Electroacupuncture Inhibits Myelin Sheath Injury in the Internal Capsule After Focal Cerebral Infarction in Rats Through the Nogo-A/NgR Signaling Pathway.

BACKGROUND Ischemic stroke is usually accompanied by white matter damage. The effect of electroacupuncture (EA) on ameliorating white matter damage is still unclear. The purpose of this study was to explore the precise mechanism of EA in treating ischemic white matter. MATERIAL AND METHODS In this study, 40 Sprague-Dawley rats were randomly divided into 4 groups: normal group, the sham-operated group, model group, and EA group. The stroke model was established by right middle cerebral artery occlusion, and EA was performed 24 h after the operation for 30 min per day. After 14 days of treatment, brain tissue samples were collected. Hematoxylin and eosin and Luxol fast blue staining were used to observe the changes of white matter damage in the internal capsule (IC). The expression levels of myelin basic protein (MBP), Nogo-A, and Nogo-A receptor (NgR) were detected by immunohistochemistry and western blot. RESULTS Compared with the sham-operated group, the model group had decreased expression of MBP and significantly increased expression of Nogo-A and NgR (P<0.05). Compared with the model group, the IC damage was alleviated in the EA group. Immunohistochemistry and western blot analysis showed that EA significantly increased the expression of MBP in white matter (P<0.05) and downregulated the expression levels of Nogo-A and NgR (P<0.05). CONCLUSIONS The results of this study indicate that EA can inhibit the expression of Nogo-A/NgR and promote myelin sheath regeneration.

Fasudil alleviates brain damage in rats after carbon monoxide poisoning through regulating neurite outgrowth inhibitor/oligodendrocytemyelin glycoprotein signalling pathway.

Carbon monoxide (CO) poisoning can lead to many serious neurological symptoms. Currently, there are no effective therapies for CO poisoning. In this study, rats exposed to CO received hyperbaric oxygen therapy, and those in the Fasudil group were given additional Fasudil injection once daily. We found that the escape latency in CO poisoning group (CO group) was significantly prolonged, the T1 /Ttotal was obviously decreased, and the mean escape time and the active escape latency were notably extended compared with those in normal control group (NC group, P < 0.05). After administration of Fasudil, the escape latency was significantly shortened, T1 /Ttotal was gradually increased as compared with CO group (>1 week, P < 0.05). Ultrastructural damage of neurons and blood-brain barrier of rats was serious in CO group, while the structural and functional integrity of neuron and mitochondria maintained relatively well in Fasudil group. Moreover, we also noted that the expressions of neurite outgrowth inhibitor (Nogo), oligodendrocyte-myelin glycoprotein (OMgp) and Rock in brain tissue were significantly increased in CO group, and the elevated levels of the three proteins were still observed at 2 months after CO poisoning. Fasudil markedly reduced their expressions compared with those of CO group (P < 0.05). In summary, the activation of Nogo-OMgp/Rho signalling pathway is associated with brain injury in rats with CO poisoning. Fasudil can efficiently down-regulate the expressions of Nogo, OMgp and Rock proteins, paving a way for the treatment of acute brain damage after CO poisoning.

The protective effect of the active components of ERPC on diabetic peripheral neuropathy in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Euonymus alatus, Radix trichosanthis, Panax notoginseng and Coptis chinensis are popular plants used in traditional Chinese medicine to treat diabetes. AIM OF THE STUDY: The aim of the study is to investigate the therapeutic effect of the active components of Euonymus alatus, Radix trichosanthis, Panax notoginseng and Coptis chinensis (cERPC) on diabetic peripheral neuropathy in the rats and explore the underlying mechanism involved. METHODS: After diabetes was induced in rats for 20 weeks, cERPC or water was administered for 12 weeks. After a hot plate test, motor nerve conduction velocity and sciatic nerve blood flow were determined; the sciatic nerves were isolated for toluidine blue staining; and the fibre area, fibre diameter, axon area, axon diameter and myelin thickness were evaluated. The levels of the myelin basic protein, myelin protein zero, Oct6 and Krox20 were measured by western blot or immunofluorescence. RESULTS: cERPC was efficient in reducing the response latency, increasing motor nerve conduction velocity, enhancing sciatic nerve blood flow and ameliorating the pathological changes in diabetic rats. cERPC also had a role in increasing the levels of myelin basic protein and myelin protein zero and improving the expression of Oct6 and Krox20 in sciatic nerves of diabetic rats. CONCLUSIONS: cERPC ameliorates diabetic peripheral neuropathy by attenuating electrophysiological, circulatory and morphological alterations, which is mediated by the Oct6-Krox20 pathway.

