Neurons In Spinal Cord Injury Research Articles

UBC9-mediated SUMOylation of Lamin B1 enhances DNA-damage-induced nuclear DNA leakage and autophagy after spinal cord injury.

Recent studies have shown that nucleophagy can mitigate DNA damage by selectively degrading nuclear components protruding from the nucleus. However, little is known about the role of nucleophagy in neurons after spinal cord injury (SCI).Western blot analysis and immunofluorescence were performed to evaluate the nucleophagy after nuclear DNA damage and leakage in SCI neurons in vivo and NSC34 expression in primary neurons cultured with oxygen-glucose deprivation (OGD) in vitro, as well as the interaction and colocalization of autophagy protein LC3 with nuclear lamina protein Lamin B1. The effect of UBC9, a Small ubiquitin-related modifier (SUMO) E2 ligase, on Lamin B1 SUMOylation and nucleophagy was examined by siRNA transfection or 2-D08 (a small-molecule inhibitor of UBC9), immunoprecipitation, and immunofluorescence.In SCI and OGD injured NSC34 or primary cultured neurons, neuronal nuclear DNA damage induced the SUMOylation of Lamin B1, which was required by the nuclear Lamina accumulation of UBC9. Furthermore, LC3/Atg8, an autophagy-related protein, directly bound to SUMOylated Lamin B1, and delivered Lamin B1 to the lysosome. Knockdown or suppression of UBC9 with siRNA or 2-D08 inhibited SUMOylation of Lamin B1 and subsequent nucleophagy and protected against neuronal death.Upon neuronal DNA damage and leakage after SCI, SUMOylation of Lamin B1 is induced by nuclear Lamina accumulation of UBC9. Furthermore, it promotes LC3-Lamin B1 interaction to trigger nucleophagy that protects against neuronal DNA damage.

Mesenchymal stem cell attenuates spinal cord injury by inhibiting mitochondrial quality control-associated neuronal ferroptosis

Ferroptosis is a newly discovered form of iron-dependent oxidative cell death and drives the loss of neurons in spinal cord injury (SCI). Mitochondrial damage is a critical contributor to neuronal death, while mitochondrial quality control (MQC) is an essential process for maintaining mitochondrial homeostasis to promote neuronal survival. However, the role of MQC in neuronal ferroptosis has not been clearly elucidated. Here, we further demonstrate that neurons primarily suffer from ferroptosis in SCI at the single-cell RNA sequencing level. Mechanistically, disordered MQC aggravates ferroptosis through excessive mitochondrial fission and mitophagy. Furthermore, mesenchymal stem cells (MSCs)-mediated mitochondrial transfer restores neuronal mitochondria pool and inhibits ferroptosis through mitochondrial fusion by intercellular tunneling nanotubes. Collectively, these results not only suggest that neuronal ferroptosis is regulated in an MQC-dependent manner, but also fulfill the molecular mechanism by which MSCs attenuate neuronal ferroptosis at the subcellular organelle level. More importantly, it provides a promising clinical translation strategy based on stem cell-mediated mitochondrial therapy for mitochondria-related central nervous system disorders.

Therapeutic effects of KCC2 chloride transporter activation on detrusor overactivity in mice with spinal cord injury.

