Absence of μ opioid receptor mRNA expression in astrocytes and microglia of rat spinal cord

Cumulating evidence has demonstrated that μ opioid receptor (MOR) agonists promote spinal glial activation, lead to synthesis and release of proinflammatory cytokines and chemokines, and contribute to opioid-induced hyperalgesia and development of opioid tolerance and dependence. However, whether these MOR agonists directly or indirectly act on spinal cord astrocytes and microglial cells in vivo is unclear. In the present study, by combining the techniques of in-situ hybridization of MOR mRNA with immunohistochemistry of glial fibrillary acidic protein (GFAP; an astrocyte marker) and Iba1 (a microglial marker), we examined expression and distribution of GFAP, Iba1, and MOR mRNA in the spinal cord of rats under chronic morphine tolerance conditions. Intrathecal injections of morphine twice daily for 7 days reduced morphine analgesic effect and increased both GFAP and Iba1 immunostaining densities in the spinal cord. Surprisingly, neither GFAP nor Iba1 colocalized with MOR mRNA in spinal cord cells. Our findings indicate that MOR expression is absent from spinal cord astrocytes and microglia, suggesting that these cell types are indirectly activated by MOR agonists under chronic opioid tolerance conditions.     

Is the arginine-nitric oxide pathway involved in the pathogenesis of schizophrenia?

The reciprocal regulation of arginase and nitric oxide synthase (NOS) in L-arginine-metabolizing pathways has been demonstrated. There are various evidences of the role of the nitric oxide (NO) in several neuropsychiatric disorders including schizophrenia. However, there is no study which has investigated the role of arginase as an important part of the arginine regulatory system affecting NOS activity in schizophrenia. This study aims to investigate arginase, manganese (Mn) and total nitrite levels (a metabolite of NO) and their relationship to the arginine-NO pathway in patients with schizophrenia. Arginase activities, Mn and total nitrite levels were measured in plasma from 46 patients with schizophrenia and 32 healthy control subjects. Plasma arginase activities and Mn were found to be significantly lower and total nitrite level higher in patients with schizophrenia compared with controls. Our results suggest that the arginine-NO pathway is involved in the pathogenesis of schizophrenia.     

Src/p38 MAPK pathway in spinal microglia is involved in mechanical allodynia induced by peri-sciatic administration of recombinant rat TNF-α

Our previous work has shown that peri-sciatic administration of recombinant rat TNF-α (rrTNF) induces mechanical allodynia and up-regulation of TNF-α in the spinal dorsal horn of rats; however, the underlying mechanisms remain unknown. In the current study, we found that the levels of phosphorylated Src-family kinases (p-SFKs) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were significantly increased in bilateral lumbar spinal dorsal horn on day 3 after rrTNF administration. Double immunofluorescence staining revealed that p-SFKs and p-p38 MAPK were nearly restricted to the microglia. Intrathecal delivery of SFKs inhibitor PP2 or p38 MAPK inhibitor SB203580, started 30 min before rrTNF administration and given once daily thereafter for 7 days, blocked mechanical allodynia in bilateral hind paws and increase of TNF-α expression in the spinal dorsal horn. Moreover, PP2 inhibited the up-regulation of p-p38 MAPK induced by rrTNF. We also found that intrathecal injection of TNF-α neutralization antibody alleviated mechanical allodynia in bilateral hind paws and suppressed up-regulation of p-SFKs and p-p38 MAPK. These results suggest that activation of the SFKs/p38 MAPK pathway in microglia and subsequent TNF-α expression in the spinal dorsal horn may contribute to the mechanical hyperalgesic state induced by peri-sciatic administered rrTNF.     

Is oxidative stress involved in the developmental neurotoxicity of chlorpyrifos?