Capparis ovata treatment suppresses inflammatory cytokine expression and ameliorates experimental allergic encephalomyelitis model of multiple sclerosis in C57BL/6 mice.

Since ancient times, Capparis species have been widely used in traditional medicine to treat various diseases. Our recent investigations have suggested Capparis ovata's potential anti-neuroinflammatory application for the treatment of multiple sclerosis (MS). The present study was designed to precisely determine the underlying mechanism of its anti-neuroinflammatory effect in a mouse model of MS. C. ovata water extract (COWE) was prepared using the plant's fruit, buds, and flower parts (Turkish Patent Institute, PT 2012/04,093). We immunized female C57BL/6J mice with MOG35-55/CFA. COWE was administered at a daily dose of 500mg/kg by oral gavage either from the day of immunization (T1) or at disease onset (T2) for 21days. Gene expression analysis was performed using a Mouse Multiple Sclerosis RT² Profiler PCR Array, and further determinations and validations of the identified genes were performed using qPCR. Whole-genome transcriptome profiling was analyzed using Agilent SurePrint G3 Mouse GE 8X60K microarrays. Immunohistochemical staining was applied to brain sections of the control and treated mice to examine the degree of degeneration. COWE was further fractionated and analyzed phytochemically using the Zivak Tandem Gold Triple Quadrupole LC/MS-MS system. COWE remarkably suppressed the development of EAE in T1, and the disease activity was completely inhibited. In the T2 group, the maximal score was significantly reduced compared with that of the parallel EAE group. The COWE suppression of EAE was associated with a significantly decreased expression of genes that are important in inflammatory signaling, such as TNFα, IL6, NF-κB, CCL5, CXCL9, and CXCK10. On the other hand, the expression of genes involved in myelination/remyelination was significantly increased. Immunohistochemical analysis further supported these effects, showing that the number of infiltrating immune cells was decreased in the brains of COWE-treated animals. In addition, differential expression profiling of the transcriptome revealed that COWE treatment caused the down regulation of a group of genes involved in the immune response, inflammatory response, antigen processing and presentation, B-cell-mediated immunity and innate immune response. Collectively, these results suggest anti-neuroinflammatory mechanisms by which COWE treatment delayed and suppressed the development of EAE and ameliorated the disease in mice with persistent clinical signs.

[Effect of Electroacupuncture Intervention Combined with Gastrodin Administration on Neurological Function, Nogo-A and NgR Expression in the Frontal Lobe Cortex of Focal Cerebral Ischemia Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention combined with medication (Gastrodin) on changes of neurological function and expression of Nogo-A and Nogo-A receptor (NgR) in the frontal lobe cortex around the ischemic loci of focal cerebral ischemia (FCI) rats, so as to explore its mechanism underlying improvement of neuroregeneration of FC. METHODS: Fifty male Sprague-Dawley rats were randomly divided into normal control, model, EA, medication and EA+ medication groups (n = 10 in each group). The FCI model was induced by occlusion of the middle cerebral artery (MCAO) with thread embolus. EA was applied to the left "Quchi" (LI 11) and "Hegu" (Li 4) for 30 min, once daily for 14 days after MCAO. For rats of the medication group, Gastrodin (10 mg/kg) was administrated by intraperitoneal injection, once daily for 14 days. The neurological impairment was assessed by Zea Longa's scoring. The expression of Nogo-A and NgR in the frontal lobe cortex around the ischemic loci was detected by immunohistochemistry. RESULTS: In comparison with the normal control group, cerebro- cortical Nogo-A and NgR expression levels of the model group vere significantly increased (P < 0.05). Compared with the model group, the Zea Longa's score and Nogo-A and NgR expression levels were evidently down-regulated in the EA, medication and EA + medication groups (P < 0.05). The Zea Longa's score and Nogo-A and NgR expression levels were significantly lower in the EA + medication group than in the EA and medication groups (P < 0.05). CONCLUSION: EA intervention and Gastrodin administration can down-regulate cerebro-cortical Nogo-A and NgR protein expression in FCI rats, which may contribute to their action in improving neurological impairment. The effect of EA+ Gastrodin is better than simple EA or Gastrodin treatment.

