The Limited Effects of Estradiol Administration Immediately after Spinal Cord Injury.

OBJECTIVE: We aimed to investigate estradiol (E2) as a therapeutic drug for spinal cord injury (SCI) and elucidate the disagreement in the field about the use of this hormone after an injury. METHODS: Eleven animals underwent surgery (laminectomy at the T9-T10 levels) followed by an intravenous injection (100 µg) of an E2 bolus and the implantation of 0.5cm of Silastic tubing containing 3 mg of E2 (sham E2 + E2 bolus) immediately after the laminectomy. The SCI control animals received a moderate contusion using the Multicenter Animal SCI Study impactor device over the exposed spinal cord followed by an intravenous bolus injection of sesame oil and were implanted with empty Silastic tubing (injury SE + vehicle); treated rats received a bolus of E2 and a Silastic implant with 3 mg of E2 (injury E2 + E2 bolus). Functional locomotor recovery and fine motor coordination were assessed by the Basso, Beattie, and Bresnahan (BBB) open field test and grid-walking tests, respectively, from the acute (7 days post-injury [DPI]) to the chronic stages (35 DPI). Anatomical studies of the cord were performed using Luxol fast blue staining followed by densitometric analysis. RESULTS: As observed in the BBB open field and the grid-walking tests, E2 post-SCI did not improve locomotor function but instead increased spared white matter tissue, in the rostral region. CONCLUSION: Estradiol post-SCI, at the dose and route of administration used in this study, failed to promote locomotor recovery but partially restored spared white matter tissue.

Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma.

Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y2 receptor (P2Y2R) to modulate its downstream signaling. P2Y2R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y2R signaling and its pro-survival actions in the 1321N1 astrocytoma cells mechanical injury model system. KD of Cav-1 decreased its expression in 1321N1 cells devoid of or expressing hHAP2Y2R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y2R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y2R) revealed a P2Y2R-dependent temporal increase in both kinases. These temporal increases in pERK1/2 and pAkt were significantly decreased in Cav-1 KD 1321N1 (devoid of or expressing a hHAP2Y2R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y2R-mediated pERK1/2 and pAkt kinases' activity, respectively. These early-onset hHAP2Y2R-mediated signaling responses in Cav-1-expressing and Cav-1 KD 1321N1 correlated with changes in cell viability (via a resazurin-based method) and apoptosis (via caspase-9 expression). In Cav-1-positive 1321N1 cells, expression of hHAP2Y2R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y2R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y2R. These findings support the importance of Cav-1 in modulating P2Y2R signaling during mechanical injury and its protective actions in a human astrocytoma cell line, whilst shedding light on potential new venues for brain injury or trauma interventions.

Continuous tamoxifen delivery improves locomotor recovery 6h after spinal cord injury by neuronal and glial mechanisms in male rats.

No treatment is available for patients with spinal cord injury (SCI). Patients often arrive to the hospital hours after SCI suggesting the need of a therapy that can be used on a clinically relevant window. Previous studies showed that Tamoxifen (TAM) treatment 24h after SCI benefits locomotor recovery in female rats. Tamoxifen exerts beneficial effects in male and female rodents but a gap of knowledge exists on: the therapeutic window of TAM, the spatio-temporal mechanisms activated and if this response is sexually dimorphic. We hypothesized that TAM will favor locomotor recovery when administered up-to 24h after SCI in male Sprague-Dawley rats. Rats received a thoracic (T10) contusion using the MACSIS impactor followed by placebo or TAM (15mg/21days) pellets in a therapeutic window of 0, 6, 12, or 24h. Animals were sacrificed at 2, 7, 14, 28 or 35days post injury (DPI) to study the molecular and cellular changes in the acute and chronic stages. Immediate or delayed therapy (t=6h) improved locomotor function, increased white matter spared tissue, and neuronal survival. TAM reduced reactive gliosis during chronic stages and increased the expression of Olig-2. A significant difference was observed in estrogen receptor alpha between male and female rodents from 2 to 28 DPI suggesting a sexually dimorphic characteristic that could be related to the behavioral differences observed in the therapeutic window of TAM. This study supports the use of TAM in the SCI setting due to its neuroprotective effects but with a significant sexually dimorphic therapeutic window.

