George M. Smith

Laminin-coated poly(L-lactide) filaments induce robust neurite growth while providing directional orientation

Cellular channels during development and after peripheral nerve injury are thought to provide guidance cues to growing axons. In tissue culture where these cues are absent, neurites from dorsal root ganglion neurons grow with a radial distribution. To induce directional axonal growth and to enhance the rate of axonal growth after injury, we have designed microfilaments of poly(L-lactide). We demonstrate that dorsal root ganglia grown on these filaments in vitro extend longitudinally oriented neurites in a manner similar to native peripheral nerves. The extent of neurite growth was significantly higher on laminin-coated filaments compared with uncoated and poly-L-lysine-coated filaments. As high as 5.8 +/- 0.2 mm growth was observed on laminin-coated filaments compared with 2.0 +/- 0.2 mm on uncoated and 2.2 +/- 0.3 mm on poly-L-lysine-coated filaments within 8 days. Schwann cells were found to grow on all types of filaments. They were, however, absent in the leading edges of growth on laminin-coated filaments. Photolysis of Schwann cells caused a significant reduction in the neurite length on all types of filaments. Laminin-coated filaments, however, induced significantly longer neurites compared with uncoated and/or poly-L-lysine-coated filaments even in the absence of Schwann cells. Our results suggest that laminin-coated poly(L-lactide) filaments are suitable for inducing directional and enhanced axonal growth. Implants designed by arranging these microfilaments into bundles should aid regenerating axons by providing guidance cues and channels to organize matrix deposition, cell migration, axon growth, and improve functional recovery.

Macrophage/Microglia Regulation of Astrocytic Tenascin: Synergistic Action of Transforming Growth Factor-β and Basic Fibroblast Growth Factor

After injury to the CNS, extracellular matrix molecules such as tenascin are upregulated around the injury site and may be involved in inhibition of axon growth. In the present study, astrocytes were investigated to determine which cell types, growth factors, or cytokines are responsible for the injury-induced regulation of tenascin. The addition of activated macrophage- or microglial-conditioned medium increased astrocytic expression of tenascin 2.5-fold, as determined by Northern and Western blot analysis and ELISA. Of the cytokines and growth factors examined, only transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (bFGF) significantly induced an increase in the production of astrocytic tenascin. Examination of macrophage and microglial supernatants showed the presence of TGF-β1 but not bFGF; however, the TGF-β1 concentration in supernatants was lower than that expected to induce an increase in astrocytic tenascin similar to that seen with recombinant TGF-β1. Western blot analysis of astrocytes showed only the presence of bFGF. Compared with the responses of the individual growth factors, tenascin production by astrocytes was dramatically potentiated when grown in the presence of a combination of both TGF-β1 and bFGF. A similar synergistic effect was observed after the addition of either TGF-β1 or bFGF to macrophage-conditioned medium. Northern analysis also showed concomitant increases in TGF-β1, bFGF, and tenascin after CNS injury to animals 14 d of age or older. These results show that the regulation of astrocytic tenascin is mediated by the synergistic action of TGF-β1 and bFGF in vitro and after injury in vivo.

Altered apolipoprotein E secretion in cytokine treated human astrocyte cultures

Apolipoprotein E (ApoE), postulated to be a major lipid carrier protein in brain, is synthesized and secreted primarily by astrocytes and is involved in brain development and repair. We have analyzed its secretion in primary cultures of older (high passage) slowly dividing and younger (lower passage) rapidly dividing fetal human astrocytes exposed to various inflammatory and anti-inflammatory cytokines, alone and in combination. ApoE secretion was reduced in high passage astrocytes when compared to lower passage astrocytes. A further reduction in ApoE secretion in high passage cells was consistently produced by the combination of cytokines interleukin 1 (IL-1) alpha and beta and interferon (IFN-gamma) cytokines or by the basic fibroblast growth factor (basic-FGF) alone. Epidermal growth factor (EGF) increased ApoE secretion. The combination of these cytokine effects in chronically degenerating brain regions of Alzheimers disease and other neurodegenerative diseases could reduce the amount of ApoE available for neuronal regeneration. EGF, or agents inducing EGF, could ameliorate these ApoE deficiencies.

