Targeted Retrograde Gene Delivery into the Injured Spinal Cord Using Recombinant Adenovirus Vector Carrying Neurotrophic Factor Gene

Fig. 16.1

Targeted retrograde gene delivery of recombinant adenovirus vector-mediated LacZ gene through the sternomastoid muscle innervated by spinal accessory nerves. (a) Lower magnification, (b–d) high-power photomicrograph of the box area

However, it is not clear whether the AdV-BDNF suppresses neuronal and glial cell apoptosis in the acute spinal cord injury model, and there is no report that describes cell death, particularly apoptosis, following retrograde neurotrophin gene transfection. The present study was designed to investigate the effects of retrograde AdV-BDNF gene delivery on apoptosis of neurons and glial cells (oligodendrocytes) after traumatic spinal cord injury.

16.1.1 Animal Model of Spinal Cord Injury and Targeted AdV-BDNF Gene Delivery

AdV-mediated BDNF was prepared using the instruction provided with the Adenovirus Expression Vector Kit (Takara Biomedical, Otsu, Japan). As a control marker gene, a recombinant adenovirus vector coding for bacterial β-galactosidase cDNA was isolated using the same procedure (AdV-LacZ). The final titers of the adenovirus vector contained 5.0 × 10⁸ plaque forming units/mL.

Experiments were conducted in adult male Sprague–Dawley rats (Clea, Tokyo), aged 8–10 weeks. At the C4 vertebral level (C5 cord segment), the dorsal dural surface of the spinal cord was compressed extradurally using a 50 g static load (custom-made rod, 2 × 3 mm in diameter) for 5 min, as we described previously [19]. Immediately after the injury, the sternomastoid muscles were exposed on both sides while the rat was positioned in supine position using a stereotaxic surgical frame, taking utmost care in preserving branches of the spinal accessory nerves that innervate the muscles. Using a microsyringe (Hamilton, Reno, NV), each 100 μL of AdV encoding LacZ (5.0 × 10⁸ PFU) or BDNF (5.0 × 10⁸ PFU) was injected carefully and slowly into the middle belly of the superficial layer of the bilateral sternomastoid muscles simultaneously. AdV-LacZ injection without spinal cord injury (only operation for laminectomy) was used as normal groups (i.e., positive controls).

16.1.2 Distribution of β-Galactosidase Expression in the Spinal Cord by Retrograde Gene Delivery

To assess the distribution of β-galactosidase following retrograde gene delivery into the normal (Fig. 16.2a) and injured (Fig. 16.2e) spinal cord, immunofluorescence staining was performed. β-Galactosidase-positive cells were observed mainly in the gray matter including motoneurons and interneurons in the normal spinal cord on axial (Fig. 16.2b–d) and sagittal sections (Fig. 16.2i, j) of positive controls, while they were observed in all (gray matter and white matter) of the injured spinal cord on axial (Fig. 16.2f–h) and sagittal (Fig. 16.2k, l) sections.

Fig. 16.2

Photomicrographs showing immunofluorescence for β-galactosidase in axial and sagittal sections of normal (a–d) and (i, j) and injured (e–h) and (k, l) spinal cord at 1 week after AdV-LacZ gene injection. (a, e) Luxol fast blue (LFB) staining. (b, f) DAPI. (c, g) β-galactosidase. (d, h–l) merged. Scale bar = 500 μm (a, b, e, and f), 1 mm (i, k), 100 μm (j, l) (Reprinted, with permission, from [20])

16.1.3 Apoptosis of Neurons and Oligodendrocytes

Histological evaluation and immunoblot analysis were applied to assess the effects of retrograde BDNF gene delivery on apoptotic cell death in the injured spinal cord. A number of TUNEL-positive cells were found in both gray and white matters of the cord after AdV-LacZ gene transfection. In comparison, TUNEL-positive cells were markedly decreased in the injured cord after treatment with AdV-BDNF gene transfection. The number of TUNEL-positive cells per cross section significantly decreased in the AdV-BDNF-injected group compared to the AdV-LacZ-injected group at 3 days, 1 and 2 weeks after injury in the rostral and caudal adjacent site (Fig. 16.3a, c), and at 1 week after the injury in the epicenter (Fig. 16.4b).

