Expression Of Regeneration-associated Genes Research Articles

Oral Administration of a Specific p300/CBP Lysine Acetyltransferase Activator Induces Synaptic Plasticity and Repairs Spinal Cord Injury.

TTK21 is a small-molecule activator of p300/creb binding protein (CBP) acetyltransferase activity, which, upon conjugation with a glucose-derived carbon nanosphere (CSP), can efficiently cross the blood-brain barrier and activate histone acetylation in the brain. Its role in adult neurogenesis and retention of long-term spatial memory following intraperitoneal (IP) administration is well established. In this study, we successfully demonstrate that CSP-TTK21 can be effectively administered via oral gavage. Using a combination of molecular biology, microscopy, and electrophysiological techniques, we systematically investigate the comparative efficacy of oral administration of CSP and CSP-TTK21 in wild-type mice and evaluate their functional effects in comparison to intraperitoneal (IP) administration. Our findings indicate that CSP-TTK21, when administered orally, induces long-term potentiation in the hippocampus without significantly altering basal synaptic transmission, a response comparable to that achieved through IP injection. Remarkably, in a spinal cord injury model, oral administration of CSP-TTK21 exhibits efficacy equivalent to that of IP administration. Furthermore, our research demonstrates that oral delivery of CSP-TTK21 leads to improvements in motor function, histone acetylation dynamics, and increased expression of regeneration-associated genes (RAGs) in a spinal injury rat model, mirroring the effectiveness of IP administration. Importantly, no toxic and mutagenic effects of CSP-TTK21 are observed at a maximum tolerated dose of 1 g/kg in Sprague-Dawley (SD) rats via the oral route. Collectively, these results underscore the potential utility of CSP as an oral drug delivery system, particularly for targeting the neural system.

CBP/p300 activation promotes axon growth, sprouting, and synaptic plasticity in chronic experimental spinal cord injury with severe disability.

The interruption of spinal circuitry following spinal cord injury (SCI) disrupts neural activity and is followed by a failure to mount an effective regenerative response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here, we have investigated whether the epigenetic stimulation of the regenerative gene expression program can overcome the current inability to promote neurological recovery in chronic SCI with severe disability. We delivered the CBP/p300 activator CSP-TTK21 or vehicle CSP weekly between week 12 and 22 following a transection model of SCI in mice housed in an enriched environment. Data analysis showed that CSP-TTK21 enhanced classical regenerative signalling in dorsal root ganglia sensory but not cortical motor neurons, stimulated motor and sensory axon growth, sprouting, and synaptic plasticity, but failed to promote neurological sensorimotor recovery. This work provides direct evidence that clinically suitable pharmacological CBP/p300 activation can promote the expression of regeneration-associated genes and axonal growth in a chronic SCI with severe neurological disability.

Recovering the regenerative potential in chronically injured nerves by using conditioning electrical stimulation.

Chronically injured nerves pose a significant clinical challenge despite surgical management. There is no clinically feasible perioperative technique to upregulate a proregenerative environment in a chronic nerve injury. Conditioning electrical stimulation (CES) significantly improves sensorimotor recovery following acute nerve injury to the tibial and common fibular nerves. The authors' objective was to determine if CES could foster a proregenerative environment following chronically injured nerve reconstruction. The tibial nerve of 60 Sprague Dawley rats was cut, and the proximal ends were inserted into the hamstring muscles to prevent spontaneous reinnervation. Eleven weeks postinjury, these chronically injured animals were randomized, and half were treated with CES proximal to the tibial nerve cut site. Three days later, 24 animals were killed to evaluate the effects of CES on the expression of regeneration-associated genes at the cell body (n = 18) and Schwann cell proliferation (n = 6). In the remaining animals, the tibial nerve defect was reconstructed using a 10-mm isograft. Length of nerve regeneration was assessed 3 weeks postgrafting (n = 16), and functional recovery was evaluated weekly between 7 and 19 weeks of regeneration (n = 20). Three weeks after nerve isograft surgery, tibial nerves treated with CES prior to grafting had a significantly longer length of nerve regeneration (p < 0.01). Von Frey analysis identified improved sensory recovery among animals treated with CES (p < 0.01). Motor reinnervation, assessed by kinetics, kinematics, and skilled motor tasks, showed significant recovery (p < 0.05 to p < 0.001). These findings were supported by immunohistochemical quantification of motor endplate reinnervation (p < 0.05). Mechanisms to support the role of CES in reinvigorating the regenerative response were assessed, and it was demonstrated that CES increased the proliferation of Schwann cells in chronically injured nerves (p < 0.05). Furthermore, CES upregulated regeneration-associated gene expression to increase growth-associated protein-43 (GAP-43), phosphorylated cAMP response element binding protein (pCREB) at the neuronal cell bodies, and upregulated glial fibrillary acidic protein expression in the surrounding satellite glial cells (p < 0.05 to p < 0.001). Regeneration following chronic axotomy is impaired due to downregulation of the proregenerative environment generated following nerve injury. CES delivered to a chronically injured nerve influences the cell body and the nerve to re-upregulate an environment that accelerates axon regeneration, resulting in significant improvements in sensory and motor functional recovery. Percutaneous CES may be a preoperative strategy to significantly improve outcomes for patients undergoing delayed nerve reconstruction.

