Enhanced Motor Function by Training in Spinal Cord Contused Rats Following Radiation Therapy

Weight-bearing stepping, without supraspinal re-connectivity, can be attained by treadmill training in an animal whose spinal cord has been completely transected at the lower thoracic level. Repair of damaged tissue and of supraspinal connectivity/circuitry following spinal cord injury in rat can be achieved by specific cell elimination with radiation therapy of the lesion site delivered within a critical time window, 2-3 weeks postinjury. Here we examined the effects of training in the repaired spinal cord following clinical radiation therapy. Studies were performed in a severe rat spinal cord contusion injury model, one similar to fracture/crush injuries in humans; the injury was at the lower thoracic level and the training was a combined hindlimb standing and stepping protocol. Radiotherapy, in a similar manner to that reported previously, resulted in a significant level of tissue repair/preservation at the lesion site. Training in the irradiated group, as determined by limb kinematics tests, resulted in functional improvements that were significant for standing and stepping capacity, and yielded a significant direct correlation between standing and stepping performance. In contrast, the training in the unirradiated group resulted in no apparent beneficial effects, and yielded an inverse correlation between standing and stepping performance, e.g., subject with good standing showed poor stepping capacity. Further, without any training, a differential functional change was observed in the irradiated group; standing capacity was significantly inhibited while stepping showed a slight trend of improvement compared with the unirradiated group. These data suggest that following repair by radiation therapy the spinal circuitries which control posture and locomotor were modified, and that the beneficial functional modulation of these circuitries is use dependent. Further, for restoring beneficial motor function following radiotherapy, training seems to be crucial

Therapeutic strategy for acute spinal cord contusion injury: cell elimination combined with microsurgical intervention.

No cure is available for human spinal cord injury. Cell elimination by localized radiation therapy that is timed within 2-3 weeks postinjury can facilitate repair of structure and function in transected rat spinal cord. In pilot studies in contusion spinal cord injury, a model similar to crush/fracture injury in human, we did not observe the expected beneficial effects of radiation therapy. Long forgotten data show that in contusion/crush injury, fluid accumulation from hemorrhage is critical. Alfred Reginald Allen observed that the most devastating sequelae in contusive injury are secondary to fluid accumulation which could be alleviated by surgical intervention, midline slits (myelotomy) at the lesion site.Here, we tested whether release of fluid buildup by microsurgery (partial myelotomy) would affect the structural outcome of radiation therapy in the severely contused rat spinal cord. Surgical intervention alone significantly enhanced tissue and functional preservation in the contused cord, thus confirming Allen's observations. Combining partial myelotomy with radiation therapy that is specifically timed postinjury elicited substantial beneficial therapeutic outcome; it led to significant increase in tissue repair/preservation compared with the group that received surgical intervention only, as determined by histology and in vivo MRI. Altogether, the combined treatments led to a 1.8 fold increase in tissue repair/preservation as compared with the contused group.The data suggest that a clinical protocol could be developed to treat acute human spinal cord injury through conventional clinical procedures, a combination of microsurgical manipulation and radiation therapy. These also suggest it is imperative to first prevent the secondary damage caused by fluid accumulation for a cure to be possible

Enhanced motor function by training in spinal cord contused rats following radiation therapy.

Weight-bearing stepping, without supraspinal re-connectivity, can be attained by treadmill training in an animal whose spinal cord has been completely transected at the lower thoracic level. Repair of damaged tissue and of supraspinal connectivity/circuitry following spinal cord injury in rat can be achieved by specific cell elimination with radiation therapy of the lesion site delivered within a critical time window, 2-3 weeks postinjury. Here we examined the effects of training in the repaired spinal cord following clinical radiation therapy. Studies were performed in a severe rat spinal cord contusion injury model, one similar to fracture/crush injuries in humans; the injury was at the lower thoracic level and the training was a combined hindlimb standing and stepping protocol. Radiotherapy, in a similar manner to that reported previously, resulted in a significant level of tissue repair/preservation at the lesion site. Training in the irradiated group, as determined by limb kinematics tests, resulted in functional improvements that were significant for standing and stepping capacity, and yielded a significant direct correlation between standing and stepping performance. In contrast, the training in the unirradiated group resulted in no apparent beneficial effects, and yielded an inverse correlation between standing and stepping performance, e.g., subject with good standing showed poor stepping capacity. Further, without any training, a differential functional change was observed in the irradiated group; standing capacity was significantly inhibited while stepping showed a slight trend of improvement compared with the unirradiated group. These data suggest that following repair by radiation therapy the spinal circuitries which control posture and locomotor were modified, and that the beneficial functional modulation of these circuitries is use dependent. Further, for restoring beneficial motor function following radiotherapy, training seems to be crucial

Acute fluid buildup and its detrimental outcome at the contusion lesion site.

