Reconstruction of the transected cat spinal cord following NeuroGel (TM) implantation: axonal...

Int. J. Devl. Neuroscience 19 (2001) 63–83

Reconstruction of the transected cat spinal cord followingNeuroGel™ implantation: axonal tracing, immunohistochemical

and ultrastructural studies

Stephane Woerly a,*, van Diep Doan a, Norma Sosa b, Jean de Vellis b,Araceli Espinosa b

a Organogel Canada Ltee, 1400 Parc Technologique Bl6d, Quebec, Canada G1P 4R7b UCLA, 760 Westwood Bl6d, Los Angeles, CA 90024, USA

Received 3 May 2000; received in revised form 12 October 2000; accepted 18 October 2000

Abstract

This study examined the ability of NeuroGel™, a biocompatible porous poly [N-(2-hydroxypropyl) methacrylamide] hydrogel,to establish a permissive environment across a 3 mm gap in the cat spinal cord in order to promote tissue reconstitution andaxonal regeneration across the lesion. Animals with NeuroGel™ implants were compared to transection-only controls andobserved for 21 months. The hydrogel formed a stable bridge between the cord segments. Six months after reconstructive surgery,it was densely infiltrated by a reparative tissue composed of glial cells, capillary vessels and axonal fibres. Axonal labelling anddouble immunostaining for neurofilaments and myelin basic protein, showed that descending supraspinal axons of the ventralfuniculus and afferent fibres of the dorsal column regenerated across the reconstructed lesion. Fifteen months after reconstructivesurgery, axons had grown, at least, 12 mm into the distal cord tissue, and in the rostral cord there was labelling of neurons ofthe intermediate gray matter. Electron microscopy showed that after 9 months, most of the regenerating axons were myelinated,principally by Schwann cells. Newly formed neurons presumably from precursor cells of the ependyma and/or migrating neuronswere observed within the reparative tissue after 21 months. Results indicate that functional deficit, as assessed by treadmilltraining, and morphological changes following double transection of the spinal cord can be modified by the implantation ofNeuroGel™. This technology offers the potential to promote the formation of a neural tissue equivalent via a reparativeneohistogenesis process, that facilitates and supports regenerative growth of axons. © 2001 ISDN. Published by Elsevier ScienceLtd. All rights reserved.

Keywords: NeuroGel; Axonal tracing

www.elsevier.nl/locate/ijdevneu

1. Introduction

There has been considerable interest in trying toreverse the deficit induced by transection of the spinalcord by restoring neural pathways across the lesion.Because recovery depends on regrowth and reconnec-tion of nerve fibres, the major question is whether it ispossible to reconstitute the cellular components of thetransection site of the spinal cord, so that regrowingaxons have an appropriate micro-environment for cell(neuron-glial) and molecular interactions (accessibility

of neurotrophic molecules) that are required for elonga-tion. Although the inability of the transected adultmammalian spinal cord to regenerate after injury hasbeen demonstrated on a large number of experimentalmodels (Fernandez et al., 1991), recent cellular andmolecular studies have highlighted its potential forrepair following injury. Injury triggers complex cellularhealing mechanisms including cellular invasion and re-organization at the lesion site (Guth et al., 1985; Beattieet al., 1997; Brook et al., 1998), ependymal cell prolifer-ation and differentiation (Johansson et al., 1999;Namiki and Tator, 1999), axonal regeneration andsprouting of intact axons (Richardson et al., 1980; Liand Raisman, 1997), remyelination of axons (Salgado-

* Corresponding author. Tel.: +1-418-6504478; fax: +1-418-6504479.

E-mail address: [email protected] (S. Woerly).

0736-5748/01/$20.00 © 2001 ISDN. Published by Elsevier Science Ltd. All rights reserved.PII: S 0 7 3 6 -5748 (00 )00064 -2

S. Woerly et al. / Int. J. De6l Neuroscience 19 (2001) 63–8364

Ceballos et al., 1998), and upregulation of myelin basicprotein by reactive oligodendrocytes (Bartholdi andSchwab, 1998). In addition, axotomy triggers a neu-ronal regeneration program for the surviving neurons,which includes the activation of genes for severalproteins associated with axonal outgrowth and cy-toskeleton formation (Schwaiger et al., 1998). Likewise,molecules that influence axonal guidance and neuronalconnectivity are re-expressed in the adult regeneratingCNS, suggesting that axotomized neurons can partiallyre-activate their ontogenic program by recapitulatingsome molecular processes of axonal development andconnectivity (Ard et al., 1993). Finally, neurotrophicmolecules are released by the reactive tissue borderingthe lesion (Blottner and Baumgarten, 1994; Logan etal., 1994; Mocchetti et al., 1996). Nevertheless, undernormal conditions, the natural healing process, insteadof restoring structure and function to the lesionedspinal cord, results in fibrous tissue substitution andabortion of the regenerative growth of lesioned axons(Reier et al., 1983). The scar tissue does, however,exhibits some properties for axonal growth (Frisen etal., 1993; Risling et al., 1993). Therefore, there is a needto direct localized cellular regeneration events by creat-ing a suitable environment for migrating tissue repaircells and regenerating axons. This can be achieved byintroducing a three-dimensional substrate into the siteof injury, which provide a structural continuity acrossthe lesion (Woerly et al., 1990). This concept arisesfrom studies of the role of extracellular matrices (ECM)which provide a skeletal tissue framework in the con-struction of tissue structure during development, re-modelling and regeneration (Adams and Watt, 1993).ECM form macromolecular networks of glycoproteinsand heteropolysaccharides which, in living tissue, formhighly hydrated gel-like structures due to interactionswith extracellular fluids. These ECM provides a hy-drated environment for cell proliferation and supracel-lular organization, and for diffusion of neuroactivefactors essential for cell survival and differentiation.Comparative anatomy of CNS regeneration in adultnon-mammalian vertebrates shows the importance ofECM for successful repair and axonal regeneration.For example, following spinal cord transection andprior to the migration of regenerating axons, a pre-ex-isting cellular/extracellular framework is elaborated atthe site of injury. This is formed by reactive ependymaland glial cells prior to the migration of regeneratingaxons. These cellular/extracellular matrices give polar-ity to axons and allow growth across and beyond theinjury site, and this allow some degree of functionalrecovery (Larner et al., 1995; Chernoff, 1996). Accord-ingly, and with an obvious clinical perspective, mucheffort has been directed towards providing a substrateto facilitate cellular reconstitution and to promote neu-ral growth across the cut spinal cord, so allowingreconnection of lost axonal pathways.

Various materials have been used to experimentallybridge spinal cord lesions and to establish a growth-promoting environment for developing axons. Collagenmatrices, either alone (Marchand and Woerly, 1990;Spilker et al., 1997) or in combination with neuroactiveagents (Goldsmith and de la Torre, 1992; Howeling etal., 1998) or cell grafts (Bernstein and Goldberg, 1986),have been used to bridge transected rat spinal cord.Tubes, cylinders and guidance channels of poly (acry-lonitrile–vinylchloride) (Xu et al., 1995), polycarbonate(Harvey et al., 1994; Montgomery et al., 1996), nitrocel-lulose (Harvey et al., 1993), poly (a-hydroxyacids)(Gautier et al., 1998) or silicone (Borgens, 1999), whichcan contain Schwann (Xu et al., 1995) or olfactory cells(Ramon-Cueto et al., 1998), or electrodes (Borgens,1999) have also been developed. These promote variousdegree of axonal regeneration from spinal segments.The water-soluble polymer, polyethylene glycol, hasalso been proposed for the reconnection of the tran-sected spinal cord (Shi et al., 1999).

Hydrogels form another group of potentially usefulsubstrates. They consist of a crosslinked network ofhydrophilic co-polymers that swell in water, in culturemedium or in biological fluids, and which may providethree-dimensional substrates for cell attachment andgrowth (Woerly et al., 1996). Hydrogels are well toler-ated by living tissues and have been already used in awide range of biomedical applications (Ratner andHoffman, 1976; Corkhill et al., 1990). Because of theirability to retain a substantial amount of water withrespect to network density, hydrogels allow the trans-port of small molecules. In addition, viscoelastic be-haviour, low interfacial tension with biological fluids,and structural stability make porous hydrogels suitablefor implantation in soft tissue. Recently, pioneeringwork in the application of biocompatible polymer hy-drogels as prosthetic devices with neural specificity,which encourage tissue repair and axonal regenerationhas been reported (Woerly et al., 1990; Woerly, 1997).It has been shown that these polymer hydrogel ma-trices, by providing a structural continuity across le-sions of the brain and spinal cord, encouraged tissueformation that form a suitable environment for axonalregrowth (Woerly et al., 1990, 1998, 1999a). We havedeveloped such hydrogels with physical and chemicalcharacteristics that match the biological requirementsfor tissue formation (Woerly, 1996, 1997). More re-cently, we synthesized NeuroGel™ a biocompatiblehydrogel of poly [N-2-(hydroxypropyl) methacry-lamide] (PHPMA) for structural and functional repairof damaged area of the central nervous system (CNS).This hydrogel has a multimodal pore size distribution,comprising micropores (B2 nm), mesopores (\2 nm–B50 nm) and macropores (\50 nm B300 mm) (Wo-erly et al., 1998, 1999a). Micropores allow the transportof small molecules, and mesopores the transport of

S. Woerly et al. / Int. J. De6l Neuroscience 19 (2001) 63–83 65

larger molecules, while the macropores allow both freetransport of large molecules and migration of cells andblood vessels. The pore structure of this hydrogel per-mits the establishment of an interface with the hosttissue, which allows mobilization of host tissue cells andstimulation of tissue remodelling and organizationwithin the polymer network. The tissue-building prop-erties of NeuroGel™ rely also on its large surface tovolume ratio and its high fractional porosity (Woerly etal., 1998). In addition, diffusion properties of this hy-drogel allow it to maintain a physiological environmentin the implantation site, facilitating diffusion of neuro-trophic molecules that are released from the reactivecells of host CNS tissue (Woerly et al., 1999a). Otherimportant features of NeuroGel™ are its viscoelasticcharacteristics, its low interfacial tension for biologicalfluids, and its structural stability. These characteristicshave been shown to favour tissue formation and axonalregeneration within the polymer matrix in the rat spinalcord following transection (Woerly et al., 1998, 1999a)or compression injury (Peduzzi et al., 1999), and in therat brain after lesioning the fimbria–fornix pathway(Duconseille et al., 1998).