Ten-eleven translocation methylcytosine dioxygenase 3-loaded microspheres penetrate neurons in vitro causing active demethylation and neurite outgrowth.

SummaryEpigenetic processes, such as DNA methylation and other chromatin modifications, are believed to be largely responsible for establishing a reduced capacity for growth in the mature nervous system. Ten‐eleven translocation methylcytosine dioxygenase 3 (Tet3)‐, a member of the Tet gene family, plays a crucial role in promoting injury‐induced DNA demethylation and expression of regeneration‐associated genes in the peripheral nervous system. Here, we encapsulate Tet3 protein within a clinically tolerated poly(lactide‐co‐glycolide) microsphere system. Next, we show that Tet3‐loaded microspheres are internalized into mHippoE‐18 embryonic hippocampal cells. We compare the outgrowth potential of Tet3 microspheres with that of commonly used nerve growth factor (NGF)‐loaded microspheres in an in vitro injury model. Tet3‐containing microspheres increased levels of nuclear 5‐hydroxymethylcytosine indicating active demethylation and outperformed NGF‐containing microspheres in measures of neurite outgrowth. Our results suggest that encapsulated demethylases may represent a novel avenue to treat nerve injuries.

Role of Myc Proto-Oncogene as a Transcriptional Hub to Regulate the Expression of Regeneration-Associated Genes following Preconditioning Peripheral Nerve Injury.

Preconditioning peripheral nerve injury enhances the intrinsic growth capacity of DRGs sensory axons by inducing transcriptional upregulation of the regeneration-associated genes (RAGs). However, it is still unclear how preconditioning injury leads to the orchestrated induction of many RAGs. The present study identified Myc proto-oncogene as a transcriptional hub gene to regulate the expression of a distinct subset of RAGs in DRGs following the preconditioning injury. We demonstrated that c-MYC bound to the promoters of certain RAGs, such as Jun, Atf3, and Sprr1a, and that Myc upregulation following SNI preceded that of the RAGs bound by c-MYC. Marked DNA methylation of the Myc exon 3 sequences was implicated in the early transcriptional activation and accompanied by open histone marks. Myc deletion led to a decrease in the injury-induced expression of a distinct subset of RAGs, which were highly overlapped with the list of RAGs that were upregulated by Myc overexpression. Following dorsal hemisection spinal cord injury in female rats, Myc overexpression in DRGs significantly prevented the retraction of the sensory axons in a manner dependent on its downstream RAG, June Our results suggest that Myc plays a critical role in axon regeneration via its transcriptional activity to regulate the expression of a spectrum of downstream RAGs and subsequent effector molecules. Identification of more upstream hub transcription factors and the epigenetic mechanisms specific for individual hub transcription factors would advance our understanding of how the preconditioning injury induces orchestrated upregulation of RAGs.

Ascorbic Acid Promotes Functional Restoration after Spinal Cord Injury Partly by Epigenetic Modulation.

