MiR-137-3p rescue motoneuron death by targeting calpain-2.

Brachial plexus root avulsion (BPRA) is a type of injury that leads to motor function loss as a result of motoneurons (MNs) degeneration. Here we identified that the reduced expression of rat miR-137-3p in the ventral horn of spinal cord was associated with MNs death. However, the pathophysiological role of miR-137-3p in root avulsion remains poorly understood. We demonstrated that the calcium-activated neutral protease-2 (calpain-2) was a direct target gene of miR-137-3p with miR-137-3p binding to the 3'-untranslated region of calpain-2. Silencing of calpain-2 suppressed the expression of neuronal nitric oxide synthase (nNOS), a primary source of nitric oxide (NO). After avulsion 2 weeks, up-regulation of miR-137-3p in the spinal cord reduced calpain-2 levels and nNOS expression inside spinal MNs, resulting in an amelioration of the MNs death. These events provide new insight into the mechanism by which upregulation of miR-137-3p can impair MN survival in the BPRA.

Neuroprotective Effects of Methyl 3,4-Dihydroxybenzoate against TBHP-Induced Oxidative Damage in SH-SY5Y Cells.

This study investigated the neuroprotective effects of methyl 3,4-dihydroxybenzoate (MDHB) against t-butyl hydroperoxide (TBHP) induced oxidative damage in SH-SY5Y (human neuroblastoma cells) and the underlying mechanisms. SH-SY5Y were cultured in DMEM + 10% FBS for 24 h and pretreated with different concentrations of MDHB or N-acetyl-l-cysteine (NAC) for 4 h prior to the addition of 40 µM TBHP for 24 h. Cell viability was analyzed using the methylthiazolyltetrazolium (MTT) and lactate dehydrogenase (LDH) assays. An annexin V-FITC assay was used to detect cell apoptosis rates. The 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay was used to determine intracellular ROS levels. The activities of antioxidative enzymes (GSH-Px and SOD) were measured using commercially available kits. The oxidative DNA damage marker 8-OHdG was detected using ELISA. Western blotting was used to determine the expression of Bcl-2, Bax, caspase 3, p-Akt and Akt proteins in treated SH-SY5Y cells. Our results showed that MDHB is an effective neuroprotective compound that can mitigate oxidative stress and inhibit apoptosis in SH-SY5Y cells.

Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.

Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.

Longitudinal Assessments of Normal and Perilesional Tissues in Focal Brain Ischemia and Partial Optic Nerve Injury with Manganese-enhanced MRI.

Although manganese (Mn) can enhance brain tissues for improving magnetic resonance imaging (MRI) assessments, the underlying neural mechanisms of Mn detection remain unclear. In this study, we used Mn-enhanced MRI to test the hypothesis that different Mn entry routes and spatiotemporal Mn distributions can reflect different mechanisms of neural circuitry and neurodegeneration in normal and injured brains. Upon systemic administration, exogenous Mn exhibited varying transport rates and continuous redistribution across healthy rodent brain nuclei over a 2-week timeframe, whereas in rodents following photothrombotic cortical injury, transient middle cerebral artery occlusion, or neonatal hypoxic-ischemic brain injury, Mn preferentially accumulated in perilesional tissues expressing gliosis or oxidative stress within days. Intravitreal Mn administration to healthy rodents not only allowed tracing of primary visual pathways, but also enhanced the hippocampus and medial amygdala within a day, whereas partial transection of the optic nerve led to MRI detection of degrading anterograde Mn transport at the primary injury site and the perilesional tissues secondarily over 6 weeks. Taken together, our results indicate the different Mn transport dynamics across widespread projections in normal and diseased brains. Particularly, perilesional brain tissues may attract abnormal Mn accumulation and gradually reduce anterograde Mn transport via specific Mn entry routes.

Nitric oxide synthase inhibitor attenuates number of regenerating spinal motoneurons in adult rats.