The present study, which continues our previouswork, aimed to investigate the potential of NeuroGel™to promote tissue restoration within, and axonal regen-eration across, the transected cat spinal cord. We usedaxonal tracing, immunocytochemical and ultrastruc-tural techniques, and a study of recovery of treadmilllocomotion. We provide evidences of the beneficialeffects of NeuroGel™ on tissue repair within the spinalcord. This hydrogel: (i) provided a bridge across the

lesion cavity; (ii) promoted and sustained tissue forma-tion and angiogenesis; (iii) supported and directed re-generating ascending and descending axons through thereconstructed lesion; and (iv) allowed axonal remyelina-tion. In addition, tissue restoration and axonal regener-ation was accompanied by functional recovery that wasassessed by treadmill training. Reparative surgery usingNeuroGel™ opens new possibilities for reconstructionand regeneration of CNS lesions by inducing and sus-taining endogeneous cell regeneration processes. A pre-liminary account of this work has been presented inabstract form (Woerly et al., 1999b).

2. Experimental procedures

2.1. Materials

Biotinylated dextran amine (BDA) was obtainedfrom Molecular Probes, Oregon (USA). Wheat GermAgglutinin-Horseradish Peroxidase (WGA-HRP) andVectastain ABC Elite kit were purchased from VectorLaboratories, Burlingam (USA). 3,3%-diaminobenzidinetetrahydrochloride (DAB, Sigma Fast), antibodiesagainst Neurofilaments (NF) and Glial FibrillaryAcidic Protein (GFAP), and Normal Goat Serum werefrom Sigma Chemicals (Canada). Microkitt was fromM.E.C.A. Ltee, Montreal (Canada). Triton X-100 andTri-buffer from Laboratories MAT, Quebec (Canada).Antibodies against Myelin Basic Protein (MBP) andNeurofilament 160 were purchased from BoehringerManheim (Germany).

2.2. Surgery

Twenty-six, 5-month-old, female cats (average weight2.5 kg), were obtained from Liberty Research Inc.(New York). Animals were identified by a tattoo on theinner face of the ear. On arrival, all animals wereexamined by a qualified veterinarian. Cats were vacci-nated against feline rhinotracheitis, calici andpanoleukopenia. In order to accustom the animals tothe laboratory environment, an acclimation period of 3weeks was allowed before the initiation of the experi-ment. The animals were housed in a 150 sq. ft gangroom (21°C, humidity 45–60%, light cycle: 12 h photo-period, 15 changes of air/h).

2.2.1. Preoperati6e careCats were assigned to the following treatment

groups: one shame operated, eight injury only controls,and 16 experimental cats that received NeuroGel™implants (Table 1). One cat was left intact as ananatomical control for axonal labelling and immunocy-tochemistry. Prior to surgery, animals were given acomplete veterinary health check. The cats were pre-

Table 1Group protocol

Number ofGroups Surival timeanimal (Months)

Shame surgery control 1 17

Spinalization 2 0a

1 61 91 15

172191

Spinalization and 0a26implantation of 1

NeuroGel™ 2 91 131 155 172 192 21

a Animals lost during the surgery procedure.


medicated (i.m.) with Ketamine hydrochloride (13 mg/kg), Butorphanol (0.3 mg/kg) and Atropine Sulphate(0.05 mg/kg) to prevent excessive salivation induced byKetamine, and sedated with Acepromazine Maleate(0.3 mg/kg) to reduce the bradycardial effects of Ke-tamine and to provide muscle relaxation. Anaesthesiawas induced with isoflurane by endotracheal intubation(rate 0.6–2 l/min, concentration 0.5–4%). Once anaes-thetised, fur was shaved from the surgical site and theskin washed with hexachlorophene soap and rinsedthoroughly with water. The surgical area was scrubbedwith 70% isopropyl alcohol and then with Betadine(10% iodine). Animals ready for surgery were placed onsterile drapes over a heating pad. Surgery was carriedout under sterile conditions. An i.v. drip of LactateRinger’s solution was maintained throughout surgeryto maintain an open line for injection and for hydrationduring surgery (10 ml/kg/h).

2.2.2. Spinalization and implantation of NeuroGel™A dorsal midline incision was made from T5–T10

and the paravertebral muscles retracted. A laminectomywas performed at the junction T6–T7 in which thespinous processes were removed. The laminectomy wasextended laterally to the articular surfaces that were leftintact. Bipolar electrocauterization was used to mini-mize bleeding of the peridural veins. Surgicel was usedfor hemostasis. The dura was opened longitudinallyalong the midline under a Leica surgical microscopeand retracted laterally to expose the dorsal surface ofthe cord. Prior to transection, the spinal cord wasirrigated with chilled saline solution and the animalgiven Mannitol 20% (30 ml, i.v.) and corticosteroide(Solu-Delta-Cortef 15 mg/kg, i.v.). The spinal cord wastransected using microscissors at two points, approxi-mately 3 mm apart, and the segment was removedusing a microdissector. The spinal ventral artery wasnot preserved. A NeuroGel™ implant was inserted intothe lesion cavity using ophthalmic sponges and thenhydrated with saline solution. For controls the cavitywas left empty. In both treatment groups, the dura wassutured using 6-0 monofilament nylon and a duraplastywas performed using Gelfim. A fixature made ofmonofilament surgical steel wire was used to providedynamic fixation of the vertebral column, while avoid-ing involvement of the spinal canal. This structureformed an S-shaped loop between the bases of the T5and T8 spinal processes. The loop was fastened to thespinal column in order to prevent retraction of thespinal stumps. All manipulations were recorded with avideo camera attached to the surgical microscope.

2.2.3. Postoperati6e careFollowing reconstructive surgery, animals were kept

under heat lamps for 12–24 h and respiration rate,temperature and reflexes were monitored. Cats were

then returned to the colony and were rehydrated for 2days with Lactate Ringer’s solution (20 ml i.v.). Blad-der evacuation was done manually. Cats were placed ondry pads for comfort and for absorption of urine.When handling, care was taken to support and keep thespine in anatomic alignment to prevent trauma to thesurgical site. In order to prevent swelling of the spinalcord, cats received Mannitol 20% (30 ml, i.v.) 30 minand 3 h after surgery. Prednisolone orally (4 mg/kg, peros), and Buprenorphine (0.01 mg/kg, s.c., twice) wereadministered to reduce pain. The antibiotic Clavamoxwas given orally (62.5 mg). From the second day,animals were given Prednisolone orally twice a day for2 weeks and then once a day for 1 week. Clavamox,was given twice a day for 1 week. When it occurred,pyuria was treated with Lactate Ringer (60 cc, s.c.,twice a day for 2 days), Clavamox (62.5 mg, per os),and Prednisolone (5 mg, twice a day for 10–15 days)without further complications.

Cats underwent physiotherapy which consisted oftreadmill exercising of the hindlimbs to maintain theintegrity of the neuromuscular system below the level ofthe lesion. The ankle joints were manipulated throughtheir full range of movement to prevent ankylosys, andpassive exercises of flexor–extensor muscles of thehindlimbs were performed. Throughout the treatmentand recovery period, animals were examined twice aday for any signs of illness or potential reaction to thetreatment.

2.3. Treadmill training

All animals were trained to walk on a motor-driventreadmill at a speed 0.1–0.4 m/s for 3 weeks prior tospinalization. Two days after spinalization, daily exer-cise on the treadmill was re-initiated. The forepaws ofthe cat were placed on the treadmill belt, while theanimal was stabilized by a mesh jacket around thehindquarters. Each cat was exercised for up to 5 min at0.1 m/s. Stepping was initiated by movements of thetreadmill belt at speeds between 0.1 and 0.4 m/s. Step-ping could be enhanced by sensory stimuli, such assqueezing the tail, rubbing the perianal region, squeez-ing the abdomen and/or pinching the leg or foot. Thelength of each session, and the speed of treadmill (up to0.4 m/s) was gradually increased. Subsequently, the catswere tested for their ability to perform full-weight-bear-ing steps, and the highest speed they were able toaccommodate was recorded. When the mesh jacket wasremoved, the animal was supported by holding the tailabove the level of the hip joints to provide assistance inmaintaining lateral stability if needed, but for maxi-mum benefits it was important that as little verticalsupport as possible was given during the stepping. Noother assistance was permitted. Treadmill training wasexpected to maintain the integrity of the neuromuscular


system below the level of the lesion. Functional analysisof the injured and grafted animals was assessed usingsix separable states based on Blight (Blight, 1983):1. those unable to stand spontaneously or to move the

hindlimbs in a functionally related manner;2. those capable of standing spontaneously on their

knees with weight supported movements of theirhindlimbs but without sustained locomotorybehaviour;

3. those capable of standing spontaneously on theirknees with weight supported movements of theirhindlimbs with sustained and co-ordinated locomo-tory behaviour;

4. those capable of standing on their hindlimbs andwalking spontaneously with external support andoccasional co-ordination with nonetheless obviousabnormalities of gait;

5. those capable of standing on their hindlimbs andconsistent weight supported plantar stepping withoccasional external support only, and consistent co-ordinated gait;

6. those capable of locomotory behaviour without ob-vious deficits.

2.4. Anatomical study

Anatomical study of spinal cords included axonallabelling, histochemistry, immunocytochemistry andtransmission electron microscopy. These were carriedout from 6 to 21 months after surgery (see Table 1).

