Partial restoration of spinal cord neural continuity via vascular pedicle hemisected spinal cord transplantation using spinal cord fusion technique

Abstract Aims Our team tested spinal cord fusion (SCF) using the neuroprotective agent polyethylene glycol (PEG) in different animal (mice, rats, and beagles) models with complete spinal cord transection. To further explore the application of SCF for the treatment of paraplegic patients, we developed a new clinical procedure for SCF called vascular pedicle hemisected spinal cord transplantation (vSCT) and tested this procedure in eight paraplegic participants. Methods Eight paraplegic participants (American Spinal Injury Association, ASIA: A) were enrolled and treated with vSCT (PEG was applied to the sites of spinal cord transplantation). Pre‐ and postoperative pain intensities, neurologic assessments, electrophysiologic monitoring, and neuroimaging examinations were recorded. Results Of the eight paraplegic participants who completed vSCT, objective improvements occurred in motor function for one participant, in electrophysiologic motor‐evoked potentials for another participant, in re‐establishment of white matter continuity in three participants, in autonomic nerve function in seven participants, and in symptoms of cord central pain for seven participants. Conclusions The postoperative recovery of paraplegic participants demonstrated the clinical feasibility and efficacy of vSCT in re‐establishing the continuity of spinal nerve fibers. vSCT could provide the anatomic, morphologic, and histologic foundations to potentially restore the motor, sensory, and autonomic nervous functions in paraplegic patients. More future clinical trials are warranted.


| INTRODUC TI ON
Spinal cord injury (SCI) is a disease with a high morbidity and mortality rate. It is estimated that 27 million people worldwide are paralyzed by SCI, and 500,000 new cases occur each year. Patients with paraplegia are confined to a wheelchair and subjected to a lifetime of multiple medical comorbidities. 1 In addition to greatly reducing the quality of life of patients, paraplegia also brings a huge social burden due to high medical costs and loss of productivity in the workplace. 2 Therefore, treatment to restore motor function in these patients would be a great advance.
In our proposed neurologic foundations of spinal cord fusion (SCF), which is called GEMINI, polyethylene glycol (PEG) was used as a neuroprotective fusogen to fuse two approximated stumps of the transected spinal cord. [3][4][5] PEG is an inexpensive, stable, nontoxic, fully biocompatible, and water-soluble linear polymer that is synthesized by the living anionic ring-opening polymerization of ethylene oxide and has molecular weights ranging from 0.4 to 100 kDa. 6 PEG is known to promote the fusion of plant protoplasts. 7 In 1975, Pontecorvo reported that PEG could be used as a fusogen to fuse mammalian cells to produce hybrid cells. 7 In 1986, Bittner et al. 8 showed that PEG has the ability to fuse and reseal the membranes of severed axon membranes in vitro under controlled conditions. Subsequently, in 1999, Shi et al 9 demonstrated that PEG could fuse completely transected strips of isolated white matter in vitro. These studies suggest that PEG might be used to reconstruct not only peripheral nerves but also severed spinal cord axons in vivo. More recently, a number of molecular agents have been tested in different models of SCI; they include astaxanthin, 10 and Chitosan combined therapy. 11 Since 2016, our team has found that PEG could restore the motor function of hind limbs in different animal models of paraplegia (complete spinal cord transection at T10), such that mice and rats regained the ability to stand and crawl using their hind legs about 1 month after surgery, 12,13 and beagles regained the ability to stand and crawl about 2 months after surgery. 14,15 In addition to PEG, the cortico-trunco-reticulo-propriospinal (CTRPS) pathway mentioned in GEMINI also plays an important role in SCF. [3][4][5] The CTRPS is the phylogenetically oldest motor and sensory command system. As the species evolved, the pyramidal tract, a faster command system, developed allowing for rapid transmission of volitional signals. After spinal cord transection, however, even if no pyramidal tract axon could be fused by PEG. The propriospinal neurons of the CTRPS along with others in proximity that were not damaged by the extra-sharp blade used to transect the spinal cord can regrow (sprout) their fibers immediately and re-establish contacts between the apposed interfaces. 3,5 The entire motor and sensory recovery would thus hinge on the CTRPS pathway. 16,17 In the GEMINI and our previous animal experiments, paraplegic models were constructed by quickly and acutely transecting the spinal cord with an extremely sharp surgical blade that imparted minimal local trauma. There was no gap between the adjacent spinal cord stumps. However, clinical paraplegic patients often have extensive spinal cord contusion and fibrous scarring, with numerous cysts and fibrous connective tissue response in the area of SCI. Therefore, we developed several clinical translation models of SCF for paraplegic patients and conducted a clinical trial (http://www.chictr.org.cn/ showp roj.aspx?proj=50526, ChiCTR2000030788) of SCF. In this report, we focus on one of the SCF clinical translation models: vascular pedicle hemisected spinal cord transplantation (vSCT). Prior to this clinical trials, we used beagle dogs as animal models to verify the feasibility of vSCT and the efficacy of PEG-600 (see Appendix S1).
Herein, we present the preliminary postoperative results of eight participants treated with vSCT in the clinic. This paper demonstrates that vSCT, a clinical translational model of SCF, appears to be a potential therapeutic approach in the field of SCI.

