Granulocyte colony‐stimulating factor improves neuron survival in experimental spinal cord injury by regulating nucleophosmin‐1 expression

Granulocyte colony‐stimulating factor (G‐CSF) and its related mechanisms were investigated to assess the potential for this factor to exert neuroprotective effects against spinal cord injury in mice. Recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) was injected into mice spinal cord hemisection models. Locomotor activity was assessed by using the Basso‐Bettie‐Bresnahan scale. Neurons isolated from spinal cords were cultured in vitro and used in a neuronal mechanical injury model. Three treatment groups were compared with this model, 1) G‐CSF, 2) G‐CSF + NSC348884 (a nucleophosmin 1‐specific inhibitor), and 3) NSC348884. Immunofluorescence staining and Western blotting were performed to analyze the expression of G‐CSF and nucleophosmin 1 (Npm1). TUNEL staining was performed to analyze apoptosis after G‐CSF treatment. We found that the G‐CSF receptor (G‐CSFR) and Npm1 were expressed in neurons and that Npm1 expression was induced after G‐CSF treatment. G‐CSF inhibited neuronal apoptosis. NSC348884 induced p53‐dependent cell apoptosis and partially blocked the neuroprotective activity of G‐CSF on neurons in vitro. G‐CSF promoted locomotor recovery and demonstrated neuroprotective effects in an acute spinal cord injury model. The mechanism of G‐CSF's neuroprotection may be related in part to attenuating neuronal apoptosis by NPM1. © 2014 Wiley Periodicals, Inc.     

MicroRNA‐mediated non‐cell‐autonomous regulation of cortical radial glial transformation revealed by a Dicer1 knockout mouse model

Radial glia (RG), as neurogenic progenitors and neuronal migration scaffolds, play critical roles during cortical neurogenesis. RG transformation into astrocytes, marking the transition from developmental to physiological function of these cells, is an important step during cortical development. In this study, we aim to determine the roles of microRNAs (miRNAs) during this biological process. In a conditional Dicer1‐null mouse where Dicer1 is deleted in both RG and their neuronal progeny, we observe delayed RG transformation as revealed by the persistence of their radial processes, and reduced number and complexity of translocated RG cell bodies in the postnatal cerebral cortex. Downregulation of Notch1 signaling is crucial to RG transformation, and consistently we find that Notch1 signaling is enhanced in the Dicer1‐null cerebral cortex. In addition, we show that, among the Notch1 ligands, Jagged2 (Jag2) is preferentially upregulated in the postnatal Dicer1‐null cerebral cortex as well as primary embryonic cortical cultures with instant Dicer1 deletion. Functionally, Dicer1‐deleted postnatal cerebellar cells with elevated Jag2 expression stimulate a stronger Notch1 signaling in a RG clone L2.3 when co‐cultured than control cells. Therefore, we unravel a novel non‐cell‐autonomous mechanism that regulates RG transformation by modulating Notch1 signaling via miRNA‐mediated suppression of the Nocth1 ligand Jag2. Furthermore, we validate Jag2 as a miR‐124 target gene and demonstrate in vitro that Jag2 expression is highly sensitive to Dicer1 deletion. Finally, we propose a new concept of MiRNA‐Sensitive target genes, identification of which may unravel a unique mode of miRNA‐mediated gene expression regulation. GLIA 2015;63:860–876     

Depletion of SASH1, an astrocyte differentiation‐related gene,contributes to functional recovery in spinal cord injury

AimsThis study aimed to evaluate the effects of the depletion of SAM and SH3 domain‐containing protein 1 (SASH1) on functional recovery after spinal cord injury (SCI) and to investigate the possible mechanism of SASH1 knockdown in astrocytes facilitating axonal growth.MethodsSCI model was established in adult rats. SASH1 small interfering RNA (siSASH1) was used to investigate its function. Hindlimb motor function was evaluated by the Basso‐Bresnahan‐Beattie (BBB) assay. The gene expressions were evaluated by the methods of qRT‐PCR, Western‐blotting, ELISA, and immunohistochemistry.ResultsSASH1 knockdown improved the BBB scores after SCI and significantly reduced GFAP expression. In cultured spinal astrocytes, siSASH1 treatment decreased interferon‐γ release and increased brain‐derived neurotrophic factor (BDNF) release. When cocultured with SASH1‐knockdown astrocytes, axonal growth increased. The neuronal tropomyosin receptor kinase B (BDNF receptor) expression increased, especially in the axonal tips. SASH1 expression increased while NSCs differentiated into glial cells, instead of neurons. After SASH1 depletion, differentiated NSCs maintained a higher level of Nestin protein and an increase in BDNF release.ConclusionsThese results indicate that SASH1 acts as an astrocytic differentiation‐maintaining protein, and SASH1 downregulation limits glial activation and contributes toward functional recovery after SCI.     

