Experimental Traumatic Spinal Cord Injury Research Articles

Minocycline-Loaded Poly(α-Lipoic Acid)-Methylprednisolone Prodrug Nanoparticles for the Combined Anti-Inflammatory Treatment of Spinal Cord Injury.

PurposeTraumatic spinal cord injury (TSCI) induces a powerful inflammatory response that can significantly exacerbate the extent and severity of neural damage (termed as “secondary injury”). Thus, the suppression of inflammation is crucial for reducing neurological dysfunction following TSCI. However, the conventional anti-inflammatory drugs show limited efficacy because of poor penetration and release kinetics at the injury site. This study describes the design, synthesis, release kinetics, biosafety, and preclinical efficacy of minocycline (MC)-loaded poly(α-lipoic acid)–methylprednisolone (PαLA-MP) prodrug nanoparticles (NPs) for the combined anti-inflammatory treatment of TSCI.MethodsNPs were produced by conjugating MP to PαLA and then loading MC. The NP structure was confirmed through 1H nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy, ultraviolet–visible absorption spectroscopy, gel permeation chromatography, dynamic light scattering, and transmission electron microscopy. Drug-loading content and efficacy were measured using high-performance liquid chromatography (HPLC) or 1H NMR and release kinetics through HPLC. Biosafety was examined using the MTT assay, cell penetration efficiency using confocal microscopy, and flow cytometry using Cyanine5 (Cy5)-labeled MC-PαLA-MP NPs, effects on injury-induced pro-inflammatory cytokine release using enzyme-linked immunosorbent assays and immunofluorescence, and treatment efficacy by measuring motor recovery in a rat model of TSCI.ResultsThe MC-PαLA-MP NPs exhibited high biocompatibility and released 81% MC and 54% MP within 24 h under TSCI-like conditions, effectively reducing 40% of pro-inflammatory cytokine release both in cultures and injured rat spinal cord tissues. Systemic injection increased the Basso, Beattie, Bresnahan score of TSCI rats from 2.33 ± 0.52 to 8.83 ± 1.83 in 8 weeks, providing effective neuroprotection and enhanced exercise recovery in the TSCI rats.ConclusionThe MC-PαLA-MP NPs can mitigate secondary inflammation and preserve motor function following experimental TSCI, which suggests their potential for clinical application.

Azathioprine as a Neuroprotective Agent in Experimental Traumatic Spinal Cord Injury.

To evaluate the protective effects of azathioprine, a macrophage-inhibiting agent, on secondary injury in spinal cord trauma. A total of 40 Wistar rats were randomly divided into 4 groups. All the animals had undergone T8-10 laminectomy. Except in group I (control), all the animals were exposed to spinal cord trauma at the T9 level. Animals in group II (trauma) received no treatment following trauma. Animals in group 3 (treatment) and group IV (vehicle) were given intraperitoneal azathioprine 4 mg/kg and saline 2 ml, respectively, 30 minutes after the trauma. Half of the animals in each group were sacrificed 24 hours after injury and specimens were used for biochemical and immunohistochemical evaluations. The rest of the animals were followed-up for 4 weeks in terms of neurological functions and were also sacrificed to perform the histopathological analysis. Significant decrease in apoptotic cells and improved neurological function were observed in the animals treated with azathioprine. Biological and immunohistochemical analysis also showed less oxidative stress in this group compared to those without treatment. Azathioprine, a potent macrophage-inhibiting agent, has been shown to decrease the extent of secondary injury following spinal cord trauma.

Neuroprotective Effects of Lacosamide in Experimental Traumatic Spinal Cord Injury in Rats.

To evaluate the effects of lacosamide on traumatic spinal cord injury (SCI) in rats. A total of 28 male Wistar albino rats, each weighing 300-350 g, were included. They were randomly assigned to four groups. In Group 1, only a laminectomy was performed; in Group 2, SCI was performed after laminectomy; in Group 3, SCI was performed after laminectomy followed by lacosamide administration, and in Group 4, SCI was performed after laminectomy followed by physiological saline administration. After 48 hours, all animals were sacrificed, blood samples were drawn, and their spinal cords were removed. The serum levels of catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured, and the spinal cord specimens were examined for neuronal degeneration (PND). The MDA level was the lowest and the antioxidant enzyme levels were the highest in Group 3. There were statistically significant differences between Group 3 and the others in their PND score, serum MDA, SOD, GPX and catalase levels (p < 0.05). Lacosamide has a neuroprotective effect in SCI in rats that is related to its ability to decrease the production of reactive oxygen species by increasing antioxidant enzyme expression, inhibit lipid peroxidation and attenuate glial cell activation.