Tamoxifen: an FDA approved drug with neuroprotective effects for spinal cord injury recovery.

Spinal cord injury (SCI) is a condition without a cure, affecting sensory and/or motor functions. The physical trauma to the spinal cord initiates a cascade of molecular and cellular events that generates a non-permissive environment for cell survival and axonal regeneration. Among these complex set of events are damage of the blood-brain barrier, edema formation, inflammation, oxidative stress, demyelination, reactive gliosis and apoptosis. The multiple events activated after SCI require a multi-active drug that could target most of these events and produce a permissive environment for cell survival, regeneration, vascular reorganization and synaptic formation. Tamoxifen, a selective estrogen receptor modulator, is an FDA approved drug with several neuroprotective properties that should be considered for the treatment of this devastating condition. Various investigators using different animal models and injury parameters have demonstrated the beneficial effects of this drug to improve functional locomotor recovery after SCI. Results suggest that the mechanism of action of Tamoxifen administration is to modulate anti-oxidant, anti-inflammatory and anti-gliotic responses. A gap of knowledge exists regarding the sex differences in response to Tamoxifen and the therapeutic window available to administer this treatment. In addition, the effects of Tamoxifen in axonal outgrowth or synapse formation needs to be investigated. This review will address some of the mechanisms activated by Tamoxifen after SCI and the results recently published by investigators in the field.

Modulation of Vascular ACE by Oxidative Stress in Young Syrian Cardiomyopathic Hamsters: Therapeutic Implications.

Increased vascular angiotensin-converting enzyme (ACE) activity and oxidative stress are present in young Syrian cardiomyopathic hamsters (SCH) before the clinical manifestation of heart failure (HF). The developmental time-course of these alterations and their potential interactions, however, are still unknown. We evaluated mRNA and protein levels of ACE, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) in the vasculature of SCH from one to four months of age. Total RNA and proteins were quantified with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. The role of nitric oxide (NO) on vascular ACE activity was also assessed. ACE mRNA and protein levels were up-regulated in SCH at two months of age compared with controls (CT) (p < 0.05). At this two-month stage, eNOS protein levels were lower in SCH (87%) than in CT (100%) (p < 0.05), although iNOS protein levels increased significantly (482%) compared to CT (100%; p < 0.05). In addition, ACE mRNA expression and activity were modulated by NO at two months of age. Thus, the combination of low eNOS and high iNOS protein levels may underlie vascular renin-angiotensin system (RAS) over-activation. Altogether, these factors may contribute to the development of endothelial dysfunction and vascular hyper-reactivity in the early stages of heart failure, and eventually trigger cardiac deterioration in this animal model of HF.

Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells.

Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides, ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y2 nucleotide receptor (P2Y2R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y2R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y2R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y2R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, co-localize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y2R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y2R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca(2+) concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca(2+) responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y2R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y2R/Cav-1 interactions represent a novel target for controlling P2Y2R function after CNS injury.

Tamoxifen Administration Immediately or 24 Hours after Spinal Cord Injury Improves Locomotor Recovery and Reduces Secondary Damage in Female Rats.

Spinal cord injury (SCI) is a condition with no available cure. The initial physical impact triggers a cascade of molecular and cellular events that generate a nonpermissive environment for cell survival and axonal regeneration. Spinal cord injured patients often arrive at the clinic hours after the initial insult. This indicates the need to study and develop treatments with a long therapeutic window of action and multiactive properties, which target the complex set of events that arise after the initial trauma. We provide evidence that tamoxifen (TAM), a drug approved by the Food and Drug Administration, exerts neuroprotective effects in an animal model when applied up-to 24 h after SCI. We hypothesized that continuous TAM administration will improve functional locomotor recovery by favoring myelin preservation and reducing secondary damage after SCI. Adult female Sprague-Dawley rats (∼230 g) received a moderate contusion to the thoracic (T9-T10) spinal cord, using the MASCIS impactor device. To determine the therapeutic window available for TAM treatment, rats were implanted with TAM pellets (15 mg) immediately or 24 h after SCI. Locomotor function (Basso, Beattie, Bresnahan open field test, grid walk, and beam crossing tests) was assessed weekly for 35 days post-injury. TAM-treated rats showed significant functional locomotor recovery and improved fine movements when treated immediately or 24 h after SCI. Further, TAM increased white matter preservation and reduced secondary damage caused by astrogliosis, axonal degeneration, and cell death after trauma. These results provide evidence for TAM as a potential therapeutic agent to treat SCI up to 24 h after the trauma.

