Nogo-A Protein Expression Research Articles

MiR-182-5p Regulates Nogo-A Expression and Promotes Neurite Outgrowth of Hippocampal Neurons In Vitro.

Nogo-A protein is a key myelin-associated inhibitor of axonal growth, regeneration, and plasticity in the central nervous system (CNS). Regulation of the Nogo-A/NgR1 pathway facilitates functional recovery and neural repair after spinal cord trauma and ischemic stroke. MicroRNAs are described as effective tools for the regulation of important processes in the CNS, such as neuronal differentiation, neuritogenesis, and plasticity. Our results show that miR-182-5p mimic specifically downregulates the expression of the luciferase reporter gene fused to the mouse Nogo-A 3′UTR, and Nogo-A protein expression in Neuro-2a and C6 cells. Finally, we observed that when rat primary hippocampal neurons are co-cultured with C6 cells transfected with miR-182-5p mimic, there is a promotion of the outgrowth of neuronal neurites in length. From all these data, we suggest that miR-182-5p may be a potential therapeutic tool for the promotion of axonal regeneration in different diseases of the CNS.

Xuesaitong exerts long-term neuroprotection for stroke recovery by inhibiting the ROCKII pathway, in vitro and in vivo

Ethnopharmacological relevanceXuesaitong (XST) is a traditional Chinese medicine injection with neuroprotective properties and has been extensively used to treat stroke for many years. The main component of XST is Panax notoginseng saponins (PNS), which is the main extract of the Chinese herbal medicine Panax notoginseng. Aim of the studyIn this study, we investigated whether XST provided long-term neuroprotection by inhibiting neurite outgrowth inhibitor-A (Nogo-A) and the ROCKII pathway in experimental rats after middle cerebral artery occlusion (MCAO) and in SH-SY5Y cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R). Materials and methodsRats with permanent MCAO were administered XST, Y27632, XST plus Y27632, and nimodipine for 14 and 28 days. Successful MCAO onset was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Neurological deficit score (NDS) was used to assess neurological impairment. Hematoxylin-eosin (HE) staining and immunohistochemical (IHC) analysis of synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) were performed to evaluate cerebral ischemic injury and the neuroprotective capability of XST. Nogo-A levels and the ROCKII pathway were detected by IHC analysis, western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR) to explore the protective mechanism of XST. OGD/R model was established in SH-SY5Y cells. Cell counting kit 8 (CCK8) was applied to detect the optimum OGD time and XST concentration. The expression levels Nogo-A and ROCKII pathway were determined using western blotting. ResultsOur results showed that XST reduced neurological dysfunction and pathological damage, promoted weight gain and synaptic regeneration, reduced Nogo-A mRNA and protein levels, and inhibited the ROCKII pathway in MCAO rats. CCK8 assay displayed that the optimal OGD time and optimal XST concentration were 7 h and 20 μg/mL respectively in SH-SY5Y cells. XST could evidently inhibit OGD/R-induced Nogo-A protein expression and ROCKII pathway activation in SH-SY5Y cells. ConclusionsThe present study suggested that XST exerted long-term neuroprotective effects that assisted in stroke recovery, possibly through inhibition of the ROCKII pathway.

The Effect of Mild Hypothermia on Nogo-A and Neurological Function in the Brain after Cardiopulmonary Resuscitation in Rats

Objective We investigated the dynamic changes of Nogo-A protein in brain and the effects of mild therapeutic hypothermia (MTH) on its expression after cardiopulmonary resuscitation (CPR). Methods: Western-blotting and neurological scoring of 45 rats subjected to cardiac arrest and CPR with and without MTR were performed to investigate the changes in the expression of Nogo-A protein in the hippocampus and cortex over a period of time ranging from 6 h to 72 h after restoration of spontaneous circulation (ROSC). Results: Nogo-A expression levels were increased at 6 h after CPR in the hippocampus and cortex, peaked at 24 h in the cortex, and at 48 h in the hippocampus. The expression of Nogo-A in the MTR group was significantly lower at 12 h (p < 0.05) compared to those with no MTR after ROSC. Conclusions: MTR blunts the expression of Nogo-A protein in the hippocampus and cortex after cardiac arrest and resuscitation, and MTR may provide cerebral protection after ischemia.

