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Restoration of motor function after operative reconstruction of the acutely transected spinal cord in the canine model

  • Author Footnotes
    † These authors contributed equally.
    Zehan Liu
    Footnotes
    † These authors contributed equally.
    Affiliations
    Hand and Microsurgery Center, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China

    State-Province Key Laboratories of Biomedicine-Pharmaceutics, Harbin Medical University, Nangang District, Harbin, China

    Heilongjiang Medical Science Institute, Harbin Medical University, Harbin, China
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  • Author Footnotes
    † These authors contributed equally.
    Shuai Ren
    Footnotes
    † These authors contributed equally.
    Affiliations
    Hand and Microsurgery Center, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China

    State-Province Key Laboratories of Biomedicine-Pharmaceutics, Harbin Medical University, Nangang District, Harbin, China

    Heilongjiang Medical Science Institute, Harbin Medical University, Harbin, China
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  • Kuang Fu
    Affiliations
    Department of MR Diagnosis, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China
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  • Qiong Wu
    Affiliations
    Department of MR Diagnosis, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China
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  • Jun Wu
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China
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  • Liting Hou
    Affiliations
    Department of Anesthesia, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China
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  • Hong Pan
    Affiliations
    Department of Anesthesia, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China
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  • Linlin Sun
    Affiliations
    Department of Pharmacology, Harbin Medical University, Nangang District, Harbin, China
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  • Jian Zhang
    Affiliations
    Hand and Microsurgery Center, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China

    State-Province Key Laboratories of Biomedicine-Pharmaceutics, Harbin Medical University, Nangang District, Harbin, China

    Heilongjiang Medical Science Institute, Harbin Medical University, Harbin, China
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  • Bingjian Wang
    Affiliations
    Hand and Microsurgery Center, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China

    State-Province Key Laboratories of Biomedicine-Pharmaceutics, Harbin Medical University, Nangang District, Harbin, China

    Heilongjiang Medical Science Institute, Harbin Medical University, Harbin, China
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  • Qing Miao
    Affiliations
    Hand and Microsurgery Center, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China

    State-Province Key Laboratories of Biomedicine-Pharmaceutics, Harbin Medical University, Nangang District, Harbin, China

    Heilongjiang Medical Science Institute, Harbin Medical University, Harbin, China
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  • Guiyin Sun
    Affiliations
    Hand and Microsurgery Center, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China

    State-Province Key Laboratories of Biomedicine-Pharmaceutics, Harbin Medical University, Nangang District, Harbin, China

    Heilongjiang Medical Science Institute, Harbin Medical University, Harbin, China
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  • Vincenzo Bonicalzi
    Affiliations
    HEAVEN/GEMINI International Collaborative Group, Turin, Italy
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  • Sergio Canavero
    Affiliations
    HEAVEN/GEMINI International Collaborative Group, Turin, Italy
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  • Author Footnotes
    † These authors contributed equally.
    Xiaoping Ren
    Correspondence
    Corresponding author. Hand and Microsurgery Center, the Second Affiliated Hospital of Harbin Medical University, Xuefu Road 246, Nangang District, Harbin 150081, China. (X. Ren).
    Footnotes
    † These authors contributed equally.
    Affiliations
    Hand and Microsurgery Center, the Second Affiliated Hospital of Harbin Medical University, Nangang District, Harbin, China

    State-Province Key Laboratories of Biomedicine-Pharmaceutics, Harbin Medical University, Nangang District, Harbin, China

    Heilongjiang Medical Science Institute, Harbin Medical University, Harbin, China

    Department of Molecular Pharmacology and Therapeutics, Stritch School of Medicine, Loyola University Chicago, Chicago, IL, USA
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  • Author Footnotes
    † These authors contributed equally.
Published:December 06, 2017DOI:https://doi.org/10.1016/j.surg.2017.10.015

      Abstract

      Background

      Cephalosomatic anastomosis or what has been called a “head transplantation” requires full reconnection of the respective transected ends of the spinal cords. The GEMINI spinal cord fusion protocol has been developed for this reason. Here, we report the first randomized, controlled study of the GEMINI protocol in large animals.

      Methods

      We conducted a randomized, controlled study of a complete transection of the spinal cord at the level of T10 in dogs at Harbin Medical University, Harbin, China. These dogs were followed for up to 8 weeks postoperatively by assessments of recovery of motor function, somato-sensory evoked potentials, and diffusion tensor imaging using magnetic resonance imaging.

      Results

      A total of 12 dogs were subjected to operative exposure of the dorsal aspect of the spinal cord after laminectomy and longitudinal durotomy followed by a very sharp, controlled, full-thickness, complete transection of the spinal cord at T10. The fusogen, polyethylene glycol, was applied topically to the site of the spinal cord transection in 7 of 12 dogs; 0.9% NaCl saline was applied to the site of transection in the remaining 5 control dogs. Dogs were selected randomly to receive polyethylene glycol or saline. All polyethylene glycol-treated dogs reacquired a substantial amount of motor function versus none in controls over these first 2 months as assessed on the 20-point (0–19), canine, Basso-Beattie-Bresnahan rating scale (P<.006). Somatosensory evoked potentials confirmed restoration of electrical conduction cranially across the site of spinal cord transection which improved over time. Diffusion tensor imaging, a magnetic resonance permutation that assesses the integrity of nerve fibers and cells, showed restitution of the transected spinal cord with polyethylene glycol treatment (at-injury level difference: P<.02).