Fig. 16.3

Bar graphs show results of quantitative analysis of TUNEL-positive cells per cross section following AdV-LacZ or AdV-BDNF transfection at rostral (a), epicenter (b), and caudal (c) sites of the injured cervical cord. Data are mean ± SD. *p < 0.05 (Reprinted, in part, with permission, from [20])

Fig. 16.4

Immunoblot analysis for the expression of active caspase-3 following AdV-LacZ or AdV-BDNF gene transfection into the injured cervical cord (Reprinted, in part, with permission, from [20])

Figure 16.4 shows the results of immunoblot analysis and double immunofluorescence staining carried out to investigate the effect of retrograde gene transfection on the expression of active caspase-3. A band of active caspase-3 was evident from 3 h to 1 week after spinal cord injury in AdV-LacZ-treated rats, while the band appeared 3–12 h later in AdV-BDNF gene-injected animals.

16.1.4 Effects of Retrograde AdV-BDNF Gene Delivery on Endogenous Oligodendrocyte Progenitor Cells

Expression of NG2 (precursor of oligodendrocyte lineage) in the white matter of the cord was investigated at 4 weeks after retrograde injection of AdV-BDNF. Abundant NG2-positive cells were noted in the white matter of AdV-BDNF- than AdV-LacZ-injected rats in transaxial (Fig. 16.5a, b) sections. Quantitative analysis of NG2-positive area in AdV-BDNF-treated rats showed significant increase in all three anatomic sites (Fig. 16.5c).

Fig. 16.5

Quantitative analysis of the NG2-positive area in the axial sections showed a significant increase of NG2 expression after AdV-BDNF gene injection. Data are mean ± SD. *p < 0.05 (Reprinted, in part, with permission, from [20])

16.2 Discussion

The delivery of neuroprotective genes such as neurotrophic factors can be potentially beneficial for restoration of neural tissue function after spinal cord injury [9, 10, 13, 16]. Development of virus vector for gene delivery is a practically feasible tool for transduction of neurotrophic peptides [22]. However, effective administration of neuroprotective genes into the injured site of the cord remains a major challenge. Direct routes for administration of neuroprotective genes have been used for gene delivery. However, there are difficulties and serious concerns regarding the possible spread of traumatic neural injury and worsening of neural insult, including necrosis, and apoptosis as well as cell death [23]. In contrast, targeted retrograde gene delivery through the peripheral nervous system or by muscle injection of adenovirus vectors seems a less invasive method that can be conducted repeatedly [16].

In a series of experiments, our group evaluated the feasibility and efficacy of retrograde gene delivery into the injured cervical spinal cord using recombinant adenovirus vector through the sternomastoid muscle, which is innervated by the spinal accessory nerve [18–21], applying modified wheat germ agglutinin-horseradish peroxidase labeling method of cervical motoneurons pool [9]. β-Galactosidase (AdV-LacZ) gene expression was found in the cervical spinal cord from 4 to 6 weeks after injection of adenovirus vector, reaching peak expression level at 1–2 weeks and exhibiting transduction efficacy (survival rate of cervical spinal cord motoneurons: 87.5–98.9 % within 3 days after injection) [18, 19]. Transduction of interneurons and glial cells (microglia, reactive astrocytes, and oligodendrocytes) induced by primary or secondary wave of injury could be verified with X-gal staining at early time after the trauma. The efficiency of retrograde delivery of neuropeptide genes could be influenced by various factors [24], but our group has confirmed that AdV-LacZ is transported via retrograde axonoplasmic flow rather than through systemic circulation because no gene expression was observed in various peripheral organs [20]. Accordingly, targeted retrograde AdV-LacZ gene and hopefully AdV-BDNF gene delivery is considered feasible from a methodological point of view.

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