Glial Fibrillary Acidic Protein Protein Research Articles

Changes of mitochondrial dynamics related genes expression in a glaucoma rat model

Objective To investigate the ultrastructural changes of mitochondria and dynamic changes of mitochondrial dynamics related genes in chronic ocular hypertension rat models. Methods Forty Sprague-Dawley (SD) rats were randomized into normal control group, modeling 3 day-group, modeling 7 day-group and modeling 14 day-group.Glaucoma models were induced by translimbal laser photocoagulation of trabecular meshwork in rats.The number of survival retinal ganglion cells was evaluated by retrogradely labeling of FluoroGold.The morphological change of mitochondria in optical nerve was observed under the transmission microscope.The dynamic changes of mitochondrial dynamic-related gene expression in the retina in gene and protein levels were detected by real-time fluorescence quantitative PCR and Western blot assay, respectively.The experiment complied ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Experimentation Ethics Committee of the Eye and ENT Hospital of Fudan University. Results The intraocular pressure was significantly higher in the modeling 3-day group, modeling 7-day group and modeling 14-day group than that in the normal control group (all at P<0.01). Compared with the normal control group, the immunoreactivity of glial fibrillary acidic protein (GFAP) was increased in the Muller cells in all modeling retinas, and the relative expressing levels of GFAP protein were significantly mcreased in all modeling groups (F=91.47, 17.58, both at P<0.01). Compared to the normal control group, the number of mitochondria was increased, and the mitochondrial health degree in the optic nerve and the nearest neighbor distance between adjacent mitochondria were decreased in the modeling 3- day group and 14-day group (all at P<0.05). The expression levels of dynamics-related protein 1(Drp1), parkin protein, PINK1, KIF5B and Miro1 mRNA in the retina were significantly elevated in the modeling 3-day group (all at P<0.05). In addition, the expression levels of optineurin, parkin and OPA1 proteins were higher in the modeling 3-, 7- and 14-day groups than those in the normal control group (all at P<0.05). Conclusions Dysfunction of mitochondria and changes of mitochondrial dynamics exist in RGCs of glaucomatous rats, including increased mitochondrial fission and fusion as well as the activation of mitophagy pathway, which probably are the mechanism of RGC damage in glaucoma. Key words: Mitochondria; Glaucoma; Fission; Fusion; Mitophagy; Retinal ganglion cells; Animal models; SD rats

Deep brain stimulation electrode insertion and depression: Patterns of activity and modulation by analgesics

BackgroundAn initial antidepressant effect when using deep brain stimulation (DBS) of the subcallosal area of the cingulate cortex (Cg25) to treat resistant depression that could be the result of electrode insertion has been described. We previously showed that electrode insertion into the infralimbic cortex (ILC; the Cg25 rodent correlate) provokes a temporally limited antidepressant-like effect that is counteracted by non-steroidal anti-inflammatory drugs, such as those routinely used for pain relief. ObjectiveWe characterized the effect of electrode insertion using functional neuroimaging and evaluated the impact of different analgesics on this effect. MethodsThe effect of electrode insertion into the ILC was evaluated by positron emission tomography. The effect of analgesics (ibuprofen, tramadol and morphine) on the behavioral effect induced by electrode insertion were evaluated through the forced swimming test and the novelty suppressed feeding test. Furthermore, glial fibrillary acidic protein (GFAP) and p11 expression were measured. ResultsElectrode implantation produces an antidepressant- and anxiolytic-like effect, a local decrease in glucose metabolism, and changes in several brain regions commonly related to depression and the antidepressant response. Ibuprofen counteracted the behavioral and molecular changes produced by electrode insertion (changes in GFAP and p11 protein expression). However, analgesics with no anti-inflammatory properties (e.g., tramadol) neither counteract the behavioral effects of electrode implantation nor the molecular mechanisms triggered. ConclusionsAnalgesics without anti-inflammatory properties may not limit the transient benefit produced by electrode insertion reducing the time required to achieve remission in depressive DBS patients.

