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Autism, a neurodevelopmental disorder affecting between 0.2% and 0.5% of the population, is characterized by deficits in communication and behavior. Although the cause of autism is unknown, studies strongly suggest a genetically heritable risk factor (reviewed in Geschwind & Levitt, 2007). Epilepsy, particularly seizure disorders of the frontal and temporal lobes (reviewed in Gedye, 1991; Salmond et al., 2005), is highly comorbid with autism (Clarke et al., 2005; Kagan-Kushnir et al., 2005), although the potential interactions between autistic symptoms and epileptiform activity are still under investigation (Deonna & Roulet, 2006). Mutations in a number of candidate genes have been suggested based on chromosomal linkage analysis, but causative roles have remained elusive (Maestrini et al., 1999; Newbury et al., 2002). Recently, however, single nucleotide polymorphisms in the autism susceptibility region 2q 34 of the human genome coding for neuropilin 2 (NPN2; also known as NRP2) have been identified and strongly associated with autism (Wu et al., 2007). NPN2 is a receptor for the chemorepulsive axon guidance mediator semaphorin 3F (Sema3F; a member of the class III semaphorins) (Xiang et al., 1996) and is part of a family of axon guidance cues that are involved in responses to brain injury and environment changes (Tessier-Lavigne & Goodman, 1996; Song & Poo, 2001; Pasterkamp & Verhaagen, 2006), as well as during neuronal migration and differentiation in development (Marin et al., 2001; Pozas et al., 2001; Dent et al., 2003; Fenstermaker et al., 2004; Niell et al., 2004; Pascual et al., 2004). Further, this signaling system is highly expressed in adult hippocampus (Chen et al., 2000; Barnes & Slevin, 2003; Barnes et al., 2003; Sahay et al., 2003; Pascual et al., 2004; Sahay et al., 2005; Yang et al., 2005). In rodent studies, neuropilin expression levels are increased by exposure to enriched environments (Cao et al., 2004) and decreased by chemically induced status epilepticus (SE) and/or N-methyl D-aspartate (NMDA) receptor blockade (Barnes et al., 2003; de Wit & Verhaagen, 2003; Holtmaat et al., 2003; O'Donnell et al., 2003; Yang et al., 2005) (but see Shimakawa et al., 2002). Importantly, genetic lesioning studies knocking out different components of this signaling system (NPN2, Sema3F, or plexin A3) have resulted in animals with extended infrapyramidal mossy fiber axonal pathways and spontaneous seizures (Giger et al., 2000; Sahay et al., 2005), similar to those found in the epileptic brain (Cavazos & Cross, 2006; Holtkamp & Meierkord, 2007; Nadler et al., 2007). These data suggest that semaphorin signaling is normally associated with experience-dependent neuronal activity and that experimental manipulations decreasing this signaling pathway's function are closely allied with hyperexcitability and abnormal neuritic outgrowth in the hippocampus. However, NPN2 has pleiotropic roles in guidance, differentiation, and neuritogenesis, and it is not known to what degree developmental alterations contribute to these phenotypes. Here, we used a NPN2 knockout murine model to assess the consequences of deficits in this signaling system to behavioral convulsions, hippocampal excitability, neuronal cell type/number, ultrastructural morphology (neurite length and complexity as well as dendritic spine type and number), and synaptic protein content. Our results strongly suggest that a deficit in NPN2 during development critically affects interneuron migration and differentiation in the hippocampus and that this has downstream consequences, negatively impacting seizure susceptibility and hippocampal network excitability. Given earlier work showing NPN2's association with autism, this model system that appears to recapitulate at least the seizure susceptibility aspects may be ideal for investigating NPN2's putative role in this neurodevelopmental disease and may aid in defining new therapeutic interventions for autism and autism-related epilepsy.

Neurons are continuously added to the hippocampal dentate gyrus throughout life. These neurons must develop dendritic arbors and spines by which they form synapses for making functional connections with existing neurons. The molecular mechanisms that regulate dendritic development and synaptic formation of postnatal-born granular neurons in the dentate gyrus are largely unknown. Hippocampal dentate gyrus (HDG) has been shown to express high level of brain-derived neurotrophic factor (BDNF). Here we reported that when BDNF is conditionally knockout in the postnatal-born granular neurons of the HDG, the mutant neurons exhibit aberrant morphological development with less dendritic branches, shorter dendritic length, and lower density of dendritic spines, while their primary dendrites are not obviously affected. Even though, these BDNF-deficient granular neurons develop immature dendritic spines to initiate synaptic contacts with afferent axons, they fail to develop or maintain mature spine structures. Thus, these postnatal-born neurons have fewer numbers of synapses, particularly mature synaptic spines. These results suggest that BDNF plays an important role during dendritic development, synaptic formation and synaptic maturation in postnatal-born granular neurons of the HDG in vivo.

To assess the role of white matter inhibition as a barrier to neurite outgrowth in vivo, we unilaterally transected three consecutive lumbar dorsal roots (L4-L6), incised the spinal cord, and transplanted the peripheral stump of L4 either medially onto the white matter of the dorsal columns or laterally, just superficial to the gray matter of the dorsal horn at the level of L5. Three weeks to seven months later, the translocated root was retransected, and its central stump was anterogradely labeled with HRP. The staining pattern demonstrated that regenerating sensory axons had entered the spinal cord from both medially and laterally placed roots. Axonal staining from medially placed dorsal roots (onto the white matter of the dorsal columns) was sparse and limited to the white matter. Staining of laterally placed roots revealed a small subpopulation of regenerating axons which had entered the gray matter and formed terminal arbors. Successful axonal regeneration into the gray matter, albeit minimal, was associated with a localized and limited inflammatory response near the sites of axonal ingrowth.