The increasing use of chlorpyrifos (CPF) has elicited concern about neurotoxic effects on the fetus and neonate. CPF targets a number of events specific to brain development, over and above the ability of its active metabolite, CPF oxon, to inhibit cholinesterase. We used PC12 cells, a model system which displays many of the neurodevelopmental effects of CPF, in order to examine whether oxidative stress underlies the direct effects of CPF on development. Production of reactive oxygen species (ROS) was measured with a fluorescent intracellular dye. When PC12 cell suspensions were treated acutely with CPF for 10 min, ROS generation was increased in a concentration-dependent manner; CPF oxon was much less effective than the native compound. CPF also increased the ROS production in response to an acute sodium nitroprusside challenge, indicating sensitization of the cells to other oxidant stressors. Next, PC12 cells were grown in an undifferentiated state in the presence of CPF or CPF oxon for extended time periods, under conditions in which CPF inhibits mitosis, and the cells were then washed and ROS production measured. Neither compound elicited a significant change in ROS production. Finally, differentiation was initiated with nerve growth factor and the cells were exposed continuously to CPF or CPF oxon over a 72 h period; under these conditions, CPF inhibits neurite outgrowth. When the cells were washed and evaluated for ROS production, no significant differences were seen. These results indicate that CPF, but not CPF oxon, has the ability to elicit acute increases in ROS production. However, the effect disappears immediately once CPF exposure is terminated, possibly reflecting cellular defense mechanisms that lessen the impact of oxidant injury.     

Is the cerebellum involved in the visuo-locomotor associative learning?

The role played by cerebellar circuits in visuo-motor associative learning is still unclear. The aim of the present study was to analyse cerebellar involvement using a visuo-locomotor associative learning paradigm that did not require spatial competences. Hemicerebellectomized (HCbed) and Control rats were tested in a visual discrimination task. First, both groups of rats had to learn that a reward was associated with an object that had a specific colour and shape (Experiment 1). Then, the shape but not the colour of the rewarded object was modified to verify whether the animals were able to transfer the rule of rewarding or whether they had to acquire a new association (Experiment 2). In the first sessions of the Experiment 1, HCbed animals displayed a tendency toward peripheral circling and a delay of about three sessions in reaching the criterion of correct choices compared to Controls. This delay has to be correlated to the need to overcome the procedural impairment elicited by the HCb. Once the HCbed animals put efficient procedural abilities into action, they exhibited a similar increase in percentages of successes from the fourth session onward as Controls. The results of Experiment 2 confirm the intact associative abilities of HCbed animals, as demonstrated by their progressive increase in successful associative responses, which, at the end of the transfer phase, were not significantly different from those of the Control group. The present findings indicate that the presence of a cerebellar lesion delays but does not prevent visuo-locomotor associative learning and that stimulus generalisation is performed without difficulty even in the presence of a cerebellar lesion.     

Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase Ⅱ in spinal cord injury rats

Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase(ROCK) signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan(GV3), Dazhui(GV14), Zusanli(ST36) and Ciliao(BL32) and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the m RNA and protein expression of Rho-A and Rho-associated kinase Ⅱ(ROCKⅡ) of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKⅡ. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKⅡ. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of Rho A and ROCKⅡ. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.     

Is the midbrain involved in the manifestation of gait disturbance in idiopathic normal-pressure hydrocephalus?

Gait disturbance is the most common symptom in idiopathic normal-pressure hydrocephalus (iNPH). However, its pathophysiology in iNPH has not been clarified. Some researchers have hypothesized that the mesencephalic locomotor region, which is a functionally defined area in the brainstem playing an important role in locomotion, is involved in the development of gait disturbance in iNPH. The purpose of the study was to investigate whether the midbrain is involved in the manifestation of gait disturbance in iNPH. Twenty-one iNPH patients who showed clinical improvements after shunt surgery were studied. Brain magnetic resonance imaging (MRI) was performed and clinical symptoms were assessed before and 1 year after surgery. Gait disturbance was assessed by the Timed Up and Go test and gait subcategory of the iNPH Grading Scale, a validated assessment tool for iNPH symptoms. Anteroposterior, left-to-right diameter and cross-sectional areas of the midbrain were measured at the inferior collicular level of axial images in MRI. The diameters and cross-sectional area of the midbrain at baseline did not show significant correlation with gait assessments at baseline (Spearman’s correlation). The midbrain measurement did not show significant difference between the baseline and postoperative values (paired t test), and its change rates did not show significant correlation with the change (rates) of the gait assessments. In this study there were no findings to suggest involvement of the midbrain in the manifestation of gait disturbance in iNPH. The hypothesis that the mesencephalic locomotor region is involved in the manifestation of gait disturbance in iNPH needs to be reconsidered.     

