Bromocriptine protects perilesional spinal cord neurons from lipotoxicity after spinal cord injury

Recent studies have revealed that lipid droplets accumulate in neurons after brain injury and evoke lipotoxicity,damaging the neurons.However,how lipids are metabolized by spinal cord neurons after spinal cord injury remains unclear.Herein,we investigated lipid metabolism by spinal cord neurons after spinal cord injury and identified lipid-lowering compounds to treat spinal cord injury.We found that lipid droplets accumulated in perilesional spinal cord neurons after spinal cord injury in mice.Lipid droplet accumulation could be induced by myelin debris in HT22 cells.Myelin debris degradation by phospholipase led to massive free fatty acid production,which increased lipid droplet synthesis,β-oxidation,and oxidative phosphorylation.Excessive oxidative phosphorylation increased reactive oxygen species generation,which led to increased lipid peroxidation and HT22 cell apoptosis.Bromocriptine was identified as a lipid-lowering compound that inhibited phosphorylation of cytosolic phospholipase A2 by reducing the phosphorylation of extracellular signal-regulated kinases 1/2 in the mitogen-activated protein kinase pathway,thereby inhibiting myelin debris degradation by cytosolic phospholipase A2 and alleviating lipid droplet accumulation in myelin debris-treated HT22 cells.Motor function,lipid droplet accumulation in spinal cord neurons and neuronal survival were all improved in bromocriptine-treated mice after spinal cord injury.The results suggest that bromocriptine can protect neurons from lipotoxic damage after spinal cord injury via the extracellular signal-regulated kinases 1/2-cytosolic phospholipase A2 pathway.

Effects of brain-derived neurotrophic factor on synapsin expression in rat spinal cord anterior horn neurons cultured in vitro

Brain-derived neurotrophic factor （BDNF） promotes synaptic formation and functional maturation by upregulating synapsin expression in cortical and hippocampal neurons. However, it remains controversial whether BDNF affects synapsin expression in spinal cord anterior horn neurons. Wistar rat spinal cord anterior horn neurons were cultured in serum-supplemented medium containing BDNF, BDNF antibody, and Hank＇s solution for 3 days, and then synapsin I and synaptophysin protein and mRNA expression was detected. Under serum-supplemented conditions the number of surviving neurons in the spinal cord anterior horn was similar among BDNF, anti-BDNF, and control groups （P 〉 0.05）. Synapsin I and synaptophysin protein and mRNA expressions were increased in BDNF-treated neurons, but decreased in BDNF antibody-treated neurons （P 〈 0.01）. These results indicated that BDNF significantly promotes synapsin I and synaptophysin expression in in vitro-cultured rat spinal cord anterior horn neurons.

Pulsed electrical stimulation protects neurons in the dorsal root and anterior horn of the spinal cord after peripheral nerve injury

Most studies on peripheral nerve injury have focused on repair at the site of injury, but very few have examined the effects of repair strategies on the more proximal neuronal cell bodies. In this study, an approximately 10-mm-long nerve segment from the ischial tuberosity in the rat was transected and its proximal and distal ends were inverted and sutured. The spinal cord was subjected to pulsed electrical stimulation at T10 and L3, at a current of 6.5 m A and a stimulation frequency of 15 Hz, 15 minutes per session, twice a day for 56 days. After pulsed electrical stimulation, the number of neurons in the dorsal root ganglion and anterior horn was increased in rats with sciatic nerve injury. The number of myelinated nerve fibers was increased in the sciatic nerve. The ultrastructure of neurons in the dorsal root ganglion and spinal cord was noticeably improved. Conduction velocity of the sciatic nerve was also increased. These results show that pulsed electrical stimulation protects sensory neurons in the dorsal root ganglia as well as motor neurons in the anterior horn of the spinal cord after peripheral nerve injury, and that it promotes the regeneration of peripheral nerve fibers.

Erythropoietin inhibits ferroptosis and ameliorates neurological function after spinal cord injury

Ferroptosis is one of the critical pathological events in spinal cord injury.Erythropoietin has been reported to improve the recovery of spinal cord injury.However,whether ferroptosis is involved in the neuroprotective effects of erythropoietin on spinal cord injury has not been examined.In this study,we established rat models of spinal cord injury by modified Allen’s method and intraperitoneally administered 1000 and 5000 IU/kg erythropoietin once a week for 2 successive weeks.Both low and high doses of erythropoietin promoted recovery of hindlimb function,and the high dose of erythropoietin led to better outcome.High dose of erythropoietin exhibited a stronger suppressive effect on ferroptosis relative to the low dose of erythropoietin.The effects of erythropoietin on inhibiting ferroptosis-related protein expression and restoring mitochondrial morphology were similar to those of Fer-1(a ferroptosis suppressor),and the effects of erythropoietin were largely diminished by RSL3(ferroptosis activator).In vitro experiments showed that erythropoietin inhibited RSL3-induced ferroptosis in PC12 cells and increased the expression of xCT and Gpx4.This suggests that xCT and Gpx4 are involved in the neuroprotective effects of erythropoietin on spinal cord injury.Our findings reveal the underlying anti-ferroptosis role of erythropoietin and provide a potential therapeutic strategy for treating spinal cord injury.