This study aimed to clarify whether downregulation of K+-Cl- cotransporter 2 (KCC2) in the sacral parasympathetic nucleus (SPN) of the lumbosacral spinal cord, from which the efferent pathway innervating the bladder originates, causes cellular hyperexcitability and triggers detrusor overactivity (DO) in spinal cord injury (SCI). SCI was produced by Th8-9 spinal cord transection in female C57BL/6 mice. At 4 wk after SCI, CLP290, a KCC2 activator, was administered, and cystometry was performed. Thereafter, neuronal activity with c-fos staining and KCC2 expression in cholinergic preganglionic parasympathetic neurons in the SPN was examined using immunohistochemistry. Firing properties of neurons in the SPN region were evaluated by extracellular recordings in the spinal cord slice preparations. DO evident as nonvoiding contractions was significantly reduced by CLP290 treatment in SCI mice. The number of c-fos-positive cells and coexpression of c-fos in choline acetyltransferase-positive cells were decreased in the SPN region of the SCI CLP290-treated group versus the SCI vehicle-treated group. KCC2 immunoreactivity was present on the cell membrane of SPN neurons and normalized fluorescence intensity of KCC2 in choline acetyltransferase-positive SPN neurons was decreased in the SCI vehicle-treated group versus the spinal intact vehicle-treated group but recovered in the SCI CLP290-treated group. Extracellular recordings showed that CLP290 suppressed the high-frequency firing activity of SPN neurons in SCI mice. These results indicated that SCI-induced DO is associated with downregulation of KCC2 in preganglionic parasympathetic neurons and that activation of KCC2 transporters can reduce DO, increase KCC2 expression in preganglionic parasympathetic neurons, and decrease neuronal firing of SPN neurons in SCI mice.NEW & NOTEWORTHY This study is the first report to suggest that activation of the Cl- transporter K+-Cl- cotransporter 2 may be a therapeutic modality for the treatment of spinal cord injury-induced detrusor overactivity by targeting bladder efferent pathways.

Oxidative stress disrupts the cytoskeleton of spinal motor neurons.

Traumatic spinal cord injury (SCI) is a common and devastating central nervous disease, the treatment of which faces many challenges to the medical community and society as a whole. Treatment measures based on oxidative stress of spinal motor neurons during SCI are expected to help restore biological functions of neurons under injury conditions. However, to date, there are no systematic reports regarding oxidative stress on spinal motor neuron injury. Our aim is to better understand and explain the influences and mechanisms of oxidative stress on spinal motor neurons during SCI. We first exposed VSC4.1 motor neurons to hydrogen peroxide (H2 O2 ) and evaluated the effects on cell viability, morphology, cycling, and apoptosis, with an emphasis on the changes to the cytoskeleton and the effect of N-acetyl-l-cysteine (NAC) on these changes. Then, we investigated the effects of NAC on these cytoskeletal changes in vitro and in vivo. We found that H2 O2 caused severe damage to the normal cytoskeleton, leading to a reduction in neurite length and number, rearrangement of the actin cytoskeleton, and disorder of the microtubules and neurofilaments in VSC4.1. Importantly, NAC attenuated the oxidative damage of spinal motor neurons in vitro and in vivo, promoting the recovery of hindlimb motor ability in mice with SCI at the early stage of injury. This study shows that oxidative stress plays an important role in the cytoskeleton destruction of spinal motor neurons in SCI, and treatment of SCI on this basis is a promising strategy. These findings will help to elucidate the role of oxidative stress in spinal motor neuron injury in SCI and provide references for further research into the study of the pathology and underlying mechanism of SCI.

Availability of neuregulin-1beta1 protects neurons in spinal cord injury and against glutamate toxicity through caspase dependent and independent mechanisms

Spinal cord injury (SCI) causes sensorimotor and autonomic impairment that partly reflects extensive, permanent loss of neurons at the epicenter and penumbra of the injury. Strategies aimed at enhancing neuronal protection are critical to attenuate neurodegeneration and improve neurological recovery after SCI. In rat SCI, we previously uncovered that the tissue levels of neuregulin-1beta 1 (Nrg-1β1) are acutely and persistently downregulated in the injured spinal cord. Nrg-1β1 is well-known for its critical roles in the development, maintenance and physiology of neurons and glia in the developing and adult spinal cord. However, despite this pivotal role, Nrg-1β1 specific effects and mechanisms of action on neuronal injury remain largely unknown in SCI. In the present study, using a clinically-relevant model of compressive/contusive SCI in rats and an in vitro model of glutamate toxicity in primary neurons, we demonstrate Nrg-1β1 provides early neuroprotection through attenuation of reactive oxygen species, lipid peroxidation, necrosis and apoptosis in acute and subacute stages of SCI. Mechanistically, availability of Nrg-1β1 following glutamate challenge protects neurons from caspase-dependent and independent cell death that is mediated by modulation of mitochondria associated apoptotic cascades and MAP kinase and AKT signaling pathways. Altogether, our work provides novel insights into the role and mechanisms of Nrg-1β1 in neuronal injury after SCI and introduces its potential as a new neuroprotective target for this debilitating neurological condition.

