大鼠海马胶质细胞培养牵张损伤模型的建立

目的 建立大鼠海马胶质细胞培养牵张损伤模型.方法 提取出生时间24h到48h的SD大鼠海马组织行星形胶质细胞培养,采用CIC Ⅱ型细胞损伤装置、根据Ellis方法建立改良的大鼠海马胶质细胞体外牵张损伤模型,损伤程度分轻、中、重三级.对照组不予损伤.分别在2h和24h检测细胞乳酸脱氧酶释放量,并通过碘化丙啶荧光染色观察细胞损伤情况.结果 细胞培养液中乳酸脱氧酶释放量随着损伤程度加重而增高.PrI红染细胞数随着牵张损伤的程度增高而增加.结论 本实验建立的牵张损伤模型使用方便,可重复性好,适于进行神经细胞机械件体外损伤的研究.     

Differential subcellular distribution and colocalization of the microsomal and soluble epoxide hydrolases in cultured neonatal rat brain cortical astrocytes

The microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH) enzymes exist in a variety of cells and tissues, including liver, kidney, and testis. However, very little is known about brain epoxide hydrolases. Here we report the expression, localization, and subcellular distribution of mEH and sEH in cultured neonatal rat cortical astrocytes by immunocytochemistry, subcellular fractionation, Western blotting, and radiometric enzyme assays. Our results showed a diffuse immunofluorescence pattern for mEH, which colocalized with the astroglial cytoskeletal marker glial fibrillary acidic protein (GFAP). The GFAP-positive cells also expressed sEH, which was localized mainly in the cytoplasm, especially in and around the nucleus. Western blot analyses revealed a distinct protein band with a molecular mass of approximately 50 kDa, the signal intensity of which increased about 1.5-fold in the microsomal fraction over the whole-cell lysate and other subcellular fractions. The polyclonal anti-human sEH rabbit serum recognized a protein band with a molecular mass similar to that of the affinity-purified sEH protein (approximately 62 kDa), the signal intensity of which increased over 1.7-fold in the 105,000g supernatant fraction over the cell lysate. Furthermore, the corresponding enzyme activities measured by using mEH- and sEH-selective substrates generally corroborated the immunocytochemical and Western blotting data. These results suggest that rat brain cortical astrocytes differentially coexpress mEH and sEH enzymes. The differential subcellular localization of mEH and sEH may play a role in the cerebrovascular functions that are known to be affected by brain-derived vasoactive epoxides.     

Modulation of the piriform cortex activity by the basal forebrain: an optical recording study in the rat

The piriform cortex (PCx) is the main projection area of the olfactory bulb. It further receives afferents from neurons located in the horizontal limb of the diagonal band (HDB) and the magnocellular preoptic nucleus (MCPO) of the basal forebrain. Using an optical recording technique, we examined the influence of electrical stimulation of the HDB-MCPO complex upon the PCx reactivity to electrical stimulation of the olfactory bulb. We observed an inhibitory modulation which was stronger on the intrinsic activity than on the afferent one. This effect was not homogeneously distributed within the whole PCx. The neurotransmitter involved and its synaptic target remain to be determined. The present findings are discussed in view of anatomical and functional data.     

Different response of astrocytes and Bergmann glial cells to portacaval shunt: an immunohistochemical study in the rat cerebellum.

The present study was performed in order to follow the response of rat cerebellum astroglial cells (Bergmann glial cells and astrocytes) to long-term portacaval shunt (PCS), by means of glial fibrillary acidic protein (GFAP) and vimentin immunoreactivities. Bergmann glia accumulated GFAP in response to PCS, whereas astrocytes decreased GFAP immunoreactivity when compared to control rats. The increase of GFAP occurs in cells located in the cerebellar layer where glutamate is mainly released. Since the vimentin content remained unaltered in response to PCS, when compared to control rats, it can be concluded that only the GFAP filaments are affected by PCS. Nevertheless, GFAP immunoreactivity presents regional differences in the cerebellar astroglial population, and the factors responsible for these variations are still unknown.     