Dysregulation of the neuregulin‐1–ErbB network modulates endogenous oligodendrocyte differentiation and preservation after spinal cord injury

Spinal cord injury (SCI) results in degeneration of oligodendrocytes that leads to demyelination and axonal dysfunction. Replacement of oligodendrocytes is impaired after SCI, owing to the improper endogenous differentiation and maturation of myelinating oligodendrocytes. Here, we report that SCI‐induced dysregulation of neuregulin‐1 (Nrg‐1)–ErbB signaling may underlie the poor replacement of oligodendrocytes. Nrg‐1 and its receptors, ErbB‐2, ErbB‐3, and ErbB‐4, play essential roles in several aspects of oligodendrocyte development and physiology. In rats with SCI, we demonstrate that the Nrg‐1 level is dramatically reduced at 1 day after injury, with no restoration at later time‐points. Our characterisation shows that Nrg‐1 is mainly expressed by neurons, axons and oligodendrocytes in the adult spinal cord, and the robust and lasting decrease in its level following SCI reflects the permanent loss of these cells. Neural precursor cells (NPCs) residing in the spinal cord ependyma express ErbB receptors, suggesting that they are responsive to Nrg‐1 availability. In vitro, exogenous Nrg‐1 enhanced the proliferation and differentiation of spinal NPCs into oligodendrocytes while reducing astrocyte differentiation. In rats with SCI, recombinant human Nrg‐1β1 treatment resulted in a signifcant increase in the number of new oligodendrocytes and the preservation of existing ones after injury. Nrg‐1β1 administration also enhanced axonal preservation and attenuated astrogliosis, tumor necrosis factor‐α release and tissue degeneration after SCI. The positive effects of Nrg‐1β1 treatment were reversed by inhibiting its receptors. Collectively, our data provide strong evidence to suggest an impact of Nrg‐1–ErbB signaling on endogenous oligodendrocyte replacement and maintenance in the adult injured spinal cord, and its potential as a therapeutic target for SCI.     

Apolipoprotein E expression after spinal cord injury in the mouse

Apolipoprotein E (apo-E), a protein involved in lipid metabolism and cholesterol transport, has been found to be up-regulated in CNS injury and is associated with Alzheimer's disease in humans. In this study, we show that apo-E is also up-regulated after complete spinal cord transection in the C57BL/6 mouse. In the uninjured cord, the cellular localization of apo-E protein is in astrocytes, in individual neurons throughout the laminae except for the dorsal horn, and in endothelial cells of capillaries in the immediate vicinity of those neurons. After injury, RNA levels are elevated as early as 4 days and reach a maximal level between 1 and 2 weeks. Protein levels follow closely but remain up-regulated beyond 3 weeks. Early on, the protein can be found in neutrophils and macrophages at the injury site and only at later times in astrocytes during the remodeling of white matter tracts, most prominently in degenerating parts of the fasciculus gracilis.     

Blood-spinal cord barrier after spinal cord injury: relation to revascularization and wound healing

Spinal cord injury produces prominent disruption of the blood-spinal cord barrier. We have defined the blood-spinal cord barrier breakdown to the protein luciferase (61 kDa) in the acutely injured murine spinal cord and during revascularization. We show that newly formed and regenerating blood vessels that have abnormal permeability exhibit differential expression of the glucose-1 transporter (Glut-1), and that its expression is dependent on astrocytes. There was overt extravasation of luciferase within the first hour after injury, a period that coincided with marked tissue disruption within the epicenter of the lesion. Although there was a significant reduction in the number of blood vessels relative to controls by 24 hr after injury, abnormal barrier permeability remained significantly elevated. A second peak of abnormal barrier permeability at 3-7 days postinjury coincided with prominent revascularization of the epicenter. The barrier to luciferase was restored by 21 days postinjury and vascularity was similar to that of controls. During wound-healing process, the cord was reorganized into distinct domains. Between 14 and 21 days postinjury, each domain consisted primarily of nonneuronal cells, including macrophages. Astrocytes were limited characteristically to the perimeter of each domain. Only blood vessels affiliated closely with astrocytes in the perimeter expressed Glut-1, whereas blood vessels within each domain of the repairing cord did not express it. Together, these data demonstrate that both injured and regenerating vessels exhibit abnormal permeability and suggest that Glut-1 expression during revascularization is dependent on the presence of astrocytes.     

