We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0–15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 μm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30–50 μm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1–10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.
axonal tracing; biodegradable polymers; Fast Blue; Schwann cells; spinal cord injury
The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively compa0ring biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ε-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF+ hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF+ polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.
OPF; PLGA; PCLF; axon regeneration; spinal cord injury; Schwann cell
Biodegradable polymer scaffolds provide an excellent approach to quantifying critical factors necessary for restoration of function after a transection spinal cord injury. Neural stem cells (NSCs) and Schwann cells (SCs) support axonal regeneration. This study examines the compatibility of NSCs and SCs with the poly-lactic-co-glycolic acid polymer scaffold and quantitatively assesses their potential to promote regeneration after a spinal cord transection injury in rats. NSCs were cultured as neurospheres and characterized by immunostaining for nestin (NSCs), glial fibrillary acidic protein (GFAP) (astrocytes), βIII-tubulin (immature neurons), oligodendrocyte-4 (immature oligodendrocytes), and myelin oligodendrocyte (mature oligodendrocytes), while SCs were characterized by immunostaining for S-100. Rats with transection injuries received scaffold implants containing NSCs (n = 17), SCs (n = 17), and no cells (control) (n = 8). The degree of axonal regeneration was determined by counting neurofilament-stained axons through the scaffold channels 1 month after transplantation. Serial sectioning through the scaffold channels in NSC- and SC-treated groups revealed the presence of nestin, neurofilament, S-100, and βIII tubulin–positive cells. GFAP-positive cells were only seen at the spinal cord–scaffold border. There were significantly more axons in the NSC- and SC- treated groups compared to the control group. In conclusion, biodegradable scaffolds with aligned columns seeded with NSCs or SCs facilitate regeneration across the transected spinal cord. Further, these multichannel biodegradable polymer scaffolds effectively serve as platforms for quantitative analysis of axonal regeneration.
Regeneration of endogenous axons through a Schwann cell (SC)-seeded scaffold implant has been demonstrated in the transected rat spinal cord. The formation of a cellular lining in the scaffold channel may limit the degree of axonal regeneration. Spinal cords of adult rats were transected and implanted with the SC-loaded polylactic co-glycollic acid (PLGA) scaffold implants containing seven parallel-aligned channels, either 450-μm (n=19) or 660-μm in diameter (n=14). Animals were sacrificed after 1, 2, and 3 months. Immunohistochemistry for neurofilament-expression was performed. The cross-sectional area of fibrous tissue and regenerative core was calculated. We found that the 450-μm scaffolds had significantly greater axon fibers per channel at the one month (186 ± 37) and three month (78 ± 11) endpoints than the 660-μm scaffolds (90 ± 19 and 40 ± 6, respectively) (P=0.0164 & 0.0149, respectively). The difference in the area of fibrous rim between the 450-μm and 660-μm channels was most pronounced at the one month endpoint, at 28,046 μm2 ± 6,551 and 58,633 μm2 ± 7,063, respectively (P=0.0105). Our study suggests that fabricating scaffolds with smaller diameter channels promotes greater regeneration over larger diameter channels. Axonal regeneration was reduced in the larger channels due to the generation of a large fibrous rim. Optimization of this scaffold environment establishes a platform for future studies of the effects of cell types, trophic factors or pharmacological agents on the regenerative capacity of the injured spinal cord.
Biomedical Engineering; Tissue Development and Growth; Central Nervous System; Polymeric Scaffolds
This study describes the use of oligo [(polyethylene glycol) fumarate] (OPF) hydrogel scaffolds as vehicles for sustained delivery of dibutyryl cyclic adenosine monophosphate (dbcAMP) to the transected spinal cord. dbcAMP was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within the scaffolds architecture. Functionality of the released dbcAMP was assessed using neurite outgrowth assays in PC12 cells and by delivery to the transected spinal cord within OPF seven channel scaffolds, which had been loaded with Schwann cells or mesenchymal stem cells (MSCs). Our results showed that encapsulation of dbcAMP in microspheres lead to prolonged release and continued functionality in vitro. These microspheres were then successfully incorporated into OPF scaffolds and implanted in the transected thoracic spinal cord. Sustained delivery of dbcAMP inhibited axonal regeneration in the presence of Schwann cells but rescued MSC-induced inhibition of axonal regeneration. dbcAMP was also shown to reduce capillary formation in the presence of MSCs, which was coupled with significant functional improvements. Our findings demonstrate the feasibility of incorporating PLGA microsphere technology for spinal cord transection studies. It represents a novel sustained delivery mechanism within the transected spinal cord and provides a platform for potential delivery of other therapeutic agents.