Axonal regeneration after spinal cord injury (SCI) is difficult to achieve, and no fundamental treatment can be applied in clinical settings. DNA methylation has been suggested to play a role in regeneration capacity and neuronal growth after SCI by controlling the expression of regeneration-associated genes (RAGs). The aim of this study was to examine changes in neuronal DNA methylation status after SCI and to determine whether modulation of DNA methylation with ascorbic acid can enhance neuronal regeneration or functional restoration after SCI. Changes in epigenetic marks (5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC)); the expression of Ten-eleven translocation (Tet) family genes; and the expression of genes related to inflammation, regeneration, and degeneration in the brain motor cortex were determined following SCI. The 5hmC level within the brain was increased after SCI, especially in the acute and subacute stages, and the mRNA levels of Tet gene family members (Tet1, Tet2, and Tet3) were also increased. Administration of ascorbic acid (100 mg/kg) to SCI rats enhanced 5hmC levels; increased the expression of the Tet1, Tet2, and Tet3 genes within the brain motor cortex; promoted axonal sprouting within the lesion cavity of the spinal cord; and enhanced recovery of locomotor function until 12 weeks. In conclusion, we found that epigenetic status in the brain motor cortex is changed after SCI and that epigenetic modulation using ascorbic acid may contribute to functional recovery after SCI.

Knocking Out Non-muscle Myosin II in Retinal Ganglion Cells Promotes Long-Distance Optic Nerve Regeneration

SUMMARYIn addition to altered gene expression, pathological cytoskeletal dynamics in the axon are another key intrinsic barrier for axon regeneration in the central nervous system (CNS). Here, we show that knocking out myosin IIA and IIB (myosin IIA/B) in retinal ganglion cells alone, either before or after optic nerve crush, induces significant optic nerve regeneration. Combined Lin28a overexpression and myosin IIA/B knockout lead to an additive promoting effect and long-distance axon regeneration. Immunostaining, RNA sequencing, and western blot analyses reveal that myosin II deletion does not affect known axon regeneration signaling pathways or the expression of regeneration-associated genes. Instead, it abolishes the retraction bulb formation and significantly enhances the axon extension efficiency. The study provides clear evidence that directly targeting neuronal cytoskeleton is sufficient to induce significant CNS axon regeneration and that combining altered gene expression in the soma and modified cytoskeletal dynamics in the axon is a promising approach for long-distance CNS axon regeneration.

Motor Recovery after Chronic Spinal Cord Transection in Rats: A Proof-of-Concept Study Evaluating a Combined Strategy.

The chronic phase of Spinal Cord (SC) injury is characterized by the presence of a hostile microenvironment that causes low activity and a progressive decline in neurological function; this phase is non-compatible with regeneration. Several treatment strategies have been investigated in chronic SC injury with no satisfactory results. OBJECTIVE- In this proof-of-concept study, we designed a combination therapy (Comb Tx) consisting of surgical glial scar removal plus scar inhibition, accompanied with implantation of mesenchymal stem cells (MSC), and immunization with neural-derived peptides (INDP). This study was divided into three subsets, all in which Sprague Dawley rats were subjected to a complete SC transection. Sixty days after injury, animals were randomly allocated into two groups for therapeutic intervention: control group and animals receiving the Comb-Tx. Sixty-three days after treatment we carried out experiments analyzing motor recovery, presence of somatosensory evoked potentials, neural regeneration-related genes, and histological evaluation of serotoninergic fibers. Comb-Tx induced a significant locomotor and electrophysiological recovery. An increase in the expression of regeneration-associated genes and the percentage of 5-HT+ fibers was noted at the caudal stump of the SC of animals receiving the Comb-Tx. There was a significant correlation of locomotor recovery with positive electrophysiological activity, expression of GAP43, and percentage of 5-HT+ fibers. Comb-Tx promotes motor and electrophysiological recovery in the chronic phase of SC injury subsequent to a complete transection. Likewise, it is capable of inducing the permissive microenvironment to promote axonal regeneration.

The nerve conditioning lesion: A strategy to enhance nerve regeneration.

By altering the intrinsic metabolism of the cell, including the upregulation of regeneration-associated genes (RAGs) and the production of structural proteins for axonal outgrowth, the conditioning lesion sets up an environment highly conducive to regeneration. In this review, we assess 40 years of research to provide a comprehensive overview of the conditioning lesion literature, directed at (1) discussing the mechanisms of and barriers to nerve regeneration that can be mitigated by the conditioning lesion, (2) describing the cellular and molecular pathways implicated in the conditioning lesion effect, and (3) deliberating on how these insights might be applied clinically. The consequential impact on regeneration is profound, with a conditioned nerve demonstrating longer neurite extensions in vitro, enhanced expression of RAGs within the dorsal root ganglia, early assembly and transportation of cytoskeletal elements, accelerated axonal growth, and improved functional recovery in vivo. Although this promising technique is not yet feasible to be performed in humans, there are potential strategies, such as conditioning electrical stimulation that may be explored to allow nerve conditioning in a clinically safe and well-tolerated manner. Ann Neurol 2018;83:691-702.