PPAR-γ agonist rosiglitazone reduces autophagy and promotes functional recovery in experimental traumaticspinal cord injury

BackgroundSecondary damage is often more important in determining the functional outcome and provides a practical target for therapeutic intervention. Rosiglitazone (ROSG) is a potent PPAR-γ agonist and has been shown to induce neuroprotection in animal models of spinal cord injury (SCI). However, it is still unclear whether this PPAR-γ agonist can mediate neuronal autophagy after SCI. MethodsSCI was induced by application of vascular clips (force of 24g) to the dura via a four-level T5–T8 laminectomy. The role of the PPAR-γ agonist ROSG on neuronal autophagy induced by SCI was investigated. ResultsThe expression of autophagy-related proteins, including microtubule-associated protein 1 light chain 3 type II (LC3-II), beclin-1, and cathepsin D, increased significantly after SCI. ROSG downregulated autophagy-related protein expression and improved the locomotor function after SCI. GW9662 (a PPAR-γ inhibitor) significantly antagonized the effect of ROSG and abolished the protective effect on SCI. ConclusionsOur results clearly demonstrated that the administration of ROSG after SCI reduced autophagy and promoted functional recovery after SCI in rats.

Aescin reduces oxidative stress and provides neuroprotection in experimental traumatic spinal cord injury

Aescin has many physiological functions that are highly relevant to spinal cord injury (SCI), including anti-inflammation, anti-oxidation, anti-oedema, and enhancing vascular tone. The present study investigated the putative therapeutic value of aescin in SCI, with a focus on its neuroprotective, anti-inflammatory, and anti-oxidative properties. Sodium aescinate (1.0mg/kg body weight) or equivalent volume of saline was administered 30min after injury by intravenous injection, with an additional dose daily for seven consecutive days after moderate SCI in rats. After contusion injury of the 8th thoracic (T8) spinal cord, aescin-treated rats developed less severe hind limb weakness than saline controls, as assayed by the Basso-Beattie-Bresnahan scale, the beam walking test, and a footprint analysis. The improved locomotor outcomes in aescin-treated rats corresponded to markedly decreased immune response, oxidative stress, neuronal loss, axon demyelination, spinal cord swelling, and cell apoptosis, measured around T8 after impact. Our data suggest aescin treatment as a novel, early, neuroprotective approach in SCI. Given the known safety of aescin in clinical applications, the results of this study suggest that it is a good candidate for SCI treatment in humans.

Hyperbaric oxygen treatment in the experimental spinal cord injury model

Background contextSpinal cord trauma is a major cause of mortality and morbidity. Although no known treatment for spinal cord injury exists, a limited number of effective treatment modalities and procedures are available that improve secondary injury. Hyperbaric oxygen (HBO) treatment has been used to assist in neurologic recovery after cranial injury or ischemic stroke. PurposeTo report the findings on the effectiveness of HBO treatment on rats with experimental traumatic spinal cord injury. Improvement was evaluated through motor strength assessment and nitrite level assay testing. Study designWe randomly distributed 40 rats among 5 groups of 8 rats each: sham incurable trauma, induced trauma, HBO treatment begun at the 1st hour, HBO treatment begun at the 6th hour, and HBO treatment begun at the 24th hour. MethodThe HBO treatment was administered to rats in three of the groups and conducted in two 90-minute sessions, under an absolute atmospheric pressure of 2.4 at 100% oxygen for 5 days. In the motor strength evaluations, all the rats were observed during the inclined plane test and clinical motor examination on the first, third, and fifth days. In addition, the nitrite levels of spinal cord tissues on the sixth day were also studied. ResultsResults from the inclined plane levels and motor strength test from all the three groups undergoing HBO treatment were higher than those from Group 2. It was also determined that early HBO treatment resulted in higher recovery rates (groups 3 and 4). The highest levels were seen in the group in which the HBO treatments were started in the first hour (Group 3). It was noted that nitrite levels of rats in the group exposed to trauma increased, compared with the sham group, but increased levels also diminished after HBO treatments. Again, the greatest decrease in nitrite levels was evident in the group where the HBO treatment was started the earliest (Group 3). ConclusionsPrompt HBO treatment after trauma significantly contributed to the clinical, histopathologic, and biochemical recovery of the rats.

