Neural stem cell therapy in conjunction with curcumin loaded in niosomal nanoparticles enhanced recovery from traumatic brain injury

Abstract

Despite a great amount of effort, there is still a need for reliable treatments of traumatic brain injury (TBI). Recently, stem cell therapy has emerged as a new avenue to address neuronal regeneration after TBI. However, the environment of TBI lesions exerts negative effects on the stem cells efficacy. Therefore, to maximize the beneficial effects of stem cells in the course of TBI, we evaluated the effect of human neural stem/progenitor cells (hNS/PCs) and curcumin-loaded niosome nanoparticles (CM-NPs) on behavioral changes, brain edema, gliosis, and inflammatory responses in a rat model of TBI. After TBI, hNS/PCs were transplanted within the injury site and CM-NPs were orally administered for 10 days. Finally, the effect of combination therapy was compared to several control groups. Our results indicated a significant improvement of general locomotor activity in the hNS/PCs + CM-NPs treatment group compared to the control groups. We also observed a significant improvement in brain edema in the hNS/PCs + CM-NPs treatment group compared to the other groups. Furthermore, a significant decrease in astrogliosis was seen in the combined treatment group. Moreover, TLR4-, NF-κB-, and TNF-α- positive cells were significantly decreased in hNS/PCs + CM-NPs group compared to the control groups. Taken together, this study indicated that combination therapy of stem cells with CM-NPs can be an effective therapy for TBI.

Figure 1

Culture and characterization of neural stem/progenitor cells (NS/PCs) derived from a human fetus. (a) Phase-contrast images of hNS/PCs on 5–7 days. hNS/PCs at 70–80% confluency were also shown. (b) Immunocytochemistry of hNS/PCs. Nestin as a neural stem cell marker was conjugated with FITC and nuclei were stained with PI.

Figure 2

Characterization of CM-NPs. (a) Transmission electron microscopy (TEM) image of CM-NPs showed particles with spherical morphology and an average size of 60 nm. (b) The HPLC system was used to evaluate the ability of penetration of CM-NPs into the brain tissue. The representative micrograph shows different concentrations of curcumin in nanoparticles (i.e., 25, 50, and 100 mg/kg; n = 3). To find out an optimum dose of CM-NPs, some behavioral assessments, such as rotarod, mNSS, and OF were performed to evaluate the advanced locomotor function, sensory-motor function, and general loco-motor activity, respectively. (c) The rotarod test was performed before TBI as well as during predetermined time points after TBI. There was no significant difference for mean latency to fall between groups in the rotarod test. (d) The mNSS test was performed before TBI and predetermined time points after TBI in different groups. The mNSS score in the treated group with 50 mg/kg of CM-NPs was significantly decreased compared to 25 mg/kg of CM-NPs on day 7 after TBI. (e) Evaluation of total distance traveled in the open field in different groups on days 7–28 after TBI. The statistical analysis showed no significant difference for total distance traveled in the OF between groups. Data represented as the mean ± SEM and * indicates P < 0.05 (n = 6).

Figure 3

Brain water content was measured at 72 h after TBI in different groups. (a) Data from ipsilateral hemispheres have been shown that the CM-NPs group had a significantly lower brain water content than that of the control and PBS groups. (b) The statistical analysis showed no significant difference in brain water content in the contralateral hemispheres between groups. (c) The brain water content in the total cerebrum was significantly reduced in CM-NPs and hNS/PCs + CM-NPs treatment groups compared to the control and PBS groups. Data are presented as the mean ± SEM (n = 3).

Figure 4

Rotarod, mNSS, and OF were assessed to evaluate the advanced locomotor function, sensory-motor function, and general locomotor activity in different groups following TBI. (a) The mean latency to fall on the rod showed no significant difference between groups in the rotarod test. (b) The statistical analysis showed no significant difference in mNSS scores between groups. (c) In the OF task, total distance traveled significantly increased in hNS/PCs and hNS/PCs + CM-NPs groups in comparison with the control, PBS, and CM-NPs groups on days 7 and 14. The data are shown as the mean ± SEM and * indicates P < 0.05 (n = 6).

Figure 5

The mean lesion volume at the injury site after TBI in different groups. (a) Lesion areas are stained by hematoxylin–eosin (HE) in different groups. (b) The bar graph indicates the lesion volume in different experimental groups on day 28 after TBI. Data represented as lesion volume mean ± SEM (n = 6).

Figure 6

Immunohistochemistry staining of GFAP- and Iba-1-positive cells at the injury site after TBI and cell transplantation in rats. (a) GFAP-positive cells indicated brown stains are shown within the injury site after 28 days of TBI. (b) The mean number of GFAP-positive cells significantly decreased in the hNS/PCS + CM-NPs group compared to the control and PBS groups. Furthermore, the mean number of GFAP-positive cells in the hNS/PCS + CM-NPs group was significantly lower than the hNS/PCs group. (c) Immunohistochemistry slides of Iba-1-positive cells as indicated brown stain. (d) Bar graphs show the mean number of Iba-1-positive cells in the lesion area after 28 days of TBI in different study groups. The bar graphs indicate a significantly lower expression of Iba-1-positive cells in the CM-NPs group than in the control and PBS groups. Moreover, the mean numbers of Iba-1-positive cells in the CM-NPs group were significantly lower than that of the hNS/PCs group. Data are expressed as the mean ± SEM (n = 6).

Figure 7

Immunohistochemistry staining of TLR4-, NF-κB-, and TNF-α-positive cells at the injury site after TBI and cell transplantation in experimental groups. (a) Representative images of TLR4-positive cells (i.e., brown stain) within the injury site on day 28 after induction of TBI. (b) The mean number of TLR4-positive cells significantly decreased in the CM-NPs, hNS/PCs, and hNS/PCS + CM-NPs groups compared to the control and PBS groups. (c) Representative images of immunohistochemistry slides of NF-κB-positive cells as shown brown stain. (d) Bar graphs show the mean number of NF-κB-positive cells in the lesion area after 28 days of TBI in different experimental groups. The bar graphs indicate a significantly lower expression of NF-κB-positive cells in the CM-NPs, hNS/PCs, and hNS/PCS + CM-NPs groups compared to the control and PBS groups. (e) TNF-α-positive cells indicated brown stains are shown within the injury site after 28 days of TBI. (f) The mean number of TNF-α-positive cells significantly decreased in the CM-NPs and hNS/PCS + CM-NPs groups compared to the control group. Data are presented as the mean ± SEM (n = 6).

Figure 8

An overview of experimental design. Rats were trained 3 days before TBI to adapt to behavioral tests. Stem cell transplantation was performed 10 min after TBI in rats. The rotarod test was performed before TBI as well as on 5, 10, 15, 20, and 25 days, open field (OF) test was performed on 7, 14, 21, and 28 days, and mNSS test was performed Pre-TBI as well as 1, 7, 14, 21, and 28 days after TBI. In the CM-NPs and hNS/PCs + CM-NPs groups, 50 mg/kg CM-NPs were dissolved in PBS and gavaged daily. To evaluate brain water content, the brains were weighed on day 3 after TBI and incubated at 100 °C for 24 h and then were reweighed. In addition, HPLC analysis was performed to find out the optimal dose of CM-NPs on day 10 after TBI. After a 28-day treatment period, rats were sacrificed to assess lesion volume, gliosis, and TLR4/NF-κB inflammatory pathway by HE staining and IHC method, respectively.