Neuregulin‐1 positively modulates glial response and improves neurological recovery following traumatic spinal cord injury

Spinal cord injury (SCI) results in glial activation and neuroinflammation, which play pivotal roles in the secondary injury mechanisms with both pro‐ and antiregeneration effects. Presently, little is known about the endogenous molecular mechanisms that regulate glial functions in the injured spinal cord. We previously reported that the expression of neuregulin‐1 (Nrg‐1) is acutely and chronically declined following traumatic SCI. Here, we investigated the potential ramifications of Nrg‐1 dysregulation on glial and immune cell reactivity following SCI. Using complementary in vitro approaches and a clinically‐relevant model of severe compressive SCI in rats, we demonstrate that immediate delivery of Nrg‐1 (500 ng/day) after injury enhances a neuroprotective phenotype in inflammatory cells associated with increased interleukin‐10 and arginase‐1 expression. We also found a decrease in proinflammatory factors including IL‐1β, TNF‐α, matrix metalloproteinases (MMP‐2 and 9) and nitric oxide after injury. In addition, Nrg‐1 modulates astrogliosis and scar formation by reducing inhibitory chondroitin sulfate proteoglycans after SCI. Mechanistically, Nrg‐1 effects on activated glia are mediated through ErbB2 tyrosine phosphorylation in an ErbB2/3 heterodimer complex. Furthermore, Nrg‐1 exerts its effects through downregulation of MyD88, a downstream adaptor of Toll‐like receptors, and increased phosphorylation of Erk1/2 and STAT3. Nrg‐1 treatment with the therapeutic dosage of 1.5 μg/day significantly improves tissue preservation and functional recovery following SCI. Our findings for the first time provide novel insights into the role and mechanisms of Nrg‐1 in acute SCI and suggest a positive immunomodulatory role for Nrg‐1 that can harness the beneficial properties of activated glia and inflammatory cells in recovery following SCI.     

Treatment with the neurosteroid dehydroepiandrosterone promotes recovery of motor behavior after moderate contusive spinal cord injury in the mouse

The neurosteroid dehydroepiandrosterone (DHEA) has neuroprotective properties after ischemic and excitatory insults to the brain. In the developing embryo, it is produced in discrete regions of the central nervous system (CNS), where it specifically promotes axonal growth of differentiated neurons. To test if DHEA could be beneficial after spinal cord injury (SCI), we used a model of moderate contusive SCI developed and characterized in the mouse. Immediately after surgery, we applied treatment with DHEA or with vehicle only and compared treatment groups (n = 12 in each group) over a 42-day period. Locomotor recovery was assessed in an open field using a standardized 21-point scale, according to gait analysis on paw print recordings and using foot fault analyses on an inclined ladder beam. The DHEA-treated group showed improved function compared to vehicle-treated animals in these tests. More strikingly, DHEA enhanced recovery of left-right coordination and fine motor control. In an attempt to correlate functional recovery with spinal cord neuropathology in the different experimental groups, we studied the area of spared white matter at the epicenter and reactive gliosis/scar formation 42 days post-injury (DPI). DHEA significantly increased the area of white matter spared at the epicenter and reduced the area of reactive gliosis surrounding the lesion. These data demonstrate the effectiveness of DHEA in promoting functional recovery in the adult murine injured spinal cord.     

Role of spinal 5‐HT2 receptor subtypes in quipazine‐induced hindlimb movements after a low‐thoracic spinal cord transection