A controlled study to evaluate a new technique for spinal rod fixation after spinal cord injury in rats. Alignment of implanted tissue-engineered scaffolds was assessed radiographically and by magnetic resonance imaging.
To evaluate the stability of implanted scaffolds and the extent of kyphoscoliotic deformities after spinal fixation.
Summary of Background Data
Biodegradable scaffolds provide an excellent platform for the quantitative assessment of cellular and molecular factors that promote regeneration within the transected cord. Successful delivery of scaffolds to the damaged cord can be hampered by malalignment following transplantation, which in turn, hinders the assessment of neural regeneration.
Radio-opaque barium sulfate-impregnated poly-lactic-co-glycolic acid scaffolds were implanted into spinal transection injuries in adult rats. Spinal fixation was performed in one group of animals using a metal rod fixed to the spinous processes above and below the site of injury, while the control group received no fixation. Radiographic morphometry was performed after 2 and 4 weeks, and 3-dimensional magnetic resonance microscopy analysis 4 weeks after surgery.
Over the course of 4 weeks, progressive scoliosis was evident in the unfixed group, where a Cobb angle of 8.13 ± 2.03° was measured. The fixed group demonstrated significantly less scoliosis, with a Cobb angle measurement of 1.89 ± 0.75° (P = 0.0004). Similarly, a trend for less kyphosis was evident in the fixed group (7.33 ± 1.68°) compared with the unfixed group (10.13 ± 1.46°). Quantitative measurements of the degree of malalignment of the scaffolds were also significantly less in the fixed group (5 ± 1.23°) compared with the unfixed group (11 ± 2.82°) (P = 0.0143).
Radio-opaque barium sulfate allows for visualization of scaffolds in vivo using radiographic analysis. Spinal fixation was shown to prevent scoliosis, reduce kyphosis, and reduce scaffold malalignment within the transected rat spinal cord. Using a highly optimized model will increase the potential for finding a therapy for restoring function to the injured cord.
spine fixation; transection spinal cord injury; scaffold; scoliosis
Therapeutic strategies following spinal cord injury must address the multiple barriers that limit regeneration. Multiple channel bridges have been developed that stabilize the injury following implantation and provide physical guidance for regenerating axons. These bridges have now been employed as a vehicle for localized delivery of lentivirus. Implantation of lentivirus loaded multiple channel bridges produced transgene expression that persisted for at least 4 weeks. Expression was maximal at the implant at the earliest time point, and decreased with increasing time of implantation, as well as rostral and caudal to the bridge. Immunohistochemical staining indicated transduction of macrophages, Schwann cells, fibroblasts, and astrocytes within the bridge and adjacent tissue. Subsequently, the delivery of lentivirus encoding the neurotrophic factors NT3 or BDNF significantly increased the extent of axonal growth into the bridge relative to empty scaffolds. In addition to promoting axon growth, the induced expression of neurotrophic factors led to myelination of axons within the channels of the bridge, where the number of myelinated axons was significantly enhanced relative to control. Combining gene delivery with biomaterials to provide physical guidance and create a permissive environment can provide a platform to enhance axonal growth and promote regeneration.
This study was designed to assess a new composite implant to induce regeneration of injured spinal cord in paraplegic rats following complete cord transection. Neuronal xenogeneic cells from biopsies of adult nasal olfactory mucosa (NOM) of human origin, or spinal cords of human embryos, were cultured in two consecutive stages: stationary cultures in a viscous semi-solid gel (NVR-N-Gel) and in suspension on positively charged microcarriers (MCs). A tissue-engineered tubular scaffold, containing bundles of parallel nanofibers, was developed. Both the tube and the nanofibers were made of a biodegradable dextran sulphate–gelatin co-precipitate. The suturable scaffold anchored the implant at the site of injury and provided guidance for the regenerating axons. Implants of adult human NOM cells were implanted into eight rats, from which a 4 mm segment of the spinal cord had been completely removed. Another four rats whose spinal cords had also been transected were implanted with a composite implant of cultured human embryonic spinal cord cells. Eight other cord-transected rats served as a control group. Physiological and behavioral analysis, performed 3 months after implantation, revealed partial recovery of function in one or two limbs in three out of eight animals of the NOM implanted group and in all the four rats that were implanted with cultured human embryonic spinal cord cells. Animals of the control group remained completely paralyzed and did not show transmission of stimuli to the brain. The utilization of an innovative composite implant to bridge a gap resulting from the transection and removal of a 4 mm spinal cord segment shows promise, suggesting the feasibility of this approach for partial reconstruction of spinal cord lesions. Such an implant may serve as a vital bridging station in acute and chronic cases of paraplegia.
