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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-.

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Encouraging Regeneration of Host Neurones: The Use of Peripheral Nerve Bridges, Glial Cells or Biomaterials

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Introduction

Recent results challenged the dogma that regeneration of CNS axons is impossible. These findings stimulated the interest of experimental neurobiologists and led to research that improved our understanding of the rules that control regeneration of structures in the mammalian spinal cord after injury or disease.

Therapeutic approaches can target either the acute management of the injured spinal cord to protect it from the secondary, autodestructive lesions, such as edema, inflammation, necrosis etc. and/or after the acute phase of the injury the functional restoration of the patients' lost functions.1 This restoration can be at present attempted only by using functional methods or restorative neurology.

In experimental conditions of partial or complete spinal cord injury the restoration of lost spinal cord function was thought to be possible by using neuronal grafts to injured spinal cord to act as so-called “relay-bridges” or to release neurotransmitters (for details see Chapters 3 and 5). The evaluation of the effects of various cellular components taken from either the PNS or the CNS on regeneration are also discussed in this chapter.

Nonneuronal PNS elements and various materials have been used for decades to establish proper connections between lesioned and disconnected parts of the spinal cord and to restore the anatomical continuity and possibly the functional integrity between the separated elements of the cord. Most of these efforts simply wanted to build a “bridge” between the disconnected parts of the severed cord along which nerve fibres could pass and bridge the injured area. These experiments focused on encouraging the limited regenerative capacity of spinal cord neurones. Though initially there were some promising results obtained by several laboratories, the number of different approaches shows how desperate these attempts were to achieve any improvement in the sad outcome of a trauma to the spinal cord.

In this chapter we discuss the results of these attempts and their possible use for the management of patients with injured spinal cord.

Grafting Peripheral Nerves Into Spinal Cord

Intraspinal Nerve Bridges

The idea to use peripheral nerve segments as intraspinal grafts to improve regeneration in severed cords has its origin in the early decades of this century. Since Ramon y Cajal and Tello2,3 proposed to use peripheral nerve bridges to promote axonal regeneration an increasing number of original articles have appeared often reporting fairly controversial effects of peripheral nerves implanted into transected spinal cords. The fact that this controversy could persist for several years was possibly due to inefficient functional analysis of spinal cord locomotion, possibly unsatisfactory surgical techniques, inadequate silver staining methods and rough electrical stimulation techniques in these studies.

The first pioneers of this promising grafting technique were Sugar and Gerard4 whose classical experiments unfortunately could not be reproduced. They reported functional recovery after inserting degenerated sciatic nerve pieces in between the stumps of a transected cord. The recovered animals were reported to perform voluntary hindleg toe movements during walking and “peculiar hopping of both hindlegs alternated with stepping movements”. Electrical stimulation of the brainstem elicited rhythmic stepping or extension of hindlimbs. Also, excellent correlation was claimed to exist between physiological recovery and anatomical restoration where newly formed axons connected the separated cord stumps via the grafted degenerated nerves. However, the source of these regenerating fibres was not established.

Later, other studies using the same grafting technique could neither reproduce nor confirm the above results. Barnard and Carpenter in 19505 used fresh or degenerated peripheral nerve auto- or homotransplants to reconnect the transected cord of rats. They observed no functional improvement in grafted animals though, morphologically ten days after grafting few axons penetrated the implanted nerve but none of these axons could be traced throughout the graft. Similarly negative results were reported by Brown and McCouch6 as well as Feigin et al7 who found no difference between grafted and control animals. Moreover, the anatomical analysis showed relatively poor fibre growth into the implant. Crude electrical stimulation of the brainstem and the cortex elicited either no or only a weak motor response in the hindlimbs in both control and grafted animals.6,7 Accordingly, these authors claimed that no regeneration can occur through an intraspinal nerve bridge and concluded that Sugar and Gerard experiments were either based on incomplete spinal cord transection methods or misinterpreted (see also for earlier reviews: Windle 1956,8 Nornes et al, 19849).

These early experiments also suggested that the surgical technique used for either inflicting an injury or placing a graft into the spinal cord was very important. These studies all reported a connective tissue/glial scar formation at the nerve graft-spinal cord junction although its significance in the failure of regeneration was interpreted differently. Brown and McCouch6 thought that this scar tissue was the impediment to axonal growth whilst Barnard and Carpenter5 suggested on the basis of careful histological analysis that regeneration would not have been better in the absence of the scar. Later, electron microscopic and immunohistochemical investigations added significant amount of information towards elucidating this problem. It became evident that following transplantation of a peripheral nerve into the CNS the junction between the PNS and CNS tissue consisted of glial cells both of peripheral and central origin.10 Astroglial cells separated the grafted nerve from the CNS environment but did not invade the graft significantly. On the contrary, Schwann cells from the nerve penetrated the host tissue for several millimeters and it was suggested that the axonal regeneration observed through the astroglial scar around the implanted nerve was probably due to the presence of Schwann cells.10-12 Immunostaining to neurofilament proteins revealed that axonal regeneration was not confined to the implant but also occurred around the grafted tissue i.e., at sites where Schwann cells were present.11 Thus it became clear that although glial and/or connective tissue scarring at the nerve-spinal cord junction is important but it is not solely responsible for the abortive regeneration and many other factors that may impede regeneration after such spinal cord injury have to be considered (for review see Guth et al, 198313).