A role of serotonin receptors (5‐HTRs) in spinal rhythmogenesis has been proposed several years ago based mainly upon data showing that bath‐applied 5‐HT could elicit locomotor‐like rhythms in in vitro isolated spinal cord preparations. Such a role was partially confirmed in vivo after revealing that systemically administered 5‐HTR₂ agonists, such as quipazine, could induce some locomotor‐like movements (LM) in completely spinal cord‐transected (Tx) rodents. However, given the limited binding selectivity of currently available 5‐HTR₂ agonists, it has remained difficult to determine clearly if one receptor subtype is specifically associated with LM induction. In situ hybridization, data using tissues from L1–L2 spinal cord segments, where critical locomotor network elements have been identified in mice, revealed greater 5‐HTR_2A mRNA levels in low‐thoracic Tx than non‐Tx animals. This expression level remained elevated for several days, specifically in the lateral intermediate zone, where peak values were detected at 1 week post‐Tx and returned to normal at 3 weeks post‐Tx. Behavioral and kinematic analyses revealed quipazine‐induced LM in 1‐week Tx mice either non‐pretreated or pretreated with selective 5‐HTR_2B and/or 5‐HTR_2C antagonists. In contrast, LM completely failed to be induced by quipazine in animals pretreated with selective 5‐HTR_2A antagonists. Altogether, these results provide strong evidence suggesting that 5‐HTR_2A are specifically associated with spinal locomotor network activation and LM generation induced by quipazine in Tx animals. These findings may contribute to design drug treatments aimed at promoting locomotor function recovery in chronic spinal cord‐injured patients.     

Neuronal release and successful astrocyte uptake of aminoacidergic neurotransmitters after spinal cord injury in lampreys

In contrast to mammals, the spinal cord of lampreys spontaneously recovers from a complete spinal cord injury (SCI). Understanding the differences between lampreys and mammals in their response to SCI could provide valuable information to propose new therapies. Unique properties of the astrocytes of lampreys probably contribute to the success of spinal cord regeneration. The main aim of our study was to investigate, in the sea lamprey, the release of aminoacidergic neurotransmitters and the subsequent astrocyte uptake of these neurotransmitters during the first week following a complete SCI by detecting glutamate, GABA, glycine, Hu and cytokeratin immunoreactivities. This is the first time that aminoacidergic neurotransmitter release from neurons and the subsequent astrocytic response after SCI are analysed by immunocytochemistry in any vertebrate. Spinal injury caused the immediate loss of glutamate, GABA and glycine immunoreactivities in neurons close to the lesion site (except for the cerebrospinal fluid‐contacting GABA cells). Only after SCI, astrocytes showed glutamate, GABA and glycine immunoreactivity. Treatment with an inhibitor of glutamate transporters (DL‐TBOA) showed that neuronal glutamate was actively transported into astrocytes after SCI. Moreover, after SCI, a massive accumulation of inhibitory neurotransmitters around some reticulospinal axons was observed. Presence of GABA accumulation significantly correlated with a higher survival ability of these neurons. Our data show that, in contrast to mammals, astrocytes of lampreys have a high capacity to actively uptake glutamate after SCI. GABA may play a protective role that could explain the higher regenerative and survival ability of specific descending neurons of lampreys. GLIA 2014;62:1254–1269     

.：pendymal cell proliferation and apoptosis following acute spinal cord injury in the adult rat

BACKGROUND： Studies have reported that spinal cord injury can induce the reactive proliferation of ependymal cells and secondarily cause the apoptosis of nerve cells. However, there is no generally accepted theory on the apoptotic characteristics of ependymal cells in the injured spinal cord. OBJECTIVE： To observe the reactive proliferation and apoptosis of ependymal cells in adult rats following acute spinal cord injury. DESIGN, TIME AND SETTING： A randomized control study based on neuropathology was performed in the Third Military Medical University of Chinese PLA between 2005 and 2007. MATERIALS： Forty healthy, adult, Wistar rats were included in the present study. METHODS： Moderate spinal cord injury was established in twenty rats using Feeney＇s method, while the remaining 20 rats served as controls and were only treated with laminectomy. All rats were injected intraperitoneally with 1.25 mL of BrdU solution （10 mg BrdU/mL saline） 3 times at 4 hours intervals during the 12 hours prior to sacrifice. MAIN OUTCOME MEASURES： Ependymal cell proliferation and apoptosis in the rat spinal cord were determined by BrdU and nestin immunofluorescence double-labeling, as well as the TUNEL method, at 1, 3, 7, and 14 days after operation. RESULTS： In the moderate spinal cord injury rats, nestin expression was observed in the cytoplasm of ependymal cells. One day immediately following surgery, ependymal cells were BrdU-labeled. The number of BrdU-positive cells increased at 3 days, reached a peak at 7 days, and gradually reduced thereafter. The ependyma developed from a constitutive monolayer cells to a multi-layer cell complex. Some BrdU/Nestin double-positive ependymal cells migrated out from the ependyma. TUNEL-positive cells were also detected in the ependyma in the central region, as well as ischemic regions of the injured spinal cord. In addition, TUNEL-positive cells were visible in the ependyma. No TUNEL-positive ependymal cells were observed in the normal spinal cord. CONCLUSION： Proliferating     

