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The ependyma of the adult spinal cord is a latent stem cell niche that is reactivated by spinal cord injury contributing new cells to the glial scar. The cellular events taking place in the early stages of the reaction of the ependyma to injury remain little understood. Ependymal cells are functionally heterogeneous with a mitotically active subpopulation lining the lateral domains of the central canal (CC) that are coupled via gap junctions. Gap junctions and connexin hemichannels are key regulators of the biology of neural progenitors during development and in adult neurogenic niches. Thus, we hypothesized that communication via connexins in the CC is developmentally regulated and may play a part in the reactivation of this latent stem cell niche after injury. To test these possibilities, we combined patch-clamp recordings of ependymal cells with immunohistochemistry for various connexins in the neonatal and the adult (P > 90) normal and injured spinal cord of male and female mice. We find that coupling among ependymal cells is downregulated as postnatal development proceeds but increases after injury, resembling the immature CC. The increase in gap junction coupling in the adult CC was paralleled by upregulation of connexin 26, which correlated with the resumption of proliferation and a reduction of connexin hemichannel activity. Connexin blockade reduced the injury-induced proliferation of ependymal cells. Our findings suggest that connexins are involved in the early reaction of ependymal cells to injury, representing a potential target to improve the contribution of the CC stem cell niche to repair.SIGNIFICANCE STATEMENTEpendymal cells in the adult spinal cord are latent progenitors that react to injury to support some degree of endogenous repair. Understanding the mechanisms by which these progenitor-like cells are regulated in the aftermath of spinal cord injury is critical to design future manipulations aimed at improving healing and functional recovery. Gap junctions and connexin hemichannels are key regulators of the biology of neural progenitors during development and in adult neurogenic niches. We find here that connexin signaling in the ependyma changes after injury of the adult spinal cord, functionally resembling the immature active-stem cell niche of neonatal animals. Our findings suggest that connexins in ependymal cells are potential targets to improve self-repair of the spinal cord.

Focal traumatic brain injury (TBI) induces astrogliosis, a process essential to protecting uninjured brain areas from secondary damage. However, astrogliosis can cause loss of astrocyte homeostatic functions and possibly contributes to comorbidities such as posttraumatic epilepsy (PTE). Scar-forming astrocytes seal focal injuries off from healthy brain tissue. It is these glial scars that are associated with epilepsy originating in the cerebral cortex and hippocampus. However, the vast majority of human TBIs also present with diffuse brain injury caused by acceleration-deceleration forces leading to tissue shearing. The resulting diffuse tissue damage may be intrinsically different from focal lesions that would trigger glial scar formation. Here, we used mice of both sexes in a model of repetitive mild/concussive closed-head TBI, which only induced diffuse injury, to test the hypothesis that astrocytes respond uniquely to diffuse TBI and that diffuse TBI is sufficient to cause PTE. Astrocytes did not form scars and classic astrogliosis characterized by upregulation of glial fibrillary acidic protein was limited. Surprisingly, an unrelated population of atypical reactive astrocytes was characterized by the lack of glial fibrillary acidic protein expression, rapid and sustained downregulation of homeostatic proteins and impaired astrocyte coupling. After a latency period, a subset of mice developed spontaneous recurrent seizures reminiscent of PTE in human TBI patients. Seizing mice had larger areas of atypical astrocytes compared with nonseizing mice, suggesting that these atypical astrocytes might contribute to epileptogenesis after diffuse TBI.SIGNIFICANCE STATEMENTTraumatic brain injury (TBI) is a leading cause of acquired epilepsies. Reactive astrocytes have long been associated with seizures and epilepsy in patients, particularly after focal/lesional brain injury. However, most TBIs also include nonfocal, diffuse injuries. Here, we showed that repetitive diffuse TBI is sufficient for the development of spontaneous recurrent seizures in a subset of mice. We identified an atypical response of astrocytes induced by diffuse TBI characterized by the rapid loss of homeostatic proteins and lack of astrocyte coupling while reactive astrocyte markers or glial scar formation was absent. Areas with atypical astrocytes were larger in animals that later developed seizures suggesting that this response may be one root cause of epileptogenesis after diffuse TBI.

Fidgetin is a microtubule-severing protein that pares back the labile domains of microtubules in the axon. Experimental depletion of fidgetin results in elongation of the labile domains of microtubules and faster axonal growth. To test whetherfidgetinknockdown assists axonal regeneration, we plated dissociated adult rat DRGs transduced using AAV5-shRNA-fidgetinon a laminin substrate with spots of aggrecan, a growth-inhibitory chondroitin sulfate proteoglycan. This cell culture assay mimics the glial scar formed after CNS injury. Aggrecan is more concentrated at the edge of the spot, such that axons growing from within the spot toward the edge encounter a concentration gradient that causes growth cones to become dystrophic and axons to retract or curve back on themselves.Fidgetinknockdown resulted in faster-growing axons on both laminin and aggrecan and enhanced crossing of axons from laminin onto aggrecan. Strikingly, axons from within the spot grew more avidly against the inhibitory aggrecan concentration gradient to cross onto laminin, without retracting or curving back. We also tested whether depleting fidgetin improves axonal regenerationin vivoafter a dorsal root crush in adult female rats. Whereas control DRG neurons failed to extend axons across the dorsal root entry zone after injury, DRG neurons in whichfidgetinwas knocked down displayed enhanced regeneration of axons across the dorsal root entry zone into the spinal cord. Collectively, these results establish fidgetin as a novel therapeutic target to augment nerve regeneration and provide a workflow template by which microtubule-related targets can be compared in the future.SIGNIFICANCE STATEMENTHere we establish a workflow template from cell culture to animals in which microtubule-based treatments can be tested and compared with one another for their effectiveness in augmenting regeneration of injured axons relevant to spinal cord injury. The present work uses a viral transduction approach to knock downfidgetinfrom rat neurons, which coaxes nerve regeneration by elevating microtubule mass in their axons. Unlike previous strategies using microtubule-stabilizing drugs,fidgetinknockdown adds microtubule mass that is labile (rather than stable), thereby better recapitulating the growth status of a developing axon.

Spinal cord injury (SCI) induces a centralized fibrotic scar surrounded by a reactive glial scar at the lesion site. The origin of these scars is thought to be perivascular cells entering lesions on ingrowing blood vessels and reactive astrocytes, respectively. However, two NG2-expressing cell populations, pericytes and glia, may also influence scar formation. In the periphery, new blood vessel growth requires proliferating NG2+pericytes; if this were also true in the CNS, then the fibrotic scar would depend on dividing NG2+pericytes. NG2+glial cells (also called oligodendrocyte progenitors or polydendrocytes) also proliferate after SCI and accumulate in large numbers among astrocytes in the glial scar. Their effect there, if any, is unknown. We show that proliferating NG2+pericytes and glia largely segregate into the fibrotic and glial scars, respectively; therefore, we used a thymidine kinase/ganciclovir paradigm to ablate both dividing NG2+cell populations to determine whether either scar was altered. Results reveal that loss of proliferating NG2+pericytes in the lesion prevented intralesion angiogenesis and completely abolished the fibrotic scar. The glial scar was also altered in the absence of acutely dividing NG2+cells, displaying discontinuous borders and significantly reduced GFAP density. Collectively, these changes enhanced edema, prolonged hemorrhage, and impaired forelimb functional recovery. Interestingly, after halting GCV at 14 d postinjury, scar elements and vessels entered the lesions over the next 7 d, as did large numbers of axons that were not present in controls. Collectively, these data reveal that acutely dividing NG2+pericytes and glia play fundamental roles in post-SCI tissue remodeling.SIGNIFICANCE STATEMENTSpinal cord injury (SCI) is characterized by formation of astrocytic and fibrotic scars, both of which are necessary for lesion repair. NG2+cells may influence both scar-forming processes. This study used a novel transgenic mouse paradigm to ablate proliferating NG2+cells after SCI to better understand their role in repair. For the first time, our data show that dividing NG2+pericytes are required for post-SCI angiogenesis, which in turn is needed for fibrotic scar formation. Moreover, loss of cycling NG2+glia and pericytes caused significant multicellular tissue changes, including altered astrocyte responses and impaired functional recovery. This work reveals previously unknown ways in which proliferating NG2+cells contribute to endogenous repair after SCI.