Enhanced Neural Regeneration with a Concomitant Treatment of Framework Nucleic Acid and Stem Cells in Spinal Cord Injury.

Spinal cord injury (SCI), began with a primary injury including contusion and compression, is a common disease caused by various pathogenesis. Characterized disruption of axons and irreversible loss of neurons in SCI, and further damage in spinal cord tissue caused by following secondary injuries, such as the formation of glial scar and inflammation, makes it even harder to recover for affected patients. Tetrahedral framework nucleic acid (tFNA), which possesses the capability of promoting neuroprotection and neuroregeneration in vitro, might alleviate the injuries, and facilitate the neural tissue regeneration in experimental animal models of SCI. Here, we developed a concomitant treatment of tFNA and neural stem cells (NSCs) for the synergistic therapy in treating the injury of the spinal cord. We first observed that tFNA could promote cell proliferation of NSCs then verified that the concomitant treatment of tFNA and NSCs showed the neuroprotective actions by increasing the survival of transplanted NSCs. Furthermore, the recovery of motor function and the tissue regeneration in the lesion site of the spinal cord achieved the best performance in the SCI rats treated with the combination of tFNA and NSCs than others, and the formation of glial scar was the least. Our findings provide novel evidence of a promising strategy for synergistic treatment of SCI in the future.

Restoring Motor Neurons in Spinal Cord Injury With Induced Pluripotent Stem Cells.

Spinal cord injury (SCI) is a devastating neurological disorder that damages motor, sensory, and autonomic pathways. Recent advances in stem cell therapy have allowed for the in vitro generation of motor neurons (MNs) showing electrophysiological and synaptic activity, expression of canonical MN biomarkers, and the ability to graft into spinal lesions. Clinical translation, especially the transplantation of MN precursors in spinal lesions, has thus far been elusive because of stem cell heterogeneity and protocol variability, as well as a hostile microenvironment such as inflammation and scarring, which yield inconsistent pre-clinical results without a consensus best-practice therapeutic strategy. Induced pluripotent stem cells (iPSCs) in particular have lower ethical and immunogenic concerns than other stem cells, which could make them more clinically applicable. In this review, we focus on the differentiation of iPSCs into neural precursors, MN progenitors, mature MNs, and MN subtype fates. Previous reviews have summarized MN development and differentiation, but an up-to-date summary of technological and experimental advances holding promise for bench-to-bedside translation, especially those targeting individual MN subtypes in SCI, is currently lacking. We discuss biological mechanisms of MN lineage, recent experimental protocols and techniques for MN differentiation from iPSCs, and transplantation of neural precursors and MN lineage cells in spinal cord lesions to restore motor function. We emphasize efficient, clinically safe, and personalized strategies for the application of MN and their subtypes as therapy in spinal lesions.

Role of axon resealing in retrograde neuronal death and regeneration after spinal cord injury

Spinal cord injury leads to persistent behavioral deficits because mammalian central nervous system axons fail to regenerate. A neuron’s response to axon injury results from a complex interplay of neuron-intrinsic and environmental factors. The contribution of axotomy to the death of neurons in spinal cord injury is controversial because very remote axotomy is unlikely to result in neuronal death, whereas death of neurons near an injury may reflect environmental factors such as ischemia and inflammation. In lampreys, axotomy due to spinal cord injury results in delayed apoptosis of spinal-projecting neurons in the brain, beyond the extent of these environmental factors. This retrograde apoptosis correlates with delayed resealing of the axon, and can be reversed by inducing rapid membrane resealing with polyethylene glycol. Studies in mammals also suggest that polyethylene glycol may be neuroprotective, although the mechanism(s) remain unclear. This review examines the early, mechanical, responses to axon injury in both mammals and lampreys, and the potential of polyethylene glycol to reduce injury-induced pathology. Identifying the mechanisms underlying a neuron’s response to axotomy will potentially reveal new therapeutic targets to enhance regeneration and functional recovery in humans with spinal cord injury.

Quantification of surviving neurons after contusion, dislocation, and distraction spinal cord injuries using automated methods.