Yonkenafil: a novel phosphodiesterase type 5 inhibitor induces neuronal network potentiation by a cGMP-dependent Nogo-R axis in acute experimental stroke.

Yonkenafil is a novel phosphodiesterase type 5 (PDE5) inhibitor. Here we evaluated the effect of yonkenafil on ischemic injury and its possible mechanism of action. Male Sprague-Dawley rats underwent middle cerebral artery occlusion, followed by intraperitoneal or intravenous treatment with yonkenafil starting 2h later. Behavioral tests were carried out on day 1 or day 7 after reperfusion. Nissl staining, Fluoro-Jade B staining and electron microscopy studies were carried out 24h post-stroke, together with an analysis of infarct volume and severity of edema. Levels of cGMP-dependent Nogo-66 receptor (Nogo-R) pathway components, hsp70, apaf-1, caspase-3, caspase-9, synaptophysin, PSD-95/neuronal nitric oxide synthases (nNOS), brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) and nerve growth factor (NGF)/tropomyosin-related kinase A (TrkA) were also measured after 24h. Yonkenafil markedly inhibited infarction and edema, even when administration was delayed until 4h after stroke onset. This protection was associated with an improvement in neurological function and was sustained for 7d. Yonkenafil enlarged the range of penumbra, reduced ischemic cell apoptosis and the loss of neurons, and modulated the expression of proteins in the Nogo-R pathway. Moreover, yonkenafil protected the structure of synapses and increased the expression of synaptophysin, BDNF/TrkB and NGF/TrkA. In conclusion, yonkenafil protects neuronal networks from injury after stroke.

Effect of high-dose 1.25 dihydroxyvitamin D3 on remyelination in the cuprizone model.

Vitamin D supplementation is increasingly recommended to patients with multiple sclerosis (MS). To study the effect of high-dose vitamin D on remyelination, female C57Bl/6 mice were demyelinated with dietary 0.2% cuprizone for 7 weeks. The mice received intraperitoneal injections of 1.25-dihydroxyvitamin D3 (calcitriol) or placebo (vehicle) injections twice a week, from week 6, throughout week 9. Mice that received calcitriol had initially increased demyelination (p = 0.021), astrocytosis (p = 0.043), and microglia activation. However, levels of astrocytosis and microglia activation dropped below those of the placebo group during the remyelination phase. There was a significant increase in myelination in the calcitriol group throughout the remyelination phase (p = 0.041), while the remyelination in the placebo group was not significant (p = 0.317). After 3 weeks of remyelination, the calcitriol group had more myelin than the placebo group (p = 0.001). The calcitriol group had a higher density of NOGO-A positive cells throughout the remyelination phase, and the number of NOGO-A positive cells was significantly higher in the calcitriol group at one week of remyelination (p = 0.019). There were no significant differences in extent of T-lymphocyte infiltration. High-dose calcitriol seems to be safe regarding remyelination. Our results indicate that this treatment could actually promote the repair process, possibly through a stimulating effect on oligodendrocyte maturation and astrocyte activation. The potential of calcitriol to stimulate the remyelination process should be investigated further in functional studies.

[Effect of electric acupuncture on the expression of NgR in the cerebral cortex, the medulla oblongata, and the spinal cord of hypertensive rats after cerebral infarction].