There are few studies of neural implants in spinal cord injury (SCI) focused on supporting directed axon growth. In this study, we fabricated a macroporous poly (lactic acid) (PLA) foam with oriented inner channels. Amorphous foam without linear channels served as a control in an acute SCI injury model, and the effectiveness of foam with linear channels was further investigated in a chronic SCI model. Implants were placed into a 2 mm hemisection lesion cavity at the T8 spinal cord level in adult rats. Two weeks post-implantation, tissue sections including the implants were examined using antibodies against GFAP, p75, ED-1, laminin, GAP-43, and CGRP. Foam implants were well-integrated with the host spinal cord. In linear foams, numerous DAPI-stained cells were found within the inner channels. Schwann cells but not astrocytes had migrated within the channels. Intense laminin staining was observed throughout the extracellular matrix substrate. GAP-43- and CGRP-positive axons grew through the implants following the linear channels. In the amorphous control foams, DAPI staining distributed evenly through the pores. However, the growth of GAP-43 or CGRP-positive axons was misguided and impeded at the entrance area of the foam. Higher numbers of GAP-43 and CGRP-positive axons grew into linear foam implants after chronic SCI than acute SCI. These results suggest the potential application of linear foam implants in cell and axon guidance for SCI repair, especially for chronic SCI.

Axonal growth cones are guided to their targets by contact-dependent mechanisms or by diffusible chemotropic factors. Axon guidance by these factors typically involves culturing neurons on an acellular substrate which may not represent the in vivo biological environment. We developed two novel in vitro methods to create patterned gene expression of guidance molecules in a physiologically-relevant cellular environment. In the Matrigel™ assay, a droplet of adenovirus-Matrigel™ suspension was placed on astrocytes grown in Matrigel™. The adenovirus diffused through the gel and transduced underlying astrocytes, creating a radial infection gradient within a localized area. In the second model, recombinant adenovirus was bound to an anti-hexon antibody adsorbed onto stripe patterns of nitrocellulose. Once the cells were added, only those contacting the adenovirus were infected. The outgrowth pattern of chick DRG neurons on NGF, semaphorin 3A and brevican were studied. As expected, results showed robust axonal growth toward NGF as opposed to either secreted Sema 3A or membrane bound brevican, however subtle differences in axonal growth responses were observed in comparison to those obtained with less physiologically-relevant methods. Novel to this technology, the location and area of molecule expression can be controlled and manipulated in an intricate cellular environment.

After injury to the adult central nervous system (CNS), numerous cytokines and growth factors are released that contribute to reactive gliosis and extracellular matrix production. In vitro examination of these cytokines revealed that the presence of transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) greatly increased the production of several chondroitin sulfate proteoglycans (CSPG) by astrocytes. Treatment of astrocytes with other EGF-receptor (ErbB1) ligands, such as TGF-alpha and HB-EGF, produced increases in CSPG production similar to those observed with EGF. Treatment of astrocytes, however, with heregulin, which signals through other members of the EGF-receptor family (ErbB2, ErbB3, ErbB4), did not induce CSPG upregulation. The specificity of activation through the ErbB1 receptor was further verified by using a selective antagonist (AG1478) to this tyrosine kinase receptor. Western blot analysis of astrocyte supernatant pre-digested with chondroitinase ABC indicated the presence of multiple core proteins containing 4-sulfated or 6-sulfated chondroitin. To identify some of these CSPGs, Western blots were screened using antibodies to several known CSPG core proteins. These analyses showed that treatment of astrocytes with EGF increased phosphacan expression, whereas treatment with TGF-beta1 increased neurocan expression. Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of these molecules in vivo, which result in increased expression of TGF-beta1, EGF-receptor, neurocan, and phosphacan after injury to the brain. These data begin to elucidate some of the injury-induced growth factors that regulate the expression of CSPGs which could be targeted in the future to modulate CSPG production after injury to the central nervous system.

The host response to neural injury, which can include axonal sprouting and synaptic reorganization is likely to be under tight genetic regulatory control at the level of the genome and may be implicated in epileptogenesis. Despite its importance, however, the molecular basis of synaptic reorganization is unclear. We have studied the development of synaptic reorganization, semaphorin gene expression, and epileptogenesis in hippocampus of epileptogenic sensitive (FVB/NJ) and epileptogenic resistant (C57BL/6J) mice (i.e. distinct genetic backgrounds) after kainic acid-induced status epilepticus. Our results support the hypothesis that disruption of transcriptional regulation of axon guidance genes leads to a differential loss of tonic neuropilin-2 dependent activation of semaphorin 3F receptors on hippocampal neurons on distinct genetic backgrounds. This results in rearranged synaptic circuitry and thus promotes epileptogenesis. These findings may define biologic principles underlying the role of semaphorin signaling which may broadly apply to other systems undergoing neural regeneration.

Axon regeneration in vivo is blocked at boundaries between Schwann cells and astrocytes, such as occur at the dorsal root entry zone and around peripheral nerve or Schwann cell grafts. We have created a tissue culture model of these boundaries in Schwann cell - astrocyte monolayer co-cultures. Axon behaviour resembles that in vivo, with axons showing a strong preference for Schwann cells over astrocytes. At boundaries between the two cell types, axons growing on astrocytes cross readily onto Schwann cells, but only 15% of axons growing on Schwann cells are able to cross onto astrocytes. Treatment with chondroitinase or chlorate to reduce inhibition by proteoglycans did not change this behaviour. The neural adhesion molecule L1 is present on Schwann cells and not astrocytes, and manipulation of L1 by application of an antibody, L1-Fc in solution, or adenoviral transduction of L1 into astrocytes increased the proportion of axons able to cross onto astrocytes to 40-50%. Elevating cAMP levels increased crossing from Schwann cells onto astrocytes in live and fixed cultures, and had a co-operative effect with NT-3 but not with NGF. Inactivation of Rho with a cell-permeant form of C3 exoenzyme also increased crossing from Schwann cells to astrocytes. Our experiments indicate that the preference of axons for Schwann cells is largely mediated by the presence of L1 on Schwann cells but not astrocytes, and that manipulation of growth cone signalling pathways can allow axons to disregard boundaries between the two cell types.

