Adult Rat Thoracic Spinal Cord Research Articles

Development of a chemically extracted acellular muscle scaffold seeded with amniotic epithelial cells to promote spinal cord repair

Bridging strategies are essential for spinal cord repair in order to provide a physical substrate allowing axons to grow across the site of spinal cord lesions. In this study, we have evaluated the therapeutic effects of adding amniotic epithelial cells to a unidirectionally oriented acellular muscle scaffold and have compared this with the effect of a scaffold alone. Chemically extracted acellular muscles, with or without amniotic epithelial cells, were implanted into the lateral hemisected adult rat thoracic spinal cord. Control rats were similarly injured. After 4 weeks, the acellular muscle scaffolds were found to be well integrated with the host tissue. The chemically extracted acellular muscle scaffold seeded with amniotic epithelial cells promoted axonal growth in a distinctly organized and linear fashion, induced sprouting of calcitonin gene-related peptide positive axons, and was not associated with an astrocyte response. Compared with acellular muscle scaffolds alone, the addition of amniotic epithelial cells further promoted the remyelination of nerve fibers, sprouting of 5-hydroxytryptamine nerve fibers, relays of cortical motor-evoked potential and cortical somatosensory-evoked potential, and functional recovery. All these data together suggest that co-implantation of chemically extracted acellular muscle with amniotic epithelial cells may constitute a valuable approach to study and/or develop therapies for spinal cord injury.

Chemically extracted acellular muscle: A new potential scaffold for spinal cord injury repair

Extracellular matrix is the gold standard for tissue regeneration. In this study, we directly made the extracellular matrix of the tissue or organ into scaffold for spinal cord injuries, a strategy that is seldomly tried in spinal cord engineering. The aim of this study was to determine if the chemically extracted acellular muscle could be a potential scaffold for spinal cord injury. The chemically extracted acellular muscle was implanted in the lateral hemisected adult rat thoracic spinal cord. Control rats were similarly injured. After 1 and 4 weeks, scaffold integration and biocompatibility, axon sprouting, and myelination were evaluated. The chemically extracted acellular muscle scaffolds were found to be well integrated with the host tissue. Sprouting axons grew into the full length of the scaffold in a strikingly parallel and linear fashion. A few remyelinated axons were also detected in the scaffolds. The tracing results in another six rats showed that labeled fibers entered the chemically treated muscle grafts. Furthermore, there were no apparent quantitative differences in the ED-1 and glial fibrillary acidic protein positive cells between groups. Neuron counting showed more surviving neurons in the acellular muscle treated group than those of the injured only group. Vascularization of the grafts was also confirmed. These findings clearly demonstrated that chemically extracted acellular muscle grafts provided useful biomatrices to enhance axon sprouting in the injured spinal cord.

Novel combination strategies to repair the injured mammalian spinal cord

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.

Reducing macrophages to improve bone marrow stromal cell survival in the contused spinal cord

We tested whether reducing macrophage infiltration would improve the survival of allogeneic bone marrow stromal cells (BMSC) transplanted in the contused adult rat thoracic spinal cord. Treatment with cyclosporine, minocycline, or methylprednisolone all resulted in a significant decrease in macrophage infiltration at 3 days postinjury. However, when BMSC were injected at that time point, survival 7 days later was similar between treatment groups and saline-injected controls. In fact, we found that the presence of BMSC resulted in a significant increase in macrophage infiltration into the contusion.

Bone Marrow Stromal Cells Elicit Tissue Sparing after Acute but Not Delayed Transplantation into the Contused Adult Rat Thoracic Spinal Cord

Bone marrow stromal cells (BMSC) transplanted into the contused spinal cord may support repair by improving tissue sparing. We injected allogeneic BMSC into the moderately contused adult rat thoracic spinal cord at 15 min (acute) and at 3, 7, and 21 days (delayed) post-injury and quantified tissue sparing and BMSC survival up to 4 weeks post-transplantation. BMSC survival within the contusion at 7 days post-transplantation was significantly higher with an acute injection (32%) and 3-day delayed injection (52%) than with a 7- or 21-day delayed injection (9% both; p < 0.01). BMSC survival at 28 days post-transplantation was close to 0 in all paradigms, indicating rejection. In contused rats without a BMSC transplant (controls), the volume of spared tissue gradually decreased until 46% (p < 0.001) of the volume of a comparable uninjured spinal cord segment at 49 days post-injury. In rats with BMSC, injected at 15 min, 3, or 7 days post-injury, spared tissue volume was significantly higher in grafted rats than in control rats at the respective endpoints (i.e., 28, 31, and 35 days post-injury). Acute and 3-day delayed but not 7- and 21-day delayed injection of BMSC significantly improved tissue sparing, which was strongly correlated (r = 0.79-1.0) to BMSC survival in the first week after injection into the contusion. Our data showed that neuroprotective effects of BMSC transplanted into a moderate rat spinal cord contusion depend strongly on their survival during the first week post-injection. Acutely injected BMSC elicit more tissue sparing than delayed injected BMSC.