OBJECTIVE: To observe the effect of electric acupuncture (EA) on the Nogo receptors (NgR) protein expression in the cerebral cortex, the medulla oblongata, and the spinal cord of cerebral ischemia-reperfusion (I/R) stroke-prone renovascular hypertensive rats (RHRSP) with middle cerebral artery occlusion (MCAO) at different time points, and to investigate its possible mechanisms for remote-organ injury of acute cerebral infarction (ACI). METHODS: The RHRSP model was duplicated in male SPF grade SD rats. Then the MCAO model was prepared by a thread stringing method. Rats were divided into the hypertension group,the sham-operation group, the MCAO group, the EA group, and the sham-acupoint group by random number table method, 60 in each group. Rats in the MCAO group only received MCAO reperfusion treatment. Those in the sham-operation group only received surgical trauma. Baihui (DU20) and Dazhui (DU14) were needled in the EA group, once daily for a total of 28 days.The needles were acupunctured at the skin one cun distant from Baihui (DU20) and Dazhui (DU14) and then the same EA treatment was performed in the sham-acupoint group. At day 1, 7, 14, 28 after treatment, six rats were executed from each group, and their right cortex and medulla oblongata, and the left spinal cord were isolated. The infarct volume was detected by Nissl's staining method. The NgR expression was detect by Western blot. RESULTS: (1) In the cortex area: compared with the hypertension group,the NgR expression increased in the MCAO group at day 1,7,14,and 28 after MCAO (P < 0.05). Compared with the MCAO group, the NgR expression of the EA group and the sham-acupoint group were equivalent at 1 day af ter MCAO (P > 0.05). At day 7, 14,and 28 after MCAO, the NgR expression decreased in the EA group (P < 0.05), it was quite similar to that in the sham-acupoint group (P > 0.05). (2) In the medulla oblongata area: compared with the hypertension group, the NgR expression was equivalent in the sham-operation group. the MCAO group,the EA group, and the sham-acupoint group at 1 day after MCAO (P > 0.05). At day 7.14, and 28 after MCAO, the NgR expression increased in the MCAO group (P < 0.05). Compared with the MCAO group,the NgR expression decreased in the EA group at day 7, 14, and 28 after MCAO (P < 0.05), whereas it was similar in the sham-acupoint group (P > 0.05). (3) In the spinal cord area: compared with the hypertension group, the NgR expression was equivalent in the sham-operation group, the MCAO group,the EA group, and the sham-acupoint group at day 1 and 7 after MCAO (P > 0.05). At day 14 and 28 after MCAO, the NgR expression increased in the MCAO group (P < 0.05). Compared with the MCAO group, the NgR expression decreased in the EA group at day 14 and 28 after MCAO (P < 0.05), whereas it was equivalent in the sham-acupoint group (P > 0.05). CONCLUSIONS: Increased NgR expression in the cerebral cortex, the medulla oblongata, and the spinal cord of cerebral infarct rats was an important reason for involving remote-organ injury of ACI. The protective effect of EA on hypertensive I/R cerebral injury rats might be closely related to down-regulating central nervous system myelin growth inhibition mediated factors Nogo-A receptor NgR protein expression.

[Effects of electroacupuncture on the ultrastructure and the Nogo-A expressions in the cerebral cortex in rats with cerebral ischemia-reperfusion].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on the expressions of Nogo-A and the ultrastructure in the cerebral cortex at different time points after the cerebral ischemia-reperfusion in rats. METHODS: One hundred and thirty male Sprague Dawley (SD) rats were randomly divided into the EA group (n = 30), the sham-EA group (n = 30), the model group (n = 30), the sham-operation group (n = 30), and the blank group (n = 10). The modified ZeaLonga method was used to prepare the left middle cerebral artery occlusion (MCAO) model in the first three groups. After the operation Baihui (DU20) and Dazhui (DU14) were daily needled in the EA group. One inch beside Baihui (DU20) and Dazhui (DU14) were daily needled in the sham-EA group. Rats in the model group were only treated with MCAO ischemia/reperfusion. Rats in the sham-operation group only received surgical wound. No treatment was given to rats in the blank group. The ultrastructures of ischemic cells and the intervention of the Nogo-A expressions were observed using the immunohistochemical staining and the transmission electron microscope 1, 7, and 28 days after EA. RESULTS: (1) In the EA group, the damage of ultrastructures of neurons, gliocytes, and blood brain barrier in the ischemic region was alleviated when compared with that of the sham-EA group and the model group. (2) On the 1st, 7th and 28th day after the cerebral ischemia-reperfusion, the expressions of Nogo-A in the ischemic cortex in the EA group was lower when compared with those in the sham-EA group and the model group at the corresponding time points, showing significant difference (P < 0.05). But there was no statistical difference between the sham-EA group and the model group at the same time point (P > 0.05). CONCLUSION: The mechanism of EA for protecting cerebral ischemia/reperfusion might be closely associated with alleviating the damage on the ultrastructures of brain cells, and down-regulating the expressions of Nogo-A.

Preclinical retinal neurodegeneration in a model of multiple sclerosis.