Neuronal survival and death-related effects of nitric oxide synthase are widely studied, yet its potential involvement in regeneration remains largely unexplored. In the present study, the regenerative role of nitric oxide synthase in injured motoneurons was investigated. A ventral root was avulsed and a piece of peripheral nerve was implanted into the spinal cord. Results showed that nitric oxide synthase inhibitor reduced the number of regenerating motoneurons to half compared with sham-operated control at 2 weeks and 4 weeks after injury, but the rate of axonal regeneration was not affected. Our study adds a new line of evidence that expression of nitric oxide synthase is beneficial to the axonal regeneration of the injured spinal motoneurons.

Reactivated astrocytes as a possible source of oligodendrocyte precursors for remyelination in remitting phase of experimental autoimmune encephalomyelitis rats.

Multiple sclerosis (MS) is ademyelinating disease in the central nervous system (CNS). Majority of the MS patients show relapsing-remitting disease course. Evidences show that oligodendrocyte precursor cells (OPCs), which remain relatively quiescent in normal adult CNS, play a key role in the remitting phase by proliferation and remyelination. In the present study, we found that spinal cord astrocytesco-expressed progenitor cell marker and oligodendroglial lineage markers in the remittance phase in adult rat experimental autoimmune encephalomyelitis (EAE) model. We suggest that activated astrocyte could de-differentiate into OPCs and re-differentiate into mature oligodendrocytes, raising the possibility that astrocytes can be a potential source of OPCs in the adult demyelinated spinal cord.

PI3K/Akt signaling pathway is involved in the neurotrophic effect of senegenin.

Neurodegenerative diseases are frequently associated with the loss of synapses and neurons. Senegenin, extracted from the Chinese herb Polygala tenuifolia Willd, was previously found to promote neurite outgrowth and neuronal survival in primary cultured rat cortical neurons. The aim of the present study was to investigate the underlying mechanisms of senegenin-induced neurotrophic effects on rat cortical neurons. Primary cortical rat neurons were treated with various pharmacological antagonists and with or without senegenin, and subjected to MTT and western blot analysis to explore the effects of senegenin on cell survival as well as the activation of signaling pathways. Neurite outgrowth and neuronal survival induced by senegenin were significantly inhibited by A2A receptor antagonist ZM241385 and specific phosphoinositide-3 kinase (PI3K) inhibitor LY294002, but not by tropomyosin receptor kinase A receptor inhibitor K252a, mitogen-activated protein kinase kinase inhibitor PD98059 or protein kinase C inhibitor GÖ6976. Furthermore, senegenin enhanced the phosphorylation of Akt, which was blocked by LY294002. The present study revealed that the PI3K/Akt signaling pathway may be involved in the neurotrophic effects of senegenin.

Comparison between self-assembling peptide nanofiber scaffold (SAPNS) and fibrin sealant in neurosurgical hemostasis.

RADA16-I is a synthetic type I self-assembling peptide nanofiber scaffold (SAPNS) which may serve as a novel biocompatible hemostatic agent. Its application in neurosurgical hemostasis, however, has not been explored. Although RADA16-I is nontoxic and nonimmunogenic, its intrinsic acidity may potentially provoke inflammation in the surgically injured brain. We conducted an animal study to compare RADA16-I with fibrin sealant, a commonly used agent, with the hypothesis that the former would be a comparable alternative. Using a standardized surgical brain injury model, 30 Sprague-Dawley rats were randomized into three treatment groups: RADA16-I, fibrin sealant or gelatin sponge (control). Animals were sacrificed on day 3 and 42. Astrocytic and microglial infiltrations within the cerebral parenchyma adjacent to the operative site were significantly lower in the RADA16-I and fibrin sealant groups than control. RADA16-I did not cause more cellular inflammatory response despite its acidity when compared with fibrin sealant. Immunohistochemical studies showed infiltration by astrocytes and microglia into the fibrin sealant and RADA16-I grafts, suggesting their potential uses as tissue scaffolds. RADA16-I is a promising candidate for further translational and clinical studies that focus on its applications as a safe and effective hemostat, proregenerative nanofiber scaffold as well as drug and cell carrier.