This study proposes and validates an automated method for counting neurons in spinal cord injury (SCI) and then uses it to examine and compare the surviving cells in common types of SCI mechanisms. Moderate contusion, dislocation, and distraction SCIs were surgically induced in Sprague Dawley male rats (n = 6 for each type of injury). Their spinal cords were harvested 8 weeks post injury with 5 normal weight-matched rats. The spinal cords were cut, stained with anti-NeuN antibody and fluorescent Nissl, and imaged in the dorsal and ventral horns at various distances to the epicenter. Neurons in the images were automatically counted using an algorithm that was designed to filter non-soma-like objects based on morphological characteristics (size, solidity, circular pattern) and check the remaining objects for the double-stained nucleus/cell body features (brightness variation, brightness distribution, color). To validate the automated method, some of the images were randomly selected for manual counting. The number of surviving cells that were automatically measured by the algorithm was found to be correlated with the values that were manually measured by 2 observers (P < .001) with similar differences (P > .05). Neurons in the dorsal and ventral horns were reduced after the SCIs (P < .05). Dislocation and distraction, respectively, caused the most severe damage to the ventral horn neurons especially near the epicenter and the most extensive and uniform damage to the dorsal horn neurons (P < .05). Our method was proved to be reliable, which is suitable for studying different types of SCI.

Protective effect of docosahexaenoic acid on lipotoxicity-mediated cell death in Schwann cells: Implication of PI3K/AKT and mTORC2 pathways.

Background and AimDocosahexaenoic acid (DHA) exhibits neuroprotective properties and has been shown to preserve nerve cells following trauma and ischemic injury. Recently, we showed that DHA pretreatment improved locomotion and reduced neuropathic pain after acute spinal cord injury in adult rats. These improvements were associated with an increase in the levels of AKT in spinal cord injury neurons. In this study, we investigate the implication of PI3K/AKT and mTOR pathway in DHA‐mediated protection of primary cultured Schwann cells (pSC) undergoing palmitic acid‐induced lipotoxicity (PA‐LTx).MethodsPrimary cultured Schwann cells were treated with PA (PA:BSA, 2:1) in the presence or absence of DHA (1–200 µM) for 24–48 hr. Cell viability was determined by crystal violet staining and nuclear morphology was examined using Hoechst staining.ResultsWe found that pSC cultures exposed to palmitic acid (PA) overload showed chromatin condensation, a decrease in cell viability and an inhibition of AKT phosphorylation in a time‐dependent manner. Next, pSC exposed to PA overload were treated with DHA. The data show that co‐treatment with DHA inhibited the loss of cell viability and apoptosis caused by PA. Moreover, treatment with DHA inhibited chromatin condensation, significantly stimulated p‐AKT phosphorylation under PA‐LTx condition, and DHA alone increased AKT phosphorylation. Additionally, when these pSC cultures were treated with PI3K inhibitors LY294002 and, BKM120 and mTOR inhibitors Torin 1 (mTORC1/mTORC2), but not rapamycin (mTORC1), the protective effects of DHA were not observed.ConclusionThese findings suggest PI3K/AKT and mTORC2 kinase pathways are involved in the protective function (s) of DHA in PA‐induced Schwann cell death.

