Glial Fibrillary Acidic Protein Staining Research Articles

Effect of Chlorogenic Acid Supplementation in MPTP-Intoxicated Mouse.

Oxidative stress and neuroinflammation play a key role in dopaminergic (DA) neuronal degeneration, which results in the hindrance of normal ongoing biological processes in the case of Parkinson’s disease. As shown in several studies, on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, different behavioral parameters have suggested motor impairment and damage of antioxidant defence. Thus, some specific biological molecules found in medicinal plants can be used to inhibit the DA neuronal degeneration through their antioxidant and anti-inflammatory activities. With this objective, we studied chlorogenic acid (CGA), a naturally occurring polyphenolic compound, for its antioxidant and anti-inflammatory properties in MPTP-intoxicated mice. We observed significant reoccurrence of motor coordination and antioxidant defence on CGA supplementation, which has been in contrast with MPTP-injected mice. Moreover, in the case of CGA-treated mice, the enhanced expression of tyrosine hydroxylase (TH) within the nigrostriatal region has supported its beneficial effect. The activation of glial cells and oxidative stress levels were also estimated using inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) immunoreactivity within substantia nigra (SN) and striatum of MPTP-injected mice. Administration of CGA has prevented the neuroinflammation in SN by regulating the nuclear factor-κB expression in the MPTP-induced group. The significant release of certain pro-inflammatory mediators such as tumor necrosis factor-α and interleukin (IL)-1β has also been inhibited by CGA with the enhanced expression of anti-inflammatory cytokine IL-10. Moreover, reduced GFAP staining within the nigrostriatal region has supported the fact that CGA has significantly helped in the attenuation of astrocyte activation. Hence, our study has shown that CGA supplementation shows its therapeutic ability by reducing the oxidative stress and neuroinflammation in MPTP-intoxicated mice.

Continuous remote ischemic conditioning attenuates cognitive and motor deficits from moderate traumatic brain injury.

While studies show that single-dose remote ischemic conditioning (RIC) improves outcomes, the effect of continuous (daily) RIC is unknown. Thus, we aimed to investigate the role of continuous RIC on cognitive and motor function following traumatic brain injury (TBI). We subjected 24 male C57BL mice to a cortical-controlled TBI. Two hours after TBI, the animals were randomly allocated to the RIC group (n = 12) or the sham group (n = 12). Remote ischemic conditioning was induced by noninvasive external compression of the hind limb using an occlusive band (six 4-minute cycles/24 hours) for six consecutive days. Before TBI, a baseline rotarod test and novel object recognition were performed. Post-TBI rotarod and novel object recognition tests were performed on Days 1 to 5, 7, 14, and 21. After the animals were sacrificed on Day 21, brain sections were analyzed using hematoxylin and eosin and glial fibrillary acidic protein staining to evaluate the hippocampal CA1 area for neuronal injury. Both the RIC and sham groups had lower latency to fall compared with the baseline post-TBI. The RIC animals had a higher latency to fall compared with the sham animals at all time points, statistically significant after Day 3, until Day 21 post-TBI. Both the RIC and sham groups had lower recognition index compared with the baseline post-TBI. The RIC animals had a significantly higher recognition index than the sham animals after Day 1, until Day 21 post-TBI. Hematoxylin and eosin and glial fibrillary acidic protein staining of the brain samples of the sham group revealed that more neurons in the hippocampal CA1 area appeared shrunken with eosinophilic cytoplasm and pyknotic nuclei compared with the brain samples of the RIC group. Postinjury continuous RIC resulted in improved cognitive functions and motor coordination in a mouse model of moderate TBI. Further studies are required to determine optimum dosage and frequency of this novel therapy to maximize its beneficial effects following TBI.

Protective effects of a novel drug RC28-E blocking both VEGF and FGF2 on early diabetic rat retina.

