Giles W. Plant

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.

Inhibitory Proteoglycan Immunoreactivity Is Higher at the Caudal Than the Rostral Schwann Cell Graft-Transected Spinal Cord Interface

To begin to evaluate the influence that proteoglycans may have on the success of Schwann cell (SC) transplants to induce axonal regrowth across a complete transection lesion and beyond, we determined the pattern of expression of inhibitory chondroitin sulfate proteoglycans (CSPGs) 3 weeks after transplantation into completely transected adult rat thoracic spinal cord. Using immunohistochemistry, we observed that: (1) CSPGs recognized by CS-56 antibody are present on astrocytes, fibroblasts, and SCs in the distal graft, and at lesion and cystic cavity borders; (2) CS-56 immunoreactivity (IR) is greater at the caudal SC graft-host cord interface than the rostral interface; (3) phosphacan-IR, also greater at the caudal interface, is associated with astrocytes, fibroblasts, as yet unidentified cells, and extracellular matrix; (4) neurocan-IR is present on astrocytes and as yet unidentified cells in grey and white matter; and (5) NG2-IR is associated with matrix near SC grafts, unidentified cells mainly in white matter, and lesion borders and cysts. Neither oligodendrocytes nor activated macrophages/microglia were immunostained. In sum, the CSPGs studied are increased at 3 weeks, especially at the caudal SC graft-cord interface, possibly contributing to an inhibitory molecular barrier that precludes regrowing descending axons from entering the caudal host cord.

Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord

Implantation of olfactory ensheathing glia (OEG) is a promising strategy to augment long-distance regeneration in the injured spinal cord. In this study, implantation of OEG following unilateral hemisection of the dorsal cervical spinal cord was combined with ex vivo gene transfer techniques. We report, to our knowledge for the first time, that purified cultures of primary OEG are capable of expressing a foreign gene following adenoviral (AdV) and lentiviral (LV) vector-mediated gene transfer. OEG implants subjected to AdV vector-mediated gene transfer expressed high levels of transgenic protein in both intact and lesioned spinal cord at 7 days after implantation. However, the levels of transgene expression gradually declined between 7 and 30 days after implantation in lesioned spinal cord. Infection with LV vectors resulted in stable transduction of primary OEG cultures and transgene expression persisted for at least 4 months after implantation. Genetic engineering of OEG opens the possibility of expressing additional neurotrophic genes and create optimal ‘bridging’ substrates to support spinal axon regeneration. Furthermore, stable transduction of OEG allows us to reliably study the behaviour of implanted cells and to obtain better understanding of their regeneration supporting properties.

Poly(α-hydroxyacids) for application in the spinal cord: Resorbability and biocompatibility with adult rat schwann cells and spinal cord

Future surgical strategies to restore neurological function in the damaged human spinal cord may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. We have studied the in vitro and in vivo degradability of various aliphatic polyesters as well as their effects on rat Schwann cells in vitro and on spinal cord tissue in vivo. In vitro, cylinders made of poly(D,L-lactic-co-glycolic acid) 50:50 (PLA25GA50) started to degrade at 7 days, compared with 28 days for cylinders made of poly(D,L-lactic acid) (PLA50). This faster degradation of PLA25GA50 was reflected by a much higher absorption of water. In vivo, after implantation of PLA25GA50 or PLA50 cylinders between the stumps of a completely transected adult rat spinal cord, the decrease in molecular weight of both polymers was similar to that found in vitro. In vitro degradation of poly(L-lactic acid) (PLA100) mixed with increasing amounts of PLA100 oligomers also was determined. The degradation rate of PLA100 mixed with 30% oligomers was found to be similar to that of PLA50. In vitro, PLA25GA50 and the breakdown products had no adverse effect on the morphology, survival, and proliferation of cultured rat Schwann cells. In vivo, PLA25GA50 cylinders were integrated into the spinal tissue 2 weeks after implantation, unlike PLA50 cylinders. At all time points after surgery, the glial and inflammatory response near the lesion site was largely similar in both experimental and control animals. At time points later than 1 week, neurofilament-positive fibers were found within PLA25GA50 cylinders or the remains thereof. Growth-associated protein 43, which is indicative of regenerating axons, was observed in fibers in the vicinity of the injury site and in the remains of PLA25GA50 cylinders. The results suggest that poly(alpha-hydroxyacids) are likely candidates for application in spinal cord regeneration paradigms involving Schwann cells.