The effect of mild hypothermia on Nogo-A and neurological function in the brain after cardio-pulmonary resuscitation in rats

The aim of the current study was to investigate dynamic changes in brain Nogo-A protein in rats, and the effects of mild hypothermia therapy on its expression after Cardio-Pulmonary Resuscitation (CPR). Western-blotting and neurological scoring of 45 rats subjected to cardiac arrest and CPR were performed to investigate the expression of Nogo-A protein in the hippocampus and cortex 6 h, 12 h, 24 h, 48 h, and 72 h after restoration of spontaneous circulation (ROSC). Nogo-A expression levels gradually increased from 6 h after CPR in the brain. In the hippocampus and cortex, Nogo-A exhibited a unimodal waveform-first it increased, but then it decreased. It peaked at 24 h in the hippocampus, and at 48 h in the cortex. The expression of Nogo-A in the mild hypothermia group was significantly lower at 12 h (p<0.05). Mild hypothermia can reduce the expression of Nogo-A protein in the hippocampus and cortex after CPR, which may be one of the mechanisms that play a role in cerebral protection.

Role of Nogo-A in the regulation of hepatocellular carcinoma SMMC-7721 cell apoptosis

Nogo-A has been identified as an inhibitor of neurite outgrowth specific to the central nervous system. However, little is known about the role of Nogo-A in hepatocellular carcinoma (HCC), the most common primary malignant tumor with a high mortality rate. This study aimed to investigate the role of endogenous Nogo-A in human liver cancer cells. Reverse transcription polymerase chain reaction was used to detect the expression of Nogo-A in four liver cancer cell lines. A lentivirus vector was then constructed to mediate RNA interference (RNAi) targeting of Nogo‑A (LV‑Nogo-A‑siRNA) and was confirmed to successfully suppress the expression of the Nogo-A gene in SMMC-7721 cells. Furthermore, Nogo-A was observed to be highly expressed in liver cancer cell lines. RNAi of Nogo-A using the LV‑Nogo-A‑siRNA construct significantly decreased Nogo-A protein expression and specifically inhibited the growth of SMMC-7721 cells. This growth inhibitory effect may be attributed to an increase in G2/M phase arrest and apoptosis in SMMC-7721 cells containing Nogo-A‑siRNA. The results of this study demonstrate that Nogo-A may represent a novel therapeutic target for the treatment of liver cancer, in addition to its potent roles in neural systems.

Protection of NEP1-40 on retinal cells following retinal ischemia reperfusion injury