Neural Progenitor Cells Promote Axonal Growth and Alter Axonal mRNA Localization in Adult Neurons.

The inhibitory environment of the spinal cord and the intrinsic properties of neurons prevent regeneration of axons following CNS injury. However, both ascending and descending axons of the injured spinal cord have been shown to regenerate into grafts of embryonic neural progenitor cells (NPCs). Previous studies have shown that grafts composed of glial-restricted progenitors (GRPs) and neural-restricted progenitors (NRPs) can provide a permissive microenvironment for axon growth. We have used cocultures of adult rat dorsal root ganglion (DRG) neurons together with NPCs, which have shown significant enhancement of axon growth by embryonic rat GRP and GRPs/NRPs, both in coculture conditions and when DRGs are exposed to conditioned medium from the NPC cultures. This growth-promoting effect of NPC-conditioned medium was also seen in injury-conditioned neurons. DRGs cocultured with GRPs/NRPs showed altered expression of regeneration-associated genes at transcriptional and post-transcriptional levels. We found that levels of GAP-43 mRNA increased in DRG cell bodies and axons. However, hepcidin antimicrobial peptide (HAMP) mRNA decreased in the cell bodies of DRGs cocultured with GRPs/NRPs, which is distinct from the increase in cell body HAMP mRNA levels seen in DRGs after injury conditioning. Endogenous GAP-43 and β-actin mRNAs as well as reporter RNAs carrying axonally localizing 3'UTRs of these transcripts showed significantly increased levels in distal axons in the DRGs cocultured with GRPs/NRPs. These results indicate that axon growth promoted by NPCs is associated not only with enhanced transcription of growth-associated genes but also can increase localization of some mRNAs into growing axons.

Regulating Axonal Responses to Injury: The Intersection between Signaling Pathways Involved in Axon Myelination and The Inhibition of Axon Regeneration.

Following spinal cord injury (SCI), a multitude of intrinsic and extrinsic factors adversely affect the gene programs that govern the expression of regeneration-associated genes (RAGs) and the production of a diversity of extracellular matrix molecules (ECM). Insufficient RAG expression in the injured neuron and the presence of inhibitory ECM at the lesion, leads to structural alterations in the axon that perturb the growth machinery, or form an extraneous barrier to axonal regeneration, respectively. Here, the role of myelin, both intact and debris, in antagonizing axon regeneration has been the focus of numerous investigations. These studies have employed antagonizing antibodies and knockout animals to examine how the growth cone of the re-growing axon responds to the presence of myelin and myelin-associated inhibitors (MAIs) within the lesion environment and caudal spinal cord. However, less attention has been placed on how the myelination of the axon after SCI, whether by endogenous glia or exogenously implanted glia, may alter axon regeneration. Here, we examine the intersection between intracellular signaling pathways in neurons and glia that are involved in axon myelination and axon growth, to provide greater insight into how interrogating this complex network of molecular interactions may lead to new therapeutics targeting SCI.

CCL2 Mediates Neuron–Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

CNS neurons in adult mammals do not spontaneously regenerate axons after spinal cord injury. Preconditioning peripheral nerve injury allows the dorsal root ganglia (DRG) sensory axons to regenerate beyond the injury site by promoting expression of regeneration-associated genes. We have previously shown that peripheral nerve injury increases the number of macrophages in the DRGs and that the activated macrophages are critical to the enhancement of intrinsic regeneration capacity. The present study identifies a novel chemokine signal mediated by CCL2 that links regenerating neurons with proregenerative macrophage activation. Neutralization of CCL2 abolished the neurite outgrowth activity of conditioned medium obtained from neuron-macrophage cocultures treated with cAMP. The neuron-macrophage interactions that produced outgrowth-promoting conditioned medium required CCL2 in neurons and CCR2/CCR4 in macrophages. The conditioning effects were abolished in CCL2-deficient mice at 3 and 7 d after sciatic nerve injury, but CCL2 was dispensable for the initial growth response and upregulation of GAP-43 at the 1 d time point. Intraganglionic injection of CCL2 mimicked conditioning injury by mobilizing M2-like macrophages. Finally, overexpression of CCL2 in DRGs promoted sensory axon regeneration in a rat spinal cord injury model without harmful side effects. Our data suggest that CCL2-mediated neuron-macrophage interaction plays a critical role for amplification and maintenance of enhanced regenerative capacity by preconditioning peripheral nerve injury. Manipulation of chemokine signaling mediating neuron-macrophage interactions may represent a novel therapeutic approach to promote axon regeneration after CNS injury.