2.4.1. Axonal labellingThe ability of ascending and supraspinal descending

fibres to enter, cross and leave the NeuroGel-recon-structed region of the spinal cord were assessed usinganterograde (BDA, 5%) and retrograde (WGA-HRP4%) axonal labelling. A total of 10 cats were used foraxonal tracing at the following time points after recon-structive surgery: 15 months (one experimental cat andone control), 17 months (three experimental cats), 19months (two experimental cats and one control) and 21month (one experimental cat). This included also oneintact 15 month-old cat. The selected animals wereanaesthetised and a laminectomy performed as de-scribed above, at the same level as the previous surgery.To label ascending axonal fibres WGA-HRP was pres-sure injected at four sites, using a 100 ml Hamiltonmicrosyringe fitted with a 30 G

12 needle secured in a

stereotaxic frame. The tracer was delivered bilaterallyinto the dorsal funiculus at 1.5 mm from the midline, at2–3 mm caudal to the NeuroGel/host junction, and toa depth of 1.5–2 mm vertically. A total of 3 ml ofWGA-HRP were injected, at a rate of 7.5 ml/min. Inorder to prevent leakage of tracer along the penetrationtrack, it was allowed to penetrate for 5 min beforewithdrawal of the micropipette. Supraspinal descending

pathways of the ventrolateral funiculus were labelled bylowering the syringe 4 mm vertically into the spinalcord, 2–3 mm rostral to the NeuroGel/host junctionand 1.5 mm from the midline. A total of 20 ml of BDA(in saline solution of 0.9%) was pressured injectedbilaterally, at a rate of 5 ml/min. The aspect of thevarious spinal cords was recorded using a video cameraattached to the surgical microscope.

After 72 h animals were anaesthetised with Ketamine(i.m.), perfused intracardiacally with 1 l of a cold salinesolution (0.9%) containing heparin (1:500), followed by1 l of a fixative solution (4% paraformaldehyde and0.4% glutaraldehyde in 0.1 M phosphate buffer (PB) atpH 7.4). The spinal cord was dissected out from thevertebrate column and their envelops, and a segment ofapproximately 3 cm including the implantation site wascut for anatomical analysis. The spinal cord segmentswere inspected under a Tesovar microscope at 6.5×magnification. After 24 h post-fixation in the samefixative, the cords were transferred to a 30% sucrosesolution in PB for 24 h at 4°C, rinsed in PB, andembedded in 25% gelatine. The blocks were hardenedat 4°C, and then kept in 10% formalin solution untilsectioning. For each spinal cord, a complete series of 30mm horizontal sections were cut from the ventral to thedorsal surface on a freezing microtome and collected in24-well plates with PB. For the staining of BDA-la-belled axons, selected sections were incubated overnightin avidin-biotin-peroxidase solution (Vectastain ABCElite kit) in 0.1 M PB with 1% bovine serum albuminfraction V and 0.1% Triton X-100 at 4°C, and washedin 0.1 M PB (2×15 min) and then in Tris buffer (0.05M, pH 7.6, 2×15 min). The reaction product wasdetected using 0.05% 3,3%-diaminobenzidine tetrahy-drochloride in Tris–HCl buffer with 0.02% CoCl2, for25 min at room temperature. The reaction was stoppedby washing in Tris–HCl buffer (2×10 min). For HRPhistochemistry, the tissue sections stained for BDA andadjacent unstained sections were washed (2×10 min)in PB and incubated in a solution of 3% hydrogenperoxide. After washing in PB (3×10 min) and then inTris–HCl 0.05 M (2×10 min), the tissue sections wereincubated for 10–15 min at room temperature in 3,3%-diaminobenzidine tetrahydrochloride until a dark reac-tion product could be detected in the sections. Thesections were mounted on gelatine-coated slides, airdried at room temperature, coverslipped using Mi-crokitt, and examined using Nomarsky optics (Zeiss).

For quantification of axonal density of supraspinaldescending tracts, tissue sections were selected fromthree anatomically distinct levels of the spinal cord.Level A: ventral and ventrolateral funiculi that includethe reticulo-vestibulo- and tectospinal pathways; levelB: lateral white matter that include propriospinal nervefibres; level C: dorsolateral white matter that includecortico- and rubrospinal pathways.


For each level, axon density was determined bycounting the intersections of DAB-labelled axons witha straight line 3 mm rostral to the NeuroGel implantjunction (or proximal-transection site junction in tran-sected-only controls); 3 mm caudal to the NeuroGelimplant (and distal to transection site in transected-onlycontrols), and within the implantation site on horizon-tal sections using a 20× objective.

2.4.2. HistochemistryGelatin-embedded tissue sections from animals in-

jected with BDA and WGA-HRP were evaluated histo-logically using the Mallory-PTAH technique for glia,the Holmes technique (silver staining) to detect nervefibers and Hematoxylin–Phloxin–Safran (HPS) forgeneral pathology. Spinal cords that were not used foraxonal tracing were embedded in paraffin and serialsections cut on a rotatory microtome at 10 mm andstained using the above technique. Certain paraffin-em-bedded sections were also processed for immunocyto-chemical analysis (see below).

2.4.3. ImmunocytochemistryThirty micron, free-floating, longitudinal sections of

spinal cord that had been injected with BDA andWGA-HRP were processed for immunocytochemistryusing neurofilaments (NF) and Glial Fibrillary AcidicProtein (GFAP) antibodies. Selected sections werewashed in PB (3× 15 min) and permeabilized for 30min at room temperature in PB containing 0.2% TritonX-100. They were then incubated and blocked with PBcontaining 0.2% Triton X-100 and 1% Normal GoatSerum at room temperature for 30 min. Incubationwith the primary antibodies rabbit anti-GFAP (1/100)and rabbit anti-NF 200 (1/500), were carried out for 24h and 48 h at 4°C, respectively. After washing in PB(3×15 min) and in PB plus 0.2% Triton X-100 (30min), sections were incubated with the secondary anti-body, TRITC conjugated goat anti-rabbit (1/80), for 3h at room temperature. After washing in PB (3×15min), sections were mounted on slides and coverslippedwith Citifluor.

For double immunocytochemical labelling againstNF and MBP, paraffin embedded sections of nineadditional spinal cords, including two control-injuredonly and one intact cord, were used. Sections wererehydrated and rinsed three times in PB prior to im-munocytochemical analysis. The primary antibodies in-cluded a monoclonal, a rabbit anti-rat anti-MBP(1/100), and an anti-NF 160 (1/100). Normal mouseand rabbit sera were used as negative controls. Fluores-cein iso-thiocyanate conjugated goat anti-mouse IgGwas used as the secondary antibody to show NF local-ization (green fluorescence) and Texas Red goat anti-rabbit antibody was used to show MBP (redfluorescence) (Espinosa de los Monteros et al., 1997).

Immunostained sections were examined with a NikonFX microscope equipped with appropriate filters andbarriers.

Detailed analysis of the immunostained sections wasperformed using a Biorad confocal microscope. Imageswere acquired using a single channel to visualise eitherthe Texas red, TRITC or Fluorescein label. For doubleimmunostaining, the two images were merged and re-sized for preparation of the composites. All imageswere taken using a fluorescence/phase-2 objectiveNikon microscope, 20× .

2.4.4. Transmission electron microscopySpinal cords used for axonal labelling and/or im-

munocytochemistry were also used for ultrastructuralstudies. For each spinal cord (eight from the experimen-tal group and two controls) a small tissue block (ap-proximately 3×1×1 mm) was excised from thereconstructed tissue, post-fixed in 2.5% glutaraldehydein 0.1 M sodium cacodylate for one hour at roomtemperature, dehydrated in acetone, and embedded inEpon 812. Semi-thin sections (1 mm) were cut with aReichert-Jung NOVA ultramicrotome and stained withtoluidine blue, and ultrathin sections of selected areaswere contrasted with uranyl acetate and lead citrate.Observations and photomicrographs of selected ultra-thin sections were made with a JEOL-1200 CX electronmicroscope operating at 60 kV.

3. Results

3.1. Spinalization and NeuroGel implantation

Transection of the spinal cord produced two cleansections 2–3 mm apart, and separated a tissue fragmentof approximately of 70–110 mm3 (Ard et al., 1993).However, there was some variability in the operativeprocedure, which was due to excessive bleeding andfastidious hemostasis in some animals, or when theexcised segment was removed in fragments, in bothcases additional manipulations of the spinal stumpswere necessary. The NeuroGel™ implant adhered veryrapidly to the cut surface of the spinal cord providing asolid and intimate contact with the ends of the spinalstumps. This restablished the anatomical continuity ofthe organ, and because of its viscoelastic and bioadhe-sive properties, the gel molded into the shape of theimplantation cavity and entirely filled the volume avail-able. Twenty-four hours after surgery, the NeuroGel™was well approximated to the host spinal cord and hadmerged with the cut surface of the spinal stumps (Fig.1A). After surgery all cats showed a pronounced loco-motor and postural deficit of the hindquarters, and theywere unable to support their hindquarters or walk withtheir hindlimbs. All cats were ambulatory, though with


Fig. 1. (A) Aspect of cat spinal cord 24 h after reconstructive surgery.The transection at T7 (arrows) is filled with NeuroGel™ which hasanchored to both spinal stumps, and merged with the surface of thehost neuropile. (B) Aspect of the spinal cord 13 months after repara-tive surgery using NeuroGel™. The dura (d) is opened longitudinally,showing the dorsal surface of the transection/implantation site. Thetransection is no longer discernible (compare with Fig. 1A) and thespinal cord has a near normal appearance. Arrows show the suture ofthe dura matter. Scale bars: A and B=2.5 mm.

perineal or abdominal cutaneous stimulations, or bygently squeezing the tail. Cats in the experimentalgroup recovered variable degrees of locomotion: threefell into category 2, three into category 3, one cat intocategory 4, and two cats into category 5. The latter twoshowed the highest level of achievement in locomotioncapability. Eight months after reconstructive surgery,they displayed quadrupedal locomotion at speeds of upto 0.4 m/s. These cats were able to walk for at least 10min on the moving belt without the need of stimula-tion. They showed good co-ordination of the homolat-eral fore-hindlimb coupling and had control of theequilibrium of their hindquarter needing little supportof the tail. Videotape analysis indicated that locomo-tion of the four limbs had a near-normal gait comparedto that of an intact cat, with proper paw placement onthe plantar surface when standing. The other animalsreached speed of 0.2 m/s, and plateaued after 9–11months. In some individuals, the hindlimbs were fullyextended or unusually flexed and assistance in main-taining balance of their hindquarters, by holding thetail, was necessary. The shame operated cat fell intocategory 6, showing normal quadripedal locomotion onthe treadmill. After injection of BDA and HRP aboveand below the gel implant, the locomotion of these catsbecame visibly impaired or lost. The volumes injectedwere sufficient to cause focal axotomy and limitedtissue necrosis as shown by histology.