Effect of orotic acid on the metabolism of cerebral cortical astrocytes during hypoxia and reoxygenation: An NMR spectroscopy study

Astrocytes were incubated under normoxic or hypoxic conditions in Dulbecco's minimum essential medium containing [12-¹³C]acetate, unlabeled glucose and in some cases orotic acid, an intermediate in pyrimidine biosynthesis. After 12 hr the medium was replaced by fresh medium without drug and incubation was continued for 17 hr in a normal oxygen atmosphere (reoxygenation). Thereafter, medium was removed, cell extracts were prepared, and metabolism in the treatment group was compared to the untreated hypoxia group and to control. ¹³C and H NMR spectra revealed that ¹³C enrichment in citrate and glutamine C-4 in the initial medium were increased in the presence of orotic acid, compared to the untreated hypoxia group but lower than control. The drug increased acetate utilization during hypoxia to normoxic levels. Thus it appears that the treatment group had a more active mitochondrial metabolism, which was also reflected in higher intracellular uridine diphosphoryl sugars and ADP concentrations. Glutamine labeling was increased in the cell extracts in the presence of orotic acid. Thus it appears that, in the presence of the pyrimidine nucleotide precursor, astrocytes are capable of normal metabolism during hypoxia which might have implications for neuronal survival during low oxygen insults, since neurons are dependent on astrocyte produced precursors for their neurotransmitter synthesis.     

Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors

ATP is the dominant messenger for astrocyte-to-astrocyte calcium-mediated communication. Definition of the exact ATP/P2 receptors in astrocytes and of their coupling to intracellular calcium ([Ca(2+)](i)) has important implications for brain physiology and pathology. We show that, with the only exception of the P2X(6) receptor, primary rat cortical astrocytes express all cloned ligand-gated P2X (i.e., P2X(1-5) and P2X(7)) and G-protein-coupled P2Y receptors (i.e., P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(12)). These cells also express the P2Y-like UDP-glucose receptor, which has been recently recognized as the P2Y(14) receptor. Single-cell image analysis showed that only some of these receptors are coupled to [Ca(2+)](i). While ATP induced rapid and transient [Ca(2+)](i) increases (counteracted by the P2 antagonists suramin, pyridoxal-phosphate-6-azophenyl-2'-4'-disulfonic acid and oxidized ATP), the P2X(1)/P2X(3) agonist alphabetameATP produced no changes. Conversely, the P2X(7) agonist BzATP markedly increased [Ca(2+)](i); the presence and function of the P2X(7) receptor was also confirmed by the formation of the P2X(7) pore. ADP and 2meSADP also produced [Ca(2+)](i) increases antagonized by the P2Y(1) antagonist MRS2179. Some cells also responded to UTP but not to UDP. Significant responses to sugar-nucleotides were also detected, which represents the first functional response reported for the putative P2Y(14) receptor in a native system. Based on agonist preference of known P2 receptors, we conclude that, in rat astrocytes, ATP-induced calcium rises are at least mediated by P2X(7) and P2Y(1) receptors; additional receptors (i.e., P2X(2), P2X(4), P2X(5), P2Y(2), P2Y(4), and P2Y(14)) may also contribute.     

Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity

As the two major glial cell types in the brain, astrocytes and microglia play pivotal but different roles in maintaining optimal brain function. Although both cell types have been implicated as major targets of methylmercury (MeHg), their sensitivities and adaptive responses to this metal can vary given their distinctive properties and physiological functions. This study was carried out to compare the responses of astrocytes and microglia following MeHg treatment, specifically addressing the effects of MeHg on cell viability, reactive oxygen species (ROS) generation and glutathione (GSH) levels, as well as mercury (Hg) uptake and the expression of NF-E2-related factor 2 (Nrf2). Results showed that microglia are more sensitive to MeHg than astrocytes, a finding that is consistent with their higher Hg uptake and lower basal GSH levels. Microglia also demonstrated higher ROS generation compared with astrocytes. Nrf2 and its downstream genes were upregulated in both cell types, but with different kinetics (much faster in microglia). In summary, microglia and astrocytes each exhibit a distinct sensitivity to MeHg, resulting in their differential temporal adaptive responses. These unique sensitivities appear to be dependent on the cellular thiol status of the particular cell type.     