      Conclusion

      A sharply and fully transected spinal cord at the level of T10 can be reconstructed with restoration of many aspects of electrical continuity in large animals following the GEMINI spinal cord fusion protocol, with objective evidence of motor recovery and of electrical continuity across the site of transection, opening the way to the first cephalosomatic anastomosis. (Surgery 2017;160:XXX-XXX.)
      In 1986 and in many studies thereafter by this group, a novel approach to the repair of transected peripheral nerves has been studied by Bittner et al, i.e., reapposing freshly and sharply transected peripheral nerves bathed in polyethylene glycol (PEG), a substance acting as a fusogen that has the ability to literally “fuse” transected membranes of both neuronal cell bodies and nerve fibers.
      • Canavero S.
      HEAVEN: the head anastomosis venture project outline for the first human head transplantation with spinal linkage (GEMINI).
      • Ye Y.
      • Kim C.Y.
      • Miao Q.
      • Ren X.
      Fusogen-assisted rapid reconstitution of anatomophysiologic continuity of the transected spinal cord.
      • Bamba R.
      • Waitayawinyu T.
      • Nookala R.
      • et al.
      A novel therapy to promote axonal fusion in human digital nerves.
      We have been interested in using this technology to restore electrical continuity (and thus function) across a fully transected spinal cord. The GEMINI spinal cord fusion protocol was first described in 2013.
      • Canavero S.
      HEAVEN: the head anastomosis venture project outline for the first human head transplantation with spinal linkage (GEMINI).
      The protocol recognizes that sensorimotor function in mammals is subserved by a cellular “highway” embedded in the gray matter of the spinal cord and that protecting the neurons that make up this pathway at the point of operative transection would allow sprouting of new neural connections between the stumps of the transected spinal cord. PEG seems to act as a cytoprotectant by resealing the damaged membranes and simultaneously “fusing” other transected axons in the white matter.
      • Canavero S.
      HEAVEN: the head anastomosis venture project outline for the first human head transplantation with spinal linkage (GEMINI).
      • Canavero S.
      • Ren X.
      • Kim C.Y.
      • Rosati E.
      Neurologic foundations of spinal cord fusion (GEMINI).
      In 2016, proof-of-principle animal studies confirmed the feasibility of fusing a sharply transected, cervical spinal cord with objective restoration of electrical transmission and recovery of ambulation, first in mice,
      • Ye Y.
      • Kim C.Y.
      • Miao Q.
      • Ren X.
      Fusogen-assisted rapid reconstitution of anatomophysiologic continuity of the transected spinal cord.
      • Kim C.Y.
      PEG-assisted reconstruction of the cervical spinal cord in rats: effects on motor conduction at 1 h.
      • Kim C.Y.
      • Oh H.
      • Hwang I.K.
      • Hong K.S.
      GEMINI: initial behavioral results after full severance of the cervical spinal cord in mice.
      then in rats,
      • Kim C.Y.
      • Sikkema W.K.
      • Hwang I.K.
      • et al.
      Spinal cord fusion with PEG-GNRs (TexasPEG): neurophysiological recovery in 24 hours in rats.
      and finally in a single dog.
      • Kim C.Y.
      • Hwang I.K.
      • Kim H.
      • Jang S.W.
      • Kim H.S.
      • Lee W.Y.
      Accelerated recovery of sensorimotor function in a dog submitted to quasi-total transection of the cervical spinal cord and treated with PEG.
      Preliminary histologic analysis revealed that the 2 approximated stumps of the transected spinal cord regrew nerve fibers that reestablished the gray matter sensorimotor highway.
      • Kim C.Y.
      • Ren X.
      • Canavero S.
      Immunohistochemical evidence of axonal regrowth across polyethylene glycol-fused cervical cords in mice.
      An adequately powered, randomized, controlled study in rats confirmed these initial findings.
      • Ren S.
      • Liu Z.H.
      • Wu Q.
      • et al.
      Polyethylene glycol-induced motor recovery after total spinal transection in rats.
      Before advancing with possible clinical application in humans, we think that it was necessary to replicate the findings in large animals. Here, our aim was to provide robust, objective evidence in a scientifically appropriate number of dogs that the GEMINI spinal cord fusion protocol can restore motor function after a sharp, complete spinal cord transection at the level of T10 with immediate PEG treatment. Objective evidence of rapid recovery of electrical continuity was supported both by partial recovery from paralysis and by neurophysiologic assessment of cranial electrical transmission of distal sensory input by somatosensory evoked potentials (SSEP) across the site of spinal cord transection as well as evidence on diffusion tensor imaging (DTI) of magnetic resonance imaging of spinal cord restitution of neural continuity.