Bone marrow stromal cell survival, migration, and differentiation following acute and delayed transplantation into the moderately contused adult rat thoracic spinal cord

Bone marrow stromal cell survival, migration, and differentiation following acute and delayed transplantation into the moderately contused adult rat thoracic spinal cord

Novel Combination Strategies to Repair the Injured Mammalian Spinal Cord

Summary: 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.

Early necrosis and apoptosis of Schwann cells transplanted into the injured rat spinal cord

Poor survival of cells transplanted into the CNS is a widespread problem and limits their therapeutic potential. Whereas substantial loss of transplanted cells has been described, the extent of acute cell loss has not been quantified previously. To assess the extent and temporal profile of transplanted cell death, and the contributions of necrosis and apoptosis to this cell death following spinal cord injury, different concentrations of Schwann cells (SCs), lentivirally transduced to express green fluorescent protein (GFP), were transplanted into a 1-week-old moderate contusion of the adult rat thoracic spinal cord. In all cases, transplanted cells were present from 10 min to 28 days. There was a 78% reduction in SC number within the first week, with no significant decrease thereafter. Real-time polymerase chain reaction showed a similar 80% reduction in GFP-DNA within the first week, confirming that the decrease in SC number was due to death rather than decreased GFP transgene expression. Cells undergoing necrosis and apoptosis were identified using antibodies against the calpain-mediated fodrin breakdown product and activated caspase 3, respectively, as well as ultrastructurally. Six times more SCs died during the first week after transplantation by necrosis than apoptosis, with the majority of cell death occurring within the first 24 h. The early death of transplanted SCs indicates that factors present, even 1 week after a moderate contusion, are capable of inducing substantial transplanted cell death. Intervention by strategies that limit necrosis and/or apoptosis should be considered for enhancing acute survival of transplanted cells.

Temporal‐spatial dynamics in oligodendrocyte and glial progenitor cell numbers throughout ventrolateral white matter following contusion spinal cord injury

The aim of this study was to systematically determine the spatial distribution of oligodendrocytes (CC1(+)) and glial progenitor cells (NG2(+)) throughout the ventral lateral funiculi (VLF) of adult rat thoracic spinal cord white matter over the course of spontaneous hindlimb locomotor recovery following moderate contusion injury. We used the optical fractionator technique to establish an unbiased estimate of total CC1(+) and NG2(+) cell numbers throughout equivalent segments of VLF from normal and injured spinal cords at designated time points. The results demonstrated a greater than 60% loss of oligodendrocytes and a 50% increase in NG2(+) cells 2 days postinjury. Subsequently, there was a significant increase in oligodendrocytes 7 days postinjury that continued throughout the time course of our experiments (42 days) when the total numbers recovered to 80% of controls. Conversely, NG2(+) cell numbers progressively declined after 2 days postinjury but remained significantly higher than controls throughout the experiments. The pattern of acute loss and repopulation of oligodendrocytes in the VLF paralleled the initial recovery of hindlimb weight-bearing function. Whether such improvement is directly related to NG2(+) cell differentiation into functional oligodendrocytes is uncertain. However, of critical importance is that significant cellular dynamics occurred primarily distal to the injury, and these changes were mirrored by significant alterations in the expression of mature myelin proteins. This infers that site-specific genetic or cellular interventions designed to enhance locomotor recovery by fostering remyelination of spared and/or newly established relay circuits may need to target not only the injury site but also rostrocaudal regions.