Astrogliosis after spinal cord injury (SCI) is a major impediment to functional recovery. More than half of new astrocytes generated after SCI are derived from ependymal zone stem cells (EZCs). We demonstrate that expression of β1-integrin increases in EZCs following SCI in mice. Conditional knock-out of β1-integrin increases GFAP expression and astrocytic differentiation by cultured EZCs without altering oligodendroglial or neuronal differentiation. Ablation of β1-integrin from EZCsin vivoreduced the number of EZC progeny that continued to express stem cell markers after SCI, increased the proportion of EZC progeny that differentiated into GFAP+ astrocytes, and diminished functional recovery. Loss of β1-integrin increased SMAD1/5/8 and p38 signaling, suggesting activation of BMP signaling. Coimmunoprecipitation studies demonstrated that β1-integrin directly interacts with the bone morphogenetic protein receptor subunits BMPR1a and BMPR1b. Ablation of β1-integrin reduced overall levels of BMP receptors but significantly increased partitioning of BMPR1b into lipid rafts with increased SMAD1/5/8 and p38 signaling. Thus β1-integrin expression by EZCs reduces movement of BMPR1b into lipid rafts, thereby limiting the known deleterious effects of BMPR1b signaling on glial scar formation after SCI.

Adult progenitor cells proliferate in the acutely injured spinal cord and their progeny differentiate into new oligodendrocytes (OLs) that remyelinate spared axons. Whether this endogenous repair continues beyond the first week postinjury (wpi), however, is unknown. Identifying the duration of this response is essential for guiding therapies targeting improved recovery from spinal cord injury (SCI) by enhancing OL survival and/or remyelination. Here, we used two PDGFRα-reporter mouse lines and rats injected with a GFP-retrovirus to assess progenitor fate through 80 d after injury. Surprisingly, new OLs were generated as late as 3 months after injury and their processes ensheathed axons near and distal to the lesion, colocalized with MBP, and abutted Caspr+ profiles, suggesting newly formed myelin. Semithin sections confirmed stereotypical thin OL remyelination and few bare axons at 10 wpi, indicating that demyelination is relatively rare. Astrocytes in chronic tissue expressed the pro-OL differentiation and survival factors CNTF and FGF-2. In addition, pSTAT3+ NG2 cells were present through at least 5 wpi, revealing active signaling of the Jak/STAT pathway in these cells. The progenitor cell fate genes Sox11, Hes5, Id2, Id4, BMP2, and BMP4 were dynamically regulated for at least 4 wpi. Collectively, these data verify that the chronically injured spinal cord is highly dynamic. Endogenous repair, including oligodendrogenesis and remyelination, continues for several months after SCI, potentially in response to growth factors and/or transcription factor changes. Identifying and understanding spontaneous repair processes such as these is important so that beneficial plasticity is not inadvertently interrupted and effort is not exerted to needlessly duplicate ongoing spontaneous repair.

NG2 is purportedly one of the most growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) produced after spinal cord injury. Nonetheless, once the severed axon tips dieback from the lesion core into the penumbra they closely associate with NG2+ cells. We asked if proteoglycans play a role in this tight cell—cell interaction and whether overadhesion upon these cells might participate in regeneration failure in rodents. Studies using varying ratios of CSPGs and adhesion molecules along with chondroitinase ABC, as well as purified adult cord-derived NG2 glia, demonstrate that CSPGs are involved in entrapping neurons. Once dystrophic axons become stabilized upon NG2+ cells, they form synaptic-like connections bothin vitroandin vivo. In NG2 knock-out mice, sensory axons in the dorsal columns dieback further than their control counterparts. When axons are double conditioned to enhance their growth potential, some traverse the lesion core and express reduced amounts of synaptic proteins. Our studies suggest that proteoglycan-mediated entrapment upon NG2+ cells is an additional obstacle to CNS axon regeneration.

When the brain or spinal cord is injured, glial cells in the damaged area undergo complex morphological and physiological changes resulting in the formation of the glial scar. This scar contains reactive astrocytes, activated microglia, macrophages and other myeloid cells, meningeal cells, proliferating oligodendrocyte precursor cells (OPCs), and a dense extracellular matrix. Whether the scar is beneficial or detrimental to recovery remains controversial. In the acute phase of recovery, scar-forming astrocytes limit the invasion of leukocytes and macrophages, but in the subacute and chronic phases of injury the glial scar is a physical and biochemical barrier to axonal regrowth. The signals that initiate the formation of the glial scar are unknown. Both canonical and noncanonical signaling Wnts are increased after spinal cord injury (SCI). Because Wnts are important regulators of OPC and oligodendrocyte development, we examined the role of canonical Wnt signaling in the glial reactions to CNS injury. In adult female mice carrying an OPC-specific conditionally deleted β-catenin gene, there is reduced proliferation of OPCs after SCI, reduced accumulation of activated microglia/macrophages, and reduced astrocyte hypertrophy. Using an infraorbital optic nerve crush injury, we show that reducing β-catenin-dependent signaling in OPCs creates an environment that is permissive to axonal regeneration. Viral-induced expression of Wnt3a in the normal adult mouse spinal cord induces an injury-like response in glia. Thus canonical Wnt signaling is both necessary and sufficient to induce injury responses among glial cells. These data suggest that targeting Wnt expression after SCI may have therapeutic potential in promoting axon regeneration.

Transplantation of Schwann cells (SCs) is a promising therapeutic strategy for spinal cord repair. SCs introduced into lesions support axon regeneration, but because these axons do not exit the transplant, additional approaches with SCs are needed. Here, we transplanted SCs genetically modified to secrete a bifunctional neurotrophin (D15A) and chondroitinase ABC (ChABC) into a subacute contusion injury in rats. We examined the effects of these modifications on graft volume, SC number, degradation of chondroitin sulfate proteoglycans (CSPGs), astrogliosis, SC myelination of axons, propriospinal and supraspinal axon numbers, locomotor outcome (BBB scoring, CatWalk gait analysis), and mechanical and thermal sensitivity on the hind paws. D15A secreted from transplanted SCs increased graft volume and SC number and myelinated axon number. SCs secreting ChABC significantly decreased CSPGs, led to some egress of SCs from the graft, and increased propriospinal and 5-HT-positive axons in the graft. SCs secreting both D15A and ChABC yielded the best responses: (1) the largest number of SC myelinated axons, (2) more propriospinal axons in the graft and host tissue around and caudal to it, (3) more corticospinal axons closer to the graft and around and caudal to it, (4) more brainstem neurons projecting caudal to the transplant, (5) increased 5-HT-positive axons in the graft and caudal to it, (6) significant improvement in aspects of locomotion, and (7) improvement in mechanical and thermal allodynia. This is the first evidence that the combination of SC transplants engineered to secrete neurotrophin and chondroitinase further improves axonal regeneration and locomotor and sensory function.

