Replication-incompetent HSV-1 vector, HSV-HGF (HGF group) or HSV-LacZ (LacZ group), was injected into the spinal cord at thoracic (T)10 level after laminectomy in adult female Sprague–Dawley rats. To examine the distribution and amount of HGF protein in uninjured spinal cord after gene delivery, the spinal cord tissues were harvested and processed for an ELISA and HGF immunostaining at 3 days after the HSV-1 vectors injection. While HGF immunoreactivity (IR) showed a remarkable expansion putatively in the extracellular matrix in the HGF group at 3 days after injection, very little HGF-IR was observed in the LacZ group (data not shown). Injection of the HSV-1 vectors resulted in a significantly higher amount of HGF protein in the HGF group (11.5 ± 0.8 ng/g tissue), compared with that in the LacZ group (3.4 ± 0.1 ng/g tissue), at 3 days after injection (data not shown) [25].

Based on these findings, at 3 days after HSV-1 vectors injection, contusive SCI was induced at the injection site using the Infinite Horizon impactor (200 kdyn, Precision Systems, Lexington, KY) to investigate therapeutic effects of HGF on injured spinal cord.

To determine the effects of exogenous HGF on the injured spinal cord, we performed several quantitative histological analyses. First, the cavity area of the injured spinal cord at 6 weeks after SCI was significantly smaller in the HGF group than the LacZ group (Fig. 14.2a). Second, the HGF group obviously had more preserved myelinated areas than the LacZ group at 6 weeks after SCI. Of note, the HGF group exhibited a significantly spared rim of white matter, even at the lesion epicenter, whereas the LacZ group exhibited severely demyelinated white matter throughout the lesion epicenter (Fig. 14.2b). Next, to determine the effect of HGF on motoneurons, the numbers of ChAT-positive motoneurons in the ventral horns were quantified at 6 weeks after SCI. Although almost all the ChAT-positive motoneurons disappeared at the lesion epicenter in both groups, a significantly larger number of ChAT-positive motoneurons were observed at the site rostral to the epicenter in the HGF group, compared with that in the LacZ group (Fig. 14.2c). These findings suggested that HGF exerted protective effects on motoneurons and oligodendrocytes and contributed to tissue sparing after SCI [25].

Next, to determine whether HGF inhibited the activation of caspase-3 after SCI, immunoblotting analyses using anti-cleaved caspase-3 antibody were performed at 1, 3, and 7 days after SCI. Cleaved caspase-3 was strongly induced after SCI and was most detectable at 3 days after SCI in both the HGF and LacZ groups (data not shown). Quantitative analysis revealed that the induction of cleaved caspase-3 was significantly attenuated in the HGF group, compared with the LacZ group, at 3 days after SCI (data not shown). Furthermore, double immunostaining using anti-cleaved caspase-3 antibody and antibodies for neurons or oligodendrocytes showed that the numbers of NeuN and cleaved caspase-3 double-positive motoneurons in the ventral horns and GST-π and cleaved caspase-3 double-positive oligodendrocytes were obviously reduced in the HGF group, compared with the LacZ group (data not shown). These results suggested that HGF significantly reduced the levels of cleaved caspase-3 activation in neurons and oligodendrocytes after SCI, thereby promoting their survival [25].

To examine the effect of HGF on vascular endothelial cells after SCI, immunostaining with anti-RECA-1 antibody was performed. In intact thoracic spinal cord, the vessels had delicate walls composed of homogeneously stained endothelial cells. While most of the vessels disappeared at the epicenter at 1 week after SCI, several vessels were stained intensely and showed abnormally large lumina with thick wall, which were not observed in the intact spinal cord (data not shown). We found that significantly larger number of RECA-1-positive vessels with lumina larger than 20 μm, representing newly formed vessels [8], were observed in the HGF group compared with the LacZ group at the epicenter and at 4 mm rostral to the epicenter at 1 week after SCI (data not shown) [25].

To determine the effects of HGF on the axonal growth after SCI, axial sections of injured spinal cords were immunostained with anti-5HT antibody at 1 week and 6 weeks after SCI. 5HT-positive raphe-spinal serotonergic fibers were observed mainly in the gray matter in each group. Quantitative analyses revealed that while 5HT-positive fibers were almost undetectable in either group at 1 week after SCI, a significantly greater abundance of 5HT-positive fibers was detected, even in an area 4 mm caudal to the epicenter, in the HGF group as compared with that in the LacZ group at 6 weeks after SCI (data not shown). Furthermore, at 1 week after SCI, the 5HT-positive fibers also showed c-Met-IR, and at 6 weeks after SCI, they expressed GAP-43, which has been used as a marker of axonal regeneration [27–30], even in a region 4 mm caudal to the epicenter (data not shown).

The contusive SCI resulted in complete paralysis followed by gradual recovery, reaching a plateau (BBB score 6.6 ± 1.1) at 6 weeks after SCI in the LacZ group. Significant differences in the BBB scores were observed between the two groups from 7 days after SCI (data not shown). We believe that the difference between a BBB score of 8 (sweeping of hindlimbs) and BBB score of 9 (weight support on hindlimbs) is clinically substantial. From a clinical perspective, the recovery of the HGF group to weight-supported plantar steps (BBB score 10.1 ± 0.6) was noteworthy [25].