The histopathological reactions after spinal cord injury and following immediate grafting were analysed by Kao and his colleagues.14-18 Their main observation was that transected spinal cords undergo a so called “autotomy” which leads to the disruption of a segment of both the rostral and caudal stumps.14 Transplantation of various nervous tissue such as nodose ganglion, sciatic nerve, cultured cerebral tissue14 or spinal cord strips19 has not prevented this necrotic process. Sometimes the graft itself disappeared several weeks after grafting or a cysts was formed at the graft-cord interface. Only peripheral nerve grafts were able to establish a structural continuity between the transected stumps but still necrotic changes developed in the transected stumps of the spinal cord rostral and caudal to the graft.14 Further analysis of the pathological events after spinal cord transection, which was considered the least destructive method, revealed that the autolysis seen in the injured cord is chiefly due to lysosomal digestion of the separated cord stumps.16-18 Kao et al concluded that spinal cord injury is followed by an imperfect wound healing resulting in initial microcyst formation, later cavitation and the necrotic tissue is replaced by a connective tissue scar. Electron microscopical investigations revealed that possibly regenerating axons form so-called terminal clubs as the result of abortive regeneration.17 Accordingly, immediate grafting of a peripheral nerve segment between the transected stumps of a spinal cord which later undergoes partial necrosis will result in the degeneration of the graft and formation of dense collagenous scar between the implanted nerve and spinal cord.

Many of these problems were avoided by introducing more sophisticated surgical techniques and delayed grafting.15,18,20 The introduction of a delicate transection method in dogs and rodents left the pia-arachnoid tubing intact and one week later, when the necrotic changes were complete, the necrotic tissue was carefully removed and grafting carried out.18 The use of these procedures prevented to some extent the formation of connective tissue but not that of cavitation. Delayed grafting itself resulted in the formation of glial basement membrane at the stumps of the transected cord but this did not prevent some axonal regeneration across the graft-cord interface.15 Although several months after grafting the grafted peripheral nerve contained both some thinly myelinated and unmyelinated regenerating axons the reinnervation of the graft still appeared poor. From these results it could be concluded that in the case of delayed grafting the main impediment to regeneration was the separation of the graft tissue from the host cord by the glial basement membrane. Later an important improvement has been achieved by Wrathall et al21,22 who used cultured, nonneuronal peripheral cells (mainly Schwann cells) to fill the microscopic spaces between the contused host spinal cord and the grafted peripheral nerve. Thus the grafted tissue had several encouraging effects upon axonal growth into the graft. The cografted nonneuronal cells migrated both into the spinal cord and the peripheral nerve segment and induced a superior wound healing to that where only nerve segments were grafted.

Moreover, in cords where nonneuronal cells were cografted along with peripheral nerves the axonal growth was significantly promoted. Axons were observed in the grafted nerve as early as seven days after the delayed grafting and by this time these axons enwrapped by Schwann cells penetrated at least one mm into the nerve. However, the effect of these nonneuronal cells was temporary for one year after surgery no obvious difference could be observed between the reinnervation pattern of nerves grafted with or without nonneuronal peripheral cells.

Further factors that may be important for improvement of the axonal growth into peripheral neve grafts have been explored recently. Senoo et al (1998) showed that grafts taken from the proximal stump of peripheral nerves ligated 7 days before grafting suppressed astrocytic scar formation at the host-graft interface and the number of regenerating axons was 10 time greater in the predegenerated grafts than in control animals with untreated grafts.23 The glial environment of the host cord is also an important factor for regenerating axons. Sims et al (1999) found that host animals, whose spinal cord was x-ray irradiated at postnatal day 3, developed less complete astrocytic scar at the host-graft interface and Schwann cells intermingled with the host tissue. In nonirradiated animals many axon terminals, that traversed the peripheral nerve graft, ended blindly in the astrocytic scar.24

Notwithstanding the importance of the above attempts to restore the anatomical continuity of a transected or contused spinal cord these studies left several intriguing questions open. It was evident that some regenerating axons entered the intraspinally implanted nerve and possibly elongated till they reached the other end of the graft but the source of these fibres and their capacity to penetrate into the host spinal cord and establish synapses there remained unanswered. Most of the animals where evidence for morphologically successful regeneration into the graft was established were not analysed for recovery of function.

The quantitative analysis of reinnervation of intraspinal nerve grafts was performed by Richardson and his colleagues.25-27 Injections of HRP into the transplanted spinal cord rostrally and caudally to the graft resulted in some retrograde labelling of spinal cord neurones in the vicinity of the other end of the graft.25-27 However, the number of these neurones was relatively low suggesting that many fibres might have entered the graft but only few reentered the host spinal cord. According to combined autoradiographic and HRP retrograde labelling experiments the dorsal root ganglion cells that were close to the graft contributed significantly to the reinnervation of the transplanted nerve segment but not to that of the rostral spinal cord.26 There was also a difference in the ability of axons of different origin to penetrate into the graft: axons of neurones near the implanted nerves entered the graft and some of these were able to reenter the spinal cord. The cell bodies were, however always a few mm away from the graft. Nerve segments bridging the brainstem and the cervical cord attracted regenerating neurites from more distant cell bodies: these grafts were reinnervated by neurones in the brainstem and thoracic cord as far as 2-3.5 cm rostral and 4-5 cm caudal from the nerve implant.27 In contrast, neither bulbospinal nor corticospinal descending fibres invaded the graft suggesting that the long fibre tracts have a limited capacity to regenerate.26

Disappointingly, the grafts did not produce any obvious functional improvement or changes of primitive stepping movements. Considering the relatively low and varying number of neurones that were able to establish connections between the transected cord stumps via the nerve bridge this finding was not surprising particularly in view of the fact that none of the long descending pathways did enter the graft.26

Regeneration into grafts that replaced the dorsal columns of the cat spinal cord dorsal columns by a segment of the radial nerve was studied by Wilson.28-30 It was suggested by the authors that partial injury of the spinal cord with preservation of the ventral half of the cord would not cause mechanical distraction and autolysis between the graft and the cord and better regeneration would occur into the graft as found in previous experiments. Although the presence of good nerve-graft junction and that of many axons traversing the nerve bridge was reported, HRP injections into the central portion of the graft resulted only in very low numbers of retrogradely labelled neurones in the spinal cord stumps and neighbouring dorsal root ganglia.29,30 Evoked potentials could be recorded from the dorsal columns caudal and rostral to the graft in response to stimulation of the sciatic nerve demonstrating some degree of transmission through the graft. Along with these electrophysiological results good sensation, proprioception and muscular coordination were reported five months after grafting together with the regained ability to walk and use of the hind legs. Although this implantation technology tried to challenge the concept of “spinal cord autotomy” and improve regeneration into peripheral nerve grafts, unfortunately, in fact it did not achieve better results than reconnection of fully transected cords by others.