Autoantibodies in the serum of post-stroke patients with different clinical outcome

Natural autoantibodies (auto-Abs) of IgG class against nervous tissue proteins, as well as corresponding anti-idiotypic antibodies (AIAbs) are presented in serum of each healthy adult and their content are relatively constant among individuals. In opposite, in patients with disorders of the nervous system serum levels of such AIAbs were often found beyond the normal ranges. Characteristic changes of auto-Abs against protein S100, glial fibrillary acidic protein (GFAP), MP-65 protein and nervous growth factor (NGF) and corresponding immune reactivity were investigated in serum samples obtained from patients with ischemic stroke. Samples for investigation were collected at 3, 10, 21 days after stroke and 6 months later. Forty-two post-stroke patients were examined in acute period of disease and 6 months later. The follow-up was based NIHSS score. Outcomes were assessed with the Bartel Index at 6 month. Severity of post-stroke dementia was defined according to the DSM-IV and NINDS-AIREN. Auto-Abs against S100, GFAP, MP-65 and NGF as well as corresponding AIAbs were determined in patient serum by standard immuno-ferment assay (ELISA). In acute period of disease in 100% of post-stroke patients the serum immune reactivity of auto-Abs and AIAbs against proteins S100, GFAP, MP-65 and NGF were deviated compare to the same characteristics in serum samples of healthy persons ( P < 0.001). Sixth months later the deviations of serum immune reactivity were revealed in most part of post-stroke patients and relations between changed immune reactivity and clinical spate were found. The most characteristic feature of patients with post-stroke dementia was deviations of normal balance between pairs of specific auto-Abs and corresponding AIAbs. Changes in the serum content of auto-Abs against nervous tissue proteins and corresponding AIAbs in post-stroke patients may reflect intensity of reparative processes and probably could be used for prognostic aims of rehabilitation program success.

Effect of Acupuncture on Expression of Glial Fibrillary Acidic Protein in Hippocampus and Prefrontal Cortex and Serum Interleukin-10 in Chronic Restraint Stress Depression Rats

To observe the effect of acupuncture on the expression of glial fibrillary acidic protein (GFAP) in hippocampus and prefrontal cortex and the level of serum interleukin 10 (IL-10) in rats with depression, so as to explore its mechanism underlying improvement of depression. Thirty-two male SD rats were randomly divided into four groups: control, model, acupuncture, and medication (Fluoxetine, Flu) (n=8 rats in each). The depression model was established by using chronic restraint stress (constraint, fasting, water deprivation, etc.) combined with solitary raising for 28 days. Acupuncture was applied to "Baihui" (GV 20) and "Yintang" (GV 29), and bilateral "Sanyinjiao" (SP 6) for 20 min, once daily for 28 days. Fluoxetine (1.8 mg/kg) was given to rats of the medication group by gavage once every day for 28 days. Sucrose consumption test and open field test (crossing and rearing locomotion) were carried out to evaluate the behavioral changes. Western blot was used to detect the expression of GFAP in the hippocampus and prefrontal cortex, and the content of serum IL-10 was detected by enzyme linked immunosorbent assay (ELISA). After modeling, the sucrose consumption, the crossing numbers and rearing times, hippocampal GFAP protein expression and serum IL-10 content were significantly decreased and prefrontal GFAP protein expression was up-regulated markedly in the model group relevant to the control group (P<0.01). After the treatment, the decreased levels of sucrose consumption and crossing numbers, hippocampal GFAP protein expression and serum IL-10 content and the increased prefrontal GFAP protein expression were considerably suppressed in both medication and acupuncture groups compared with the model group (P<0.05, P<0.01). No significant differences were found between the acupuncture and medication groups in increasing the rats' locomotion, sucrose consumption, hippocampal GFAP protein expression (P>0.05) and in down-regulating prefrontal GFAP protein expression (P>0.05 ) except up-regulation of IL-10 level. Acupuncture intervention plays a positive role in anti-depression in rats, which may be related to its effects in regulating the expression of GFAP in the hippocampal and prefrontal astrocytes, and in increasing the content of serum anti-inflammatory cytokine IL-10.