When the CNS is injured, damaged axons do not regenerate. This failure is due in part to the growth-inhibitory environment that forms at the injury site. Myelin-associated molecules, repulsive axon guidance molecules, and extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs) found within the glial scar inhibit axon regeneration but the intracellular signaling mechanisms triggered by these diverse molecules remain largely unknown. Here we provide biochemical and functional evidence that atypical protein kinase C (PKCζ) and polarity (Par) complex proteins mediate axon growth inhibition. Treatment of postnatal rat neuronsin vitrowith the NG2 CSPG, a major component of the glial scar, activates PKCζ, and this activation is both necessary and sufficient to inhibit axonal growth. NG2 treatment also activates Cdc42, increases the association of Par6 with PKCζ, and leads to a Par3-dependent activation of Rac1. Transfection of neurons with kinase-dead forms of PKCζ, dominant-negative forms of Cdc42, or mutant forms of Par6 that do not bind to Cdc42 prevent NG2-induced growth inhibition. Similarly, transfection with either a phosphomutant Par3 (S824A) or dominant-negative Rac1 prevent inhibition, whereas expression of constitutively active Rac1 inhibits axon growth on control surfaces. These results suggest a model in which NG2 binding to neurons activates PKCζ and modifies Par complex function. They also identify the Par complex as a novel therapeutic target for promoting axon regeneration after CNS injury.

Astrogliosis following spinal cord injury (SCI) involves an early hypertrophic response that serves to repair damaged blood–brain barrier and a subsequent hyperplastic response that results in a dense scar that impedes axon regeneration. The mechanisms regulating these two phases of astrogliosis are beginning to be elucidated. In this study, we found that microRNA-21 (miR-21) increases in a time-dependent manner following SCI in mouse. Astrocytes adjacent to the lesion area express high levels of miR-21 whereas astrocytes in uninjured spinal cord express low levels of miR-21. To study the role of miR-21 in astrocytes after SCI, transgenic mice were generated that conditionally overexpress either the primary miR-21 transcript in astrocytes or a miRNA sponge designed to inhibit miR-21 function. Overexpression of miR-21 in astrocytes attenuated the hypertrophic response to SCI. Conversely, expression of the miR-21 sponge augmented the hypertrophic phenotype, even in chronic stages of SCI recovery when astrocytes have normally become smaller in size with fine processes. Inhibition of miR-21 function in astrocytes also resulted in increased axon density within the lesion site. These findings demonstrate a novel role for miR-21 in regulating astrocytic hypertrophy and glial scar progression after SCI, and suggest miR-21 as a potential therapeutic target for manipulating gliosis and enhancing functional outcome.

Astrocytes are both detrimental and beneficial for repair and recovery after spinal cord injury (SCI). These dynamic cells are primary contributors to the growth-inhibitory glial scar, yet they are also neuroprotective and can form growth-supportive bridges on which axons traverse. We have shown that intrathecal administration of transforming growth factor α (TGFα) to the contused mouse spinal cord can enhance astrocyte infiltration and axonal growth within the injury site, but the mechanisms of these effects are not well understood. The present studies demonstrate that the epidermal growth factor receptor (EGFR) is upregulated primarily by astrocytes and glial progenitors early after SCI. TGFα directly activates the EGFR on these cellsin vitro, inducing their proliferation, migration, and transformation to a phenotype that supports robust neurite outgrowth. Overexpression of TGFαin vivoby intraparenchymal adeno-associated virus injection adjacent to the injury site enhances cell proliferation, alters astrocyte distribution, and facilitates increased axonal penetration at the rostral lesion border. To determine whether endogenous EGFR activation is required after injury, SCI was also performed onVelvet(C57BL/6J-EgfrVel/J) mice, a mutant strain with defective EGFR activity. The affected mice exhibited malformed glial borders, larger lesions, and impaired recovery of function, indicating that intrinsic EGFR activation is necessary for neuroprotection and normal glial scar formation after SCI. By further stimulating precursor proliferation and modifying glial activation to promote a growth-permissive environment, controlled stimulation of EGFR at the lesion border may be considered in the context of future strategies to enhance endogenous cellular repair after injury.

Chondroitin sulfate proteoglycans (CSPGs) are upregulated after CNS lesions, where they inhibit axon regeneration. In order for axon growth and regeneration to occur, surface integrin receptors must interact with surrounding extracellular matrix molecules. We have explored the hypothesis that CSPGs inhibit regeneration by inactivating integrins and that forcing integrins into an active state might overcome this inhibition. Using cultured rat sensory neurons, we show that the CSPG aggrecan inhibits laminin-mediated axon growth by impairing integrin signaling via decreasing phosphorylated FAK (pFAK) and pSrc levels, without affecting surface integrin levels. Forcing integrin activation and signaling by manganese or an activating antibody TS2/16 reversed the inhibitory effect of aggrecan on mixed aggrecan/laminin surfaces, and enhanced axon growth from cultured rat sensory neurons (manganese) and human embryonic stem cell-derived motoneurons (TS2/16). The inhibitory effect of Nogo-A can also be reversed by integrin activation. These results suggest that inhibition by CSPGs can act via inactivation of integrins, and that activation of integrins is a potential method for improving axon regeneration after injury.

Mature retinal ganglion cells (RGCs) cannot normally regenerate axons into the injured optic nerve but can do so after lens injury. Astrocyte-derived ciliary neurotrophic factor and leukemia inhibitory factor have been identified as essential key factors mediating this effect. However, the outcome of this regeneration is still limited by inhibitors associated with the CNS myelin and the glial scar. The current study demonstrates that Taxol markedly enhanced neurite extension of mature RGCs and PC12 cells by stabilization of microtubules and desensitized axons toward myelin and chondroitin sulfate proteoglycan (CSPG) inhibitionin vitrowithout reducing RhoA activation.In vivo, the local application of Taxol at the injury site of the optic nerve of rats enabled axons to regenerate beyond the lesion site but did not affect the intrinsic regenerative state of RGCs. Furthermore, Taxol treatment markedly increased lens injury-mediated axon regenerationin vivo, delayed glial scar formation, suppressed CSPG expression, and transiently reduced the infiltration of macrophages at the injury site. Thus, microtubule-stabilizing compounds such as Taxol might be promising candidates as adjuvant drugs in the treatment of CNS injuries particularly when combined with interventions stimulating the intrinsic regenerative state of neurons.