Genetic manipulation of the intraspinal implants has also been attempted to improve the axonal growth-promoting capacity of these grafts.31,32 Intercostal nerve grafts, transfected with an adenovirus encoding neurotrophin-3, promoted the growth of more corticospinal axons through the nerves into the distal grey matter than in controls. The effect of NT-3 expression in Schwann cells on axonal growth also improved functional recovery in grafted animals.

One of the most interesting findings in this field was described by Cheng et al.33 In their model the spinal cord of adult rats was completely transected and the proximal white matter bundles were connected by intercostal nerve grafts to the grey matter of the distal stump. The grafts were stabilized with fibrin glue containing acidic fibroblast growth factor. The neurotrophic and/or neuroprotective properties of this factor have not been fully revealed yet. Compared with untreated rats, animals with aFGF-peripheral nerve grafts showed regeneration of axons into the distal grey matter and better locomotor recovery of hindlimbs. Though this study reported functional connections between the transected spinal cord stumps, the rats showed limited locomotor recovery. Clinical and experimental studies revealed that remarkably few spinal axons are needed (cc 10%) for injured humans or animals to recover. This suggest that some minimal axonal connection between transected stumps may induce functional recovery.

Taken together, these experiments highlighted the fact that the concept of abortive regeneration can be challenged. Although it is evident that grafting a peripheral nerve into a severed spinal cord is neither morphologically nor functionally able to replace the damaged spinal cord segment but may induce significant regeneration of axotomized neurones of the host spinal cord and neighbouring PNS tissue (for more details see section: Extraspinal nerve bridges).

Extraspinal Nerve Bridges

The possibility of reconnecting separated parts of the central nervous system by using peripheral nerve grafts outside the CNS has been considered for a long time.2,3,8 These ideas had the clear cut advantage that the grafted nerve was not affected by the degenerative events in the lesioned CNS and the ends of the graft could be placed into intact segments of the host spinal cord. Therefore their use offered an even more promising outcome and better regeneration than that of intraspinal grafts. In the early experiments the brain was the main target for such experiments and attempts were made to eliminate the glial barrier formed after a lesion and/or implantation of peripheral nerve grafts into the brain.34 Initial attempts to bridge a complete lesion in the spinal cord were made few years later with surprisingly good outcome.35,36 The transected spinal cord was bridged with a intercostal nerve one-two months after the first operation so that the ventral and dorsal roots of the nerve were left intact and its distal stump was implanted into the spinal cord beyond the lesion site. 25 dogs out of 30 were reported to have developed first signs of “reflex standing and walking” within 10-14 days after grafting. Moreover, resection of the bridge led to loss of the above functional improvements within two-three days. Although no morphological evidence of regenerating fibres traversing from the transected stumps via the scar tissue was found considerable number of regenerated fibres could be traced within the graft from the upper part of the spinal cord and the dorsal root ganglion.36 In dogs which did not show any functional improvement very little axonal ingrowth was observed. It is difficult to evaluate these results, even with a significantly increased knowledge of 35 years after these pioneering experiments. The reconnection achieved by the ingrowing fibres was not quantified in these early experiments. Therefore it is difficult to accept in view of previously listed experiments with intraspinal nerve grafts14-18,25-27 that such functional improvement was solely due to a relatively weak connection established by an intercostal nerve. Another possibility is that Schwann cells from the inserted nerve and from the lesion site invaded the injured spinal cord areas and promoted regeneration of intrinsic spinal cord axons and dorsal root afferents into the spinal cord. Ultrastructural studies by Lampert and Cressman37 (1964) reported such ingrowth of axons along Schwann cells that invaded the lesioned cord and vessels. However, the growth of these regenerated fibres was not sustained for a sufficient length of time and they degenerated later. Unfortunately, this model of extraspinal nerve grafting was not used later so that the possible use this technique is unknown.

In another series of investigations initiated by Aguayo and his colleagues sciatic nerve segments were used to bridge segments of an intact spinal cord or connect the brainstem to certain parts of the cord.38-40 This system had several advantages for the study of spinal cord regeneration.38 The cord remained intact with minimal lesioning at the sites of grafting allowing long-term survival of the animals. Moreover, the origin and length of regenerated axons could also be documented using electrophysiological investigations. Nevertheless, no functional analysis could be performed as the grafting did not produce loss of neurological function. Nerve bridges connecting the medulla and the spinal cord contained regenerating fibres invading the nerves for several cm from both the cord and medulla as shown by retrograde labelling with HRP.38,39 The distance of the cell bodies with regenerating axons from the graft appeared a very important factor. Only neurones in the vicinity of the graft and neighbouring dorsal root ganglia were able to grow their axons into the nerve implant.40 Usually there were more retrogradely labelled cells caudally rather than rostrally to the graft and accordingly, the rostrocaudal extension of labelled neurones was greater caudally. In the cat, the rostral extension of regenerated axons was approximately 30 mm whilst caudally numerous labelled neurones were found when their axons were labelled, as far as 75 mm from the nerve implant.41 On the other hand, despite this propensity of local spinal cord and dorsal root ganglion-derived neurones to grow into peripheral nerve grafts long tract fibres did not show such an ability to regenerate. Similarly to previous experiments on grafted Schwann cells (see section: Transplantation of Schwann cells) long descending axons rarely entered midthoracic or lumbar implants and ascending axons from the lumbar segments failed to regenerate into high cervical grafts.40

Electrophysiological recording from the peripheral nerves grafted into the medulla showed that the regenerated axons had the ability to propagate action potentials so that they originated from a functionally active CNS neurone.42 Some of the axons had both spontaneous (synchronously active with the respiratory cycle) and induced (responding to sciatic nerve stimulation) activity which, at least in some cases resembled that of a normal cell present in the region of implantation. On the other hand, many axons in the graft remained silent. There was also evidence to suggest that some central neurones projecting into the graft responded to both excitatory and inhibitory transynaptic influences. However, it was suggested that most neurones projecting into peripheral nerve grafts had reduced or altered synaptic input. Accordingly, it seemed conceivable that regenerated CNS neurones may be able to establish a simple neuronal circuitry in the lesioned CNS if a more complex experimental reconstruction is aimed.42

All these above studies raised a further question. What is the response of these regenerated central neurones to an injury of their axons in the grafted nerve? Normally only motoneurones or preganglionic sympathetic cells project their axons from the CNS into peripheral nerves and usually these axons have a long course in PNS environment. Lesioning a peripheral nerve results in rapid regeneration but is it true for any central neurone which just regenerated into a peripheral nerve?