Overexpression of the monocyte chemokine CCL2 in dorsal root ganglion neurons causes a conditioning-like increase in neurite outgrowth and does so via a STAT3 dependent mechanism

Neuroinflammation plays a critical role in the regeneration of peripheral nerves following axotomy. An injury to the sciatic nerve leads to significant macrophage accumulation in the L5 DRG, an effect not seen when the dorsal root is injured. We recently demonstrated that this accumulation around axotomized cell bodies is necessary for a peripheral conditioning lesion response to occur. Here we asked whether overexpression of the monocyte chemokine CCL2 specifically in DRG neurons of uninjured mice is sufficient to cause macrophage accumulation and to enhance regeneration or whether other injury-derived signals are required. AAV5-EF1α-CCL2 was injected intrathecally, and this injection led to a time-dependent increase in CCL2 mRNA expression and macrophage accumulation in L5 DRG, with a maximal response at 3 weeks post-injection. These changes led to a conditioning-like increase in neurite outgrowth in DRG explant and dissociated cell cultures. This increase in regeneration was dependent upon CCL2 acting through its primary receptor CCR2. When CCL2 was overexpressed in CCR2-/- mice, macrophage accumulation and enhanced regeneration were not observed. To address the mechanism by which CCL2 overexpression enhances regeneration, we tested for elevated expression of regeneration-associated genes in these animals. Surprisingly, we found that CCL2 overexpression led to a selective increase in LIF mRNA and neuronal phosphorylated STAT3 (pSTAT3) in L5 DRGs, with no change in expression seen in other RAGs such as GAP-43. Blockade of STAT3 phosphorylation by each of two different inhibitors prevented the increase in neurite outgrowth. Thus, CCL2 overexpression is sufficient to induce macrophage accumulation in uninjured L5 DRGs and increase the regenerative capacity of DRG neurons via a STAT3-dependent mechanism.

Regeneration-associated genes decline in chronically injured rat sciatic motoneurons.

Chronic nerve injuries are notorious for their poor regenerative outcomes. Here, we addressed the question of whether the established reduced ability of injured motoneurons to regenerate their axons with time of disconnection with targets (chronic axotomy) is associated with a failure of injured motoneurons to express and sustain their expression of regeneration-associated genes. Sciatic motoneurons were prevented from regenerating by ligation of the transected nerves (chronic axotomy), and then subjected to a second nerve transection (acute axotomy) to mimic the clinical surgical procedure of refreshing the proximal nerve stump prior to delayed nerve repair. The expression of α1-tubulin, actin and GAP-43 mRNA was analysed in axotomized sciatic motoneurons by the use of in situ hybridization followed by autoradiography and silver grain quantification. The expression of these regeneration-associated genes by naive (acutely) axotomized motoneurons declined exponentially, to reach baseline levels within 6 months. These chronically injured motoneurons responded to a refreshment axotomy by elevating the expression of the genes to the same levels as in acutely (i.e. for the first time) axotomized sciatic motoneurons. However, the expression of these declined more rapidly than after acute axotomy. We conclude that a progressive decline in the expression of the regeneration-associated genes in chronically axotomized motoneurons and the even more rapid decline in their expression in response to a refreshment axotomy may explain why the regenerative capacity of chronically axotomized neurons declines with time.