Gene therapy and transplantation in CNS repair: The visual system

Normal visual function in humans is compromised by a range of inherited and acquired degenerative conditions, many of which affect photoreceptors and/or retinal pigment epithelium. As a consequence the majority of experimental gene- and cell-based therapies are aimed at rescuing or replacing these cells. We provide a brief overview of these studies, but the major focus of this review is on the inner retina, in particular how gene therapy and transplantation can improve the viability and regenerative capacity of retinal ganglion cells (RGCs). Such studies are relevant to the development of new treatments for ocular conditions that cause RGC loss or dysfunction, for example glaucoma, diabetes, ischaemia, and various inflammatory and neurodegenerative diseases. However, RGCs and associated central visual pathways also serve as an excellent experimental model of the adult central nervous system (CNS) in which it is possible to study the molecular and cellular mechanisms associated with neuroprotection and axonal regeneration after neurotrauma. In this review we present the current state of knowledge pertaining to RGC responses to injury, neurotrophic and gene therapy strategies aimed at promoting RGC survival, and how best to promote the regeneration of RGC axons after optic nerve or optic tract injury. We also describe transplantation methods being used in attempts to replace lost RGCs or encourage the regrowth of RGC axons back into visual centres in the brain via peripheral nerve bridges. Cooperative approaches including novel combinations of transplantation, gene therapy and pharmacotherapy are discussed. Finally, we consider a number of caveats and future directions, such as problems associated with compensatory sprouting and the reformation of visuotopic maps, the need to develop efficient, regulatable viral vectors, and the need to develop different but sequential strategies that target the cell body and/or the growth cone at appropriate times during the repair process.

Neural tissue engineering: from polymer to biohybrid organs

This investigation reports on the immobilization of neuronal and glial cells (Schwann cells and astrocytes) within N-(2-hydroxypropyl) methacrylamide (HPMA) polymer hydrogels for the production of cell-based polymer hybrid devices. Cells were included within HPMA polymer networks by gel-entrapment, and these biogels were maintained in vitro for up to 6 days. Cell viability and differentiation were studied using immunocytochemical methods and image analysis techniques. The polymer structure and its relationships with cells were examined by scanning electron microscopy. A proportion of the cell population was viable, expressing its own antigenic profile throughout the period of gel incubation, as cells do in conventional culture conditions, and some cells exhibited behaviour such as spreading or process outgrowth and secretion of laminin. The result of the present study allows us to envisage tissue replacement in the central nervous system by means of such cell-based polymer constructs.

Hydrogels containing peptide or aminosugar sequences implanted into the rat brain: influence on cellular migration and axonal growth.

Biocompatible polymer matrices for implantation into lesion sites in the brain were synthesized by incorporating peptide or aminosugar sequences into N-(2-hydroxypropyl)methacrylamide (HPMA) hydrogels. RGD peptide sequences were chemically linked to the hydrogel backbone via a glycylglycine spacer; aminosugars were glucosamine (NHGlc) or N-acetylglucosamine residues. Unmodified or sequence containing HPMA hydrogels were implanted into the lesioned optic tract or cerebral cortex of juvenile (17- to 19-day-old) or adult rat brains, respectively. After 10-12 months host animals were perfused and the brains were processed for immunohistochemistry using antibodies to neurofilaments (RT97), laminin, glial fibrillary acidic protein (GFAP), carbonic anhydrase II (CAII), S100 protein, macrophages (ED1), and myelin basic protein (MBP). Unmodified (control) HPMA hydrogels contained no cellular infiltration or axonal growth. Peptide (RGD)- and aminosugar-modified hydrogels showed increased adhesion properties with host neural tissue, were vascularized, and were infiltrated by host nonneuronal cells. Astrocytes (GFAP+) and macrophages (ED1(+)) were the major cell types seen within modified HPMA hydrogels, the largest numbers being found in RGD-containing polymers. CAII+ oligodendroglia were not seen within any of the hydrogel matrices. RT97(+)/MBP- axons grew into both the RGD and NHGlc hydrogel matrices for small distances. The number of axons was greatest in hydrogels implanted into cerebral cortex but in both cortex and optic tract implants the highest density of axons was seen in polymers containing RGD. The findings of this study are discussed in the context of CNS tissue replacement and the construction of bioactive scaffolds to promote regenerative axonal growth across areas of injury in the brain and spinal cord.