Olfactory mucosa; Spinal cord; Transection; Transplantation
This review highlights current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury. The concept of developing 3-dimensional polymer scaffolds for placement into a spinal cord transection model has recently been more extensively explored as a solution for restoring neurologic function after injury. Given the patient morbidity associated with respiratory compromise, the discrete tracts in the spinal cord conveying innervation for breathing represent an important and achievable therapeutic target. The aim is to derive new neuronal tissue from the surrounding, healthy cord that will be guided by the polymer implant through the injured area to make functional reconnections. A variety of naturally derived and synthetic biomaterial polymers have been developed for placement in the injured spinal cord. Axonal growth is supported by inherent properties of the selected polymer, the architecture of the scaffold, permissive microstructures such as pores, grooves or polymer fibres, and surface modifications to provide improved adherence and growth directionality. Structural support of axonal regeneration is combined with integrated polymeric and cellular delivery systems for therapeutic drugs and for neurotrophic molecules to regionalize growth of specific nerve populations.
Spinal cord injury; Axonal regeneration; Polymer scaffold; Tissue engineering; Neurotrophins
In fresh-water turtles, the bridge connecting the proximal and caudal stumps of transected spinal cords consists of regenerating axons running through a glial cellular matrix. To understand the process leading to the generation of the scaffold bridging the lesion, we analyzed the mitotic activity triggered by spinal injury in animals maintained alive for 20–30 days after spinal cord transection. Flow cytometry and bromodeoxyuridine (BrdU)-labeling experiments revealed a significant increment of cycling cells around the lesion epicenter. BrdU-tagged cells maintained a close association with regenerating axons. Most dividing cells expressed the brain lipid-binding protein (BLBP). Cells with BrdU-positive nuclei expressed glial fibrillary acidic protein. As spinal cord regeneration involves dynamic cell rearrangements, we explored the ultra-structure of the bridge and found cells with the aspect of immature oligodendrocytes forming an embryonic-like microenvironment. These cells supported and ensheathed regenerating axons that were recognized by immunocytological and electron-microscopical procedures. Since functional recovery depends on proper impulse transmission, we examined the anatomical axon-glia relationships near the lesion epicenter. Computer-assisted three-dimensional models revealed helical axon-glial junctions in which the intercellular space appeared to be reduced (5–7 nm). Serial-sectioning analysis revealed that fibril-containing processes provided myelinating axon sheaths. Thus, disruption of the ependymal layer elicits mitotic activity predominantly in radial glia expressing BLBP on the lateral aspects of the ependyma. These cycling cells seem to migrate and contribute to the bridge providing the main support and sheaths for regenerating axons.
Spinal cord; Cell proliferation; Regeneration; Radial glia; Re-myelination; Turtle; Trachemys dorbignyi (Chelonia)
The difficulty in spinal cord regeneration is related to the inhibitory factors for axon growth and the lack of appropriate axon guidance in the lesion region. Here we developed scaffolds with aligned nanofibers for nerve guidance and drug delivery in spinal cord. Blended polymers including Poly (l-lactic acid) (PLLA) and Poly (lactide-co-glycolide) (PLGA) were used to electrospin nanofibrous scaffolds with two-layer structure: aligned nanofibers in the inner layer and random nanofibers in the outer layer. Rolipram, a small molecule that can enhance cAMP activity in neurons and suppress inflammatory responses, was immobilized onto nanofibers. To test the therapeutic effects of nanofibrous scaffolds, the nanofibrous scaffolds loaded with rolipram were used to bridge the hemisection lesion in 8-week old athymic rats. The scaffolds with rolipram increased axon growth through the scaffolds and in the lesion, promoted angiogenesis through the scaffold, and decreased the population of astrocytes and chondroitin sulfate proteoglycans in the lesion. Locomotor scale rating analysis showed that the scaffolds with rolipram significantly improved hindlimb function after 3 weeks. This study demonstrated that nanofibrous scaffolds offered a valuable platform for drug delivery for spinal cord regeneration.