What we did first in marmoset study was to investigate the distribution of motoneurons in intact spinal cord that innervate the hand muscles by injecting cholera toxin B subunit (CTB) into the forearm flexor and extensor muscles. Immunohistological analysis using anti-CTB antibody revealed that the wrist and finger extensor motoneurons were mainly located in lamina IX of the cervical (C)4–C7 segments and wrist flexor motoneurons were in lamina IX of the C6–T1 segments (data not shown) [31]. Based on this finding, we induced contusive SCI at C5 level and almost all of the wrist extensor and flexor motoneurons were located at a site caudal to the lesion epicenter. After a laminectomy at the C5 level, the dura mater was exposed and a 20-g weight was dropped from a height of 50 mm onto the dura using a modified NYU impactor as reported previously [32–34] in adult female common marmosets (n = 6 for the rhHGF group; n = 5 for the PBS group). Right after contusive SCI at the C5 level, a C7 laminectomy was carried out and an intrathecal catheter was inserted from the C7 level. An osmotic mini pump, filled with 400 μg rhHGF (HGF group) or PBS (control group), was connected to a catheter and placed in the subcutaneous space on the right side of the animal’s back [31]. The pump was left in place to deliver the total 400 μg dose of rhHGF for 4 weeks; dosage was based on previous results from intrathecal rhHGF administration in ALS rats [24], which have about the same body weight as marmosets.

All animals showed severe quadriplegia 1 day after injury; they lay on the floor in a prone position, with little limb movement, and could not roll over or move forward by themselves. After C5-level SCI, irrespective of therapeutic intervention, the animals showed progressive recovery of motor function. To access the recovery of motor function and evaluate the therapeutic effects of intrathecal rhHGF, we established an original open-field rating scale with three main categories: upper limbs, lower limbs, and trunk stability (Table 14.1). This study’s injury model, cervical SCI, requires a detailed analysis of upper limb function to precisely evaluate motor function recovery. Thus, the upper limb category was divided into three subdivisions: weight bearing, reach-and-grasp performance, and somatosensory function (Table 14.1) [31].

The hand position (below shoulder, between shoulder and head, or above head) and placement in walking (dorsal placement, ulnar placement but not pronated, pronated but no palmar placement, or palmar placement) were highlighted in the subdivision of weight bearing (Table 14.1, Fig. 14.3c, d). The assessment of reach-and-grasp performance highlighted finger flexion and extension, wrist extension, forearm pronation, and shoulder flexion (Table 14.1, Fig. 14.3a, b). To access somatosensory function, we evaluated the distance the limb was dropped through gaps in the cage, as follows: lower limbs up to the thigh, knee, or foot only and upper limbs up to the humerus, elbow, or hand only (Table 14.1, Fig. 14.3e, f). Because there were various combinations of improvement in multiple subdivisions and attributes, we adopted a point-addition scoring system in this original open-field rating scale, unlike the system used in the BBB scale [26]. Details of motor function recovery over time and precise category definitions of the original open-field rating scale are described in [31].

The evaluation of motor function by the original open-field scale showed significant differences between the two groups (Fig. 14.4a–c). Animals in both groups gradually recovered, reaching a plateau around 8 weeks after SCI, and the original score of the upper limbs (pre-injury score = 20) recovered to 15.9 ± 0.8 and 7.8 ± 1.8 in the rhHGF and PBS groups, respectively, 12 weeks after SCI (Fig. 14.4a). Significant differences between the two groups were observed at most time points from 14 days onward after SCI in the open-field upper limb scores (Fig. 14.4a) [31].

Twelve weeks after injury, the ChAT-positive motoneurons around the lesion epicenter had almost disappeared, consistent with our rat study. However, we did not detect any significant differences in the number of surviving C2–T1 ChAT-positive motoneurons between the two groups (data not shown) [32]. We next investigated the pattern of the corticospinal tract (CST) pathway and its terminations in common marmosets. Axial sections of intact and injured spinal cords were immunostained with an anti-calmodulin-dependent kinaseIIα (CaMKIIα) antibody [35]. We previously found that CaMKIIα immunoreactivity (IR) labels the CST in the marmoset spinal cord [32, 34, 36], although its specificity remains to be demonstrated. In this study, we made the following observations. First, in the intact spinal cord, CaMKIIα-IR was detected in the lateral column of white matter, the dorsal horn, and the intermediate zone (IMZ) of the gray matter (Fig. 14.5a), suggesting that the CST in the common marmoset is located in lateral columns, unlike in rodents, in which the CST projects mainly to dorsal horn neurons and premotor spinal circuits [37]. Second, we did not detect CaMKIIα-IR in either the ventral white matter column or the ventral gray matter horn, suggesting that, as in rodents, the proportion of the ventral CST extending into the white matter is small and that the CST does not project directly to cervical motoneurons in the ventral horns [31].