Amine-modified single-walled carbon nanotubes protect neurons from injury in a rat stroke model

Abstract

Stroke results in the disruption of tissue architecture and is the third leading cause of death in the United States. Transplanting scaffolds containing stem cells into the injured areas of the brain has been proposed as a treatment strategy, and carbon nanotubes show promise in this regard, with positive outcomes when used as scaffolds in neural cells and brain tissues. Here, we show that pretreating rats with amine-modified single-walled carbon nanotubes can protect neurons and enhance the recovery of behavioural functions in rats with induced stroke. Treated rats showed less tissue damage than controls and took longer to fall from a rotating rod, suggesting better motor functions after injury. Low levels of apoptotic, angiogenic and inflammation markers indicated that amine-modified single-walled carbon nanotubes protected the brains of treated rats from ischaemic injury.

Figure 1. Pretreatment with a-SWNT-protected animals following ischaemia–reperfusion

One week before MCAO, animals received either PBS or a-SWNTs. Animals were killed 1 day after reperfusion. a, Coronal brain sections (stained with triphenyltetrazolium chloride (TTC) solution) of sham-operated control and PBS- or a-SWNT-treated rats. Brain slices were arranged in sequence. White areas represent the infarcted region after MCAO. b, Quantification of the ischaemic lesion of brain sections in a. Data are means±s.e.m. *P <, 0.001 versus the PBS group. c, Schematic of injection and training schedule. d, Graph showing that pretreatment with a-SWNTs reduced neurological damage after ischaemia. Neurological score was evaluated by the Rota-Rod treadmill test. Data are means±s.e.m. *P <, 0.001 versus pre-MCAO.

Figure 2. Reduction of apoptosis in a-SWNT-treated animals

a–f, Representative photomicrographs showing TUNEL staining in the penumbral region of brain sections from the sham-operated group (a,b), PBS group (c,d) and a-SWNTs group (e,f) 7 days after MCAO (n = 5). Images in b,d,f, are magnified areas from a,c,e, respectively. Scale bars, 200 μm. g, Western blot analysis of cerebral cortex tissue 7 days after MCAO, showing lowered expression of pro-apoptotic proteins (p53 and Bax) in a-SWNT-treated rats. β-actin is the loading control. Data represent the results of three independent experiments.

Figure 3. Immunohistochemical analysis of GFAP and Iba-1 expression after MCAO

a–l, Representative photomicrographs of GFAP and Iba-1 expression in ipsilateral (a–f) and contralateral (g–l) hemispheres of sham-operated, PBS-treated and a-SWNT-treated rats following MCAO (n = 5). Scale bars, 200 μm. m,n, Bar graphs showing quantification of GFAP- (m) and Iba-1- (n) positive cells on the ipsilateral side. Data are the mean number of cells from five random fields. Data are presented as means±s.e.m. *P <, 0.001 versus the PBS group. o,p, Effects of a-SWNTs on the production of proinflammatory cytokine IL-1b (o) and TNF-α (p). Cytokines, determined by ELISA, were normalized against the cortex of the contralateral hemisphere. Immunohistochemical analysis and ELISA were performed 7 days after MCAO. Data are expressed as means±s.e.m. *P < 0.001 versus the sham-operated group; **P < 0.001 versus the PBS group; n = 6.

Figure 4. Western blot analysis of cerebral cortex lysates after MCAO in PBS- or a-SWNT-treated rats

a,c, Lanes 1–4 show data from individual rats of the PBS group; lanes 5–9 show data from individual rats of the a-SWNTs group. b,d, Bar graphs showing quantification of the expression level of each protein compared with the expression of β-actin or the non-phosphorylated isoform. Data are expressed as means±s.e.m. Western blots are representative of four independent experiments.