Fatty Acid Binding Protein 5 Modulates Docosahexaenoic Acid-Induced Recovery in Rats Undergoing Spinal Cord Injury.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) promote functional recovery in rats undergoing spinal cord injury (SCI). However, the precise molecular mechanism coupling n-3 PUFAs to neurorestorative responses is not well understood. The aim of the present study was to determine the spatiotemporal expression of fatty acid binding protein 5 (FABP5) after contusive SCI and to investigate whether this protein plays a role in n-3 PUFA-mediated functional recovery post-SCI. We found that SCI resulted in a robust spinal cord up-regulation in FABP5 mRNA levels (556 ± 187%) and protein expression (518 ± 195%), when compared to sham-operated rats, at 7 days post-injury (dpi). This upregulation coincided with significant alterations in the metabolism of fatty acids in the injured spinal cord, as revealed by metabolomics-based lipid analyses. In particular, we found increased levels of the n-3 series PUFAs, particularly docosahexaenoic acid (DHA; 22:6 n-3) and eicosapentaenoic acid (EPA; 20:5 n-3) at 7 dpi. Animals consuming a diet rich in DHA and EPA exhibited a significant upregulation in FABP5 mRNA levels at 7 dpi. Immunofluorescence showed low basal FABP5 immunoreactivity in spinal cord ventral gray matter NeuN(+) neurons of sham-operated rats. SCI resulted in a robust induction of FABP5 in glial (GFAP(+), APC(+), and NG2(+)) and precursor cells (DCX(+), nestin(+)). We found that continuous intrathecal administration of FABP5 silencing with small interfering RNA (2 µg) impaired spontaneous open-field locomotion post-SCI. Further, FABP5 siRNA administration hindered the beneficial effects of DHA to ameliorate functional recovery at 7 dpi. Altogether, our findings suggest that FABP5 may be an important player in the promotion of cellular uptake, transport, and/or metabolism of DHA post-SCI. Given the beneficial roles of n-3 PUFAs in ameliorating functional recovery, we propose that FABP5 is an important contributor to basic repair mechanisms in the injured spinal cord.

Tamoxifen and Src kinase inhibitors as neuroprotective/neuroregenerative drugs after spinal cord injury.

Spinal cord injury (SCI) is a devastating condition that produces significant changes in the lifestyle of patients. Many molecular and cellular events are triggered after the initial physical impact to the cord. Two major phases have been described in the field of SCI: an acute phase and late phase. Most of the therapeutic strategies are focused on the late phase because this provides an opportunity to target cellular events like apoptosis, demyelination, scar formation and axonal outgrowth. In this mini-review, we will focus on two agents (tamoxifen and a Src kinase family inhibitor known as PP2) that have been shown in our laboratory to produce neuroprotective (increase cell survival) and/or regenerative (axonal outgrowth) actions. The animal model used in our laboratory is adult female rat (~250 g) with a moderate contusion (12.5 mm) to the spinal cord at the T10 level, using the MASCIS impactor device. Tamoxifen or PP2 was administered by implantation of a 15 mg pellet (Innovative Research of America, Sarasota, FL, USA) or by intraperitoneal injections (1.5 mg/kg, every 3 days), respectively, to produce a long-term effect (28 days). Tamoxifen and the Src kinase inhibitor, PP2, are drugs that in rats with a moderate spinal cord injury promote functional locomotor recovery, increase spared white matter tissue, and stimulate axonal outgrowth. Moreover, tamoxifen reduces the formation of reactive oxygen species. Therefore, these drugs are possible therapeutic agents that have a neuroprotective/regenerative activity in vertebrates with SCI.