Neutral sphingomyelinase (NSMase) has been proposed to mediate interleukin (IL)-1beta signaling in liver. In this paper, we used adenovirus-mediated gene transfer to inducibly express FLAG-tagged mouse NSMase-2 in primary rat hepatocytes in order to further elucidate the molecular nature of the NSMase involved. Initial studies confirmed that the EST clone used in these experiments encoded a Mg2+-dependent NSMase. The in vitro activity of the heterologously expressed enzyme was inhibited in the presence of 0.5% Triton or 50 mM EDTA. In addition, the expression of this NSMase-2 clone in primary hepatocytes led to increased cellular levels of ceramide, indicating that the enzyme is active in situ. Immunofluorescence studies in Hep G2 cells infected with NSMase-2 expressing adenoviruses showed that the FLAG-tagged NSMase-2 was localized to the plasma membrane. Cell viability remained unchanged 72 h following infection and induction. The effect of NSMase-2 expression on IL-1beta-induced activation of c-Jun N-terminal kinase (JNK) was tested. Expression of NSMase-2 increased JNK phosphorylation between 1.5- and 2-fold over the basal level. Furthermore, NSMase-2 was found to strongly increase the ability of IL-1beta to phosphorylate JNK. This potentiation was mediated by a phosphatase from the PP2A family, possibly by modulating the phosphorylation pattern of IL-1beta receptor-associated kinase (IRAK). In conclusion, the data presented suggest that NSMase-2 could be involved in IL-1beta-induced JNK activation in hepatocytes.

The success of entubulation for peripheral nerve regeneration is still limited, especially with long lesion gaps. In this study, we examined if regeneration could be en- hanced by constructing implants to both align axonal growth and promote Schwann cell proliferation and migra- tion. Silicone implants were used to bridge a 1.4-cm gap in the rat sciatic nerve. Adult female Sprague-Dawley rats were divided into four groups of tubes containing either 1) Matrigel; 2) Matrigel and heregulin; 3) Matrigel and poly(l- lactic acid) (PLLA) microfilaments; or 4) Matrigel, PLLA microfilaments, and heregulin. Ten weeks postimplantation, the number of axons and Schwann cells were measured at the distal end of implants. Implants with microfilaments displayed better tissue cable formation, increased Schwann cell migration, and regeneration of anti-calcitonin gene-re- lated peptide-positive axons, but not RMDO95-positive ax- ons compared with nonfilament-containing groups. Heregu- lin treatment caused an increase in Schwann cell number, but it demonstrated no significant improvement in either tissue cable formation or axon number. Extensive regenera- tion was observed through implants containing Matrigel, microfilaments, and heregulin, which induced significant improvements in the number and longitudinal organization of both Schwann cells and axons. These results indicate that physical guidance of microfilaments and the Schwann cell growth factor, heregulin, act synergistically to improve nerve regeneration across long lesion gaps. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 247-258, 2004

Increased expression of NGF after spinal cord injury induces sprouting of primary nociceptive axons. Exogenous application of NGF also results in extensive sprouting of these axons and causes chronic pain in uninjured animals. During development, semaphorin3A is thought to act as a repulsive guidance cue for NGF-responsive nociceptive afferents, restricting their projections to the superficial dorsal horn. We investigated the ability of semaphorin3A to selectively reduce NGF-induced sprouting and neuropathic pain in adult rats. The chemorepulsive effect of virus-mediated semaphorin3A expression was shown to counteract the sprouting induced by NGF in a dose-dependent manner, bothin vitroand in adult rat spinal cords. Coexpression of semaphorin3A and NGF at moderate to low concentrations within the adult spinal cord reduced sprouting of calcitonin gene-related peptide and substance P-containing axons compared with GFP and NGF coexpression controls. At high expression levels of NGF, there was no difference in sprouting between the semaphorin3A-treated and control groups. The distribution of endogenous primary nociceptive afferents in the spinal cord appeared to be unaffected by semaphorin3A treatment in these experiments. Behavioral assessment shows that semaphorin3A coexpression with NGF led to decreased mechanical allodynia but no significant reductions in thermal hyperalgesia. These findings demonstrate directly that mature sensory afferents maintain their responsiveness to semaphorin3A, suggesting that this molecule might be used therapeutically to control aberrant sensory sprouting involved in pain or autonomic dysfunction.

The loss of NF2 gene function leads to vestibular nerve schwannoma formation in humans. The NF2 gene product, Merlin/Schwannomin, has recently been found to interact with the two PDZ domains containing protein EBP50/NHE-RF, which is itself known to interact with the PDGF receptor (PDGFR) in several cell types. In this study, an up-regulation of both PDGFR and EBP50/NHE-RF, and an interaction of both proteins were found in primary human schwannoma tissue. Furthermore, using an adenoviral vector mediated gene transfer technique, changes in the phenotypic characteristics after NF2 gene restoration in a newly established NF2 gene-mutated human schwannoma cell line (HEI 193) were investigated. The overexpression of Merlin/Schwannomin in HEI 193 led to an inhibition of cell proliferation under serum-free conditions. Upon PDGF stimulation in culture, Merlin/Schwannomin appeared to inhibit the activation of the MAPK and PI3K signaling pathways, impinging on the phosphorylation of Erk 1/2 and Akt, respectively. The data also show that PDGFR is more rapidly internalized by the schwannoma cells overexpressing NF2. Therefore, this process is suggested as a model for a mechanism of Merlin/Schwannomin tumor suppressor function, which intermediates acceleration of the cell surface growth factor degradation.