In a series of experiments extraspinal nerve grafts bridging the medulla with the spinal cord were crushed close to the nerve-spinal cord junction 6-42 weeks after grafting, left to regenerate for further 4-11 weeks and labelled with HRP approximately 10 mm distally from the site of crush.43 Retrograde labelling with HRP revealed a distribution and number of spinal cord cells similar to that seen in previous experiments using the same grafting and labelling procedure. Most of the labelled spinal cord neurones were intrinsic cells whose axons do not normally project into peripheral nerves. To prove the origin of regenerated fibres after crush injury, neurones injured by the crush were first labelled with Fast Blue and two weeks later the regenerated axons with Nuclear Yellow. The presence of double-labelled neurones (Fast Blue + Nuclear Yellow) suggested that neurones which regenerated into the peripheral nerve were able to regrow their injured processes and the regrowth was not due to collateral sprouting. This indicated that spinal cord neurones in general possess considerable regenerative capacity if a favourable axonal environment is provided. Nevertheless, in another series of experiments by Richardson and Verge44 it became evident that regeneration and regrowth of central axons of otherwise peripheral dorsal root ganglion (DRG) neurones into a peripheral nerve graft is enhanced by a deleterious rather than a known encouraging event applied to the peripheral process. Thus, aggressive treatment with colchicine or section of the peripheral processes of DRG neurones enhanced regeneration of their central processes into the nerve graft while for example nerve growth factor had no such effect.44

Grafting peripheral nerves into a chronic spinal cord lesion was also able to induce recovery of certain populations of injured neurones.45 Tibial nerves inserted into an aspiration cavity four weeks after the lesion induced many chronically injured neurones to extend their axons into the nerve graft. The distribution and size of cells was not uniform: from the neighbouring dorsal root ganglia mainly small cells regenerated whilst labelled spinal cord neurones originated from laminae III-VIII and X. This finding suggested that some chronically injured neurones may lose their potential to regenerate whilst others are able to retain it after a long delay and repeated injury. Recently it has been shown, that neurons of Clarke' nucleus showed increased survival when a peripheral nerve was grafted into the hemisected thoracic spinal cord.46

In view of the above results it can be concluded that spinal cord neurones have a tremendous potential to regenerate even after a repeated physical damage. These regenerating neurones are also functionally active, but have limited or altered synaptic contacts probably due to the absence of their target.42

Although this capacity of lesioned neurones to regenerate into a peripheral nerve is encouraging the functional reconstruction of severely lesioned spinal cords is probably not possible by reconnecting the lesioned segments with extraspinal peripheral nerve bridges. Instead, the regenerative capacity of the spinal cord neurones should probably be enhanced in other ways, using all the practical and theoretical experience accumulated in these studies.

Transplantation of Schwann Cells and Olfactory Ensheathing Cells Into Lesioned Spinal Cord to Enhance Regeneration

Transplantation of Schwann Cells

Contrary to the rapid and effective regeneration in the PNS after a lesion to a peripheral nerve, injured axons in the CNS of adult mammals are not capable of considerable regeneration. Accordingly, the ability of axotomized spinal cord neurones to penetrate the lesion site in adult animals is limited but they are able to extend some neurites around the site of the lesion. This phenomenon is called “abortive regeneration”. The causes for the abortive nature of the axonal growth are not understood. Nevertheless, there are few factors which have been considered to hinder axonal regeneration in the CNS. These include a, the possible presence of inhibitory molecules on the surface of oligodendrocytes and myelin b, glial scarring or c, the lack of molecules stimulating axonal growth. Recent findings highlighted the fact that axotomized CNS neurones are able to grow processes, given the right external environment such as peripheral nerves implanted into the CNS (see Chapter 2). An increasing weight of evidence suggests that Schwann cells are the elements in the PNS which support axonal growth.47 Accordingly, Kromer and Cornbrooks48 reported considerable regeneration of the lesioned septohippocampal pathway after grafting cultured Schwann cells into the lesion (1985). Based on the success of these experiments Schwann cells were implanted into lesioned spinal cords with the possibility in mind that (a) Schwann cells may promote axonal regeneration of lesioned CNS nerve fibres by neutralizing the inhibitory effects present in the CNS and providing a favourable microenvironment for growing axons and (b) Schwann cell implants may cause a regression of the astrocytic gliosis at the site of the lesion.

In a number of studies Schwann cells were grafted into an injured cord where the injured portion of the spinal cord was free of axons and in the case of delayed transplantation astroglial-connective tissue mixed scar occupied the damaged part of the cord. The lesion was produced either photochemically,49 by compressing the cord with an inflatable microballoon50,51 or by creating a suction cavity in the corticospinal tract.52 In spite of the different lesioning methods the outcome of Schwann cell grafting was very similar for each model. Moreover, the fact that the grafted Schwann cells were mixed in some studies with DRG cells52,53 or collagen gel49 did not seem to influence the effects of grafted Schwann cells on axonal regeneration. Syngeneic grafts of purified or mixed Schwann cells easily invaded the injured spinal cord and the area occupied by the grafted cells depended on the number of implanted cells. Cell-poor grafts produced only clusters of Schwann cell invasion intermingled with the remnants of scar tissue.50 Larger number of grafted cells filled the lesion cavity completely and produced a very smooth interface between the host and grafted tissues.50-52 The Schwann cell invasion was detected either by S-100 protein immunostaining or by prelabelling the grafted Schwann cells with an E.Coli galactosidase gene51 so that the extent of repair by Schwann cells and the ingrowth of axons into the graft could be assessed.