Evaluation of [3H]paroxetine as an in vivo ligand for serotonin uptake sites: A quantitative autoradiographic study in the rat brain

Paroxetine, a selective inhibitor of serotonin uptake and an antidepressant, was used in conjunction with quantitative ex vivo autoradiography to study the feasibility of imaging serotonin terminals in the living brain. Tritiated paroxetine was injected in the rat tail vein, and the brain was processed for quantitative autoradiography 3 hours later. Animals received either [³H]paroxetine alone (100 μCi/animal) or a mixture of labeled paroxetine (100 μCi) and an excess of unlabeled drug (0.5 or 2 mg/kg intravenously [i.v.]). Computerized image analysis of the resulting autoradiograms revealed high densities of radioactivity in brain regions known to contain high densities of serotonergic terminals and high specific binding of [³H]paroxetine in vitro, such as the raphe nuclei, interpeduncular nucleus, basolateral amygdala, substantia nigra, and some hypothalamic nuclei. Radioactivity uptake in these brain regions was effectively blocked (50-72%) by coadministration of excess unlabeled paroxetine. However, cortical and hippocampal binding of paroxetine in vivo was moderately high, in contrast to the relatively sparse serotonergic innervation in these regions. Only a relatively small proportion of cortical and hippocampal binding (20-40%) could be blocked by excess unlabeled paroxetine, indicating that most of the radioactivity in these regions is not associated with serotonin terminals or uptake sites. The usefulness of [³H]paroxetine as an in vivo ligand for imaging serotonin terminals in the human brain is limited by these nonserotonergic binding sites. © 1993 Wiley-Liss, Inc.     

Striatal perfusion of indomethacin attenuates dopamine increase in immature rat brain exposed to anoxia: an in vivo microdialysis study

Using in vivo microdialysis and HPLC, we examined the effects of indomethacin on extracellular dopamine (DA) in the striatum of immature rats submitted to anoxia. Rat pups in two indomethacin groups received intrastriatal perfusion of either 1 mM or 5 mM indomethacin throughout the experiment. The DA level reached 1185+/-400% of the basal level during anoxia; in contrast, the peak levels of DA were only 307+/-63%, 153+/-35% in indomethacin groups (p<0.05). We consider that this suppression would be one of the mechanisms of the protective effect of indomethacin on hypoxic ischemic encephalopathy.     

Effect of dopamine content in rat brain striatum on anesthetic requirement: an in vivo microdialysis study

This study investigated the changes in anesthetic requirement caused by administration of two different concentrations (0.1 μM and 1.0 μM/h) of dopamine into the rat striatum. During the measurement of minimum alveolar concentration (MAC), each dopamine solution was continuously injected directly into rat striatum by microdialysis technique. During perfusion of the lower dose, MAC did not change. During the higher dose of dopamine, MAC decreased by approximately 30%.     

Differential susceptibility of cortical and subcortical inhibitory neurons and astrocytes in the long term following diffuse traumatic brain injury