After injury, axonal regeneration occurs across short gaps in the peripheral nervous system, but regeneration across larger gaps remains a challenge. To improve regeneration across extended nerve defects, we have fabricated novel microfilaments with the capability for drug release to support cellular migration and guide axonal growth across a lesion. In this study, we examine the nerve repair parameters of non-loaded filaments. To examine the influence of packing density on nerve repair, wet-spun poly(L-Lactide) (PLLA) microfilaments were bundled at densities of 3.75, 7.5, 15, and 30% to bridge a 1.0-cm gap lesion in the rat sciatic nerve. After 10 weeks, nerve cable formation increased significantly in the filament bundled groups when compared to empty-tube controls. At lower packing densities, the number of myelinated axons was more than twice that of controls or the highest packing density. In a consecutive experiment, PLLA bundles with lower filament-packing density were examined for nerve repair across 1.4- and 1.8-cm gaps. After 10 weeks, the number of successful regenerated nerves receiving filaments was more than twice that of controls. In addition, nerve cable areas for control groups were significantly less than those observed for filament groups. Axonal growth across 1.4- and 1.8-cm gaps was more consistent for the filament groups than for controls. These initial results demonstrate that PLLA microfilaments enhance nerve repair and regeneration across large nerve defects, even in the absence of drug release. Ongoing studies are examining nerve regeneration using microfilaments designed to release neurotrophins or cyclic AMP.

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.

Excitatory agonists can induce significant smooth muscle contraction under constant free Ca(2+) through a mechanism called Ca(2+) sensitization. Considerable evidence suggests that free arachidonic acid plays an important role in mediating agonist-induced Ca(2+)-sensitization; however, the molecular mechanisms responsible for maintaining and regulating free arachidonic acid level are not completely understood. In the current study, we demonstrated that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is expressed in vascular smooth muscle tissues. Inhibition of the endogenous iPLA(2) activity by bromoenol lactone (BEL) decreases basal free arachidonic acid levels and reduces the final free arachidonic acid level after phenylephrine stimulation, without significant effect on the net increase in free arachidonic acid stimulated by phenylephrine. Importantly, BEL treatment diminishes agonist-induced Ca(2+) sensitization of contraction from 49 +/- 3.6 to 12 +/- 1.0% (p0.01). In contrast, BEL does not affect agonist-induced diacylglycerol production or contraction induced by Ca(2+), phorbol 12,13-dibutyrate (a protein kinase C activator), or exogenous arachidonic acid. Further, we demonstrate that adenovirus-mediated overexpression of exogenous iPLA(2) in mouse portal vein tissue significantly potentiates serotonin-induced contraction. Our data provide the first evidence that iPLA(2) is required for maintaining basal free arachidonic acid levels and thus is essential for agonist-induced Ca(2+)-sensitization of contraction in vascular smooth muscle.

Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function.

Various types of tracers are available for use in axon regeneration, but they require an extra operational tracer injection, time-consuming immunohistochemical analysis and cause non-specific labeling. Considerable efforts over the past years have explored other methodologies, especially the use of viral vectors, to investigate axon regeneration after injury. Recent studies have demonstrated that self-complementary Adeno-Associated Virus (scAAV) induced a high transduction efficiency and faster expression of transgenes. Here, we describe for the first time the use of scAAV2-GFP to label long-projection axons in the corticospinal tract (CST), rubrospinal tract (RST) and the central axons of dorsal root ganglion (DRG) in the normal and lesioned animal models. We found that scAAV2-GFP could efficiently transduce neurons in the sensorimotor cortex, red nucleus and DRG. Strong GFP expression could be transported anterogradely along the axon to label the numerous axon fibers from CST, RST and central axons of DRG separately. Comparison of the scAAV2 vector with single-stranded (ss) AAV2 vector in co-labeled sections showed that the scAAV2 vector induced a faster and stronger transgene expression than the ssAAV2 vector in DRG neurons and their axons. In both spinal cord lesion and dorsal root crush injury models, scAAV-GFP could efficiently label the lesioned and regenerated axons around the lesion cavity and the dorsal root entry zone (DREZ) respectively. Further, scAAV2-GFP vector could be combined with traditional tracer to specifically label sensory and motor axons after spinal cord lesion. Thus, we show that using scAAV2-GFP as a tracer is a more effective and efficient way to study axon regeneration following injury.

Slow axonal transport conveys cytoskeletal proteins from cell body to axon tip. This transport provides the axon with the architectural elements that are required to generate and maintain its elongate shape and also generates forces within the axon that are necessary for axon growth and navigation. The mechanisms of cytoskeletal transport in axons are unknown. One hypothesis states that cytoskeletal proteins are transported within the axon as polymers. We tested this hypothesis by visualizing individual cytoskeletal polymers in living axons and determining whether they undergo vectorial movement. We focused on neurofilaments in axons of cultured sympathetic neurons because individual neurofilaments in these axons can be visualized by optical microscopy. Cultured sympathetic neurons were infected with recombinant adenovirus containing a construct encoding a fusion protein combining green fluorescent protein (GFP) with the heavy neurofilament protein subunit (NFH). The chimeric GFP-NFH coassembled with endogenous neurofilaments. Time lapse imaging revealed that individual GFP-NFH-labeled neurofilaments undergo vigorous vectorial transport in the axon in both anterograde and retrograde directions but with a strong anterograde bias. NF transport in both directions exhibited a broad spectrum of rates with averages of approximately 0.6-0.7 microm/sec. However, movement was intermittent, with individual neurofilaments pausing during their transit within the axon. Some NFs either moved or paused for the most of the time they were observed, whereas others were intermediate in behavior. On average, neurofilaments spend at most 20% of the time moving and rest of the time paused. These results establish that the slow axonal transport machinery conveys neurofilaments.