Lithium chloride reinforces the regeneration-promoting effect of chondroitinase ABC on rubrospinal neurons after spinal cord injury.

After spinal cord injury, enzymatic digestion of chondroitin sulfate proteoglycans promotes axonal regeneration of central nervous system neurons across the lesion scar. We examined whether chondroitinase ABC (ChABC) promotes the axonal regeneration of rubrospinal tract (RST) neurons following injury to the spinal cord. The effect of a GSK-3beta inhibitor, lithium chloride (LiCl), on the regeneration of axotomized RST neurons was also assessed. Adult rats received a unilateral hemisection at the seventh cervical spinal cord segment (C7). Four weeks after different treatments, regeneration of RST axons across the lesion scar was examined by injection of Fluoro-Gold at spinal segment T2, and locomotor recovery was studied by a test of forelimb usage. Injured RST axons did not regenerate spontaneously after spinal cord injury, and intraperitoneal injection of LiCl alone did not promote the regeneration of RST axons. Administration of ChABC at the lesion site enhanced the regeneration of RST axons by 20%. Combined treatment of LiCl together with ChABC significantly increased the regeneration of RST axons to 42%. Animals receiving combined treatment used both forelimbs together more often than animals that received sham or single treatment. Immunoblotting and immunohistochemical analysis revealed that LiCl induced the expression of inactive GSK-3beta as well as the upregulation of Bcl-2 in injured RST neurons. These results indicate that in vivo, LiCl inhibits GSK-3beta and reinforces the regeneration-promoting function of ChABC through a Bcl-2-dependent mechanism. Combined use of LiCl together with ChABC could be a novel treatment for spinal cord injury.

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A.

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca(2+) content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.

Rat model of intracerebral hemorrhage permitting hematoma aspiration plus intralesional injection.

A combination of hematoma aspiration and local delivery of chemicals may be more effective than either therapy in intracerebral hemorrhage (ICH). The aim of the present study was to develop a rat model of hematoma aspiration plus intralesional injection after ICH. ICH was induced in adult Sprague-Dawley rats by an intrastriatal injection of bacterial collagenase IV. Hematoma aspiration was performed 3.5 h after ICH onset. Following aspiration, normal saline was injected into the lesion cavity. Hematoma aspiration with or without subsequent saline injection significantly reduced the hematoma volume, lesion volume, and perihematomal neutrophil infiltration. Hematoma aspiration plus subsequent intralesional injection is simple, feasible, and safe. This ICH model can be used to assess the effectiveness of hematoma removal plus local delivery of chemicals.

Elevated blood pressure aggravates intracerebral hemorrhage-induced brain injury.

Elevated blood pressure (BP) is commonly seen in patients with intracerebral hemorrhage (ICH), and is independently associated with poor functional outcomes. Little is known about how elevated BP influences ICH-related brain injury. In the present study, we investigated the physiological and brain histological changes, as well as functional recovery following ICH in renovascular hypertensive rats. Renovascular hypertension (RVHT) was achieved by applying a silver clip onto the left renal artery of adult Sprague-Dawley rats. ICH was induced by an intrastriatal injection of bacterial collagenase IV about 5-6 weeks after left renal artery clipping or the sham operation. Following induction of ICH, both the normotensive and RVHT rats demonstrated an ultra-acute elevation in BP. Elevated BP increased hematoma volume, brain swelling, and apoptosis in the perihematomal areas. Brain degeneration, including local atrophy and lateral ventricle enlargement, was greater in the RVHT rats. In addition, many proliferating cells were seen over the ipsilateral striatum in the RVHT rats after ICH. The modified limb placing tests were done weekly for 3 weeks. In line with the histological damage, elevated BP worsened neurological deficits. These results suggest that ICH in the hypertensive rats mimics the clinical scenario of hypertensive ICH and may provide a platform to study the mechanisms of ICH-induced brain injury and potential therapies for ICH.

