Administration of Tamoxifen Can Regulate Changes in Gene Expression during the Acute Phase of Traumatic Spinal Cord Injury.

Traumatic spinal cord injury (SCI) causes irreversible damage leading to incapacity. Molecular mechanisms underlying SCI damage are not fully understood, preventing the development of novel therapies. Tamoxifen (TMX) has emerged as a promising therapy. Our aim was to identify transcriptome changes in the acute phase of SCI and the effect of Tamoxifen on those changes in a rat model of SCI. Four groups were considered: (1) Non-injured without TMX (Sham/TMX-), (2) Non-injured with TMX (Sham/TMX+), (3) injured without TMX (SCI/TMX-), and (4) injured with TMX (SCI/TMX+). Tamoxifen was administered intraperitoneally 30 min after injury, and spinal cord tissues were collected 24 h after injury. Clariom S Assays Array was used for transcriptome analysis. After comparing Sham/TMX- versus SCI/TMX-, 708 genes showed differential expression. The enriched pathways were the SCI pathway and pathways related to the inflammatory response. When comparing SCI/TMX- versus SCI/TMX+, only 30 genes showed differential expression, with no pathways enriched. Our results showed differential expression of genes related to the inflammatory response after SCI, and Tamoxifen seems to regulate gene expression changes in Ccr2 and Mmp12. Our study contributes data regarding the potential value of tamoxifen as a therapeutic resource for traumatic SCI during the acute phase.

Evaluation of Traumatic Spinal Cord Injury in a Rat Model Using 99mTc-GA-5 as a Potential In Vivo Tracer.

Spinal cord injury (SCI) refers to the damage suffered in the spinal cord by any trauma or pathology. The purpose of this work was to determine whether 99mTc-GA-5, a radiotracer targeting Glial Fibrillary Acidic Protein (GFAP), can reveal in vivo the reactivation of astrocytes in a murine model with SCI. A method for the 99mTc radiolabeling of the mouse anti-GFAP monoclonal antibody GA-5 was implemented. Radiochemical characterization was performed, and radioimmunohistochemistry assays were used to evaluate the integrity of 99mTc-GA-5. MicroSPECT/CT was used for in vivo imaging to trace SCI in the rats. No alterations in the GA-5's recognition/specificity ability were observed after the radiolabeling. The GA-5's radiolabeling procedure implemented in this work offers a practical method to allow the in vivo following of this monoclonal antibody to evaluate its biodistribution and specificity for GFAP receptors using SPECT/CT molecular imaging.

High-resolution Micro-CT Myelography to Assess Spinal Subarachnoid Space Changes After Spinal Cord Injury in Rats.

BACKGROUND AND PURPOSE: The spinal subarachnoid space (SSAS) is vital for neurologic function. Although SSAS alterations are known to occur after spinal cord injury (SCI), there is a lack of high-resolution imaging studies of the SSAS after SCI in rodents. Therefore, the aim here was to assess changes in the SSAS of rats subjected to graded SCI, using high-resolution micro-CT myelography. METHODS: Long-Evans adult rats were subjected to mild or severe spinal cord contusion at T9. Imaging studies of SSAS features were carried out in injured rats at acute (day 1) and subacute (day 15) stages postinjury, as well as in control rats, using high-resolution micro-CT myelography with a contrast-enhanced digital subtraction protocol. We studied a total of 33 rats randomly allocated into five experimental groups. Micro-CT myelograms were assessed by expert observers using both qualitative and quantitative criteria. RESULTS: Qualitative and quantitative analyses showed that SCI induces changes in the SSAS that vary as a function of both injury severity and time elapsed after injury. SSAS blockage was the main alteration detected. Moreover, the method used here allowed fine details to be observed in small animals, such as variations in the preferential pathways for contrast medium flow, neuroimaging nerve root enhancement, and leakage of contrast medium due to tearing of the dural sac. CONCLUSION: Micro-CT myelography provides high-resolution images of changes in the SSAS after SCI in rats and is a useful tool for further experimental studies involving rat SCI in vivo.

Pharmacokinetics and anti-inflammatory effect of naproxen in rats with acute and subacute spinal cord injury.