| Participant selection and evaluation
Our inclusion criteria were 1) age ≤50 years old; 2) a traumaticchronic or acute-complete type of SCI; 3) SCI located in the thoracic fibers. vSCT could provide the anatomic, morphologic, and histologic foundations to potentially restore the motor, sensory, and autonomic nervous functions in paraplegic patients. More future clinical trials are warranted.

K E Y W O R D S
clinic trial, GEMINI, polyethylene glycol, spinal cord fusion, spinal cord injury segment; 4) able to understand and complete the trial; and 5) normal cardiopulmonary function to complete the prolonged operation

| Preoperative preparation for surgery
Before surgery, the surgical team discussed the SCI condition with the participants. Participants completed routine examinations before the surgery, including laboratory examination, electrocardiogram, and ultrasonography. Participants stopped smoking 2 weeks before the operation. Tilopidine and clopidogrel, aspirin, and nonsteroidal anti-inflammatory drugs were discontinued 10, 7, and 2 days before the operation, respectively. Conditions, such as malnutrition, hypertension, and high blood sugar were addressed and controlled before the surgery. Prophylactic antibiotics were administered 30 min before the surgery. Tranexamic acid was administered to minimize bleeding during the surgery.

| Vascular pedicle hemisected spinal cord transplantation (vSCT)
The participant was placed in a prone position under the general anesthesia. The skin and muscles overlying the thoracic spinal column were incised. A laminectomy was performed at the SCI level with a cutting ultrasonic scalpel (BoneScalpel ® , Misonix) to expose the dura mater, which was then opened to expose the spinal cord.
vSCT was performed, which is a new surgical treatment protocol that we originated and independently developed. The main surgical procedures of this operation are shown in Figure 1.

| Safety evaluation
After the surgery, the participant's body temperature was measured three times a day. Blood routine examinations were performed every 3 days, and the surgical incision was observed daily to assess for postoperative infection. We also observed whether the participant had any symptoms of headache and dizziness, to evaluate whether there was a leakage of cerebrospinal fluid. Potential adverse reactions to PEG, such as urticaria, dermatitis, and even anaphylactic shock, were monitored daily.

| Immunohistochemistry
The removed spinal cord tissue was fixed with 4% paraformaldehyde for 24 h and then embedded in paraffin and horizontally cut into transverse slices of 7 μm thickness.
After removing the paraffin, the slices were quenched with endogenous peroxidase activity in 3% methanol hydrogen peroxide for 0.5 h.
The antigen (https://www.abcam.com/proto cols/ihc-fixat ion-protocol) was fixed in the citrate buffer (pH 6.0) in a microwave for 5 min. The The myelin sheaths were stained by an antibody specific for MBP.