To investigate protective effects of a novel recombinant decoy receptor drug RC28-E on retinal damage in early diabetic rats. The streptozotocin (STZ)-induced diabetic rats were randomly divided into 6 groups: diabetes mellitus (DM) group (saline, 3 µL/eye); RC28-E at low (0.33 µg/µL, 3 µL), medium (1 µg/µL, 3 µL), and high (3 µg/µL, 3 µL) dose groups; vascular endothelial growth factor (VEGF) Trap group (1 µg/µL, 3 µL); fibroblast growth factor (FGF) Trap group (1 µg/µL, 3 µL). Normal control group was included. At week 1 and 4 following diabetic induction, the rats were intravitreally injected with the corresponding solutions. At week 6 following the induction, apoptosis in retinal vessels was detected by TUNEL staining. Glial fibrillary acidic protein (GFAP) expression was examined by immunofluorescence. Blood-retinal barrier (BRB) breakdown was assessed by Evans blue assay. Ultrastructural changes in choroidal and retinal vessels were analyzed by transmission electron microscopy (TEM). Content of VEGF and FGF proteins in retina was measured by enzyme linked immunosorbent assay (ELISA). The retinal expression of intercellular cell adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), VEGF and FGF genes was examined by quantitative polymerase chain reaction (qPCR). TUNEL staining showed that the aberrantly increased apoptotic cells death in diabetic retinal vascular network was significantly reduced by treatments of medium and high dose RC28-E, VEGF Trap, and FGF Trap (all P<0.05), the effects of medium and high dose RC28-E or FGF Trap were greater than VEGF Trap (P<0.01). GFAP staining suggested that reactive gliosis was substantially inhibited in all RC28-E and VEGF Trap groups, but the inhibition in FGF Trap group was not as prominent. Evans blue assay demonstrated that only high dose RC28-E could significantly reduce vascular leakage in early diabetic retina (P<0.01). TEM revealed that the ultrastructures in choroidal and retinal vessels were damaged in early diabetic retina, which was ameliorated to differential extents by each drug. The expression of VEGF and FGF2 proteins was significantly upregulated in early diabetic retina, and normalized by RC28-E at all dosages and by the corresponding Traps. The upregulation of ICAM-1 and TNF-α in diabetic retina was substantially suppressed by RC28-E and positive control drugs. Dual blockade of VEGF and FGF2 by RC28-E generates remarkable protective effects, including anti-apoptosis, anti-gliosis, anti-leakage, and improving ultrastructures and proinflammatory microenvironment, in early diabetic retina, thereby supporting further development of RC28-E into a novel and effective drug to diabetic retinopathy (DR).

Morphology and quantification of sheep pineal glands at pre-pubertal, pubertal and post-pubertal periods.

The pineal gland is a neuroendocrine organ associated with photoperiodic regulation in mammals. The aim of this study was to evaluate the pineal gland at the pre-pubertal, pubertal and post-pubertal periods by means of morphology and stereology. The study examined at total of 24 ovine pineal glands collected from healthy female Akkaraman breed. Thick sections (40μm) were cut and treated with synaptophysin. Following each thick section, six consecutive sections at a thickness of 5μm were cut. Each thin section was stained with one of the following dyes: Crossman's modified triple dye, glial fibrillary acidic protein (GFAP), melatonin marker, periodic acid-Schiff, Von Kossa and AgNOR. The pineal gland volume was measured using Cavalieri's method. The optical fractionator was used to estimate the total number of pinealocytes. The percentage of parenchyma and connective tissue and degree of vascularization were estimated by the area fraction fractionator method. The pineal gland volumes in the pre-pubertal, pubertal and post-pubertal groups were 7.53±1.715mm3 , 11.20±1.336mm3 and 17.75±1.188mm3 , respectively (p<.5). The number of pinealocytes in the pre-pubertal, pubertal and post-pubertal groups was 3,244,000±228,076, 4,438,000±243,610, 7,381,766±406,223, respectively (p<.05). The glands of the post-pubertal group contained the highest amount of connective tissue (11.49±2.103%; p<.5) and the largest GFAP staining area (p<.05). The melatonin staining density was the highest in the pubertal group. The density of lipofuscin staining was higher in the pubertal and post-pubertal groups.

Different behavioral and pathological changes between epilepsy-associated depression and primary depression models