Endothelial cell loss is not a major cause of neuronal and glial cell death following contusion injury of the spinal cord

Contusion of the spinal cord causes an immediate local loss of neurons and disruption of vasculature; additional loss continues thereafter. To explore the possibility of a causal link between delayed endothelial cell (EC) death and secondary neural cell loss, we evaluated neural and endothelial cell survival, and measured inflammatory cell infiltration, at times up to 48 h after contusion injury to the adult rat thoracic spinal cord. Female Fischer rats (200 g), subjected to moderate (10 g × 12.5 mm) weight drop injuries by the MASCIS (NYU) impactor, were analyzed at 15 min and at 1, 8, 24 and 48 h. ECs, neurons, astrocytes, oligodendrocytes, neutrophils and activated macrophages/microglia were counted in transverse sections. At the injury site, 90% of all neurons died within 48 h of injury; no medium–large diameter neurons survived beyond 48 h. EC death occurred with kinetics similar to glial cell death. Because, in the injury site, most cell death occurred before 8 h, it preceded inflammatory cell infiltration. Three millimeters rostral and caudal to the injury epicenter neuronal numbers were stable for 8 h, and a sharp decrease in neuronal numbers beginning at 8 h strongly correlated with the onset of inflammatory cell infiltration. Glial and blood vessel numbers remained relatively stable in these areas up to 48 h. These results suggest that the loss of ECs during the first 48 h after a contusion injury is not a major cause of neuronal and glial cell death and, in tissue adjacent to the epicenter, inflammatory cell infiltration leads to neuronal loss.

Poly ( d, l-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord

Freeze-dried poly( d, l-lactic acid) macroporous scaffold filled with a fibrin solution containing Schwann cells (SCs) lentivirally transduced to produce and secrete D15A, a bi-functional neurotrophin with brain-derived neurotrophic factor and neurotrophin-3 activity, and to express green fluorescent protein (GFP) were implanted in the completely transected adult rat thoracic spinal cord. Control rats were similarly injured and then implanted with scaffolds containing the fibrin solution with SCs lentivirally transduced to produce express GFP only or with the fibrin solution only. Transgene production and biological activity in vitro, SC survival within the scaffold in vitro and in vivo, scaffold integration, axonal regeneration and myelination, and hind limb motor function were analyzed at 1, 2, and 6 weeks after implantation. In vitro, lentivirally transduced SCs produced 87.5 ng/24 h/10 6 cells of D15A as measured by neurotrophin-3 activity in ELISA. The secreted D15A was biologically active as evidenced by its promotion of neurite outgrowth of dorsal root ganglion neurons in culture. In vitro, SCs expressing GFP were present in the scaffolds for up to 6 h, the end of a typical surgery session. Implantation of SC-seeded scaffolds caused modest loss of spinal nervous tissue. Reactive astrocytes and chondroitin sulfate glycosaminoglycans were present in spinal tissue adjacent to the scaffold. Vascularization of the scaffold was ongoing at 1 week post-implantation. There were no apparent differences in scaffold integration and blood vessel formation between groups. A decreasing number of implanted (GFP-positive) SCs were found within the scaffold during the first 3 days after implantation. Apoptosis was identified as one of the mechanisms of cell death. At 1 week and later time points after implantation, few of the implanted SCs were present in the scaffold. Neurofilament-positive axons were found in the scaffold. At 6 weeks post-grafting, myelinated axons were observed within and at the external surface of the scaffold. Axons did not grow from the scaffold into the caudal cord. All groups demonstrated a similar improvement of hind limb motor function. Our findings demonstrated that few seeded SCs survived in vivo, which could account for the modest axonal regeneration response into and across the scaffold. For the development of SC-seeded macroporous scaffolds that effectively promote axonal regeneration in the injured spinal cord, the survival and/or total number of SCs in the scaffold needs to be improved.

Freeze-dried poly( d, l-lactic acid) macroporous guidance scaffolds impregnated with brain-derived neurotrophic factor in the transected adult rat thoracic spinal cord