Minocycline Treatment Inhibits Lipid Peroxidation, Preserves Spinal Cord Ultrastructure, and Improves Functional Outcome After Traumatic Spinal Cord Injury in the Rat

A prospective, randomized experimental research. To evaluate the short- and long-term neuroprotective effects of minocycline on the secondary injury process of an experimental traumatic spinal cord injury (SCI) model. Traumatic SCI is a devastating problem of health that results in high morbidity and mortality rates. The loss of function after SCI results from both the primary mechanical insult and the subsequent, multifaceted secondary response. A total of 80 adult male Spraque-Dawley rats (breeded by the Baskent University Animal Research Center) were randomly divided into 4 groups. A T10 contusion injury was produced by using modified Allen technique in all groups except the control group. No medication was administered to the rats in the trauma group. Minocycline was administered intraperitoneally and intravenously to the treatment groups. Short-term and/or long-term neuroprotective effects of minocycline on the lipid peroxidation (malondialdehyde, glutathione), apoptosis (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate-biotin nick end labeling), ultrastructure of spinal cord (tissue electron microscopy), and behavioral assessments (Basso-Beattie-Bresnahan) were evaluated. As compared with the trauma group, tissue malondialdehyde and glutathione levels demonstrated that minocycline significantly diminishes lipid peroxidation. Electromicroscopic study showed that minocycline preserves the ultrastructure of spinal cord tissue in the early post-traumatic period. Minocycline treatment significantly reduced the number of terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate-biotin nick end labeling positive cells both 1 day and 28 days after SCI. Behavioral assessments showed significant improvement in the hind limb functions of minocycline receiving rats starting 7 days after the SCI. Any statistically significant difference was not found between intraperitoneal or intravenous routes for minocycline injection. Minocycline is neuroprotective and contributes to functional improvement after traumatic SCI by eliminating the destructive process of secondary injury. Having both satisfying anti-inflammatory and antiapoptotic effects in experimental models, it promises to be of therapeutic use in human SCI.

Kallikrein 6 signals through PAR1 and PAR2 to promote neuron injury and exacerbate glutamate neurotoxicity

CNS trauma generates a proteolytic imbalance contributing to secondary injury, including axonopathy and neuron degeneration. Kallikrein 6 (Klk6) is a serine protease implicated in neurodegeneration, and here we investigate the role of protease-activated receptors 1 (PAR1) and PAR2 in mediating these effects. First, we demonstrate Klk6 and the prototypical activator of PAR1, thrombin, as well as PAR1 and PAR2, are each elevated in murine experimental traumatic spinal cord injury (SCI) at acute or subacute time points. Recombinant Klk6 triggered extracellular signal-regulated kinase (ERK1/2) signaling in cerebellar granule neurons and in the NSC34 spinal cord motoneuron cell line, in a phosphoinositide 3-kinae and MEK-dependent fashion. Importantly, lipopeptide inhibitors of PAR1 or PAR2, and PAR1 genetic deletion, each reduced Klk6-ERK1/2 activation. In addition, Klk6 and thrombin promoted degeneration of cerebellar neurons and exacerbated glutamate neurotoxicity. Moreover, genetic deletion of PAR1 blocked thrombin-mediated cerebellar neurotoxicity and reduced the neurotoxic effects of Klk6. Klk6 also increased glutamate-mediated Bim signaling, poly-ADP-ribose polymerase cleavage and lactate dehydrogenase release in NSC34 motoneurons and these effects were blocked by PAR1 and PAR2 lipopeptide inhibitors. Taken together, these data point to a novel Klk6-signaling axis in CNS neurons that is mediated by PAR1 and PAR2 and is positioned to contribute to neurodegeneration.

Neuroprotective Effect of Anthocyanin on Experimental Traumatic Spinal Cord Injury

We investigated the neuroprotective effect of anthocyanin, oxygen radical scavenger extracted from raspberries, after traumatic spinal cord injury (SCI) in rats. THE ANIMALS WERE DIVIDED INTO TWO GROUPS : the vehicle-treated group (control group, n=20) received an oral administration of normal saline via stomach intubation immediately after SCI, and the anthocyanin-treated group (AT group, n=20) received 400 mg/kg of cyanidin 3-O-β-glucoside (C3G) in the same way. We compared the neurological functions, superoxide expressions and lesion volumes in two groups. At 14 days after SCI, the AT group showed significant improvement of the BBB score by 16.7±3.4%, platform hang by 40.0±9.1% and hind foot bar grab by 30.8±8.4% (p<0.05 in all outcomes). The degree of superoxide expression, represented by the ratio of red fluorescence intensity, was significantly lower in the AT group (0.98±0.38) than the control group (1.34±0.24) (p<0.05). The lesion volume in lesion periphery was 32.1±2.4 µL in the control and 24.5±2.3 µL in the AT group, respectively (p<0.05), and the motor neuron cell number of the anterior horn in lesion periphery was 8.3±5.1 cells/HPF in the control and 13.4±6.3 cells/HPF in the AT group, respectively (p<0.05). Anthocyanin seemed to reduce lesion volume and neuronal loss by its antioxidant effect and these resulted in improved functional recovery.