The grafts had several effects, which could be taken to be “beneficial”, on the lesioned cord. Immediate grafting after the injury resulted in more sprouting and ingrowth of host fibres (see later) but caused cyst formation in the grafted cord and the reduction of gliosis was only moderate.52 Grafting performed between 2 and 4 days after injury led to poor survival of the grafted tissue probably because of release of cytotoxic factors in the lesioned cord.51 Delayed grafting (five or more days after injury) improved the survival and the integration of the graft into the host spinal cord and often resulted in a very good fusion between implant and host cord with minimal glial scarring and no cyst formation. In order to achieve such results relatively large number of Schwann cells had to be implanted.52 In these areas central glia, Schwann cells and myelin were intermingled.52

An important effect of grafted Schwann cells is, however, that they encourage the growth and regeneration of lesioned fibres of the host cord.49-53 Martin et al50,51 have reported ingrowth of peptidergic fibres into the Schwann cell clusters and their growth was probably strictly related to the presence of the implant because these axons were always accompanied by Schwann cells. Immunohistochemical stainings to neurotransmitters suggested that the majority of these fibres arose from dorsal root afferents and only a few of them were descending supraspinal afferents.50,51 These latter rarely invaded the graft but ran along the margins of the implant. Paino and Bunge49 using a silver stain detected ingrowth of myelinated and unmyelinated fibres into a Schwann cell-collagen implant as early as 14 days after grafting. The number of the invading axons increased with time and reached its maximum 28 days after implantation. These axons became myelinated by the grafted Schwann cells and often followed parallel paths within the graft. However, profuse axonal branching was characteristic only at the host-graft interface but not in the graft. Similar results were reported by Kuhlengel et al in 1990.52,53 In their studies the corticospinal tract of neonatal rats was injured and the cavity filled with a mixed Schwann cell-neurone implant. Several months later the corticospinal tract was traced anterogradely with WGA-HRP injected into the motor cortex. Regenerated corticospinal fibres were found growing in fascicles along the border of the implant in the host grey matter but never in the graft. Immediate grafting after the injury improved the fasciculation and regeneration of the corticospinal tract whilst delayed grafting somehow resulted in lesser fasciculation. According to functional analysis implanted rats had no improved locomotion and functional recovery compared with lesioned control animals which also exhibited substantial recovery two weeks after injury. Human Schwann cells, as potential therapeutic cells in human CNS injuries, have also been used by Guest et al. Similarly to rat Schwann cell, human cells alone did not promote the regeneration of injured corticospinal axons,54,55 while additional therapy with a monoclonal antibody (IN-1) raised against a myelin-associated, neurite growth inhibitory protein or with aFGF-fibrin glue supported regeneration of some fibres into the graft (fig. 1).55 These results indicated that Schwann cells alone are not able to alter the growth-inhibitory environment of the injured spinal cord.

Figure 1. Corticospinal tract (CST) terminations in Schwann cell-grafted animals supplemented with aFGF-fibrin glue 35 days following graft implantation (rostral at right).

Figure 1

Corticospinal tract (CST) terminations in Schwann cell-grafted animals supplemented with aFGF-fibrin glue 35 days following graft implantation (rostral at right). A) A representative area of the cortex to demonstrate the strong labelling of neurons by (more...)

Similar results were obtained with Schwann cells that were genetically modified to overexpress BDNF or NGF.56,57 These Schwann cells showed similar phenotype as nontransfected cells and promoted the growth of more axons into the spinal stump distal to the trail of grafted cells than untreated Schwann cells.57 Some axons from several supraspinal nuclei have also reached the distal portion of the cord. The presence of supraspinal axons in the grafts as well as in the distal stump of the spinal cord is encouraging for the establishment of locomotion.

Another factor influencing the effect of Schwann cells on regeneration was the presence of a demyelinating lesion. When both demyelination and axotomy were induced in the spinal cord, the Schwann cells spread 6-7 mm throughout the region of demyelisation where growth cones were also present.58 Axotomy without demyelination did not induce such growth of severed axons. These findings suggest that Schwann cell transplantation combined with demyelinisation facilitates long distance axonal growth in the injured adult spinal cord.

Li and Raisman studied the effect of implanted Schwann cells in a minimal spinal cord lesion.59 Small, circumscribed lesions in the corticospinal tract were filled with Schwann cells harvested from neonatal sciatic nerve so as to form a bolus. The Schwann cells induced sprouting of the lesioned fibres and some collateral fibres entered the superficial parts of the Schwann cell graft. Although this “minimal lesion” model is in no way comparable to human spinal cord injuries, it may suggest that Schwann cells may induce more regeneration where the secondary degenerative changes are also minimized.

Further attempts to establish a proper “bridge” between the stumps of the transected cord involved application of mini-guidance channels. Resorbable collagen rolls, poly-lactic acid or other polymers (e.g., PAN/PVC) seeded with purified Schwann cells were implanted into the lesioned cord.60-64 Schwann cells survived in any of these channels and myelinated the ingrowing axons, but host astrocytes failed to enter the tubes. The ingrowing axons were mainly immunorecative for CGRP, but not for monoaminergic transmitters.61 Poly-lactic acid guidance channels induced the ingrowth of high number of axons from the host cord but after a peak at 2 month following grafting the fibre ingrowth and myelination decreased in these channels.63 Bamber et al infused neurotrophins into the spinal cord caudal to the PAN/PVC channels seeded with Schwann cells.64 They found that BDNF and NT-3 treatment did not increase the number of myelinated axons within the channel, but enhanced the ingrowth of these fibres into the host gray matter distal to the graft (fig. 2). This effect of the above neurotrophins was not observed when the tube was filled with Matrigel, but had no Schwann cells inside.