This study characterized gene transfer into both normal and injured adult rat dorsal spinal cord using first (E1-/E3-) or second (E1-/E2A125/E3-, temperature-sensitive; ts) generation of replication-defective adenoviral (Ad) vectors. A novel immunosuppressive regimen aimed at blocking CD4/CD45 lymphocytic receptors was tested for improving transgene persistence. In addition, the effect of gene transfer on nociception was also evaluated. Seven days after treatment, numerous LacZ-positive cells were observed after transfection with either viral vector. By 21 days after transfection, beta-galactosidase staining was reduced and suggestive of ongoing cytopathology in both Ad-treated groups, despite the fact that the immunogenicity of LacZ/Adts appeared less when compared with that elicited by the LacZ/Ad vector. In contrast, immunosuppressed animals showed a significant (P < or = 0.05) increase in the number of LacZ-positive cells not displaying cytopathology. In these animals, a concomitant reduction in numbers of macrophages/microglia and CD4 and CD8 lymphocytes was observed. Only animals that received LacZ/Adts and immunosuppression showed transgene expression after 60 days. Similar results were observed in animals in which the L4-L5 dorsal roots were lesioned before transfection. Gene transfer into the dorsal spinal cord did not affect nociception, independent of the adenovirus vector. These results indicate that immune blockade of the CD4/CD45 lymphocytic receptors enhanced transgene stability in adult animals with normal or injured spinal cords and that persistent transgene expression in the spinal cord does not interfere with normal neural function.

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.

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 bFGFin vitroand after injuryin vivo.

During development, semaphorins (collapsin, fasciclin) mediate repulsive and inhibitory guidance of neurons1–4. Semaphorin III, a secretable member of this family, is expressed by the ventral spinal cord at the time corresponding to projection of sensory afferents from the dorsal root ganglion (DRG) into the spinal cord6. The inhibitory effect of E14 ventral cord is active only on nerve growth factor (NGF)-responsive sensory afferents (smalldiameter A-delta and C fibers subserving sensations of temperature and pain)5. Similarly, COS cells secreting recombinant semaphorin III are able to selectively repel DRG afferents whose growth is stimulated by NGF and not NT-3 (ref. 7). However, it is not known whether these molecules can exert a functional role in the fully developed adult peripheral nervous system. In this study, we demonstrated that gene gun transfection and production of semaphorin III in corneal epithelial cells in adult rabbits in vivo can cause repulsion of established A-delta and C fiber trigeminal sensory afferents. In addition, it is shown that, following epithelial wounding and denervation, semaphorin III is able to inhibit collateral nerve sprouts from innervating the reepithelialized tissue. These findings are significant in that they provide direct evidence that small-diameter adult sensory neurons retain the ability to respond to semaphorin III. In addition, the corneal gene gun technique may be generally used to study the in vivo effects of neural growth modulators by quantifying the amount of sensory nerve growth.

Smith, George M., Richard L. Berry, Jay Yang, and Darrell Tanelian. Electrophysiological analysis of dorsal root ganglion neurons pre- and post-coexpression of green fluorescent protein and functional 5-HT3receptor. J. Neurophysiol. 77: 3115–3121, 1997. Aequorea green fluorescent protein (GFP) is an excellent marker to examine genetically altered live cells in whole animals or culture. Its potential use in identifying genetically modified neurons, however, has not been investigated extensively. To examine the usefulness, toxicity, and potential electrophyiological effects of GFP expression in neurons, we generated adenovirus containing the mGFP4 cDNA. One week after virus transfection of dorsal root ganglion neurons (DRG), 10% of postnatal DRG neurons appeared brightly fluorescent, labelling the soma and neurites. Temporal examination of these neurons demonstrated no toxicity to DRG neurons even after several weeks in culture with repeated daily epifluorescent exposure. Electrophysiological analysis and comparison of control and viral exposed (GFP− and GFP+) DRG neurons did not demonstrate any differences in whole cell resistance, resting potential, action potential (AP) threshold, AP duration, AP amplitude, or whole cell capacitance. To investigate the usefulness of GFP as a marker for identifying neurons genetically altered to express a novel neurotransmitter receptor, a second adenovirus construct was generated containing both GFP and serotonin type 3 (5-HT3) receptor cDNAs. Transfection of DRG neurons with this virus produced an inward current in the presence of serotonin only in DRG neurons that were GFP-positive. It is concluded that adenoviral transfection of neurons with GFP, for cellular labeling, and coexpression of GFP-neurotransmitter constructs are safe, nontoxic, methods for electrophysiologically investigating neurons over several weeks. The uniqueness of the vector used in these experiments is that it was constructed to express GFP in a second cassette so that it would label the transduced cells, but have no potential for interfering with the function of the foreign 5-HT3receptor.

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 Alzheimer's 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.

