Anatomical mechanism of spontaneous recovery in regions caudal to thoracic spinal cord injury lesions in rats

Animals

In this study, we used adult Sprague–Dawley rats (200–220 g). All rats were allowed to acclimate to the new environment for seven days before the start of any experimental procedures. The rats were housed on a 12 h light-dark cycle with food and water provided ad libitum. All rats were deeply anesthetized before any surgical procedures were performed (10% chloral hydrate, 35 ml/kg). After surgery, antibiotics (Penicillin, 128,000 UI/kg) and 10 ml of sterile saline were administered subcutaneously each day during the first week. After each operation, the rat was placed in a separate cage for seven days before it was housed with other rats. The rats in the study were obtained from the Vital River Company. Ethical approval was obtained from the Beijing Neurosurgical Institute Laboratory Animals Ethics Committee in China.

In the first part of study, there were 50 rats in total and 10 rats died throughout the study; the mortality was 20%. In the second part of study, there were 125 rats in total and 16 rats died throughout the study; the mortality was 12.8%.

Groups

In the first part of the study, we investigated if regenerated axons coursed ipsilaterally or contralaterally. All rats underwent two operations on T7 spinal cord and were divided into 5 groups (Figs. 1A–1E). The first group was a sham operation group (N = 10) in which both operations were sham. Groups 2–5 all first received a hemisection of the left side of spinal cord at T7, but had different second operations. The second group was the control group (N = 6), in which the second operation was sham, consisted a midline cut in the spinal cord at T7. The third group was the ipsilateral experimental group (N = 8), in which a second hemisection was conducted on the ipsilateral side. The fourth group was the contralateral group (N = 8) in which a second hemisection was made on the contralateral side. The fifth was the transection group (N = 8), which received a full transection at T7 in the second operation.

In the second part of the study, we investigated the innervation of vertebrae downstream of the lesion site to determine if regenerated axons target single or multiple vertebrae. These experiments comprised three main groups, each of which were divided into five smaller sub-groups defined according to the site of the second operation: T8–T12. The first group was the operation group (N = 37, Figs. 2A–2E), in which all rats underwent 2 hemisection operations. The first operation was at T7 on the left side; eight weeks later the second operation was carried out ipsilaterally at a single vertebra from T8–T12 depending on sub-group. The second group was the control group (N = 37, Figs. 2F–2J). All received a hemisection at T7 on the left side, and eight weeks later underwent a sham operation at a single vertebra at T8–T12 depending on sub-group. The third was the sham operation group (N = 35, Figs. 2K–2O), in which rats in all sub-groups received a sham operation at both time points.

Hemisection operation

Basso, Beattie, Bresnahan (BBB) score tests in both hindlimbs

Motor performance was scored were performed according to the well-known Open Field BBB locomotor scale (Basso, Beattie & Bresnahan, 1995). It was given each week after each hemisection operation All of the BBB score tests, each lasting 5 min, were performed in an open field (diameter 150 cm) with a wood floor. When we monitored the movement of the hindlimbs, all rats moved freely without any disturbance. The paw placement, joint movements, weight bearing, and coordination among the limbs were used to evaluate the BBB locomotion scale.

Electrophysiological examinations

Before beginning electrophysiological examinations each rat was anesthetized. An electrophysiological examination was given before and after each hemisection operation.

Motor-evoked potential (MEP) studies on the body surface

MEP examination and analyses were performed mainly according to previously published methods (Shen et al., 2016; Yin et al., 2015; Ziegler et al., 2011). Before the examination, the rat was in a relaxed state. Intramuscular electrode needles were implanted in the anterior tibial muscle (TA) and little toe abductor muscle (LTA) on both sides. There were five wires used for MEP examination, and each wire was connected to a stainless steel pin. The rostral-caudal locations of the wires were as follows: the first needle was in the midline 5 mm from the nose; the second needle was located subcutaneously in the midline of the head; the third needle was located subcutaneously in the mid-belly; the fourth needle was located in the middle of the muscle being tests; and the fifth needle was located in the tail 3 cm from the root. Stimulations of 10 mA at 1 Hz were administered once per time point: for 1 ms per stimulus. Each muscle received three standard stimuli, and the interval time was 30 s.

Criteria for excluding animals

Rats were excluded from the research according to the following criteria: (1) death during or after the operation; (2) signs of autophagia and/or a serious skin infection; (3) an edematous hindlimb that would affect the BBB score test; (4) death during the electrophysiological recordings.