3.3. Gross pathology

Because a laminectomy was performed at the site ofthe previous surgery in order to inject the tracers, thelesioned/reconstructed spinal segment could be in-spected in the living animal under the surgical micro-scope. The spinal cords were examined 6–21 monthsafter the reconstructive surgery. The dorsal surface ofthe spinal cord showed thickening of the dura matterand a normal aspect of the spinal tissue immediatelyadjacent to the reconstructed segment. In particularthere was no atrophy of the spinal stumps. In all cases,a high degree of integration between NeuroGel™ andthe host neuropil was achieved. When compared toimages recorded at the time of implantation, the Neu-roGel™ structure was no longer apparent and therostral and caudal polymer/spinal cord junctions weredifficult to distinguish. As a result, the reconstructedspinal cord segment showed a macroscopic aspect thatwas comparable to that of an intact spinal cord. Newvascular connections were observed at the surface ofthe transected/reconstructed spinal cord as well aswithin the gel. In contrast, control spinal cord wasfragile, irregular in shape, showing a pronounced atro-phy and extrusion of cystic cavities.

Macroscopic examination of the fixed spinal cordsegments revealed a thickening of the dura matter by

their forelimbs only. Four cats died either during orshortly after surgery, and during the study some catshad pyuria with in a few cases, hematuria. After treat-ment, animals recovered without complications.

3.2. Treadmill training

To evaluate post-surgical improvement, 14 cats (oneshame, four controls and nine experimental) partici-pated in treadmill exercises. The other cats were elimi-nated from treadmill training because of aggressivebehaviour and/or no participation. The performance ofcats during the treadmill training was a function oftheir motivation, and variability in performance for asingle animal was frequently observed during a giventraining period. Evaluation of cats in the control groupshowed that animals fell into category 1 or 2: they wereonly able to ambulate with their forelimbs and draggedthe hindlimbs, with the knee and ankle in extension andthe hip in flexion. Typically, the hindlimbs steppedspontaneously in an alternate fashion even in resting.However, stepping pattern was frequently elicited by


dense fibrous tissue, and leptomeninges were adherentto the dorsal surface of the reconstructed cord segment.After opening the dura matter the reconstructed spinalcord segment showed restoration of the spinal cordcontinuity. Careful examination of spinal cords confi-rmed that the lesion/implantation site was occupied bya tissue replacement, and that the structure of the gelwas no longer discernible (Fig. 1B). On both sides ofthe reconstructed lesion, the cord showed a normalaspect with the usual anatomical landmarks. Spinalcord from control group animals showed a markedatrophy and deformation by a dense scar tissue andmulticystic cavities.

3.4. Histology

The extent of the transection was verified on paraffinembedded horizontal tissue sections stained with HPS,at low magnification. On average, the transection sitemeasured between 2 and 2.5 mm ventrally, and 3 and 4mm dorsally. However, it was the final aspect of thelesion that was examined, and therefore, it did notnecessarily reflect the dimensions of the initial trauma.At 6 months post-surgery (the earliest time point exam-ined) and at later time points, hydrogel grafted spinalcord showed the reconnection of the spinal stumps bythe NeuroGel™ implant and the associated newlyformed tissue (Fig. 2B). The appearance of the spinalcords at later time points was similar. In a few casesthere was formation of small cavities in the tissueadjacent of the experimental cords, but in no cases didthis occur at the level of the reconstructed spinal seg-ment. This contrasts markedly with control spinal cord,which showed formation of dense collageneous scartissue oriented perpendicularly to the longitudinal axisof the spinal cord, dividing the transection site by arostral and caudal cavity (Fig. 2A). Formation of large,ball-like cavities was frequently observed in the spinalstumps. These cavities were lined with thin layer of glialtissue, connected to the central canal and extended intoboth spinal stumps. In two cases, implantation of Neu-roGel™ failed, resulting in a poor integration of thepolymer with the concomitant formation of scar tissuearound the implant.

Microscopy examination of horizontal, Mallorystained, paraffin sections at the mid-point of the Neu-roGel™ implant showed that the polymer containedmainly glial and vascular features. Three zones could bedistinguished rostro-caudally: (1) a central zone thatwas characterised by the presence of polymer aggre-gates on a collagen background, newly formed bloodvessels, interlaced neuroglia processes and disseminatedcells (meningeal cells and fibroblasts); (2) an intermedi-ate zone containing polymer aggregates and a vascular-ized glio-connective tissue; and (3) the junctional zoneswith the rostral and caudal spinal stumps characterised

by reactive changes in the host tissue. The latter in-cluded intense cell proliferation, predominantly of hy-pertrophied stellate astrocytes with elongated processes,that infiltrated the implantation site. From both inter-faces, darkly blue stained glial fibres formed a loose-to-dense network around the polymer aggregates andfollowed the microgeometry of the polymer network. Amild foreign body giant cell reaction was also observed.The histological features were essentially the same atlater time points after reconstructive surgery. Therewere striking differences with control injured-onlyspinal cord, that showed changes of the microarchitec-ture of the spinal segments proximal to the site ofinjury. The histologic hallmarks included cell rarefac-tion, vacuolization of the white matter, disorganizationof the gray matter, axonal degeneration and presence of

Fig. 2. Low-power photomicrographs of HPS-stained horizontal tis-sue sections of a 15 month-old transected-only spinal cord (A), and a13 month-old spinal cord after complete transection and reconstruc-tion with NeuroGel™ (B). (A) Histopathological features of controlspinal cord showing formation of a dense collageneous scar tissue(Sc) transversally filling the lesion cavity and cystic cavities (asterisks)in the adjacent spinal segments and degeneration of the white andgray matter. Note the dilatation of the ependymal canal (arrows), andthe pale, non-uniform staining of the white matter, which is indicativeof the severity of the post-traumatic degenerative changes. (B) Thetransection site (arrows) is occupied by a reparative tissue that hasdeveloped from the host neuropile into the polymer implant. Note theabsence of cavitation and the integrity of the white and gray matterin the adjacent cord segments (uniform staining). Asterisk is anartefact during sectioning. Scale bars: A and B=1.5 mm.


Fig. 3. Holmes-stained horizontal sections of spinal cord 17 monthsafter reconstructive surgery with NeuroGel™. (A) Stained perikariaof neurones located in the gray matter immediately adjacent to thecaudal junction of the transection/implantation site (S) and the hostneuropile, showing a normal appearance and long processes (arrows)which have crossed the junction zone and entered the reparativetissue. Other neurites from neurons which are out with the plane offocus are also seen extending into the implantation site. (B) Photomi-crograph taken at the midpoint of the reconstructed lesion with thegel implant, showing a tangle of regenerating nerve fibres. Scale bars:A=50 mm; B=25 mm.

sections containing the polymer implant revealed thepresence of numerous argyrophilic processes compat-ible with axonal regrowth into the reconstructed zone(Fig. 3B). These were often arranged in fascicles, andwere seen to follow paths in the reparative tissue andaround polymer aggregates.

3.5. Axonal labelling

The quality of BDA and HRP labelling was verifiedon the spinal cord of the intact animal. Tissue sectionstreated with cobalt-BDA yielded a profuse blue–blackanterograde labelling of axons of the ventromedial andventrolateral funiculi that carry the vestibulo- and theresticulospinal pathways and descending propriospinalaxonal fibres. BDA staining of fibres of the dorsolateralfuniculus was consistently observed as a consequence oflabelling of the cortico- and rubrospinal tracts. Thiswas due to the rhythmic breathing movements of thecat during the injection procedure. There was alsolabelling of the ventral and intermediate gray matter asshown by the retrograde labelling of spinal neurons ofthe different laminae. The core of the injection site wascharacterised by tissue necrosis surrounded by denselabelling of somata in the gray matter and their pro-cesses. WGA-HRP yielded a typical brownish-goldgranular reaction product which, except for the nu-cleus, filled the cell soma.

Of the seven animals in the experimental group,histology showed the lack of integration of the Neu-roGel™ implant and ineffective labelling in one spinalcord (Table 3). Among the remaining six spinal cords,that showed good integration of NeuroGel™, injectionof BDA resulted in adequate labelling of four spinalcords. However, for the two cases where labellingfailed, neurofilament immunostaining of adjacent sec-tions revealed immuno-positive axons at the implanta-tion site. Spinal cords were examined in a series ofhorizontal sections at the level of the rostral and caudalsegment, and at the implantation site.

Control injured only. Longitudinal sections of the twocontrol cats revealed axonal labelling in the rostral cordsegment but no labelling caudal to the transection site.Likewise, we did not observe HRP neuronal labelling inthe rostral gray matter. A representative example of acontrol spinal cord is shown in Fig. 2A.

