feasibility and safety of neural tissue transplantation in patients with syringomyelia

JOURNAL OF NEUROTRAUMAVolume 18, Number 9, 2001Mary Ann Liebert, Inc.

Feasibility and Safety of Neural Tissue Transplantation inPatients with Syringomyelia

EDWARD D. WIRTH III,1,6 PAUL J. REIER,1,2,6 RICHARD G. FESSLER,1,2,6

FLOYD J. THOMPSON,1,6 BASIM UTHMAN,3,6,7 ANDREA BEHRMAN,5,6

JOELLA BEARD,4 CHARLES J. VIERCK,1,6 and DOUGLAS K. ANDERSON1,2,6,7

ABSTRACT

Transplantation of fetal spinal cord (FSC) tissue has demonstrated significant potential in animalmodels for achieving partial anatomical and functional restoration following spinal cord injury(SCI). To determine whether this strategy can eventually be translated to humans with SCI, a pi-lot safety and feasibility study was initiated in patients with progressive posttraumatic syringomyelia(PPTS). A total of eight patients with PPTS have been enrolled to date, and this report presentsfindings for the first two patients through 18 months postoperative. The study design included de-tailed assessments of each subject at multiple pre- and postoperative time points. Outcome datawere then compared with each subject’s own baseline. The surgical protocol included detethering,cyst drainage, and implantation of 6–9-week postconception human FSC tissue. Immunosuppres-sion with cyclosporine was initiated a few days prior to surgery and continued for 6 months post-operatively. Key outcome measures included: serial magnetic resonance imaging (MRI) exams, stan-dardized measures of neurological impairment and functional disability, detailed pain assessment,and extensive neurophysiological testing. Through 18 months, the first two patients have been sta-ble neurologically and the MRIs have shown evidence of solid tissue at the graft sites, without evi-dence of donor tissue overgrowth. Although it is still too soon to draw any firm conclusions, thefindings from the initial two patients in this study suggest that intraspinal grafting of human FSCtissue is both feasible and safe.

Key words: fetal tissue; human; magnetic resonance imaging; spinal cord injury; syringomyelia; trans-plant

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Departments of 1Neuroscience, 2Neurological Surgery, 3Neurology, and 4Orthopedics and Rehabilitation, University of FloridaCollege of Medicine; 5Department of Physical Therapy, University of Florida College of Health Professions; 6Evelyn F. & WilliamL. McKnight Brain Institute of the University of Florida, Gainesville, Florida.

7Malcom Randall Veterans Administration Medical Center, Gainesville, Florida.

INTRODUCTION

OVER THE LAST TWO DECADES, numerous studies haveexamined the capacity of fetal spinal cord (FSC) tis-

sue to facilitate structural and functional repair of the in-jured spinal cord in a variety of animal models (Ander-son et al., 1995; Reier et al., 1994). Early studies in this

field have demonstrated successful reconstruction ofacute resection lesions in terms of graft survival, growth,differentiation, host-graft integration, and connectivity(Itoh et al., 1996; Jakeman and Reier, 1991; Privat et al.,1988; Reier et al., 1986; Tessler et al., 1988). Other in-vestigations showed that fetal tissue grafts could fill ex-pansive regions of cystic cavitation in chronic contusion

lesions, which more closely mimic the histopathology ofmost human spinal cord injuries (Anderson et al., 1991;Houle and Reier, 1988; Reier et al., 1992).

These encouraging initial results led to considerationof the functional impact of such grafts in the rat and catspinal cord. In adult rats, FSC transplants have beenshown to assist recovery of locomotor function (Bern-stein and Goldberg, 1987; Bregman et al., 1993; Ribottaet al., 1998; Stokes and Reier, 1992) and to modulatelumbar motor neuron excitability (Thompson et al., 1993,1996). Improvement in hindlimb motor function has alsobeen observed in the cat (Howland et al., 1995, 1996),although the degree of recovery was reduced when thetime interval between injury and grafting exceeded 3–4months (Anderson et al., 1995).

Although more rigorous, quantitative studies in animalmodels are still needed to fully elucidate the mechanismsof graft-mediated functional recovery, the consistently fa-vorable results in rats and cats suggests that cellular trans-plantation will likely be part of an effective repair strat-egy for human spinal cord injury (SCI). Even thoughalternative sources of tissue for SCI repair have beenidentified recently (McDonald et al., 1999; Park et al.,1999; Whittemore 1999), the substantial collective expe-rience with fetal tissue presents a window of opportunityto test whether intraspinal grafting can be successfullytranslated to the clinical level. In addition, it can be read-ily appreciated that a judicious, limited clinical experi-ence could be instrumental in guiding the direction ofbench science toward the more rapid development of anoptimal clinical strategy.

To move forward as systematically as possible, thebasic safety and feasibility of intraspinal transplantationinto human SCI subjects first needs to be addressed bylooking at fundamental issues of graft survival andgrowth. Accordingly, preclinical studies have demon-strated the capacity of human FSC xenografts to sur-vive and fill both acute and chronic injury cavities inthe rat spinal cord (Akesson et al., 1998; Gilerovich etal., 1991; Giovanini et al., 1997). In addition, the donorhuman FSC tissue in these xenograft models did notshow any evidence of uncontrolled growth or inductionof an inflammatory response in the adjacent hostparenchyma.

Patient selection for a safety and feasibility study mustalso be given careful consideration. Transplantation offetal tissue into SCI patients with stable deficits wouldbe ill advised until more information is known about therisk of causing further neurological impairment. A moresuitable patient population for initial study would bethose with deficits that continue to worsen due to a pro-gressive expansion of the lesion cavity (i.e., progressiveposttraumatic syringomyelia) and, thus, who require sur-

gical intervention to stabilize the syrinx and prevent fur-ther tissue destruction.

Progressive posttraumatic syringomyelia (PPTS) is arelatively infrequent, but potentially disastrous compli-cation of SCI that can cause intractable pain and loss ofupper extremity function in paraplegic patients and maybe life-threatening if allowed to extend upward into thebrainstem (Biyani and el Masry, 1994; Piatt, 1996). Thereported incidence of progressive cystic degeneration fol-lowing spinal cord trauma has traditionally ranged from0.3% to 3.4% (Barnett et al., 1966; Biyani and el Masry,1994; Umbach and Heilporn, 1991). However, with thewidespread availability of MRI, the true incidence ofPPTS is now thought to be as high as 20% of all SCIcases (Edgar and Quail, 1994; Nielsen et al., 1999).