      Methods

      Animals

      All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Harbin Medical University (HMUIRB-2008-06) and the Institute of Laboratory Animal Science of China (A5655-01) and were in accordance with Directive 2010/63/EU of the European Parliament. Female beagles (8 kg) were used in this study because of their docility and greater resistance to urinary tract infections. Animals obtained from Qingdao Agricultural University (Qingdao, Shandong Province, PRC) were housed comfortably with a light/dark cycle of 12 h/12 h and fed ad libitum. The experimental group (PEG-treated) consisted of 7 experimental animals and 5 controls treated with only 0.9% NaCl. The initial goal of this study as presented here was to assess the recovery of motor function after a sharp, complete transection of the spinal cord in the presence of PEG treatment as demonstrated in rodents previously.
      • Ye Y.
      • Kim C.Y.
      • Miao Q.
      • Ren X.
      Fusogen-assisted rapid reconstitution of anatomophysiologic continuity of the transected spinal cord.
      • Kim C.Y.
      PEG-assisted reconstruction of the cervical spinal cord in rats: effects on motor conduction at 1 h.
      • Kim C.Y.
      • Oh H.
      • Hwang I.K.
      • Hong K.S.
      GEMINI: initial behavioral results after full severance of the cervical spinal cord in mice.
      • Kim C.Y.
      • Sikkema W.K.
      • Hwang I.K.
      • et al.
      Spinal cord fusion with PEG-GNRs (TexasPEG): neurophysiological recovery in 24 hours in rats.
      The ultimate goal of our experimental program will be to assess outcomes over the life span of treated animals, including the recovery of both motor and sensory and autonomic function, and importantly the question of possible onset of central pain (a form of neuropathic pain that may follow spinal injury
      • Canavero S.
      • Bonicalzi V.
      Central Pain Syndrome.
      ) that would compromise a human CSA. In accordance with the ethical international guidelines above, our experimental protocol assured that should any dog displaying any severe neuropathic (but not nociceptive) pain syndrome or uncontrollable, recurrent infections would have been euthanized.

      Operation of spinal cord transection

      After establishing intravenous (IV) access, general anesthesia was induced by ketamine (0.1 mL/kg given intramuscularly) followed by intubation and artificial ventilation. Propofol (10 mg/kg ⋅ h), remifentanil (0.2 µg/kg ⋅ min), and vecuronium bromide (0.1 mg/kg) were administered intravenously for maintenance. Animals were kept in a prone position during the operation. The skin and muscles overlying the thoracic spinal column were incised, and a laminectomy was performed at T10 with standard neurosurgical instrumentation. With the assistance of a surgical microscope, the dura mater was opened, and the spinal cord was lifted gently using a piece of Kirchner wire molded into a hook. The spinal cord was then transected with an ultrasharp microsurgical blade (Sharpoint, Surgical Specialties, Mexico). Prior to transection, cold 0.9% NaCl at 4°C containing 0.1% epinephrine was applied to the transection site to induce vasoconstriction and decrease intraoperative bleeding. Immediately thereafter, animals were randomized according to computer-generated random numbers to receive either 0.9% NaCl (2 mL) or PEG-600 (2 mL) applied topically to the site of spinal cord transection via a syringe and left in situ at the point of transection. Standard closure by layers followed.
      An IV solution containing Cefoperazone Sodium and Sulbactam Sodium (25 mg/kg; Harbin General Pharmaceutical Factory's Sales Company, Harbin, Heilongjiang, PRC) was administered to all dogs for 3 consecutive days. Urine from the bladder was expressed using abdominopelvic compression twice a day until the voiding reflex was restored or, in the absence of recovery, throughout the period of observation postoperatively. Animals were fed dog food (Bridge PeCare Co., Ltd, Shanghai, China, PRC), along with free access to water, or if necessary because of inability to eat or drink, intravenous hydration with electrolyte solutions was provided for the first 2 postoperative weeks, after which a normal diet was instituted. The dogs were inspected for signs infection, dehydration, and other potential complications related to the operation according to the criteria of the Appropriate Care and Use of Laboratory Animals.
      • Garber J.C.
      • Barbee R.W.
      • Bielitzki J.T.
      • et al.
      Guide for the Care and Use of Laboratory Animals.
      Although the lower extremities were paralyzed, the upper extremities were functional, so the dogs could move albeit with some difficulty. Hip massage and lower limb rehabilitation using range of motion exercise were carried out 4 times a day. Pressure sores and skin irritation were minimized or prevented by supporting the animals in a custom-made wheelchair until they regained useful voluntary motor function. All 12 animals survived the operation and were able to complete the study without obvious undue subjective discomfort.