Despite tremendous effort, glioblastoma (GBM) remains the most pervasive and lethal of all brain malignancies. One factor that contributes to this exceedingly poor prognosis is the highly invasive character of the tumor. GBM is characterized by microscopic infiltration of tumor cells throughout the otherwise healthy brain, whereas non-neural metastases, as well as select lower-grade gliomas develop as self-contained and clearly delineated lesions. Illustrated by rodent xenograft tumor models as well as pathological human patient specimens, we present evidence that one fundamental switch between these two distinct pathologies - invasion and non-invasion - is mediated through the tumor extracellular matrix (ECM). Specifically, non-invasive lesions are associated with a rich matrix containing substantial amounts of glycosylated chondroitin sulfate proteoglycans (CSPGs), whereas glycosylated CSPGs are essentially absent from diffusely infiltrative tumors. CSPGs, acting as a central organizer of the tumor microenvironment, dramatically influence resident reactive astrocytes, inducing their exodus from the tumor mass and the resultant encapsulation of non-invasive lesions. Additionally, CSPGs induce activation of tumor-associated microglia. We demonstrate that the astrogliotic barrier can directly inhibit tumor invasion, and its absence from GBM presents an environment favorable to diffuse infiltration. We also identify the LAR phosphatase receptor as a putative intermediary between extracellular CSPGs and the tumor cells themselves. In all, we present CSPGs as critical regulators of brain tumor histopathology and help to clarify the role of the tumor microenvironment in brain tumor invasion. Citation Format: Daniel J. Silver, Florian A. Siebzehnrubl, Michela J. Schildts, Anthony T. Yachnis, George M. Smith, Amy A. Smith, Bjorn Scheffler, Brent A. Reynolds, Jerry Silver, Dennis A. Steindler. Chondroitin sulfate proteoglycans potently inhibit invasion and serve as a central organizer of the brain tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2013-1639

An investigation of the transport of solids in combined sewers during both dry weather flow (DWF) periods and storms is described. The study was based on data obtained from a number of sites in the combined sewer system of Dundee, Scotland. The relationship between hydraulic conditions in a combined sewer and the transport of solids in suspension was examined. The aim was to arrive at a methodology by which an appropriate model could be selected or developed which would predict solids transport rates given information on hydraulic conditions. It was found that for individual sites, site-specific regression equations could be developed separately for dry weather and storm conditions. A non-site-specific regression equation was also developed, which was found to be preferable to the site specific equations, in terms of accuracy and ease of use. More important, however, were the fundamental procedures (ie the methodology) developed by which the model type was in each case selected and subsequently developed.

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cochlear development. Whereas tissue specific differences in cochlear ODC activity have been demonstrated, cellular localization of ODC protein in the inner ear of the immature rat has not. ODC was localized in inner ear structures using an ODC polyclonal antibody and the effects of cycloheximide on ODC immunoreactivity and enzymatic activity were determined. Tissues demonstrating elevated enzymatic activity contained cells with the strong immunoreactivity. ODC activity was highest in the organ of Corti and lateral wall followed by the cochlear nerve. Immunoreactivity was demonstrated throughout the cochlea with intense staining of the hair cells, pillar cells, Deiter's cells, inner sulcus cells, basilar membrane, stria vascularis, spiral ganglion cell bodies and cochlear nerve fibers. Cycloheximide rapidly diminished cochlear ODC activity and expression of ODC protein. The half-life of cochlear ODC was 30 min. Localization of cellular sites of ODC is important in understanding the role of the ODC-polyamine pathway in cochlear development and will be a valuable marker for tissue damage from ototoxic agents.

Tight junctions may play an important role in maintaining the integrity of the blood-brain barrier. These junctions can be individually visualized using electron microscopy but no current technique is able to provide a more global picture of the presence and density of tight junctions in central nervous system tissue. We used an antibody that recognizes a high molecular weight protein (ZO-1) associated with tight junctions, to identify these specialized junctions within the rat brain and spinal cord. Immunofluorescent labeling showed a network of tight junctions between cells in the brain vasculature, leptomeninges and choroid plexus, and between tanycytes lining the floor of the third ventricle and the central canal of the spinal cord. Anti-ZO-1 labeled the majority of cells associated with the blood-brain barrier and may prove a useful marker, possibly in conjunction with functional dye studies, in evaluating the anatomical and functional integrity of the blood-brain barrier.

The surgical repair of peripheral nerve injuries is possible through the ability of neurons to regenerate axons successfully within the environment of the peripheral nervous system (PNS).1 Unfortunately in humans as well as other mammals, the mature central nervous system (CNS) demonstrates only a limited capacity for regeneration.1 Thus, surgical attempts to reconstruct pathways within the mammalian CNS have so far met with little, if any, success.

Lesioning of the developing cerebral midline and corpus callosum of mice results in the formation of bilateral masses of axons known as Probst's longitudinal bundles. These ectopic axons were examined using an in vitro brain slice technique, and they were found to be functional and to have electrical properties that were similar to those of the unlesioned corpus callosum at a comparable age. When a nitrocellulose bridge is properly oriented at the site of the callosal lesion in neonates, the implant will support the migration of glianates, in turn, promote the redirection of callosal axons across the midline. These axons were also analyzed by in vitro brain slice techniques, and they were found to be functional and to have electrical properties that were similar to the unlesioned corpus callosum with respect to conduction velocity and chronaxie but dissimilar with respect to charge threshold and rheobase current.