The effects of poly( d, l-lactic acid) macroporous guidance scaffolds (foams) with or without brain-derived neurotrophic factor (BDNF) on tissue sparing, neuronal survival, axonal regeneration, and behavioral improvements of the hindlimbs following implantation in the transected adult rat thoracic spinal cord were studied. The foams were embedded in fibrin glue containing acidic-fibroblast growth factor. One group of animals received fibrin glue with acidic-fibroblast growth factor only. The foams were prepared by a thermally induced polymer-solvent phase separation process and contained longitudinally oriented macropores connected to each other by a network of micropores. Both foams and fibrin only resulted in a similar gliotic and inflammatory response in the cord–implant interfaces. With BDNF foam, up to 20% more NeuN-positive cells in the spinal nervous tissue close to the rostral but not caudal spinal cord–implant interface survived than with control foam or fibrin only at 4 and 8 weeks after implantation. Semithin plastic sections and electron microcopy revealed that cells and axons more rapidly invaded BDNF foam than control foam. Also, BDNF foam contained almost twice as many blood vessels than control foam at 8 weeks after implantation. Tissue sparing was similar in all three implantation paradigms; approximately 42% of tissue was spared in the rostral cord and approximately 37% in the caudal cord at 8 weeks post grafting. The number of myelinated and unmyelinated axons was low and not different between the two types of foams. Many more axons were found in the fibrin only graft. Serotonergic axons were not found in any of the implants and none of the axons regenerated into the caudal spinal cord. The behavioral improvements in the hindlimbs were similar in all groups. These findings indicated that foam is well tolerated within the injured spinal cord and that the addition of BDNF promotes cell survival and angiogenesis. However, the overall axonal regeneration response is low. Future research should explore the use of poly( d, l-lactic acid) foams, with or without axonal growth-promoting factors, seeded with Schwann cells to enhance the axonal regeneration and myelination response.

Delayed transplantation of olfactory ensheathing glia promotes sparing/regeneration of supraspinal axons in the contused adult rat spinal cord.

The aim of this study was to determine the preferred time and environment for transplantation of olfactory ensheathing glia (OEG) into the moderately contused adult rat thoracic spinal cord. Purified OEG were suspended in culture medium with or without fibrinogen and injected into the contused cord segment at 30 min or 7 days after injury. Control animals received a contusion injury only or injection of only medium 7 days after contusion. The effects on axonal sparing/regeneration and functional recovery were evaluated 8 weeks after injury. The grafts largely filled the lesion site, reducing cavitation, and appeared continuous with the spinal nervous tissue. Whereas in 7d/medium only animals, 54% of spinal tissue within a 2.5-mm-long segment of cord centered at the injury site was spared, significantly more tissue was spared in 0 d/OEG-medium (73%), 0 d/OEG-fibrin (66%), 7 d/OEG-medium (70%), and 7 d/OEG-fibrin (68%) grafted animals. Compared with controls, the grafted animals exhibited more serotonergic axons within the transplant, the surrounding white matter, and the spinal cord up to at least 20 mm caudal to the graft. Retrograde tracing revealed that all but the 0 d/OEG-fibrin graft promoted sparing/regeneration of supraspinal axons compared with controls. Overall, the 7 d/OEG-medium group resulted in the best response, with twice as many labeled neurons in the brain compared with 7 d/medium only controls. Of the labeled neurons, 68% were located in the reticular formation, and 4% in the red, 4% in the raphe, and 5% in the vestibular nuclei. Hindlimb performance was modestly but significantly improved in the 7 d/OEG-medium group. Our results demonstrate that transplantation of OEG into the moderately contused adult rat thoracic spinal cord promotes sparing/regeneration of supraspinal axons and that 7 d transplantation is more effective than acute transplantation of OEG. Our results have relevant implications for future surgical repair strategies of the contused spinal cord.

Schwann cell but not olfactory ensheathing glia transplants improve hindlimb locomotor performance in the moderately contused adult rat thoracic spinal cord.

Cultured adult rat Schwann cells (SCs) or olfactory ensheathing glia (OEG), or both, were transplanted in the adult Fischer rat thoracic (T9) spinal cord 1 week after a moderate contusion (10 gm, 12.5 mm, NYU impactor). Rats received either a total of 2 x 10(6) cells suspended in culture medium or culture medium only (controls). At 12 weeks after injury, all grafted animals exhibited diminished cavitation. Although in medium-injected rats 33% of spinal tissue within a 5-mm-long segment of cord centered at the injury site was spared, significantly more tissue was spared in SC (51%), OEG (43%), and SC/OEG (44%) grafted animals. All three types of glial grafts were filled with axons, primarily of spinal origin. SC grafts contained more myelinated axons than SC/OEG and OEG grafts. Both types of SC-containing grafts expressed more intense staining for glial fibrillary acidic protein and chondroitin sulfate proteoglycan compared with OEG-only grafts. Retrograde tracing demonstrated that the number of propriospinal and brainstem axons reaching 5-6 mm beyond the grafted area was significantly higher with SC and SC/OEG grafts but not with OEG-only grafts compared with controls. Corticospinal fibers terminated closer to the lesion epicenter in all grafted animals than in controls. With SC-only grafts, a modest but statistically significant improvement in hindlimb locomotor performance was detected at 8-11 weeks after injury. Thus, in addition to this functional improvement, our results show that an SC graft is more effective in promoting axonal sparing/regeneration than an SC/OEG or OEG graft in the moderately contused adult rat thoracic spinal cord.