Comparison of Two Methods of Estradiol Replacement: their Physiological and Behavioral Outcomes.

Fluctuating sex steroids during the estrous or menstrual cycle of mammalian females make it difficult to determine their role on behaviors and physiology. To avoid this, many investigators ovariectomize their animals and administer progesterone, estradiol or a combination of both. Several different strategies are used to administer estradiol, which confounds interpretation of results. This study compared two methods of estradiol replacement implants: Silastic tubes filled with crystalline estradiol benzoate (E2) and commercially available estradiol benzoate pellets. Implants were placed subcutaneously in adult ovariectomized (OVX) rats and blood samples obtained weekly. Control OVX rats received empty Silastic tubes or placebo pellets. Our data shows that E2 plasma levels from rats with Silastic implants peaked after one week and decreased slowly thereafter. In contrast, plasma E2 from commercial pellets peaked after two weeks, increasing and decreasing over time. To validate hormone release, body weight was monitored. All E2 treated animals maintained a similar body weight over the four weeks period whereas an increase in body weight over time was observed in the OVX group that received empty implants, confirming E2 release and supporting the role of E2 in the regulation of body weight. Furthermore, the effects of E2 on basal locomotor activity were assessed using animal activity cages. Results showed no difference between E2 and control group in several locomotor activities. These results indicate that Silastic implants achieve more stable plasma estradiol levels than pellets and thus are a better alternative for studies of estradiol on brain function and behavior.

Tamoxifen and estradiol improved locomotor function and increased spared tissue in rats after spinal cord injury: their antioxidant effect and role of estrogen receptor alpha.

17ß-Estradiol is a multi-active steroid that imparts neuroprotection via diverse mechanisms of action. However, its role as a neuroprotective agent after spinal cord injury (SCI), or the involvement of the estrogen receptor-alpha (ER-α) in locomotor recovery, is still a subject of much debate. In this study, we evaluated the effects of estradiol and of Tamoxifen (an estrogen receptor mixed agonist/antagonist) on locomotor recovery following SCI. To control estradiol cyclical variability, ovariectomized female rats received empty or estradiol filled implants, prior to a moderate contusion to the spinal cord. Estradiol improved locomotor function at 7, 14, 21, and 28 days post injury (DPI), when compared to control groups (measured with the BBB open field test). This effect was ER-α mediated, because functional recovery was blocked with an ER-α antagonist. We also observed that ER-α was up-regulated after SCI. Long-term treatment (28 DPI) with estradiol and Tamoxifen reduced the extent of the lesion cavity, an effect also mediated by ER-α. The antioxidant effects of estradiol were seen acutely at 2 DPI but not at 28 DPI, and this acute effect was not receptor mediated. Rats treated with Tamoxifen recovered some locomotor activity at 21 and 28 DPI, which could be related to the antioxidant protection seen at these time points. These results show that estradiol improves functional outcome, and these protective effects are mediated by the ER-α dependent and independent-mechanisms. Tamoxifen׳s effects during late stages of SCI support the use of this drug as a long-term alternative treatment for this condition.

Long-term treatment with PP2 after spinal cord injury resulted in functional locomotor recovery and increased spared tissue.

The spinal cord has the ability to regenerate but the microenvironment generated after trauma reduces that capacity. An increase in Src family kinase (SFK) activity has been implicated in neuropathological conditions associated with central nervous system trauma. Therefore, we hypothesized that a decrease in SFK activation by a long-term treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2), a selective SFK inhibitor, after spinal cord contusion with the New York University (NYU) impactor device would generate a permissive environment that improves axonal sprouting and/or behavioral activity. Results demonstrated that long-term blockade of SFK activation with PP2 increases locomotor activity at 7, 14, 21 and 28 days post-injury in the Basso, Beattie, and Bresnahan open field test, round and square beam crossing tests. In addition, an increase in white matter spared tissue and serotonin fiber density was observed in animals treated with PP2. However, blockade of SFK activity did not change the astrocytic response or infiltration of cells from the immune system at 28 days post-injury. Moreover, a reduced SFK activity with PP2 diminished Ephexin (a guanine nucleotide exchange factor) phosphorylation in the acute phase (4 days post-injury) after trauma. Together, these findings suggest a potential role of SFK in the regulation of spared tissue and/or axonal outgrowth that may result in functional locomotor recovery during the pathophysiology generated after spinal cord injury. Our study also points out that ephexin1 phosphorylation (activation) by SFK action may be involved in the repulsive microenvironment generated after spinal cord injury.