Previous studies have shown that immature but not mature astrocytes have the capacity to suppress glial scar formation and enhance axon outgrowth when transplanted into the adult mouse brain. We report here that glial scar formation is suppressed following transplantation of purified immature but not mature cultured type-1 rat cortical astrocytes into the adult rat brain. To examine the fate of transplanted cells, cultured astrocytes were labeled with either fluorescent beads or BSA-conjugated colloidal gold and traced after transplantation using both light and electron microscopy. While both immature and mature astrocytes survived transplantation, mature astrocytes appeared more susceptible to phagocytosis by cells of the immune system than immature astrocytes. Furthermore, while mature astrocytes were restricted to the region of the implant, immature astrocytes migrated into the surrounding CNS and became closely associated with host blood vessels. Such blood vessels were impermeable to the diffusion of systematically applied Evans blue dye. To determine whether immature astrocytes were intrinsically more motile than mature astrocytes, their rate of translocation was compared in vitro. Immature astrocytes translocated more than twice as fast as mature astrocytes. This ability of immature astrocytes to translocate throughout the host CNS and become associated with blood vessels may be a major factor in their ability to suppress glial scar formation in the adult animal.

Summary The Wood Research Group at Dundee Institute of Technology is currently investigating a number of parameters associated with methodology for) the preservation of timber and the detection of decay organisms within timber. Application of specific methodologies allowing the in situ treatment of timbers with preservative agents and the relatively non-destructive testing of timbers for decay organisms are particularly appropriate to conservation strategies for wooden structures. Currently used procedures for remedial treatment of such structures require visual identification of decayed wood followed by removal and replacement of defective timbers. An alternative strategy is required where artefacts of historical or archaeological importance are under threat. The development of immunological methods for the detection of incipient decay (i.e. the presence of decay organisms before they have caused damage to timber) and the use of water soluble diffusible biocides such as boron based salts will be discussed in this presentation. Applications of these methods to two historical artefacts present in Dundee, viz. the UNICORN, the oldest surviving European built warship still afloat, and the DISCOVERY, a research ship used in Antarctic exploration in the early part of this century, will also be discussed.

To overcome obstacles to the regeneration of crushed dorsal root fibers at the dorsal root entry zone, we have employed specially designed Millipore implants coated with embryonic astrocytes to serve as a substrate for axonal growth. This strategy was successful in promoting the growth of crushed dorsal root axons into the grey matter of the adult mammalian spinal cord in a small number of animals. Fiber ingrowth into the spinal cord was closely associated with the surface of the polymer implant. In addition, unique terminal arbor malformations, not normally present, were seen in several animals. A consistent finding was the presence of a limited inflammatory response in regions immediately adjacent to the implant where axons penetrate the spinal cord. Our findings suggest that providing the dorsal root entry zone with an embryonic milieu can stimulate a limited amount of axonal regeneration into the adult mammalian spinal cord.

In the developing mammalian central nervous system astrocytes have been proposed as an important substrate for axon growth. In the adult central nervous system following injury, astrocytes are a major component of the gliotic response which has been proposed to block axon growth. Experimental transplantation studies using cultured astrocytes have suggested that immature but not mature cultured astrocytes have the capacity to support axon outgrowth when transplanted into the adult rodent CNS. These observations suggest that astrocyte maturation is accompanied by changes in the functional capacity of these cells to support axon outgrowth. To determine whether this functional change reflects an intrisic astrocyte property, the extent and molecular bases of neurite outgrowth from embryonic rat cortical and chick retinal neurons on cultures of purified immature and mature astrocytes have been compared in vitro. The rate and extent of neurite outgrowth from both neuronal populations are consistently greater over the surface of immature than over the surface of mature astrocytes. Furthermore, antibodies to NCAM and G4/L1 significantly reduce neurite outgrowth on immature but not mature astrocytes, while antibodies to the integrin B1 receptor reduced outgrowth on both immature and, to a lesser extent, mature astrocytes. These results suggest that in vitro mature astrocytes have a reduced capacity and different molecular bases for supporting neurite outgrowth compared to immature astrocytes and are consistent with the proposal that functional changes during astrocyte maturation may partially contribute to regulating axon growth in the mammalian CNS.

Devonian and Lower Mississippian volcano-plutonic complexes and related basinal strata that extend from California to British Columbia lie outboard of coeval North American shelf rocks. Remarkable similarities between these complexes indicate that they form the remnants of a mid-Paleozoic east Pacific fringing arc system. Arc basement was composite. It is characterized in part by high initial Sr ratios, radiogenic Pb, and −ɛ_(Nd) values, and in part by low initial Sr ratios, various initial Pb ratios, and +ɛ_(Nd) values. These distinctive isotopic signatures, together with the presence of Proterozoic average ages for detrital zircon in ultimately continent-derived sedimentary rocks and Proterozoic upper-intercept (inheritance) ages on zircon from arc- and rift-related magmatic rocks, imply that basement was composed of both continental crust and transitional or oceanic crust overlain by continent-derived sedimentary rocks. These data suggest that the arc system formed adjacent to a continental landmass. The initial geometry of the arc has been highly modified by subsequent crustal thickening, extension, associated uplift, and transport along strike-slip faults.

In the article on how Brca2 deficiency sensitizes cells to PARP inhibition in the November 15, 2005 issue of Cancer Research ( 1), the grant support in the Acknowledgment should have read as follows: Association for International Cancer Research grant no. 03–336, KuDOS Pharmaceuticals Ltd., and Wales Gene Park.

Within the last 10 years the development of tools used to manipulate DNA have shown promise in the generation of a new form of therapeutic intervention known as gene therapy. Gene therapy involves the introduction of genetic material into cells to replace or supplement defective genes or to induce the expression of a novel gene to help alleviate a disease. These manipulations can be done in cells either outside the body and transplanted back in (ex vivo gene therapy) or directly within a specific target organ (in vivo gene therapy). To target and transfer DNA or RNA to cells a variety of techniques have been developed, including the use of replication-defective viruses, lipid bilayers that encapsulate DNA (liposomes), and DNA-coated gold particles that are ballistically shot into cells (gene gun). Most of these topics will be discussed in articles to follow in this series.