Previous reports have warned about the influence of spinal cord injury (SCI) on the pharmacokinetics of various drugs. However, the role of SCI in the efficacy and safety of pharmacotherapy remains unknown. Thereby, our aim was to explore the role of SCI on pharmacokinetics and anti-inflammatory effect of naproxen in response to a local inflammatory challenge. Rats received a severe contusive SCI at T9 or sham injury. Pharmacokinetics of a single intravenous dose of naproxen (10 mg kg-1) was studied at days 1 and 15 post-surgery. For the anti-inflammatory assessment, carrageenan was subcutaneously injected in forelimb and hindlimb paws at the same post-surgery periods, and naproxen efficacy was evaluated measuring paw swelling. Plasma protein concentrations and body weight changes were also determined. Plasma naproxen levels and pharmacokinetic parameters were unchanged by acute injury, but subacute injury generated alterations in volume of distribution, clearance, and bioavailability, resulting in significantly reduced plasma naproxen concentrations, in the absence of changes in plasma proteins. Assessment of naproxen anti-inflammatory activity during the acute stage of injury could not be determined because of carrageenan failure to elicit swelling. During the subacute stage, naproxen anti-inflammatory effect on forelimbs (above injury) was similar to that observed in sham-injured animals, while it was almost absent in paralyzed hindlimbs. Under conditions of SCI and peripheral inflammation, pharmacokinetics and anti-inflammatory activity of naproxen vary according to post-injury timing and neurological status of the assessed region.

Micro-CT myelography using contrast-enhanced digital subtraction: feasibility and initial results in healthy rats.

PURPOSE: The spinal subarachnoid space (SSAS) is vital for neural performance. Although models of spinal diseases and trauma are used frequently, no methods exist to obtain high-resolution myelograms in rodents. Thereby, our aim was to explore the feasibility of obtaining high-resolution micro-CT myelograms of rats by contrast-enhanced dual-energy (DE) and single-energy (SE) digital subtraction. METHODS: Micro-CT contrast-enhanced DE and SE imaging protocols were implemented with live adult rats (total of 18 animals). For each protocol, contrast agents based on iodine (Iomeron® 400 and Fenestra® VC) and gold nanoparticles (AuroVist™ 15 nm) were tested. For DE, images at low- and high-energy settings were acquired after contrast injection; for SE, one image was acquired before and the other after contrast injection. Post-processing consisted of region of interest selection, image registration, weighted subtraction, and longitudinal alignment. RESULTS: High-resolution myelograms were obtained with contrast-enhanced digital subtraction protocols. After qualitative and quantitative (contrast-to-noise ratio) analyses, we found that the SE acquisition protocol with Iomeron® 400 provides the best images. 3D contour renderings allowed visualization of SSAS and identification of some anatomical structures within it. CONCLUSION: This in vivo study shows the potential of SE contrast-enhanced myelography for imaging SSAS in rat. This approach yields high-resolution 3D images without interference from adjacent anatomical structures, providing an innovative tool for further assessment of studies involving rat SSAS.

Methylprednisolone Administration Following Spinal Cord Injury Reduces Aquaporin 4 Expression and Exacerbates Edema.

Spinal cord injury (SCI) is an incapacitating condition that affects motor, sensory, and autonomic functions. Since 1990, the only treatment administered in the acute phase of SCI has been methylprednisolone (MP), a synthetic corticosteroid that has anti-inflammatory effects; however, its efficacy remains controversial. Although MP has been thought to help in the resolution of edema, there are no scientific grounds to support this assertion. Aquaporin 4 (AQP4), the most abundant component of water channels in the CNS, participates in the formation and elimination of edema, but it is not clear whether the modulation of AQP4 expression by MP plays any role in the physiopathology of SCI. We studied the functional expression of AQP4 modulated by MP following SCI in an experimental model in rats along with the associated changes in the permeability of the blood-spinal cord barrier. We analyzed these effects in male and female rats and found that SCI increased AQP4 expression in the spinal cord white matter and that MP diminished such increase to baseline levels. Moreover, MP increased the extravasation of plasma components after SCI and enhanced tissue swelling and edema. Our results lend scientific support to the increasing motion to avoid MP treatment after SCI.

Creation of an intramedullary cavity by hemorrhagic necrosis removal 24 h after spinal cord contusion in rats for eventual intralesional implantation of restorative materials.