Background The retina ischemia reperfusion injury (RIRI) can lead to apoptosis,which is associated with many genes.It is very significant to explore the protection of drugs on RIRI-induced apoptosis.Objective This study was to explore the relationship between the expression of Nogo-A and the cell apoptosis as well as the therapeutic effect of NEP1-40 in RIRI retina.Methods The device of raising intraocular pressure (IOP)was used to elevate the IOP for 60 minutes and then restore the IOP to normal for the establishment of RIRI models.Seventy-eight SD rats were randomized to the normal group,RIRI group and NEP1-40 group.PBS of 5 ml/(kg · d)was injected intraperitoneally in the rats of the RIRI group,and NEP1-40 of 8 mg/(L · kg) was used in the same way in the NEP1-40 group.The rats were sacrificed in overdose anesthesia at 6 hours,12 hours and 1 day,2,3,7 days after RIRI to prepare the retinal specimens.The ultrastructure of rat retinas was examined under the transmission electron microscope.Cell apoptosis was assessed by the TUNEL method,and the apoptotic index (AI) was calculated.The expressions of Nogo-A protein and mRNA in rat retinas were detected by immunohistochemistry and semiquantitative reverse transcription PCR (RT-PCR).Results The ultrastructure of retinal cells were normal in the normal group.Retinal cell organelle dissolution,mitochondria swelling,cavitation,chromatin edge heterochromatin and apoptotic body were seen in the rats of the RIRI group from 12 hours through 7 days after injection.However,only the slight loose of outer membranous disk,inner and outer nuclear layer and retinal ganglion cells,the nucleus gap broadening,shortness of some mitochondrial cristae in the NEP1-40 group.TUNEL-positive cells appeared 6 hours after RIRI and reached peak in 1 day,and gradually declined after that in the RIRI group.A similar pattern was seen in the rats of the NEP1-40 group with a more mild manifestation.Significant differences were seen in the AI values among the different groups at various time points (Fgroup =100.850,P =0.000 ; Ftime =34.309,P =0.000),and the AI values were significantly higher in the RIRI group and NEP1-40 group compared with normal group;while the AI values in the NEP1-40 group was lower than those of the RIRI group (all at P＜0.05).The expressions of Nogo-A protein and mRNA showed a coincident pattern with apoptosis procedure,with a gradually elevated level from 6 hours through 7 days after RIRI and a peak in 2 days,and those in the NEP1-40 group were weaker in comparison with the RIRI group in various time points.Significant differences were detected in the expression of Nogo-A protein and the expression of Nogo-A mRNA among different groups and various time points(protein:Fgroup =164.139,P =0.000;Ftime =21.772,P =0.000.mRNA:Fgroup =93.889,P =0.000 ; Ftime =6.349,P =0.000).Conclusions Nogo-A probably plays an important role in RIRI.NEP1-40 can protect retinal cells from apoptosis following RIRI through down-regulating the expression of Nogo-A. Key words: NEP1-40; Retina; Ischemia reperfusion injury; Nogo-A; Apoptosis

Down-regulation of Nogo-A by collagen scaffolds impregnated with bone marrow stromal cell treatment after traumatic brain injury promotes axonal regeneration in rats

Nogo-A is a major form of growth inhibitory molecule (growth-IM) which inhibits axonal regeneration and neurite regrowth after neural injury. Bone marrow stromal cells (MSCs) have been shown to inhibit Nogo-A expression in vitro and in cerebral ischemic animal models. The present study was designed to investigate the effects of treatment with human MSCs (hMSCs) impregnated into collagen scaffolds on the expression of Nogo-A and axonal plasticity after traumatic brain injury (TBI). Adult male Wistar rats were injured with controlled cortical impact and treated either with saline, hMSCs-alone or hMSCs impregnated into collagen scaffolds (scaffold+hMSC) transplanted into the lesion cavity 7 days after TBI. Rats were sacrificed 14 days after TBI and brain tissues were harvested for immunohistochemical studies, Western blot analysis, laser capture microdissections and qRT-PCR to evaluate axonal density and Nogo-A protein and gene expressions. Our data showed that treatment of TBI with scaffold+hMSC significantly decreased TBI-induced Nogo-A protein expression and increased axonal density compared to saline and hMSC-alone treatments. In addition, scaffold+hMSC transplantation decreased Nogo-A transcription in oligodendrocytes after TBI. Scaffold+hMSC treatment was superior to hMSC-alone treatment in suppressing Nogo-A expression and enhancing axonal regeneration after TBI. Our data suggest that transplanting hMSCs with scaffolds down-regulates Nogo-A transcription and protein expression which may partially contribute to the enhanced axonal regeneration after TBI.

Methylprednisolone inhibits Nogo-A protein expression after acute spinal cord injury.

Oligodendrocyte-produced Nogo-A has been shown to inhibit axonal regeneration. Methylprednisolone plays an effective role in treating spinal cord injury, but the effect of methylprednisolone on Nogo-A in the injured spinal cord remains unknown. The present study established a rat model of acute spinal cord injury by the weight-drop method. Results showed that after injury, the motor behavior ability of rats was reduced and necrotic injury appeared in spinal cord tissues, which was accompanied by increased Nogo-A expression in these tissues. After intravenous injection of high-dose methylprednisolone, although the pathology of spinal cord tissue remained unchanged, Nogo-A expression was reduced, but the level was still higher than normal. These findings implicate that methylprednisolone could inhibit Nogo-A expression, which could be a mechanism by which early high dose methylprednisolone infusion helps preserve spinal cord function after spinal cord injury.