Neurodegeneration plays a major role in multiple sclerosis (MS), in which it is thought to be the main determinant of permanent disability. However, the relationship between the immune response and the onset of neurodegeneration is still a matter of debate. Moreover, recent findings in MS patients raised the question of whether primary neurodegenerative changes can occur in the retina independent of optic nerve inflammation. Using a rat model of MS that frequently leads to optic neuritis, we have investigated the interconnection between neurodegenerative and inflammatory changes in the retina and the optic nerves with special focus on preclinical disease stages. We report that, before manifestation of optic neuritis, characterized by inflammatory infiltration and demyelination of the optic nerve, degeneration of retinal ganglion cell bodies had already begun and ultrastructural signs of axon degeneration could be detected. In addition, we observed an early activation of resident microglia in the retina. In the optic nerve, the highest density of activated microglia was found within the optic nerve head. In parallel, localized breakdown in the integrity of the blood-retinal barrier and aberrations in the organization of the blood-brain barrier marker aquaporin-4 in the optic nerves were observed during the preclinical phase, before onset of optic neuritis. From these findings, we conclude that early and subtle inflammatory changes in the retina and/or the optic nerve head reminiscent of those suggested for preclinical MS lesions may initiate the process of neurodegeneration in the retina before major histopathological signs of MS become manifest.

Effect of tonifying liver and kidney-essence herbs on expression of Nogo-A and p75(NTR) in cerebral ischemic stroke rats model.

OBJECTIVE: To observe the effect of tonifying liver and kidney-essence herbs on expression of a nerve regeneration inhibitor, Nogo for neuron A (Nogo-A), and its associated signaling molecule, low-affinity neurotrophin receptor p75 (p75(NTR)), in rats with cerebral ischemic stroke (CIS), with the aim of exploring the possible mechanism of tonifying liver and kidney-essence herbs in recovery following injury to the central nervous system. METHODS: A cerebral ischemic stroke model in SD rats was established with the suture-occlusion method. Successful model rats were divided into placebo and herb groups at random; sham-operated and control groups were set up simultaneously. Each of these groups was divided into six subgroups at random. Expression of Nogo-A and p75(NTR) was evaluated with immunofluorescence microscopy at days 3, and weeks 1, 2, 3, 4 and 8 after administration. RESULTS: Tonifying liver and kidney-essence herbs suppressed the expression of Nogo-A and p75(NTR) (P < 0.05 and P < 0.01, respectively). CONCLUSION: Suppressing the expression of Nogo-A and p75(NTR) is possibly one of the mechanisms underlying the ability of tonifying liver and kidney-essence herbs to promote recovery of the injured central nervous system.

Postmortem and imaging based analyses reveal CNS decreased myelination in restless legs syndrome.

BACKGROUND: Restless legs syndrome (RLS) is a neurological disorder characterized by a strong urge to move the legs and has been shown in many studies with abnormally low brain iron. Iron deficiency is associated with hypomyelination in brains of animals. Therefore we hypothesized that a myelin deficit should be present in the brains of patients with RLS. METHODS: We performed Western blot analysis on myelin isolated from RLS (n=11) and control (n=11) brain tissue obtained at autopsy for the expression of the integral myelin proteins, myelin basic protein (MBP), and proteolipid protein (PLP) and the oligodendrocyte specific enzyme 3'5'-cyclic nucleotide phosphohydrolase (CNPase). To expand the postmortem findings to in vivo, we analyzed the brains of RLS patients (n=23) and controls (n=23) using voxel-based morphometry (VBM). RESULTS: The expression of MBP, PLP and CNPase in the myelin from RLS was decreased by approximately 25% (p<0.05) compared to controls. The amounts of transferrin (Tf) and H-ferritin (H-Frt) in the myelin fraction were also significantly decreased in RLS compared to controls. The imaging analysis revealed significant small decreases in white matter volume in RLS patients compared to controls in the corpus callosum, anterior cingulum and precentral gyrus. CONCLUSION: A decrease in myelin similar to that reported in animal models of iron deficiency was found in the brains of individuals with RLS. The evidence for less myelin and loss of myelin integrity in RLS brains, coupled with decreased ferritin and transferrin in the myelin fractions, is a compelling argument for brain iron insufficiency in RLS. These data also indicate the need to look beyond the sensorimotor symptoms that typically define the syndrome and its assumed relation to the dopaminergic system. Understanding the full range of RLS pathology may help us better understand the complex, intermittent nature and diversity of the clinical features of RLS and expand our consideration of treatment options for RLS.

