensorimotor and vestibulomotor deficits at 24 h and 48 h after permanent focal cerebral ischemia. (a) Left forelimb sensorimotor deficits were assessed by the forelimb placing test. Data are presented as mean±SE, and analyzed by Student’s t-test. rhMFG-E8 treatment significantly reduced the sensorimotor deficit at 24 h post MCAO compared with vehicle (n = 5, *p<0.05 vs. Vehicle). (b) Left hindlimb sensorimotor deficit was measured by the hindlimb placing test. Data are presented as mean±SE, and analyzed by Student’s t-test. There was no statistical difference in hindlimb sensorimotor deficit between rhMFG-E8 treatment and vehicle at 24 h post-MCAO (n = 5). (c) Vestibulomotor deficit was assessed at 24 h after onset of cerebral ischemia by the beam balance test. Data are presented as mean±SE, and analyzed by Student’s t-test. Compared with vehicle, rhMG-E8 treatment decreased the vestibulomotor deficit at 24 h post-MCAO (n = 5, *p<0.05 vs. Vehicle).(d) Vestibulomotor deficit was assessed at 48 h after onset of cerebral ischemia by the beam balance test. Data are presented as mean±SE, and analyzed by Student’s t-test. The vestibulomotor deficit was significantly reduced at 48 h post-MCAO by rhMFG-E8 treatment compared with vehicle (n = 5, *p<0.05 vs. Vehicle).

The effect of rhMFG-E8 on infarct size after 24 h and 48 h permanent focal cerebral ischemia. 2 mm thick coronal slices of fresh brain tissue were stained with triphenyl tetrazolium chloride (TTC) and digitally analyzed with NIH ImageJ software. Data are presented as mean±SE, and analyzed by Student’s t-test. (a) Treatment with rhMFG-E8 decreased infarct size at 24 h post-MCAO compared with Vehicle (n = 6, *p<0.05 vs. Vehicle). (b) rhMFG-E8 treatment decreased infarct size at 48 h post-MCAO compared with Vehicle (n = 6, *p<0.05 vs. Vehicle). (c) Representative TTC staining of rostro-caudally sectioned rat brains at 48 h post-MCAO shows that the vehicle grouped developed larger cerebral infarcts.

Histopathological changes after 24 h permanent cerebral ischemia. Hematoxylin-eosin (H&E) stained slides of rat brain sections were examined under bright field microscopy. (a) Slides were first examined at 40× original magnification to delineate the ischemic core from the penumbra (delineation indicated by dashed cyan line) [Sham (A), Vehicle (C), and rhMFG-E8 (E)]. Scale bar = 100 μm. An area was then selected in the penumbra and examined at a higher magnification of 100× to show intact basophilic neurons (green arrow heads) and necrotic eosinophilic neurons (blue short arrows). Sham (B) shows intact basophilic neurons and rhMFG-E8 (F) shows more intact neurons in the penumbra compared with Vehicle (D). Scale bar = 100 μm. (c) Intact neuron (basophilic neuron) counts were done at a higher magnification of 400×. The average number of intact neurons in six images taken from six fields in the penumbra per slide, one slide per animal, was determined. Data are presented as mean±SE, and analyzed by Kruskal-Wallis one-way ANOVA. Cerebral ischemia (MCAO) caused significant reduction in number of intact neurons in both Vehicle and rhMFG-E8 treated animals compared with Sham animals. rhMFG-E8 treatment protected neurons against necrosis compared with vehicle (n = 6, *p<0.05 vs. Sham, # p <0.05 vs. Vehicle).

The effect of rhMFG-E8 on inflammatory cytokine secretion, neutrophil infiltration, and PPARγ levels in 24 h cerebral ischemia. (a) Cerebral IL-6 levels were measured by ELISA at 24 h post-MCAO. Data are presented as mean±SE, and analyzed by one-way ANOVA and Student Newman Keul’s method. Cerebral ischemia (MCAO) caused elevation of IL-6 levels in both Vehicle and rhMFG-E8 treated animals compared with Sham animals. Treatment with rhMFG-E8 downregulated IL-6 expression compared with Vehicle (n = 6, *p<0.05 vs. Sham, #p <0.05 vs. Vehicle). (b) IL-6 ELISA was performed on supernatants obtained from BV2 microglia 24 h oxygen-glucose deprivation (OGD), without re-oxygenation. Cells were plated in triplicates in three independent experiments. Data is presented as the mean (of the averages of the three experiments)±SE, and analyzed by one-way ANOVA and Student Newman Keul’s method. 24 h-OGD resulted in elevation of IL-6 levels, and rhMFG-E8 significantly decreased IL-6 secretion in all three experiments compared with vehicle (n = 3, *p<0.05 vs. Sham, #p<0.05 vs. Vehicle). c) Brain tissue was immunohistochemically stained for( TNF-α and examined at 400× original magnification under bright field microscopy. Vehicle (B) and rhMFG-E8 (C) treated animals showed increased expression of TNF-α compared with Sham animals (A). rhMFG-E8 treatment (C) decreased TNF-α expression compared with Vehicle (B). Scale bar = 50 μm. (d) Brain tissue immunohistochemically stained for the neutrophil marker, myeloperoxidase, and examined under bright field microscopy at 400× original magnification. Sham (A) animals showed no staining for myeloperoxidase (no neutrophil infiltration) whereas Vehicle (B) and rhMFG-E8 (C) treated animals showed staining for myeloperoxidase. rhMFG-E8 treatment (C) after cerebra ischemia decreased myeloperoxidase staining compared with Vehicle (B). Scale bar = 50 μm. (f) Quantification of cerebral neutrophil infiltration by myeloperoxidase immunohistochemistry. Neutrophils were identified as small, round, myeloperoxidase-staining cells on bright field microscopy at 400× original magnification. The average number of neutrophils in six random fields per slide was determined as neutrophil count/40× high power field (hpf). Data are presented as mean±SE, and analyzed by one-way ANOVA and Student Newman Keul’s method. Compared with Sham animals, Vehicle and rhMFG-E8 treated animals showed increased neutrophil infiltration. rhMFG-E8 treatment decreased cerebral neutrophil infiltration compared with vehicle (n = 4, *p<0.05 vs. Sham, #p<0.05, vs. Vehicle). (f) Cerebral ICAM-1 gene expression was measured by RT-PCR. Data are presented as mean±SE, and analyzed by one-way ANOVA and Student Newman Keul’s method. Cerebral ischemia resulted in upregulation of ICAM-1 expression in Vehicle compared with Sham. rhMFG-E8 treatment decreased ICAM-1 expression, even though not significant compared with Vehicle (n = 4–6, *p<0.05 vs. Sham). (b) PPAR-γ protein levels were determined by Western blot at 24 h post-MCAO. Data are presented as mean±SE, and analyzed by Kruskal-Wallis one-way ANOVA. Compared with Sham, cerebral ischemia downregulated PPAR-γ in the Vehicle group. rhMFG-E8 treatment upregulated PPAR-γ expression compared with Vehicle (n = 6, *p<0.05 vs. Sham, #p<0.05 vs. Vehicle).