The precise pathophysiology of PPTS is still somewhatcontroversial, and, thus, there is no single surgical tech-nique for its treatment (Falci et al., 1999; Hida et al.,1994; Lee et al., 2000; Levi and Sonntag, 1998; Peerlessand Durward, 1983; Schwartz et al., 1999a; Sgouros andWilliams, 1995; Silberstein and Hennessy, 1992; Smalland Sheridan, 1996; Tator et al., 1982; Wiart et al., 1995;Williams 1990). Although clinical symptoms improve inover 75% of patients following surgery, only about 50%of patients have sustained improvement after 1 year, re-gardless of the surgical approach used (Anton andSchweigel, 1986; Aschoff and Kunze, 1993; La Haye andBatzdorf, 1988; Ronen et al., 1999; Rossier et al., 1985;Schaller et al., 1999; Sgouros and Williams, 1995; Suzukiet al., 1985).

As noted above, FSC grafts can completely fill post-traumatic cysts in rats and cats, and fuse extensively withthe host spinal tissue. Therefore, it was readily envisionedthat FSC grafts might at least partially obliterate thesecavities in human patients. Although it was not the ob-jective of this study to develop another surgical treatmentfor PPTS, the potential for clinical benefit clearly exists.

The first reported clinical attempt of human FSC graftsfor obliteration of syringomyelia cysts was by Blago-datskii et al. (1994). Five patients received implants ofFSC tissue. No complications were observed and the au-thors reported that cyst obliteration was confirmed for upto 11 months after surgery by delayed-contrast computedtomography (CT) and ultrasound. Subsequently, Falci etal. (1997) reported preliminary MRI evidence of a sur-viving graft 7-months postoperative in a single patient.In this individual, a small portion (about 6 cm) of the cystwas occluded by the fetal tissue.

Although these two studies provide preliminarydemonstration of feasibility and safety, many questionsregarding long-term graft survival, growth, and safetystill needed to be addressed. Thus, a primary goal of ourongoing study is to evaluate the safety of this procedure

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with respect to both possible early complications andlong-term consequences. This investigation is also in-tended to establish a more comprehensive diagnostic pro-tocol that spans the continuum from monitoring subclin-ical function on neurophysiological tests to standardizedquestionnaires regarding subjects’ quality of life. Here,we present data obtained from the first two patients en-rolled in this study who have been evaluated at multiplepostoperative intervals extending to 18 months aftertransplantation surgery. Some of these findings have beensummarized previously (Reier et al., 2000; Thompson etal., 1999; Wirth et al., 1999).

MATERIALS AND METHODS

Study Design and Inclusion Criteria

Patients were considered for participation in the studyif they presented with both clinical and radiological evi-dence of progressive syringomyelia, with the syrinx lo-cated primarily in the thoracic spinal cord (extension upto the C6 vertebral level was acceptable), and were freeof serious medical complications that would increase therisk of surgery and/or immunosuppression. Additional in-clusion criteria were age of $18 years and grade A–Con American Spinal Injury Association (ASIA) impair-ment scale or grade D with significant loss of function.Excluded were patients with MRI incompatible implants,history of intraspinal neoplasm or congenital spinal de-fect, or serious medical disease (e.g., AIDS, hepatitis)that would be complicated by immunosuppression. At theinitial visit, patients were evaluated by the neurosurgeon(R.G.F.), who also discussed the study and obtained in-formed consent. Each patient then began the initial com-prehensive assessment protocol: ASIA neurologicalexam, functional independence measure (FIM) for dis-ability, comprehensive pain assessment, psychosocial as-sessment, MRI scan, electrical studies of spinal conduc-tion and reflexes, and quantitative testing for spasticity.

Patients returned 1 month after the initial visit for asecond round of testing, so that an average baseline scorecould be computed for each outcome measure. Approx-imately 1 month after the second round of preoperativetests were completed, patients were admitted for syrinxdrainage and implantation of FSC tissue. Follow-up clin-ical, neurophysiological, and MRI evaluations were per-formed before discharge and at 1.5, 3, 6, 9, 12, and 18months after discharge.

Patient Histories

Patient 1. This patient is a 44-year-old man who re-ceived a T6 laminectomy and resection of an in-

tramedullary “mass,” possibly an arteriovenous malfor-mation, in 1979. He recovered immediately and was freeof any neurological impairment until 1991, when he de-veloped chest wall pain and weakness in his legs. AnMRI at that time revealed a large syrinx in his thoracicspinal cord, and he was treated with a syringosubarach-noid shunt and two subsequent shunts between 1992 and1995. Despite these three operations, the pain, weaknessand spasticity in his lower extremities continued toworsen. At the time of enrollment into this study, he wasseverely paraparetic with no voluntary bowel or bladdercontrol. In addition, he reported having constant tinglingpain from his upper abdomen to his toes bilaterally.

Patient 2. This patient is a 51-year-old man who wasinjured in a motorcycle accident in 1977. He suffered aT6 vertebral body fracture and immediate T6 level spinalcord injury with paralysis below this level, but withpreservation of sensation. His neurological status re-mained stable until early in 1994 when he began experi-encing pain in his back, arms, and neck, and weaknessbilaterally in his hands. A MRI scan at that time revealeda syrinx extending from the level of his injury up to thecraniocervical junction. He was treated with a syringo-subarachnoid shunt in May, 1995, and although thestrength in his hands returned, the pain in his back didnot improve. Approximately 7 months after shunt place-ment, he noted increasing numbness in the left C7-T1dermatomes, and subsequent MRI exam demonstrated re-turn of the syrinx from C2-C8. The patient then under-went a CT-guided drainage at T3/4, which removed ap-proximately 12 mL of fluid from the syrinx. He hadpartial relief of his symptoms immediately following thisprocedure, but the symptoms returned their previous lev-els within a few weeks. He was subsequently stable ex-cept for progressive worsening of the numbness in hisupper back and neck.