Intramedullary hemorrhagic necrosis occurs early after spinal cord injury at the site of injury and adjacent segments. It is considered harmful because of its potential to aggravate secondary injury, and to interfere with axonal regeneration; it might also lead to an unfavorable environment for intralesional implants. Removal of hemorrhagic necrosis has been attempted before with variable results. The invasive nature of these procedures carries the risk of exacerbating damage to the injured cord. The overall objective for this study was to test several strategies for non-damaging removal of hemorrhagic necrosis and characterize the resulting cavity looking for a space for future intralesional therapeutic implants in rats with acute cord injury. Rats were subjected to graded cord contusion, and hemorrhagic necrosis was removed after 24h. Three grades of myelotomy (extensive, medium sized, and small) were tested. Using the small surgical approach to debridement, early and late effects of the intervention were determined by histology and by analytical and behavioral analysis. Appearance and capacity of the resulting cavity were characterized. Satisfactory removal of hemorrhagic necrosis was achieved with all three surgical approaches to debridement. However, bleeding in spared cord tissue was excessive after medium sized and extensive myelotomies but similar to control injured rats after small cord surgery. Small surgical approach to debridement produced no swelling nor acute inflammation changes, nor did it affect long-term spontaneous locomotor recovery, but resulted in modest improvement of myelination in rats subjected to both moderate and severe injuries. Cavity created after intervention was filled with 10 to 15 µL of hydrogel. In conclusion, by small surgical approach to debridement, removal of hemorrhagic necrosis was achieved after acute cord contusion thereby creating intramedullary spaces without further damaging the injured spinal cord. Resulting cavities appear suitable for future intralesional placement of pro-reparative cells or other regenerative biomaterials in a clinically relevant model of spinal cord injury.

Temporal changes of spinal subarachnoid space patency after graded spinal cord injury in rats.

INTRODUCTION: Disturbances in spinal subarachnoid space (SSAS) patency after SCI have been reported as an incidental finding, but there is a lack of information on its in vivo extent and time course. For substances and cells carried in the cerebrospinal fluid (CSF) to reach damaged neural tissue and promote reparative processes, CSF must be able to flow freely in SASS. OBJECTIVE: To characterise the extent and time course of SSAS patency disruption in vivo in a rat model after graded SCI. MATERIALS AND METHODS: Anaesthetised rats were subjected to mild or severe cord contusion at T9. Estimation of SSAS patency was carried out at 1h and 1, 3, 7, 15, 30 and 90 days postinjury, as well as in naïve rats, by quantifying the passage of superparamagnetic beads injected into the CSF at the cisterna magna and recovered at spinal level L2. CSF volume recovery was measured simultaneously. Data were analysed by the two-way ANOVA test. RESULTS: Estimation of SSAS patency revealed nearly complete blockage early after contusion that was unevenly restored entering the chronic stages. Volume of CSF recovered was also significantly decreased early after injury compared to naïve rats, but was fully restored by 1 month postinjury. Overall, although modestly different from each other, changes in both parameters were more pronounced after severe rather than mild injuries for each time point examined. CONCLUSIONS: SCI alters SSAS patency. Its extent is a function primarily of time elapsed after lesion and secondly of injury severity. It is reasonable to expect that disturbances in SASS patency might alter CSF dynamics and impair self-reparative mechanisms and intrathecal therapeutics, making SSAS patency blockage a key target for SCI management.

Superparamagnetic beads for estimation of spinal subarachnoid space permeability in rats.

BACKGROUND: Human spinal pathological processes have been linked to a loss of spinal subarachnoid space (SSAS) permeability, which has therefore become a target for therapy. Hence, it has become important to measure SSAS patency in rat models of these human disorders. NEW METHOD: The estimation of in vivo rat SSAS patency is described by quantifying passage of streptavidin-covered superparamagnetic beads (SPMB) in cerebrospinal fluid (CSF). Beads are injected into the cisterna magna and recovered at spinal level L2. They are then coated with biotynilated horseradish peroxidase for enzymatically based colorimetric measurement, after removal of bloody CSF to avoid interference with the colorimetric readings. The procedure was tested in intact rats and in rats 24 h after T9 laminectomy. Residual beads in SSAS were viewed by histology. RESULTS: Average bead recovery from intact rats was 6.4% of amount initially administered, in a mean CSF volume of 126 µL; in laminectomized rats, it was 1%, in a mean CSF volume of 39.2 µL. COMPARISON WITH EXISTING METHOD(S): Unlike in vivo imaging techniques, such as myelography (used here to validate our method) and near infrared fluorescence technology for qualitative rat SSAS patency viewing, our SPMB-based method allows for an in vivo quantitative estimation of the permeability of this space. CONCLUSIONS: A novel method has been established to reliably determine SSAS permeability in rats. The method is reproducible and has the required sensitivity to detect an 84.4% reduction in bead recovery, as seen in laminectomized rats compared to intact animals.

Spatial and temporal morphological changes in the subarachnoid space after graded spinal cord contusion in the rat.