Figure 2. Axonal penetration into the distal host spinal cord after BDNF infusion.

Figure 2

Axonal penetration into the distal host spinal cord after BDNF infusion. A) Schematic drawing showing the lateral view of the spinal cord and graft. Dashed lines indicate the positions of a cross-section of the graft (B) and a horizontal section of the (more...)

On the basis of experiments with successful Schwann cell grafting it can be concluded that transplanted Schwann cells have several effects which do influence the restorative process of the injured host spinal cord. They reduce glial scarring and are able to enhance to some extent the otherwise abortive axonal growth and regeneration. Unfortunately, grafts of Schwann cells cannot form functioning bridges between the injured parts of the spinal cord since long descending fibres do not usually enter the graft. Nevertheless, they are able to render the surrounding host spinal cord more permissive to axonal growth. Grafting Schwann cells to stimulate regeneration as opposed to remyelination in the spinal cord has not resulted in functional improvement and their use for spinal cord injury patients is not yet applicable. Although purified and cultured Schwann cells can be maintained in tissue cell banks and are available from host peripheral nerves, they are still considered as alien elements in the spinal cord under normal circumstances and their long-term behaviour is not known. Their almost unlimited capacity to proliferate within a CNS environment may therefore be dangerous.

Transplantation of Olfactory Ensheathing Cells

Olfactory ensheathing cells (OECs) from the olfactory bulb have some common phenotypic properties with Schwann cells and astrocytes. They express glial fibrillary acidic protein (GFAP, an astrocyte marker) and form end-feet around blood vessels. They also express the low-affinity NGF receptor and produce laminin, characteristic for Schwann cells. However, they should be considered as a distinct glial cell type in the CNS. In the adult mammalian olfactory bulb, where olfactory ensheathing cells are present, normal and injured olfactory axons are able to elongate and establish synaptic contacts with other neurones throughout lifetime. These features indicated that grafted olfactory cells may be able to support regeneration in other parts of the CNS.

The pioneering studies of Ramon-Cueto and Nieto-Sampedro showed that olfactory ensheathing cells indeed promoted the growth of axotomised dorsal root axons into the spinal cord.65 The dorsal roots were cut, attached to the dorsal surface of the cord and the gap between the root and the cord was bridged with olfactory glia cells. Ensheathing cells readily migrated into the spinal cord and were followed by regenerating dorsal root axons.65 In the absence of OECs regenerating axons never reached the spinal cord suggesting that presence of these cells in the spinal cord was a prerequisite of successful regeneration. Spinal reflex restitution was studied in experiments where multiple lumbar (L3 to L6) rhizotomies were performed and the gap bridged with olfactory glial transplants.66 The H reflex and withdrawal reflex returned by 60 days after grafting in most of the grafted animals while no recovery was observed in control animals.

Further studies followed to explore the growth-promoting effects of olfactory glia cells in the spinal cord. Li et al electrolytically injured the corticospinal tract and filled the lesion site with olfactory glia cells. Grafted animals showed extensive axonal growth and sprouting as well as improved forepaw reaching tasks compared with untreated animals. Further morphological studies revealed that the grafts contained at least two types of remyelinating olfactory glia cells, although their different role in the regenerative process is not clear.67,68 The regenerating axons further penetrated the caudal host cord stump and later became myelinated by oligodendrocytes. This suggests that the environment around the lesion is permissive for axonal growth for a long time after injury. However, as in case of Schwann cell grafts, the “minimal lesioning” method introduced by Li et al did not allow to determine to what extent olfactory ensheathing cells enhance regeneration in a severely damaged spinal cord.

In another series of experiments Ramon-Cueto et al removed a 4 mm segment from the spinal cord, replaced it with a Schwann cell-seeded guidance channel and then injected olfactory glia suspensions into the proximal and distal spinal cord stumps.69,70 OECs promoted the long-distance regeneration of both ascending and descending axons which readily entered the glial scars at the transected stumps and grew for several centimeters. Grafted olfactory glia cells migrated from the injection sites toward more rostral and caudal directions. When the transected cord stumps were injected with olfactory glia cells as above, but without Schwann cell-filled guidance channels, ensheathing cells induced significant functional recovery in paralysed animals. From 3 to 7 months after grafting, grafted animals supported their body weight, presented voluntary hindlimb movements and their hindlimbs responded to sensory stimuli. This functional improvement suggests that OECs may be the future choice of cells to be grafted into injured human spinal cords. However, the availability of purified olfactory glia cells raises more problems than that of Schwann cells. A potential source could be the nasal olfactory lamina propria of the individual which contains OECs.71 This technology would make possible a kind of autologous transplantation provided that from the nasal mucosal surface, which is very small in humans, useful amount of tissue can be obtained. Alternatively, transgenic cells from large animals (such as pigs) can be used to obtain reasonable amount of tissue. Imaizumi et al72 (2000) have shown that pig ensheathing cells genetically altered to reduce the hyperacute responses in humans are able to induce axonal elongation and restore impulse conduction in the transected spinal cord.

Implantation of Various Materials into the Spinal Cord

Implantation of Collagen

After early experiments had reported some success in reversing the abortive regeneration of lesioned spinal cord axons by grafting peripheral nerves, smooth muscle or fetal CNS cells some authors tried to use various biological materials, such as collagen gels, coated nitrocellulose or Millipore filters to form bridges between lesioned parts of the spinal cord and establish compatible environment for growing axons. Highly purified collagen matrices derived from animals appeared very promising for this purpose since recent results suggested that type I collagen inhibits the glial proliferation in vitro,73 so that implanted collagen might decrease glial scar formation at the site of the lesion. Moreover, collagen matrices were reported to promote growth of severed central and peripheral axons and sustain the ingrowth of vascular elements.74,75 On the basis of these results it seemed feasible that the use of a scaffolding structure placed into a damaged spinal cord could influence the cellular mechanisms of wound healing and facilitate axonal growth.