Genetic Deletion of the Transcriptional Repressor NFIL3 Enhances Axon Growth In Vitro but Not Axonal Repair In Vivo

Axonal regeneration after injury requires the coordinated expression of genes in injured neurons. We previously showed that either reducing expression or blocking function of the transcriptional repressor NFIL3 activates transcription of regeneration-associated genes Arg1 and Gap43 and strongly promotes axon outgrowth in vitro. Here we tested whether genetic deletion or dominant-negative inhibition of NFIL3 could promote axon regeneration and functional recovery after peripheral nerve lesion in vivo. Contrary to our expectations, we observed no changes in the expression of regeneration-associated genes and a significant delay in functional recovery following genetic deletion of Nfil3. When NFIL3 function was inhibited specifically in dorsal root ganglia prior to sciatic nerve injury, we observed a decrease in regenerative axon growth into the distal nerve segment rather than an increase. Finally, we show that deletion of Nfil3 changes sciatic nerve lesion-induced expression in dorsal root ganglia of genes that are not typically involved in regeneration, including several olfactory receptors and developmental transcription factors. Together our findings show that removal of NFIL3 in vivo does not recapitulate the regeneration-promoting effects that were previously observed in vitro, indicating that in vivo transcriptional control of regeneration is probably more complex and more robust against perturbation than in vitro data may suggest.

Intrathecal delivery of IL-6 reactivates the intrinsic growth capacity of pyramidal cells in the sensorimotor cortex after spinal cord injury.

We have previously demonstrated the growth-promoting effect of intrathecal delivery of recombinant rat IL-6 immediately after corticospinal tract (CST) injury. Our present study aims to further clarify whether intrathecal delivery of IL-6 after CST injury could reactivate the intrinsic growth capacity of pyramidal cells in the sensorimotor cortex which project long axons to the spinal cord. We examined, by ELISA, levels of cyclic adenosine monophosphate (cAMP), adenylyl cyclase (AC, which synthesizes cAMP), phosphodiesterases (PDE, which degrades cAMP), and, by RT-PCR, the expression of regeneration-associated genes in the rat sensorimotor cortex after intrathecal delivery of IL-6 for 7 days, started immediately after CST injury. Furthermore, we injected retrograde neuronal tracer Fluorogold (FG) to the spinal cord to label pyramidal cells in the sensorimotor cortex, layers V and VI, combined with βIII-tubulin immunostaining, then we analyzed by immunohistochemisty and western blot the expression of the co-receptor gp-130 of IL-6 family, and pSTAT3 and mTOR, downstream IL-6/JAK/STAT3 and PI3K/AKT/mTOR signaling pathways respectively. We showed that intrathecal delivery of IL-6 elevated cAMP level and upregulated the expression of regeneration-associated genes including GAP-43, SPRR1A, CAP-23 and JUN-B, and the expression of pSTAT3 and mTOR in pyramidal cells of the sensorimotor cortex. In contrast, AG490, an inhibitor of JAK, partially blocked these effects of IL-6. All these results indicate that intrathecal delivery of IL-6 immediately after spinal cord injury can reactivate the intrinsic growth capacity of pyramidal cells in the sensorimotor cortex and these effects of IL-6 were partially JAK/STAT3-dependent.

Effect of local application of transforming growth factor-β at the nerve repair site following chronic axotomy and denervation on the expression of regeneration-associated genes. Laboratory investigation.

Although peripheral nerves can regenerate after traumatic injury, functional recovery is often suboptimal, especially after injuries to large nerve trunks such as the sciatic nerve or brachial plexus. Current research with animal models suggests that the lack of functional recovery resides in the lack of sufficient mature axons reaching their targets due to the loss of neurotrophic support by Schwann cells in the distal stump of injured nerves. This study was designed to investigate the effect of one-time application of transforming growth factor-β (TGF-β) at the repair site of chronically injured nerve. The authors used the rat tibial nerve injury and repair model to investigate the effects of application of physiological concentrations of TGF-β plus forskolin or forskolin alone in vivo at the repair site on gene and protein expression and axon regeneration at 6 weeks after nerve repair. They used gene expression profiling and immunohistochemical analysis of indicative activated proteins in Schwann cells to evaluate the effects of treatments on the delayed repair. They also quantified the regenerated axons distal to the repair site by microscopy of paraffin sections. Both treatment with forskolin only and treatment with TGF-β plus forskolin resulted in increased numbers of axons regenerated compared with saline-only control. There was robust activation and proliferation of both Schwann cells and macrophages reminiscent of the processes during Wallerian degeneration. The treatment also induced upregulation of genes implicated in cellular activation and growth as detected by gene array. Addition of TGF-β plus forskolin to the repair after chronic nerve injury improved axonal regeneration, probably via upregulation of required genes, expression of growth-associated protein, and reactivation of Schwann cells and macrophages. Further studies are required to better understand the mechanism of the positive effect of TGF-β treatment on old nerve injuries.