Astrocytes Infected with Replication-Defective Adenovirus Containing a Secreted Form of CNTF or NT3 Show Enhanced Support of Neuronal Populationsin Vitro

Neurotrophic factors have been shown to ameliorate neuronal death in several in vitro and in vivo models of neurodegenerative disease. However, delivery of polypeptide growth factors to compromised neurons in the CNS is problematic as the blood-brain barrier prevents systemic delivery, and chronic in-dwelling cannulae are required for intraparenchymal delivery. To circumvent these problems and specifically target neurotrophic factors to the environment surrounding degenerating neurons in the CNS, we have generated replication-defective adenovirus (Ad) vectors that contain a secretable form of ciliary neurotrophic factor (sCNTF) or neurotrophin-3 (NT-3). In this study, we demonstrate that sCNTF/Ad and NT-3/Ad can efficiently infect primary astrocytes, resulting in gene transcription and the production of functional protein. Using Northern blot analysis, dose-dependent expression of sCNTF or NT-3 mRNA was detected 7 days after infection. The levels of mRNA expressed in transgenic astrocytes was dependent on virus titer and increased with increasing virus concentration. sCNTF or NT-3 protein was also detected in astrocyte supernatants by immunoblot analysis and 2-site ELISA. ELISA indicated that astrocytes infected with sCNTF/Ad or NT-3/Ad secreted neurotrophic factors at a rate of approximately 120 pg/10(6) cells/h and 350 pg/10(6) cells/h, respectively. To test for secretion of bioactive sCNTF or NT-3 protein, E8 chick ciliary ganglion or nodose ganglion neurons were grown in medium conditioned by control astrocytes or astrocytes treated with sCNTF/Ad or NT-3/Ad, showing a robust and dose-dependent increase in neuronal survival when compared to control supernatant. In addition, motor neurons plated onto astrocyte monolayers pretreated with sCNTF/Ad showed a two- to fourfold increase in ChAT activity when compared to those grown on astrocytes pretreated with Lac-Z/Ad. This study demonstrates that, using replication-defective adenovirus, primary astrocytes can be efficiently engineered to secrete bioactive sCNTF or NT-3, resulting in enhanced survival of responsive peripheral and central neuronal populations.

Visualization of axonally transported horseradish peroxidase using enhanced immunocytochemical detection: A direct comparison with the tetramethylbenzidine method

Visualization of the neuronal tract tracer horseradish peroxidase (HRP) is commonly achieved through the histochemical detection of its enzymatic activity using 3,3′,5,5′-tetramethylbenzidine (TMB) as a chromogen. However, the TMB product is unstable and is incompatible with tissue processing methods that render the enzyme inactive, or when a combination of HRP tract tracing with neuronal phenotype identification is required. In this study we evaluated the applicability of the immunocytochemical detection method for horseradish peroxidase (HRP) visualization using an enhanced detection method based on the Elite ABC peroxidase amplification protocol. The results provide evidence for the immunocytochemical visualization of both anterograde and transganglionic HRP transport in the rat spinal cord. This immunocytochemical method not only showed similar sensitivity to the TMB protocol in detecting HRP-labeled motor neuron perikarya but provided enhanced resolution in the identification of individual neuronal fibers compared to the TMB method. Immunodetection of the HRP tracer also allowed its co-localization with specific neuronal markers using double immunofluorescence techniques. These results offer the first demonstration that sensitive identification of axonally transported HRP can be achieved by immunocytochemistry and provides further support for its use in HRP tract tracing studies.

Bioresorbable filaments enhance nerve regeneration

Although axonal regeneration does occur after injury in the peripheral nervous system (PNS), growth across a gap and functional restoration is as yet an unmet challenge. To improve axonal and functional nerve regeneration, the authors examined bioresorbable neural stents designed to organize cellular migration and direct axon growth across a lesion. Extruded and drawn (6:1) poly(L-lactide) (PLLA) (MW 200kD) filaments were bundled, placed into silicone tubes (2 mm O.D., 1.5 mm I.D.) and used to bridge a 10-mm gap in the rat sciatic nerve. Empty silicone tubes served as controls. In a companion experiment, filament packing densities of 0% (0 filament), 3.75% (16), 7.5% (32), 15% (64), and 30% [128] with wet-spun PLLA filaments (MW 200kD) were tested. After 10 weeks, animals were perfused and implants were prepared for histological analyses. Light microscopy and TEM both showed improved regeneration for extruded and wet-spun filaments versus controls, in terms of nerve cable formation and number of myelinated axons bridging the gap. In addition, the morphology of the regenerated nerve segment was similar to the multifascicular structure of uninjured nerve. Filament packing densities of 3.75-7.5% gave the best improvement of nerve regeneration. The authors demonstrate that PLLA filament bundles enhance the extent and consistency of nerve regeneration across a gap. The results suggest that the authors PLLA filament bundles might be used to establish functional connectivity after nerve injury.