Scar formation in the nervous system begins within hours after traumatic injury and is characterized primarily by reactive astrocytes depositing proteoglycans that inhibit regeneration. A fundamental question in CNS repair has been the identity of the initial molecular mediator that triggers glial scar formation. Here we show that the blood protein fibrinogen, which leaks into the CNS immediately after blood–brain barrier (BBB) disruption or vascular damage, serves as an early signal for the induction of glial scar formation via the TGF-β/Smad signaling pathway. Our studies revealed that fibrinogen is a carrier of latent TGF-β and induces phosphorylation of Smad2 in astrocytes that leads to inhibition of neurite outgrowth. Consistent with these findings, genetic or pharmacologic depletion of fibrinogen in mice reduces active TGF-β, Smad2 phosphorylation, glial cell activation, and neurocan deposition after cortical injury. Furthermore, stereotactic injection of fibrinogen into the mouse cortex is sufficient to induce astrogliosis. Inhibition of the TGF-β receptor pathway abolishes the fibrinogen-induced effects on glial scar formationin vivoandin vitro. These results identify fibrinogen as a primary astrocyte activation signal, provide evidence that deposition of inhibitory proteoglycans is induced by a blood protein that leaks in the CNS after vasculature rupture, and point to TGF-β as a molecular link between vascular permeability and scar formation.

Astrogliosis following spinal cord injury (SCI) involves an early hypertrophic response that is beneficial and a subsequent formation of a dense scar. We investigated the role of bone morphogenetic protein (BMP) signaling in gliosis after SCI and find that BMPR1a and BMPR1b signaling exerts opposing effects on hypertrophy. Conditional ablation of BMPR1a from glial fibrillary acidic protein (GFAP)-expressing cells leads to defective astrocytic hypertrophy, increased infiltration by inflammatory cells, and reduced axon density. BMPR1b-null mice conversely develop “hyperactive” reactive astrocytes and consequently have smaller lesion volumes. The effects of ablation of either receptor are reversed in the double knock-out animals. These findings indicate that BMPR1a and BMPR1b exert directly opposing effects on the initial reactive astrocytic hypertrophy. Also, BMPR1b knock-out mice have an attenuated glial scar in the chronic stages following injury, suggesting that it has a greater role in glial scar progression. To elucidate the differing roles of the two receptors in astrocytes, we examined the effects of ablation of either receptor in serum-derived astrocytesin vitro. We find that the two receptors exert opposing effects on the posttranscriptional regulation of astrocytic microRNA-21. Further, overexpression of microRNA-21 in wild-type serum-derived astrocytes causes a dramatic reduction in cell size accompanied by reduction in GFAP levels. Hence, regulation of microRNA-21 by BMP signaling provides a novel mechanism for regulation of astrocytic size. Targeting specific BMPR subunits for therapeutic purposes may thus provide an approach for manipulating gliosis and enhancing functional outcomes after SCI.

The transplantation of neural stem/progenitor cells (NPCs) is a promising therapeutic strategy for spinal cord injury (SCI). However, to date NPC transplantation has exhibited only limited success in the treatment of chronic SCI. Here, we show that chondroitin sulfate proteoglycans (CSPGs) in the glial scar around the site of chronic SCI negatively influence the long-term survival and integration of transplanted NPCs and their therapeutic potential for promoting functional repair and plasticity. We targeted CSPGs in the chronically injured spinal cord by sustained infusion of chondroitinase ABC (ChABC). One week later, the same rats were treated with transplants of NPCs and transient infusion of growth factors, EGF, bFGF, and PDGF-AA. We demonstrate that perturbing CSPGs dramatically optimizes NPC transplantation in chronic SCI. Engrafted NPCs successfully integrate and extensively migrate within the host spinal cord and principally differentiate into oligodendrocytes. Furthermore, this combined strategy promoted the axonal integrity and plasticity of the corticospinal tract and enhanced the plasticity of descending serotonergic pathways. These neuroanatomical changes were also associated with significantly improved neurobehavioral recovery after chronic SCI. Importantly, this strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. For the first time, we demonstrate key biological and functional benefits for the combined use of ChABC, growth factors, and NPCs to repair the chronically injured spinal cord. These findings could potentially bring us closer to the application of NPCs for patients suffering from chronic SCI or other conditions characterized by the formation of a glial scar.

We previously demonstrated that activated ED1+ macrophages induce extensive axonal dieback of dystrophic sensory axonsin vivoandin vitro. Interestingly, after spinal cord injury, the regenerating front of axons is typically found in areas rich in ED1+ cells, but devoid of reactive astrocyte processes. These observations suggested that another cell type must be present in these areas to counteract deleterious effects of macrophages. Cells expressing the purportedly inhibitory chondroitin sulfate proteoglycan NG2 proliferate in the lesion and intermingle with macrophages, but their influence on regeneration is highly controversial. Ourin vivoanalysis of dorsal column crush lesions confirms the close association between NG2+ cells and injured axons. We hypothesized that NG2+ cells were growth promoting and thereby served to increase axonal stability following spinal cord injury. We observed that the interactions between dystrophic adult sensory neurons and primary NG2+ cells derived from the adult spinal cord can indeed stabilize the dystrophic growth cone during macrophage attack. NG2+ cells expressed high levels of laminin and fibronectin, which promote neurite outgrowth on the surface of these cells. Our data also demonstrate that NG2+ cells, but not astrocytes, use matrix metalloproteases to extend across a region of inhibitory proteoglycan, and provide a permissive bridge for adult sensory axons. These data support the hypothesis that NG2+ cells are not inhibitory to regenerating sensory axons and, in fact, they may provide a favorable substrate that can stabilize the regenerating front of dystrophic axons in the inhibitory environment of the glial scar.

Because there currently is no treatment for spinal cord injury, most patients are living with long-standing injuries. Therefore, strategies aimed at promoting restoration of function to the chronically injured spinal cord have high therapeutic value. For successful regeneration, long-injured axons must overcome their poor intrinsic growth potential as well as the inhibitory environment of the glial scar established around the lesion site. Acutely injured axons that regenerate into growth-permissive peripheral nerve grafts (PNGs) reenter host tissue to mediate functional recovery if the distal graft–host interface is treated with chondroitinase ABC (ChABC) to cleave inhibitory chondroitin sulfate proteoglycans in the scar matrix. To determine whether a similar strategy is effective for a chronic injury, we combined grafting of a peripheral nerve into a highly relevant, chronic, cervical contusion site with ChABC treatment of the glial scar and glial cell line-derived neurotrophic factor (GDNF) stimulation of long-injured axons. We tested this combination in two grafting paradigms: (1) a peripheral nerve that was grafted to span a chronic injury site or (2) a PNG that bridged a chronic contusion site with a second, more distal injury site. Unlike GDNF–PBS treatment, GDNF–ChABC treatment facilitated axons to exit the PNG into host tissue and promoted some functional recovery. Electrical stimulation of axons in the peripheral nerve bridge induced c-Fos expression in host neurons, indicative of synaptic contact by regenerating fibers. Thus, our data demonstrate, for the first time, that administering ChABC to a distal graft interface allows for functional axonal regeneration by chronically injured neurons.