      Motor assessment

      Open-field locomotion, coordination, trunk position, and stability were evaluated by 2, trained, blinded examiners for at least 5 minutes at each time point (see below for the schedule) and scored using the 20-point (0–19) canine Basso-Beattie-Bresnahan rating scale (a Canine locomotor rating scale [cBBB] score of 0 means paraplegia; a cBBB score of 19 means normal function; Table).
      • Song R.B.
      • Basso D.M.
      • da Costa R.C.
      • Fisher L.C.
      • Mo X.
      • Moore S.A.
      Adaptation of the Basso-Beattie-Bresnahan locomotor rating scale for use in a clinical model of spinal cord injury in dogs.
      Scoring sessions occurred at 3, 10, 17, 24, 31, 38, 45, 52, 59 days postoperatively. The Mann-Whitney U test was used to compare outcome measurements between experimental and control animals.
      TableCanine locomotor rating scale
      cBBBScore description
      0=No observable HL movement
      1=Slight movement of 1 or 2 joints
      2=Extensive movement of 1 joint, or extensive movement of 1 joint and slight movement of 1 other joint
      3=Extensive movement of 2 joints
      4=Slight movement of all 3 joints of the HL
      5=Slight movement of 2 joints and extensive movement of the third
      6=Extensive movement of 2 joints and slight movement of the third
      7=Extensive movement of all 3 joints in the HL
      8=Plantar placement of the paw with no weight support
      9=Plantar placement of the paw with weight support only when stationary, or occasional, frequent or consistent weight-supported dorsal stepping and no plantar stepping
      10=Occasional weight-supported plantar steps; no FL-HL coordination
      11=Frequent to consistent weight-supported plantar steps and no FL-HL coordination
      12=Frequent to consistent weight-supported plantar steps and occasional FL-HL coordination
      13=Frequent to consistent weight-supported plantar steps and frequent FL-HL coordination
      14=Consistent weight-supported plantar steps, consistent FL-HL coordination, and predominant paw position is externally rotated when it makes initial contact as well as just before it is lifted off; or frequent plantar stepping, consistent FL-HL coordination, and occasional dorsal stepping
      15=Consistent plantar stepping and consistent FL-HL coordination and no toe clearance or occasional toe clearance; predominant paw position is parallel to the body or internally rotated at initial contact
      16=Consistent plantar stepping and consistent FL-HL coordination and toe clearance occurs frequently; predominant paw position is parallel or internally rotated at initial contact and externally rotated at liftoff
      17=Consistent plantar stepping and consistent FL-HL coordination and toe clearance occurs frequently; predominant paw position is parallel or internal at initial contact and at liftoff
      18=Consistent plantar stepping and consistent FL-HL coordination and toe clearance occurs consistently; predominant paw position is parallel or internal at initial contact and at liftoff. Trunk instability is present
      19=Consistent plantar stepping and consistent FL-HL coordination and toe clearance occurs consistently during forward limb advancement; predominant paw position is parallel or internal at initial contact and at liftoff. Trunk instability is not observed
      FL, forelimb; HL, hindlimb. From Song RB, Basso DM, da Costa RC, Fisher LC, Mo X, Moore SA. Adaptation of the Basso-Beattie-Bresnahan locomotor rating scale for use in a clinical model of spinal cord injury in dogs. J Neurosci Methods 2016;268:117–24.
      • Song R.B.
      • Basso D.M.
      • da Costa R.C.
      • Fisher L.C.
      • Mo X.
      • Moore S.A.
      Adaptation of the Basso-Beattie-Bresnahan locomotor rating scale for use in a clinical model of spinal cord injury in dogs.

      In vivo electrophysiology

      Cortical somatosensory evoked potentials (SSEP) in response to electrical stimulation of the sciatic nerve (a sensory site distal to the area supplied by the spinal cord proximal to the site of spinal cord transection) were recorded from the scalp overlying the sensory cortex using standard apparatus (AD Instruments, Bellavista, Australia) with the following parameters: 30 mA, 200 ms, 0.25 Hz stimuli). Results were analyzed with LabChart software (AD Instruments). Data were acquired before, during, and at 2 months after operation. Postoperative recordings were performed under sedation (Xylazine 0.1 mL/kg intramuscularly; Qingdao Hanhe Animal and Plant Pharmaceutical Co. Ltd., Qingdao, PRC).

      Imaging studies

      All animals were subjected to MRI and DTI using a 3.0 T MRI system (Achieva 3, Philips, Amsterdam, The Netherlands) in the prone position. Sagittal, T2-weighted, fast spin-echo (TR = 1,700 ms; TE = 100 ms; slice thickness = 3 mm; slice gap = 0.1; NSA = 4) and axial single-shot echo-planar DTI (TR = 6,100 ms; TE = 93 ms; voxel size = 2 mm x 2 mm; slice thickness = 2 mm; slice gap = 0; NSA = 2; diffusion direction number = 15) sequences were acquired twice at 2 and 4 weeks postoperatively in all animals.

      Results

      Behavioral assessment

      All animals enrolled in the study including all 7 experimental and 5 control animals survived the operation. Sphincter control of the urinary bladder did not display sufficient recovery in either the experimental or control animals at 2 months after the spinal cord transection and required continued assistance. Defecation needed twice daily massage. Despite intense attempts to prevent pressure sores, some pressure sores were observed in both groups; these pressure sores cleared after 2 weeks of standard care in the PEG-treated group as this group recovered voluntary muscle activity. Control animals continued to experience pressure sores even with standard care; none of these pressure sores were serious enough to warrant shortening the study period according to our strict criteria for discontinuing the study duration in any animal (obvious discomfort, severe infection). Because the study is still in progress, we are reporting findings from the first 2 months' observation after the operative full transection of the thoracic spinal cord. This initial report focuses primarily on the motor function recovery at 2 months.
      Prior to the spinal cord transection, all animals had normal cBBB scores (score: 19) as determined using the methods describe earlier. As expected, complete paraplegia (score: 0) was observed in all animals immediately after the transection of the thoracic spinal cord. All PEG-treated animals showed the first signs of recovery within 3 days: both hind limbs would twitch in response to light pinching of the abdominal wall. No sign of recovery was observed in any control animals for the duration of the experiment (2 months). In contrast, in the PEG- treated group, recovery was steady without plateaus. The progress of recovery is shown in Fig 1 for each dog. At two months, one dog scored 18, one 15, one 9, one 8, two 7 and one 5. These measurements show that no experimental animal was completely paralyzed, all had reacquired movements in the lower extremities with 3 dogs able support their weight to stand and 2 of these dogs being able to ambulate almost normally (Fig 1, A see online video).
      Fig. 1
      Fig. 1(A) An illustrative case: paraplegia is seen in the first 3 days (A1) with motor recovery of the hips at 1 week (A2), knees at about 3 weeks (A3), ankles at about 5 weeks (A4), and paws at about 7 weeks (CA5/6); independent gait is attained eventually at the study end. (B) Mann-Whitney U test at all time points of follow-up (–1/59 days) and functional evaluation of cBBB score system at the indicated time points. Animals in the PEG treatment group had a median score of 8 (range: 5–18) versus 3 (range: 2–4) in controls at 2 months (Mann-Whitney U test; P < .0058). CTL, controls.
      Results of the cBBB quantitative evaluation of motor activity were statistically significant at all time points compared to the control animals (Fig 1, B). In particular, animals in the PEG treatment group had a median score of 8 (range: 5–18) versus 3 (range: 2–4) in controls at 2 months (Mann-Whitney U test; P < .006).