The effect of electrical stimulation and testosterone on the expression of regeneration‐associated genes

Despite robust regenerative potential following peripheral nerve injury, suboptimal outcomes often result in clinical situations. Our laboratory has recently shown that electrical stimulation (ES) and testosterone propionate (TP) can target two different phases of the regeneration process: delay time before sprout formation and regeneration rate, respectively. Furthermore, our laboratory has recently published that the combinatorial treatment of ES and TP significantly increased the expression of several regeneration‐associated genes (RAGs) compared to untreated animals after facial nerve crush injury. Since lower limb nerve injuries following trauma comprise a significant portion of peripheral nerve injuries, we investigated whether the combinatorial treatment of ES and TP improves axonal regeneration after injury to the sciatic nerve. We examined whether the expression profile of RAGs seen after facial nerve crush injury also occurred after sciatic nerve crush injury. To test this, rats received a crush injury to the sciatic nerve. Following injury, animals received either no treatment or treatment with ES and/or TP. After treatment, animals were euthanized at 6 hrs and 1, 2, 7, and 21 days post‐crush. Differences in mRNA expression of RAGs between the different treatment groups was determined by qPCR.Grant Funding Source: Indiana University Collaborative Research Grant

The Neuregulin‐Rac‐MKK7 pathway regulates antagonistic c‐jun/Krox20 expression in Schwann cell dedifferentiation

Schwann cells respond to nerve injury by dedifferentiating into immature states and producing neurotrophic factors, two actions that facilitate successful regeneration of axons. Previous reports have implicated the Raf-ERK cascade and the expression of c-jun in these Schwann cell responses. Here we used cultured primary Schwann cells to demonstrate that active Rac1 GTPase (Rac) functions as a negative regulator of Schwann cell differentiation by upregulating c-jun and downregulating Krox20 through the MKK7-JNK pathway, but not through the Raf-ERK pathway. The activation of MKK7 and induction of c-jun in sciatic nerves after axotomy was blocked by Rac inhibition. Microarray experiments revealed that the expression of regeneration-associated genes, such as glial cell line-derived neurotrophic factor and p75 neurotrophin receptor, after nerve injury was dependent on Rac but not on ERK. Finally, the inhibition of ErbB2 signaling prevented MKK7 activation, c-jun induction, and Rac-dependent gene expression in sciatic nerve explant cultures. Taken together, our results indicate that the neuregulin-Rac-MKK7-JNK/c-jun pathway regulates Schwann cell dedifferentiation following nerve injury.

Neurotrophins and Nerve Regeneration-Associated Genes are Expressed in the Cornea After Lamellar Flap Surgery

To determine the in vivo expression of neurotrophins (NTs) and nerve regeneration-associated genes (RAGs) after surgically creating a hinged lamellar corneal flap in thy1-YFP mice. Lamellar corneal flaps with multiple hinges were created in thy1-YFP mice. Mice were killed at weeks 2, 4, and 8. Quantitative polymerase chain reaction was performed to determine the expression of NTs and RAGs in the corneas after lamellar transection. Nerve growth factor (Ngf), brain-derived neurotrophic factor (Bdnf), glial cell-derived neurotrophic factor (Gdnf), neurotrophin 3, neurotrophin 5, small proline-rich repeat protein 1A (Sprr1a), growth-associated protein 43 (Gap43), and beta III tubulin (Tubb3) gene expressions were analyzed. Whole-mount confocal immunofluorescence and Western analyses were performed for localization and abundance of robustly expressed genes. Sprouts of fine YFP-positive fronds emanating from transected (injured) nerve bundles were seen in the flap area at 2 weeks onward. Bdnf and Sprr1a were robustly and significantly expressed at 2 weeks postoperatively (>2-fold increase in expression; P<0.05). Bdnf localized to thy1-YFP+ cells in operated corneas. Sprr1a localized to corneal epithelial cell membranes. At 8 weeks, none of the NTs and RAGs had increased expression. Bdnf (ρ=0.73, P=0.001) and Sprr1a (ρ=0.76, P=0.001) showed a significant positive correlation with beta III tubulin. The neurotrophin Bdnf and RAG Sprr1a are robustly and significantly expressed during corneal nerve regeneration in vivo.