The Effect of Umbilical Cord Blood Cells on Outcomes After Experimental Traumatic Spinal Cord Injury

A cytokine expression profile of umbilical cord blood (UCB) derived multipotential stem cells (MPSC) was produced. We then transplanted MPSCs into a rat model of spinal cord injury (SCI) and assessed neurologic function as well as spinal cord histology. To determine if MPSCs transplanted into a rat model of acute SCI would lead to a beneficial neurologic effect. Conditioned medium from UCB contains factors that could promote healing of endogenous neural tissues. Previously, our laboratory has demonstrated that UCB hematopoietic cells can develop into MPSCs capable of differentiating into multiple cell types including oligodendrocyte-like cells. We cultured MPSCs from UCB cells using fibroblast growth factor 4, stem cell factor and fms-like tyrosine kinase receptor-3 ligand supplemented serum-free medium. Using a cytokine antibody array, we produced a cytokines expression profile of MPSCs. We then transplanted MPSCs into an immunosuppressed rat model of SCI and assessed neurologic function weekly for 6 weeks by the Basso, Beattie, and Bresnahan locomotor test. The spinal cords were examined histologically and lesion areas quantified. We detected elevated levels of cytokines and growth factors with known neuroprotective, angiogenic, and anti-inflammatory effects in the MPSC conditioned media. The SCI rats treated with MPSCs showed a significant improvement in Basso, Beattie, and Bresnahan scores after 6 weeks compared with the group that received vehicle only. Immunohistochemistry revealed transplanted human cells were present in the injured spinal cord after 1 week, but were no longer present by 6 weeks. There was a trend for the lesion size in treated rats to be smaller than that of the control group. We conclude that UCB MPSCs improve neurologic function of rats with acute SCI, possibly by the release of factors that reduce secondary injury.

A comparison of adenosine A2A agonism and methylprednisolone in attenuating neuronal damage and improving functional outcome after experimental traumatic spinal cord injury in rabbits

Steroid agents remain the lone pharmacological treatment in widespread use for acute spinal cord injury (SCI), although their utility remains in dispute in the neurotrauma literature. Adenosine A2A receptor activation with ATL-146e, a selective A2A agonist, has shown potential benefit in treating SCI; however, it has not been compared with the gold standard, methylprednisolone. The authors of this study evaluated ATL-146e and methylprednisolone for their ability to preserve neuronal viability and motor function in experimental SCI. New Zealand White rabbits sustained SCI or sham injury via the Allen weight-drop technique. Ten minutes postinjury, animals received ATL-146e (ATL group, 0.06 microg/kg/min intravenously for 3 hours), methylprednisolone (steroid group, 30 mg/kg intravenously), or saline (trauma control group). Hindlimb motor function was recorded every 12 hours using the Tarlov motor grading scale (0, paralysis-5, normal hop). At 48 hours, fixed spinal cord tissue was evaluated for neuronal viability. Hindlimb motor function in animals treated with ATL-146e was equivalent to that of sham-injured animals and was significantly better than that of trauma control animals at all time points and that of steroid-treated animals at 12 hours (p = 0.05). Motor function in steroid-treated animals was worse than in those given ATL-146e and better than that of trauma control animals at later time points, but was not statistically significant (both p > 0.05). Neuronal viability (measured in neurons/hpf) was significantly higher in both treatment groups compared with the trauma control group (12.1 +/- 1.4 neurons/hpf for the ATL and 13.3 +/- 1.4 neurons/hpf for the steroid group compared with 7.5 +/- 1.5 neurons/hpf for the trauma control group; both p < 0.04). Neuronal viability did not differ among ATL-146e-treated, steroid-treated, and sham-injured groups. The use of ATL-146e is at least as effective as methylprednisolone in preserving function and is equivalent to methylprednisolone in preserving the structure of spinal cord tissue after blunt SCI. Adenosine A2A receptor activation may be an effective treatment for acute SCI while avoiding the adverse effects of steroid agents.