Leukotriene synthases and the receptors induced by peripheral nerve injury in the spinal cord contribute to the generation of neuropathic pain

Leukotrienes (LTs) belong to a large family of lipid mediators, termed eicosanoids, which are derived from arachidonic acids and released from the cell membrane by phospholipases. LTs are involved in the pathogenesis of inflammatory diseases, such as asthma, rheumatoid arthritis, and peripheral inflammatory pain. In the present study, we examined whether LTs were implicated in pathomechanism of neuropathic pain following peripheral nerve injury. Using the spared nerve injury (SNI) model in rats, we investigated the expression of LT synthases (5‐lipoxygenase; 5‐LO, Five lipoxygenase activating protein; FLAP, LTA4 hydrolase; LTA4h and LTC4 synthase; LTC4s) and receptors (BLT1, 2 and CysLT1, 2) mRNAs in the rat spinal cord. Semi‐quantitative RT‐PCR revealed that 5‐LO, FLAP, LTC4s, BLT1, and CysLT1 mRNAs increased following SNI, but not CysLT2 mRNAs. Using double labeling analysis of in situ hybridization with immunohistochemistry, we observed that 5‐LO, FLAP, and CysLT1 mRNAs were expressed in spinal microglia. LTA4h and LTC4s mRNAs were expressed in both spinal neurons and microglia. BLT1 mRNA was expressed in spinal neurons. The p38 mitogen‐activated protein kinase inhibitor, but not MEK inhibitor, reduced the increase in 5‐LO in spinal microglia. Continuous intrathecal administration of the 5‐LO inhibitor or BLT1 and CysLT1 receptor antagonists suppressed mechanical allodynia induced by SNI. Our findings suggest that the increase of LT synthesis in spinal microglia produced via p38 MAPK plays a role in the generation of neuropathic pain. © 2009 Wiley‐Liss, Inc.     

Endogenous expression of interleukin‐4 regulates macrophage activation and confines cavity formation after traumatic spinal cord injury

Traumatic spinal cord injury (SCI) triggers inflammatory reactions in which various types of cells and cytokines are involved. Several proinflammatory cytokines are up‐regulated after SCI and play crucial roles in determining the extent of secondary tissue damage. However, relatively little is known about antiinflammatory cytokines and their roles in spinal cord trauma. Recent studies have shown that an antiinflammatory cytokine, interleukin‐4 (IL‐4), is expressed and exerts various modulatory effects in CNS inflammation. We found in the present study that IL‐4 was highly expressed at 24 hr after contusive SCI in rats and declined thereafter, with concurrent up‐regulation of IL‐4 receptor subunit IL‐4α. The majority of IL‐4‐producing cells were myeloperoxidase‐positive neutrophils. Injection of neutralizing antibody against IL‐4 into the contused spinal cord did not significantly affect the expression levels of proinflammatory cytokines such as IL‐1β, IL‐6, and tumor necrosis factor‐α or other antiinflammatory cytokines such as IL‐10 and transforming growth factor‐β. Instead, attenuation of IL‐4 activity led to a marked increase in the extent of ED1‐positive macrophage activation along the rostrocaudal extent at 7 days after injury. The enhanced macrophage activation was preceded by an increase in the level of monocyte chemoattractant protein‐1 (MCP‐1/CCL2). Finally, IL‐4 neutralization resulted in more extensive cavitation at 4 weeks after injury. These results suggest that endogenous expression of antiinflammatory cytokine IL‐4 regulates the extent of acute macrophage activation and confines the ensuing secondary cavity formation after spinal cord trauma. © 2010 Wiley‐Liss, Inc     

Functional recovery after spinal cord injury in mice through activation of microglia and dendritic cells after IL-12 administration

We have previously reported that the transplantation of dendritic cells (DCs) brings about functional recovery after spinal cord injury in mice through the activation of endogenous microglia/macrophages and neural stem/progenitor cells. In this study, the effect of interleukin-12 (IL-12), which is secreted from DCs, was evaluated for the treatment of spinal cord injury in mice. Administration of IL-12 into the injured site significantly increased the number of activated microglia/macrophages and DCs as well as the expression of brain-derived neurotrophic factor surrounding the lesion site. Immunohistochemical analyses showed that de novo neurogenesis and remyelination were induced by IL-12 treatment. Furthermore, an open field test using Basso-Beattie-Brenham scoring revealed a significant improvement of locomotor function in mice treated with IL-12. These results suggest that IL-12 administration into the injured spinal cord results in a functional recovery through the activation of microglia/macrophages and DCs.     