Repeated electroacupuncture treatment attenuated hyperalgesia through suppression of spinal glial activation in chronic neuropathic pain rats

BackgroundCumulated evidence reveals that glial cells in the spinal cord play an important role in the development of chronic neuropathic pain and are also complicated in the analgesic effect of EA intervention. But the roles of microgliacytes and astrocytes of spinal cord in the process of EA analgesia remain unknown.MethodsA total of 120 male Wistar rats were used in the present study. The neuropathic pain model was established by chronic constrictive injury (CCI) of the sciatic nerve. The rats were randomly divided into sham group, CCI group, and sham CCI + EA group, and CCI + EA group. EA was applied to bilateral Zusanli (ST36)-Yanlingquan (GB34). The mechanical (both time and force responses) and thermal pain thresholds (PTs) of the bilateral hind-paws were measured. The number of microgliacytes and activity of astrocytes in the dorsal horns (DHs) of lumbar spinal cord (L4–5) were examined by immunofluorescence staining, and the expression of glial fibrillary acidic protein (GFAP) protein was detected by western blot.ResultsFollowing CCI, both mechanical and thermal PTs of the ipsilateral hind-paw were significantly decreased beginning from the 3rd day after surgery (P < 0.05), and the mechanical PT of the contralateral hind-paw was considerably decreased from the 6th day on after surgery (P < 0.05). CCI also significantly upregulated the number of Iba-1 labeled microgliacytes and the fluorescence intensity of glial fibrillary acidic protein (GFAP) -labeled astrocyte in the superficial laminae of DHs on bilateral sides (P < 0.05). After repeated EA, the mechanical and thermal PTs at bilateral hind-paws were significantly relieved (P < 0.05). The increased of number of microgliacytes was markedly suppressed by 2 days’ EA intervention, and the average fluorescence intensity was suppressed by 2 weeks’ EA. The expression of GFAP protein were down-regulated by 1 and 2 weeks’ EA treatment, respectively (P < 0.05).ConclusionsRepeated EA can relieve neuropathic pain and mirror-image pain in chronic neuropathic pain rats, which is probably associated with its effect in downregulating glial cell activation of the lumbar spinal cord, the microgliacyte first and astrocyte later.

Calcineurin A beta deficiency ameliorates HFD-induced hypothalamic astrocytosis in mice

ᅟAstrocytosis is a reactive process involving cellular, molecular, and functional changes to facilitate neuronal survival, myelin preservation, blood brain barrier function and protective glial scar formation upon brain insult. The overall pro- or anti-inflammatory impact of reactive astrocytes appears to be driven in a context- and disease-driven manner by modulation of astrocytic Ca2+ homeostasis and activation of Ca2+/calmodulin-activated serine/threonine phosphatase calcineurin. Here, we aimed to assess whether calcineurin is dispensable for astrocytosis in the hypothalamus driven by prolonged high fat diet (HFD) feeding.Global deletion of calcineurin A beta (gene name: Ppp3cb) led to a decrease of glial fibrillary acidic protein (GFAP)-positive cells in the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and arcuate nucleus (ARC) of mice exposed chronically to HFD. The concomitant decrease in Iba1-positive microglia in the VMH further suggests a modest impact of Ppp3cb deletion on microgliosis. Pharmacological inhibition of calcineurin activity by Fk506 had no impact on IBA1-positive microglia in hypothalami of mice acutely exposed to HFD for 1 week. However, Fk506-treated mice displayed a decrease in GFAP levels in the ARC. In vivo effects could not be replicated in cell culture, where calcineurin inhibition by Fk506 had no effect on astrocytic morphology, astrocytic cell death, GFAP, and vimentin protein levels or microglia numbers in primary hypothalamic astrocytes and microglia co-cultures. Further, adenoviral overexpression of calcineurin subunit Ppp3r1 in primary glia culture did not lead to an increase in GFAP fluorescence intensity.Overall, our results point to a prominent role of calcineurin in mediating hypothalamic astrocytosis as response to acute and chronic HFD exposure. Moreover, discrepant findings in vivo and in cell culture indicate the necessity of studying astrocytes in their “natural” environment, i.e., preserving an intact hypothalamic microenvironment with neurons and non-neuronal cells in close proximity.