Statistics

The statistical analysis was performed using SPSS database (version 19.0; SPSS Inc., Chicago, IL, USA). The BBB scores and electrophysiological examination data are shown as means ±  SEM. When the data agreed with the Bartley Ball Test, the repeated measures general linear model test was used to determine the overall differences in the different test times after the operation (1 week to 6, 8, or 16 weeks), followed by LSD (least significant difference) tests to make comparisons among groups. P values less than 0.05 were considered statistically significant.

Part 1: determination of an ipsilateral versus contralateral course

BBB score s after the first hemisection operation at T7

After the first operation, none of the rats could move their left hindlimb or perform weight-bearing movements, while the right hindlimb could move slightly. The most significant and rapid improvements in BBB scores of both hindlimbs occurred over the first two weeks. BBB scores continued to increase through the 3rd week after the operation and then reached a plateau phase in the 4th week that persisted through the end of the evaluation period at week 8 (Fig. 4A).

Electrophysiological examinations in part 1 of the study

After the second hemisection at T7, the latency periods in both TA muscles and both LTA muscles between rats in the ipsilateral group and control group were not significantly different (p < 0.05, Figs. 5A–5D). However, they were longer in the contralateral group than in the control group (p < 0.05). There were no significant differences between the contralateral group and the transection group (p < 0.05). All experimental groups had significantly longer latency periods than the sham group (p < 0.05).

There were not significant differences in latency periods in the spinal cord between the sham, control, and ipsilateral groups (p < 0.05, Figs. 6A, 6B and 3A–3F). However, the latency period and wave amplitude disappeared in the contralateral and transection groups after the second hemisection (p < 0.05, Figs. 6A, 6B, 3G, 3H, 3I and 3J).

Part 2: determination of the involvement of vertebrae T8–T12

BBB scores after a second hemisection operation at T8–T12

In the T8 second hemisection group, BBB scores for the left hindlimb decreased slightly, while BBB scores of the right hindlimb were barely affected. Approximately three weeks later, BBB scores of both hindlimbs recovered to the level before the operation (p < 0.05, Fig. 7A). In comparisons of BBB scores six weeks after the second hemisection, the left hindlimbs in T8–T12 sub-groups displayed poorer and poorer improvement (p < 0.05, Figs. 7A–7E). Seven of eight rats in T12 operation sub-groups exhibited no left hindlimb movement 6 weeks after the operation.

Compared to the operation group, BBB scores for both hindlimbs in the T8–T12 sub-groups in the control group decreased significantly after the second hemisection (p < 0.05, Figs. 7A–7E and 7F–7J).

BBB scores of both hindlimbs in all T8–T11 sub-groups in the sham operation group recovered completely by the second week after the second hemisection (p < 0.05, Figs. 7K–7N). Compared to the T12 sub-group, the T8–T11 sub-groups displayed better improvement (p < 0.05, Figs. 7K–7O).

Electrophysiological examinations in part 2 of the study

After the second hemisection in the operation group, the latency period in left TA muscles and left LTA muscles became progressively longer from the T8 sub-group to T12 sub-group (p < 0.05, Figs. 8A and 8C).

Effects of a second hemisection operation at T7

Many previous animal experiments have shown that after a hemisection operation of the spinal cord, the transplantation of stem cells or various engineered tissue materials in the injury region can improve movement of both hindlimbs. Immunohistochemical examinations have also shown that the nerve fibers in regions rostral to the injury site increased and deeply innervated the lesion site. This indicates that nerves fibers penetrating into the area of injury probably play important roles in recovery after spinal cord injury (Estrada et al., 2014; Fan et al., 2010; Jee et al., 2012; McCall, Weidner & Blesch, 2012; Wright et al., 2011).

However, some studies using animal models have reached conflicting conclusions. An important example is the work of Courtine et al. (2008) in which they carried out two successive lesions of the rat spinal cord and showed that the contralateral but not ipsilateral side was essential for recovery.

However, Courtine et al. (2008) did not performed a second hemisection at the injury region, so whether nerve regeneration within the injury region was attributable to hindlimb movements recovery could not be ruled out. Accordingly, in this study, we added an additional experimental group that received a second hemisection in the region of injury.