Astrocytes in the CNS respond to tissue damage by becoming reactive. They migrate, undergo hypertrophy, and form a glial scar that inhibits axon regeneration. Therefore, limiting astrocytic responses represents a potential therapeutic strategy to improve functional recovery. It was recently shown that the epidermal growth factor (EGF) receptor is upregulated in astrocytes after injury and promotes their transformation into reactive astrocytes. Furthermore, EGF receptor inhibitors were shown to enhance axon regeneration in the injured optic nerve and promote recovery after spinal cord injury. However, the signaling pathways involved were not elucidated. Here we show that in cultures of adult spinal cord astrocytes EGF activates the mTOR pathway, a key regulator of astrocyte physiology. This occurs through Akt-mediated phosphorylation of the GTPase-activating protein Tuberin, which inhibits Tuberin's ability to inactivate the small GTPase Rheb. Indeed, we found that Rheb is required for EGF-dependent mTOR activation in spinal cord astrocytes, whereas the Ras–MAP kinase pathway does not appear to be involved. Moreover, astrocyte growth and EGF-dependent chemoattraction were inhibited by the mTOR-selective drug rapamycin. We also detected elevated levels of activated EGF receptor and mTOR signaling in reactive astrocytesin vivoin an ischemic model of spinal cord injury. Furthermore, increased Rheb expression likely contributes to mTOR activation in the injured spinal cord. Interestingly, injured rats treated with rapamycin showed reduced signs of reactive gliosis, suggesting that rapamycin could be used to harness astrocytic responses in the damaged nervous system to promote an environment more permissive to axon regeneration.

In the injured spinal cord, a glial scar forms and becomes a major obstacle to axonal regeneration. Formation of the glial scar involves migration of astrocytes toward the lesion. Matrix metalloproteinases (MMPs), including MMP-9 and MMP-2, govern cell migration through their ability to degrade constituents of the extracellular matrix. Although MMP-9 is expressed in reactive astrocytes, its involvement in astrocyte migration and formation of a glial scar is unknown. Here we found that spinal cord injured, wild-type mice expressing MMPs developed a more severe glial scar and enhanced expression of chondroitin sulfate proteoglycans, indicative of a more inhibitory environment for axonal regeneration/plasticity, than MMP-9 null mice. To determine whether MMP-9 mediates astrocyte migration, we conducted a scratch wound assay using astrocytes cultured from MMP-9 null, MMP-2 null, and wild-type mice. Gelatin zymography confirmed the expression of MMP-9 and MMP-2 in wild-type cultures. MMP-9 null astrocytes and wild-type astrocytes, treated with an MMP-9 inhibitor, exhibited impaired migration relative to untreated wild-type controls. MMP-9 null astrocytes showed abnormalities in the actin cytoskeletal organization and function but no detectable untoward effects on proliferation, cellular viability, or adhesion. Interestingly, MMP-2 null astrocytes showed increased migration, which could be attenuated in the presence of an MMP-9 inhibitor. Collectively, our studies provide explicit evidence that MMP-9 is integral to the formation of an inhibitory glial scar and cytoskeleton-mediated astrocyte migration. MMP-9 may thus be a promising therapeutic target to reduce glial scarring during wound healing after spinal cord injury.

Injured axons of the adult CNS undergo lengthy retraction from the initial site of axotomy after spinal cord injury. Macrophage infiltration correlates spatiotemporally with this deleterious phenomenon, but the direct involvement of these inflammatory cells has not been demonstrated. In the present study, we examined the role of macrophages in axonal retraction within the dorsal columns after spinal cord injuryin vivoand found that retraction occurred between days 2 and 28 after lesion and that the ends of injured axons were associated with ED-1+ cells. Clodronate liposome-mediated depletion of infiltrating macrophages resulted in a significant reduction in axonal retraction; however, we saw no evidence of regeneration. We used time-lapse imaging of adult dorsal root ganglion neurons in anin vitromodel of the glial scar to examine macrophage–axon interactions and observed that adhesive contacts and considerable physical interplay between macrophages and dystrophic axons led to extensive axonal retraction. The induction of retraction was dependent on both the growth state of the axon and the activation state of the macrophage. Only dystrophic adult axons were susceptible to macrophage “attack.” Unlike intrinsically active cell line macrophages, both primary macrophages and microglia required activation to induce axonal retraction. Contact with astrocytes had no deleterious effect on adult dystrophic axons, suggesting that the induction of extensive retraction was specific to phagocytic cells. Our data are the first to indicate a direct role of activated macrophages in axonal retraction by physical cell–cell interactions with injured axons.

Peptide amphiphile (PA) molecules that self-assemblein vivointo supramolecular nanofibers were used as a therapy in a mouse model of spinal cord injury (SCI). Because self-assembly of these molecules is triggered by the ionic strength of thein vivoenvironment, nanoscale structures can be created within the extracellular spaces of the spinal cord by simply injecting a liquid. The molecules are designed to form cylindrical nanofibers that display to cells in the spinal cord the laminin epitope IKVAV at nearly van der Waals density. IKVAV PA nanofibers are known to inhibit glial differentiation of cultured neural stem cells and to promote neurite outgrowth from cultured neurons. In this work,in vivotreatment with the PA after SCI reduced astrogliosis, reduced cell death, and increased the number of oligodendroglia at the site of injury. Furthermore, the nanofibers promoted regeneration of both descending motor fibers and ascending sensory fibers through the lesion site. Treatment with the PA also resulted in significant behavioral improvement. These observations demonstrate that it is possible to inhibit glial scar formation and to facilitate regeneration after SCI using bioactive three-dimensional nanostructures displaying high densities of neuroactive epitopes on their surfaces.

The Ig superfamily adhesion molecule CHL1, the close homolog of the adhesion molecule L1, promotes neurite outgrowth, neuronal migration, and survivalin vitro. We tested whether CHL1, similar to its close homolog L1, has a beneficial impact on recovery from spinal cord injury using adult CHL1-deficient (CHL1−/−) mice and wild-type (CHL1+/+) littermates. In contrast to our hypothesis, we found that functional recovery, assessed by locomotor rating and video-based motion analyses, was improved inCHL1−/−mice compared with wild-type mice at 3–6 weeks after compression of the thoracic spinal cord. Better function was associated with enhanced monoaminergic reinnervation of the lumbar spinal cord and altered pattern of posttraumatic synaptic rearrangements around motoneurons. Restricted recovery of wild-type mice was likely related to early and persistent (3–56 d after lesion) upregulation of CHL1 in GFAP-positive astrocytes at the lesion core. In both the intact spinal cord and cultured astrocytes, enhanced expression of CHL1 and GFAP was induced by application of basic fibroblast growth factor, a cytokine involved in the pathophysiology of spinal cord injury. This upregulation was abolished by inhibitors of FGF receptor-dependent extracellular signal-regulated kinase, calcium/calmodulin-dependent kinase, and phosphoinositide-3 kinase signaling pathways. In homogenotypic and heterogenotypic cocultures of neurons and astrocytes, reduced neurite outgrowth was observed only if CHL1 was simultaneously present on both cell types. These findings and novelin vitroevidence for a homophilic CHL1–CHL1 interaction indicate that CHL1 is a glial scar component that restricts posttraumatic axonal growth and remodeling of spinal circuits by homophilic binding mechanisms.