Hippocampal astrocyte atrophy in a mouse depression model induced by corticosterone is reversed by fluoxetine instead of benzodiazepine diazepam

Astrocytes have become promising new agents against major depressive disorders (MDD) primarily due to the crucial role they play in the pathogenesis of such disorders. However, a simple and reliable animal model that can be used to screen for astrocyte-targeting antidepressants has not yet been developed. In this study, we utilized a repeated corticosterone (CORT) injection paradigm to develop a mouse depression model wherein we examined the occurrence of alterations in hippocampal astrocyte population by using two astrocytic markers, namely, glial fibrillary acidic protein (GFAP) and S100β. Moreover, we determined the effects of fluoxetine and diazepam on CORT-induced astrocytic alterations to assess the predictive validity. Results showed that repeated CORT injections showed no effects on the number of GFAP+ and S100β+ astrocytes, but they decreased the protrusion length of GFAP+ astrocytes and GFAP protein expression in the hippocampus. Furthermore, repeated CORT injections produced a sustained increase of S100β protein levels in the entire hippocampus of male mice. CORT-induced hippocampal astrocyte disruption was antagonized by chronic fluoxetine treatment. By contrast, the anxiolytic drug diazepam was ineffective in the same experimental setting. All these findings suggest that the repeated CORT injection paradigm produces the astrocytic alterations similar to those in MDD and can serve as a useful mouse model to screen antidepressants meant to target astrocytes. These observations can also help in further discussing the underlying mechanisms of CORT-induced astrocytic alterations.

Aberrant astrocyte Ca2+ signals "AxCa signals" exacerbate pathological alterations in an Alexander disease model.

Alexander disease (AxD) is a rare neurodegenerative disorder caused by gain of function mutations in the glial fibrillary acidic protein (GFAP) gene. Accumulation of GFAP proteins and formation of Rosenthal fibers (RFs) in astrocytes are hallmarks of AxD. However, malfunction of astrocytes in the AxD brain is poorly understood. Here, we show aberrant Ca2+ responses in astrocytes as playing a causative role in AxD. Transcriptome analysis of astrocytes from a model of AxD showed age-dependent upregulation of GFAP, several markers for neurotoxic reactive astrocytes, and downregulation of Ca2+ homeostasis molecules. In situ AxD model astrocytes produced aberrant extra-large Ca2+ signals "AxCa signals", which increased with age, correlated with GFAP upregulation, and were dependent on stored Ca2+ . Inhibition of AxCa signals by deletion of inositol 1,4,5-trisphosphate type 2 receptors (IP3R2) ameliorated AxD pathogenesis. Taken together, AxCa signals in the model astrocytes would contribute to AxD pathogenesis.

Protective effect of epigenetic silencing of CyclinD1 against spinal cord injury using bone marrow-derived mesenchymal stem cells in rats.

This study focuses on the protective effect of epigenetic silencing of CyclinD1 against spinal cord injury (SCI) using bone marrow-derived mesenchymal stem cells (BMSCs) in rats. Eighty-eight adult female Wistar rats were randomly assigned into the sham group, the control group, the si-CyclinD1 + BMSCs group and the BMSCs group. CyclinD1 protein and mRNA expressions after siRNA transfection were detected by Western blotting and qRT-PCR. The siRNA-CyclinD1 BMSCs were transplanted into rats in the si-CyclinD1 + BMSCs group using stereotaxic method 6 hr after SCI. Hindlimb locomotor performance was determined using inclined plane test and Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Expressions of glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) were detected by immunohistochemistry. Inclined plane and BBB scores in the control, si-CyclinD1 + BMSCs, and BMSCs groups were significantly lower than the sham group, but these scores were evidently decreased in the control group and increased in the si-CyclinD1 + BMSCs group compared with the BMSCs group. The repair degree of spinal cord tissues of rats in the si-CyclinD1 + BMSCs group was obvious than the BMSCs group. GFAP and NGF protein expressions were markedly decreased in the control, si-CyclinD1 + BMSCs and BMSCs groups when compared with the sham group. GFAP- and NGF-positive cells were significantly increased in the si-CyclinD1 + BMSCs group while decreased in the control group. Our study provides evidence that epigenetic silencing of CyclinD1 using BMSCs might accelerate the repair of SCI in rats.