De la Torre76,77 used first a cell-free bovine collagen matrix in a delayed grafting model. Collagen matrices implanted into the spinal cord 10 days after transection injury established a tight structural continuity between the transected ends and this natural protein integrated well with the host tissue. Newly formed blood vessels entered the implant and the new vessels were able to anastomose with the vascular supply of the spinal cord. Apart from new blood vessel formation within the implant, numerous and heterogenous cells invaded the biomatrix, including Schwann cells, macrophages, meningeal cells, fibroblasts and this latter actively produced new collagen.76,78,79 However, in the first few days after implantation astrocytes rarely migrated into the collagen but later they penetrated into the biomatrix.78,79 Microcyst formation (cavitation) was occasionally found in the bioimplant, mainly close to its proximal junction with the cord. De la Torre reported a limited axonal ingrowth of catecholaminergic fibres into the implant and some of these axons reentered the spinal cord.76,77 Nevertheless, this limited regeneration did not cause any functional improvement, the treated animals did not regain sensory functions and showed no coordinated walking ability.

Other authors used slightly modified collagen gels but could not achieve better fibre ingrowth. Although there was observed only a moderate gliosis at the implant-host tissue interface79 many axons did not enter the collagen but remained “dormant” at the interface. Gelderd78 labelled the neurones projecting into the collagen matrix with HRP six weeks after implantation and found a higher number of labelled cells only rostral to the bioimplant than in control (transection only) animals.

Thus, several signs indicated that the structure of the collagen gel was of particular importance for wound healing and axonal regeneration. Gels treated with glyoxal upon implantation showed a good stability and dense texture formation whilst untreated gels became disorganized by massive infiltration of fibroblasts.79 Paino and Bunge49 used a different, three-dimensional collagen matrix which did not promote axonal growth at all and the authors suggested that this failure was possibly due to the unusual structure. More recently, Marchand et al80 have reported an improved stability and durability of collagen implants by chemical cross-linking treatment. Cross-linked collagen gels have survived for at least six months after implantation and favoured regeneration because numerous axons extended into the implant and reentered the spinal cord.80 Liu et al81,82 used collagen guidance channels made of human placental collagen (type IV/IVoX) to bridge avulsed ventral roots and the spinal cord. In an elegantly designed experiment the collagen tube was implanted into the ventral horn of the spinal cord of marmosets and the avulsed C6 ventral root was inserted into the distal end of the tube. Collagen tubes alone or with an autologous peripheral nerve graft inside the tube induced the regeneration of numerous motoneurones into the denervated ventral root. Reinnervation by the growing axons of motoneurones induced considerable functional reinnervation in the biceps brachii muscle. It should be noted that the number of reinnervating motoneurones was nearly 7 times greater in the case of combined collagen tube-peripheral nerve implants compared with the use of collagen tubes alone.

However, the collagen gel implantation allowed further manipulations. Goldsmith and de la Torre83 used in cats neurotrophic-like substances mixed in the gel before implantation into the transection gap and the implant was covered with an omental pedicle to improve blood supply of the biomatrix.77 Two of the substances, 4-aminopyridine and laminin together with collagen matrix-omentum grafts induced tremendous axonal growth arising from descending monoaminergic tracts into the caudal stump as far as 90 mm below the lesion site. Retrograde labelling with Fluoro Gold showed labelled neurones in the brainstem nuclei which probably contributed to the reinnervation of the spinal cord. Consistent with these morphological findings coordinated forehindlimb locomotion was reported in cats treated with 4-aminopyridine or laminin.

Although, these findings are encouraging and have considerable clinical implications, they should be treated with caution until further studies will be conducted to determine the usefulness of these biomatrices.83

Implantation of Other Biosynthetic Materials

Apart from the studies on implantation of collagen biomatrix into injured spinal cord several other attempts have been made to bridge lesion cavities or rather just promote axonal regeneration by using bioimplants. Nitrocellulose treated with biological substances is known to support neurite growth in vitro.84 Similarly, when nitrocellulose implants were placed into the spinal cord of newborn rats84 to obstruct the growth of the corticospinal tract, the growing fibres which reached the implant several days (six-eight days) after implantation were able to grow and penetrate the implant. Successful regeneration was reported only in cases where the nitrocellulose filter was coated with laminin or had been kept previously in vitro with cultured spinal cord tissue for three-four days. Implantation of untreated nitrocellulose or exposure of the filter to cultures of cerebral cortex or spinal cord tissues longer than eight days before grafting did not support axonal growth of the corticospinal tract. The preliminary success with the coated nitrocellulose was considered to be due to the presence of certain adhesion molecules which are not present in cerebral or mature spinal cord cultured cells. Houlé et al85-87 implanted a strip of nitrocellulose paper treated with nerve growth factor (NGF) in conjunction with a fetal spinal cord tissue transplant in order to enhance ingrowth of injured dorsal root fibres into the cord. They reported nearly 3 times enhanced axonal outgrowth compared to controls with untreated nitrocellulose implants. Interestingly, it was observed that the regenerating fibres were separated from the NGF-treated implant by a continuous layer of macrophages and astrocytes whilst untreated nitrocellulose was surrounded only with scattered macrophages and some astrocytes.86 Thus the effect of NGF on axonal growth must have been indirect, probably mediated by the nonneuronal cell lining of the implant. Regenerating axons readily entered the rostral host spinal cord indicating that ascending axons were primarily promoted to grow across the injury site.87 However, it could be that a combination of structural and biochemical effects mediated by astrocytes derived from the fetal spinal cord and other nonneuronal cells associated with the NGF-treated implant was responsible for the enhanced regeneration of dorsal root axons.