Nanofibers; spinal cord; drug delivery; regeneration
Spinal Wistar Hannover rats injected with olfactory ensheathing glia (OEG) have been shown to recover some bipedal stepping and climbing abilities. Given the intrinsic ability of the spinal cord to regain stepping with pharmacological agents or epidural stimulation after a complete mid-thoracic transection, we asked if functional recovery after OEG injections is due to changes in the caudal stump or facilitation of functional regeneration of axons across the transection site. OEG were injected rostral and caudal to the transection site immediately after transection. Robotically assisted step training in the presence of intrathecal injections of a 5-HT2A receptor agonist (quipazine) was used to facilitate recovery of stepping. Bipedal stepping as well as climbing abilities were tested over a 6-month period post-transection to determine any improvement in hindlimb functional due to OEG injections and/or step training. The ability for OEG to facilitate regeneration was analyzed electrophysiologically by transcranially stimulating the brainstem and recording motor evoked potentials (MEP) with chronically implanted intramuscular EMG electrodes in the soleus and tibalis anterior with and without intrathecal injections of noradrenergic, serotonergic, and glycinergic receptor antagonists. Analyses confirmed that along with improved stepping ability and increased use of the hindlimbs during climbing, only OEG rats showed recovery of MEP. In addition the MEP signals were eliminated after a re-transection of the spinal cord rostral to the original transection and were modified in the presence of receptor antagonists. These data indicate that improved hindlimb function after a complete transection was coupled with OEG-facilitated functional regeneration of axons.
We aimed to investigate whether an innovative growth factor-laden scaffold composed of acellular sciatic nerve (ASN) and brain-derived neurotrophic factor (BDNF) could promote axonal regeneration and functional recovery after spinal cord injury (SCI).
Following complete transection at the thoracic level (T9), we immediately transplanted the grafts between the stumps of the severed spinal cords. We evaluated the functional recovery of the hindlimbs of the operated rats using the BBB locomotor rating scale system every week. Eight weeks after surgery, axonal regeneration was examined using the fluorogold (FG) retrograde tracing method. Electrophysiological analysis was carried out to evaluate the improvement in the neuronal circuits. Immunohistochemistry was employed to identify local injuries and recovery.
The results of the Basso-Beattie-Bresnahan (BBB) scale indicated that there was no significant difference between the individual groups. The FG retrograde tracing and electrophysiological analyses indicated that the transplantation of ASN-BDNF provided a permissive environment to support neuron regeneration.
The ASN-BDNF transplantation provided a promising therapeutic approach to promote axonal regeneration and recovery after SCI, and can be used as part of a combinatory treatment strategy for SCI management.
Neurotrophins have been shown to promote axonal growth and regeneration after spinal cord injury. The therapeutic utility of neurotrophins may be enhanced by using a controlled delivery system to increase the duration of neurotrophin availability following injury. Such a delivery system can be incorporated into a bioactive scaffold to serve as a physical bridge for regeneration. This study assessed the effect of controlled delivery of neurotrophin-3 (NT-3) from fibrin scaffolds implanted in spinal cord lesions immediately following 2-mm ablation injury in adult rats. Nine days after injury, fibrin scaffolds containing the delivery system and NT-3 (1000 ng/mL) elicited more robust neuronal fiber growth into the lesion than did control scaffolds or saline (1.5- to 3-fold increase). Implantation of fibrin scaffolds resulted in a dramatic reduction of glial scar formation at the white matter border of the lesion. Hindlimb motor function of treated animals did not improve relative to controls at 12 weeks post-injury. Thus, controlled delivery of NT-3 from fibrin scaffolds enhanced the initial regenerative response by increasing neuronal fiber sprouting and cell migration into the lesion, while functional motor recovery was not observed in this model.