Upregulated expression of GAP-43 mRNA and protein in anterior horn motoneurons of the spinal cord after brachial plexus injury.

BACKGROUND AND AIMS: This study is concerned with the expressions of growth-associated protein-43 (GAP-43) mRNA and protein in the anterior horn of the spinal cord after brachial plexus injury. METHODS: Animals were killed 1, 7, 14 days after injury and were divided into three injury groups: group 1, right C(7) ventral motor root avulsion; group 2, right C(7) ventral motor root avulsion and cut right C(5)-T(1) dorsal sensitive roots; and group 3, right C(7) ventral motor root avulsion plus right hemisection between C(5) and C(6) segment of the spinal cord. The combined behavioral scores (CBS) 1, 7 and 14 days after surgery were used in behavioral testing. Expressions of both GAP-43 mRNA and protein were analyzed using QRT-PCR and immunohistochemistry 14 days after surgery. RESULTS: Among the injury groups, rats in group 3 had the highest score and those in group 1, the lowest score. On day 14 after surgery, the expressions of GAP-43 mRNA and protein were evidently up-regulated compared to the control group, with the highest in group 3 and the lowest in group 1, showing significant differences among the three injury groups (p <0.01). CONCLUSIONS: Our study suggests that the expressions of GAP-43 mRNA and protein may be upregulated after brachial plexus injury, and GAP-43 protein is possibly associated with the axon regeneration and function reconstruction.

Early detection of neurodegeneration in brain ischemia by manganese-enhanced MRI.

This study aims to employ in vivo manganese-enhanced MRI (MEMRI) to detect neurodegenerative changes in two models of brain ischemia, photothrombotic cortical injury (PCI) and transient middle cerebral artery occlusion (MCAO) in rodents. After systemic Mn(2+) injection to both ischemic models, a close pattern of T1-weighted hyperintensity was observed throughout different brain regions in comparison to the distribution of GFAP, MnSOD and GS immunoreactivities, whereby conventional MRI could hardly detect such. In addition, the infarct volumes in the posterior parts of the brain had significantly reduced after Mn(2+) injection to the MCAO model. It is suggested that exogenous Mn(2+) injection may provide enhanced MEMRI detection of oxidative stress and gliosis early after brain ischemia. Manganese may also mediate infarctions at remote brain regions in transient focal cerebral ischemia before delayed secondary damage takes place.

LINGO-1 antagonist promotes functional recovery and axonal sprouting after spinal cord injury.

LINGO-1 is a CNS-specific protein and a functional component of the NgR1/p75/LINGO-1 and NgR1/TAJ(TROY)/LINGO-1 signaling complexes that mediate inhibition of axonal outgrowth. These receptor complexes mediate the axonal growth inhibitory effects of Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp) via RhoA activation. Soluble LINGO-1 (LINGO-1-Fc), which acts as an antagonist of these pathways by blocking LINGO-1 binding to NgR1, was administered to rats after dorsal or lateral hemisection of the spinal cord. LINGO-1-Fc treatment significantly improved functional recovery, promoted axonal sprouting and decreased RhoA activation and increased oligodendrocyte and neuronal survival after either rubrospinal or corticospinal tract transection. These experiments demonstrate an important role for LINGO-1 in modulating axonal outgrowth in vivo and that treatment with LINGO-1-Fc can significantly enhance recovery after spinal cord injury.

Survival, regeneration and functional recovery of motoneurons after delayed reimplantation of avulsed spinal root in adult rat.