Protective effect of thyroid hormone on spinal cord injury neurons in rats

Objective To investigate the protective effect of thyroid hormone on spinal cord injury neurons and its molecular mechanism. Methods A DMEM culture medium with a volume fraction of 10% fetal bovine serum was cultured with the dorsal ridge neurons of RN- dsc rats. The neurons were inoculated in the culture plate after the digestion of trypsin and treated differently as follows: (1) control group: DMEM treatment with no drugs or serum; (2) H2O2 group: serum-free DMEM treatment containing 100 μmol/L H2O2; (3) H2O2+ 10-6mol/L triiodothyronine (T3) group: serum-free DMEM treatment containing 100 μmol/L H2O2 and 10-6mol/L T3 ; (4) H2O2+ 10-5mol/L T3 group: serum-free DMEM treatment containing 100 μmol/L H2O2 and 10-5mol/L T3; (5) negative control group: transfection of negative control mimics with Lipofectamine™2000 reagent; (6) miR-210 group: transfection of miR-210 mimics with Lipofectamine™2000 reagent. Cell viability, apoptosis number, and expressions of nuclear factor E2 correlation factor 2 (Nrf-2) antioxidant pathway molecules and miR-210 were determined. After transfection of miR-210 mimics and negative control mimics, expressions of Nrf-2 antioxidant pathway molecules were determined. Results The cell proliferation activity and protein expressions of Nrf-2, antioxidant reaction elements (ARE), superoxide dismutase 2 (SOD2), and heme oxygenase (HO-1) in H2O2 group (0.39±0.06, 0.52±0.08, 0.31±0.08, 0.25±0.05, respectively) were significantly lower than those in control group (1.00±0.15, 1.00±0.17, 1.00±0.13, 1.00±0.11, respectively) (P<0.05), while the apoptosis numbers and the expressions of miR-210 were significantly higher than those in control group (P<0.05). The cell proliferation activity and protein expressions of Nrf-2, ARE, SOD2, HO-1 in H2O2+ 10-6mol/L T3 group and H2O2+ 10-5mol/L T3 group were significantly higher than those in the H2O2 group (P<0.05), while apoptosis numbers and expressions of miR-210 were significantly lower than those in H2O2 group (P<0.05). The cell proliferation activity and protein expressions of Nrf-2, ARE, SOD2, HO-1 in H2O2+ 10-5mol/L T3 group (0.88±0.14, 0.84±0.12, 0.72±0.09, 0.69±0.09) were significantly higher than those in H2O2+ 10-6mol/L T3 group (0.73±0.09, 0.71±0.08, 0.58±0.09, 0.52±0.08)(P<0.05), while apoptosis numbers and expressions of miR-210 were significantly lower than those in H2O2+ 10-6mol/L T3 group. The protein expressions of Nrf-2, ARE, SOD2, and HO-1 in miR-210 group (0.37±0.06, 0.24±0.05, 0.45±0.08, 0.49±0.07, respectively) were significantly lower than those in negative control group (1.00±0.13, 1.00±0.19, 1.00±0.15, 1.00±0.14, respectively) (P<0.05). Conclusion Thyroid hormone can inhibit the expression of Nrf-2 in oxidative stress injury process of neurons by inhibiting the expression of miR-210, and hence reduce the oxidative stress injury of spinal cord neurons. Key words: Triiodothyronine; Spinal cord injuries; Nf-E2-related factor 2

Neuroprotective effects of curcumin on spinal cord injury mice by nuclear factor E2-related factor 2 pathway

Objective To investigate the neuroprotective effect of curcumin (CCM) on spinal cord injury (SCI) mice by nuclear factor related factor 2 (Nrf2)/ARE pathway. Methods The SCI mouse model was prepared by modified Allen’s hitting method. At 3 days after SCI, the number of necrotic cells near the injured area was detected by propidium iodide (PI) staining. Hydroethidine (HEt) staining was used to detect reactive oxygen species (ROS) production. enzyme linked immunosorbent assay (ELISA) kit was used to detect the expression of tumor necrosis factor-α (TNF-α) and interleukin- 10 (IL- 10). At 28 days after SCI, Basso Mouse Scale (BMS) score was used to detect the hindlimb motor function. Sucorse preference test was used to evaluate the depressive status of mice. The expression of Nrf2, SOD1 and quinone oxidoreductase gene (NQO1) were detected by Western blotting. Results (1) The spinal cord water content (75.540±1.564) in the CCM group was significantly lower than that in the Veh group (85.500±1.297), the difference was statistically significant (t=4.903, P=0.001). (2) BMS scores showed that CCM treatment group 7, 14, 21 and 28 d scores of hindlimb locomotor function [(2.400±0.894), (3.400±0.894), (4.200±0.837) and (4.800±0.837) scores] compared with the Veh Group [(1.000±0.707), (1.800±0.447), (2.200±0.837), (3.000±0.707) scores, P= 0.004]. (3) Sucorse preference test showed that the difference between the CMM treatment group and the Veh group was statistically significant [(55.330±1.124)% and (45.740±1.965)%, t=4.314, P=0.002]. (4) Compared with Veh group, CCM group of anti- inflammatory cytokines IL- 10 expression (674.20±6.94) was significantly higher than that of group Veh (525.00±6.43), and proinflammatory factor TNF- α (763.20±16.24) was significantly lower than that of Veh group (1 091.00±29.02), P=0.000, 0.000. (5) The fluorescence intensity of ROS in CCM group (1 377.00±27.06) was significantly lower than Veh group (1 750.00±42.45), the difference was statistically significant (t=7.410, P=0.000). (6) Compared with Veh group (72.00±3.81), the number of necrotic cells in CCM group was significantly decreased (53.60±3.20), and the difference was statistically significant (t=3.690, P=0.006). (7) Compared with Veh group, the expression of SOD1、Nrf2 and NQO1 in CCM group were up- regulated (0.763±0.039, 0.442±0.026, 0.636±0.023 and 0.937±0.030, 0.692±0.034, 0.898±0.025, P=0.025, 0.004, 0.002). Conclusion Curcumin protects neurons in spinal cord injury by Nrf2 pathway. Key words: Curcumin; Spinal cord injury; Nuclear factor related factor 2 pathway; Neuroprotection