controlled release; growth factor; nerve regeneration
Axons of the adult central nervous system exhibit an extremely limited ability to regenerate after spinal cord injury. Experimentally generated patterns of axon growth are typically disorganized and randomly oriented. Support of linear axonal growth into spinal cord lesion sites has been demonstrated using arrays of uniaxial channels, templated with agarose hydrogel, and containing genetically engineered cells that secrete brain-derived neurotrophic factor (BDNF). However, immobilizing neurotrophic factors secreting cells within a scaffold is relatively cumbersome, and alternative strategies are needed to provide sustained release of BDNF from templated agarose scaffolds. Existing methods of loading the drug or protein into hydrogels cannot provide sustained release from templated agarose hydrogels. Alternatively, here it is shown that pH-responsive H-bonded poly(ethylene glycol)(PEG)/poly(acrylic acid)(PAA)/protein hybrid layer-by-layer (LbL) thin films, when prepared over agarose, provided sustained release of protein under physiological conditions for more than four weeks. Lysozyme, a protein similar in size and isoelectric point to BDNF, is released from the multilayers on the agarose and is biologically active during the earlier time points, with decreasing activity at later time points. This is the first demonstration of month-long sustained protein release from an agarose hydrogel, whereby the drug/protein is loaded separately from the agarose hydrogel fabrication process.
The ability of a substrate bound
neurotrophic factor to promote growth of
ascending sensory axons across a complete
transection lesion of the rat spinal cord was
examined in a transplantation model. Aspiration
lesions created a 3 mm long cavity in the upper
lumbar spinal cord of adult rats. Five weeks
after injury two strips of nerve growth factortreated
nitrocellulose, were implanted, each in a
medio-lateral position, and apposed to the
rostral and caudal surfaces of the cavity.
Control animals received untreated nitrocellulose
implants. Fetal spinal cord tissue was
transplanted alongsideand between these strips.
Six weeks post transplantation, animals were
sacrificed and vibratome sections through the
grafts were processed for immunocytochemical
demonstration of ingrowing axons expressing
calcitonin gene-related peptide (CGRP-IR),
Immunolabeled axons were abundant at the
caudal interface between host tissue and the
NGF-treated nitrocellulose implants, with dense
fascicles of fibers abutting the grafts. As the
distance from the caudal surface increased some
CGRP-IR fibers extended into the fetal tissue
although most appeared to remain oriented in a
longitudinal course adjacent to the nitrocellulose.
Labeled axons were evident along the
entire length of the nitrocellulose and appeared
to aggregate at the rostral tip of the implant,
with many fibers extending into the host spinal
cord rostral to the lesion/transplant site. When
untreated nitrocellulose was implanted, fewer
labeled axons appeared to extend beyond the
caudal host-graft interface. Most CGRP-IR
axons displayed limited association or contact
with the untreated nitrocellulose in this
condition. Computer-assisted quantitative
analysis indicated that NGF-treated nitrocellulose
supported regrowing host axons for
nearly three times the length exhibited by axons
associated with non-treated nitrocellulose
implants. These results indicate that substrate
bound nerve growth factor has the capacity to
enhance the regrowth of ascending sensory
axons across a traumatic spinal cord injury site.
The potential to reestablish functional contacts
across such a lesion may be heightened by the
ability of neurotrophic factors to promote more
extensive axonal regrowth.
Due to the varied and numerous changes in spinal cord tissue following injury, successful treatment for repair may involve strategies combining neuroprotection (pharmacological prevention of some of the damaging intracellular cascades that lead to secondary tissue loss), axonal regeneration promotion (cell transplantation, genetic engineering to increase growth factors, neutralization of inhibitory factors, reduction in scar formation), and rehabilitation. Our goal has been to find effective combination strategies to improve outcome after injury to the adult rat thoracic spinal cord. Combination interventions tested have been implantation of Schwann cells (SCs) plus neuroprotective agents and growth factors administered in various ways, olfactory ensheathing cell (OEC) implantation, chondroitinase addition, or elevation of cyclic AMP. The most efficacious strategy in our hands for the acute complete transection/SC bridge model, including improvement in locomotion [Basso, Beattie, Bresnahan Scale (BBB)], is the combination of SCs, OECs, and chondroitinase administration (BBB 2.1 vs 6.6, 3 times more myelinated axons in the SC bridge, increased serotonergic axons in the bridge and beyond, and significant correlation between the number of bridge myelinated axons and functional improvement). We found the most successful combination strategy for a subacute spinal cord contusion injury (12.5–mm, 10–g weight, MASCIS impactor) to be SCs and elevation of cyclic AMP (BBB 10.4 vs 15, significant increases in white matter sparing, in myelinated axons in the implant, and in responding reticular formation and red and raphe nuclei, and a significant correlation between the number of serotonergic fibers and improvement in locomotion). Thus, in two injury paradigms, these combination strategies as well as others studied in our laboratory have been found to be more effective than SCs alone and suggest ways in which clinical application may be developed.