BBB scores for both hindlimbs decreased significantly after the first hemisection at T7, and 4 weeks later, the improvement reached a plateau. When the second hemisection operation was performed on the injury region, it had almost no effect on movement of either hindlimb (p < 0.05, Figs. 4C, 4D, 3C, 3D, 3E and 3F). The MEP results from body surface and spinal cord also displayed no significant differences (p < 0.05, Figs. 5A–5D, 6A, 6B, 3E, 3F, 3G and 3H). However, in the contralateral and transection groups, hindlimb movement on both sides disappeared after the second operation and there were no significant difference between groups at 8 weeks post-injury (p < 0.05, Figs. 4E and 4F). Moreover, MEP in the spinal cord disappeared after the second operation in both groups (Figs. 6A, 6B, 3G, 3H, 3I and 3J). By contrast, we found that, after the spinal cord transection operation MEP activities were still observed at the body surface. For this reason, MEP examination of the spinal cord is a more accurate indicator of injury and recovery than MEP at the body surface.

We thus conclude that the nerve repair in the injury region has no effect after the hemisection operation at T7 on the ipsilateral side, and reparative responses involved nerve fibers on the contralateral side.

Effects of a second hemisection operation at T8–T12 on the ipsilateral side

Repair processes occurring rostral to the lesion

Many findings have shown that, although the direct conduction pathway was destroyed in rats with a spinal cord injury, commands from brain conducted by the corticospinal tract (CST) can still be transmitted to the lumbar spinal cord below the lesion on the ipsilateral side (Bareyre et al., 2004; Courtine et al., 2008; Jankowska & Edgley, 2006; Kerschensteiner et al., 2004; Van den Brand et al., 2012). After injury, an important mechanism of the spontaneous recovery process is thus that the structure and course of nerve fibers in the CST are remodeled such that they make contact with propriospinal neurons that form detour pathways bypassing the lesion (Nishimura & Isa, 2012; Pierrot-Deseilligny, 2002; Rosenzweig et al., 2010; Zaaimi et al., 2012). However, the CST is not the only descending tract that affects movement and is not be the only projection system that conveys functional recovery (Han et al., 2013; Hurd, Weishaupt & Fouad, 2013). Spared reticulospinal fibers play an important role in the recovery process through spontaneous compensatory sprouting and increases in density after injury; they may also operate caudal to the lesion by enhancing indirect access to reticulospinal commands (Ballermann & Fouad, 2006; Zorner et al., 2014). Therefore, we hypothesize that the nerve fiber circuit underlying repair in this study was composed of CST and reticulospinal fibers and propriospinal neurons. We speculate that around the T12 vertebra, which was the most important for nerve repair, there were more nerve fibers relative to the other vertebrae that crossed the midline from the contralateral side to the injury side. However, more research, particularly nerve fiber tracing experiments, is needed to confirm this.

Repair processes occurring caudal to the lesion

Tillakaratne et al. (2010) showed that rats exhibited spontaneous recovery via a step-wise process after a complete transection of the mid-thoracic spinal cord, even though the region caudal to the spinal cord lesion did not make any connections to the brain and in absence of descending tracts passing through the lesion. Thus, roles of activity of a locally acting central pattern generator (CPG), which is present in many species, are important to consider (Deliagina et al., 1999; Ekeberg & Pearson, 2005; Grillner, 1985). While our research argues for the presence of significant spontaneous locomotor recovery resulting from new forms of dynamic control in the spinal CPG from newly generated or remodeled descending tracts, the local CPG still may play the primary role in recovery following spinal cord injury (Rossignol et al., 2007).

The CPG of the spinal cord is located in the lumbar enlargement at about the T1–12 segments (Magnuson et al., 1999). The second part of this study showed that areas closer to T12 vertebra are more important for nerve repair. Therefore, we could conceivably use various treatments to reinforce the role of CPG and the nerve fibers that connect to it to promote recovery. However, given that a second hemisection operation at T9 on the ipsilateral side could also impair hindlimb movements, which could not recover to pre-operation levels, the CPG is likely not the only factor playing a role in the recovery process.

Study limitations

These studies assessed spontaneous recovery from spinal cord injury, but not the impact of any treatment. Further research is needed to confirm if similar results are observed in the case of using a therapeutic intervention within the same experimental injury paradigm; potential therapies include targeting neuroinflammation, transplantation of engineered tissue materials, stem cells, neurotrophic factors, or genetic modifications. We also did not directly assess the path of newly projected nerve fibers to more conclusively and precisely define their course.