Grafted spinal cord. Observations of sections troughthe ventro-lateral funiculus and rostral to the implanta-tion site, revealed a profuse labelling of small- andlarge-diameter axons that formed long unbranched andparallel tracts which extended into the ventral fu-niculus. Axon density was higher at the core of theinjection site and at the rostral reconstructed lesionjunction. This decreased gradually with distance fromthe injection site, however, a few long axons could befollowed up to the rostral extremity of the spinal seg-

numerous microcysts. Foamy (lipid-laden) microglialcells were also present in the interstices between surviv-ing axonal fibres. Throughout the scar, there werenumerous haemosiderin-laden phagocytes. The overallcytoarchitecture of the gray matter was disorganisedwith multiple ‘retraction ball’ formations of severedaxons. Immunostaining of longitudinal sections andconfocal microscopy showed that the degree and theextent of myelin damage were considerable (see below).In contrats, in some areas of the experimental cordsspinal motor neurons of the ventral horn proximal anddistal to the transection/implantation site were clearlyvisible without signs of chromatolysis, and could havesent processes across the interface into the recon-structed lesion (Fig. 3A). Also, the extent of myelindamage was less marked in the experimental spinalcords. In both animal groups, the central canal inregions adjacent to the injury/implantation site wasdilated and ependymal cells showed hypertrophy andproliferation. At higher magnification, silver stained


ment on serial sections. Interestingly, labelled bipolarcells with long fine processes extending along the longi-tudinal axis of the cord were frequently observed. Thesecells were isolated and branched along labelled axonaltracts of the white matter. Close to the rostral junctionof the lesion/implantation site, axon morphologychanged, showing small dilatations or multiple varicosi-ties along their segments, while other axons showedswellings suggestive of boutons en passant, and axonterminal varicosities or growth-cone-like structures. Ax-ons terminating with an endbulb that emitted a finesprout were also frequently observed. At this point,axon density was lower and the organization of theoriented bundles was lost. Labelled fibres were observedgiving rise to fine short and long collateral projections,some of these crossed the long rostro-caudal axis of thecord, while others entered the implantation site. On thesame sections, intracellular HRP labelling of neurons ofthe thoracic nucleus of Clarke and/or interneurons andlabelled axon collaterals were observed in the medialpart of lamina VII, rostral to the implantation site (Fig.4A). These neurons were multipolar or triangular-shaped and of medium size, around 20 mm, and ori-ented along the cranio-caudal axis of the spinal cord.Three experimental spinal cords exhibited labelled neu-rons in the intermediate gray matter, with a maximumof 14 HRP-labelled neurons in one individual. Someneurons were also retrogradely labelled after BDA in-jection, showing a Golgi-like clarity and extensive fillingof their dendrites. There was a gradual loss of DAB-la-belled axons at the level of the dorsolateral funiculus.This was due to the increased vertical distance from thesite of injection.

Observation of the implantation/reconstructed areaunder Nomarsky optics revealed the presence of poly-mer aggregates and stained axonal fibres. These oc-curred either individually or arranged in fascicles withgrowth cones (Fig. 4B). The fibres followed complexpaths around the polymer aggregates that resemble thepattern of the stroma of the ingrowing tissue observedin adjacent histological sections (e.g. Mallory staining).

At the level of the caudal segment of the spinal cord,long or short irregular projections of BDA-labelledaxons extended into the white matter of the ventral andventrolateral funiculi. The general staining patternshowed a gradual decrease in the number of labelledaxons in the white matter with increased distance fromthe junction with the reconstructed lesion. The highestdensity of axons was on ventral sections, and decreasedorsally to the dorsolateral funiculus. Fig. 4C–E showan example of axonal labelling from three differentregions of the caudal white matter. Close to the junc-tion with the lesion/implantation site, there was profuselabelling of axons leaving the implantation site (Fig.4C), these showed varicosities, terminal bulbs andgrowth cones. Moving further into the white matter,

half way between the junction with the lesion/implanta-tion site and the extremity of the spinal cord, individualaxons with terminal growth cones were observed (Fig.4D). These labelled axons could be followed up to theextremity of the caudal segment of the histologicalpreparation (approximately 12 mm) (Fig. 4E). Thisstaining pattern of regenerating descending axon tractswas observed on consecutive longitudinal sections, withan increased axonal density on the more ventral sec-tions. Retrograde BDA labelling of neuronal perikaryain the gray matter was observed on the same sections.

Heavily labelled neurons with profuse somato-den-dritic labelling were consistently observed throughoutthe intermediate gray matter at the level of laminaeVIand VII immediately adjacent to the caudal and to therostral interface. These had single or complex dendritictrees with a horizontotranverse orientation extendinginto the white matter (Fig. 4F). Labelled cells withnumerous fine longitudinal processes extending into thewhite matter were also frequently observed along thepath of regenerating axonal fibres. These cells were notobserved in intact spinal cord. Adjacent sections stainedwith silver (Holmes) indicated that not all ingrowingaxons that traversed the implantation site were labelled(see Fig. 3A).

Table 2 shows the number of BDA-labelled axons atthree levels through the spinal cord white matter. Tissuesections were selected on the basis of their quality andof the labelling when compared to an intact controlspinal cord that had been injected with BDA in theventral funiculus. This table includes the four experi-mental cats for which BDA injection yielded adequate

Table 2Counts of axons that were anterogradely labelled with BDA(Mean9SD) of the four spinal cords that showed adequate labelling(see Section 2 and Table 3)*

Longidutinal axisVentrodorsalaxis

Distal (n)ReconstructedRostral (n)lesion (n)

2797 (5)a 2698 (5) 3397 (5)Level A9295 (3)b n/a 103917 (3)42910 (3)c 59921 (3) 4597 (3)

n.d. n.d. n.d.

4296 (10)aLevel B 2796(10) 59912 (10)43910 (5)b 66920 (5)1094 (5)

23910(6)39911 (6)48912(6)c

1592 (4)d 2694 (4) 2598 (4)

44913 (4)aLevel C 1898 (4) 6798 (4)5396 (7)b 1793 (7) 3294 (7)84930(4)c 78917 (4) 1096 (4)2996 (8)d 2295 (8) 4798 (8)

* Survival time after reconstructive surgery: a, c, d, 17 months; b,15 months; n.d., not determined.


Fig. 4. A series of photomicrograph showing biotin dextran (BDA) labelling of spinal axons and HRP neuronal staining on longitudinal sectionsof 15 months after complete spinal cord transection and reconstruction using NeuroGel™ (A–F), and fluoresence immunocytochemistry for NFand GFAP 17 months after reconstruction surgery (G and H). (A) Example intracellularlabelling with WGA-HRP of neurons of the dorsalnucleus of Clarke (laminae VII). Note also labelled nerve fibres of the gray matter. (B) Anterograde axonal labelling by BDA of Supraspinaldescending axons extending into the implantation/reconstruction site. There are in contact with both the polymer aggregates and the associatednewly-formed tissue. (C) Photomicrograph taken 1 mm caudal to the reconstructed transection site, showing numerous BDA-stained axons withvaricosities and terminal bulbs that have entered the white matter of the distal cord segment and which are running in parallel. (D)Photomicrograph taken at the midpoint between the distal cord junction and the extremity of the spinal cord section, showing BDA-labelled axonsextending longitudinally in the white matter. (E) Photomicrograph taken at the extremity of the spinal cord section, 12 mm from the junction withthe reconstruction site showing labelled axons, one of which has a growth cone. (F) Heavily labelled neurones with profuse somato-dendriticlabelling, inimediately adjacent to the rostral junction with the reconstructed lesion, showing single or complex dendritic trees with anhorizontotranverse orientation and extending into the white matter. G and H are fluorescence confocal microscopy photomicrographs of sectionsinimunostained for NF and GFAP, showing numerous axons (G) and processes of reactive astrocytes (H) at the rostral junction of the cord withthe implantation/reconstruction site and extending into the site of tissue reconstruction. Scale bars: A=25 mm; C–E, G, H=50 mm; B, F=80mm.


Table 3Summary of axonal labelling and corellation with NeuroGel™ integration

Number of animals BDA labellingb (caudal segment)Integration of NeuroGel™a HRP labellingb (rostral segment)

+c +cIntact animal (1)−cControl (2) −c

4+d/2−c6+/1− 3+e/3−cNeuroGel™ (7)

a Integration means absence of intervening scar between the polymer implant and the host neuropile.b Evaluated on the six experimental spinal cords that showed good integration of NeuroGel™.c (+), integration and labelling; (−), lack of integration and of labelling.d Positive labelling at 15 and 17 months after reconstructive surgery.e Positive labelling at 17 months after reconstructive surgery.

axonal labelling. In all cases, axons were seen to crossthe reconstructed lesion site.

3.6. Immunocytochemistry

Tissue sections adjacent to those stained for BDAwere immunostained with NF and GFAP antibodies.There was a good correlation between BDA axonallabelling and neurofilament immunocytochemistry.Fluorescence microscopy showed numerous and in-tensely NF-immunostained axons from the rostral andcaudal lesion junctions entering or leaving the implan-tation site. Fig. 4G, shows an example of immunos-tained axons that entered the transected/reconstructedsite and extended throughout the reconstructed segmentas small fascicles. These followed a longitudinal direc-tion, however, some crossed the rostrocaudal axis ofthe spinal cords. Some of these axons emitted sproutsperpendicular to their cylindraxes. Immunoreactive ax-ons showed staining dots corresponding to multiplevaricosities along their length, and growth-cone-likestructures. This immunostaining pattern was observedon consecutive longitudinal sections through the ven-tro-dorsal diameter of the spinal cord. However, be-cause of their tortuous route, it was difficult to followaxons throughout the reconstructed site and thus todetermine if fibres re-entered the opposite spinal seg-ments. Nevertheless, retrograde labelling indicated thatregeneration of axons throughout the reconstructedlesion occurred. Strongly GFAP-immunostained astro-cytes were seen at both interfaces but a gradual loss ofthe immunostaining pattern from rostral and caudalreconstructed lesion junctions was observed, this wasdue to the limited extension of their processes withinthe reconstructed lesion (Fig. 4H). This immunostain-ing profile contrasted with Mallory-stained sections,which revealed the presence of a loose network ofastrocyte processes across the implant/reconstructed le-sion, that were moulded onto the geometry of thepolymer aggregates. For control spinal cord, there wasa strongly GFAP-positive glial tissue lining the transec-tion site which extended into the rostral and caudalspinal segments, while NF immunocytochemistry

showed that a few axons entered the distal zone of theglial tissue and extended some distance along the bor-der of the cavity (Table 3)

3.7. Double immunofluorescence and confocal imaging

The use of both anti-neurofilaments and anti-myelinbasic protein antibodies on the intact cat spinal cordclearly showed the continuity of axons at the level ofthe white matter. This technique provided a reliableway of observing axons and myelin by double immu-nofluorescence in the injured-treated and injured butnon-treated spinal cords. Examination of each samplewas performed from the distal caudal region, progress-ing rostrally.