The role of the miR-99b-5p/mTOR signaling pathway in neuroregeneration in mice following spinal cord injury.

The present study aimed to investigate the role of microRNA (miR)-99b-5p in spinal cord injury (SCI). Reverse transcription-quantitative polymerase chain reaction demonstrated that, compared with control mice, the expression levels of miR-99b-5p were upregulated in the mouse spinal cord following SCI. Mechanistic target of rapamycin (mTOR) was predicted to be the possible target of miR-99b-5p according to TargetScan and microrna databases. Dual-luciferase reporter assay verified that miR-99b-5p was able to target mTOR. Furthermore, the results of an apoptosis analysis demonstrated that there were few apoptotic neurons in the control group, whereas SCI induced a significant increase in the number of apoptotic cells. Conversely, apoptosis was inhibited following transfection with a miR-99b-5p inhibitor. The effects of miR-99b-5p on neurite growth were also evaluated. The results of an immunofluorescence analysis indicated that neurite growth was normal in the control group, whereas SCI induced a reduction in neurite growth, which was rescued following transfection with a miR-99b-5p inhibitor. The protein expression levels of mTOR were detected in the three groups by western blotting. The results demonstrated that, compared with the control group, the protein expression levels of mTOR were significantly reduced in SCI neurons, whereas transfection with a miR-99b-5p inhibitor suppressed the SCI-induced reduction of mTOR. In conclusion, treatment with a miR-99b-5p inhibitor may attenuate SCI-induced harmful alterations in spinal cord neurons via the regulation of mTOR expression.

New Non-Invasive Way to Rescue Neurons in Spinal Cord Injury via Intranasal Administration of Nerve Growth Factor: Pilot Study

New Non-Invasive Way to Rescue Neurons in Spinal Cord Injury via Intranasal Administration of Nerve Growth Factor: Pilot Study

Administration of low dose estrogen attenuates gliosis and protects neurons in acute spinal cord injury in rats.

Spinal cord injury (SCI) is a debilitating condition with neurological deficits and loss of motor function that, depending on the severity, may lead to paralysis. The only treatment currently available is methylprednisolone, which is widely used and renders limited efficacy in SCI. Therefore, other therapeutic agents must be developed. The neuroprotective efficacy of estrogen in SCI was studied with a pre-clinical and pro-translational perspective. Acute SCI was induced in rats that were treated with low doses of estrogen (1, 5, 10, or 100 μg/kg) and compared with vehicle-treated injured rats or laminectomy control (sham) rats at 48 h post-SCI. Changes in gliosis and other pro-inflammatory responses, expression and activity of proteolytic enzymes (e.g., calpain, caspase-3), apoptosis of neurons in SCI, and cell death were monitored via Western blotting and immunohistochemistry. Negligible pro-inflammatory responses or proteolytic events and very low levels of neuronal death were found in sham rats. In contrast, vehicle-treated SCI rats showed profound pro-inflammatory responses with reactive gliosis, elevated expression and activity of calpain and caspase-3, elevated Bax:Bcl-2 ratio, and high levels of neuronal death in lesion and caudal regions of the injured spinal cord. Estrogen treatment at each dose reduced pro-inflammatory and proteolytic activities and protected neurons in the caudal penumbra in acute SCI. Estrogen treatment at 10 μg was found to be as effective as 100 μg in ameliorating the above parameters in injured animals. Results from this investigation indicated that estrogen at a low dose could be a promising therapeutic agent for treating acute SCI. Experimental studies with low dose estrogen therapy in acute spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes. Estrogen has been found to ameliorate several degenerative pathways following SCI. Thus, such early protective effects may even lead to functional recovery in long term injury. Studies are underway in chronic SCI in a follow up manuscript.