Gene expression profiling of experimental traumatic spinal cord injury as a function of distance from impact site and injury severity

Changes in gene expression contribute to pathophysiological alterations following spinal cord injury (SCI). We examined gene expression over time (4 h, 24 h, 7 days) at the impact site, as well as rostral and caudal regions, following mild, moderate, or severe contusion SCI in rats. High-density oligonucleotide microarrays were used that included approximately 27,000 genes/ESTs (Affymetrix RG-U34; A, B and C arrays), together with multiple analyses (MAS 5.0, dChip). Alterations after mild injury were relatively rapid (4 and 24 h), whereas they were delayed and prolonged after severe injury (24 h and 7 days). The number and magnitude of gene expression changes were greatest at the injury site after moderate injury and increased in rostral and caudal regions as a function of injury severity. Sham surgery resulted in expression changes that were similar to mild injury, suggesting the importance of using time-linked surgical controls as well as naive animals for these kinds of studies. Expression of many genes and ESTs was altered; these were classified functionally based on ontology. Overall representation of these functional classes varied with distance from the site of injury and injury severity, as did the individual genes that contributed to each functional class. Different clustering approaches were used to identify changes in neuronal-specific genes and several transcription factors that have not previously been associated with SCI. This study represents the most comprehensive evaluation of gene expression changes after SCI to date. The results underscore the power of microarray approaches to reveal global genomic responses as well as changes in particular gene clusters and/or families that may be important in the secondary injury cascade.

Induced hypothermia in experimental traumatic spinal cord injury: an update

The use of induced hypothermia in the treatment of traumatic spinal cord injury (SCI) has been studied extensively between the 1960s and 1970s. Although the treatment showed some promise, it became less popular by the 1980s, mainly because of its adverse effects. However, a revival of hypothermia in the treatment of traumatic brain injury (TBI) in the last decade has encouraged neuroscientists to conduct experiments to reevaluate the potential benefits of hypothermia in traumatic SCI. All laboratory investigations studying the mechanisms of action and/or the efficacy of induced hypothermia in treating experimental traumatic SCI published in the last decade were reviewed. Although efficacy of hypothermia in improving functional outcome of mild to moderate traumatic SCI has been demonstrated, hypothermia may not be protective against severe traumatic SCI. At present, induced hypothermia has yet to be recognized or approved as a potential treatment having therapeutic value for traumatic SCI in humans. The continued search for a possible synergistic effect of induced hypothermia and pharmacological therapy may yield some promise. It has also been deduced from these laboratory studies that hyperthermia is deleterious and rigorous measures to prevent hyperthermia should be taken to minimize the propagation of secondary neuronal damage after traumatic SCI.

Effects of theN-Methyl-d-Aspartate Receptor Blocker MK-801 on Neurologic Function After Experimental Brain Injury

Pharmacologic inhibition of excitatory amino acid (EAA) neurotransmission attenuates cell death in models of global and focal ischemia and hypoglycemia and improves neurologic outcome after experimental traumatic spinal cord injury. The present study examined the effects of the noncompetitive N-methyl-D-aspartate (NMDA) receptor blocker MK-801 on cardiovascular and neurologic function after experimental fluid-percussion (FP) brain injury in the rat. Animals received either an intravenous bolus of MK-801 (1 mg/kg) or saline (equal volume) 15 min prior to FP brain injury or 15 min following FP brain injury. MK-801 pretreatment significantly improved postinjury cardiovascular variables and attenuated postinjury neurologic dysfunction. Postinjury treatment with MK-801 also significantly improved cardiovascular variables, but had little effect on postinjury neurologic scores. These results suggest that EAA neurotransmitters may be involved in the pathophysiological sequelae of traumatic brain injury and that noncompetitive blockade of the NMDA receptor prior to brain injury may reduce EAA-induced damage and limit neurologic dysfunction.

A method for estimating edema in experimental traumatic spinal cord injury

In this experiment a method was developed to estimate spinal cord edema following experimental trauma. After the intravenous administration of Evans Blue solution and 100 μc of iodinated I-131 serum albumin, the spinal cords of rhesus monkeys were traumatized with a 600 g cm injury. Edema at the injury site was evaluated at 6 hr after injury and compared to a section of uninjured spinal cord from each animal. The I-131 serum albumin acted as an excellent marker to estimate the extravasation of serum proteins into the neural extracellular spaces. In the future this method will be used to test the effect of various drugs on post-traumatic edema formation.