Changes in expression of Nogo-A protein in dorsal root ganglion and spinal dorsal horn in a rat model of inflammatory pain

Objective To evaluate the changes in the expression of Nogo-A protein in the dorsal root ganglion (DRG) and spinal dorsal horn in a rat model of inflammatory pain.Methods One hundred and twenty male Sprague-Dawley rats,weighing 250-300 g,were randomly divided into 2 groups (n =60 each):control group (group C) and formalin group (group F).The inflammatory pain was induced by injection of 3％ formalin 100 μl into the plantar surface of left hindpaw in group F.The equal volume of normal saline was given instead of formalin in group C.Mechanical withdrawal threshold to yon Frey filament stimulation was measured at 1 h and 1,2,3 and 7 days after injection.Twelve rats in each group were chosen at each time point and sacrificed.The L5 DRG and L4,5 segment of spinal cord on the operated side were removed for determination of Nogo-A protein expression by immunofluorescence and Western blot.Remlts Compared with group C,the mechanical withdrawal threshold was significantly decreased at 1 h,1,2,3 and 7 days after injection,and the expression of Nogo-A protein in the DRG and L4,5 segment of spinal cord was up-regulated at 1 h and 2,3 and 7 days after injection in group F (P ＜0.05).Conclusion Up-regulation of Nogo-A protein in the DRG and spinal dorsal horn may play an important role in the development of formalin-induced inflammatory pain in rats. Key words: Infammation; Pain; Spinal cord; Ganglia, spinal; Nogo-A protein

Time Course and Spatial Profile of Nogo-A Expression in Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice

Inhibition of the myelin-associated neurite outgrowth inhibitor Nogo-A has been found to be beneficial in experimental autoimmune encephalomyelitis (EAE), but there are little data on its expression dynamics during the disease course. We analyzed Nogo-A mRNA and protein during the course of EAE in 27 C57BL/6 mice and in 8 controls. Histopathologic and molecular analyses were performed on Day 0 (naive), preclinical (Day 10), acute (Days 18-22) and chronic (Day 50) time points. In situ hybridization and real-time polymerase chain reaction analyses revealed reduced Nogo-A mRNA expression at preclinical (p < 0.0001) and acute phases (p < 0.0001), followed by upregulation during the chronic phase (p < 0.0001). Nogo-A mRNA was expressed in neurons and oligodendrocytes. By immunohistochemistry and Western blot, there was increased Nogo-A protein expression (p < 0.001) in the chronic phase. Moreover, spatial differences were observed within EAE lesions. The pattern of Nogo-A protein expression inversely correlated with axonal regeneration growth-associated protein 43-positive axons (60% of which were Nogo-A contact-free during the acute phase) and axonal injury (β-amyloid precursor protein-positive axons). Cortical Nogo-66 receptor protein and mRNA levels increased during the chronic phase. The results indicate that Nogo-A and Nogo receptor are actively regulated in EAE lesions; this may indicate a specific time window for localized axonal regeneration in the acute phase of EAE.

P-380 - Spatial cognition in nogo-a-deficient transgenic rats as an animal model: effects of ageing and behavioral conditions

Introduction Research into mechanisms of interaction between growth and inhibitory proteins of the CNS and behavioral expressions of healthy and disordered brain is one of the contemporary topics. A knockdown model with decreased expression of Nogo-A protein in neurons was developed on the genetic background of Sprague-Dawley wildtypes. Disruption of this inhibitory factor was hypothesized to result in behavioral abnormalities, which were studied both in young, middle age (6 months) and aged (12 months) rats. Methods Animals were tested in a battery of Carousel maze variants with various demands for segregation of spatial information and flexibility; animals avoided an unmarked sector of either stable or rotating arena; moreover the sector could be defined in room- or arena-frame. A shortened Carousel maze battery and Morris water maze (MWM) including one- trial matching-to-place and reversal configurations was used. Results Nogo-A-deficient rats were impaired in the final phases of the Carousel maze battery but their spatial working memory tested in the MWM was intact. Middle-aged and aged groups were differently affected in the battery, but deficits in young animals were observed not to be worsened with ageing. Concept of multidirectional age-related alterations in this animal model is further supported by biochemical brain changes. Conclusion Nogo-A-deficient rats may serve as an extremely useful model of neurodevelopmental deficit, which may manifest by behavioral changes accessible to phenotyping and in-depth analysis. Relevance of this approach for animal models of neuropsychiatric models will be discussed.