Intranasal nerve growth factor bypasses the blood-brain barrier and affects spinal cord neurons in spinal cord injury.

The purpose of this work was to investigate whether, by intranasal administration, the nerve growth factor bypasses the blood-brain barrier and turns over the spinal cord neurons and if such therapeutic approach could be of value in the treatment of spinal cord injury. Adult Sprague-Dawley rats with intact and injured spinal cord received daily intranasal nerve growth factor administration in both nostrils for 1 day or for 3 consecutive weeks. We found an increased content of nerve growth factor and enhanced expression of nerve growth factor receptor in the spinal cord 24 hours after a single intranasal administration of nerve growth factor in healthy rats, while daily treatment for 3 weeks in a model of spinal cord injury improved the deficits in locomotor behaviour and increased spinal content of both nerve growth factor and nerve growth factor receptors. These outcomes suggest that the intranasal nerve growth factor bypasses blood-brain barrier and affects spinal cord neurons in spinal cord injury. They also suggest exploiting the possible therapeutic role of intranasally delivered nerve growth factor for the neuroprotection of damaged spinal nerve cells.

Magnetic manipulation of bacterial magnetic nanoparticle-loaded neurospheres

Specific targeting of cells to sites of tissue damage and delivery of high numbers of transplanted cells to lesion tissue in vivo are critical parameters for the success of cell-based therapies. Here, we report a promising in vitro model system for studying the homing of transplanted cells, which may eventually be applicable for targeted regeneration of damaged neurons in spinal cord injury. In this model system, neurospheres derived from human neuroblastoma SH-SY5Y cells labeled with bacterial magnetic nanoparticles were guided by a magnetic field and successfully accumulated near the focus site of the magnetic field. Our results demonstrate the effectiveness of using an in vitro model for testing bacterial magnetic nanoparticles to develop successful stem cell targeting strategies during fluid flow, which may ultimately be translated into in vivo targeted delivery of cells through circulation in various tissue-repair models.

Direct Evidence for Calpain Involvement in Apoptotic Death of Neurons in Spinal Cord Injury in Rats and Neuroprotection with Calpain Inhibitor

To demonstrate calpain involvement in neurodegeneration in rat spinal cord injury (SCI), we examined SCI segments for DNA fragmentation, neurons for calpain overexpression, neuronal death, and neuroprotection with calpain inhibitor (E-64-d). After the induction of SCI (40 g cm force) on T12, rats were treated within 15 min with vehicle (DMSO) or E-64-d. Sham animals underwent laminectomy only. Animals were sacrificed at 24 h, and five 1-cm long spinal cord segments were collected: two rostral (S1 and S2), one lesion (S3), and two caudal segments (S4 and S5). Agarose gel electrophoresis of DNA samples isolated from the SCI segments showed both random and internucleosomal DNA fragmentation indicating occurrence of necrosis as well as apoptosis mostly in the lesion, moderately in caudal, and slightly in rostral segments from SCI rats. Treatment of SCI rats with E-64-d (1 mg/kg) reduced DNA fragmentation in all segments. The lesion and adjacent caudal segments (S3 and S4) were further investigated by in situ double-immunofluorescent labelings that showed increase in calpain expression in neurons in SCI rats and decrease in calpain expression in SCI rats treated with E-64-d. In situ combined TUNEL and double-immunofluorescent labelings directly detected co-localization of neuronal death and calpain overexpressin in SCI rats treated with only vehicle while attenuation of neuronal death in SCI rats treated with E-64-d. Previous studies from our laboratory indirectly showed neuroprotective effect of E-64-d in SCI rats. Our current results provide direct in situ evidence for calpain involvement in neuronal death and neuroprotective efficacy of E-64-d in lesion and penumbra in SCI rats.