Establishment and evaluation of a rat model of complete transected spinal cord injury*☆

BACKGROUND： The establishment of a rat model of complete transected spinal cord injury lacks technological specifications. The current models lack concordance and reliability, and the death rate of the experimental animals is high. Therefore, there is a great need for a reliable model to apply clinical applications of therapy. OBJECTIVE： To construct a rat model of complete transected spinal cord injury characterized by stability, reproducibility, and a high animal survival rate. DESIGN： Completely randomized controlled study. SETTING： Department of Neurosurgery, Xiangya Hospital of Central South University. MATERIALS： Fifty-five healthy specific pathogen free grade adult female Sprague Dawley rats were provided by the Experimental Animal Department, Xiangya Medical College, Central South University. Olympus BX51 imaging collecting analytic system was provided by Olympus Company, Japan; and SEN-7203 Nihon-Kohden electrical stimulator by Nihon Kohden, Japan. METHODS： This study was performed at the Laboratory of Neurosurgery, Xiangya Hospital of Central South University from April to June 2006. Experimental grouping： 55 rats were randomly divided into model group （n = 40） and sham surgery group （n = 15）. In the model group, a self-made sliver hook was passed through the ventral side to support the spinal cord at the T12 segment and to shear it off. A complete transected spinal cord, 2 mm in length, was resected. In the sham surgery group, the spinal cord was identically exposed. The dura mater of the spinal cord was cut open, but the spinal cord was not damaged. MAIN OUTCOME MEASURES： Histopathological changes after spinal cord injury at L2 segment were observed subsequent to hematoxylin and eosin staining under optical microscopy. Olympus BX51 imaging collecting analytic system was used to count spinal cord ventral horn neurons. Motor function of rat hindlimb was evaluated with the Basso, Beattie and Bresnahan （BBB） scale. Paraplegia was evaluated as 0 point, and complete normality as     

Is the Saitohin gene involved in neurodegenerative diseases?

Recently, a single nucleotide polymorphism that results in an amino acid change (Q7R) was identified in a previously undescribed gene, named saitohin, nested within the tau gene. We analyzed the distribution of this polymorphism in 499 patients with Alzheimer's disease, 91 patients with frontotemporal dementia, and 402 controls. This polymorphism was in complete disequilibrium with the well-defined extended tau haplotype. We failed to replicate the association between the RR genotype and late-onset Alzheimer's disease, but we found a trend toward an association between the QQ genotype and frontotemporal dementia. Thus, the saitohin Q allele, which is a novel determinant of the tau H1 haplotypes, might represent a causative factor involved in the determinism of several tauopathies.     

Is spinal cord isolation a good model of muscle disuse?

The patterns of normal daily activity that are required to maintain normal skeletal muscle properties remain unknown. The present study was designed to determine whether spinal cord isolation can be used as a reliable experimental model of neuromuscular inactivity, that is, as a baseline for the absence of activity. Electromyograms (EMGs) were recorded from selected hindlimb muscles of unanesthetized rats over 24-hour periods before and 7, 30, 60, and 90 days after surgical isolation of the lumbar spinal cord. Our data indicate that some rat slow muscle fibers pre-surgery were activated for less than 3 hours per day. Spinal cord isolation (SI) reduced the mean daily integrated EMG (IEMG) and daily EMG duration in the primary slow extensor muscle (soleus) to <1% of control, and in the primary fast extensor muscles [medial gastrocnemius (MG) and vastus lateralis (VL)] to <2% of control. These parameters were decreased to <8% and 3% of control, respectively, in a primary fast flexor muscle, the tibialis anterior (TA). From 30 to 90 days post-SI, the mean amplitudes of the spontaneous EMG bursts were relatively normal in the soleus, increased approximately 2-fold in the MG and VL, and increased approximately 4-fold in the TA. Some evidence of the normal antagonistic flexor-extensor relationship was apparent in the brief periods of recorded activity post-SI. These results indicate that SI eliminates nearly all of the normal EMG activity in the hindlimb muscles in the presence of relatively normal muscle innervation and functional intraspinal neural circuitry.     