| Neurophysiological assessment
Somatosensory-evoked potential and MEP were recorded before surgery and at 1, 3, and 6 months after surgery and analyzed with the NIM-ECLIPSE ® System (Medtronic).

| Neuroimaging assessment
Participants were subjected to MRI and DTI using a 1.5 T MRI were processed to produce color DTI maps.

| Pain assessment
We elected to use the visual analog scale (VAS), because it is one of the most widely used methods to give valid and reliable assessments on experimental pain, as well as acute and chronic pain, in patients. 23 The VAS consisted of a 10 cm horizontal line on a card with the words "no pain" and "worst pain ever" placed at the left-and right-hand extremes of the line, respectively. The participants were instructed to mark the line at a point representing their pain intensity. 24

| Statistical analysis
All data were analyzed in SPSS Statistics software (SPSS 20.0, IBM).
The Kolmogorov-Smirnov test and Shapiro-Wilk test for normality were used to assess the data distribution. All data exhibited a normal distribution (p > 0.05). Data are presented as the mean ± SD. Paired sample t tests were used for statistical analysis in the clinical trial.
Statistical significance was set at a p < 0.05.

| Participant enrollment
We enrolled eight participants (six men and two women) into the clinical trial (Table 1, Figure 2). All participants were classified as ASIA Impairment Scale grade A and had complete injuries in the thoracic spinal cord segment.
The entire operation of vSCT took approximately 4 h. The mean length of spinal cord removed was 5.5 cm (

| Immunohistochemistry
The

| Neurophysiological assessment
Preoperatively, the SSEPs of bilateral median nerve were normal in these participants with paraplegia, and the SSEPs of bilateral posterior tibial nerve were completely absent. In addition, the MEPs recorded at the abductor pollicis brevis muscles in the bilateral upper extremities were normal, and the MEPs recorded at both tibialis anterior and abductor hallucis muscles in the bilateral lower extremities were completely absent.

ASIA impairment scale
A Complete. No sensory or motor function is preserved in the sacral segments S4-S5.

B
Incomplete. Sensory but not motor function is preserved below the neurologic level and includes the sacral segments S4-S5.
C Incomplete. Motor function is preserved below the neurologic level, and more than half of the key muscles below the neurologic level have a muscle grade less than 3.

D
Incomplete. Motor function is preserved below the neurologic level, and at least half of key muscles below the neurologic level have a muscle grade greater than or equal to 3. The preoperative SSEPs and MEPs of participant SCF006 were similar to those of the other seven participants (Figures 4 and 5).
Although postoperative SSEPs of bilateral posterior tibial nerve did not show significant recovery compared with preoperative SSEPs in participant SCF006, the restoration of MEPs recorded at left tibialis anterior was noted at 1 month after the surgery (the right MEPs did not recover) (Figures 4 and 5).

| Neuroimaging assessment
Preoperatively, T2-weighted MRI scans from all participants showed markedly abnormal signal intensity in the SCI area ( Figure 6A1,C1,E1). In addition, DTI showed no continuity of nerve fibers in the proximal and distal spinal cords of these participants ( Figure 6A2,C2,E2,E3).
MRI and DTI were repeated on all participants after the surgery.
T2-weighted MRI scans of all participants showed that the vascularized transplanted spinal cord could be observed at the original SCI site ( Figure 6B1,D1,F1). In addition, the DTI of three participants also showed important positive results, including the fibers tracked at the operating site and the overlapped three bundles of nerve fibers above and below the operating site ( Figure 6B2,D2,F2). The fibers were tracked at two selected planes above and below the operating site to show the fibers that crossed two transectional sites (arrows in Figure 6B3,D3,F3). The DTI of participants SCF002, SCF005, and SCF008 showed reconnection of some nerve fibers, restoring some neural continuity of the spinal cord ( Figure 6B3,D3,F3).

