Functional motor recovery from brain ischemic insult by carbon nanotube-mediated siRNA silencing

Abstract

Stroke is the second cause of death worldwide with ischemic stroke accounting for 80% of all stroke insults. Caspase-3 activation contributes to brain tissue loss and downstream biochemical events that lead to programmed cell death after traumatic brain injury. Alleviation of symptoms following ischemic neuronal injury can be potentially achieved by either genetic disruption or pharmacological inhibition of caspases. Here, we studied whether silencing of Caspase-3 using carbon nanotube-mediated in vivo RNA interference (RNAi) could offer a therapeutic opportunity against stroke. Effective delivery of siRNA directly to the CNS has been shown to normalize phenotypes in animal models of several neurological diseases. It is shown here that peri-lesional stereotactic administration of a Caspase-3 siRNA (siCas 3) delivered by functionalized carbon nanotubes (f-CNT) reduced neurodegeneration and promoted functional preservation before and after focal ischemic damage of the rodent motor cortex using an endothelin-1 induced stroke model. These observations illustrate the opportunity offered by carbon nanotube-mediated siRNA delivery and gene silencing of neuronal tissue applicable to a variety of different neuropathological conditions where intervention at well localized brain foci may offer therapeutic and functional benefits.

Fig. 1.

Neuronal uptake of f-CNT: Alexa Fluor 546-labeled siNEG (siNEG- AF 549) complex in vitro. (A) Chemical structure of f-CNT and TEM images of f-CNT dispersed in 5% dextrose at 250 μg/mL final concentration. (B) Epifluorescence and confocal laser scanning microscopy images of primary neuronal culture isolated from mouse brain motor cortex and incubated with media containing free uncomplexed siNEG-AF 549 (100 nM) or f-CNT:siNEG- AF 549 complex (8∶1 mass ratio) for 24 h. siRNA uptake is shown in red.

Fig. 2.

Silencing of Caspase-3 in murine Neuro2A (N2a) neuroblastoma cell line by f-CNT:siCaspase-3 (siCas 3) complexes. (A) mRNA expression levels of full length caspase-3 (35 kDa) in N2a cells was analyzed by real time PCR (n = 3) after treatment with siCas 3 alone, f-CNT:siCas 3 or f-CNT:siNEG complexes. Cells were incubated with the complexes for 4 h at 37 °C, and analyzed for mRNA expression after 24 or 48 h posttransfection. Both β-actin and total RNA were used as internal controls. Full length Caspase-3 mRNA level was reduced only in cells treated with f-CNT:siCas 3 complexes at both time points. Error bars indicate means + SEM (B) Western blot of N2a cells treated as in (A) and assessed 48 h posttransfection. Full length Caspase-3 was detected by a Caspase-3 antibody. Caspase-3 silencing was seen when N2a cells were pretreated with f-CNT:siCas 3 but not with uncomplexed siCas 3 or f-CNT:siNEG. No difference in band intensity was seen for GAPDH housekeeping gene in all treated groups. (C) Quantification of full length Caspase-3 silencing, the ordinate shows the normalized band intensity of full length Caspase-3 which was significantly reduced compared to its level in siCas 3 alone or f-CNT:siNEG treated groups (*, p ≤ 0.05, n = 3).

Fig. 3.

Internalization of f-CNT by neuronal tissue in vivo. TEM of brain cortical parenchyma at 48 h after stereotactic administration of f-CNT showing nanotubes internalization into brain cells (A). Cells are identified by their morphological characteristics; neurons are marked by a dotted line and zoomed in with internalized f-CNT marked in a sqaure. G, M, and N stands for Golgi apparatus, mitochondria, and nucleus respectively (B–D).

Fig. 4.

Caspase-3 silencing in vivo by f-CNT:siCas 3 complexes. (A) Dosage regimen used in the preischemia treatment protocol and Endothelin-1 (ET-1) administration in mice. C57Bl/6 mice were pretreated with 5% dextrose, siCas 3 alone (4.7 pmol), f-CNT:siCas 3, or f-CNT:siNEG complexes (8∶1 mass ratio) at 24 h before exposure to ET-1 (30 pmol) injury. Mice were killed 24 h postlesion (ischemia) induction, and brains were sectioned and stained for TUNEL. Quantification of TUNEL-positive cells/mm² from CLSM shown in (B), showed that f-CNT:siCas 3 treatment group showed significantly lower number of apoptotic cells compared to 5% dextrose treatment group after lesion induction. (B) TUNEL staining of brain sections, apoptotic cells are stained in green (TUNEL) and all nuclei are counterstained in red (propidium iodide). Data were collected from mice killed in two to three independent experiments (*, p ≤ 0.05, n = 7–12).

Fig. 5.

Neuroprotective effect of f-CNT:siCas 3 complex injected 1 h after ET-1 focal stroke induction in mouse motor cortex. The number of apoptotic cells per mm² were detected 24 h after treatment delivery, by TUNEL staining of 50 microns fixed tissue slices (number of slices per case = 4, number of animals per group n > 5). The number of apoptotic cells in the group treated with f-CNT:siCas 3 complex is reduced if compared with the values measured in animals treated with 5% dextrose+ ET-1 or with siCas 3 alone+ET-1, however this apparent trend did not quite meet statistical significance (NS: not significant).

Fig. 6.

Behavioral analysis using “skilled reaching” test in rats. Functional improvement in ET-1 ischemic rat forelimb function with or without pretreatment was measured. Rats were pretreated with 5% dextrose, siCas 3 alone (4.7 pmol), f-CNT:siCas 3 or f-CNT:siNEG complexes (8∶1 mass ratio) 24 h before exposure to ET-1 (30 pmol) leading to unilateral lesions made contralateral to the preferred (for reaching) forelimb. During recall, rats treated with f-CNT:siCas 3 performed a significant higher number of positive trials in food pellet reaching compared to all other treatment groups. (*, p ≤ 0.05; **, p ≤ 0.01, n = 4) and reaching prelesion levels