Electrical stimulation of cortical neurons promotes oligodendrocyte development and remyelination in the injured spinal cord

Background and early studies： Endogenous tri-potential neural stem cells （NSCs） exist in the adult mammalian central nervous system （CNS）. In the spinal cord, NSCs distribute throughout the entire cord, but exist predominately in white matter tracts. The phenotypic fate of these cells in white matter is glial, largely oligodendrocyte, but not neuronal.

Protective effect of sodium valproate on motor neurons in the spinal cord following sciatic nerve injury in rats

BACKGROUND: Sodium valproate (VPA) is used to be an effective anti-epileptic drug. VPA possesses the characteristics of penetrating rapidly through the blood-brain barrier (BBB) and increasing levels of Bcl-2 and growth cone-associated protein (GAP) 43 in spinal cord. OBJECTIVE: To observe the effect of VPA on Bcl-2 expression and motor neuronal apoptosis in spinal cord of rats following sciatic nerve transection. DESIGN: Randomized controlled experiment. SETTING: Department of Hand Surgery and Microsurgery, Wuhan Puai Hospital. MATERIALS: A total of 30 male healthy SD rats of clean grade and with the body mass of 180-220 g were provided by Experimental Animal Center of Medical College of Wuhan University. Sodium Valproate Tablets were purchases from Hengrui Pharmaceutical Factory, Jiangsu. METHODS: The experiment was performed in the Central Laboratory of Wuhan Puai Hospital and Medical College of Wuhan University from February to May 2006. Totally 30 rats were randomly divided into two groups: treatment group (n =15) and model group (n =15). Longitudinal incision along backside of right hind limbs of rats was made to expose sciatic nerves, which were sharply transected 1 cm distal to the inferior margin of piriform muscle after nerve liberation under operation microscope to establish sciatic nerve injury rat models. Sodium Valproate Tablets were pulverized and diluted into 50 g/L suspension with saline. On the day of operation, the rats in the treatment group received 6 mL/kg VPA suspension by gastric perfusion, once a day, whereas model group received 10 mL/kg saline by gastric perfusion, once a day. L4-6 spinal cords were obtained at days 1, 4, 7, 14 and 28 after operation, respectively. Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) technique and immunohistochemical method (SP method) were used to detect absorbance (A) of neurons with positive Bcl-2 expression. Apoptotic rate of cells (number of apoptotic cells/total number of cells×100%) was calculated. MAIN OUTCOME MEASURES: A value of neurons with positive Bcl-2 expression and apoptotic rate in spinal cord of rats in the two groups. RESULTS: A total of 30 SD rats were involved in the result analysis. ①expression of positive Bcl-2 neurons: A value of positive Bcl-2 neurons were 0.71±0.02, 0.86±0.04, 1.02±0.06 at days 4, 7 and 14, respectively after operation in the treatment group, which were obviously higher than those in the model group (0.62±0.03, 0.71±0.05, 0.89±0.04, t = 3.10-4.50, P < 0.05). ②apoptotic result of motor neurons: Apoptotic rate of motor neurons in spinal cord was (6.91±0.89)% and (15.12±2.34)% at days 7 and 14 in the treatment group, which was significantly lower than those in the model group [(9.45±1.61)%, (19.35±0.92)%, t = 2.39, 3.03. P < 0.05]. CONCLUSION: VPA can increase expression of Bcl-2 in spinal cord and reduce neuronal apoptosis in rats following sciatic nerve injury, and has protective effect on motor neuron in spinal cord of rats.

Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury

Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.

Transplanting neural progenitors to build a neuronal relay across the injured spinal cord

Cellular transplantation for repair of spinal cord injury is a prom- ising therapeutic strategy that includes the use of a variety of neural and non-neural cells isolated or derived from embryonic and adult tissue as well as embryonic stem cells and induced plu- ripotent stem cells. In particular, transplants of neural progenitor cells （NPCs） have been shown to limit secondary injury and scar formation and create a permissive environment in the injured spinal cord through the provision of neurotrophic molecules and growth supporting matrices that promote growth of injured host axons. Importantly, transplants of NPC are unique in their poten- tial to replace lost neural cells - including neurons, astrocytes,

The Double Projecting Neuron of the Cat Spinal Dorsal Horn——A Study with Double Retrograde Fluorescent Tracing Technique

The new double projecting neurons were found in the cat spinal dorsal horn by the double retrograde fluorescent tracing technique.Fast Blue(FB)was injected into unilateral dorsal column nucleus(DCN)of adult cats anesthetized with pentobarbital.Nuclear Yellow(NY)was injected ipsilaterally into the lateral cervical nucleus(LCN)8-9 days later.After an additional 18-30 hrs.