Six months after surgery. In the distal caudal part ofthe injured-only spinal cord, there was severe damageto the white matter. Myelin was sclerosed and therewere yellow/red bubble-like structures and very fewNF-positive fragmented fibres. This indicated the ab-sence of intact axons. Lipid vesicle debris andmacrophages were abundant within the severely injuredmilieu. The structure of the caudal cord segment wascompletely disorganised, lacking the characteristic par-allel fibre pattern and MBP staining. Very few short,hair-like, NF-positive MBP-negative fibres could beseen. The rostral portion of the spinal cord was lessdamaged and a thin continuous MBP immunoreactivitywas observed along the axons.

Spinal cord implanted with NeuroGel™ showedstriking differences in NF and MBP immunostainingwhen compared to controls. There was a clear reduc-tion in secondary cell damage in the distal caudalregion of the spinal cord with considerably less scle-rosed myelin, and a few axons still showed MBP im-munostaining. The presence of yellow/orange lipid-likedroplets and debris was also reduced. Within the gelimplant, we observed a few long but incompletely en-sheathed axons organized along a caudo-rostral axiswithin the gel. The newly formed tissue within the gelappeared clean with little debris and few spheres con-taining NF-positive material. More rostral to the lesion,the tissue showed a reduced disorganization and only a


few areas of sclerosed myelin when compared to equiv-alent regions of the injured-only spinal cord. Overall,tissue preservation was improved in the experimentalspinal cords as shown by a considerable reduction insclerosed myelin. However, a few NF- or MBP-contain-ing vesicles were still present.

Nine months post-surgery. In the distal caudal region,NF-positive immunostained axons of the injured-onlyspinal cord were either covered with sclerosed myelin,naked or transected. The lipid bubbles seen at 6 monthswere no longer present. The edges of the lesion werecharacterised by amorphous tissue region (Fig. 5B) withpractically no cells nor myelin. Only a few very shortMBP-positive sprouts, and abundant lipid inclusions orspheres containing NF and myelin debris were found.In contrast, the rostral junction with the transection siteshowed a network of newly formed myelinated fibres.More distally in the rostral segment of the spinal cord,sclerosed myelin with very few myelinated axons, and afew incompletely ensheathed axons were observed. Itwas not clear if this was newly formed or pre-existingmyelin. No lipid inclusions were found at this level.

Nine months after tissue reconstruction using Neu-roGel™, differences with injured-only non-treatedspinal cords were evident. In both the caudal androstral portions distal to the lesion, well-structured andmyelinated fibres were observed in parallel alignmentalong the longitudinal axis of the cord. The caudalwhite matter distant from the implantation site waslargely devoid of sclerosed myelin and naked axons.Moreover, at the caudal interface with the recon-structed region, axonal fibres were well myelinated asshown by their strongly positive MBP immunostaining.Neither sclerosed myelin nor naked axons were ob-served (Fig. 5D). Within the reconstructed region con-taining the gel implant, blood vessels were identifiable(see Fig. 5D). Fig. 4G shows the supportive and per-missive effect of the gel implant, demonstrated by thepresence of numerous axonal fibres that had grown intothe mid-region of the reconstructed lesion. MBP-posi-tive immunostaining showed that they were myelinated(Fig. 5A). At the rostral gel-tissue interface with theimplant region newly myelinated axonal fibres had pen-etrated the gel (Fig. 5C). Some thin fibres were not yetmyelinated and a few small lipid inclusions were stillpresent within the gel. In the ventral region of the gel,short NF-positive, MBP-negative sprouts were also vis-ible. The immunostaining pattern of the rostral distalregion of the spinal cord resembled that of the intactspinal cord (positive control), showing well organizedaxonal fibres immunostained for NF and MBP. Scle-rosed myelin was not found and only a few lipidinclusions remained. In summary, at 9 months numer-ous regenerating axons with newly formed myelin werefound at both interfaces of the reconstructed region andwithin it.

Se6enteen months post-surgery. The white matter atthe distal caudal portion appeared fairly normal, andwas clear of all traces of sclerosis, debris and lipidinclusions (Fig. 5H). The caudal gel-tissue interfaceshowed a myriad of axonal fibres which exhibited bothMBP and NF immunoreactivity and that had enteredthe reconstructed region containing the gel implant(Fig. 5F). Thinner NF-positive MBP-negative fibreswere also present. Within the gel and in regions corre-sponding to the ventral white matter, all fibres werearranged longitudinally and were NF and MBP positive(Fig. 5G). In the same region, we observed for the firsttime, various NF immunostained neuronal cell bodies(Fig. 5E). At the interface and still in the portion of thegel corresponding to the ventral white matter, NF andMBP co-localized in axonal fibres. At the same levelbut more dorsally, some bundles of fibres disappearedto give rise to long thin NF-positive fibres. The mostremarkable finding was that all the fibres, myelinated ornot, were oriented in a histotypic manner along the gel.Still within the gel in what would correspond to thegray matter region, neuronal cell bodies were conspicu-ous and myelinated axonal fibres with NF-positivesprouts were present (Fig. 5E and G). Adjacent to thisarea and in the gray matter-like structure of the gel,bundles of thicker and well myelinated fibres werepresent. In a more rostral plan and adjacent to theinterface, short and long fibres were present within thegel. The most rostral part of the ventral white matterappeared nearly normal. Thus, 17 months after receiv-ing the gel implant animals showed sustained axonalregeneration as assessed by the length of the axonalfibres and the presence of MBP along them. In addi-tion, immunostained neuronal cell bodies were ob-served in the newly formed tissue of the implant region.

3.8. Electron microscopy

Ultra thin sections of spinal cords that were used foraxonal labelling and/or NF and MBP immunocyto-chemistry were examined by transmission electron mi-croscopy. Irrespective of the time of implantation, ultrathin sections through the implantation area showed acomplex cellular structure, and processes of neuroglialand meningothelial origin associated with polymer ag-gregates. These formed the reparative tissue that hadgrown within the polymer implant. As a result of cellinfiltration, the structure of the polymer gel was disor-ganized and formed clusters of polymer microspheresup to 5 mm in diameter which were scattered within theimplantation area. There was no evidence of polymerdegradation but deformation of polymer aggregates bysurrounding cells was frequently observed. Glial cellswith dark nuclei infiltrated the polymer by sending outcytoplasmic expansions that molded the surface micro-geometry of the polymer network and formed cell


bridges between polymer particles (Fig. 6A). Manycapillary vessels of 10–20 mm were seen in the area ofthe reparative tissue in contact with polymer aggre-gates. These exhibited relatively thick walls and a slight

hypertrophy of the endothelial cells. There was a goodcorellation between BDA axonal labelling, NF andMBP double immunostaining, and ultrastructural ob-servations at the various time points examined. At 6

Fig. 5. A series of fluorescence confocal photomicrographs of cat spinal cord sections double immunostained for NF and MPB, 9 months (A, Cand D) and 17 months (E–H) after reconstructive surgery. (A) Photomicrograph taken at the midpoint of the reconstructed lesion area showingseveral myelinated axonal fibres of significant length running in parallel along the longitudinal axis of the cord. (B) Control animal (injured-only)showing the transection site where only debris and vesicles containing NF and MBP are observed. At the rostral (C) and caudal (D) junctionswith the cord segment, several newly myelinated axons are seen at the interfaces either entering (C) or leaving (D) the implantation site to extendin parallel into the distal white matter. (E) Photomicrograph taken in the area of tissue reconstruction which shows immunostained neuronal cellbodies within the gel implant (arrowhead, see also the insert at higher magnification) associated with numerous myelinated axons and NF-positivesprouts, as well as NF and MBPpositive thin and thicker fibres. In another example (G) axonal fibres followed a longitudinal pattern, numerousNF-positive cell bodies are also present. (F) Caudal interface, showing a myriad of thin myelinated axonal fibres and thin NF-positive fibresleaving the area of tissue reconstruction and entering the distal white matter. (H) View of the most distal cord segment; there is virtually nodamage and all fibres appear myelinated indicating the repair of the distal portion of the spinal cord following transection and implantation ofNeuroGel™.