      Neurophysiologic assessment

      SSEPs to assess electrophysiologic conduction in the posterior columns of the cord across the site of spinal cord transection showed that recovery of the electrical signal across the site of spinal cord transection was evident even before closure of the dura mater at the end of operation in all animals with PEG topical application (Fig. 2, Fig. 3); no such recovery was evident in the control dogs. By 2 months, almost normal waves were seen in all the PEG-treated animals compared to the control animals in which no evoked waves in response to peripheral electrical stimulation were evident (Fig 2).
      Fig. 2
      Fig. 2SSEP: changes in the waveform of PEG-treated and control groups before spinal cord transection, immediately after transection, and 2 months postoperatively. Traces from 2 representative animals (PEG and control) taken at different time-points have been assembled into one single tracing for maximal explanatory power. Upper traces: normal waveform before transection (1); low-amplitude wave in PEG-treated animals immediately after transection with PEG application (2); quasi-normal waveform prior to closure (3); waveform at 2 months postoperatively (4). Lower traces: normal waveform prior to transection in controls (5); total absence of any waves after transection and application of the control solution of the 0.9 NaCl (6) and irregular, unpatterned waves at 2 months (7) (vertical calibration bar: 400 µV; horizontal calibration bar: 20 ms).

      Neuroimaging assessment

      In the neuroimaging study, T2-weighted MRI scans showed normal or near normal signal intensity in PEG-animals at the site of transection (Fig 3, A and B). Because the standard T2-WI scan could not reveal the fine structures of the nervous system, it was uninformative regarding the nature of the fusion process. In contrast, DTI, which measures the integrity of nerve fiber tracts, was able to show fiber regrowth across the plane of transection in the PEG-treated group versus none in the control group (Fig 3, C and E). In general, the extent of fiber regrowth correlated well with motor recovery; the animals with the best level of recovery had the most intense regrowth. In the PEG-treated group, the mean value of fractional anisotropy (FA) at the level of injury, a measure of myelination) was different from controls (P<.02; Fig 4). Also, the mean value of the apparent diffusion coefficient (ADC; also called mean diffusivity, MD) distal to the site of spinal cord transection (a measure of cellularity and organization of nerve fibers) also differed from controls (P<.02; Fig 4).
      Fig. 3
      Fig. 3MRI results at 1 month postoperatively in both controls and PEG-treated animals. T2W images of control animals showing the plane of transection at T10 (A), which is not seen in any of the PEG-treated animals (B). DTI (C and D): Complete fiber transection with no spinal reconstitution in the control group. (E and F) Reestablishment of anatomic continuity in the PEG-treated group. (C and E) Fiber tracking from both ends to the transectional area. (D and F) Fiber tracking in the 2 selected planes in the above and below injury site to find fibers that cross the transectional area, some of fibers reconstitute in the PEG-treated group (F), but none in the control group (D). The color of the fibers were designed to distinguish between the 2 groups without any practical significance. (The arrow flags the point of transection and PEG application.).
      Fig. 4
      Fig. 4Datametrics for DTI (FA, ADC, also called MD). The FA measures the degree of myelination, the MD/ADC is more sensitive to cellularity. Myelination points to reconstitution of long-range fibers, because DTI cannot yet reliably assess the contribution of myelination in the process of re-sprouting of axons in the gray matter. Damage to the axonal membrane is mirrored by diffusion at that level becoming unrestricted and isotropic. In intact neurons, the FA value is closer to 1 suggesting more intact spinal nerves, while in injured neurons, the value is closer to 0. A low value for MD/ADC indicates that the imaged structure (e.g., nerve fibers) is organized, whereas a high value indicates disorganization within the fiber tracts. A lesser FA and a greater MD/ADC is expected at the level of the spinal cord transection (injury level) in the control animals and a greater FA and lesser MD/ADC in the reconstituted spinal cords of the PEG-treated animals. In this study, the FA (but not the MD/ADC values at the level of injury) were statistically different between PEG-treated and saline-treated animals; also, the MD/ADC values distal to the site of level of spinal cord transection were statistically different between groups.