Donor Tissue Procurement and Preparation

Human fetal spinal cord tissue was procured follow-ing elective abortions in accordance with federal and statelaws and ethical guidelines. Informed consent for dona-tion of the fetal tissue was obtained only after informedconsent was given for the abortion. Only donor tissue of6–9 weeks gestational age (GA) was used in this study.This gestational period spans the arbitrary boundary be-tween the embryonic and fetal development stages, whichare operationally defined as ending and beginning 8weeks postconception, respectively (O’Rahilly andMüller, 1987). Therefore, the term “fetal” used herein in-cludes some donor tissue that is still in the embryonicphase of development.

The donor tissue was collected via the low-pressure

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aspiration technique (Nauert and Freeman, 1994) into asterile container and all subsequent processing was per-formed under aseptic conditions. Donor ages were veri-fied according to the Carnegie criteria for tissue up to 8weeks GA, (O’Rahilly and Müller, 1987), or by hand andfoot length measurements for tissue of .8 weeks GA.Each fetal spinal cord and any adjacent CNS tissue wasdissected en bloc and washed in five wells containing 2mL sterile hibernation medium (Freeman and Kordower,1991; Kawamoto and Barrett, 1986). The fetal spinalcords were then transferred into a tube containing 50 mlsterile hibernation medium (HM) and this tube wasplaced into a beaker on ice. This arrangement maintainedthe HM temperature near the desired level of 8°C(Kawamoto and Barrett, 1986). After the tissue was trans-ferred to this tube, the medium from the 5th well wastransferred to a sterile container and sent for bacterial, vi-ral, and fungal cultures.

The tissue was then transported to a sterile laminar-flow hood where the FSC was dissected free from themeninges and adjacent CNS tissue. The FSC was washedin a second set of five wells containing 2 mL sterile HM,placed into a tube with 15 mL HM, and stored in a re-frigerator at 8°C. A section of the adjacent CNS tissuewas then washed in the same five wells and sent for cul-tures.

A piece of the FSC tissue approximately 2 mm inlength was removed for viability assays using an accepteddye-exclusion technique (Brundin et al., 1985). Half ofthis piece was used to check viability immediately afterthe initial dissection and viability of the remaining halfwas recorded 1–2 h prior to transplantation. Viability ofthe FSC tissue after the initial dissection ranged from71% to 81%, with a mean of 76%, whereas final viabil-ities averaged 73%. No tissue was used if the viabilityfell below 50%, although this level was exceeded in allcases. Since multiple donor cords were pooled for eachrecipient, donor tissue was procured over several days,yielding storage durations of 2–9 days prior to surgery.However, most of the tissue used for transplantation wasstored for less than 3 days.

Immediately prior to transplantation, the donor spinalcords were cut into smaller pieces, with considerationgiven to the intended graft site in each recipient. Sincethe target site in the first patient consisted of multiplesmall compartments in the inferior portion of his syrinx,the donor tissue in this case was minced into fragmentsapproximately 0.75 mm3 to facilitate multiple 100-mL in-jections of tissue. In contrast, the target sites in the sec-ond patient were a large cavity extending from C6-T5and a smaller cyst at the T7-T8 levels. Accordingly, largerpieces approximately 10 mm in length 3 1 mm in diam-eter were prepared for grafting into the upper cyst,

whereas smaller pieces 3 mm 3 0.75 mm were preparedfor the lower cyst. These larger pieces were introducedto reduce the likelihood of donor tissue floating awayfrom the graft site.

Screening for Transmissible Diseases

All screening and testing of the maternal donor and fe-tal tissue was performed in accordance with Florida lawand the FDA Guidelines for screening and testing ofdonors of tissue intended for human transplantation anddonors of reproductive tissue. Furthermore, the screen-ing procedures closely followed published methods, thathave reliably yielded uncontaminated fetal CNS tissuefor transplantation (Holt et al., 1997). To avoid trans-mission of serious medical diseases from the maternaldonors to the recipients, the maternal donor’s blood wastested for exposure to HIV-1/2, HTLV-1/2, HepatitisA/B/C, Syphilis (RPR), and herpes (HSV-1/2). Since se-rious medical disease is one of the exclusion criteria foreligibility, the recipients were also tested preoperativelyfor exposure to all the above agents. Blood samples fromevery maternal donor also were screened for IgG anti-bodies to cytomegalovirus (CMV) and toxoplasmosis(Toxo) in order to avoid grafting tissue that may be con-taminated with these agents into a CMV IgM-negative orToxo IgM-negative recipient.

As mentioned in the previous section, two sets of wash-ings for each donor FSC were sent for bacterial, fungal,and viral cultures. Since most of the donor tissue was ob-tained 48–72 h prior to surgery, some of the final cultureresults were not available until 1–5 days postoperative.Therefore, antibiotic and antifungal prophylaxis (Van-comycin, 1 g IV q12h; Cefipime, 2 g IV q12h; Diflucan,200 mg IV qd) was administered beginning immediatelyafter surgery and continued until final culture results wereavailable. All culture results were negative, and no post-operative infections were noted except for a urinary tractinfection in patient 2.

Neurosurgical Procedure

The anesthetic regimen, opening and closing proce-dures were performed in the usual fashion for conven-tional surgical treatment of syringomyelia. Briefly, thepatients were sedated and maintained under general anes-thesia with endotracheal intubation and placed in theprone position. Electrodes were placed for somatosen-sory evoked potential monitoring (SSEP), and the spinalcolumn was then exposed through a standard midline in-cision followed by a combination of blunt and sharp dis-section. From this point, the surgical approach and graft-ing method was tailored for each patient with regard totheir previous operations and the intended graft site. Af-

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ter the donor tissue was implanted and the meninges su-tured, graft placement was verified by intraoperative ul-trasound. The deep muscle layers, subcutis, and skin werethen closed in the usual sterile fashion.