Axonal growth within poly (2-hydroxyethyl methacrylate) sponges infiltrated with Schwann cells and implanted into the lesioned rat optic tract

Porous hydrophilic sponges made from 2-hydroxyethyl methacrylate (HEMA) have a number of possible biomedical applications. We have investigated whether these poly(HEMA) hydrogels, when coated with collagen and infiltrated in vitro with cultured Schwann cells, can be implanted into the lesioned optic tract and act as prosthetic bridges to promote axonal regeneration. Nineteen rats (20-21 days old) were given hydrogel/Schwann cell implants. No obvious toxic effects were seen, either to the transplanted glia or in the adjacent host tissue. Schwann cells survived the implantation technique and were immunopositive for the low affinity nerve growth factor receptor, S100 and laminin. Immunohistochemical studies showed that host non-neuronal cells (astrocytes, oligodendroglia and macrophages) migrated into the implanted hydrogels. Astrocytes were the most frequently observed host cell in the polymer bridges. RT97-positive axons were seen in about two thirds of the implants. The axons were closely associated with transplanted Schwann cells and, in some cases, host glia (astrocytes). Individual axons regrowing within the implanted hydrogels could be traced for up to 900 microns, showing that there was continuity in the network of channels within the polymer scaffold. Axons did not appear to be myelinated by either Schwann cells or by migrated host oligodendroglia. In three rats, anterograde tracing with WGA/HRP failed to demonstrate the presence of retinal axons within the hydrogels. The data indicate that poly(HEMA) hydrogels containing Schwann cells have the potential to provide a stable three-dimensional scaffold which is capable of supporting axonal regeneration in the damaged CNS.

Mitogenic Response of Adult Rat Olfactory Ensheathing Glia to Four Growth Factors

Olfactory ensheathing glia (EG) from adult rat proliferate slowly in vitro without added mitogens. The potential future use of EG in transplantation within the central nervous system to improve neural repair is dependent on identifying mitogens that will effectively expand EG without altering their phenotype. The mitogenic effects of heregulin (HRG), fibroblast growth factor 2 (FGF‐2), platelet‐derived growth factor BB (PDGF‐BB), insulin‐like growth factor 1 (IGF‐1), and forskolin (FSK) on cultured adult‐derived rat EG were monitored by tritiated‐thymidine labeling and p75 immunostaining. In serum‐containing medium, HRG, FGF‐2, PDGF‐BB, IGF‐1, and FSK were capable of stimulating EG proliferation, and the stimulation by these growth factors was potentiated by FSK. The combinations of HRG + FGF‐2, HRG + PDGF‐BB, HRG + IGF‐1, FGF‐2 + PDGF‐BB, and FGF‐2 + IGF‐1 all promoted EG proliferation in an additive manner. In serum‐free medium, HRG and FGF‐2 were mitogenic, but PDGF‐BB, IGF‐1 and FSK were not; however, FSK potentiated the stimulation by HRG and FGF‐2, and the combination of HRG + FGF‐2 promoted EG proliferation in an additive manner. This new information will be useful for the design of protocols to achieve sufficient numbers of adult‐derived EG for clinical purposes. This study also further establishes similarities between EG and Schwann cells. GLIA 33:334–342, 2001.

Cultured rat neuronal and glial cells entrapped within hydrogel polymer matrices: a potential tool for neural tissue replacement

Cultured Schwann cells, neonatal astrocytes or cells dissociated from embryonic cerebral hemispheres were dispersed within poly-[N-(2-hydroxypropyl)-methacrylamide]-based hydrogel matrices by gel entrapment and maintained in vitro for 1-6 days. Glial cells were pre-labelled with Hoechst 33342. Cell differentiation and viability were studied by immunocytochemistry. Up to 15% of Schwann cells initially entrapped within the polymer matrices were immunopositive for the low affinity nerve growth factor receptor, S100, glial fibrillary acidic protein (GFAP) and laminin; up to 10% of pre-labelled astrocytes were immunopositive for GFAP and laminin. Embryonic neurons displayed immunostaining for neurofilaments. Hydrogels containing entrapped Schwann cells were implanted into the rat neocortex. These polymers supported cellular and axonal ingrowth within parts of the polymer matrix.