Expression profile of flotillin-2 and its pathophysiological role after spinal cord injury.

Some receptors that block axonal regeneration or promote cell death after spinal cord injury (SCI) are localized in membrane rafts. Flotillin-2 (Flot-2) is an essential protein associated with the formation of these domains and the clustering of membranal proteins, which may have signaling activities. Our hypothesis is that trauma will change Flot-2 expression and interference of this lipid raft marker will promote functional locomotor recovery after SCI. Analyses were conducted to determine the spatiotemporal profile of Flot-2 expression in adult rats after SCI, using the MASCIS impactor device. Immunoblots showed that SCI produced a significant decrease in the level of Flot-2 at 2 days post-injury (DPI) that increased until 28 DPI. Confocal microscopy revealed Flot-2 expression in neurons, reactive astrocytes and oligodendrocytes specifically associated to myelin structures near or close to the axons of the cord. In the open field test and grid walking assays, to monitor locomotor recovery of injured rats infused intrathecally with Flot-2 antisense oligonucleotides for 28 days showed significant behavioral improvement at 14, 21 and 28 DPI. These findings suggest that Flot-2 has a role in the nonpermissive environment that blocks locomotor recovery after SCI by clustering unfavorable proteins in membrane rafts.

Cocaine sensitization increases I h current channel subunit 2 (HCN2) protein expression in structures of the mesocorticolimbic system.

Alteration of the biological activity among neuronal components of the mesocorticolimbic (MCL) system has been implicated in the pathophysiology of drug abuse. Changes in the electrophysiological properties of neurons involved in the reward circuit seem to be of utmost importance in addiction. The hyperpolarization-activated cyclic nucleotide current, I h, is a prominent mixed cation current present in neurons. The biophysical properties of the I h and its potential modulatory role in cell excitability depend on the expression profile of the hyperpolarization-activated cyclic nucleotide gated channel (HCN) subunits. We investigated whether cocaine-induced behavioral sensitization, an animal model of drug addiction, elicits region-specific changes in the expression of the HCN2 channel's subunit in the MCL system. Tissue samples from the ventral tegmental area, prefrontal cortex, nucleus accumbens, and hippocampus were analyzed using Western blot. Our findings demonstrate that cocaine treatment induced a significant increase in the expression profile of the HCN2 subunit in both its glycosylated and non-glycosylated protein isoforms in all areas tested. The increase in the glycosylated isoform was only observed in the ventral tegmental area. Together, these data suggest that the observed changes in MCL excitability during cocaine addiction might be associated with alterations in the subunit composition of their HCN channels.

Docosahexaenoic acid pretreatment confers protection and functional improvements after acute spinal cord injury in adult rats.

Currently, few interventions have been shown to successfully limit the progression of secondary damage events associated with the acute phase of spinal cord injury (SCI). Docosahexaenoic acid (DHA, C22:6 n-3) is neuroprotective when administered following SCI, but its potential as a pretreatment modality has not been addressed. This study used a novel DHA pretreatment experimental paradigm that targets acute cellular and molecular events during the first week after SCI in rats. We found that DHA pretreatment reduced functional deficits during the acute phase of injury, as shown by significant improvements in Basso-Beattie-Bresnahan (BBB) locomotor scores, and the detection of transcranial magnetic motor evoked potentials (tcMMEPs) compared to vehicle-pretreated animals. We demonstrated that, at 7 days post-injury, DHA pretreatment significantly increased the percentage of white matter sparing, and resulted in axonal preservation, compared to the vehicle injections. We found a significant increase in the survival of NG2+, APC+, and NeuN+ cells in the ventrolateral funiculus (VLF), dorsal corticospinal tract (dCST), and ventral horns, respectively. Interestingly, these DHA protective effects were observed despite the lack of inhibition of inflammatory markers for monocytes/macrophages and astrocytes, ED1/OX42 and GFAP, respectively. DHA pretreatment induced levels of Akt and cyclic AMP responsive element binding protein (CREB) mRNA and protein. This study shows for the first time that DHA pretreatment ameliorates functional deficits, and increases tissue sparing and precursor cell survival. Further, our data suggest that DHA-mediated activation of pro-survival/anti-apoptotic pathways may be independent of its anti-inflammatory effects.