Characteristics of the transcription factor HIF-1α expression in the rat brain during the development of a depressive state and the antidepressive effects of hypoxic preconditioning

The dynamics of HIF-1α expression during the development of stress-related depression, as well as after hypoxic preconditioning (HP), which has an antidepressant-like effect, were studied in the hippocampus, paraventricular hypothalamic nucleus, and neocortex of rats, using an immunocytochemical method. It has been found that the factor HIF-1α is induced in neurons in response to psychoemotional stress that causes the development of experimental depression in rats in the “learned helplessness” model. The profile of the stress-induced expression of HIF-1α in the hippocampus has a two-wave character: early expression on the first day and the delayed expression 10 days after the stress. No significant change was found in the neocortex. In the hypothalamus, up-regulation of HIF-1α expression was delayed (5–10 days). After HP by a moderate repetitive hypobaric hypoxia, which prevents the development of the depressive state in rats, the post-stress expression of HIF-1α was considerably altered in the brain regions studied. In the hippocampus of HP rats, the peak of the early expression lasted for about 5 days after the stress; we observed a multifold increase in its amplitude. In contrast, the HIF-1α delayed peak was eliminated. A similar but smaller effect of HP was also observed in the hypothalamus. The data obtained indicate that delayed HIF-1α expression in the hippocampus and hypothalamus was apparently involved in the mechanisms of pathogenesis of the depressive pathology. However, strong modifications in early and late post-stress expression of HIF-1α caused by HP obviously play an important role in increasing the brain’s tolerance to severe stresses and protection against the development of stress-induced depressive pathologies.     

Is the prediction of initial stability of cervical spinal osteo-synthesis possible using a finite element model?

The study is dealing with a three segmental (C4-C7) finite element model of the intact human cervical spine. Additionally, anterior cervical fusion and plating (ACFP) with Caspar-plate and bicortical screws in C5/6 was simulated. The models were loaded using pure moments of 2.5 Nm in flexion-extension, axial rotation and lateral bending. The range of motion in C5/6 was calculated and compared to the results of a biomechanical in vitro study, that used six cadaveric human spinal segments C4-C7 for analysing range of motion C5/6 in the intact state and following ACFP. The predictions of the finite element models were always within one standard deviation of the results of the in vitro study. Thus, the current model could be used for first analysis on new C-spine implants. However, the results should be interpreted as a trend and the limitations of these models should be kept in mind.     

Is frontal lobe involved in the generation of auditory evoked P50?

This study examined the functional substrate of P50 suppression. Auditory evoked potentials (AEPs) and magnetic fields (AEFs) were recorded from healthy subjects simultaneously and analyzed using spatio-temporal source analysis. The resulting equivalent dipole model for the AEP consisted of one source in the auditory cortex (AC) of each hemisphere and an radially oriented medial frontal source, both with maximum AEP activity around 50 ms. The frontal source was functionally separated from the AC sources since it peaked significantly later and showed significantly larger P50 amplitude suppression. P30m showed neither suppression nor substantial frontal activity. In sum, this study relates P50 suppression to reduction of AC source activity and is the first to yield direct evidence for frontal involvement in P50 suppression.     