Spontaneous repair or treatment-induced recovery after spinal cord injury (SCI) is very limited and might be related to extramedullary alterations that have only briefly been documented. Here we report on the morphological changes of the spinal subarachnoid space (SAS) in a clinically relevant model of SCI. Anesthetized rats were subjected either to mild or severe spinal cord contusion at T9. Spine blocks from the site of injury and adjacent segments were harvested at acute (1 h and 1 day [d]), subacute (3 and 7 d), and chronic (1 and 3 months) stages post-injury. Histopathology and morphometry at each decalcified vertebral level were assessed. At acute and subacute stages, reduction of SAS lumen was observed after both mild and severe injuries. Acutely, after severe injuries, SAS occlusion was associated mainly with cord swelling and subarachnoid hematomas; a trend for dural sac constriction was observed for mild injuries. At 7 d, cord swelling diminished in both instances, but dural sac constriction increased for severe injuries. At early stages, in the epicenter and vicinity, histopathology revealed compression of neurovascular elements within the SAS, which was more intense in severe than in mild injuries. In the chronic stage, SAS lumen increased notably, mostly from cord atrophy, despite dural sac constriction. Myelograms complemented observations made on SAS lumen permeability. Post-traumatic arachnoiditis occurred mainly in animals with severe injury. In conclusion, early extramedullary SAS changes described here might be expected to produce alterations in cerebrospinal fluid (CSF) dynamics and cord blood perfusion, thereby contributing to the pathophysiology of SCI and becoming novel targets for treatment.

New approach for graded compression spinal cord injuries in Rhesus macaque: method feasibility and preliminary observations.

BACKGROUND: Current models of spinal cord injury (SCI) have been ineffective for translational research. Primate blunt SCI, which more closely resembles human injury, could be a promising model to fill this gap. METHODS: Graded compression SCI was produced by inflating at T9 an epidural balloon as a function of spinal canal dimensions in a non-uniform group of monkeys. RESULTS: Sham injury and cord compression by canal invasion of 50-75% produced minimal morpho-functional alterations, if at all. Canal invasion of 90-100% resulted in proportional functional deficits. Unexpectedly, these animals showed spontaneous gradual recovery over a 12-week period achieving quadruped walking, although with persistent absence of foot grasping reflex. Histopathology revealed predominance of central cord damage that correlated with functional status. CONCLUSIONS: Our preliminary results suggest that this model could potentially be a useful addition to translational work, but requires further validation by including animals with permanent injuries and expansion of replicates.

Early administration of L-arginine in experimental acute spinal cord injury impairs long-term motor function recovery.

BACKGROUND: Recently, we reported that L-arginine, a nitric oxide precursor, reverses altered drug disposition induced by acute spinal cord injury (SCI) by increasing hepatic blood flow, without affecting mean arterial pressure and heart rate, whereas others have shown that it produces neuroprotection in several models of acute neurologic damage. Its use as a therapeutic agent for microcirculatory alterations associated with spinal shock seems promising. Therefore, here we have tested its influence on long-term morphofunctional neurologic outcome. METHODS: Intravenous L-arginine (300 mg/kg per dose) was administered to adult rats after SCI of moderate intensity according to the following schemes (n=6): (1) single dose at 1 hour, (2) single dose at 24 hour, and (3) repeated doses first at 24 hour and then daily for 7 days. Control injured rats received the vehicle (saline solution). RESULTS: Contrary to our expectations, locomotor function, assessed using the Basso-Beattie-Bresnahan scale for 8 weeks, was significantly worse in the L-arginine treated groups compared with the control group. Areas of both spared white matter and myelin stain at the epicenter seemed reduced in rats that received L-arginine as a single dose at 1 hour after injury but were not significantly different from the control group. CONCLUSIONS: L-arginine as used here interfered with the functional outcome of rats subjected to SCI, suggesting that L-arginine or its metabolic products may be neurotoxic. Because of its potential utility for acute SCI suggested in the past, strategies should be designed to block its apparent neurotoxicity.

Thalidomide fails to be therapeutic following contusive spinal cord injury in rats.

Mechanical damage to the spinal cord (SC) generates self-destructive processes that contribute to post-traumatic neurodegeneration. Because thalidomide apparently counteracts these effects its use clinically has been proposed enthusiastically. Nonetheless, we tested its action as a neuroprotectant in a clinically relevant model of SC injury in rats. We administered thalidomide intraperitoneally to rats subjected to thoracic SC contusion as single or repeated doses within the first 24 h after injury. Edema, neutrophil infiltration, and cord tissue preservation/destruction were assessed in the SC 24 h after injury and motor function for 7 weeks. Rats treated with thalidomide showed significant increase in SC water compared with naive rats, but not vehicle-treated rats; their neutrophil infiltration and amount of spared/destroyed cord tissue was not different from vehicle-treated rats; and in no case was motor performance improved after thalidomide. In conclusion, thalidomide failed here to be therapeutic, discouraging its use clinically for SC trauma.