      Discussion

      In this study, we confirmed that an acutely and sharply transected thoracic spinal cord in a large animal (canine) model can be “fused” in the presence of a fusogen according to the rationale and principles of the GEMINI spinal cord fusion protocol with remarkable early functional recovery of motor function distal to the site of transection; moreover, somatosensory evoked potentials are transmitted proximally (centrally) across the site of transection. Results from these observations demonstrate restoration of electrical continuity across the site of spinal cord transection within the first 2 months after spinal cord transection and replicate our previous studies in rodents
      • Ren S.
      • Liu Z.H.
      • Wu Q.
      • et al.
      Polyethylene glycol-induced motor recovery after total spinal transection in rats.
      with the notable exception in the timeline of recovery, which differs according to the animal studied: about 1 week for mice, 2 weeks for rats, and 8 weeks for dogs.
      As described in earlier reports, one of the keys to successful spinal cord fusion is an extremely sharp transection of the spinal cord that results in minimal local damage to the gray and white matter. A typical force generated by creating this type of a sharp transection is less than 10 N versus ~26,000 N experienced during spinal cord injury (SCI), a 2600x difference.
      • Sledge J.
      • Graham W.A.
      • Westmoreland S.
      • et al.
      Spinal cord injury models in non human primates: are lesions created by sharp instruments relevant to human injuries?.
      The sensory and motor recovery is supported by unequivocal, objective, electrophysiologic recordings and advanced magnetic resonance imaging scans. While it may be difficult to understand the findings based on our classic knowledge of spinal cord regeneration and repair, it may be explained by considering how the spinal cord processes motor information. Roughly about 20 million nerve fibers course through the entire spinal cord, with approximately 1 million descending pyramidal fibers.
      • Canavero S.
      • Ren X.
      • Kim C.Y.
      • Rosati E.
      Neurologic foundations of spinal cord fusion (GEMINI).
      • Canavero S.
      The “Gemini” spinal cord fusion protocol: reloaded.
      It is thought generally that the long-range corticospinal (pyramidal) fibers are mainly responsible for activating the spinal motorneurons and triggering a motor response.
      • Canavero S.
      • Ren X.
      • Kim C.Y.
      • Rosati E.
      Neurologic foundations of spinal cord fusion (GEMINI).
      • Canavero S.
      The “Gemini” spinal cord fusion protocol: reloaded.
      Data obtained from previous studies in human and animals proved that maintenance of 5–20% of these fibers are sufficient for satisfactory motor function to occur.
      • Canavero S.
      • Ren X.
      • Kim C.Y.
      • Rosati E.
      Neurologic foundations of spinal cord fusion (GEMINI).
      • Canavero S.
      The “Gemini” spinal cord fusion protocol: reloaded.
      Most importantly, this recovery is made possible largely because of the existence of a parallel cellular pathway, a gray matter-based network of interneurons extending from the brainstem to the spinal cord. In addition to the long-range fibers, this network of interneurons may simultaneously convey command signals from cortical motor areas to peripheral motoneurons.
      • Canavero S.
      • Ren X.
      • Kim C.Y.
      • Rosati E.
      Neurologic foundations of spinal cord fusion (GEMINI).
      • Canavero S.
      The “Gemini” spinal cord fusion protocol: reloaded.
      This short-fiber, cortico-truncoreticulo-propriospinal pathway embeds and links the central pattern generators located in the cervical and lumbar cord.
      • Canavero S.
      • Ren X.
      • Kim C.Y.
      • Rosati E.
      Neurologic foundations of spinal cord fusion (GEMINI).
      • Canavero S.
      The “Gemini” spinal cord fusion protocol: reloaded.
      Such anatomic configuration forms the basis of the action of PEG. The minimally disruptive (nanometers) transection of the spinal cord caused damage to a very thin layer of these interneurons whose cell membranes may have been “resealed” acutely by the topically applied PEG that seems to act as a cell membrane protectant preventing cell death.
      • Canavero S.
      HEAVEN: the head anastomosis venture project outline for the first human head transplantation with spinal linkage (GEMINI).
      • Ye Y.
      • Kim C.Y.
      • Miao Q.
      • Ren X.
      Fusogen-assisted rapid reconstitution of anatomophysiologic continuity of the transected spinal cord.
      These same interneurons along with others in proximity that were not damaged by the extra-sharp blade can regrow (sprout) their fibers immediately and reestablish contacts between the apposed interfaces. The gray matter neuropil is restored subsequently by spontaneous regrowth of the severed axons/dendrites over very short distances at the point of contact between the apposed cords. This phenomenon was demonstrated recently by an immunohistochemical study of PEG-treated mice subjected to complete transection of the cervical spinal cord.
      • Kim C.Y.
      • Ren X.
      • Canavero S.
      Immunohistochemical evidence of axonal regrowth across polyethylene glycol-fused cervical cords in mice.
      In this same context, even in multiple sclerosis which has long been regarded as the prototypic white matter (long axons) disease, it is the damage to the spinal gray matter (i.e., to the TRPS) that accounts for most of the related motor disability, even in cases without white matter loss.
      • Schlaeger R.
      • Papinutto N.
      • Panara V.
      Spinal cord gray matter atrophy correlates with multiple sclerosis disability.
      • Kearney H.
      • Schneider T.
      • Yiannakas M.C.
      • et al.
      Spinal cord grey matter abnormalities are associated with secondary progression and physical disability in multiple sclerosis.