Antisense suppression of glial fibrillary acidic protein as a treatment for Alexander disease.

Alexander disease is a fatal leukodystrophy caused by autosomal dominant gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament protein primarily expressed in astrocytes of the central nervous system. A key feature of pathogenesis is overexpression and accumulation of GFAP, with formation of characteristic cytoplasmic aggregates known as Rosenthal fibers. Here we investigate whether suppressing GFAP with antisense oligonucleotides could provide a therapeutic strategy for treating Alexander disease. In this study, we use GFAP mutant mouse models of Alexander disease to test the efficacy of antisense suppression and evaluate the effects on molecular and cellular phenotypes and non-cell-autonomous toxicity. Antisense oligonucleotides were designed to target the murine Gfap transcript, and screened using primary mouse cortical cultures. Lead oligonucleotides were then tested for their ability to reduce GFAP transcripts and protein, first in wild-type mice with normal levels of GFAP, and then in adult mutant mice with established pathology and elevated levels of GFAP. Nearly complete and long-lasting elimination of GFAP occurred in brain and spinal cord following single bolus intracerebroventricular injections, with a striking reversal of Rosenthal fibers and downstream markers of microglial and other stress-related responses. GFAP protein was also cleared from cerebrospinal fluid, demonstrating its potential utility as a biomarker in future clinical applications. Finally, treatment led to improved body condition and rescue of hippocampal neurogenesis. These results demonstrate the efficacy of antisense suppression for an astrocyte target, and provide a compelling therapeutic approach for Alexander disease. Ann Neurol 2018;83:27-39.

Brain-originated peptides as possible biochemical markers of traumatic brain injury in cerebrospinal fluid post-mortem examination.

The release of brain-originated peptides such as tau protein (MAPT), S-100β, neurofilament light chain (NFL), and glial fibrillary acidic protein (GFAP) into the cerebrospinal fluid (CSF) has been positively correlated with head injuries in clinical and basic research. In this study, we wanted to examine if selected CSF biomarkers (GFAP, NFL, and myelin basic protein - MBP) of head injury may be useful in post-mortem examination and diagnosis of forensic cases. The study was carried out using cases of head injury and cases of sudden death (cardiopulmonary failure, no injuries of the head as control group) provided by forensic pathologists at the Department of Forensic Medicine, Medical University of Warsaw. Cerebrospinal fluid was collected within 24 h after death using suboccipital puncture. The concentration of these peptides was compared using an enzyme-linked immunosorbent assay (ELISA). Brain specimens (frontal cortex) were collected during forensic autopsies. Sections were stained immunohistochemically against GFAP, MBP, NF, and amyloid-β precursor protein (APP). As aresult we documented that elevated levels of CSF, GFAP, MBP, and NFL should be considered amarker for severe and moderate traumatic brain injury. Elevated levels of those peptides combined with anegative APP staining point to their role as markers of head trauma with ashorter time span than APP (manner of minutes).

The Role of Wnt/β-Catenin Signaling Pathway in the Transdifferentiation from Periodontal Ligament Stem Cells to Schwann Cells.

This study was aimed to investigate the role of Wnt/β-catenin signaling pathway in the differentiation from periodontal ligament stem cells (PDLSCs) to Schwann cells (SCs) and the possible mechanisms. FzB was applied to inhibit the Wnt/β-catenin signaling pathway of differentiated PDLSCs (dPDLSCs), and then immunofluorescence, polymerase chain reaction (PCR), and Western blotting analysis were performed to detect SC marker genes and proteins such as S100, glial fibrillary acidic protein (GFAP), and P75NTR. Results showed that when the Wnt/β-catenin signaling pathway was inhibited, the expression of S100, GFAP, and P75NTR protein significantly decreased in dPDLSCs, p < 0.05, whereas PCR results showed that expression of SC myelinogenesis-related genes krox-20, Oct-6, P0, and PMP-22 was significantly downregulated at the same time, p < 0.05. These results showed that Wnt/β-catenin signaling pathway participated in the differentiation from PDLSCs to SCs, and inhibiting it could inhibit the differentiation process.

RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.

Scolopendra Pharmacopuncture Ameliorates Behavioral Despair in Mice Stressed by Chronic Restraint.

IntroductionPharmacopuncture, which combines acupuncture with herbal medicine, is one of the newly developed acupuncture techniques that has recently been put into use. The possible mechanisms of scolopendra pharmacopuncture, as well as its potential effects on depressive symptoms, were investigated in this study by using a mouse model of chronic immobilization stress (CIS).MethodsC57BL/6 male mice were randomly assigned into three groups: mice not stressed with restraint and injected with distilled water, mice stressed with restraint and injected with distilled water, and mice stressed with restraint injected with scolopendra pharmacopuncture at a cervical site. Behavioral tests (an open field test, tail suspension test, and forced swimming test) were carried out after two weeks of CIS and injection treatments. The expression levels of glial fibrillary acidic protein (GFAP) in the hippocampus were determined by using western blot and immunohistochemistry analyses.ResultsMice exposed to CIS showed decreased behavioral activity, while scolopendra pharmacopuncture treatment significantly protected against the depressive-like behaviors induced by CIS. Moreover, scolopendra pharmacopuncture treatment increased GFAP protein levels in the hippocampi of the mice stressed by chronic immobilization.ConclusionScolopendra pharmacopuncture has an ameliorating effect on depressive behavior, which is partially mediated through protection against glial loss in the hippocampus.

The effect of netrin-1 on activation of Mueller cell in diabetes rats

Objective To observe the effect of netrin-1 on retinal Muller cells in diabetes mellitus (DM) rats. Methods Fifty Sprague-Dawley rats were randomly divided into the normal control group (group A), normal + balanced salt solution (BSS) group (group B), normal+netrin-1 group (group C), DM+BSS group (group D) and DM+netrin-1 group (group E), with 10 rats in each group. DM rats were induced by intraperitoneal injection of Streptozotocin (60 mg/kg). The expression level of glial fibrillary acidic protein (GFAP) on retinal Muller cells was determined by immunohistochemistry, the level of GFAP mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Results Immunohistochemistry showed that GFAP was distributed in retinal ganglion cells and retinal nerve fiber layer in group A, B and C. Compared to group B, GFAP staining was brighter in the group D. There were significant differences in the expression of GFAP protein and mRNA among groups A-E (F=203.43, 72.91; P=0.00, 0.00), they were higher in group D than group A (t=−26.01, 22.26; P=0.00, 0.00), and group E (t=−10.78, 3.93; P=0.00, 0.00). They were higher in group E than group A (t=7.00, −9.82; P=0.00, 0.00). There were no significant differences in between group A and group C (t=−0.29, 0.50; P=0.77, 0.62). Conclusion The expression of GFAP in Muller cells of DM rats could be decreased by injecting netrin-1 into vitreous. Key words: Diabetic retinopathy/physiopathology; Microglia/physiology; Nerve growth factors; Animal experimentation

Oxytocin Rapidly Changes Astrocytic GFAP Plasticity by Differentially Modulating the Expressions of pERK 1/2 and Protein Kinase A.