Peripherally conditioned sensory neurons have an increased capacity to regenerate their central processes. However, even conditioned axons struggle in the presence of a hostile CNS environment. We hypothesized that combining an aggressive conditioning strategy with modification of inhibitory reactive astroglial-associated extracellular matrix could enhance regeneration. We screened potential treatments using a model of the dorsal root entry zone (DREZ). In this assay, a gradient of inhibitory chondroitin sulfate proteoglycans (CSPGs) stimulates formation of dystrophic end bulbs on adult sensory axons, which mimics regeneration failurein vivo. Combining inflammation-induced preconditioning of dorsal root gangliain vivobefore harvest, with chondroitinase ABC (ChABC) digestion of proteoglycansin vitroallows for significant regeneration across a once potently inhibitory substrate. We then assessed regeneration through the DREZ after root crush in adult rats receiving the combination treatment, ChABC, or zymosan pretreatment alone or no treatment. Regeneration was never observed in untreated animals, and only minimal regeneration occurred in the ChABC- and zymosan-alone groups. However, remarkable regeneration was observed in a majority of animals that received the combination treatment. Regenerated fibers established functional synapses, as demonstrated electrophysiologically by the presence of an H-reflex. Two different postlesion treatment paradigms in which the timing of both zymosan and ChABC administration were varied after injury were ineffective in promoting regeneration. Therefore, zymosan pretreatment, but not posttreatment, of the sensory ganglia, combined with ChABC modification of CSPGs, resulted in robust and functional regeneration of sensory axons through the DREZ after root injury.

Numerous obstacles to successful regeneration of injured axons in the adult mammalian spinal cord exist. Consequently, a treatment strategy inducing axonal regeneration and significant functional recovery after spinal cord injury has to overcome these obstacles. The current study attempted to address multiple impediments to regeneration by using a combinatory strategy after complete spinal cord transection in adult rats: (1) to reduce inhibitory cues in the glial scar (chondroitinase ABC), (2) to provide a growth-supportive substrate for axonal regeneration [Schwann cells (SCs)], and (3) to enable regenerated axons to exit the bridge to re-enter the spinal cord (olfactory ensheathing glia). The combination of SC bridge, olfactory ensheathing glia, and chondroitinase ABC provided significant benefit compared with grafts only or the untreated group. Significant improvements were observed in the Basso, Beattie, and Bresnahan score and in forelimb/hindlimb coupling. This recovery was accompanied by increased numbers of both myelinated axons in the SC bridge and serotonergic fibers that grew through the bridge and into the caudal spinal cord. Although prominent descending tracts such as the corticospinal and reticulospinal tracts did not successfully regenerate through the bridge, it appeared that other populations of regenerated fibers were the driving force for the observed recovery; there was a significant correlation between numbers of myelinated fibers in the bridge and improved coupling of forelimb and hindlimb as well as open-field locomotion. Our study tests how proven experimental treatments interact in a well-established animal model, thus providing needed direction for the development of future combinatory treatment regimens.

Spinal cord injury usually results in permanent paralysis because of lack of regrowth of damaged neurons. Here we demonstrate that adult mice lacking EphA4 (-/-), a molecule essential for correct guidance of spinal cord axons during development, exhibit axonal regeneration and functional recovery after spinal cord hemisection. Anterograde and retrograde tracing showed that axons from multiple pathways, including corticospinal and rubrospinal tracts, crossed the lesion site. EphA4-/- mice recovered stride length, the ability to walk on and climb a grid, and the ability to grasp with the affected hindpaw within 1-3 months of injury. EphA4 expression was upregulated on astrocytes at the lesion site in wild-type mice, whereas astrocytic gliosis and the glial scar were greatly reduced in lesioned EphA4-/- spinal cords. EphA4-/- astrocytes failed to respond to the inflammatory cytokines, interferon-γ or leukemia inhibitory factor,in vitro. Neurons grown on wild-type astrocytes extended shorter neurites than on EphA4-/- astrocytes, but longer neurites when the astrocyte EphA4 was blocked by monomeric EphrinA5-Fc. Thus, EphA4 regulates two important features of spinal cord injury, axonal inhibition, and astrocytic gliosis.

The inability of mature CNS neurons to regenerate injured axons has been attributed to a loss of inherent growth potential of cells and to inhibitory signals associated with myelin and the glial scar. The present study investigated two complementary issues: (1) whether mature CNS neurons can be stimulated to alter their gene expression profile and switch into a strong growth state; and (2) whether inactivating RhoA, a convergence point for multiple inhibitory signals, is sufficient to produce strong regeneration even without activating the growth state of neurons. In the mature rat, retinal ganglion cells (RGCs) normally fail to regenerate axons through the injured optic nerve but can be stimulated to do so by activating macrophages in the eye (e.g., by lens injury). To investigate underlying changes in gene expression, we retrogradely labeled RGCs with a fluorescent dye, performed optic nerve surgery with or without lens injury, and 4 d later, dissociated retinas, isolated RGCs by fluorescence-activated cell sorting, and examined their profiles of gene expression using microarrays. To investigate the effects of inactivating RhoA, we transfected RGCs with adeno-associated viruses carrying a gene for C3 ribosyltransferase. Our results show that, with appropriate stimulation, mature CNS neurons can undergo dramatic changes in gene expression comparable with those seen in regenerating neurons of the PNS, and that RhoA inactivation by itself results in moderate regeneration, and strongly potentiates axon regeneration through the mature optic nerve when the growth state of neurons is activated.

We have developed a novelin vitromodel of the glial scar that mimics the gradient of proteoglycan foundin vivoafter spinal cord injury. In this model, regenerated axons from adult sensory neurons that extended deeply into the gradient developed bulbous, vacuolated endings that looked remarkably similar to dystrophic endings formedin vivo. We demonstrate that despite their highly abnormal appearance and stalled forward progress, dystrophic endings are extremely dynamic bothin vitroandin vivoafter spinal cord injury. Time-lapse movies demonstrated that dystrophic endings continually send out membrane veils and endocytose large membrane vesicles at the leading edge, which were then retrogradely transported to the rear of the “growth cone.” This direction of movement is contrary to membrane dynamics that occur during normal neurite outgrowth. As further evidence of this motility, dystrophic endings endocytosed large amounts of dextran bothin vitroandin vivo. We now have anin vitromodel of the glial scar that may serve as a potent tool for developing and screening potential treatments to help promote regeneration past the lesionin vivo.

Reactive astrocytes are prominent in the cellular response to spinal cord injury (SCI), but their roles are not well understood. We used a transgenic mouse model to study the consequences of selective and conditional ablation of reactive astrocytes after stab or crush SCI. Mice expressing a glial fibrillary acid protein-herpes simplex virus-thymidine kinase transgene were given mild or moderate SCI and treated with the antiviral agent ganciclovir (GCV) to ablate dividing, reactive, transgene-expressing astrocytes in the immediate vicinity of the SCI. Small stab injuries in control mice caused little tissue disruption, little demyelination, no obvious neuronal death, and mild, reversible functional impairments. Equivalent small stab injuries in transgenic mice given GCV to ablate reactive astrocytes caused failure of blood-brain barrier repair, leukocyte infiltration, local tissue disruption, severe demyelination, neuronal and oligodendrocyte death, and pronounced motor deficits. Moderate crush injuries in control mice caused focal tissue disruption and cellular degeneration, with moderate, primarily reversible motor impairments. Equivalent moderate crush injuries combined with ablation of reactive astrocytes caused widespread tissue disruption, pronounced cellular degeneration, and failure of wound contraction, with severe persisting motor deficits. These findings show that reactive astrocytes provide essential activities that protect tissue and preserve function after mild or moderate SCI. In nontransgenic animals, crush or contusion SCIs routinely exhibit regions of degenerated tissue that are devoid of astrocytes. Our findings suggest that identifying ways to preserve reactive astrocytes, to augment their protective functions, or both, may lead to novel approaches to reducing secondary tissue degeneration and improving functional outcome after SCI.