L-arginine reverses alterations in drug disposition induced by spinal cord injury by increasing hepatic blood flow.

High hepatic extraction drugs--such as phenacetin, methylprednisolone, and cyclosporine--exhibit an increased bioavailability after acute spinal cord injury (SCI) due to an impaired clearance. For these drugs, metabolic clearance depends on hepatic blood flow. Thus, it is possible that pharmacokinetic alterations can be reversed by increasing liver perfusion. Therefore, we evaluated the effect of L-arginine, a nitric oxide precursor, on the pharmacokinetics of a prototype drug with high hepatic extraction, and on hepatic microvascular blood flow (MVBF) after acute SCI. Pharmacokinetics of i.v. phenacetin was studied in rats 24 h after a severe T-5 spinal cord contusion; animals being pretreated with L-arginine 100 mg/kg i.v. or vehicle. MVBF was assessed under similar experimental conditions using laser Doppler flowmetry. SCI significantly altered phenacetin pharmacokinetics. Clearance was significantly reduced, resulting in a prolonged half-life and an increase in bioavailability, while volume of distribution was decreased. Pharmacokinetic alterations were reversed when injured rats were pretreated with L -arginine. It was also observed that L-arginine significantly increased hepatic MVBF in injured rats, notwithstanding it exhibited a limited effect on sham-injured animals. Our data hence suggest that L-arginine is able to reverse SCI-induced alterations in phenacetin pharmacokinetics due to an impaired hepatic MVBF, likely by increased nitric oxide synthesis leading to vasodilation. Further studies are warranted to examine the potential usefulness of nitric oxide supplementation in a clinical setting.

Nerve root degeneration and regeneration by intrathecal phenol in rats: a morphologic approach.

Intrathecal injection of phenol (ITP) has been used to control intractable pain and spasticity. Direct caustic nerve damage has been postulated as the mechanism of analgesia. Sensation is commonly recovered, suggesting that a spontaneous regeneration process takes place. There is, however, a lack of mechanistic information on ITP therapy. To define morphologically the neurolysis and regeneration phenomena produced by ITP, anesthetized rats were subjected to laminectomy at L5; 5 microl of 22% phenol in saline solution or vehicle (control) was injected. Light and electron microscopy studies of nerve roots were performed at 2, 14, and 60 days after injection. Rats given ITP showed at the early stage a variable amount of roots with signs of infarction characterized by loss of axon-myelin units and thrombosis of intra-root vessels. At 14 days, abundance of macrophages removing debris, open vessels, and nerve sprouts was identified in damaged roots. At this time, non-myelinating glial fibrillary acidic protein-positive Schwann cells were observed in both damaged and apparently undamaged roots. At 60 days, abundance of 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive Schwann cells myelinating newly formed axons was observed in damaged roots. Control rats did not show signs of neural or vascular pathology. Attempting to prevent thrombosis, another group of rats received heparin before ITP; these anti-coagulated rats developed radicular thrombosis, neurolysis, and hemorrhage. In conclusion, neurolysis produced by ITP is associated with acute ischemia (not prevented by heparin) and is followed by vascular, nerve, and myelin regeneration. Our results help understand the lack of efficacy of and some complications by ITP clinical therapy.

Systemic microcirculation after complete high and low thoracic spinal cord section in rats.

Spinal cord injury (SCI) produces multiple systemic and metabolic alterations. Although some systemic alterations could be associated with ischemic organ damage, little is known about microvascular blood flow (MVBF) in organs other than the spinal cord after acute SCI. We used laser Doppler flowmetry in anesthetized rats to assess MVBF in several tissues before and after complete T-2 and T-9 SCI at 1 h and on days 1, 3, and 7 post-SCI. Mean arterial blood pressure (MAP), heart rate and hematologic variables also were recorded. MAP changes after T-2 injury were not significant, while MAP decreased significantly 1 h after T-9 injury. Statistically significant bradycardia occurred after T-2 injury at 7 days; statistically significant tachycardia occurred after T-9 injury at 1, 3, and 7 days. Hematocrit significantly increased at day 1 and decreased at days 3 and 7 after T-2 injury. SCI was associated with significant decreases in MVBF in liver, spleen, muscle and fore footpad skin. Changes in MVBF in hind footpad skin and kidney were not significant. Changes were more pronounced at 1 h and 1 day post-SCI. Significant differences between MVBF after T-2 and T-9 SCI occurred only in liver. MVBF significantly correlated with regional peripheral vascular resistances (assessed using the MAP/MVBF ratio), but not with MAP. In conclusion, organ-specific changes in systemic MVBF that are influenced by the level of SCI, could contribute to organ dysfunction.