The importance of astrocytes to normal brain functions and neurological diseases has been extensively recognized; however, cellular mechanisms underlying functional and structural plasticities of astrocytes remain poorly understood. Oxytocin (OT) is a neuropeptide that can rapidly change astrocytic plasticity in association with lactation, as indicated in the expression of glial fibrillary acidic protein (GFAP) in the supraoptic nucleus (SON). Here, we used OT-evoked changes in GFAP expression in astrocytes of male rat SON as a model to explore the cellular mechanisms underlying GFAP plasticity. The results showed that OT significantly reduced the expression of GFAP filaments and proteins in SON astrocytes in brain slices. In lysates of the SON, OT receptors (OTRs) were co-immunoprecipitated with GFAP; vasopressin (VP), a neuropeptide structurally similar to OT, did not significantly change GFAP protein level; OT-evoked depolarization of astrocyte membrane potential was sensitive to a selective OTR antagonist (OTRA) but not to tetanus toxin, a blocker of synaptic transmission. The effects of OT on GFAP expression and on astrocyte uptake of Bauer-Peptide, an astrocyte-specific dye, were mimicked by isoproterenol (IPT; β-adrenoceptor agonist), U0126 or PD98059, inhibitors of extracellular signal-regulated protein kinase (ERK) 1/2 kinase and blocked by the OTRA or KT5720, a protein kinase A (PKA) inhibitor. The effect of OT on GFAP expressions and its association with these kinases were simulated by mSIRK, an activator of Gβγ subunits. Finally, suckling increased astrocytic expression of the catalytic subunit of PKA (cPKA) at astrocytic processes while increasing the molecular associations of GFAP with cPKA and phosphorylated ERK (pERK) 1/2. Upon the occurrence of the milk-ejection reflex, spatial co-localization of the cPKA with GFAP filaments further increased, which was accompanied with increased molecular association of GFAP with pERK 1/2 but not with cPKA. Thus, OT-elicited GFAP plasticity is achieved by sequential activation of ERK 1/2 and PKA via OTR signaling pathway in an antagonistic but coordinated manner.

Melatonin Attenuates Pain Hypersensitivity and Decreases Astrocyte-Mediated Spinal Neuroinflammation in a Rat Model of Oxaliplatin-Induced Pain.

Neuroinflammatory response in spinal dorsal horn has been demonstrated to be a critical factor in oxaliplatin-induced pain. Melatonin has been shown to have anti-inflammatory and anti-allodynia effects in both preclinical and clinical studies. In the present study, we investigated the role of systemic administration of melatonin on oxaliplatin-induced pain. Intraperitoneal (i.p.) injection with oxaliplatin induced significantly mechanical allodynia and thermal hyperalgesia. Melatonin (i.p.) significantly alleviated mechanical allodynia and thermal hyperalgesia in the oxaliplatin but not sham-treated rats. The attenuation of nociceptive response persisted at least to 3days after melatonin injection, throughout the entire observing window. Immunohistochemistry showed that oxaliplatin induced a significant increase of glial fibrillary acidic protein (GFAP) immunodensities, which could be suppressed by melatonin. Western blotting showed that GFAP protein levels were significantly elevated in the oxaliplatin-vehicle group. Melatonin significantly decreased oxaliplatin-induced upregulation of GFAP expressions. Oxaliplatin injection also enhanced the messenger RNA (mRNA) expressions of cytokines including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and chemokines including monocyte chemoattractant protein-1 (MCP-1) and monocyte inflammatory protein-1 (MIP-1α) in the spinal dorsal horn, which could be significantly repressed by melatonin. In vitro study showed that mRNA levels of TNF-α, IL-1β, MCP-1, and MIP-1α in primarily astrocytes were significantly increased after lipopolysaccharide (LPS, 1μg/ml) stimulation. Melatonin (10 and 100μM) greatly inhibited synthesis of these inflammatory mediators, in a dose-related manner. Conclusively, our data provide a novel implication of anti-nociceptive mechanism of melatonin in chemotherapy-related pain.

Breakthrough Cancer Pain Is Associated with Spinal Gap Junction Activation via Regulation of Connexin 43 in a Mouse Model.

Breakthrough cancer pain (BTcP) is a high-intensity, short-duration, unpredictable and uncontrollable pain. Recent studies have shown that activation of gap junction (GJ) in spinal cord plays an important role in the pathogenesis of BTcP. We examined the expressions of Glial fibrillary acidic protein (GFAP), connexin (Cx) 43 protein and phosphorylation of Cx43 (p-Cx43) in the spinal cord of mice. In addition, we investigated the effects of Gap26, a selective GJ blocker, on the expressions of GFAP, Cx43 and p-Cx43 in BTcP mice. We found that the expressions of GFAP and Cx43 proteins were significantly upregulated while p-Cx43 was down-regulated in the spinal cord in a mouse model of BTcP. The overexpression of Cx43 protein in the spinal cord increased GJ formation and enhanced BTcP. The variation of the ratio of p-Cx43/T-Cx43 (total Cx43) affected the function of GJ to induce BTcP. Furthermore, BTcP was alleviated by Gap26 via reducing pain hypersensitivity. The inhibition of Cx43 and p-Cx43 by Gap26 attenuated BTcP but the p/T ratio of Cx43 remained unchanged in BTcP mice. We reveal that the expression and phosphorylation of Cx43 affected BTcP and GJ activation facilitated BTcP via a Cx43-mediated signaling in the spinal cord. The finding may provide a scientific rationale for discovery and development of novel therapeutic targets for the treatment of BTcP clinically.