rhMFG-E8 treatment suppresses apoptosis in cerebral ischemia by downregulating cleaved caspase-3, bax, and upregulating bcl-2 and bcl-2/bax ratio. All data are presented as mean±SE, and analyzed by one-way ANOVA and Student-Newman Keul’s method. (a) Cleaved caspase-3 was measured by Western blot at 24 h post-MCAO. 24 h - MACO resulted in elevation of cleaved caspase-3 in the Vehicle. rhMFG-E8 decreased cleaved caspase-3 to levels similar to sham (n = 5–6, *p<0.05 vs. Sham, #p<0.05 vs. Vehicle). (b) Cleaved caspase-3 levels at 48 h after cerebral ischemia; rhMFG-E8 showed a trend of decreasing cleaved caspase-3, even though not statistically different from vehicle (n = 5). (c) Bcl-2 levels in 24 h MCAO; There was no statistically significant difference in bcl-2 levels, even though rhMFG-E8 showed a trend of increasing bcl-2 (n = 6). (d) Bax levels in 24 h MCAO; Bax levels were similar between sham and vehicle at 24 h post-MCAO. rhMFG-E8 significantly downregulated bax compared with Sham and Vehicle (n = 6, *p<0.05 vs. Sham, #p<0.05 vs. Vehicle).(e) Bcl-2/Bax ratio at 24 h post-MCAO; The Bcl-2/Bax ratio was not different between Vehicle and Sham at 24 h post-MCAO. rhMFG-E8 treatment significantly elevated Bcl-2/Bax ratio compared with Vehicle and Sham (n = 6, *p<0.05 vs. Sham, #p<0.05). (f) Bcl-2 levels in 48 h MCAO; Bcl-2 was significantly reduced in the Vehicle group compared with Sham. rhMFG-E8 upregulated bcl-2 compared with vehicle (n = 5, *p<0.05 vs. Sham, #p<0.05 vs. Vehicle).(g) Bax levels in 48 h MCAO; Bax was significantly upregulated in the Vehicle compared with Sham. rhMFG-E8 downregulated bax compared with Vehicle (n = 5, *p<0.05 vs. Sham, #p<0.05 vs. Vehicle). (h) Bcl-2/Bax ratio at 48 h post-MCAO; Bcl-2/Bax ratio was downregulated compared with Sham. rhMFG-E8 treatment significantly upregulated the bcl-2/bax ratio (n = 5, *p<0.05 vs. Sham, #p<0.05 vs. Vehicle).

(a) TUNEL staining after cerebral ischemia. On fluorescent microscopy at 200× original magnification, TUNEL positive cells appeared as green fluorescent while propidium iodide (PI) staining (red fluorescent) showed the nuclear location of the TUNEL reaction products. The Sham group (A, B, C) showed no TUNEL positive cells. Treatment with rhMFG-E8 (G, H, I) decreased TUNEL staining compared with Vehicle (D, E, F). (b) Quantification of TUNEL staining; Eight random fields were captured at 200× original magnification for each slide. The average number of TUNEL positive cells were counted and expressed as TUNEL cells/20× high power field (hpf). Data are presented as mean±SE, and analyzed by one-way ANOVA and Student Newman Keul’s method. Treatment with rhMFG-E8 decreased the number of TUNEL positive cells compared with Vehicle group (n = 4, *p<0.05 vs. Sham, #p<0.05 vs. Vehicle).

48 h survival analysis. Survival rate was estimated by Kaplan-Meier LogRank analysis. The mortality in the Vehicle group started earlier compared with the rhMFG-E8 group. rhMFG-E8 resulted in a survival rate of 77% compared with 50% for Vehicle group (Vehicle, n = 20; rhMFG-E8, n = 13; p=0.078).