Spinal cord injuries; Schwann cells; Olfactory ensheathing cells; Axonal regeneration; Chondroitinase; Cyclic adenosine monophosphate
The amount of radioactive proline which reaches the cervical cord by axoplasmic flow after intracortical injection of label is higher in rapidly growing 3 to 6 week old rats but becomes relatively constant in unoperated control rats beyond age 10 weeks. In adult rats with spinal cord transection at T-8, however, the amount of tritiated proline detected in the cervical cord above the site of transection is markedly increased five weeks after surgery, falls to more normal levels by 14 weeks after surgery, and is significantly below normal at 25 weeks after surgery. These findings are consistent with abortive attempts to regenerate axons at five weeks after injury. Twenty-five weeks after injury neuronal death and loss of both cells and axons which would normally project to the caudal cord through the site of spinal cord transection result in a decrease in the axon label found in the cervical region. Recognition of this variability in the amount of radioactivity that reaches the cervical region after spinal cord injury forced a reconsideration of previously reported evidence for regeneration in spinal cord transected animals receiving no specific postoperative therapy. There is no evidence for regeneration in such untreated transected rats.
Newts have the remarkable ability to regenerate their spinal cords as adults. Their spinal cords regenerate with the regenerating tail after tail amputation, as well as after a gap-inducing spinal cord injury (SCI), such as a complete transection. While most studies on newt spinal cord regeneration have focused on events occurring after tail amputation, less attention has been given to events occurring after an SCI, a context that is more relevant to human SCI. Our goal was to use modern labeling and imaging techniques to observe axons regenerating across a complete transection injury and determine how cells and the extracellular matrix in the injury site might contribute to the regenerative process.
We identify stages of axon regeneration following a spinal cord transection and find that axon regrowth across the lesion appears to be enabled, in part, because meningeal cells and glia form a permissive environment for axon regeneration. Meningeal and endothelial cells regenerate into the lesion first and are associated with a loose extracellular matrix that allows axon growth cone migration. This matrix, paradoxically, consists of both permissive and inhibitory proteins. Axons grow into the injury site next and are closely associated with meningeal cells and glial processes extending from cell bodies surrounding the central canal. Later, ependymal tubes lined with glia extend into the lesion as well. Finally, the meningeal cells, axons, and glia move as a unit to close the gap in the spinal cord. After crossing the injury site, axons travel through white matter to reach synaptic targets, and though ascending axons regenerate, sensory axons do not appear to be among them. This entire regenerative process occurs even in the presence of an inflammatory response.
These data reveal, in detail, the cellular and extracellular events that occur during newt spinal cord regeneration after a transection injury and uncover an important role for meningeal and glial cells in facilitating axon regeneration. Given that these cell types interact to form inhibitory barriers in mammals, identifying the mechanisms underlying their permissive behaviors in the newt will provide new insights for improving spinal cord regeneration in mammals.
In larval lamprey, with increasing recovery times after a transection of the rostral spinal cord, there is a gradual recovery of locomotor behavior, and descending brain neurons regenerate their axons for progressively greater distances below the transection site. In the present study, spinal cord “conditioning lesions” (i.e., transections) were performed in the spinal cord at 30% body length (BL; normalized distance from the head) or 50% BL. After various “lesion delay times” (D), a more proximal spinal cord “test lesion” (i.e., transection) was performed at 10% BL, and then, after various recovery times (R), horseradish peroxidase was applied to the spinal cord at 20% BL to determine the extent of axonal regeneration of descending brain neurons. Conditioning lesions at 30% BL, lesion delay times of 2 weeks, and recovery times of 4 weeks (D-R = 2–4 group) resulted in a significant enhancement of axonal regeneration for the total numbers of descending brain neurons as well as neurons in certain brain cell groups compared to control animals without conditioning lesions. Experiments with hemiconditioning lesions, which reduce interanimal variability, confirmed that conditioning lesions do significantly enhance axonal regeneration and indicate that axotomy rather than diffusible factors released at the injury site is primarily involved in this enhancement. Results from the present study suggest that conditioning lesions “prime” descending brain neurons via cell body responses and enhance subsequent axonal regeneration, probably by reducing the initial delay and/or increasing the initial rate of axonal outgrowth.
axotomy; reticulospinal neurons; spinal cord injury; conditioning lesion; test lesion
Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.