Patient 1. The previous incision, which extended fromvertebral level T4 to T12, was reopened and extendedfrom T3 to L1. Careful sharp and blunt dissection wasthen performed until the dura was exposed and freed fromtightly adherent overlying scar tissue. The syrinx wasthen identified by ultrasound and was noted to span theentire dural exposure. In addition, the inferior portion ofthe syrinx from T11 to L1 was particularly loculated. Atthis point, the dura was opened and the underlying ad-hesions were carefully dissected to free the spinal cord.These adhesions were primarily attached to the dorsalsurface of the spinal cord, although it was noted that inthe mid-portion of the exposure the spinal cord was com-pletely adherent to the dura in a circumferential pattern.Once dissection of the intradural adhesions was complete,the previous syringopleural and two syringosubarachnoidshunts were removed.

Next, the fluid within two cysts in the loculated regionwas aspirated through an 18-gauge needle attached to a5-mL syringe. This was noted to completely decompressall of the cystic cavities. Pieces from two donor spinalcords were then drawn into an 18-gauge cannula attachedto a 500-mL syringe and injected into the syrinx at theT11 vertebral level. Similar injections were also madewith additional donor cords at T12-L1 and T10-T11.Next, the mid portion of the syrinx (T7-T8) that had beencircumferentially adherent to the dura was closed usinga 5-0 silk stitch. Just before tying this down, several largerpieces from one of the donor cords were injected. In to-tal, four injections of tissue from eight fetal spinal cords(approximately 500 mL total volume) were made.

Patient 2. The previous incision was reopened and ex-tended from T1 to just below a kyphotic hump at T5-T6.The previous laminectomy sites at T2-T4 were identified,and the laminae of T6, T7, and T8 were then removed.The dural plane underneath the scar from prior operationswas identified and was carefully dissected free. Attentionwas then turned to the severe kyphotic angulation at T5-T6, which was noted to narrow the spinal canal on pre-operative MRI scans. Using a Midas-Rex drill, right andleft facetectomies were performed at the level of thekyphotic angulation. Next, curets and tamps were usedto dissect the kyphotic angle free from the dura and topush the projecting bone edge into the vertebral body,which substantially decompressed the angle. The durawas then opened from T2-T8, and the syrinx was identi-fied by ultrasound.

For the first portion of the transplant procedure, an 18-gauge needle attached to a 3cc syringe was inserted intothe inferior portion of the syrinx below the level of theinjury. Pieces from four donor cords (approximately 100mL total volume) were then drawn up into an 18-gaugecannula attached to 500-mL syringe. This cannula wasthen inserted into the superior portion of the syrinx. Thesyrinx was then drained via aspiration through the firstsyringe, followed immediately by injection of the tissuewith the 500-mL syringe.

Next, a myelotomy was performed immediately abovethe level of spinal cord injury in the area at which thecyst wall was felt to be thinnest (approximately T4). Thecyst wall at this level was roughly 0.5 mm thick. Throughthis myelotomy numerous large pieces from four donorspinal cords (approximately 400 mL total volume) wereplaced directly into the cavity. The myelotomy was thensutured closed, and at this point the syrinx was found tobe well decompressed.

Immunosuppression

Patients were begun on cyclosporine (3 mg/kg BID)starting 3–4 days preoperative. The cyclosporine dosewas reduced to 1 mg/kg BID at 6 weeks after transplan-tation and discontinued at 6 months postoperative. Tox-icity monitoring included checks of cyclosporine bloodtrough concentration, urine analysis, blood urea nitrogen,and creatinine every 24–48 h during the first week aftersurgery, biweekly until the 6 week visit, and then every1.5 months until cessation at 6 months. The cyclosporinedose was lowered or discontinued in the event of toxic-ity or if trough blood levels exceeded 400 ng/dL.

Clinical Outcome Measures

Sensory and motor function were evaluated using themethods described in the ASIA International StandardsBooklet for Neurological and Functional Classificationof Spinal Cord Injury (Ditunno et al., 1994). Where pos-sible, all ASIA examinations were performed by the samephysician to increase the reliability of the motor and sen-sory scores. In addition, the clinical evaluators wereblinded to the MR images and results of all other out-come measures. These data were supplemented by as-sessment of temperature, proprioception, and deep pres-sure sensation, which were scored in accordance withASIA criteria. The ASIA functional independence mea-sure (FIM) score was also recorded at each pre- and post-operative evaluation point.

A detailed pain assessment was also obtained for eachtransplant recipient at every visit. The complete protocolincluded a numerical rating scale (NRS) of intensity andunpleasantness, McGill Pain Questionnaire (MPQ), and


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detailed pain drawings. The MPQ, NRS, and pain draw-ing, used together, provide a systematic comprehensiveassessment of the type of pain encountered in sy-ringomyelia, as well as response of this pain to treatment(Davidoff et al., 1987).

Magnetic Resonance Imaging

MRI examinations were performed at the initial pre-operative visit 8 weeks before surgery, and at 1.5, 3, 6,9, 12, and 18 months postoperative with a 1.5-Tesla (T)system (Vision, Siemens). The MRI protocol was de-signed to visualize the entire syrinx, and to obtain re-stricted views with higher magnification of the transplantsite. Each MRI exam began with sagittal and axial T1-weighted (TR/TE 5 600/15 msec) and T2-weighted(TR/TE 5 2,000/90 msec) spin-echo images obtained be-fore and after administration of gadolinium (Gd-DTPA).In addition, sagittal fluid attenuation by inversion recov-ery (FLAIR)–weighted images (TR/TE/TI 5 9,000/105/2,200 msec) were obtained at some time points. All im-ages were acquired with a slice thickness of 3–4 mm anda 256 3 256 matrix size. In-plane resolution varied de-

pending upon the field-of-view required to visualize theentire syrinx. Total scan time per subject at each sessionwas approximately 1.5 h.

Neurophysiological Testing

In order to detect changes in spinal cord activity thatmight help explain or confirm variations in the clinicaloutcome measures, each patient received an extensiveneurophysiological evaluation at every pre- and postop-erative visit. Details of the assessment protocol and re-sults for the first two patients in this study are providedin the accompanying paper by Thompson et al. (this is-sue).

RESULTS

Due to the substantial heterogeneity between these firsttwo patients with respect to etiology and severity of spinalcord injury, syrinx history, clinical presentation, and re-sponse to surgery, the results for each patient are pre-sented in the form of a case report.