Correlation of neural response properties with auditory thalamus subdivisions in the awake marmoset.

As the information bottleneck of nearly all auditory input that reaches the cortex, the auditory thalamus serves as the basis for establishing auditory cortical processing streams. The functional organization of the primary and nonprimary subdivisions of the auditory thalamus is not well characterized, particularly in awake primates. We have recorded from neurons in the auditory thalamus of awake marmoset monkeys and tested their responses to tones, band-pass noise, and temporally modulated stimuli. We analyzed the spectral and temporal response properties of recorded neurons and correlated those properties with their locations in the auditory thalamus, thereby forming the basis for parallel output channels. Three medial geniculate body (MGB) subdivisions were identified and studied physiologically and anatomically, although other medial subdivisions were also identified anatomically. Neurons in the ventral subdivision (MGV) were sharply tuned for frequency, preferred narrowband stimuli, and were able to synchronize to rapid temporal modulations. Anterodorsal subdivision (MGAD) neurons appeared well suited for temporal processing, responding similarly to tone or noise stimuli but able to synchronize to the highest modulation frequencies and with the highest temporal precision among MGB subdivisions. Posterodorsal subdivision (MGPD) neurons differed substantially from the other two subdivisions, with many neurons preferring broadband stimuli and signaling changes in modulation frequency with nonsynchronized changes in firing rate. Most neurons in all subdivisions responded to increases in tone sound level with nonmonotonic changes in firing rate. MGV and MGAD neurons exhibited responses consistent with provision of thalamocortical input to core regions, whereas MGPD neurons were consistent with provision of input to belt regions.

Research on the mechanism of Zuogui Pill and Yougui Pill in promoting axonal regeneration in model rats of autoimmune encephalomyelitis.

OBJECTIVE: To study the molecular mechanism of Zuogui Pill (ZGP) and Yougui Pill (YGP) on axonal regeneration in rats with experimental autoimmune encephalomyelitis (EAE). METHODS: EAE rat model was established by bilateral rear pedes subcutaneous injection of antigen made by mixing myelin basic protein (MBP) and complete Freud's adjuvant (CFA) in the volume ratio of 1:1. The pathological changes of axonal injury and regeneration in the brain and the spinal cord were observed on the 14th (the acute stage) and the 28th day (the remission stage) after modeling, with hematoxylin-eosin (HE) staining, silver stain, and immunohistochemical staining. The rats treated with prednisone acetate were taken as controls. RESULTS: Observation under the light microscope with HE staining showed a sleeve-like change in rats' cerebrospinal parenchyma with inflammatory cell infiltration around the small vessels and neuronic denaturation, while silver staining showed excessive tumefaction and abscission of axon, and immunohistochemical analysis showed decreasing of nerve growth factor (NGF) expression at the acute stage of EAE, which was even more remarkable at the remission stage, showing significant difference as compared with the normal control (P<0.05). And the expressions of Nogo A, an axon growth inhibitor, and its receptor (Nogo-66 receptor, Ng R) were significantly higher than those in the normal control at the acute stage (P<0.01). However, after the intervention of ZGP and YGP, the pathological changes and axon damage in rats' brain and spinal cord were much more alleviated, and the NGF expression was significantly higher than that in the model group at the acute stage (P<0.05). The expression of NGF was even stronger during the remission stage, and a better effect was shown by YGP. As for Nogo A and Ng R expressions, they were significantly lower than those in the model group at the acute stage (P<0.05), but a better effect was shown by ZGP. CONCLUSIONS: ZGP and YGP can prevent axonal injury and promote the axonal regeneration in rats of EAE, and the possible mechanism is to increase the expression of NGF and reduce the expression of Nogo A and its receptor. However, some differences are observed between the two Chinese preparations in their acting times and points, which provides a certain basis for revealing the modern connotation of the Chinese medicine theory on tonifying Shen ()-yin and Shen-yang.

Effect of combined therapy with ephedrine and hyperbaric oxygen on neonatal hypoxic-ischemic brain injury.