Blockade of P2 nucleotide receptors after spinal cord injury reduced the gliotic response and spared tissue.

Spinal cord injury (SCI) triggers a sequel of events commonly associated with cell death and dysfunction of glias and neurons surrounding the lesion. Although astrogliosis and glial scar formation have been involved in both damage and repair processes after SCI, their role remains controversial. Our goal was to investigate the effects of the P2 receptors antagonists, PPADS and suramin, in the establishment of the reactive gliosis and the formation of the glial scar. Molecular biology, immunohistochemistry, spared tissue, and locomotor behavioral studies were used to evaluate astrogliosis, in adult female Sprague-Dawley rats treated with P2 antagonists after moderate injury with the NYU impactor device. Semi-quantitative RT-PCR confirmed the presence of P2Y(1,) P2Y(2,) P2Y(4,) P2Y(6,) P2Y(12), and P2X(2) receptors in the adult spinal cord. Immunohistochemistry studies confirmed a significant decrease in GFAP-labeled cells at the injury epicenter as well as a decrease in spared tissue after treatment with the antagonists. Functional open field testing revealed no significant locomotor score differences between treated and control animals. Our work is consistent with studies suggesting that astrogliosis is an important event after SCI that limits tissue damage and lesion spreading.

Expression profile and role of EphrinA1 ligand after spinal cord injury.

Spinal cord injury (SCI) triggers the re-expression of inhibitory molecules present in early stages of development, contributing to prevention of axonal regeneration. Upregulation of EphA receptor tyrosine kinases after injury suggest their involvement in the nervous system's response to damage. However, the expression profile of their ephrinA ligands after SCI is unclear. In this study, we determined the expression of ephrinA ligands after contusive SCI. Adult Sprague-Dawley female rats were injured using the MASCIS impactor device at the T10 vertebrae, and levels of ephrinA mRNA and protein determined at different time points. Identification of the cell phenotype expressing the ephrin ligand and colocalization with Eph receptors was performed with immunohistochemistry and confocal microscopy. Behavioral studies were made, after blocking ephrinA1 expression with antisense (AS) oligonucleotides, to assess hindlimb locomotor activity. Real-time PCR demonstrated basal mRNA levels of ephrin (A1, A2, A3, and A5) in the adult spinal cord. Interestingly, ephrinA1 was the only ligand whose mRNA levels were significantly altered after SCI. Although ephrinA1 mRNA levels increased after 2 weeks and remain elevated, we did not observe this pattern at the protein level as revealed by western blot analysis. Immunohistochemical studies showed ephrinA1 expression in reactive astrocytes, axons, and neurons and also their colocalization with EphA4 and A7 receptors. Behavioral studies revealed worsening of locomotor activity when ephrinA1 expression was reduced. This study suggests that ephrinA1 ligands play a role in the pathophysiology of SCI.

Expression and activation of ephexin is altered after spinal cord injury.