Long‐term diffuse traumatic brain injury (dTBI) causes neuronal hyperexcitation in supragranular layers in sensory cortex, likely through reduced inhibition. Other forms of TBI affect inhibitory interneurons in subcortical areas but it is unknown if this occurs in cortex, or in any brain area in dTBI. We investigated dTBI effects on inhibitory neurons and astrocytes in somatosensory and motor cortex, and hippocampus, 8 weeks post‐TBI. Brains were labeled with antibodies against calbindin (CB), parvalbumin (PV), calretinin (CR) and neuropeptide Y (NPY), and somatostatin (SOM) and glial fibrillary acidic protein (GFAP), a marker for astrogliosis during neurodegeneration. Despite persistent behavioral deficits in rotarod performance up to the time of brain extraction (TBI = 73.13 ± 5.23% mean ± SEM, Sham = 92.29 ± 5.56%, P < 0.01), motor cortex showed only a significant increase, in NPY neurons in supragranular layers (mean cells/mm² ± SEM, Sham = 16 ± 0.971, TBI = 25 ± 1.51, P = 0.001). In somatosensory cortex, only CR⁺ neurons showed changes, being decreased in supragranular (TBI = 19 ± 1.18, Sham = 25 ± 1.10, P < 0.01) and increased in infragranular (TBI = 28 ± 1.35, Sham = 24 ± 1.07, P < 0.05) layers. Heterogeneous changes were seen in hippocampal staining: CB⁺ decreased in dentate gyrus (TBI = 2 ± 0.382, Sham = 4 ± 0.383, P < 0.01), PV⁺ increased in CA1 (TBI = 39 ± 1.26, Sham = 33 ± 1.69, P < 0.05) and CA2/3 (TBI = 26 ± 2.10, Sham = 20 ± 1.49, P < 0.05), and CR⁺ decreased in CA1 (TBI = 10 ± 1.02, Sham = 14 ± 1.14, P < 0.05). Astrogliosis significantly increased in corpus callosum (TBI = 6.7 ± 0.69, Sham = 2.5 ± 0.38; P = 0.007). While dTBI effects on inhibitory neurons appear region‐ and type‐specific, a common feature in all cases of decrease was that changes occurred in dendrite targeting interneurons involved in neuronal integration. J. Comp. Neurol. 524:3530–3560, 2016. © 2016 Wiley Periodicals, Inc.     

Age- and temperature-dependent effects of opioids on medulla oblongata respiratory activity: an in vitro study in newborn rat

Influences of post-natal age (P0–P4) and temperature (22.5°–31.5°C) on the action(s) of opioids on respiratory activities from neonatal rat brainstem–spinal cord preparations were examined in this study. A temperature-dependent μ-opioid receptor effect on respiration was found. In addition, the effect of morphine increased with postnatal age (P0–P4). Hence, age and temperature must be taken into account when performing studies on medullary respiration-related structures using the neonatal rat brainstem–spinal cord preparation.     

Monitoring the temporal and spatial activation pattern of astrocytes in focal cerebral ischemia using in situ hybridization to GFAP mRNA: comparison withsgp-2 andhsp70 mRNA and the effect of glutamate receptor antagonists

We investigated the temporo-spatial expression of astrocyte glial fibrillary acidic protein (gfap) and sulfated glycoprotein 2 (sgp-2) mRNAs in comparison to 70-kDa heat shock protein (hsp70) mRNA by in situ hybridisation in rats subjected to permanent occlusion of the middle cerebral artery (MCA).Gfap mRNA started to increase in the cingulate cortex of the lesioned hemisphere 6 h after MCA occlusion and gradually spread over the lateral part of the ipsilateral cortex and the striatum from 12 h to 3 days, peaking at 3 days after MCA occlusion.Gfap mRNA also increased in the contralateral cingulate cortex and corpus callosum at 12 and 24 h.Hsp70 mRNA increased markedly in the ipsilateral cortex adjacent to the ischemic lesion, and slightly within the lesion area from 3 to 24 h and disappeared after 3 days. By 7 days,gfap andsgp-2 mRNAs were increased markedly in the peri-infarct area, and in the ipsilateral thalamus parallel with the delayed neuronal damage, whereas the widespread increase ofgfap mRNA in the ipsilateral hemisphere declined. Post-occlusion treatment with the glutamate receptor antagonists MK-801 and NBQX slightly attenuated the induction ofgfap but did not qualitatively affect the topical expression pattern. Within the cingulate cortex MK-801 treatment resulted in a significant decrease of the signal intensity at all survival times, reflecting most likely an attenuation of lesion-induced spreading depression like depolarization waves by MK-801. The area ofhsp70 expression was reduced by both MK-801 and NBQX, most likely reflecting the decrease of the lesion area by both treatment regimens. Our study thus revealed an early and widespread increase ofgfap mRNA in the non-ischemic areas including the contralateral hemisphere starting between 3 and 6 h, and a delayed circumscribed expression in the peri-infarct border zone after 1 week. Comparison with the expression ofhsp70 mRNA suggests that the absence of an earlygfap mRNA induction in the peri-lesion zone reflects an impairment of astrocytic function which may be of importance for infarct growth during the early evolution of the pathological process.     