Schwann Cell But Not Olfactory Ensheathing Glia Transplants Improve Hindlimb Locomotor Performance in the Moderately Contused Adult Rat Thoracic Spinal Cord

Cultured adult rat Schwann cells (SCs) or olfactory ensheathing glia (OEG), or both, were transplanted in the adult Fischer rat thoracic (T9) spinal cord 1 week after a moderate contusion (10 gm, 12.5 mm, NYU impactor). Rats received either a total of 2 x 10(6) cells suspended in culture medium or culture medium only (controls). At 12 weeks after injury, all grafted animals exhibited diminished cavitation. Although in medium-injected rats 33% of spinal tissue within a 5-mm-long segment of cord centered at the injury site was spared, significantly more tissue was spared in SC (51%), OEG (43%), and SC/OEG (44%) grafted animals. All three types of glial grafts were filled with axons, primarily of spinal origin. SC grafts contained more myelinated axons than SC/OEG and OEG grafts. Both types of SC-containing grafts expressed more intense staining for glial fibrillary acidic protein and chondroitin sulfate proteoglycan compared with OEG-only grafts. Retrograde tracing demonstrated that the number of propriospinal and brainstem axons reaching 5-6 mm beyond the grafted area was significantly higher with SC and SC/OEG grafts but not with OEG-only grafts compared with controls. Corticospinal fibers terminated closer to the lesion epicenter in all grafted animals than in controls. With SC-only grafts, a modest but statistically significant improvement in hindlimb locomotor performance was detected at 8-11 weeks after injury. Thus, in addition to this functional improvement, our results show that an SC graft is more effective in promoting axonal sparing/regeneration than an SC/OEG or OEG graft in the moderately contused adult rat thoracic spinal cord.

Patch-clamp recordings from white matter glia in thin longitudinal slices of adult rat spinal cord

We developed a technique of whole cell patch-clamp recordings from white matter oligodendrocytes and astrocytes in 200–250 μm-thick horizontal slices of adult (>2 months, 240–260 g) rat thoracic spinal cord. The viability of the white matter, sectioned in Na +-free, low Ca 2+ media, and the function of axons were preserved for >8 h, as demonstrated by the propagation of TTX-sensitive compound action potentials (CAPs) and the sensitivity of their refractory period to K + channel blocker 4-aminopyridine (1 μM). Glial cells were visually identified within the slices with a 40× water immersion objective using infra-red differential interference contrast (IR-DIC) video microscopy, and the details of their morphology were further elucidated after filling the cells with Lucifer Yellow or Alexa 350 fluorescent dyes during whole-cell recording. Using voltage steps and ramps, we revealed pronounced non-linearity of I– V relationships in both oligodendrocytes and astrocytes. Both types of cells expressed TEA-sensitive outward delayed rectifier-type currents activated at positive voltages but showed little, if any, signs of inward rectification at voltages up to −140 mV. At −70 mV holding voltage, bath-applied kainic acid (100 μM) activated inward currents in both types of cells. This novel horizontal slice preparation of adult rat thoracic cord will facilitate the examination of mature glial cell physiology, glial–axonal signaling and the pathophysiology of spinal cord trauma and ischemia.

Methylprednisolone and interleukin-10 reduce gray matter damage in the contused Fischer rat thoracic spinal cord but do not improve functional outcome.