Changes in expression of Nogo-A protein in dorsal root ganglion and spinal dorsal horn in a rat model of neuropathic pain

Objective To investigate the changes in the expression of Nogo-A protein in the dorsal root ganglion (DRG) and spinal dorsal horn in a rat model of neuropathic pain (NP) .Methods Seventy-two male SD rats weighing 250-300 g were randomly divided into 3 groups ( n = 24 each) : control group (group C) , sham operation group (group S) and NP group. NP was induced by ligation and severance of tibial and common fibular nerves according to the technique described by Isabelle et al. The mechanical withdrawal threshold (MWT) to von Frey filament stimulation was measured at 1, 7, 14 and 21 days after ligation. Six rats in each group were randomly selected at each time point and sacrificed (3 for determination of Nogo-A protein expression by immunofluorescence, 3 for determination of Nogo-A protein expression by Western blot) . The L5 DRG and L4,5 segment of spinal cord on the injured side were removed for determination of Nogo-A protein expression by immunofluorescence and Western blot. Results Compared with the groups C and S, MWT was significantly decreased at 7, 14 and 21 days after ligation, the expression of Nogo-A protein in the DRG was down-regulated at 7 and 14 days after ligation and the expression of Nogo-A protein in the spinal dorsal horn was up-regulated at 14 and 21 days after ligation ( P ＜0.05) .Conclusion The Nogo-A protein in the DRG and spinal dorsal horn may play an important role in peripheral nerve injury-induced NP in rats. Key words: Neuralgia; Spinal cord; Ganglia, spinal; Nogo-A protein

The effects of thioredoxin on Nogo-A after acute spinal cord injury in rats

Objective To observe the effects of thioredoxin (Trx) perfused through subarachnoid space on Nogo-A after acute spinal cord injury (ASCI) in rats and to probe into protective effects of Trx on ASCI. Methods Fifty-six rats were randomly divided into 3 groups: sham group (group A, n=8 ) , SCI group (group B,n =24) and Trx group (group C,n =24). The ASCI model was induced using the modified Allen technique in groups B and C. A thin plastic tube was placed in subarachnoid space below the injury level for perfusion. Rats in group C received Trx (0.75 mg/kg) from the tube everyday after injury, those in group B received the equal volume of normal saline at the same time as control, and those in group A only received vertebral laminectomy and tube placement in subarachnoid space. The BBB score was recorded at the 3rd, 7th and 14th day postinjury (n=8), then the rats were sacrificed and the injured spinal cord was taken out. Immunohistochemistry was used to detect the expression of Nogo-A and positive cells were counted at the 3rd, 7th and 14th day. Results The BBB score in group B was significantly lower than that in group A at each time point (P <0.01) , but the score in group C was significantly higher than that in group B (P<0.05 or 0.01) at the 7th and 14th day postinjury. The number of positive cells was significantly more in group B than in group A at the 7th and 14th day postoperatively (P<0.01). The number of positive cells was significantly more in group C than in group B at the 7th and 14th day postoperatively (P<0.05). Conclusion The expression of Nogo-A is notably increased in injured spinal cord. Trx can inhibit the expression of Nogo-A protein obviously, which can promote the regeneration of axon after ASCI possibly. Key words: Thioredoxin; Spinal cord injury; Nogo-A; Regenaration; Axon