Serial Sampling of Serum Protein Biomarkers for Monitoring Human Traumatic Brain Injury Dynamics: A Systematic Review.

The proteins S100B, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and neurofilament light (NF-L) have been serially sampled in serum of patients suffering from traumatic brain injury (TBI) in order to assess injury severity and tissue fate. We review the current literature of serum level dynamics of these proteins following TBI and used the term "effective half-life" (t1/2) in order to describe the "fall" rate in serum. Through searches on EMBASE, Medline, and Scopus, we looked for articles where these proteins had been serially sampled in serum in human TBI. We excluded animal studies, studies with only one presented sample and studies without neuroradiological examinations. Following screening (10,389 papers), n = 122 papers were included. The proteins S100B (n = 66) and NSE (n = 27) were the two most frequent biomarkers that were serially sampled. For S100B in severe TBI, a majority of studies indicate a t1/2 of about 24 h, even if very early sampling in these patients reveals rapid decreases (1-2 h) though possibly of non-cerebral origin. In contrast, the t1/2 for NSE is comparably longer, ranging from 48 to 72 h in severe TBI cases. The protein GFAP (n = 18) appears to have t1/2 of about 24-48 h in severe TBI. The protein UCH-L1 (n = 9) presents a t1/2 around 7 h in mild TBI and about 10 h in severe. Frequent sampling of these proteins revealed different trajectories with persisting high serum levels, or secondary peaks, in patients with unfavorable outcome or in patients developing secondary detrimental events. Finally, NF-L (n = 2) only increased in the few studies available, suggesting a serum availability of >10 days. To date, automated assays are available for S100B and NSE making them faster and more practical to use. Serial sampling of brain-specific proteins in serum reveals different temporal trajectories that should be acknowledged. Proteins with shorter serum availability, like S100B, may be superior to proteins such as NF-L in detection of secondary harmful events when monitoring patients with TBI.

Repetitive Closed-Head Impact Model of Engineered Rotational Acceleration Induces Long-Term Cognitive Impairments with Persistent Astrogliosis and Microgliosis in Mice

Repeated mild traumatic brain injury (rmTBI) has been identified by epidemiology as a high-risk factor for dementia at a later stage in life. Animal models to replicate complex features of human rmTBI and/or to evaluate long-term effects on brain function have not been established. In this study, we used a novel closed-head impact model of engineered rotational acceleration (CHIMERA) to investigate the long-term neuropathological and cognitive functional consequences of rmTBI. Adult C57BL/6 male mice were subjected to CHIMERA for 3 consecutive days 24 h apart. Functional outcomes were assessed by the beam walk and Morris water maze tests. Neuropathology was evaluated by immunostaining of glial fibrillary acidic protein (GFAP), amyloid precursor protein (APP), and ionizing calcium-binding adaptor molecule-1 (Iba-1), and by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blotting of GFAP, Iba-1, and tumor necrosis factor (TNF)-α. Repeated CHIMERA (rCHIMERA) resulted in motor deficits at 3 days, and in learning and memory impairments that were sustained up to 6 months post injury. GFAP and TNF-α gene expression was increased within a week, whereas astrogliosis and microgliosis were induced starting from day 1 up to 6.5 months after rCHIMERA with upregulated GFAP and Iba-1 protein levels. rCHIMERA also induced APP deposition from day 1 to day 7, but this diminished by 1 month. In conclusion, rCHIMERA produces long-lasting cognitive impairments with astrogliosis and microgliosis in mice, suggesting that rCHIMERA can be a useful animal model to study the long-term complications, as well as the cellular and molecular mechanisms, of human rmTBI.