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FIG. 1. Preoperative T1- and T2-weighted sagittal magnetic resonance images of patient 1. The syrinx is clearly visible on theT1-weighted image (A) as an elongated region of low signal (white arrows) extending from T4-L1 and is multilocular over theT11-L1 levels. The upper end of the cyst is out of the image plane in panels A and B, but axial images (not shown) and adja-cent sagittal images revealed that the cyst extended up to the T2 vertebral level. On the T2-weighted image (B), the cyst appearshyperintense (white arrows).

Patient 1

Preoperative magnetic resonance imaging findings.Preoperative MR images (Fig. 1) revealed that his syrinxextended from vertebral levels T2-L1, and was multiloc-ular over the inferior levels (approximately T8-L1). Nointervertebral disc abnormalities or enhancing lesionswere noted following administration of Gd-DTPA. Theaxial preoperative images demonstrated that the cord atthe original surgical site (T6-T7; not shown) was cir-cumferentially adherent to the arachnoid and dura. Im-mediately cephalad to these levels, the cyst was centrallysituated with only a thin rim of cord tissue visible. FromT4 up to T2, the cyst was paracentral on the left side,roughly equidistant between the posterior and anterior as-pects of the cord. Immediately below the original surgi-cal site, a prior shunt was visible on the right side at theT8 level. This shunt appeared to be encased in scar tis-sue, with the cyst descending on the left side of the cord.Over the lower thoracic levels, the cyst was located pri-marily on the left side, although at some levels its sizeincreased such that it encompassed the entire cord exceptfor the most peripheral white matter. At levels corre-sponding to the lumbar enlargement (approximately bot-tom of T11 vertebra to top of L1 vertebra), it appearedthat some gray matter on the right was spared, whereaslittle or no gray matter was visible on the left. In the sacralcord, the cyst was multilocular and encompassed most ofthe cord down to the conus.

Postoperative course. The patient tolerated the surgi-cal procedure well and postoperatively was transferred tothe surgical intensive care unit intubated, but in hemo-dynamically stable condition. During the following threedays, continuing difficulty was encountered in attempt-ing to extubate the patient due to poor oxygenation. Fol-lowing an aggressive diuresis, the patient’s pulmonarystatus improved markedly, and he was weaned and extu-bated on postoperative day (POD) 6. The only compli-cation during the patient’s hospital course was a persis-tent CSF leak. This developed as the result of anincomplete dural closure because much of the dura wasindistinguishable from scar tissue. Two attempts at plac-ing a percutaneous lumbar CSF drain failed to producesatisfactory results, so the patient was returned to the op-erating room for placement of a thoracic subarachnoiddrain. This drain functioned well and the CSF leak re-solved by the following day.

During his hospital stay, patient 1 reported no changesin his neurological status, nor were any noted on exam-ination. Trough cyclosporine levels were 168–316 ng/mLthroughout the hospitalization with no apparent compli-cations. The remainder of his hospital course was free ofcomplications, and he was discharged to rehabilitation 18days after the transplant surgery.

At the first follow-up visit, 6 weeks after transplanta-tion, the patient reported slightly increased tinglingdysesthesias and muscle spasms in both lower extremi-ties. He also noted that his sensation of bowel and blad-der fullness had improved such that he had not experi-


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FIG. 2. American Spinal Injury Association (ASIA) neuro-logical scores of motor and sensory function for patient 1. Fluc-tuation in the sensory and motor scores is evident and appearedto be related, at least in part, to interrater variability.

FIG. 3. Ratings of overall pain intensity on a 100-point nu-merical rating scale (NRS) by patient 1. A transient decreasein overall pain was observed at 6 weeks postoperative, with asubsequent return to baseline levels by 9 months.

enced a bowel or bladder accident (i.e., an episode of in-continence) in several weeks. This was in contrast to thedaily accidents that occurred prior to surgery. From thistime through 18 months postoperative, the patient re-ported no further changes in his bowel and bladder func-tion, tingling dysesthesias, and lower extremity spasms.

Motor and sensory scores on ASIA exam were alsostable throughout the study, except for some minor in-terrater variability in the motor scores (Fig. 2). Due toscheduling limitations, three different evaluators exam-ined this patient over the course of the study, which likelycontributed to the variations observed between visits. Forexample, lower motor scores were given to some musclegroups at 12 and 18 months, when one evaluator believedthat a portion of the strength was due to spasms ratherthan volitional effort. Earlier assessments by other eval-uators did not make this distinction. Interrater variabilityfor the light-touch and pinprick assessments was less pro-nounced, and the patient’s sensory levels on both sidesremained stable within 6 1 dermatome across all timepoints.

As noted earlier, patient 1 reported painful sensationsof tingling and spasms in his lower abdomen andthroughout both lower extremities. Detailed pain assess-ments revealed a slight decrease in overall pain intensityat 6 weeks postoperative with a return to baseline levelsat 3 months (Fig. 3). No significant changes in total pain

intensity were noted thereafter. The distribution and char-acter of the tingling dysesthesias and lower extremityspasms did not differ noticeably from baseline, but pa-tient 1 did begin experiencing bladder spasms about 12months postoperative (Fig. 4).

At the initial follow-up interval, 6 weeks after surgery,sagittal T1- and T2-weighted MR images showed that thetransplant site was substantially collapsed, whereas adja-cent non-grafted levels appeared unchanged (Fig. 5A,B).Subsequent MR images acquired at every visit through18 months postoperative (Fig. 5C) demonstrated littleoverall change compared to the 6-week images. In orderto determine whether the apparent cyst collapse on thesagittal MR images resulted from variations in slice po-sition and/or partial volume averaging, consecutive trans-verse slices through the graft site were also acquired ateach evaluation interval (Fig. 6). These axial images con-firmed that the cyst was substantially obliterated at thegraft site, but relatively unaltered in non-grafted regions.Despite the clear evidence of cyst collapse at the graftsite, no specific boundaries between donor and host tis-sue were observed on T1- or T2-weighted images. Thus,it was not possible to determine definitively whether anyof the transplanted tissue had survived. It was also notedthat the MR images were unchanged subsequent to re-duction of the cyclosporine dose at 6 weeks postopera-tive or after cessation of the cyclosporine at 6 months.

