Intravenous injection of neural progenitor cells improved depression-like behavior after cerebral ischemia

Abstract

Poststroke depression (PSD) occurs in approximately one-third of stroke survivors and is one of the serious sequelae of stroke. The onset of PSD causes delayed functional recovery by rehabilitation and also increases cognitive impairment. However, appropriate strategies for the therapy against ischemia-induced depression-like behaviors still remain to be developed. Such behaviors have been associated with a reduced level of brain-derived neurotrophic factor (BDNF). In addition, accumulating evidence indicates the ability of stem cells to improve cerebral ischemia-induced brain injuries. However, it remains to be clarified as to the effect of neural progenitor cells (NPCs) on PSD and the association between BDNF level and PSD. Using NPCs, we investigated the effect of intravenous injection of NPCs on PSD. We showed that injection of NPCs improved ischemia-induced depression-like behaviors in the forced-swimming test and sucrose preference test without having any effect on the viable area between vehicle- and NPC-injected ischemic rats. The injection of NPCs prevented the decrease in the level of BDNF in the ipsilateral hemisphere. The levels of phosphorylated CREB, ERK and Akt, which have been implicated in events downstream of BDNF signaling, were also decreased after cerebral ischemia. NPC injection inhibited these decreases in the phosphorylation of CREB and ERK, but not that of Akt. Our findings provide evidence that injection of NPCs may have therapeutic potential for the improvement of depression-like behaviors after cerebral ischemia and that these effects might be associated with restoring BDNF-ERK-CREB signaling.

Figure 1

Characterization of neural progenitor cells and experimental protocol of this study. Triple staining was carried outwith 4′,6-diamidino-2-phenylindole (DAPI), (a) and for nestin (b) and musashi-1 (c) was merged (d), and indicated that cells in neurospheres, which were prepared from gestational Day 14 fetal rats, expressed the neural progenitor cell markers nestin and musashi-1 on Day 6 when cultured in vitro. The cells in the neurospheres differentiated after the withdrawal of growth factors from the medium into MAP2- (e), glial fibrillary acidic protein (GFAP)- (f) and oligodendrocytes (clone RIP)-positive cells (g). Scale bars represent 50 μm (a–d) and 10 μm (e–g). Experimental protocol in this study was depicted (h). Abbreviations: mNSS, modified Neurological Severity Scores; SPT, sucrose preference test; FST, forced-swimming test; OFT, open-field test; WB: western immunoblotting; and IHC, immunohistochemistry.

Figure 2

Time courses of changes in body weight, neurological deficits and modified Neurological Severity Score (mNSS)- of vehicle- and neural progenitor cell (NPC)-injected ischemic rats. The arrow indicates the time point when NPCs or vehicle was injected. There was no significant difference in body weight between NPC- (open circles) and vehicle-treated (closed circles) groups (a). The score of each neurological deficit was rated from 3 to 0 (from very severe to little or none). There was no significant improvement in the NPC-injected ischemic (closed circles) rats compared with the vehicle-injected ischemic (open circles) ones (b). Determination of mNSS, which is based on a series of behavioral tests, including motor, sensory, reflex and balance tests, was made. There was a significant reduction in the score of NPC-injected ischemic rats (open circles) when these rats were compared with the vehicle-injected ones (closed circles) after the embolism (c). All results are presented as the mean±s.e.m. (n=17 rats per group). ^#Statistically significant difference from vehicle-injected ischemic rats (P<0.05).

Figure 3

Effect of injection of neural progenitor cells (NPCs) on sucrose preference, immobility time in the forced-swimming test and locomotor activity. A battery of the sucrose preference test was conducted before microsphere embolism and on Days 7, 14, 21 and 28 after the embolism (a and b). Time courses of changes in sucrose preference of vehicle- (closed circles) and NPC-injected (open circles) ischemic rats are shown (a). The arrows indicate the time point when NPC or vehicle was injected. There was significant improvement in the percentage of the sucrose preference of the NPC-injected ischemic rats when these rats were compared with the vehicle-injected ischemic ones on Days 14, 21 and 28 after the embolism. There was no significant difference in total consumption between vehicle- and NPC-injected ischemic rats throughout the experiment (b). All results in the sucrose preference test are presented as the mean±s.e.m. (n=17 rats per group). The forced-swimming test was performed on Day 28 after the embolism (c). ^#Statistically significant difference from vehicle-injected ischemic rats (P<0.05). There was a significant increase in the immobility of the vehicle-injected ischemic rats when these rats were compared with the sham-operated ones (c). The immobility time was not increased in NPC-injected ischemic rats (c). The open-field test was performed on Day 28 after the embolism (d). The total locomotor activity was not significantly different among sham-operated, vehicle-injected and NPC-injected ischemic rats (d). All results in the forced-swimming test and the open-field test are presented as the mean±s.e.m. (n=14 rats per group). ^*Statistically significant difference from sham-operated rats (P<0.05). ^#Statistically significant difference from vehicle-injected ischemic rats (P<0.05).

Figure 4

Histological analysis of the neural progenitor cell (NPC)-injected brain after the embolism. Effect of injection of NPCs on the viable areas after the embolism. On Day 28 after microsphere embolism-induced cerebral ischemia (ME), coronal sections were stained with hematoxylin and eosin. Photographs of hematoxylin- and eosin-stained sections are shown for vehicle-injected (a and d) and NPC-injected (b and e) ischemic rats. Viable areas were estimated by measuring hematoxylin- and eosin-stained sections (c and f) that corresponded to coronal coordinates of bregma from 0.48 to −0.30 mm (striatal region: a–c) and from −3.80 to −4.52 mm (hippocampal region: d–f). All results are presented as the mean percentage of the contralateral hemisphere±s.e.m. (n=8–9 rats per group). There was no significant attenuation of tissue degeneration in the NPC-injected ischemic rats compared with that in the vehicle-injected ischemic rats in both regions (c and f). Green-fluorescent protein (GFP)-positive NPCs were found in the peri-infarct areas (h and j) on Day 28 after the embolism (striatal region: g and h; hippocampal region: i and j). The images (white square) in ‘g' and ‘i' are enlarged in ‘h' and ‘j', respectively. To determine the effect of injection of NPCs on the phenotype of injected cells, double immunostaining in the peri-infarct areas was conducted on Day 28 after the embolism. Some GFP-positive injected NPCs expressed mature neuronal marker NeuN (k) and astrocyte marker GFAP (l). Most GFP-positive NPCs expressed NPC marker musashi-1 (m). Scale bars represent 1 mm (g and i), 50 μm (h and j) and 10 μm (k–m).

Figure 5

Effect of neural progenitor cell (NPC) injection on the levels of brain-derived neurotrophic factor (BDNF), phosphorylated CREB, TrkB, phosphorylated ERK and phosphorylated AKT on Day 28 after the embolism. Samples obtained from sham-operated (sham), vehicle-injected ischemic (vehicle) and NPC-injected (NPC) ischemic rats (ME) were solubilized and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands corresponding to BDNF (a), phospho-CREB (b), and TrkB (c), phosphorylated ERK (d) and phosphorylated Akt (e) were scanned, and scanned bands were normalized by actin (a and c), total CREB (b), total ERK (d) or total AKT (e) on the same blot. All results are presented as mean percentages of non-operated naïve rats±s.e.m. (BDNF and phospho-CREB: n=9 rats per group; TrkB: n=5–6 rats per group; and phospho-ERK and -AKT: n=6 rats per group). ^*Statistically significant difference from sham-operated rats (P<0.05). ^#Statistically significant difference from vehicle-injected ischemic rats (P<0.05).