The effects of two antiinflammatory and neuroprotective agents, methylprednisolone (MP) and interleukin-10 (IL-10), singly and in combination on tissue damage, axonal preservation and functional recovery were studied in the contused adult Fischer rat thoracic spinal cord 12 weeks after injury. MP (30 mg/kg at 5 min, and 2 and 4 h after injury) was administered intravenously and IL-10 (15 or 30 microg/kg at 30 min after injury), intraperitoneally. MP, IL-10, or the combination significantly reduced the volume of damaged tissue (including cavities) compared to control animals. The loss of spinal tissue (cavities) was reduced after treatment with MP alone or combined with IL-10, but not with IL-10 alone. The reduction in tissue damage was confined to spinal gray matter; at the level of the lesion epicenter, the thickness of the lateral white matter columns was similar in all groups. Retrograde tracing using fast blue revealed that the number of spared propriospinal and supraspinal projections was similar in all groups at 12 weeks after the contusion. The open-field BBB-test showed no significant difference in hindlimb locomotion between groups. Our results demonstrate that all tested antiinflammatory treatments significantly increase the volume of spared spinal gray matter 3 months after a moderate contusion of the Fischer rat thoracic spinal cord, but none of the treatments improved axonal preservation or functional recovery.

Chapter 19 Bridging the transected or contused adult rat spinal cord with Schwann cell and olfactory ensheathing glia transplants

Publisher Summary This chapter compares new findings from studies of cell transplantation in a rat contusion model with those utilizing a complete transection/cellular bridge paradigm. In both cases, Schwann cells (SCs) support substantial axonal regeneration and myelination in the graft. In neither case, brainstem or cortical axons appear to enter the graft. Studying transplantation in a contusion model is clinically relevant; however, there is the challenge of distinguishing spared and sprouted fibers from fibers that have regenerated. Therefore, the chapter uses a complete transection model to obtain unambiguous results by placing a cellular bridge between the stumps of completely transected adult Fischer rat thoracic spinal cord.

Conduction of impulses by axons regenerated in a Schwann cell graft in the transected adult rat thoracic spinal cord.

Central nervous system axons regenerate into a Schwann cell implant placed in the transected thoracic spinal cord of an adult rat. The present study was designed to test whether these regenerated axons are capable of conducting action potentials. Following the transection and removal of a 4- to 5-mm segment of the thoracic spinal cord (T8-T9), a polymer guidance channel filled with a mixture of adult rat Schwann cells and Matrigel was grafted into a 4- to 5-mm-long gap in the transected thoracic spinal cord. The two cut ends of the spinal cord were eased into the guidance channel openings. Transected control animals received a channel containing Matrigel only. Three months after implantation, electrophysiological studies were performed. Tungsten microelectrodes were used for monopolar stimulation of regenerated axons within the Schwann cell graft. Glass microelectrodes were used to record responses in the spinal cord rostral to the stimulation site. Evoked responses to electrical stimulation of the axon cable were found in two out of nine Schwann cell-grafted animals. These responses had approximate latencies in the range of those of myelinated axons. No responses were seen in any of the Matrigel-grafted animals. Histological analysis revealed that the two cases that showed evoked potentials had the largest number of myelinated axons present in the cable. This study demonstrates that axons regenerating through Schwann cell grafts in the complete transected spinal cord can produce measurable evoked responses following electrical stimulation.

Axonal regeneration into Schwann cell grafts within resorbable poly( α-hydroxyacid) guidance channels in the adult rat spinal cord

Axonal growth and myelination in a SC graft contained in a resorbable tubular scaffold made of poly( d, l-lactic acid) (PLA 50) or high molecular weight poly( l-lactic acid) mixed with 10% poly( l-lactic acid) oligomers (PLA 100/10) were studied for up to 4 months after implantation in the completely transected adult rat thoracic spinal cord. The PLA 50 tubes collapsed soon after implantation and, consequently, compressed the graft inside, leading to only occasional thin cables with SCs and a low number of myelinated axons: 17±6 at 1 and 158±11 at 2 months post-grafting. The cable contained 32±23 blood vessels at 2 weeks, 55±33 at 1 month and 46±30 at 2 months after implantation. PLA 100/10 tubes, on the other hand, were found to break up into large pieces, which compressed and sometimes protruded into the tissue cable inside. At all time points studied, however, cables contained SCs and were well vascularized with 414±47 blood vessels at 2 weeks, 437±139 at 1, 609±134 at 2 and 396±95 at 4 months post-grafting. The number of myelinated axons was 712±509 at 1 month, 1819±837 at 2 months and 609±132 at 4 months post implantation. These results demonstrated that fiber growth and myelination into a SC graft contained in a resorbable PLA 100/10 tube increases over the first 2 months post-implantation but decreases thereafter. Changes in geometry of both types of polymer tubes were detrimental to axonal regeneration. Future research should explore the use of polymers that better retain the appropriate mechanical, geometrical and permeability properties over time.