OEC; spinal cord injury; norepinephrine; ChAT; cholinergic neurons; locomotion
Biomaterial scaffold architecture has not been investigated as a tunable source of influence on spinal cord regeneration. This study compared regeneration in a transected spinal cord within various designed-macro-architecture scaffolds to determine if these architectures alone could enhance regeneration. Three-dimensional (3-D) designs were created and molds were built on a 3-D printer. Salt-leached porous poly(ɛ-caprolactone) was cast in five different macro-architectures: cylinder, tube, channel, open-path with core, and open-path without core. The two open-path designs were created in this experiment to compare different supportive aspects of architecture provided by scaffolds and their influence on regeneration. Rats received T8 transections and implanted scaffolds for 1 and 3 months. Overall morphology and orientation of sections were characterized by H&E, luxol fast blue, and cresyl violet staining. Borders between intact gray matter and non-regenerated defect were observed from GFAP immunolabeling. Nerve fibers and regenerating axons were identified with Tuj-1 immunolabeling. The open-path designs allowed extension of myelinated fibers along the length of the defect both exterior to and inside the scaffolds and maintained their original defect length up to 3 months. In contrast, the cylinder, tube, and channel implants had a doubling of defect length from secondary damage and large scar and cyst formation with no neural tissue bridging. The open-path scaffold architectures enhanced spinal cord regeneration compared to the three other designs without the use of biological factors.
immunohistochemistry; in vivo studies; neural injury; polycaprolactone; regeneration; scaffold architecture; traumatic spinal cord injury
The purpose of this study was to evaluate the effects of fibrin scaffolds on subacute rat spinal cord injury (SCI). Long Evans rats were anesthetized and underwent a dorsal hemisection injury, two weeks later the injury site was re-exposed, scar tissue was removed, and a fibrin scaffold was implanted into the wound site. An effective method for fibrin scaffold implantation following subacute SCI was investigated based on the presence of fibrin within the lesion site and morphological analysis 1 week after implantation. Pre-polymerized fibrin scaffolds were found to be present within the lesion site 1 week after treatment and were used for the remainder of the study. Fibrin scaffolds were then implanted for 2 and 4 weeks, after which spinal cords were harvested and evaluated using markers for neurons, astrocytes, and chondroitin sulfate proteoglycans. Compared to untreated control, the fibrin-treated group had significantly higher levels of neural fiber staining in the lesion site at 2 and 4 weeks after treatment, and the accumulation of glial fibrillary acidic protein (GFAP) positive reactive astrocytes surrounding the lesion was delayed. These results show that fibrin is conducive to regeneration and cellular migration, and illustrates the advantage of using fibrin as a scaffold for drug delivery and cell-based therapies for SCI.
nerve regeneration; glial scar; CNS scaffold; spinal cord regeneration
Spinal cord injury studies use the presence of serotonin (5-HT)-immunoreactive axons caudal to the injury site as evidence of axonal regeneration. As olfactory ensheathing glia (OEG) transplantation improves hindlimb locomotion in adult rats with complete spinal cord transection, we hypothesized that more 5-HT-positive axons would be found in the caudal stump of OEG- than media-injected rats. Previously we found 5-HT-immunolabeled axons that spanned the transection site only in OEG-injected rats but detected labeled axons just caudal to the lesion in both media- and OEG-injected rats. Now we report that many 5-HT-labeled axons are present throughout the caudal stump of both media- and OEG-injected rats. We found occasional 5-HT-positive interneurons that are one likely source of 5-HT-labeled axons. These results imply that the presence of 5-HT-labeled fibers in the caudal stump is not a reliable indicator of regeneration. We then asked if 5-HT-positive axons appose cholinergic neurons associated with motor functions: central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more 5-HT-positive varicosities in lamina X adjacent to central canal cluster cells in lumbar and sacral segments of OEG- than media-injected rats. SMNs and partition cells are less frequently apposed. As nonsynaptic release of 5-HT is common in the spinal cord, an increase in 5-HT-positive varicosities along motor-associated cholinergic neurons may contribute to the locomotor improvement observed in OEG-injected spinal rats. Furthermore, serotonin located within the caudal stump may activate lumbosacral locomotor networks. J. Comp. Neurol. 515: 664–676, 2009.
plasticity; locomotion; OEG; spinal cord injury; 5-HT; ChAT; cholinergic neurons
Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function.