Immunohistochemistry of glial reaction after injury in the rat: Double stainings and markers of cell proliferation

The astrocytic reaction in the rat after brain injury has been studied immunohistochemically for intermediate filaments (GFAP and vimentin), also with double staining procedures, and for markers of proliferation (BrdU and PCNA). GFAP-positive reactive astrocytes appeared around the lesion, where they were vimentin-positive and at a distance. BrdU and PCNA showed a high labelling index around the wound at day 2 and scattered positive nuclei were also found at a distance in the ipsilateral side. BrdU-positive astrocytes represented a minor fraction of GFAP- and vimentin-positive astrocytes. The expression of vimentin persisted at least 15 days after the lesion. Our results could suggest that distant reactive astrocytes originate through hypertrophy while those close to lesion arise by hyperplasia from mature or immature glial cells. The hypothesis is formulated that cells of the periventricular matrix contribute to the post-traumatic proliferative activity.     

Effect of exogenousL-DOPA on behavior in the rat: an in vivo voltammetric study

L-DOPA was administered intraperitoneally (i.p.) or intraventricularly (i.v.t.) to freely moving rats to investigate the effects of exogenousL-DOPA itself on behavior. Striatal dopamine (DA) in the extracellular fluid was examined with microcomputer-controlled in vivo voltammetry, and behavioural change was observed. WhenL-DOPA was administered (i.p.) after pretreatment with benserazide, a peripheral DOPA decar☐ylase inhibitor, behavioral change was elicited before the elevation in DA and suppressed before its reduction. After pretreatment with NSD-1015, a central DOPA decar☐ylase inhibitor, behavioral change was also elicited, although DA was still not increased. WhenL-DOPA was injected (i.v.t.), the behavioral effects was manifested at once; DA was still unchanged at this time, but it increased after behavioral activity reached the maximum level.L-DOPA was also injected (i.v.t.) into rats with striatal lesions induced by 6-hydroxydopamine (i.v.t.). Behaviral change was manifested promptly after the injection. When the dose-response curves to different dosages ofL-DOPA were examined in normal rats without striatal lesions, it was found to exhibit a steeper rise than that of DA. Finally, when rats were injected (i.p. or i.v.t.) with 3-O-methyl-DOPA (3-methoxytyrosine), a major metabolite ofL-DOPA, no behavioral change was elicited, and no increase in DA was recognized. These experimental results indicated thatL-DOPA is related to the manifestation of behavioral change.     

In situ transblot and immunocytochemical comparisons of astrocytic connexin-43 responses to NMDA and kainic acid in rat brain

Intracerebral injection of kainic acid (KA) in rat brain was previously found to cause altered immunohistochemical recognition of connexin-43 (Cx43) epitopes (epitope masking) with different sequence-specific antibodies against this gap junction protein. We demonstrate here that similar alterations occur when nitrocellulose membranes containing protein transferred from fresh cryostat sections of KA-injected brain are probed with these antibodies (in situ transblotting), indicating that epitope masking is not a result of epitope alteration due to fixation conditions used in earlier studies. Alterations in immuno-recognition of astrocytic Cx43 subsequent to injections of NMDA were also observed and were similar to those seen with KA in some, but not all respects. The results provide further indications of Cx43 molecular modification in excitotoxin-lesioned tissue and suggest that the sequelae of reactions by astrocytes and their gap junctions in these tissues is dependent on cell-type susceptibility to excitotoxin action.     