PurposeComorbid depression is common in patients with epilepsy. However, the epilepsy-associated depression is generally atypical and has not been fully recognized by neurologists. This study aimed to compare the behavioral and pathological changes between the chronic lithium chloride-pilocarpine rat epilepsy model (Licl-pilocarpine model) and the Chronic Unpredictable Mild Stress rat depression model (CUMS model), to evaluate for differences between epilepsy-associated depression and primary depression. MethodsThe Licl-pilocarpine model and the CUMS model were established respectively and simultaneously. Spontaneous seizures were recorded by video monitoring. Forced swim test (FST) and sucrose consumption test (SCT) were performed to test depressive behaviors. Immobility time (IMT) and climbing time (CMT) in FST, sucrose preference rate (SPR) in SCT, and weight gain rate (WGR) were adopted to represent severity of depressive behaviors in rats. Immunofluorescent staining was conducted to measure expressions of neuronal specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), and cluster of differentiation molecule 11b (CD11b) in the hippocampus of Licl-pilocarpine model, CUMS model, and Control group. ResultsSignificantly, more prolonged IMT was observed in both the Licl-pilocarpine model (p<0.05) and the CUMS model (p<0.01) than Control group. But decreased WGR was only seen in the CUMS model. The percentage of rats with CMT greater than 100s was significantly higher in the Licl-pilocarpine model than the CUMS model (p<0.05). Increased CMT was observed in the Licl-pilocarpine model with mild depression subgroup (EMD, IMT≤100s) even compared with the Control group. Neuronal loss was both found in the Licl-pilocarpine model and the CUMS model when comparing with the Control group (p<0.05). However, the number of GFAP and CD11b staining cells was both greater in the Licl-pilocarpine model than the CUMS model and the Control group (p<0.05). ConclusionThere were some different depressive behavioral and hippocampal pathological changes between the Licl-pilocarpine and the CUMS models except for some common features. Gliosis and microglial activation might be more involved in the pathophysiology of epilepsy-associated depression than primary depression.

Astrogliosis and downregulation of EAAT2 in rats infused with Nef transfected astrocytes.

Astrocytes play a major role in homeostasis and neuronal function by preventing excitotoxicity in the CNS. During HIV infection, astrocytes are reported to produce early viral proteins such as Nef, which is known to cause neurotoxicity. Excitatory amino acid transporter 2 (EAAT2) is one of the major astroglial transporters involved in the regulation of extracellular glutamate concentration. We investigated astrocytes transfected with Nef to understand the effect on learning, astrocyte morphology and glutamate transporter expression. Primary rat astrocytes expressing Nef or GFP were infused in the right hippocampus of 30‐day‐old male Sprague Dawley rats. Learning was assessed by Morris water maze five days after infusion. Brain tissues were collected for immunofluorescence staining and the morphological assessment of astrocytes and EAAT2 expression was assessed in different areas of the hippocampus (CA3 and dentate gyrus) using Image J software. In astrocytes, glial fibrillary acidic protein (GFAP) area fraction was significantly increased (P&lt;0.05), while EAAT2 expression was significantly decreased (p&lt;0.05) in the Nef‐treated group when compared to GFP controls. No differences in the length of processes in astrocytes were found, however the number of secondary processes was increased in the Nef‐treated animals. The increase of astrogliosis as measured by GFAP staining, the number of secondary processes, and the decrease of EAAT2 correlates with the learning impairment shown by the Nef‐treated group. These results suggest that there might be an astrocyte dysfunction that affects glutamate uptake at the synaptic cleft leading to neuronal toxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The soluble guanylate cyclase stimulator IWP‐550 inhibits neuroinflammation in vitro and in vivo

ObjectiveIWP‐550 is a brain‐penetrant soluble guanylate cyclase (sGC) stimulator. The objective of this work was to evaluate the effects of IWP‐550 on neuroinflammation in vitro in rat primary neurons, mouse microglial cells, rat brain 3D microtissues (MT, InSphero™), and in an in vivo quinolinic acid (QA)‐induced neuroinflammation rat model.MethodsMouse microglial SIM‐A9 cells, rat primary neurons and rat brain MT were stimulated with IWP‐550 for 30 min at 37°C. cGMP levels were measured by LC/MS. Phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) and CREB was assayed using CisBio HTRF kits. For cytokine studies, cells or brain MT were treated with LPS and IWP‐550 for 24 h at 37°C. IL‐6 and TNFα levels in SIM‐A9 cells were determined by ELISA. Cytokines from isolated rat primary neurons and brain MT were measured by MSD multiplex assay. Neuroinflammation was induced in vivo by injecting QA in the rat striatum followed by treatment with IWP‐550 (10 mg/kg, p.o. QD) for 7 days. In the striatum, expression of ionized calcium‐binding adapter molecule 1 (Iba1), a marker of microglial activation, and glial fibrillary acidic protein (GFAP), a marker of astrogliosis, was quantified by IHC staining. Gene expression of TNFα, CD40, and MIP1α was determined by QuantiGene MultiPlex Assay (Affymetrix).ResultsIWP‐550 dose‐dependently stimulated cGMP formation (EC50 = 16 nM) and phosphorylation of CREB in isolated rat primary neurons. IWP‐550 also stimulated cGMP formation and phosphorylation of VASP in SIM‐A9 cells in synergy with the nitric oxide donor, DETA‐NONOate (DETA). In rat primary neurons, IWP‐550 decreased secreted proinflammatory cytokines, including IL‐1β, TNFα and MCP‐1 that were elevated by LPS treatment (1 μg/ml). In SIM‐A9 cells, IWP‐550 dose‐dependently decreased LPS‐mediated IL‐6 and TNFα elevation. In brain MT containing a mix of neurons, astrocytes, microglial cells and oligodendrocytes, IWP‐550 stimulated cGMP formation and phosphorylation of CREB. Moreover, IWP‐550 (1 and 10 μM) in the presence of DETA (10 μM) reduced LPS‐induced elevation of IL‐6, IFNγ, TNFα, IL‐1β, IL‐13 and CXCL1. In rat, QA administered unilaterally in the striatum caused local brain tissue injury and led to the activation of microglia and astrocytes, as demonstrated by increases in IHC staining for Iba1 and GFAP compared to the control side of striatum. Gene expression of pro‐inflammatory cytokines TNFα, CD40 and MIP1α were also increased following QA injection. Treatment with IWP‐550 (10 mg/kg) for 7 days significantly reduced Iba1 and GFAP staining, as well as gene expression of TNFα, CD40 and MIP1α, indicating a reduction of QA‐induced astrogliosis and inflammation in vivo.ConclusionIWP‐550 suppressed neuroinflammation in vitro in primary neurons, microglial cells, and brain microtissues, as well as in vivo in QA‐treated rats. These data suggest that sGC stimulation may provide potential neuroprotective effect by reducing neuroinflammation, a hallmark of neurodegenerative diseases.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Medullary Astrogliosis in Sudden Infant Death Syndrome Varies With Sleeping Environment: Evidence for Different Mechanisms of Death in Alone Versus Co-sleepers?

