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. TAMtreated 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.

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.

SorLA is an established sorting and trafficking protein in neurons with demonstrated relevance to Alzheimer’s disease (AD). It shares these roles with the caveolins, markers of membrane rafts microdomains. To further our knowledge on sorLA’s expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin-1 (cav-1), a caveolar microdomain marker. RT-PCR and immunoblots demonstrated sorLA expression in rat C6 glioma, primary cultures of rat astrocytes (PCRA), and human astrocytoma 1321N1 cells. PCRA were determined to express the highest levels of sorLA’s message. Induction of differentiation of C6 cells into an astrocyte-like phenotype led to a significant decrease in sorLA’s mRNA and protein expression. A set of complementary experimental approaches establish that sorLA and cav-1 directly or indirectly interact in glia: (1) co-fractionation in light-density membrane raft fractions of rat C6 glioma, PCRA, and human 1321N1 astrocytoma cells; (2) a subcellular co-localization distribution pattern in vesicular perinuclear compartments seen via confocal imaging in C6 and PCRA; (3) additional confocal analysis in C6 cells suggesting that the perinuclear compartments correspond to their co-localization in early endosomes and the trans-Golgi; and; (4) co-immunoprecipitation data strongly supporting their direct or indirect physical interaction. These findings further establish that sorLA is expressed in glia and that it shares its subcellular distribution pattern with cav-1. A direct or indirect cav-1/sorLA interaction could modify the trafficking and sorting functions of sorLA in glia and its proposed neuroprotective role in AD.

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.

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 spatio-temporal 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 non-permissive environment that blocks locomotor recovery after SCI by clustering unfavorable proteins in membrane rafts.