| Neurologic assessment
Six participants resumed sweating below the single neurologic level. Three participants had improved bladder and/or bowel functions. The participants could feel the fullness of their bladder and/or bowel which they were unable to do preoperatively, and stool character was improved from dry and hard to wet and soft, which they were unable to do preoperatively. One participant reexperienced the dysmenorrhea symptoms during menstruation she had lost after the SCI ( Table 3). More importantly, participant SCF008 was able to autonomously flex and extend the ankle and toe joints of both lower extremities at 1 month postoperatively and maintained this degree of motor function 6 months after the surgery (online Video S1). The ISCNSCI lower extremity motor scores in participant SCF008 were restored from 0 to 16 (normal is 50). The ASIA grade of the participant was improved from A to C ( Table 4).  Table 5, Figure 7). The difference between the VAS score before and 6 months after the surgery was statistically significant (p < 0.05; Figure 7).

| DISCUSS ION
It is well known that glial scar formation is an important part of the pathophysiological mechanism of SCI. In chronic SCI, glial scarring creates a physical and chemical barrier to axon regeneration in the injured area. 26  their fibers immediately and re-establish contacts between the apposed interfaces. 5,29 In the central nervous system, DTI can only trace and image white matter fibers. 19  In addition to complete loss of motor/sensory function below the single neurologic level, many patients, perhaps most with SCI have some degree of autonomic dysfunction. In this clinical trial, three of the participants treated with vSCT had improved bladder and/ or bowel function. In some, the skin below the single neurologic F I G U R E 7 Evaluation of cord central pain in participants with paraplegia before, 1 month, and 6 months after the surgery (visual analog scale, VAS). The VAS in participant SCF004 at 1 month after the surgery was increased compared with that before the surgery, while the VAS decreased at 6 months after the surgery. The VAS of four participants (SCF002, SCF006, SCF007, and SCF008) after the surgery were gradually decreased compared with that before the surgery. The remaining participants were unchanged (1-8 on the horizontal axis represents SCF001-SCF008, see in A). The difference between VAS before and 6 months after the surgery was statistically significant ( Based on the results of this clinical trial, we plan to try to optimize the experimental protocol before expansion of the clinical trial. We will optimize the fusogen (PEG) and apply electrical stimulation to improve surgical outcomes. 35,36 In addition, mental imagery (MI) therapy will be added to our postoperative rehabilitation program.
MI refers to the active process by which humans relive the sensations with or without external stimuli 37 ; moreover, MI as a rehabilitation method can improve cord central pain in SCI. 38 Also, we will review the operative technique to reduce surgical injury, thereby minimizing postoperative scarring. Finally, we propose allograft spinal cord transplantation as another potential treatment for SCI. 39 Theoretically, the number of nerve fibers and propriospinal neurons in an allograft of spinal cord is about twice that of vSCT. Therefore, there will be more nerve fibers to potentially reconnect, thereby leading to a more optimal, faster, and better postoperative recovery of neurologic function.

| CON CLUS ION
In summary, we demonstrated the clinical feasibility and safety of vSCT through a prior preclinical trial in beagles and a clinical trial in eight paraplegic participants. PEG treatment appears to be able to fuse the axon stumps and restore the continuity of some nerve fibers during vSCT. vSCT could provide the basis of anatomy, morphology, and histology to potentially restore the motor and sensory functions of appropriately selected patients with paraplegia. vSCT also appears able to ameliorate much of the cord central pain and improve the autonomic nervous function. In addition, we have been evaluating allograft spinal cord transplantation as another option during the SCF to treat paraplegic patients. 39

ACK N OWLED G M ENTS
We thank Dr. Michael Sarr and LetPub (www.letpub.com) for linguistic assistance and pre-submission expert review.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.