      The behavioral (motor) recovery in these animals seems to be consistent with the regrowth pattern of these short range fibers.
      • Canavero S.
      • Ren X.
      • Kim C.Y.
      • Rosati E.
      Neurologic foundations of spinal cord fusion (GEMINI).
      • Canavero S.
      The “Gemini” spinal cord fusion protocol: reloaded.
      Also, given the excellent and rapid recovery of motor function, the sprouting process in the TRPS does not appear to be haphazard.
      In contrast, direct fusion of white matter fibers is also highly likely. As shown by near normal conduction of SSEPs observed intraoperatively actually before the end of the operative procedure to transect the thoracic spinal cord (Fig. 2, Fig. 3), PEG was able to allow acute “fusion” of an unknown number of transected nerve fibers of the white matter. While no technology exists to fuse the 20 million fibers that make up the entire white matter of the spinal cord after a complete transection, our experiments show clearly that a certain number are fused apparently “blindly.” More specifically, as shown by the electrophysiology, quasi-normal sensory input (SSEP) reaches the cortex across the point of transection; there are no other extraspinal pathways to explain this electrical transmission. The dorsal columns of the spinal cord are composed of large, densely packed, well-demarcated fiber bundles, apparently making it easier for fibers to come in contact by simple apposition of the transected spinal cord for PEG to have its “fusogenic” effects to allow acute neural fusion. In contrast, the pyramidal fibers are fewer and, importantly, more interspersed with other nearby bundles
      • Nathan P.W.
      • Smith M.
      • Deacon P.
      Vestibulospinal, reticulospinal and descending propriospinal nerve fibres in man.
      and thus may fuse with axons of the other pathways.
      In the present study, we did not observe a complete recovery of sphincter control up to 8 postoperative weeks in any of the animals, a finding similar to an early report.
      • Kim C.Y.
      • Hwang I.K.
      • Kim H.
      • Jang S.W.
      • Kim H.S.
      • Lee W.Y.
      Accelerated recovery of sensorimotor function in a dog submitted to quasi-total transection of the cervical spinal cord and treated with PEG.
      Because motor control of micturition and ejaculation is subserved by a direct link between the cortex and Onuf's nucleus (located in the sacral anterior horn via descending, long-range pyramidal fibers), absence of recovery most likely signifies absence of fusion of related fibers.
      The possibility of regrowth of these long fibers resulting in motor recovery is further excluded by considering the rate of axonal regrowth which is typically about 0.5–1 mm/day. Certain signs of motor recovery were already visible in treated animals at 2–3 postoperative days, too short a time for the transected pyramidal axons to regrow and reach their respective motoneurons. Besides, recovery of sensory electrophysiologic transmission (SSEPs) is immediate, whereas motor recovery takes a few days to weeks to manifest evidence of electrophysiologic innervation.
      It seems that motor recovery hinges entirely on a gray matter mechanism, while sensory transmission in the context of the present experiment also exploits normal lemniscal transmission. The problem of misalignment of neural regrowth or fusion does not seem to be of consequence; dorsal column sensory fibers will not “fuse” with lateral quadrant motor fibers. Circuital readjustment, so called adaptation, in the spinal cord and within upper CNS stations, is a well-appreciated neural response and seems sufficient to offset this problem.
      • Canavero S.
      • Ren X.
      • Kim C.Y.
      • Rosati E.
      Neurologic foundations of spinal cord fusion (GEMINI).
      • Canavero S.
      The “Gemini” spinal cord fusion protocol: reloaded.
      DTI is a novel, “histologic-grade,” MR imaging technique that assesses the integrity of nerve fiber tracts based on the simple principle of diffusion of water molecules. DTI is currently the only means of noninvasive, in vivo assessment the microstructural integrity of the white matter fibers and related changes.
      • Ries M.
      • Jones R.A.
      • Dousset V.
      • Moonen C.T.
      Diffusion tensor MRI of the spinal cord.
      The changes in diffusion on DTI can be assessed either qualitatively by “fiber tracking techniques” (tractography)
      • Ries M.
      • Jones R.A.
      • Dousset V.
      • Moonen C.T.
      Diffusion tensor MRI of the spinal cord.
      or quantitatively by calculating DTI anisotropy indices (datametrics).
      • Canavero S.
      The “Gemini” spinal cord fusion protocol: reloaded.
      Qualitative spinal tractography can show the macroscopic orientation of fibers with dramatic representation of disruption of the tracts which, however, is seen only poorly on plain MRI. The indices of datametrics quantify any observed changes, the 2 most common being fractional anisotropy (FA) and mean diffusivity (MD; often called the apparent diffusion coefficient or ADC).
      In our study, both MR and DT tracking showed clearly a complete transection of the spinal cord on the initial postoperative assessment, but later, clear disappearance of the plane of transection at 2 and even more strongly at 4 weeks postoperatively in the experimental group (Fig 3). Datametrics clearly indicated significant differences in PEG-treated and control animals, supportive of renormalization of the cord tissue (Table 1). Our results are in line with published evidence of clinical DTI studies,
      • D'souza M.M.
      • Choudhary A.
      • Poonia M.
      • Kumar P.
      • Khushu S.
      Diffusion tensor MR imaging in spinal cord injury.
      • Song T.
      • Chen W.J.
      • Yang B.
      • et al.
      Diffusion tensor imaging in the cervical spinal cord.
      which noticed differences in FA but not in MD/ACD values in the spinal cord of normal controls and patients with injured or compressed spinal cords. This is similar to our findings.