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FIG. 4. Pain drawings by patient 1 from preoperative through 18 months postoperative. The drawings show a stable pain dis-tribution, except for the delayed onset of bladder spasms, as indicated on the 18-month drawing.

Patient 2

Preoperative magnetic resonance imaging findings.Pre-grafting MR images (Fig. 7) showed a severekyphotic angulation of the spine at the T5-6 interspace.At this level, the spinal cord narrowed considerably andappeared to be tethered. The spinal cord at this level alsoformed a partition between two distinct cysts that ex-tended above and below the T5-6 interspace. The uppersyrinx extended from this partition up to vertebral levelC6, whereas the lower syrinx extended downward to T8.Both cysts had very large diameters and were boundedby a thin rim of spared white matter. No intervertebraldisc abnormalities or enhancing lesions were noted fol-lowing administration of Gd-DTPA.

Postoperative course. This patient tolerated the oper-ation well and was extubated at 1 day after surgery. Hereported that after awakening from surgery he no longerfelt the weakness and pain in his upper back and rightarm. The remainder of his neurological status was un-changed relative to baseline.

His hospital course was notable for a low-grade feverthat developed on POD 4. A urinalysis was obtained andthis demonstrated evidence of an urinary tract infection.This was treated with antibiotics and resolved by POD9. Concurrently, this patient had persistent postoperative

nausea and vomiting, which resolved after several daysof antiemetic medication. Otherwise, the patient had nopostoperative complications and was discharged on POD12.

At follow-up visits through 18 months postgrafting, hereported no changes in motor or sensory function exceptfor an incident at 8 months, in which he noticed a “nor-mal” burning sensation on his lower abdomen wheresome hot ashes had fallen from a cigarette. The patientfurther stated that earlier episodes of hot cigarette ashescontacting his abdomen prior to the transplant surgeryhad caused only a dull, poorly localized sensation of pain.

Motor and sensory ASIA scores for patient 2 were verystable over the entire duration of the study (Fig. 8). Incontrast, assessments of overall pain intensity revealed asharp decrease at 6 weeks, followed by a progressive in-crease that exceeded baseline levels by 6 months and per-sisted through 18 months (Fig. 9). Detailed drawings andverbal descriptions by the patient revealed that he hadcomplete resolution of the pain that was present beforesurgery, and that the subsequent rise in overall total painintensity was due to the delayed onset of three new painsensations (Fig. 10). The patient described these newdysesthesias as stabbing in the left flank, grinding in hismid-thoracic spine, and burning on the left side of hischest. These newer sensations were located in der-


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FIG. 5. T1-weighted sagittal magnetic resonance (MR) images of patient 1 acquired preoperatively (A), and at 1.5 months (B)and 18 months (C) after surgery. Most of the donor tissue was injected at three sites in the T11-L1 levels (double arrow), al-though a small amount of fetal spinal cord (FSC) tissue was also implanted at T7. Serial postoperative MR images show syrinxclosure in areas where donor tissue was placed, whereas adjacent syrinx regions which did not receive donor tissue (arrowheads)refilled with cerebrospinal fluid by 6 weeks.

matomes T5-T10, and appeared to be distinct from thepatient’s original pain, which was present over the C4-T4 levels.

Serial transverse and sagittal MR images of this pa-tient’s spinal cord demonstrated several changes post-grafting that closely paralleled the temporal course anddermatome levels of his pain. For example, sagittal T1-and FLAIR-weighted MR scans acquired 6 weeks after

surgery showed that the upper cyst was almost com-pletely collapsed from C6-T2, and partially collapsedfrom T3-T5 (Fig. 11A,B). In contrast, the lower cyst pres-ent at T6-T8 appeared to be relatively unchanged. At latertime points, the upper cyst appeared to be unchanged withrespect to the 6-week MRI, except for progressive col-lapse in the transplant site at the T3 vertebral level,whereas progressive downward expansion was observed

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FIG. 6. Consecutive T1-weighted axial magnetic resonance images of the graft site shown in Fig. 5 preoperatively (a–f), andat 1.5 months (g–l) and 18 months (m–r) after surgery. Preoperatively, a thin rim of spinal cord surrounds a large hypointensecyst (arrows in a–f), whereas the cyst is much smaller after grafting (arrows in g–r).

in the lower cyst (Fig. 11C). Consecutive transverse im-ages through the graft site confirmed the progressive col-lapse at T3, but as with the MRIs of patient 1, it was notpossible to visualize definitively any boundaries betweendonor and host tissue (Fig. 12).

DISCUSSION

Feasibility of Fetal Spinal Cord Transplantationinto the Human Spinal Cord

This initial component of our clinical study has demon-strated the logistical and technical feasibility of procur-ing and storing human FSC tissue in accordance with cur-rent laws and guidelines. Storage and preparation of thedonor tissue using established methods (Freeman andKordower, 1991; Kawamoto and Barrett, 1986) yieldedup to eight donor cords with acceptable pregrafting via-bility levels per transplant procedure. Furthermore, crit-ical to the safety of the procedure, it was encouragingthat all bacterial, viral and fungal cultures of the donortissue and storage medium were negative.


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FIG. 7. Preoperative T1- and T2-weighted sagittal magnetic resonance images of patient 2. Two distinct cysts are clearly vis-ible on both the T1-weighted image (A) and the T2-weighted image (B). The upper cyst (white arrows) extends from vertebrallevel C6 to the kyphotic angle at T5-6 (black arrowhead), at which point the spinal cord appears to be compressed and tethered.The lower cyst begins just below this juncture and extends down to T8 (black arrows).

FIG. 8. American Spinal Injury Association (ASIA) neuro-logical scores for patient 2. No evidence of motor function orsensation below the level of injury was observed prior to trans-plantation, and this remained unchanged through 18 monthspostoperative.