Protein kinase C regulates neurite outgrowth in spinal cord neurons

Objective The functional roles of protein kinase C （PKC） in the neurite outgrowth and nerve regeneration remain controversial. The present study was aimed to investigate the role of PKC in neurite outgrowth, by studying their regulatory effects on neurite elongation in spinal cord neurons in vitro. Methods The anterior-horn neurons of spinal cord from embryonic day 14 （E14） Sprague-Dawley （SD） rats were dissociated, purified and cultured in the serum-containing medium. The ratio of membrane-PKC （mPKC） activity to cytoplasm-PKC （cPKC） activity （m/c-PKC） was studied at different time points during culture. Results Between 3-11 d of culture, the change of m/c-PKC activity ratio and PKC-βⅡ expression in the neurite were both significantly correlated with neurite outgrowth （r=0.95, P 〈 0.01; r=0.73, P 〈 0.01, respectively）. Moreover, PMA, an activator of PKC, induced a dramatic elevation in the m/c-PKC activity ratio, accompanied with the increase in neurite length （r=-0.99, P 〈 0.01）. In contrast, GF 109203X, an inhibitor of PKC, significantly inhibited neurite elongation, which could not be reversed by PMA. Conclusion PKC activity may be important in regulating neurite outgrowth in spinal cord neurons, and βⅡ isoform of PKC probably plays a major role in this process.

Shenfu injection attenuates neurotoxicity of bupivacaine in cultured mouse spinal cord neurons

Background Our previous in vivo study in the rat demonstrates that Shenfu injection, a clinically used extract preparation from Chinese herbs, attenuates neural and cardiac toxicity induced by intravenous infusion of bupivacaine, a local anesthetic. This study was designed to investigate whether bupivacaine could induce a toxic effect in pnmary cultured mouse spinal cord neuron and if so, whether the Shenfu injection had a similar neuroprotective effect in the cell model. Methods The spinal cords from 11 - to 14-day-old fetal mice were minced and incubated. Cytarabine was added into the medium to inhibit the proliferation of non-neuronal cells. The immunocytochemical staining of β-tubulin was used to determine the identity of cultured cells. The cultured neurons were randomly assigned into three sets treated with various doses of bupivacaine, Shenfu and bupivacaine＋Shenfu, for 48 hours respectively. Cell viability in each group was analyzed by methyl thiazoleterazolium （MTT） assay. Results The viability of the cultured neurons treated with bupivacaine at concentrations of 0.01%, 0.02%, 0.04% and 0.08% was decreased in a dose-dependent manner. Although the Shenfu injection at concentrations ranging from 1/50 to 1/12.5 （VN） had no significant influence on the viability of cultured neurons （P〈0.05 vs control）, the injection significantly increased the cellular viability of cultured neurons pretreated with 0.03% bupivacaine （P〈0.05）. Conclusion Although Shenfu injection itself has no effect on spinal neurons, it was able to reduce the bupivacaineinduced neurotoxicity in vitro.

Functional recovery after rhesus monkey spinal cord injury by transplantation of bone marrow mesenchymal-stem cell-derived neurons

Background The treatment of spinal cord injury is still a challenge. This study aimed at evaluating the therapeutical effectiveness of neurons derived form mesenchymal stem cells （MSCs） for spinal cord injury. Methods In this study, rhesus MSCs were isolated and induced by cryptotanshinone in vitro and then a process of RT-PCR was used to detect the expression of glutamic acid decarboxylase （GAD） gene. The induced MSCs were tagged with Hoechst 33342 and injected into the injury site of rhesus spinal cord made by the modified Allen method. Following that, behavior analysis was made after 1 week, 1 month, 2 months and 3 months. After 3 months, true blue chloride retrograde tracing study was also used to evaluate the reestablishment of axons pathway and the hematoxylin-eosin （HE） staining and immunohistochemistry were performed after the animals had been killed. Results In this study, the expression of mRNA of GAD gene could be found in the induced MSCs but not in primitive MSCs and immunohistochemistry could also confirm that rhesus MSCs could be induced and differentiated into neurons. Behavior analysis showed that the experimental animals restored the function of spinal cord up to grade 2 -3 of Tarlov classification. Retrograde tracing study showed that true blue chollide could be found in the rostral thoracic spinal cords, red nucleus and sensory-motor cortex. Conclusions These results suggest that the transplantation is safe and effective.