The present study provides the first evidence that signaling occurs between B-ephrins and EphB receptors in the adult CNS in response to injury. Specifically, our combined histological and biochemical data indicate that two members of the B-class of ephrins and Eph receptors, ephrin-B2 and EphB2, are expressed by astrocytes and meningeal fibroblasts, respectively, in the adult spinal cord. In response to thoracic spinal cord transection lesions, ephrin-B2 and EphB2 protein levels exhibit an initial decrease (1 d after lesion), followed by a significant increase by day 14. Immunohistochemical data indicate that ephrin-B2 is expressed by reactive CNS astrocytes, and EphB2 is present on fibroblasts invading the lesion site from the adjacent meninges. During the first 3 d after injury, there is intermingling of ephrin-B2-expressing reactive astrocytes at the lesion surface with EphB2-containing fibroblasts that is concurrent with bidirectional activation (phosphorylation) of ephrin-B2 and EphB2. By 7 d, both cell types are establishing restricted cellular domains containing dense networks of cells and interweaving processes. This astroglial-meningeal fibroblast scar is fully developed by day 14 when there is strict segregation of ephrin-B2-expressing astrocytes from EphB2-positive meningeal fibroblasts. These morphological changes are concomitant with a simultaneous decrease in ephrin-B2 and EphB2 activation. These observations provide strong evidence that cell contact-mediated bidirectional signaling between ephrin-B2 on reactive astrocytes and EphB2 on meningeal fibroblasts is an early event in the cellular cascades that result in the development of the glial scar and the exclusion of meningeal fibroblasts from the injured spinal cord.

We have addressed the question of whether a family of axon growth-promoting molecules known as the laminins may play a role during axon regeneration in the CNS. A narrow sickle-shaped region containing a basal lamina-independent form of laminin exists in and around the cell bodies and proximal portion of the apical dendrites of CA3 pyramidal neurons of the postnatal hippocampus. To understand the possible function of laminin in axon regeneration within this pathway, we have manipulated laminin synthesis at the mRNA level in a slice culture model of the lesioned mossy system. In this model early postnatal mossy fibers severed near the hilus can regenerate across the lesion and elongate rapidly within strata lucidum and pyramidale. In slice cultures of the postnatal day 4 hippocampus, 2 d before lesion and then continuing for 1-5 d after lesion, translation of the gamma1 chain product of laminin was reduced by using antisense oligodeoxyribonucleotides and DNA enzymes. In the setting of the lesioned organotypic hippocampal slice, astroglial repair of the lesion and overall glial patterning were unperturbed by the antisense or DNA enzyme treatments. However, unlike controls, in the treated, lesioned slices the vast majority of regenerating mossy fibers could not cross the lesion site; those that did were very much shorter than usual, and they took a meandering course. In a recovery experiment in which the DNA enzyme or antisense oligos were washed away, laminin immunoreactivity returned and mossy fiber regeneration resumed. These results demonstrate the critical role of laminin(s) in an axon regeneration model of the CNS.

Spinal cord injury (SCI) in the mammalian CNS results in the formation of a glial scar around the lesion site ([Fig. 1][1] A ). The scar limits axon regeneration but it also serves a protective role by sequestering inflammatory cells to the lesion center, reducing tissue damage ([Herrmann et al.

Intermediate filaments (IFs) are a major component of the cytoskeleton in astrocytes. Their role is far from being completely understood. Immature astrocytes play a major role in neuronal migration and neuritogenesis, and their IFs are mainly composed of vimentin. In mature differentiated astrocytes, vimentin is replaced by the IF protein glial fibrillary acidic protein (GFAP). In response to injury of the CNS in the adult, astrocytes become reactive, upregulate the expression of GFAP, and reexpress vimentin. These modifications contribute to the formation of a glial scar that is obstructive to axonal regeneration. Nevertheless, astrocytes in vitro are considered to be the ideal substratum for the growth of embryonic CNS axons. In the present study, we have examined the potential role of these two major IF proteins in both neuronal survival and neurite growth. For this purpose, we cocultured wild-type neurons on astrocytes from three types of knock-out (KO) mice for GFAP or/and vimentin in a neuron-astrocyte coculture model. We show that the double KO astrocytes present many features of immaturity and greatly improve survival and neurite growth of cocultured neurons by increasing cell-cell contact and secreting diffusible factors. Moreover, our data suggest that the absence of vimentin is not a key element in the permissivity of the mutant astrocytes. Finally, we show that only the absence of GFAP is associated with an increased expression of some extracellular matrix and adhesion molecules. To conclude, our results suggest that GFAP expression is able to modulate key biochemical properties of astrocytes that are implicated in their permissivity.

Injury to the CNS results in the formation of the glial scar, a primarily astrocytic structure that represents an obstacle to regrowing axons. Chondroitin sulfate proteoglycans (CSPG) are greatly upregulated in the glial scar, and a large body of evidence suggests that these molecules are inhibitory to axon regeneration. We show that the CSPG neurocan, which is expressed in the CNS, exerts a repulsive effect on growing cerebellar axons. Expression of neurocan was examined in the normal and damaged CNS. Frozen sections labeled with anti-neurocan monoclonal antibodies 7 d after a unilateral knife lesion to the cerebral cortex revealed an upregulation of neurocan around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed substantially more neurocan in the injured CNS. Western blot analysis revealed neurocan and the processed forms neurocan-C and neurocan-130 to be present in the conditioned medium of highly purified rat astrocytes. The amount detected was increased by transforming growth factor β and to a greater extent by epidermal growth factor and was decreased by platelet-derived growth factor and, to a lesser extent, by interferon γ. O-2A lineage cells were also capable of synthesizing and processing neurocan. Immunocytochemistry revealed neurocan to be deposited on the substrate around and under astrocytes but not on the cells. Astrocytes therefore lack the means to retain neurocan at the cell surface. These findings raise the possibility that neurocan interferes with axonal regeneration after CNS injury.

Chondroitin sulfate proteoglycans (CS-PGs) expressed by reactive astrocytes may contribute to the axon growth-inhibitory environment of the injured CNS. The specific potentially inhibitory CS-PGs present in areas of reactive gliosis, however, have yet to be thoroughly examined. In this study, we used immunohistochemistry, combined immunohistochemistry–in situhybridization, immunoblot analysis, and reverse transcription-PCR to examine the expression of specific CS-PGs by reactive astrocytes in anin vivomodel of reactive gliosis: that is, the glial scar, after cortical injury. Neurocan and phosphacan can be localized to reactive astrocytes 30 d after CNS injury, whereas brevican and versican are not expressed in the chronic glial scar. Neurocan is also expressed by astrocytes in primary cell culture. Relative to the amount present in cultured astrocytes or uninjured cortex, neurocan expression increases significantly in the glial scar resulting from cortical injury, including the re-expression of the neonatal isoform of neurocan. In contrast, phosphacan protein levels are decreased in the glial scar compared with the uninjured brain. Because these CS-PGs are capable of inhibiting neurite outgrowthin vitro, our data suggest that phosphacan and neurocan in areas of reactive gliosis may contribute to axonal regenerative failure after CNS injury.