Although most of the donor tissue was viable imme-diately prior to transplantation, demonstration of subse-quent postgrafting survival was clearly dependent on ad-equate delineation of the transplants on MRI scans. Thisrequired acquisition of images with sufficient spatial res-olution and contrast to discriminate between the graftsand surrounding host parenchyma. In addition, since bothpatients in this study received syrinx decompression anddetethering, it was anticipated that the MRI scans wouldshow concurrent changes in the syrinx size and/or mor-phology (Barkovich et al., 1987; Hida et al., 1994;Kochan and Quencer, 1991; Milhorat et al., 1992; Tane-ichi et al., 1994) that could complicate interpretation ofthe MR data.

Previous investigations in cats, which compared in vivoMR images of intraspinal FSC grafts to correlative post-mortem histological sections, suggested that it should befeasible to visualize viable graft tissue in the recipients(Wirth et al., 1992, 1995). Similar MR findings follow-ing intraspinal grafting of FSC tissue were reported byFalci et al. (1997) for a single human subject, althoughthe precise composition of the graft site is unknown inthe absence of postmortem confirmation.

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FIG. 9. Ratings of overall pain intensity by patient 2. He re-ported a significant decrease in overall pain at 6 weeks post-operative, followed by a progressive increase in pain through9 months. This latter progression was found to be the result ofdelayed onset of new pain sensations in his lower back and ab-domen (see Fig. 10).

FIG. 10. Pain drawings by patient 2 through 18 months postoperative. A profound change in the pain distribution and charac-ter are evident. This patient reported that the burning on his upper back and right arm was relieved almost immediately follow-ing surgery. However, he reported delayed onset of several new areas of pain in his mid- and lower torso.

Despite the high quality of the MR images in the cur-rent study, it was not possible to unequivocally confirmsurvival of the FSC grafts because specific donor–hostboundaries could not be defined. While this could meanthat no tissue survived in either patient, certain findingson the MR images suggested some degree of donor tis-sue survival. The most compelling indication for the pres-ence of a graft was that cyst collapse was confined to re-gions where the donor tissue was implanted, even thoughdetethering and decompression were performed over theentire syrinx. In addition, this restricted obliteration wasobserved in two different settings: small, contiguous mul-tilocular cysts in patient 1 and a single large cavity in pa-tient 2. Furthermore, at some levels through the graftsites, there appeared to be progressive closure of the cystfrom 6 weeks to 18 months, which could be consistentwith delayed proliferation of the FSC tissue. In our ani-mal experiments, failed or rejected grafts typically re-sulted in refilling of the injury site with CSF (Theele etal., 1996; Wirth et al., 1995). Therefore, it is unclear whatmechanism, other than the donor tissue, could accountfor this sustained obliteration over 18 months.

If some of the donor tissue did indeed survive, thenour results indicate that only minimal growth was

achieved. This finding is important with respect to long-term safety, since a theoretical long-term risk in this studywas that the donor tissue might continue to proliferate af-ter filling the cysts and possibly damage the surroundinghost white matter. Since it is unclear at this time whetherany donor tissue survived, the risk of delayed graft over-growth also remains unknown. However, irrespective ofthe transplant status, there did not seem to be any indi-cation of adverse consequences (e.g., inflammation) onthe MRI scans.

The inability to detect clear graft boundaries in thepresent study may have been due to inadequate spatialresolution or tissue contrast, and may also reflect re-stricted transplant growth. For example, the transverseMR images in our earlier studies with cats had a 2-mmslice thickness and an in-plane resolution of 0.23 3 0.23mm, which resulted in image elements (“voxels”) withvolumes of 0.11 mL (1 mL 5 1 mm3). Corresponding im-ages of the human subjects in the present study had a 4-mm slice thickness and in-plane resolution of 0.7 3 0.7mm, which resulted in 1.96-mL voxels. Thus, the spatialresolution was nearly 20-fold worse in the MR imagesof the human subjects and may have obscured thedonor–host interface through partial volume averaging.


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FIG. 11. T1- and FLAIR-weighted sagittal magnetic resonance (MR) images of patient 2 acquired preoperatively (A), and at1.5 months (B) and 18 months (C) after surgery. FLAIR images (B,C) provided sharper contrast between cerebrospinal fluid inthe syrinx and neural tissue than T1-weighted images (A), which aided visualization of the graft site (double arrow). On the pre-operative MR image, an upper syrinx is visible from C6-T5 (double arrow) and a lower cyst is present from T6-T8 (arrowheads).It was confirmed intraoperatively that these two cysts were not in communication with each other. Most of the donor tissue wasimplanted at T2-T5 (double arrow), although a small amount of fetal spinal cord tissue was injected at T7. Serial postoperativeMR images show progressive closure of the syrinx at the T3 vertebral level, although it is difficult to clearly distinguish graftfrom host tissue. In contrast, the lower cyst exhibited progressive expansion after surgery (arrowheads in B,C).

In theory, better spatial resolution could be obtained inthe future by imaging at higher magnetic fields and/or byutilizing small imaging coils situated directly over thetransplant site (Wirth et al., 1993). Contrast between hostand graft tissue might also be improved with advancedtechniques such as diffusion-weighted MRI (Schwartz etal., 1999b) or by prelabeling the donor tissue with an MRIcontrast agent (Bulte et al., 1999).

Clinical and Neurophysiological Outcomes DoNot Indicate Adverse Effects

As anticipated, the major acute risks to patients in thisstudy were related to the routine portion of the surgical

procedures, in other words, syrinx drainage and deteth-ering of the spinal cord. The only notable surgical com-plication was a CSF leak in patient 1 that was caused byincomplete dural closure. This was due to the presenceof substantial scar tissue from his previous shunt opera-tions.

Importantly, there was no contamination of the donortissue and no postoperative infections of the surgical site.In addition, the absence of acute neurological deteriora-tion in either patient suggested that FSC tissue implan-tation did not cause any direct mechanical damage orother acute insult to the surrounding host spinal tissue.