Fig. 6. Transmission electron micrographs of ultrathin sections of the cat spinal cord through the area of tissue reconstruction 6 months (B) and17 months (A, C and D) after NeuroGel™ implantation. (A) shows a cell that has sent out cytoplasmic expansions (large arrow) between polymermicrospheres (p), following the microgeometry profile of the polymer. Note a dendrite in apposition with a glial cell (small arrows). (B)Ultrastructure of the reparative tissue, showing numerous unmyelinated axons cut longitudinally (Ax), surrounding the polymer. Note a neuronalcell body (large asterisk) in this area of axonal regeneration. There is one axon with myelin extending along the polymer, showing a node ofRanvier (small arrows) and a second myelinated axon cut transversally in contact with the polymer (large arrow). One cell infiltrated the intersticeof the polymer (star). (C) This transverse section shows a representative example of the composition of the reparative tissue formed within theNeuroGel™ implant. There is a cell (presumably glia shown with arrowheads) resting on the polymer (p) and numerous axons of a diameter of1–2 mm, showing signs of continuing myelinization; they have thick compact myelin sheaths associated with a Schwann cell (Sc) resting on a basallamina. These myelinated axons are enclosed in a collagen matrix (Co) whose bundles are cut transversally or longitudinally. There is anoligodendrocyte (arrow) that is in contact with four unmyelinated fibres. Note a cluster of dendro-dendritic contacts (asterisk) and a naked axon(Ax) containing filaments in contact with dendrites. (D) is from the same animal as in (C); high magnification of a Schwann cell surrounded bya basal lamina in association with four myelinated axons, one is enclosed by Schwaxm cell. Scale bars: A, C, B=2 mm; D=500 nm.

and 9 months after reconstructive surgery, unmyeli-nated and myelinated axons that had regenerated weregrouped into fascicles in the NeuroGel™ implant/tissuereconstruction area (Fig. 6B). Axons with degeneratingmyelin sheaths were also observed. At 17 and 19months after surgery, the differences were striking, withaxons being more numerous and most of them exhib-ited compact, highly ordered thick myelin sheaths asso-ciated with, or enclosed by, Schwann cell on a basallamina at a 1:1 ratio between myelin-forming glial cellsand axons (Fig. 6C and D). Myelinated axons had amean diameter of 1–2 mm and their PNS-type myelinsheaths were about 0.5–1 mm thick, with an interlamel-lar spacing of about 17 nm. CNS-type myelin wasfound in cases of aberrant remyelination, where axons

were surrounded by a CNS-type myelin sheath inter-nally and by a PNS-type myelin sheath externally. Thecentral myelin was of about 0.1–0.2 mm thick with aninterlamellar spacing of about 12 nm. On a singlesection at 17–21 months post-surgery, all axons showeda similar degree of myelination which appeared to becompleted at this time-point. Their axoplasm showedmicotubules, neurofilaments, mitochondria and ele-ments of smooth endoplasmic reticulum along a longi-tudinal or transverse plane. Fine collagen fibrilsorientated in parallel, either in the plan of the sectionor perpendicular to the section, were usually observedin the interstitial space of the reparative tissue associ-ated with polymer aggregates and the cytoplasm ofSchwann cells. This type of collagen distribution gave


rise to an orthogonal organization of birefringent colla-gen bundles when observed after pricosirius red stainingand under polarized light (not shown). There wereprocesses similar to filopodia from axonal growth conesbut without microtubules. In some cases the processesshowed a prominent accumulation of intermediatefilaments and formed small desmosome-like junctions.Clusters of dendrites forming dendro-dendritic contactswith cross-sectional diameters of 2–2.5 mm were alsoobserved throughout the area of axonal regeneration(e.g. Fig. 6C). Dendrites typically contained ribosomes,profiles of granular endoplasmic reticulum, mitochon-dria and microtubules, had a cross-sectional diametersof 0.8–1 mm and the regions of synaptic appositionshowed symmetrical membrane thickenings. Smallnumber of vesicles were present usually close to themembrane thickening.

4. Discussion

The present study aimed to evaluate the potential ofNeuroGel™ to promote tissue repair of the transectedadult cat spinal cord and to study the long-term regen-eration of cut axons across the reconstructed lesion.Indeed, to study significant regenerative changes fol-lowing complete spinal cord transection, a long experi-mental period is required to ensure the completedegeneration and removal of the transected system andto observe regenerative changes (Salgado-Ceballos etal., 1998). Double section of the thoracic spinal cordsevers axons of the white and gray matter, and bloodvessels, and creates a cavity. Our results show thatNeuroGel™ implanted into the cavity promoted theformation of a replacement tissue with histotypic char-acteristics of the host. This was achieved by the recruit-ment of non-neuronal cells at the junction of both cordsegments, while the pre-oriented structure of the poly-mer network direct spatial cell organization.

This reparative tissue provided a micro-environmentsuitable for the regrowth of axotomized fibres acrossthe lesion as shown by anterograde and retrogradeaxonal labelling, and double immunostaining. Immu-nofluorescence allowed us to evaluate the extent ofdamage and recovery of both axonal fibres and myelin.The comparison of control and grafted cats at 6 months(earliest time point examined) resulted in two majordifferences. Control cats showed moderate axonalsprouting with short fibres growing in all directions inthe rostral portion of the lesion. In addition, the dam-age in the caudal region continued and extended afterthe initial injury and sclerosed myelin persisted. Thiscontrasts markedly with spinal cord that was repairedusing a NeuroGel™ implant. This treatment reducedthe number of damaged axons in the regions distalrostral and caudal to the lesion, with a concomitant

re-organization of growing axons organized as parallelfibres within the gel. Thus, improvement or furtherdeterioration of the myeloarchitecture of the spinalcord increased as a function of time in grafted andcontrol cats, respectively. It appears that NeuroGel™has a protective effect which considerably reduces Wal-lerian degeneration of the caudal cord segment. At 9months and at later post-implantation time point, morefibres had grown from the host rostral cord segmentthan in controls through the interface into the gel.These axons were also longer and, in addition, mostaxons were myelinated.

In order to examine regeneration of supraspinal de-scending axons across the reconstructed tissue segment,BDA was injected in the ventral spinal cord rostrally tothe implantation/lesion site where the major descendingaxonal tracts are located (Nyberg-Hansen, 1966). Ourfinding from examining consecutive tissue sections,show that a large number of BDA-labelled axons oc-curred in the caudal segment of the spinal cord. De-scending axons entered the reconstructed lesion, thecaudal cord segment via the junction zone and could befollowed up to the extremity of the histological sectionof the spinal cord. Although regenerating terminalswere from supraspinal descending projections, some ofthem may have arisen from spinal neurons. Retrogradelabelling after pressure injection of BDA labelled neu-rons in the medial gray matter of the caudal cordsegment (King et al., 1989). This suggests that fibres ofprojection neurons had regenerated across the recon-structed lesion into the rostral cord segment up to theinjection site. Consistent with this finding, the injectionof WGA-HRP in the dorsal column below the recon-structed region labelled neuronal perikarya in the tho-racic intermediate gray matter of the cord segmentrostral to the lesion/implantation site as well as un-myelinated fibres. These corresponded to either in-terneurons or neurons of the thoracic nucleus of Clarkewhich extends as bilateral columns from L4 to T2 in thecat spinal cord (Szentagothai, 1961; Loewy, 1970).Since WGA-HRP is primarly transported antero-gradelly (Mantyhy and Peschanski, 1983) but transneu-ronal transport has been also shown (Peschanski andRalston, 1985; Alstermark and Kummel, 1990), ourfinding suggest that the dorsal root afferents from thelower limb, that form an ascending projection, hadregenerated through the reconstructed lesion andformed synaptic contacts with intact neurons of theClarke nucleus distant from the implantation site. Inagreement with this finding is that, following deaf-ferentation of the cat spinal cord, replacement of lostterminal in Clarke’s nucleus occured by axonal sprout-ing from intact dorsal root afferents (Murray andGoldberger, 1986). Although, axonal regenerationacross the reconstructed lesion was demonstrated 15months after reparative surgery, which was the earliest


time point studied with BDA injection, silver impregna-tion showed the presence of a larger number of fibreswithin the implantation site 6 months after reconstruc-tive surgery. Further evidences of axon regenerationacross the reconstructed cord segment was provided bydouble immunostaining and confocal microscopy. Elec-tron microscopy showed that axons which crossed Neu-roGel™ implant were associated with collagen bundlesthat formed a loose matrix. Indeed, using picrosiriusred technique coupled with polarised light, a distinctpatterns of collagen fibre organization was observedacross the implantation site within the Neurogel, wherethey formed an orthogonal organized birefringent ma-trix (not shown), and it is most likely that this organiza-tion served as a substrate for regenerating axons.

This study showed that regenerating axons can growwhile in contact with myelinated tracts of the whitematter, which are known to be inhibitory for axonalregeneration in the adult CNS(Cadelli et al., 1992;McKerracher et al., 1994). Our data are in agreementwith other studies which have shown, that in vitrodeveloping axon growth occurs in contact with oligo-dendrocytes provided that there is an astrocytic sub-strate (Fawcett et al., 1992), and that adult regeneratingaxons can regrow in the myelinated white matter(Davies et al., 1997). In addition, results of previousstudies and the observations made in this study showthat the functional properties of the GFAP-positivereactive glial, which are of the same nature as the glialscar tissue, might assist and sustain the growth ofaxonal regeneration (Frisen et al., 1993; Risling et al.,1993; Stichel and Muller, 1994). Reactive astrocytesdisplay considerable plasticity after injury and are im-portant in the context of differentiating a tissue withproperties for ‘neuron-glial relationships’. By infiltrat-ing the hydrogel matrix, the astroglial cells, which arethe principal macroglial cells in the CNS, play a majorrole in the functionality of the new tissue. Indeed, thesecells constitute the cellular support of other CNS com-ponents and provide an environment for axonal growthby producing neural cell adhesion molecules andgrowth factors. This has been shown in in vitro and invivo studies on neuron-glia interactions and cell surfaceadhesion systems, and during development of the CNS(Hatten et al., 1990, 1991; Frisen et al., 1993; Aubert etal., 1995).

In the caudal as well as rostral spinal cord segments,isolated aligned horizontally BDA-stained cells werefound along the path of the regenerating fibres in thewhite matter. Their morphology, with long fine pro-cesses extending longitudinally in the white matter inassociation with regenerating axons, and their topologystrongly suggest that these cells form the glial frame-work of the white matter described by Li and Raisman(1997). These cells exhibited an endogenous perioxida-tive-like activity after injury (Noble et al., 1990) and

thus were labelled after incubation with DAB. As ex-pected, labelled cells were not observed in the non-in-jured intact spinal cord.