We have established that extensive reinnervation and functional recovery follow immediate reimplantation of avulsed ventral roots in adult rats. In the present study, we examined the consequences of reimplantation delayed for 2 weeks after avulsion of the C6 spinal root. Twelve and 20 weeks after delayed reimplantation, 57% and 53% of the motoneurons in the injured spinal segment survived. More than 80% of surviving motoneurons regenerated axons into the reimplanted spinal root. Cholinesterase-silver staining revealed axon terminals on endplates in the denervated muscles. The biceps muscles in reimplanted animals had atrophied less than those in animals with avulsion only, as indicated by muscle wet weight and histological appearance. After electrical stimulation of the motor cortex or the C6 spinal root, typical EMG signals were recorded in biceps of reimplanted animals. The latency of the muscle potential at 20 weeks was similar to that of sham-operated controls. Behavioral recovery was demonstrated by a grooming test and ipsilateral forepaw movements were well coordinated in both voluntary and automatic activities. These results demonstrate that ventral root reimplantation can protect severed motoneurons, enable the severed motoneurons to regenerate axons, and enhance the recovery of forelimb function even when it is delayed for 2 weeks after avulsion.

Co-transplantation of schwann cells promotes the survival and differentiation of neural stem cells transplanted into the injured spinal cord.

The present study investigates whether Schwann cells (SCs) could promote the survival and differentiation of neural stem cells in the injured spinal cord. Neural stem cells were dissociated and cloned from the hippocampal tissue of newborn rats. SCs were also dissociated and purified simultaneously from the sciatic nerves of 4-day-old rats. The results showed that the number of surviving neural stem cells and differentiated neuron-like cells was significantly increased in the co-grafted (SCs and neural stem cells) group compared with the control group (neural stem cells only). Neuron-like cells that developed axon-like processes were observed more commonly in the co-grafted group. These results demonstrate that SCs can promote the survival and differentiation of transplanted neural stem cells in the injured spinal cord.

Survival, regeneration and functional recovery of motoneurons in adult rats by reimplantation of ventral root following spinal root avulsion.

We investigated the functional recovery of motoneurons after reimplanting an avulsed ventral root in a rat model of traction injury. The eighth cervical root (C8) was avulsed by controlled traction and immediately reimplanted to the spinal cord. Spinal nerves from neighbouring segments (C5, C6, C7 and T1) were ligated and cut. After 12 or 20 weeks, the survival, regeneration and functional recovery of spinal motoneurons were evaluated by Nissl staining, retrograde labelling of motoneurons, NOS histochemistry, histological examination of muscle and nerve-muscle junction, electromyography and behavioural observation. In the control animals, about 14% or 11% of spinal motoneurons survived 12 or 20 weeks postinjury, respectively. By contrast, in animals with ventral root reimplantation, 62% and 55% of motoneurons survived at 12 or 20 weeks postinjury, respectively. Retrograde labelling and histological examination indicated that about 90% of the surviving motoneurons in the C8 segment regenerated axons into the reimplanted ventral root. Staining the muscles with silver and cholinesterase revealed new motor endplates in the reinnervated muscle. Functionally significant electromyographic responses in flexor digitorum superficialis and flexor carpi radialis were observed in experimental animals; however, the average latency of the motor action potentials was greater than normal control. The grasping test showed functional recovery of finger flexors and median nerve. In conclusion, our results indicate that spinal motoneurons can regenerate axons through reimplanted roots and reinnervate muscles to recover partial function.

Highly restricted expression of Cre recombinase in cerebellar Purkinje cells.

The Purkinje neuron, one of the most fascinating components of the cerebellar cortex, is involved in motor learning, motor coordination, and cognitive function. Purkinje cell protein 2 (Pcp2/L7) expression is highly restricted to Purkinje and retinal bipolar cells, where it has been exploited to enable highly specific, Cre recombinase-mediated, site-specific recombination. Previous studies showed that mice carrying a Cre transgene produced by insertion of Cre cDNA into a small 2.88-kb Pcp2 DNA fragment expressed Cre in Purkinje cells; however, some Cre activity was also observed outside the target tissues. Here, we used Red-mediated recombineering to insert Cre cDNA into a 173-kb BAC carrying the entire intact Pcp2 gene, and characterize the resultant BAC/Cre transgenic mice for Cre expression. We show that BAC/Cre transgenic mice have exclusive Cre expression in Purkinje and bipolar cells and nowhere else. These mice will facilitate Purkinje cell and retinal bipolar cell-specific genetic manipulation.