Perinatal hypoxic-ischemic (HI) is a major cause of brain injury in the newborn, and there is a lack of effective therapies to reduce injury-related disorders. The aim of the present study was to evaluate the effect of a combination of ephedrine and hyperbaric oxygen (HBO) on neonatal hypoxic-ischemic brain injury. 7-day-old Sprague-Dawley rat pups were randomly divided into sham operation, HI, ephedrine, HBO, and combined group. The ephedrine group was intraperitoneally injected with ephedrine, HBO group was treated for 2h at 2.5 absolute atmosphere (ATA) per day, the combined group received both ephedrine and HBO treatments, the sham operation and HI groups were intraperitoneally injected with normal saline. Rat brains at 7 days after HI, were collected to determine histopathological damage and the expression levels of Caspase-3 and Nogo-A. Four weeks after insult, animals were challenged with Morris water maze test. The expressions of Caspase-3 and Nogo-A were reduced in treating groups compared to those in HI group (P<0.01). Compared with the single treatment groups, the expression levels of Caspase-3 and Nogo-A were significantly reduced in the combined group (P<0.01). Compared with the single treatment groups, the average time of escape latency was significantly shorter (P<0.01) and the number of platform location crossing was more (P<0.05) in combined group. These findings indicate that the combination of ephedrine and HBO can enhance the neuroprotective effect in the neonatal rat HI model partially mediated by inhibiting Caspase-3 and Nogo-A pathways.

Architectonic subdivisions of neocortex in the tree shrew (Tupaia belangeri).

Tree shrews are small mammals that bear some semblance to squirrels, but are actually close relatives of primates. Thus, they have been extensively studied as a model for the early stages of primate evolution. In this study, subdivisions of cortex were reconstructed from brain sections cut in the coronal, sagittal, or horizontal planes, and processed for parvalbumin, SMI-32-immunopositive neurofilament protein epitopes, vesicle glutamate transporter 2 (VGluT2), free ionic zinc, myelin, cytochrome oxidase, and Nissl substance. These different procedures revealed similar boundaries between areas, suggesting the detection of functionally relevant borders and allowed a more precise demarcation of cortical areal boundaries. Primary cortical areas were most clearly revealed by the zinc stain, because of the poor staining of layer 4, as thalamocortical terminations lack free ionic zinc. Area 17 (V1) was especially prominent, as the broad layer 4 was nearly free of zinc stain. However, this feature was less pronounced in primary auditory and somatosensory cortex. In primary sensory areas, thalamocortical terminations in layer 4 densely express VGluT2. Auditory cortex consists of two architectonically distinct subdivisions, a primary core region (Ac), surrounded by a belt region (Ab) that had a slightly less developed koniocellular appearance. Primary motor cortex (M1) was identified by the absence of VGluT2 staining in the poorly developed granular layer 4 and the presence of SMI-32-labeled pyramidal cells in layers 3 and 5. The presence of well-differentiated cortical areas in tree shrews indicates their usefulness in studies of cortical organization and function.

Nogo-A is involved in secondary axonal degeneration of thalamus in hypertensive rats with focal cortical infarction.

We investigate whether Nogo-A is involved in the secondary axonal degeneration in the thalamus after distal middle cerebral artery occlusion (MCAO) in stroke-prone renovascular hypertensive rats (RHRSP). The expression of Nogo-A in ipsilateral ventroposterior nucleus (VPN) of the thalamus in RHRSP was observed at 1, 2 and 4 weeks after distal MCAO. In addition, intracerebroventricular infusion of NEP1-40, a Nogo-66 receptor (NgR) antagonist peptide, was administered starting 24 h after MCAO and continued for 1, 2 and 4 weeks, respectively. Axonal damage and regeneration were evaluated by analysis of the immunoreactivity (IR) of amyloid betaA4 precursor protein (APP), growth associated protein 43 (GAP-43) and microtubule associated protein 2 (MAP-2) in ipsilateral VPN of the thalamus at 1, 2 and 4 weeks after distal MCAO. Following ischemia, the expression of Nogo-A in oligodendrocytes increased persistently and its localization became redistributed around damaged axons and dendrites. Administration of NEP1-40 downregulated the expression of Nogo-A, reduced axonal injury and enhanced axonal regeneration. Our data suggest that Nogo-A is involved in secondary axonal degeneration and that inhibition of Nogo-A can reduce neuronal damage in the thalamus after distal MCAO.