Although the present study lacked control subjects, theclinical and operative histories for each subject were use-

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FIG. 12. Consecutive T1-weighted axial magnetic resonance images of the graft site shown in Fig. 11 preoperatively (a–e),and at 1.5 months (f–j) and 18 months (k–o) after surgery. Preoperatively, a thin rim of spinal cord surrounds a large hypointensecyst (arrows in a–e). Partial collapse was seen at 6 weeks (arrows in f–j) and progressive closure through 18 months was ob-served at the T2-T3 level (arrows in l–m).

ful guides in assessing long-term safety on a case-by-casebasis. In this regard, each of these two subjects had a re-turn of their cyst and clinical worsening less than 1 yearafter their previous operations. The fact that the graft sitesand neurological status have been stable in both patientsthrough 18 months in the current study suggests that theiroutcomes are at least no worse than after their previousoperations. In view of the problem with MRI identifica-tion of donor tissue, we presently defer from drawing anyconclusions about potential efficacy of this grafting pro-cedure in promoting syrinx obliteration.

Immunosuppression

Closely related to the overall question of safety is theissue of immunosuppression. This required careful con-sideration because it inevitably involved a compromisebetween accepting the known risks of toxicity, carcino-genesis, and opportunistic infections that accompany along-term immunosuppressive regimen versus the theo-retical risks associated with transplant rejection. In the-ory, rejection of an intraspinal fetal graft might (a) causedirect inflammatory damage to adjacent host tissue, (b)stimulate a more widespread autoimmune response,and/or (c) eliminate any potential benefit that the trans-planted tissue may have afforded the recipient (Ander-son et al., 1991; Nicholas and Aranson, 1989).

At the initiation of this study, it was unknown whetherimmunosuppression was required for long-term survivalof fetal allografts in the human CNS. Most studies of in-tracerebral fetal transplants in patients with Parkinson’sdisease have employed some type of immunosuppressiveregimen (Freeman et al., 1995; Lindvall et al., 1989;Spencer et al., 1992), although there is some evidencethat the host immune system may not respond to thesegrafts (Ansari et al., 1995). In addition, long-term sur-vival of both intraspinal transplants in rats and intrac-erebral grafts in humans after withdrawal of immuno-suppression has been noted (Kordower et al., 1995;Theele and Reier, 1996).

In consideration of this evidence that long-term im-munosuppression may be unnecessary and that patientswith PPTS have a heightened susceptibility to oppor-tunistic infections (e.g., via decubitus ulcers), the presentstudy employed a temporary cyclosporine regimen. It wasfelt that this strategy struck a reasonable balance betweenmaximizing patient safety and optimizing the chances forgraft survival. Furthermore, by discontinuing cyclo-sporine at 6 months postgrafting, it was possible to as-sess the MR images for signs of an inflammatory re-sponse that could be associated with transplant rejection.Since the MRI scans did not show any significant changesfollowing withdrawal of cyclosporine, it is possible thateither no viable donor tissue was present or that suffi-

cient host tolerance was achieved to prevent a detectableimmune response. Alternatively, if rejection did occurfollowing cyclosporine withdrawal, there was no evi-dence of subsequent adverse effects on the MR images.

Correlation of Magnetic Resonance Imaging with Clinical and Neurophysiological Outcome Measures

Acquisition of serial MR images also provided an op-portunity to temporally evaluate correlations betweenchanges in syrinx cavity morphology and variations inthe clinical and neurophysiological outcome measures(Milhorat et al., 1995, 1996). Since the MR images ofboth patients in this study showed collapse of only smallportions of the cysts, it was anticipated that any clinicalor neurophysiological changes would be primarily relatedto segmental activity in the spinal cord, rather than im-provement of conduction in ascending and descendinglong white matter tracts. Indeed, neither patient showedany improvement in lower extremity function on ASIAexam, nor was there any return of cortical evoked po-tentials from the lower extremities, as discussed in theaccompanying paper by Thompson et al. (this issue).However, Thompson et al. did observe an improved stim-ulus-rate dependence of the H-reflex from the right legin patient 1, which suggested partial normalization of theexcitability in the corresponding motor neurons. In con-trast, patient 2 reported significant changes in his dyses-thetic pain sensations that seemed to closely parallel theevolution of his two cysts on MRI. Specifically, his up-per cyst showed substantial collapse on MRI with a con-comitant decrease in pain in the corresponding der-matomes, whereas the delayed expansion of his lowercyst was accompanied by delayed onset of dysestheticpain in his lower back.

CONCLUSION

The data obtained to date in this ongoing study sug-gest that transplantation of FSC tissue into posttraumaticcysts in the human spinal cord is logistically feasible andprocedurally safe. However, since it is still unclearwhether any donor tissue survived, the potential long-term consequences of graft survival are uncertain. Itshould be emphasized, however, that regardless of thetransplant status, the clinical outcomes for both patientsappear to be no worse than after their previous opera-tions.

One key finding to date is that conventional MR imag-ing may not be sufficient to unequivocally determinegraft survival or failure. Given the promising results inearlier studies with cats, it seems possible that refine-


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ments in the MR hardware and/or application of more ad-vanced imaging methods could produce acceptable spa-tial resolution and tissue contrast in the near future. Sincethe same imaging issues are likely to apply to othersources of donor tissue (e.g., stem cells) for intraspinalrepair, this is an area that obviously warrants further in-vestigation.

ACKNOWLEDGMENTS

We are grateful to Michelle Forthofer, R.N. for coor-dinating the study and to Thomas Freeman, M.D. andRay Moseley, Ph.D., who were instrumental in the de-velopment of the study design. We would also like tothank Ellsworth J. Remson, M.D. for his assistance withthe ASIA exams, Don Price, Ph.D. and Michael Robin-son, Ph.D. for their help in designing the pain assessmentprotocol, and Bryan Hains and Venkatesh Nonabur, M.D.for technical support. This work was supported by NIHclinical research center grant RR00082, University ofFlorida McKnight Brain Institute and College of Medi-cine, the Mark F. Overstreet and C.M. and K.E. Over-street chairs in spinal cord regeneration, and the Foun-dation for Physical Therapy.

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Address reprint requests to:Edward D. Wirth III, M.D., Ph.D.

Department of NeuroscienceUniversity of Florida Brain Institute

P.O. Box 100244Gainesville, FL 32611-0244

E-mail: [email protected]


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feasibility and safety of neural tissue transplantation in patients with syringomyelia

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