Hippocampal Nogo-A and neo-Timm's staining in amygdala kindling rats

Purpose: Nogo-A, one of the axon regeneration inhibitors, has been shown to be up-regulated in both the experimental and human temporal lobe epilepsy. However, the role of Nogo-A in mossy fiber sprouting (MFS) relative to epileptogenesis is unknown. This work was designed to examine the relationship of the hippocampal Nogo-A protein expression with MFS during the development of amygdala kindling.Methods: Using immunohistochemistry and neo-Timm's histological procedures, we evaluated the distribution and density of Nogo-A and Nogo-66 receptor (Ng-R) expression and MFS in the bilateral hippocampus of amygdala kindling rats.Results: Nogo-A expression in the ipsilateral hippocampus gradually increased with the development of kindling in the sector CA2–3. In contrast, no increased Nogo-A expression was observed in the contralateral hippocampus as the rats advanced to stage 5 kindled seizures. Furthermore, poorer Nogo-A and Nogo-66 receptor (Ng-R) expression were observed in the dentate granule cells as aberrant MFS occurred.Conclusions: In amygdala kindling rats, generalized stage 5 seizures were not associated with increased Nogo-A expression in the contralateral hippocampus supporting the concept that seizures by themselves do not induce Nogo-A expression. Furthermore, in the ipsilateral hippocampus, the expression of Nogo-A relative to MSF suggests that this protein may partially control aberrant synaptic reorganization during epileptogenesis.

Time-dependent increase in Nogo-A expression after focal cerebral ischemia in marmoset monkeys

Nogo-A is a myelin-associated protein that has been shown to inhibit axonal sprouting after lesions to the CNS. Several studies have demonstrated that blocking the activity or expression of this inhibitor can induce structural and functional recovery after CNS lesions. However, there are limited and contradictory data on the expression of Nogo-A after CNS lesions. In the present study, marmoset monkeys received permanent occlusion of the middle cerebral artery (MCAo). Two, 3, or 4 months after the onset of injury brain sections were stained for Nogo-A protein. Two sham operated marmosets were included as a control. Nogo-A protein expression was quantified in white matter and grey matter in the areas adjacent to the lesion (or the equivalent areas in the intact side). At 2 months after injury, but not at 3 or 4 months, there was a significant increase in the number of oligodendrocytes that were Nogo-A immunopositive. This increase was observed in white matter structures that were adjacent to the lesion (e.g. corona radiate (CR)); but not in: white matter structures distal to the lesion (e.g. corpus callosum (CC)); cortical regions adjacent to the lesion; contralateral regions or in sham operated marmosets. These data suggest that Nogo-A levels are significantly increased within oligodendrocytes in areas adjacent to the lesion up to 2 months following cerebral ischeamia. Future studies will determine whether this offers the opportunity to promote plasticity by targeting Nogo-A weeks or months following stroke.

Cell size-dependent Nogo-A expression in layer V pyramidal neurons of the rat primary somatosensory cortex

Nogo-A mRNA and protein are present in the perikarya of neurons in both the intact and injured CNS. The present study focused primarily on Nogo-A protein expression in the primary somatosensory cortex of the adult rat. Coronal brain sections were probed with double-immunofluorescent labeling against Nogo-A together with NeuN, RBPC, or MAP-2 for confocal imaging. The sizes of the cell somata in pyramidal neurons and the thicknesses of neurites were measured on the captured confocal images. Nogo-A was expressed in larger pyramidal neurons and thicker neurites in layer V, but not in smaller pyramidal neurons and thinner neurites. Considering the morphological properties and the cell soma size reported in previous studies together with the present data, Nogo-A-positive neurons of layer V appear to be intrinsically bursting neurons that project axons to the subcortical regions. This suggests that intraneuronal Nogo-A may play roles in neurite growth and axonal regeneration of the corticofugal neurons, but not of columnar intrinsic neurons, in layer V of the S1 barrel cortex. Additionally, this study demonstrates a novel result, which is that layer V pyramidal neurons of the S1 barrel cortex exhibit a pattern of cell size-dependent intraneuronal Nogo-A expression.