SUMMARY Sheep cold stressed for 10.5 h had plastic coats applied then the cold stress was continued. In 9 of 10 sheep (test group) showing hypothermia at the time coats were applied, body temperatures had returned to near normal 2.5 h later and to normal in 13.5 h. One sheep severely hypothermic before the coat was applied did not improve and was killed after 2.5 h. Sheep that were cold stressed without coats (wet controls) developed marked hypothermia and were killed between 10.5 h and 21 h. Rectal temperatures remained normal in controls that were cold stressed with coats on (coated wet controls) and in the controls not subjected to wetting (dry controls). Plasma cortisol increased markedly in the cold stressed sheep until plastic coats were applied, but the levels had fallen to normal levels 20 h later. Serum thyroxine levels increased at a faster rate in the test group and in wet controls than in the dry controls and coated wet controls during the first 9 h of cold stress. In 4 sheep of the test group energy utilisation increased markedly and remained high for a variable time after coats were applied then gradually returned to pre-cold stress levels. Liver tyrosine aminotransferase levels of the wet controls were markedly elevated compared to levels found in the test group sheep killed 79.5 h after coats were applied. Histological changes in the spleen and liver of wet controls was absent or only mildly present in the test group sheep. There was severe depletion of muscle and liver glycogen in the wet controls compared with the test group sheep, which were similar to the dry controls and coated wet controls.

The mortality rates were determined in two groups of dogs following acute circumflex artery occlusion. Both groups were surgically prepared in the same manner. In a group of 20 dogs the coronary sinus was cannulated to divert the blood into the left jugular vein. Circumflex artery ligation resulted in 70 per cent mortality within one hour. In a second group of 10 dogs the coronary sinus was arterialized from the left subclavian artery and the sinus mean pressure was held at 50 mm. Hg. In this group circumflex artery ligation resulted in 100 per cent survival for one hour. Statistical analysis shows the results to be highly significant. The results strongly suggest that arterialization of the coronary sinus protects the hearts of dogs from ventricular fibrillation following coronary artery ligation.

(1929). A Newcomer in the High School—The Intramural Athletic Director. American Physical Education Review: Vol. 34, No. 4, pp. 210-212.

We have developed an in vitro model in which cells responding to trauma in the immature and mature CNS can be isolated, placed into serum-free culture, and characterized. By implanting nitrocellulose filters into the brains of neonatal and adult rats under different conditions, we are able to harvest populations of cells responding to trauma in the neonate (critical period implant), in the adult (scar implant), and in implants that have remained in vivo past the critical period (postcritical period implant). Upon placement in culture, we have found that astrocytes represent the majority of cells occupying both the critical period and postcritical period implants, whereas fibroblasts and macrophages represent the majority of cells in the glial-fibroblastic scar. The morphologies of the astrocytes on the surface of the different implants, after 3 days in culture, differs markedly--the critical period astrocytes exhibiting a more ordered distribution compared to the haphazard arrangement of astrocyte processes on the surface of the postcritical and scar implants. After migration from the implant, critical period astrocytes assume an epithelioid morphology and cluster together setting up definite boundaries between themselves and the endothelial cells. In contrast, postcritical period astrocytes exhibit a more elongated morphology under the same culture conditions and appear to be randomly dispersed among the endothelial cells. The scar astrocytes exhibit a wide range of morphologies and, although they tend to cluster, do not exhibit the ordered association seen with the critical period astrocytes. We propose that the plasticity of the neonatal astrocytes and the rapid and ordered cellular response seen in vitro reflect the ability of the immature CNS in vivo to respond to injury without the formation of a glial-fibroblastic scar.

When the cerebral midline is lesioned in the embryo or neonate, the would-be callosal axons form neuromas. We have shown that an untreated Millipore implant inserted between the neuromas in young acallosal animals can support the migration of immature astrocytes that, in turn, support the de novo growth of commissural axons between the hemispheres. Since callosal neuromas persist into adulthood, we asked whether a critical period exists after which reactive glia no longer promote axon growth. We found that a critical period does exist and have documented a variety of changes in reactive gliosis that, in part, may lead to the axon growth-refractory state. In acallosal mouse postnates given untreated implants on or prior to day 8, glial fibrillary acidic protein (GFAP)+, stellate-shaped astrocytes migrated and attached to the implant by inserting foot processes into the pores of the filter. This form of gliotic response established an axon growth-promoting substratum within 24-48 hours after implantation. During this critical stage there was no evidence of scar formation or necrosis at or around the implant surface. However, when acallosal mice were implanted on or later than postnatal day 14, extensive tissue degeneration occurred, and a mixed population of astrocytes and fibroblasts invaded the surface of the filter, producing a dense scar. Reactive cells within the scar did not promote axonal outgrowth. To determine whether glia from neonates can influence the host environment and/or induce axonal regeneration in acallosal animals after the critical period, we harvested immature astrocytes on Millipore from critical-period mouse forebrains and transplanted the glia-coated prostheses into the brains of post-critical-period acallosal animals. Such transplants reduced glial scarring in the host, inhibited extensive bleeding and secondary necrosis, and promoted axonal regeneration. Our studies suggest that when controlled with a prosthesis, gliosis during the critical period is a beneficial process that can promote the reconstruction of malformed axon pathways; that in older animals a variety of changes in reactive glia and the extracellular matrix may work together to hinder axon regeneration after the critical period; and that axonal regeneration in the postcritical CNS may be stimulated by reintroducing an immature glial environment at the lesion site.