Proteomic analysis of effects of Chinese herbs to calm the liver and suppress hyperactive yang in a rat migraine model***★

BACKGROUND: The expression of ubiquitin and energy-associated protein can provoke migraines. Studies have suggested that expression is closely linked to “hyperactivity of liver-yang theory” in Traditional Chinese Medicine (TCM), as well as the function of periphery sympathetic nerve medulla. OBJECTIVE: To observe proteomic changes in a rat migraine model with regard to hyperactivity of liver-yang when treated with Chinese herbs to calm the liver and suppress hyperactive yang compound. DESIGN, TIME AND SETTING: A randomized controlled study. This study was performed at the laboratory of Institute of Integrated Traditional Chinese and Western Medicine, Institute of Human Reproduction and Stem Cell Engineering and Key Laboratory of Cancer Proteomics of Ministry of Health, Xiangya Hospital Affiliated to Central South University between September 2006 and July 2007. MATERIALS: Thirty, male, healthy, Sprague-Dawley rats, aged eight weeks, were included in the final analysis. Aconite, to calm the liver and suppress hyperactive yang compound, was provided by the Dispensary of Traditional Chinese medicine, Xiangya Hospital, Central South University. A physiological electronic stimulator, type SDQ-1, was provided by Bengbu Practical Institute of Technology. The left trigeminal ganglion was localized and stimulated for 10 minutes, and the rats were orally administered an aconite concoction to establish a rat migraine model with hyperactivity of liver-yang. METHODS: Rats were randomly divided into a normal control group, model group, and TCM treatment group, with 10 rats in each group. The TCM treatment group was orally treated to calm the liver and suppress the hyperactive yang compound once a day for 28 days. In contrast, the model group and normal group were orally administered the same amount of distilled water once a day for 28 days. MAIN OUTCOME MEASURES: The total proteins from adrenal glands of the three groups were separated by two-dimensional gel electrophoresis (2-DE), and 2-DE images were analyzed by PDQuest 7.0 software. Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) was used to obtain peptide mass fingerprints of the differential proteins. Databases were searched to identify the proteins. RESULTS: A total of 30 rats were included in the final analysis. Reproducible 2-DE patterns from rat adrenal gland of the three groups were obtained. Compared with the normal group, nine proteins were down-regulated and five proteins were up-regulated in the model group; however, these expressions returned to normal, or near normal levels, in the TCM treatment group. A total of eight differentially expressed proteins were identified: glycogen phosphorylase, ATP synthase D chain, isovaleryl-CoA dehydrogenase, ubiquitin, Annexin-3, Annexin-A1, Peroxirdoxin-II, and heat shock protein-27. CONCLUSION: Liver calming and suppression of the hyperactive yang compound may up-regulate expression of proteins related to energy metabolism and the ubiquitin system. Compounds that are used to treat migraines may contribute to protein functions in the peripheral sympathetic nervous system. Key Words: migraine; liver yang hyperactivity; adrenal glands; pacifying liver     

Is the role of steroids in acute spinal cord injury now resolved?

Steroids have long been used in the context of acute spinal cord injury but the evidence for doing so is limited. The second National Acute Spinal Cord Injury Study trial had the potential to provide such evidence for the first time, as this was a placebo controlled, prospective, randomized trial. From the outset, however, some clinicians found the methodology and consequently the results unsatisfactory. This concern has been revisited within the evidence-based framework of critical appraisal of the accumulation of clinical studies. High-dose methylprednisolone cannot be justified as a standard treatment in acute spinal cord injury within current medical practice.     

Is electrical coupling involved in the generation of posterior hypothalamic theta rhythm?

