IL-10 promotes neuronal survival following spinal cord injury - PubMed (original) (raw)

IL-10 promotes neuronal survival following spinal cord injury

Zhigang Zhou et al. Exp Neurol. 2009 Nov.

Abstract

We have previously reported that the anti-inflammatory cytokine IL-10 induces a number of signaling cascades through the IL-10 receptor in spinal cord neurons in vitro to activate NF-kappaB transcription Bcl-2 and Bcl-x(L) and that, after exposure to glutamate IL-10, blocks cytochrome c release and caspase cleavage. In the current study we used a herpes simplex virus (HSV)-based vector to express IL-10 in spinal cord in vivo. Injection of the vector 30 minutes after lateral hemisection injury resulted in increased neuronal survival in the anterior quadrant of the spinal cord and improved motor function up to 6 weeks after injury, that correlated with translocation of p50 and p65 NF-kappaB to the nucleus and increased expression of Bcl-2 and Bcl-x(L) in anterior quadrant neurons. Inhibition of cytochrome c release and caspase 3 cleavage was seen in homogenates of injured spinal cord treated by the IL-10 vector. Taken together with in vitro studies that demonstrate direct neuroprotective effects of IL-10 acting through the neuronal IL-10 receptor, these results suggest that IL-10 may provide direct neuroprotective effects in spinal cord injury separate from and in addition to the known anti-inflammatory effects and point to the possibility that IL-10 delivery by gene transfer may be a useful adjunctive therapy for spinal cord injury.

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Figures

Figure 1

Figure 1

HSV vector QHIL10 expresses IL-10 in spinal cord neurons in vitro. Vector constructs QHIL10 (A) and QHZ (B). (C) E17 spinal cord neurons (10 DIV) 48 hr after infection with QHIL10 at MOI 1 express transgene-mediated IL-10 recognized by its HA tag (anti-βIII tubulin green; anti HA red); scale bar = 10 μm.

Figure 2

Figure 2

(A, B). Western blot of cell lysate (A) and ELISA of culture medium (B) from spinal cord neurons 48 hr after infection. (C, D) IL-10 mRNA determined by RT-PCR (C) and IL-10 protein determined by Western blot (D) of spinal cord after inoculation of vector QHIL10 or vector QHZ into adult thoracic spinal cord in vivo.

Figure 3

Figure 3

Vector-produced IL-10 protects neurons in vivo resulting in enhanced recovery following SCI. (A) Inoculation of the IL-10 expressing vector directly into spinal cord 30 minutes after T13 hemisection resulted in improved locomotor function assessed by BBB; P < 0.05 by repeated measures analysis; n = 8 animals per group. (B) The number of NeuN positive neurons in ventral horn of spinal cord around the site of injury 48 hr after spinal cord injury was also increased by injection of QHIL10 compared to animals injected with QHZ.

Figure 4

Figure 4

Inoculation of the IL-10 expressing vector reduced the amount of cleaved caspase 3 (A) and cytochrome c (B) in thoracic spinal cord homogenates taken 2.5 mm above and below T13 hemisection, 48 h after injury. Quantification of the bands by chemiluminescence (C,D); n = 5, mean ± SEM; *P<0.05 and **P<0.01.

Figure 5

Figure 5

Double label immunohistochemistry of Bcl-2 (A) and Bcl-xL (B) (both green) and the neuronal marker NeuN (red) in ventral horn 2 mm below the lesion 48 hr after T13 hemisection. Scale bar = 20 μm.

Figure 6

Figure 6

Vector-mediated IL-10 expression increases Bcl-2 in ventral horn neurons of spinal cord after SCI. (A) Western blot. (B) quantification of the Western blot; n = 5, mean ± SEM; *P<0.05.

Figure 7

Figure 7

Vector mediated IL-10 expression prevents translocation of p50 and p65 subunits of NF-κB in VH spinal neurons after SCI. Double label immunohistochemistry of p50 (A) and p65 (B) (both green) with the neuronal marker NeuN (red) in ventral horn 2 mm below the site of injury 48 h after T13 hemisection. Scale bar = 20 μm.

Figure 8

Figure 8

IL-10 receptor co-localizes with the neuronal marker Neu-N in spinal cord neurons in vivo. Bar =10 μm.

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