      Another important concern is potential of scarring after the spinal cord transection. In the current study, PEG is applied immediately after transecting the spinal cord; because of the relatively atraumatic method of transection, no substantive astrocytic scar occurs to hinder the process as would occur with a blunt, traumatic injuries. There is evidence that the astrocytic scar may actually promote axon regrowth in the early stages of SCI. Scar tissue slowly becomes nonpermissive only after the subacute stage,
      • Raposo C.
      • Schwartz M.
      Glial scar and immune cell involvement in tissue remodeling and repair following acute CNS injuries.
      • Anderson M.A.
      • Burda J.E.
      • Ren Y.
      • et al.
      Astrocyte scar formation aids central nervous system axon regeneration.
      by which time the nerve fibers would have already crossed the fusion interface and bridged the 2 stumps. As reported in our previous study,
      • Kim C.Y.
      • Ren X.
      • Canavero S.
      Immunohistochemical evidence of axonal regrowth across polyethylene glycol-fused cervical cords in mice.
      PEG does not prevent the formation of a scar and thus does not deprive the regrowth process of the beneficial effects of the early scar.
      It is worth mentioning that in the present model, some minimal retraction of the proximal and distal stumps of the transected spinal cord was seen after the spinal cord transection. Despite this retraction, PEG was able to form a bridge once in contact with the tissue. This phenomenon was first demonstrated by a German study
      • Estrada V.
      • Müller H.W.
      Bridging large gaps in the injured spinal cord: mechanical and biochemical tissue adaptation.
      • Estrada V.
      • Brazda N.
      • Schmitz C.
      • et al.
      Long-lasting significant functional improvement in chronic severe spinalcord injury following scar resection and polyethylene glycol implantation.
      in which a segment of the spinal cord in rats was removed and filled with PEG that acted as a conduit for the regrowing nerve fibers. Rats used in the study eventually regained motor function, but the time of recovery pointed at regrowth rather than fusion of the axons.
      • Estrada V.
      • Müller H.W.
      Bridging large gaps in the injured spinal cord: mechanical and biochemical tissue adaptation.
      • Estrada V.
      • Brazda N.
      • Schmitz C.
      • et al.
      Long-lasting significant functional improvement in chronic severe spinalcord injury following scar resection and polyethylene glycol implantation.
      Thus, even if approximation of the stumps is not optimal, the minimal gap filled by PEG seems to be able to act as a conduit for regrowth but possibly not to allow fusion; thus the stable reapproximation of the stumps is potentially important to allow a more rapid restoration of electrical continuity across the site of spinal cord transection. This observation may also explain why all treated animals recovered, with some with better outcomes: reapproximation may have played a role. In the context of a CSA, alignment will be total without any gap, which is expected to lead to even better outcomes.
      Finally, there has been concern raised by several investigators about the possibility of the development of a neuropathic pain syndrome in patients undergoing spinal cord regeneration/restoration.
      • Canavero S.
      • Bonicalzi V.
      Central Pain Syndrome.
      We did not observe any signs of neuropathic pain in any of the dogs (e.g., chewing of the affected area or autotomy, abnormal crying out) by the end of 8 weeks and nociceptive stimuli (with a pin prick stimulus) did not evoke markedly abnormal behavior suggestive of severe pain.
      In summary, we have shown that permanent paralysis after a complete sharp transection of the thoracic spinal cord can be prevented in part by immediate, topical, intraoperative application of a fusogen solution (PEG) in the region of the spinal cord transection. By transecting the spinal cord with a very well-controlled, ultrasharp method of transection without inflicting gross damage, fibers in the gray matter cellular core may sprout rapidly before a scar forms and reconstitute the cellular “bridge” that conveys distal electrical transmission of central motor commands. At the same time, at least a portion of long-range sensory and motor fibers is restored to support the observed functional recovery. This technique holds promise for the concept of restoration of electrical continuity in patients with traumatic spinal cord transactions. The rationale for almost all attempts at reversing spinal injury has been to counteract the detrimental effects of mechanical disruption, along with cysts and scars, by either allaying local inhibition or fostering regrowth of long axons in the damaged white matter. All these studies led to no clinically available treatment. A few surgical groups, however, removed the injured segment in paraplegics and filled the gap with collagen or peripheral nerve bridges: Partial motor recovery was seen >1 year later (up to 4).
      • Canavero S.
      • Ren X.
      • Kim C.Y.
      • Rosati E.
      Neurologic foundations of spinal cord fusion (GEMINI).
      These observations suggest that if a method could be found whereby the fresh stumps of a cord submitted to cordectomy could be made to communicate electrophysiologically (i.e., spinal cord fusion), a cure for a traumatic spinal cord injury would result. In fact, the vertebral column can be surgically shortened (by corporectomy or multiple diskectomies), and the 2 freshly severed cord stumps approximated. Our work and these other anecdotal cases make a strong case for a clinical application of this approach. Finally, this study provides further evidence in support of the feasibility of a CSA.
      • Canavero S.
      HEAVEN: the head anastomosis venture project outline for the first human head transplantation with spinal linkage (GEMINI).

      Supplementary data

      The following is the supplementary data to this article:

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