Mannitol Augments the Effects of Systemical Stem Cell Transplantation without Increasing Cell Migration in a Stroke Animal Model

Abstract

Background: Mannitol increases blood-brain barrier permeability and can improve the efficiency of systemically administered stem cells by facilitating stem cell entry from the periphery into the injured brain. The aim of this study was to elucidate the neuroprotective effects of a combination of mannitol pretreatment and stem cell transplantation on stroke-induced neural injury.

Methods: The experimental rats were randomly assigned to three groups 24 h after middle cerebral artery occlusion and reperfusion. One group received intravenous (IV) injections of phosphate-buffered saline (vehicle), another group received IV injections of human adipose-derived stem cells (hADSCs), and the last group received IV injections of hADSCs 10 min after IV mannitol injections. Neurobehavioral functions and infarct volume were compared. Immunohistochemistry (IHC) analyses were performed using antibodies against ionized calcium binding adapter-1 (IBA-1), rat endothelial antigen-1 (RECA-1), and bromodeoxyuridine/doublecortin (BrdU/DCX).

Results: PKH-26 labeling revealed no difference in the number of stem cells that had migrated into the injured brain, and hADSC transplantation did not improve the infarct volume. However, neurobehavioral functions improved in the mannitol group. IHC showed higher numbers of RECA-1-positive cells in the peri-infarcted brain and BrdU-/DCX-colocalized cells in the subventricular zone in the mannitol group. IBA-1-positive cell number decreased in the hADSC-only and mannitol-pretreatment groups compared with the vehicle group even though there was no difference between the former two groups.

Conclusion: Combinatorial treatment with mannitol and hADSC transplantation may have better therapeutic potential than hADSC monotherapy for ischemic stroke.

Keywords: Combination; Human adipose-derived stem cells; Ischemic stroke; Mannitol; Pretreatment.

Fig. 1

Demonstration of PKH-26 labeling in the hADSC-only and mannitol pretreatment groups. The number of PKH-26-positive cells (red) counted in the MCAO rat SVZs in both groups is below 10 and presents no significant difference (p > 0.05). DAPI staining (blue) indicates stained nucleus of every cell of the section. Scale bar = 20 µm. hADSC human adipose-derived stem cell, Mann mannitol pretreatment, PKH PKH-26-positive cells, MCAO middle-cerebral artery occlusion, SVZ subventricular zone, DAPI 4′,6-diamidino-2-phenylindole

Fig. 2

Representative sections of the brain stained with cresyl violet. Scale bar = 1 mm. hADSC human adipose-derived stem cell, Mann mannitol pretreatment

Fig. 3

Demonstration of the chronological change in mNSS in the experimental groups. Chronological decrease in the mNSS of the vehicle and hADSC transplantation groups after MCAO was observed. The graph demonstrates improvements in neurobehavioral function after MCAO and hADSC transplantation. *p = 0.048, **p = 0.002, ***p < 0.001, ^#p < 0.001, mNSS modified neurological severity score, hADSC human adipose-derived stem cell, MCAO middle cerebral artery occlusion, Mann mannitol pretreatment, SE standard error

Fig. 4

Immunohistochemistry in ischemic rat brains. Neurogenesis after hADSC transplantation was examined using BrdU (green) and DCX (red) immunostaining. A BrdU- and DCX-positive cells in SVZs on the 15th day after MCAO. White arrows indicate BrdU-/DCX-double-positive cells. Scale bar = 20 µm. B Counts of BrdU-/DCX- double-positive cells in SVZs. The number of BrdU-/DCX-double-positive cells is significantly higher in the hADSC transplantation (p = 0.014) and hADSC transplantation with mannitol pretreatment (p < 0.001) groups. Data are expressed as mean ± SEM. BrdU bromodeoxyuridine, DCX doublecortin, hADSC human adipose-derived stem cell, Mann mannitol pretreatment, SVZ subventricular zone, MCAO middle cerebral artery occlusion, SEM standard error of the mean

Fig. 5

Decreased microglial activation after hADSC transplantation. A Representative images of IBA-1 immunostaining in the ischemic hemisphere on the 15th day after MCAO. A decreased number of IBA-1-positive cells was observed. Scale bar = 20 µm. B Count of IBA-1-positive microglia. The number of IBA-1-positive cells decreased in the hADSC transplantation as well as in the hADSC transplantation with mannitol pretreatment groups. Data are expressed as means ± SEMs. IBA-1 ionized calcium-binding adapter molecule-1, hADSC human adipose-derived stem cell, Mann mannitol pretreatment, MCAO middle cerebral artery occlusion, SEM standard error of the mean

Fig. 6

Increased angiogenesis after hADSC transplantation. A Representative images of RECA-1-positive vessels in ischemic rat brains on the 15th day after MCAO. Scale bar = 50 µm. B Analysis of RECA-1 immunoreactivity using six cryosections. Immunostaining reveals an increased vessel area in the hADSC transplantation as well as in the hADSC transplantation with mannitol pretreatment groups. Data are expressed as percentages of immunoreactivity in the control groups compared to the vehicle group. RECA-1 rat endothelial cell antigen-1, hADSC human adipose-derived stem cell, Mann mannitol pretreatment, MCAO middle cerebral artery occlusion