Small heat shock proteins (sHSPs), a family of HSPs, are known to accumulate in the CNS, mainly in astrocytes, in several pathological conditions such as Alexander's disease, Alzheimer's disease, and Creutzfeldt-Jakob disease. sHSPs may act not only as molecular chaperones, protecting against various stress stimuli, but may also play a physiological role in regulating cell differentiation and proliferation. In the present study, we have demonstrated that transient focal ischemia in rats dramatically induced HSP27 but not α B-crystallin (αBC), both of which are members of sHSPs, in reactive astrocytes. In contrast,in vitrochemical ischemic stress induced both HSP27 and αBC in cultured glial cells to the same extent. Dibutyryl cAMP (dBcAMP) and isoproterenol, a β-adrenergic receptor (βAR) agonist, enhanced HSP27 expression but suppressed αBC, and changed the shape of the cells to a stellate form. dBcAMP and isoproterenol inhibited cell proliferation under normal conditions. An increase in βAR-like immunoreactivity was also observed in reactive astrocytesin vivo. These results, together with recent findings that βAR plays an important role in glial scar formationin vivo, raise the possibility that βAR activation modulates sHSP expression after focal ischemia and is involved in the transformation of astrocytes to their reactive form.

Traumatic spinal cord injury (SCI) results in a cascade of tissue responses leading to cell death, axonal degeneration, and glial scar formation, exacerbating the already hostile environment and further inhibiting axon regeneration. Overcoming these inhibitory cues and promoting axonal regeneration is one of the primary targets in developing a cure for SCI. Previously, we demonstrated that transplantation of bone morphogenetic protein (BMP)-induced astrocytes derived from embryonic glial-restricted precursors (GDAsBMP) promotes extensive axonal growth and motor function recovery in a rodent spinal cord injury model. Here, we identify periostin (POSTN), a secreted protein, as a key component of GDABMP-induced axonal regeneration. POSTN is highly expressed by GDAsBMPand the perturbation of POSTN expression by shRNA diminished GDABMP-induced neurite extensionin vitro. We also found that recombinant POSTN is sufficient to overcome the inhibitory effect of scar-associated molecules and promote neurite extensionin vitroby signaling through focal adhesion kinase and Akt. Furthermore, transplantation of POSTN-deficient GDAsBMPinto the injured rat spinal cord resulted in compromised axonal regeneration, indicating that POSTN plays an essential role in GDABMP-mediated axonal regeneration. This finding reveals not only one of the major mechanisms underlying GDABMP-dependent recovery from SCI, but also the potential of POSTN as a therapeutic agent for traumatic injury of the CNS.

Astroglial scars surround damaged tissue after trauma, stroke, infection, or autoimmune inflammation in the CNS. They are essential for wound repair, but also interfere with axonal regrowth. A better understanding of the cellular mechanisms, regulation, and functions of astroglial scar formation is fundamental to developing safe interventions for many CNS disorders. We used wild-type and transgenic mice to quantify and dissect these parameters. Adjacent to crush spinal cord injury (SCI), reactive astrocytes exhibited heterogeneous phenotypes as regards proliferation, morphology, and chemistry, which all varied with distance from lesions. Mature scar borders at 14 d after SCI consisted primarily of newly proliferated astroglia with elongated cell processes that surrounded large and small clusters of inflammatory, fibrotic, and other cells. During scar formation from 5 to 14 d after SCI, cell processes deriving from different astroglia associated into overlapping bundles that quantifiably reoriented and organized into dense mesh-like arrangements. Selective deletion of STAT3 from astroglia quantifiably disrupted the organization of elongated astroglia into scar borders, and caused a failure of astroglia to surround inflammatory cells, resulting in increased spread of these cells and neuronal loss. In cocultures, wild-type astroglia spontaneously corralled inflammatory or fibromeningeal cells into segregated clusters, whereas STAT3-deficient astroglia failed to do so. These findings demonstrate heterogeneity of reactive astroglia and show that scar borders are formed by newly proliferated, elongated astroglia, which organize via STAT3-dependent mechanisms to corral inflammatory and fibrotic cells into discrete areas separated from adjacent tissue that contains viable neurons.

Astrocytes in nucleus magnocellularis (NM) of the chick respond to afferent activity blockade with increased immunoreactivity for glial fibrillary acidic protein (GFAP). NM neurons respond to the same manipulations with reduced protein synthesis, ribosomal dissociation, and subsequent death of a subset of these neurons. In the present study, we sought to evaluate the relationship between these neuronal and glial responses and to determine if similar activity-dependent mechanisms mediate them. We first examined the anatomical relationship between NM neurons and astrocytic processes by electron microscopy and GFAP immunostaining. Both methods showed that NM neurons deprived of activity for 6 hr were apposed by more glial processes than active NM neurons. However, we found no preferential positioning of GFAP- immunoreactive processes near neurons of the dying or surviving populations, and there were no differences in glial process apposition to dying versus surviving neurons at the EM level. To determine whether the astrocytic response is similar to the neuronal response in age dependence, GFAP immunoreactivity was analyzed in adult chickens following unilateral afferent activity blockade. Unlike the neuronal response to activity blockade, the astrocytic response is equally strong in adult animals. These results imply an independence of the neuronal and astrocytic responses to activity blockade, raising the possibility that these two cell types may be responding to different activity-related signals. This possibility was tested using an in vitro slice preparation. Unilateral stimulation of NM was provided in three ways: orthodromically, antidromically, and orthodromically in a low- calcium medium. The regulation of astrocytic GFAP immunoreactivity by these manipulations of activity was then analyzed. The results of these experiments show that, unlike neuronal protein synthesis, astrocytic GFAP immunoreactivity can be suppressed by either presynaptic or postsynaptic neuronal activity. Therefore, the astrocytes and neurons are regulated by different activity-dependent signals and, by the present measures, their responses to activity blockade appear independent of one another.

Traumatic injury to the adult mammalian CNS results in the formation of an astroglial-mesenchymal scar that seals the wound site but blocks axonal regeneration in the process. The mechanism that leads to this inhibition of axon outgrowth has been proposed to be either a physical barrier blocking the advancement of the growth cone or chemical factors actively inhibiting axon outgrowth. At present, it is unknown whether one or both of these mechanisms are responsible for the inhibitory nature of the glial scar in vivo. Using a model of CNS trauma that allows for removal of an adult rat glial scar intact on a nitrocellulose support and placement in vitro with the upper surface exposed, we addressed the question of whether the inhibitory effects could be accounted for by chemical components at the scar surface. A purified population of rat hippocampal neurons was seeded onto the scar explants as well as onto explants taken from neonatal rat cerebral cortex, and the extent of neurite outgrowth was compared. We found that the glial scar, at best, stimulates only minimal neurite outgrowth over its surface when compared to the immature environment explanted in the same manner. This growth-inhibitory state cannot merely be explained by neuronotoxic factors or fibroblasts preventing astrocyte-mediated neurite outgrowth. The inhibition is more probably due to the expression of molecules on the surface of the adult scar that either directly inhibit growth cones or inhibit them indirectly by occluding neurite-promoting factors in the extracellular matrix or on the astrocyte surface.