Data obtained in in vitro experiments and urethane anaesthetized animals have revealed that the mechanisms responsible for the generation of hippocampal cholinergic theta rhythm are specifically affected by the administration of broad spectrum gap junctions (GJs) blocker – carbenoxolone (CBX). The aim of this study was to examine the effect of GJs modulation on the production of posterior hypothalamic theta. Specifically, we were interested in evaluating whether CBX could attenuate the theta rhythm recorded from the supramammillary nucleus and posterior hypothalamic nuclei, in both in vitro and in vivo preparations. The data we obtained from in vitro and in vivo preparations demonstrated that the administration of CBX did not suppress cholinergically induced theta in posterior hypothalamic area (PHa) slices nor the theta rhythm observed in the PHa of urethane anaesthetized rats. Moreover, the application of trimethylamine, while very effective in the enhancement of hippocampal theta rhythm, did not produce any changes in theta oscillations observed in either in vitro or in vivo posterior hypothalamic area preparations. These data show that electrical coupling via GJs is not involved in theta rhythm generation in the PHa. Surprisingly, we observed a significant enhancement of theta activity in response to the carbenoxolone administration in both in vitro and in vivo PHa preparations. The theta rhythm enhancement detected in those experiments was attenuated by the application of spironolactone (mineralocorticoid receptors antagonist). We suggest that the observed excitatory effects of CBX on posterior hypothalamic oscillatory activity in the theta band could be mediated by mineralocorticoid receptors.     

Proteomic analysis of effects of Chinese herbs to calm the liver and suppress hyperactive yang in a rat migraine model***★

BACKGROUND: The expression of ubiquitin and energy-associated protein can provoke migraines. Studies have suggested that expression is closely linked to “hyperactivity of liver-yang theory” in Traditional Chinese Medicine (TCM), as well as the function of periphery sympathetic nerve medulla. OBJECTIVE: To observe proteomic changes in a rat migraine model with regard to hyperactivity of liver-yang when treated with Chinese herbs to calm the liver and suppress hyperactive yang compound. DESIGN, TIME AND SETTING: A randomized controlled study. This study was performed at the laboratory of Institute of Integrated Traditional Chinese and Western Medicine, Institute of Human Reproduction and Stem Cell Engineering and Key Laboratory of Cancer Proteomics of Ministry of Health, Xiangya Hospital Affiliated to Central South University between September 2006 and July 2007. MATERIALS: Thirty, male, healthy, Sprague-Dawley rats, aged eight weeks, were included in the final analysis. Aconite, to calm the liver and suppress hyperactive yang compound, was provided by the Dispensary of Traditional Chinese medicine, Xiangya Hospital, Central South University. A physiological electronic stimulator, type SDQ-1, was provided by Bengbu Practical Institute of Technology. The left trigeminal ganglion was localized and stimulated for 10 minutes, and the rats were orally administered an aconite concoction to establish a rat migraine model with hyperactivity of liver-yang. METHODS: Rats were randomly divided into a normal control group, model group, and TCM treatment group, with 10 rats in each group. The TCM treatment group was orally treated to calm the liver and suppress the hyperactive yang compound once a day for 28 days. In contrast, the model group and normal group were orally administered the same amount of distilled water once a day for 28 days. MAIN OUTCOME MEASURES: The total proteins from adrenal glands of the three groups were separated by two-dimensional gel electrophoresis (2-DE), and 2-DE images were analyzed by PDQuest 7.0 software. Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) was used to obtain peptide mass fingerprints of the differential proteins. Databases were searched to identify the proteins. RESULTS: A total of 30 rats were included in the final analysis. Reproducible 2-DE patterns from rat adrenal gland of the three groups were obtained. Compared with the normal group, nine proteins were down-regulated and five proteins were up-regulated in the model group; however, these expressions returned to normal, or near normal levels, in the TCM treatment group. A total of eight differentially expressed proteins were identified: glycogen phosphorylase, ATP synthase D chain, isovaleryl-CoA dehydrogenase, ubiquitin, Annexin-3, Annexin-A1, Peroxirdoxin-II, and heat shock protein-27. CONCLUSION: Liver calming and suppression of the hyperactive yang compound may up-regulate expression of proteins related to energy metabolism and the ubiquitin system. Compounds that are used to treat migraines may contribute to protein functions in the peripheral sympathetic nervous system. Key Words: migraine; liver yang hyperactivity; adrenal glands; pacifying liver