Tissue specificity and regulation of the N-terminal diversity of reticulon 3.

Over the last few years, the widely distributed family of reticulons (RTNs) is receiving renewed interest because of the implication of RTN4/Nogo in neurite regeneration. Four genes were identified in mammals and are referred to as RTN1, 2, 3 and the neurite outgrowth inhibitor RTN4/Nogo. In the present paper, we describe the existence of five new isoforms of RTN3 that differ in their N-termini, and analysed their tissue distribution and expression in neurons. We redefined the structure of human and murine rtn3 genes, and identified two supplementary exons that may generate up to seven putative isoforms arising by alternative splicing or differential promoter usage. We confirmed the presence of five of these isoforms at the mRNA and protein levels, and showed their preferential expression in the central nervous system. We analysed rtn3 expression in the cerebellum further, and observed increased levels of several of the RTN3 isoforms during cerebellum development and during in vitro maturation of cerebellar granule cells. This pattern of expression paralleled that shown by RTN4/Nogo isoforms. Specifically, RTN3A1 expression was down-regulated upon cell death of cerebellar granule neurons triggered by potassium deprivation. Altogether, our results demonstrate that the rtn3 gene generates multiple isoforms varying in their N-termini, and that their expression is tightly regulated in neurons. These findings suggest that RTN3 isoforms may contribute, by as yet unknown mechanisms, to neuronal survival and plasticity.

HBO suppresses Nogo-A, Ng-R, or RhoA expression in the cerebral cortex after global ischemia.

Nogo-A, a myelin-associated neurite outgrowth inhibitory protein, binds with the Ng-R receptor to activate RhoA intracellular signals and inhibit the plasticity after CNS injury. We evaluated the effect of hyperbaric oxygen (HBO) on the expression of Nogo-A, Ng-R, and RhoA after transient global ischemia in a rat 2 vessel occlusion global ischemic model. Male SD rats (n=78) were randomly divided into 13 groups: 1 sham group, 6 groups of global ischemia, and 6 groups of HBO treatment after global ischemia. HBO (3ATA) was applied for 2 hr at 1 hr after global ischemia. Rats were sacrificed at 6, 12, 24, 48, and 96 hr and 7 days. Global ischemia (10 min) produced a marked increase of Nogo-A/B, Nogo-A, Ng-R, and RhoA expression. Immunohistochemistry showed increased Nogo-A/B and Nogo-A located in the myelin sheath of ischemic brain cortex. Ng-R expressed on the surface of neurons and their processes, and RhoA expressed inside the cytoplasm of neurons in ischemic brain. HBO significantly reduced neurological injury, decreased the levels of Nogo-A, Ng-R, and RhoA in ischemic injured cortex (p<0.05).

The cytoplasmic domain of the myelin P0 protein influences the adhesive interactions of its extracellular domain.

The extracellular domain of the myelin P0 protein is believed to engage in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that P0 can behave as a homophilic adhesion molecule through interactions of its extracellular domains (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.) 344:871-872). To determine if the cytoplasmic domain of P0 must be intact for the extracellular domains to adhere, we compared the adhesive capabilities of P0 proteins truncated at the COOH-terminal to the full-length P0 protein. P0 cDNAs lacking nucleotides coding for the last 52 or 59 amino acids were transfected into CHO cells, and surface expression of the truncated proteins was assessed by immunofluorescence, surface labeling followed by immunoprecipitation, and an ELISA. Cell lines were chosen that expressed at least equivalent amounts of the truncated P0 proteins at the surface as did a cell line expressing the full-length P0. The adhesive properties of these three cell lines were compared. It was found that when a suspension of single cells was allowed to aggregate for a period of 60 min, only the cells expressing the full-length P0 had formed large aggregates, while the cells expressing the truncated P0 molecules were still mostly single cells indistinguishable from the control cells. Furthermore, 25-30% of the full-length P0 was insoluble in NP40, indicative of an interaction with the cytoskeleton, whereas only 5-10% of P0 lacking 52 amino acids and none of P0 lacking 59 amino acids were insoluble. These results suggest that for the extracellular domain of P0 to behave as a homophilic adhesion molecule, its cytoplasmic domain must be intact, and most probably, it is interacting with the cytoskeleton.