Crocetin Potentiates Neurite Growth in Hippocampal Neurons and Facilitates Functional Recovery in Rats with Spinal Cord Injury

Crocetin is an ingredient of traditional Chinese medicine and has therapeutic potential in various diseases due to its pharmacological properties, such as neuroprotection, anti-oxidative stress, and anti-inflammation. These properties might benefit the treatment of spinal cord injury.In the present study, we tested the effect of crocetin on neurite growth and sensorimotor dysfunction in a rat model of spinal cord injury. We evaluated the viability of cultured hippocampal neurons with tetrazolium dye and lactate dehydrogenase assays, visualized neurites and axons with antibody staining, and monitored motor and sensorimotor functions in rats with spinal cord injury using the Basso,Beattie, and Bresnahan assay and the contact plantar placement test, respectively, and measured cytokine expression using enzyme-linked immuno-absorbent assays.We found that crocetin（1） did not alter the viability of cultured hippocampal neurons;（2） accelerated neurite growth with preference for the longest process in individual hippocampal neurons;（3） reversed the inhibition of neurite growth by chondroitin sulfate proteoglycan and Nogo A;（4） facilitated the recovery of motor and sensorimotor functions after spinal cord injury; and（5） did not inhibit pro-inflammatory responses, but restored the innervation of the descending 5-HT system in injured spinalcord. Crocetin promotes neurite growth and facilitates the recovery of motor and sensorimotor functions after spinal cord injury, likely through repairing neuronal connections.

Can Hybrid Synapse be Formed between Rat Spinal Motor Neurons and Major Pelvic Ganglion Neurons in vitro?

The purpose of this study is to determine whether synapses can be formed between spinal motor neurons(SMNs)and major pelvic ganglion(MPG)neurons of a rat in vitro.The green fluorescent protein(GFP)-labelled MPG cells were cultured together with SMNs in a specific medium.The synaptic-like contacts established between SMNs and MPG neurons were studied in co-cultures using morphologic and immunocytochemistry approaches.Phase-contrast observation of co-cultures showed apparent SMNs-MPG neurons contacts as early as three or four days in vitro.We demonstrate some evidence of synaptic contacts between SMNs and MPG neurons in vitro by immunostaining with antibody directed against postsynaptic density protein 95(PSD-95).We describe the development process of a defined SMNs-MPG neurons co-culture system.The results suggest that the hybrid synapse formation that may occur between SMNs and MPG neurons in vitro played an essential role in the mechanisms of a regenerated bladder with an artificial somatic-autonomic reflex arc.

Biomaterials for spinal cord repair

Spinal cord injury （SCI） results in permanent loss of function leading to often devastating personal, economic and social problems. A contributing factor to the permanence of SCI is that damaged axons do not regenerate, which prevents the re-establishment of axonal circuits involved in function. Many groups are working to develop treatments that address the lack of axon regeneration after SCI. The emergence of biomaterials for regeneration and increased collaboration between engineers, basic and translational scientists, and clinicians hold promise for the development of effective therapies for SCI. A plethora of biomaterials is available and has been tested in various models of SCI. Considering the clinical relevance of contusion injuries, we primarily focus on polymers that meet the specific criteria for addressing this type of injury. Biomaterials may provide structural support and/or serve as a delivery vehicle for factors to arrest growth inhibition and promote axonal growth. Designing materials to address the specific needs of the damaged central nervous system is crucial and possible with current technology. Here, we review the most prominent materials, their optimal characteristics, and their potential roles in repairing and regenerating damaged axons following SCI.

Inhibition of Autophagy by Estradiol Promotes Locomotor Recovery after Spinal Cord Injury in Rats

17β-estradiol（E2） has been shown to have neuroprotective effects in different central nervous system diseases. The mechanisms underlying estrogen neuroprotection in spinal cord injury（SCI） remain unclear. Previous studies have shown that autophagy plays a crucial role in the course of nerve injury. In this study, we showed that E2 treatment improved the restoration of locomotor function and decreased the loss of motor neurons in SCI rats. Realtime PCR and western blot analysis revealed that the protective function of E2 was related to the suppression of LC3 II and beclin-1 expression. Immunohistochemical study further confirmed that the immunoreactivity of LC3 in the motor neurons was down-regulated when treated with E2. In vitro studies demonstrated similar results that E2 pretreatment decreased the autophagic activity induced by rapamycin（autophagy sensitizer） and increased viability in a PC12 cell model. These results indicated that the neuroprotective effects of E2 in SCI are partly related to the suppression of excessive autophagy.