A similar strategy using a Millipore filter implant coated with embryonic spinal cord astrocytes was used88-90 to determine whether the regrowth of sensory axons from injured dorsal roots into the implanted spinal cord can be improved. Such implants promoted the growth of crushed dorsal root fibres into the adult cord and inhibited glial scar formation, but axons leaving the nearby regions of the implant failed to enter the surrounding white matter. Retrograde labelling of regenerating fibres with HRP revealed that many of them formed axonal terminals with boutons suggesting some degree of synaptic plasticity. Although all the above studies confirmed the active function of immature astrocytes coating nitrocellulose or Millipore implants, it remained to be determined how they interact with other cell types, such as macrophages and Schwann cells in order to promote regeneration and what the molecular mechanism of their action is.86,89

Recent studies involved the use of a number of new materials, such as PHPMA (NeuroGelTM) or polyHEMA hydrogels,91,92 poly(D,L-lactide) foams modified by PELA copolymer, fibronectin mats and carbon filaments.93-95 The hydrogels and poly(D,L-lactide) foam, when implanted into injured spinal cords, promoted axonal growth into the implant, angiogenesis and cell migration. Carbon filaments reportedly directed the growth of axons and migration of astrocytes into the bridge formed by the filaments.

In another series of studies the axonal growth-promoting effect of freeze-dried alginate has been studied.96-98 Alginate is a bioabsorbable long chain polysaccharide, isolated from brown seeweed. Suzuki and colleagues developed a novel freeze-dried alginate gel to for spinal cord implantation. Alginate gel was implanted in the gap between the stumps of the transected cords of infant and adult rats. Axons penetrated the gel implants which became infiltrated with vessels, glial cells and some macrophages, although no signs of inflammation were observed. Some axons have been remyelinated. The regenerating axons left the alginate gels and entered the host cord: axonal elongation into the host cord could be followed for 1-1.5 cm rostrally and 200-300 μm caudally (fig. 3). The morphological reinnervation of the host cord stumps was accompanied by electric activity resulting from the axons regenerating through the alginate implant and moderate functional recovery. These results suggest that alginate is a promising biomaterial in spinal cord regeneration studies.

Figure 3. (A-D) Darkfield photomicrographs of horizontally sectioned spinal cord.

Figure 3

(A-D) Darkfield photomicrographs of horizontally sectioned spinal cord. These micrographs show that HRP-labelled regenerating fibres from the dorsal funiculus grew massively through the alginate-implanted gap, reentered the rostral side, and extended (more...)

Catecholaminergic neurones in rat mesencephalon were shown by using histofluorescence techniques to exhibit a considerable capacity to regenerate by growing and sprouting99 following electrolytic lesions. Based on these results, Björklund et al used iris and mitral valve grafts for studying the regeneration of descending 5-HT and catecholaminergic fibres in the spinal cord.100 The grafts placed into a compressed spinal cord became invaded by catecholaminergic fibres and formed loose, irregular plexuses within the grafted tissue. In contrast to this finding, 5-HT fibres showed abundant sprouting around the graft but were not seen to grow into it. The pattern of reinnervating fibres, in particular in the mitral valve, often resembled that of the original innervation of these tissues. The regenerated fibres probably followed the pathways presented by peripheral glial cells in the denervated iris or mitral valve.101 However, the regeneration of these monoaminergic fibres did not occur only in the presence of the graft. Newly formed axons were able to penetrate the necrotic tissue that was formed in the absence of the graft after the compression injury.

Other tissues such as nodose ganglion and smooth muscle have also been grafted into the transected spinal cord without any particular success.14

These studies have shown that the biological materials or various nonneural tissues themselves did not induce significant regeneration when transplanted into the lesioned spinal cord. The most important finding is, however, that the cotransplanted nonneuronal cells, mainly immature astrocytes made the environment around the implant more permissive for axonal regeneration. Such role for immature glial cells is proposed elsewhere in this book (see Chapter 2) and discussed in details.

Transplantation of Genetically Modified Fibroblasts

Apart from exogenous Schwann cells or biomaterials carrying factors that may encourage the growth of host axons, genetically modified fibroblast have also been used for this purpose. It is relatively easy to culture fibroblasts and efficiently transduce them with adeno- or retroviral vectors and they have several advantageous features, such as low immunogenecity as autografts and minimal risk of tumor formation that make them appropriate for ex vivo gene transfer.102 Grafting of genetically modified fibroblasts expressing various molecules (NGF, BDNF, neural cell adhesion molecule L1, etc) into an injured spinal cord103-105 resulted in reliable synthesis of the molecules which were thought to enhance regeneration. Behavioral and morphological analysis showed that the use of genetically modified fibroblasts accelerated recovery from spinal cord injury104 and/or promoted regeneration of injured axons.103,105 However, these experiments also suggested that although fibroblasts possess features that make them candidates of cells to be grafted into an injured spinal cord, immunosuppressive therapy is needed to enhance the survival of grafts of genetically modified fibroblasts.

Application of Omental Tissue to Injured Spinal Cord

Goldsmith et al106 suggested first in 1975 that the use of transposed omental grafts onto the surface of injured spinal cord would improve the autodestructive processes in the cord. It was known well before these studies that omental grafts applied to heart or brain can increase the blood flow in the recipient tissues. Therefore, application of omental tissue to lesioned cord was expected to influence the progressive fall in spinal cord blood flow at the site of injury by adding a new source of blood supply within a reasonable time. Indeed, omental tissue placed onto the dorsal surface of intact or lesioned spinal cords initiated revascularization of the cord within three days, i.e., omental vessels anastomosed with those of the spinal cord.106,107 Moreover, the application of omentum reportedly diminished the edema at the site of injury within 24 hours and in some cats functional recovery was reported one month after surgery. However, these changes could be observed only when the omentum was applied to the cord immediately following the injury. Obviously, such rapid surgical treatment could not occur in a clinical situation. Recently, there are reports of neurological improvements in human patients following omental transposition in chronic spinal cord injuries.108 Although the efficacy of this procedure was doubtful, great number of successful operations were claimed. In fact, two prospective studies of the efficacy of omental transposition in humans have shown that this procedure either failed to improve the morphological outcome in spinal cord injury or the neurological scores became even slightly worse after omental transposition.109,110 In light of these thorough studies it can be argued that this procedure cannot be a suggested protocol in patients suffering from chronic spinal cord injury.

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