Failure of axon regeneration after traumatic spinal cord injury (SCI) is attributable in part to the presence of inhibitory molecular interactions. Recent evidence demonstrates that activation of Eph signaling pathways leads to modulation of growth cone dynamics and repulsion through the activation of ephexin, a novel guanine nucleotide exchange factor (GEF). However, little is known about the expression and modulation of Eph molecular targets in the injured spinal cord. In this study, we determined the expression profile of ephexin after a moderate spinal cord contusion at thoracic level (T10) in young adult rats. Western-blot studies showed increased protein expression in injured rats at 4 and 7 days postinjury (DPI) when compared with control animals. The protein levels returned to normal at 14 DPI and remained steady until 28 DPI. However, immunoprecipitation studies of the phosphorylated ephexin demonstrated that this protein is activated by day 2 until 14 DPI. Expression of ephexin was noticeable in neurons, axons, microglia/macrophages, and reactive astrocytes, and co-localized with EphA3, A4, and A7. These results demonstrate the presence of ephexin in the adult spinal cord and its activation after SCI. Therefore, we show, for the first time, the spatiotemporal pattern of ephexin expression and activation after contusive SCI. Collectively, our data support our previous findings on the putative nonpermissive roles of Eph receptors after SCI and the possible involvement of ephexin in the intracellular cascade of events.

P2Y2 receptor expression is altered in rats after spinal cord injury.

Spinal cord injury increases inhibitory factors that may restrict neurite outgrowth after trauma. The expression of repulsive molecules in reactive astrocytes and the formation of the glial scar at the injury site produce the non-permissive environment for axonal regeneration. However, the mechanism that triggers this astrogliotic response is unknown. The release of nucleotides has been linked to this hypertrophic state. Our goal is to investigate the temporal profile of P2Y(2) nucleotide receptor after spinal cord injury in adult female Sprague-Dawley rats. Molecular biology, immunofluorescence studies, and Western Blots were used to evaluate the temporal profile (2, 4, 7, 14, and 28 days post-injury) of this receptor in rats injured at the T-10 level using the NYU impactor device. Real time RT-PCR showed a significant increase of P2Y(2) mRNA after 2 days post-injury that continues throughout 28 days post-injury. Double labeling studies localized P2Y(2) immunoreactivity in neuronal cell bodies, axons, macrophages, oligodendrocytes and reactive astrocytes. Immunofluorescence studies also demonstrated a low level of P2Y(2) receptor in sham samples, which increased after injury in glial fibrillary acidic protein positive cells. Western Blot performed with contused spinal cord protein samples revealed an upregulation in the P2Y(2) 42 kDa protein band expression after 4 days post-injury that continues until 28 days post-injury. However, a downregulation of the 62 kDa receptor protein band after 2 days post-injury that continues up to 28 days post-injury was observed. Therefore, the spatio-temporal pattern of P2Y(2) gene expression after spinal cord injury suggests a role in the pathophysiology response generated after trauma.

Molecular, anatomical, physiological, and behavioral studies of rats treated with buprenorphine after spinal cord injury.

Acute pain is a common symptom experienced after spinal cord injury (SCI). The presence of this pain calls for treatment with analgesics, such as buprenorphine. However, there are concerns that the drug may exert other effects besides alleviation of pain. Among those reported are in vitro changes in gene expression, apoptosis, and necrosis. In this investigation, the effect of buprenorphine was assessed at the molecular, behavioral, electrophysiological, and histological levels after SCI. Rats were injured at the T10 thoracic level using the NYU impactor device. Half of the animals received buprenorphine (0.05 mg/kg) for 3 consecutive days immediately after SCI, and the other half were untreated. Microarray analysis (n = 5) was performed and analyzed using the Array Assist software. The genes under study were grouped in four categories according to function: regeneration, apoptosis, second messengers, and nociceptive related genes. Microarray analysis demonstrated no significant difference in gene expression between rats treated with buprenorphine and the control group at 2 and 4 days post-injury (DPI). Experiments performed to determine the effect of buprenorphine at the electrophysiological (tcMMEP), behavioral (BBB, grid walking and beam crossing), and histological (luxol staining) levels revealed no significant difference at 7 and 14 DPI in the return of nerve conduction, functional recovery, or white matter sparing between control and experimental groups (p > 0.05, n = 6). These results show that buprenorphine (0.05 mg/kg) can be used as part of the postoperative care to reduce pain after SCI without affecting behavioral, physiological, or anatomical parameters.