<p><i>A-C.</i> Longitudinal <i>ex vivo</i> view of the lesion site following moderate contusion injury, seen in thionin stained sagittal cord sections at 0, 1, and 4 days PI (postinjury), respectively (n = 3 per time-point). Note the edema, swelling and tissue rupture at 4 days PI. <i>D.</i> Schematic cartoon of fluid accumulation at the lesion site and its sequelae. Bar  =  2 mm.</p

Tissue preservation/repair by myelotomy and radiation therapy <i>in vivo</i>.

<p><i>A</i>. <i>In vivo</i> images of: five serial T₂-weighted axial scans of normal, and of 3 differently treated severely contused cords: no added treatment, CNTS; incision, CNTS+TOMY; and incision followed by radiation therapy, CNTS+TOMY+XRAY, at 50 days postinjury (these 3 spinal cord are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000565#pone-0000565-g003" target="_blank">Fig. 3 <i>B–D</i></a>). Each of the numbered slices was taken from the same anatomical level of the cord, e.g., slice 1 is at identical level in all 4 cords and mostly unharmed. Note, the ‘fuzziness’ of these images is an innate feature of the acquisition procedure; these are <i>in vivo</i> images that were acquired for about an hour while the animal is breathing, each showing –as a result of the field of view utilized– an enlarged cord (by x7); therefore, to obtain a better view, the figure should be held at a distance. Note, in normal cord some typical anatomical features: the butterfly shaped grey matter (in slice 1), the ventral roots (arrows, slice 2, 4), and the dorsal dark circle (arrowhead in section 1) is a blood vessel. In the injured cords, note sections 3-4 (the lesion epicenter) are abnormal and reduced in size, also the irradiated cord appears to be the least abnormal. Bar  =  2 mm. <i>B</i>. Quantitative tissue preservation by myelotomy and radiation therapy. Plotted are the normalized tissue areas in each of the serial MRI sections (2–4) of 4 groups as illustrated in panel <i>A</i> above: normal (n = 3), control (n = 6), unirradiated (n = 8), and irradiated (n = 8). Note, in the control cords tissue decay extends throughout slices 2-4 whereas in those that underwent myelotomy the damage was contained to slices 3-4. Also, tissue preservation in each of the sections is the highest in the irradiated cords in comparison with the control group. Abbreviations: contusion  =  CNTS; myelotomy  =  TOMY; and radiation therapy  =  XRAY. Note, error bars cannot be shown in a 3D plot.</p

Structural repair by radiation therapy made possible by myelotomy.

<p>Micrographs of thionin-stained sagittal sections through normal intact cord and through the epicenter of 3 differently treated severely contused cords seen 90 days postinjury: <i>A</i>. normal; <i>B</i>. control, CNTS; <i>C</i>. incision, CNTS+TOMY; and <i>D</i>. incision followed by radiation therapy, CNTS+TOMY+XRAY. <i>E.</i> Schematic cartoon of the critical role of midline slits in making repair possible (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000565#pone-0000565-g001" target="_blank">figure 1<i>D</i></a>); once the secondary damage is halted intrinsic repair can be facilitated by radiation therapy. Note, the tissue preservation and the substantial wound repair in the irradiated cord which contains in its epicenter motoneurons and other neuronal cells. Abbreviations: contusion  =  CNTS; myelotomy  =  TOMY; and radiation therapy  =  XRAY. Bar  =  3 mm.</p

Midline incision is critical for facilitating repair in severe contusion injury: <i>Ex vivo</i> quantitative data.

<p>Degree of tissue preservation/repair, as measured by area of remaining tissue, in 3 differently treated severely contused cords: control, CNTS (n = 6); incision, CNTS+TOMY (unirradiated, n = 10); and incision followed by radiation therapy, CNTS+TOMY+XRAY (irradiated, n = 7). These 3 groups were either <i>A.</i> without training or <i>B</i>. with treadmill exercised. Note, tissue preservation was enhanced by midline incision from 19% to 26.1%, and radiation therapy increased it to 35% of the normal tissue; further treadmill exercising increased it in the unirradiated from 26.1% to 35% and in the irradiated from 35% to 56.3%. Abbreviations: contusion  =  CNTS; myelotomy  =  TOMY; and radiation therapy  =  XRAY. Significance, * <i>p</i> < 0.05. Error bars, SD.</p

Midline incision is critical in mitigating the outcome in severe contusion injury.

<p>Plotted are the percent of damaged area in 5 severely contused cord-groups: control, and with midline slits at either 1, 2, 4, or 24 h postinjury (n = 8 per group). Note, myelotomy at 3 time points, 1-4 h, significantly reduced the lesion size and altered the outcome of injury: * <i>p</i> < 0.05; ** <i>p</i> < 0.01. Error bars, SEM.</p