Besides axonal damage, demyelination is a consistentpathological characteristic of the traumatically injuredspinal cord, and functional repair of the CNS requiresmyelination of regenerated axons. Myelin is the com-pact membrane that coils around the axon acting as aninsulator for a fast and accurate transmission of theelectrical impulses and is essential for normal nervoussystem function (Webster and De, 1993). Lack ofmyelin leads to CNS dysfunction even in the presenceof regenerating axons, and the functional regenerationprocess is not complete unless myelination occurs. Inthe present study, the regeneration of spinal axons wasassociated with remyelination of their cylindraxes asshown by double immunocytochemical labelling for NFand MBP, and by electron microscopy. We found thatmost of axons were myelinated by Schwann cells thatmigrated into the transection/implantation site in earlystages following injury. This is most likely to haveoccurred via breaks in the compromised glial limitants(Blackemore, 1983). Ensheathment and myelination ofaxons in the CNS by Schwann cells has been reportedto occur in a number of experimental and clinicalstudies (Blackemore, 1983; Raine and Wu, 1993). Onehypothesis that has been advanced to explain the defi-ciency of myelinogenesis by Schwann cells in experi-mental transplantation models is the lack of a stableand consistent association between the Schwann cells,the newly formed extracellular matrices, and the re-grown axons (Espinosa de los Monteros et al., 1997).The fact that, axons in the area of the gel implant weremyelinated at the various time points studied, and thepresence of associated reparative tissue, suggests thatNeuroGel™ provides the necessary conditions formyelination. In addition, Schwann cells that migratedinto the reconstructed lesion contributed to the rerout-ing of axons, because sustained growth of axons de-pends on an intimate contact with Schwann cells(Kleitman et al., 1988).

The fact that we found myelin by immunocytochem-istry and electron microscopy from between 9 months(first time point study) and 21 months (last time pointstudy) after reconstructive surgery demonstrates thatmyelinogenesis was sustained. This process which isregulated by the contact of axons with Schwann cellsurfaces (Kleitman et al., 1988), requires a period of atleast a year in other systems (Salgado-Ceballos et al.,1998). In addition, myelination by endogenous oligo-dendrocytes was occasionally observed as aberrantmyelination, a process that has previously been re-ported (Sassaki and Ide, 1991). This is in agreementwith the fact that traumatic injury of the cord inducesa reactive response for the myelination of survivingoligodendrocytes adjacent to the lesion site (Vick et al.,1992; Bartholdi and Schwab, 1998).


An important finding in this study is the presence, 17months after reconstructive surgery, of neuronal cellbodies within the implant or immediately adjacent tothe site as shown by immunocytochemistry and BDAlabelling. The neurons may have migrated into theNeuroGel™ region from the adjacent tissue or may benewly formed neurons from the ependyma. It has beenshown that ependyme can differentiate after injury intosubsets of cells that give raise to astrocytes and neuronsunder appropriate interaction with circulating growthfactors (Weiss et al., 1996; Johansson et al., 1999).Migrating neurons or precursor cells moved into theimplant site because the properties (structure and diffu-sion) of the NeuroGel™ facilitated their migration andfurther differentiation. We have previously demon-strated that the diffusion properties of NeuroGel™implanted in the rat brain has values similar to that ofthe developing rat brain (Woerly et al., 1999a). There-fore, the transport of growth factors from the CSF intothe implant is facilitated so allowing cell differentiation.

Anatomical regeneration of the lesion was accompa-nied by functional recovery which, in the best case, wascharacterized by quadripedal locomotion with inter-limbs coordination and plantar stepping. The controlgroup, however, showed only typical spinal automatismmovements elicited by perineal or abdominal cutaneousstimulations. The variable degrees of functional recov-ery were correlated with axonal regeneration across thereparative tissue, as demonstrated by axonal labelling,immunocytochemistry and electron microsocopy. It isknown that an axonal regeneration of 5–10% is suffi-cient to reverse the functional deficit concomitant withspinal cord injury (Blight, 1983; Fehlings and Tator,1995). According to our results, we may hypothesis thataxonal regeneration of the ventromedial tracts was inthis range of recovery. Because the vestibulospinal andreticulospinal pathways are known to be key pathwaysin the maintenance of postural and locomotory abilityin the hindlimbs of the cat (Eidelberg et al., 1981).However, the corticospinal pathway is also implicatedin voluntary locomotion (Brustein and Rossignol,1998), and in our study we observed labelling of axonsof the dorsolateral funiculus. In separate experiments,neurophysiological stimulation of the cerebral pedun-cles of the cat brains elicited Motor Evoked Potential(MEP) responses in the opposite hindlimb muscles, butnot in control animals (Woerly et al., 1999b), and axonsremyelinated by Schwann cells have been shown torecover electrophysiological function (Blight andYoung, 1989). The correlation of function withanatomical data implies the formation of new func-tional contacts of subpopulations of the regeneratingaxons of descending spinal tracts with target neuronsand it is reasonable to suggest that this recovery ismediated at least in part, by synaptic reorganizationand replacement. Electron microscopy showed the clus-

tering of dendro-dendritic contacts in the region ofreparative tissue structure that may represent partialsynaptic reorganization by formation of local circuits asresult of axotomy and deafferentation. Such dendriticstructures have been described in parts of the normalbrain (Groves and Linder, 1983) and also as a charac-teristic of synaptic reorganization in the lateral genicu-late nucleus of adult cats which has been deafferented;this is brought about by structural alterations of thedendritic processes of relay cells and interneurons (So-mogyi et al., 1984).

This study shows that damage of the cat spinal cordcan be restored by the host’s own cells in the presenceof a NeuroGel™ implant. Once in contact with theneural tissue, this hydrogel favoured the formation of atissue equivalent. The NeuroGel™ is a dynamic system,its expandable porous structure which orientates mi-grating wound-repair cells and blood vessels, accommo-dates developing tissue which moulds to the geometryof the gel structure. This process was also shown by adiffusion study which indicated that the volume of thegel decreased, while tortuosity increased due to cellinfiltration (Woerly et al., 1999a). The diffusion proper-ties of NeuroGel™, which have been shown to besimilar to those of the developing rat spinal cord,facilitate the transport of neuroactive factors secretedby the host tissue to stimulate neuritic growth and cellsurvival (Frisen, 1997). The success of tissue regenera-tion within the polymer matrix depends also on itsmechanical integrity, on the geometry of the surface ofthe polymer network, the pore size distribution and thedegree of pore interconnection (Woerly et al., 1998).

Beside the growth-promoting characteristics of Neu-roGel™ the nature of the lesion is important for theintegration of the polymer because its final appearancedepends on the degree of the initial trauma. The inte-gration of NeuroGel™ is facilitated when the sectionsare sharp and clean, when excessive manipulations ofthe spinal stumps are avoided, and when trauma isrestricted to the border of the lesion (Das, 1989). Inother words, and according to the intended function ofthe gel implant, the formation of a reparative tissuewithin the hydrogel implant depends on the histopatho-logical history of the lesion. In turn, the feasibility ofachieving a significant degree of tissue repair relies onthe quality of the lesion/implantation procedure.

Much effort has been directed toward increasing thepotential of the spinal cord to regenerate axons acrossa lesion and to form synapses with the opposite seg-ment. Among the strategies that have been investigated,guidance channels seeded with glial or olfactory cellshave been used by Bunge (Ramon-Cueto et al., 1998).In contrast to hydrogel systems, guidance channels (ortubes or cylinders) display a two-dimensional polarityand are therefore unable to favour tissue organization,the formation of which requires a three-dimensional


substrate (i.e. ECM) (Vracko, 1974). In this respect,NeuroGel™ with its porous structure and intrinsic dif-fusion properties for small molecules that mimic theconditions for the development of the central nervoussystem, favours tissue formation following cell migra-tion (Lehmenkuhler et al., 1993; Woerly et al., 1998,1999a). From the point of view of clinical application,NeuroGel™ is easy to handle and implant, and it isstrongly bioadhesive with the surface of the spinalstumps. In addition, NeuroGel™ has hemostatic prop-erties, facilitating local hemostasis during implantationand can be sterilised. Also due to its viscoelastic proper-ties that match the mechanical properties of the spinalcord, it fills the lesion cavity by moulding to the formof the lesion.

Most human spinal cord traumas are closed injuriescaused by a compression/decompression of the ventralpart of the cord. A similar closed injury can be pro-duced in rats by the compression of the ventral spinalcord using an inflatable subdural balloon catheter (Pe-duzzi et al., 1999), and the final pathological aspecttypically include a severe atrophy associated with theformation of a central cavity (Kakulas, 1999). In acompression injury model of the rat spinal cord, Neu-roGel™ was inserted 4 months after injury into thesyringomyelic cavity, leaving the surrounding whitematter and vasculature intact. Rats showed a significantimprovement following treatment with NeuroGel™(Beattie et al., 1997). Therefore, NeuroGel™ allows thetransformation of a previously irrevesible lesion, bethey either acute (transection) or chronic (compressioninjury), into a reversible one. Finally, the hydrogel canbe engineered with growth-promoting factors (Plant etal., 1997), developing cells of the CNS (Woerly et al.,1996), genetically engineered cells (Loh et al., 1999), orwith stem cells (Stuart et al., 2000), to provide biohy-brid system for tissue replacement when a large tissuedefect exist.

The foundation of restorative surgery of the CNS isbased on the possibility of manipulating the cascade ofregenerative cellular events within the healing responseto the initial lesion of the implantation surgery in orderto create a tissue equivalent from the egdge of theimplantation site. A biohybrid tissue is then formedwith the characteristics of both polymer and host tissue,and which is intended to become a permanent part ofthe host organ by acting as the functional analogue ofthe CNS tissue. NeuroGel™ technology opens a newavenue for restorative neurosurgery which, in the fu-ture, will aim at restoring structure and function to lostor non-functional parts of the CNS.

Acknowledgements

This work was supported by Organogel Canada Ltee.

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Reconstruction of the transected cat spinal cord following NeuroGel (TM) implantation: axonal...

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