Transplantation of galectin‐1‐expressing human neural stem cells into the injured spinal cord of adult common marmosets

Delayed transplantation of neural stem/progenitor cells (NS/PCs) into the injured spinal cord can promote functional recovery in adult rats and monkeys. To enhance the functional recovery after NS/PC transplantation, we focused on galectin‐1, a carbohydrate‐binding protein with pleiotropic roles in cell growth, differentiation, apoptosis, and neurite outgrowth. Here, to determine the combined therapeutic effect of NS/PC transplantation and galectin‐1 on spinal cord injury (SCI), human NS/PCs were transfected by lentivirus with galectin‐1 and green fluorescent protein (GFP), (Gal‐NS/PCs) or GFP alone (GFP‐NS/PCs), expanded in vitro, and then transplanted into the spinal cord of adult common marmosets, 9 days after contusive cervical SCI. The animals' motor function was evaluated by their spontaneous motor activity, bar grip power, and performance on a treadmill test. Histological analyses revealed that the grafted human NS/PCs survived and differentiated into neurons, astrocytes, and oligodendrocytes. There were significant differences in the myelinated area, corticospinal fibers, and serotonergic fibers among the Gal‐NS/PC, GFP‐NS/PC, vehicle‐control, and sham‐operated groups. The Gal‐NS/PC‐grafted animals showed a better performance on all the behavioral tests compared with the other groups. These findings suggest that Gal‐NS/PCs have better therapeutic potential than NS/PCs for SCI in nonhuman primates and that human Gal‐NS/PC transplantation might be a feasible treatment for human SCI. © 2010 Wiley‐Liss, Inc.     

Syntenin‐a promotes spinal cord regeneration following injury in adult zebrafish

In contrast to mammals, adult zebrafish recover locomotor function after spinal cord injury, in part due to the capacity of the central nervous system to repair severed connections. To identify molecular cues that underlie regeneration, we conducted mRNA expression profiling and found that syntenin‐a expression is upregulated in the adult zebrafish spinal cord caudal to the lesion site after injury. Syntenin is a scaffolding protein involved in mammalian cell adhesion and movement, axonal outgrowth, establishment of cell polarity, and protein trafficking. It could thus be expected to be involved in supporting regeneration in fish. Syntenin‐a mRNA and protein are expressed in neurons, glia and newly generated neural cells, and upregulated caudal to the lesion site on days 6 and 11 following spinal cord injury. Treatment of spinal cord‐injured fish with two different antisense morpholinos to knock down syntenin‐a expression resulted in significant inhibition of locomotor recovery at 5 and 6 weeks after injury, when compared to control morpholino‐treated fish. Knock‐down of syntenin‐a reduced regrowth of descending axons from brainstem neurons into the spinal cord caudal to the lesion site. These observations indicate that syntenin‐a is involved in regeneration after traumatic insult to the central nervous system of adult zebrafish, potentially leading to novel insights into the cellular and molecular mechanisms that require activation in the regeneration‐deficient mammalian central nervous system.     

Serotonin release variations during recovery of motor function after a spinal cord injury in rats

Current literature suggests that serotonin (5‐HT) release within the ventral horn of the spinal cord plays a role in motor function. We hypothesized that endogenous 5‐HT release is involved in the recovery of motor function after spinal cord injury. To appreciate the functional parameters of regenerating serotonergic fibers, a microdialysis probe was stereotactically implanted in the ventral horn of subhemi‐lesioned rats. Microdialysis in combination with HPLC was used to measure concentrations of 5‐HT in the lumbar ventral horn during periods of rest (90 min), treadmill run (60 min) and postexercise rest (90 min) for a 1‐month time period of recovery following the surgical lesion. Within the same period of time, 5‐HT levels varied significantly. A significant (202%) increase was observed at day 18 postlesion relative to day 8, and a 16.4% decrease was observed at day 34 relative to day 18. Treadmill exercise challenge induced a 10% decrease of 5‐HT release relative to rest at days 18 and 34. In conclusion, overtime treadmill locomotor recovery is parallel to amounts (rest basal levels) and patterns (exercise and postexercise levels) of 5‐HT release suggesting that changes in serotonergic system occurred within the same time frame than locomotor recovery using treadmill challenge. Synapse 64:855–861, 2010. © 2010 Wiley‐Liss, Inc.