The effect of sodium valproate on extracellular GABA and other amino acids in the rat ventral hippocampus: an in vivo microdialysis study

We report the effects of i.p. administration of sodium valproate (VPA) on extracellular concentrations of various amino acids in the rat ventral hippocampus studied using in vivo microdialysis, followed by HPLC with fluorometric detection. At the doses used (100, 200 and 400 mg/kg), VPA had no effect on extracellular aspartate, glutamine and taurine, whilst inducing a small, but not statistically significant increase in glutamate at 200 and 400 mg/kg. In contrast, VPA administration produced a biphasic effect on extracellular GABA levels which was dependent on the dose used. At 100 mg/kg, VPA reduced GABA concentrations by 50% when compared to basal. 200 mg/kg VPA had virtually no effect, whilst 400 mg/kg VPA raised extracellular GABA levels to 200% of basal. The results are discussed in relation to the known pharmacological and anticonvulsant actions of VPA.     

The effects of cholecystokinin (CCK-8) on dopamine-containing nerve terminals in the caudate nucleus and nucleus accumbens of the anesthetized rat: an in vivo electrochemical study

The action of cholecystokinin (CCK) on presynaptic function of dopaminergic nerve terminals has been the subject of much debate in the literature. In efforts to resolve some of the reported ambiguities, high speed in vivo electrochemical recordings were carried out in the caudate nucleus and nucleus accumbens of the urethane anesthetized rat, to determine effects of locally applied sulfated (CCK-8S) and unsulfated (CCK-8US) CCK octapeptide. Locally-applied CCK-8S and CCK-8US caused no increase in the baseline electrochemical signals recorded from either brain region. However, locally applied CCK-8S potentiated the potassium-evoked overflow of dopamine (DA) into the extracellular space in both the caudate and nucleus accumbens. In contrast, pressure ejection of CCK-8US produced no significant effects on the potassium-evoked overflow of DA in either structure. These data support a facilitatory effect of CCK-8S on potassium-evoked overflow from DA-containing nerve terminals in the urethane anesthetized rat that is likely mediated through a peripheral type CCK receptor.     

Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study

Immunocytochemical studies using a monoclonal antibody to choline acetyltransferase (ChAT) were performed on sections of rat dentate gyrus. Light microscopical analysis of the immunoreactivity revealed dense fiber networks and many punctate structures predominantly located at the interface of the granule cell layer and molecular layer. In the elctron microscope, the immunostained punctate structures were identified as synaptic boutons which formed mainly symmetrical contacts onto dendritic elements. Few ChAT-immunoreactive boutons formed axosomatic contacts.     

Pre- and postnatal development of noradrenergic projections to the rat spinal cord: an immunocytochemical study.

Using immunocytochemistry with a specific antiserum against noradrenaline, the pre- and postnatal development of noradrenergic (NA) projections to the rat spinal cord was studied from embryonic day 16 (E16) to adulthood (the day following nocturnal mating being considered as E0). In this study, pregnant animals were pre-treated with the MAO inhibitor pargyline (200 mg/kg i.p.), whereas postnatal animals received 100 mg/kg. In vibratome sections, noradrenaline-immunoreactive (NA-IR) axons were seen to invade the spinal cord at E16, at cervical and upper thoracic levels, from the ventral funiculus. At E18, small caliber NA-IR fibers were present in the ventral horn at all cord levels, and some fibers were seen in the intermediolateral cell column (IML) at thoracic level. The growth of axons towards the dorsal horn became noticeable by postnatal day 0 (P0). At P3, fine beaded and radially orientated NA-IR fibers were observed at all levels. The pattern of NA innervation of the dorsal horn was similar to that of the adult by P7. The segregation of noradrenaline immunoreactivity in the ventral and dorsal horns, the IML and the periependymal area was more obvious at all levels by P14 and P20. From P30 the NA innervation was similar to that found in the adult spinal cord. Thus, noradrenaline, like serotonin, was present early in the spinal cord before the onset of specific functions. In addition to and prior to its transmitter function, it might play a trophic role in the neurogenesis of the spinal cord.