Sudden infant death syndrome remains the leading cause of death in infants under 1 year, and underlying pathophysiological mechanisms are poorly understood. The current study investigated the hypothesis that co-sleepers die more rapidly from causes such as suffocation from overlaying by comparing levels of reactive astrogliosis in the medulla of infants who died sleeping alone to those who died co-sleeping. The amount of glial fibrillary acidic protein (GFAP) staining in alone sleepers was significantly higher than shared sleepers in 3 specific areas of the medulla, the inferior vestibular nucleus, the medial vestibular nucleus and the cochlear nucleus. Given that glial fibrillary acidic protein elevations follow a delayed time course, this suggests that death in co-sleepers was more rapid, not allowing for reactive gliosis to occur. This provides evidence of pathological differences in mechanisms of death in infants who are classified as having died from sudden infant death syndrome, suggesting potential need for refinement of categorization of these cases.

Effect of Qingxin Kaiqiao Fang on Hippocampus mRNA Expression of the Inflammation‐Related Genes IL‐1β, GFAP, and Aβ in an Alzheimer’s Disease Rat Model

Objective To investigate the effects of QKF on expression of amyloid-beta (Aβ), interleukin-1 beta (IL-1β), and glial fibrillary acidic protein (GFAP) using a rat model of AD. Materials and Methods Fifty-six male Sprague-Dawley rats were randomly divided into seven groups (eight rats each): control group, sham-operated group, AD model group, groups of AD rats administered with low, medium, and high doses of QKF, and the donepezil group. AD was established by bilateral injection of β-amyloid (Aβ) 1–40 into the hippocampus. Two days after AD was established, drugs were administered by gavage. After 14 days of treatment, we used RT-PCR, Western blotting, and immunohistochemistry to measure the transcript expression and protein abundance of Aβ, IL-1β, and GFAP, and methenamine silver staining was used to detect amyloid protein particle deposition. Results Compared to the control group, the rats from the AD model group showed significantly greater expression levels of Aβ, IL-1β, and GFAP. However, these differences in expression were abolished by treatment with QKF or donepezil. Conclusion QKF possesses therapeutic potential against AD because it downregulated Aβ, IL-1β, and GFAP in the hippocampus of AD rats. Future studies should further examine the mechanisms through which QKF produces its effects and the consequences of long-term QKF administration.

Curcumin Increase the Expression of Neural Stem/Progenitor Cells and Improves Functional Recovery after Spinal Cord Injury.

ObjectiveTo investigates the effect of curcumin on proliferation of spinal cord neural stem/progenitor cells (SC-NSPCs) and functional outcome in a rat spinal cord injury (SCI) model.MethodsSixty adult male Sprague-Dawley rats were randomly and blindly allocated into three groups (sham control group; curcumin treated group after SCI; vehicle treated group after SCI). Functional recovery was evaluated by the Basso, Beattie, and Bresnahan (BBB) scale during 6 weeks after SCI. The expression of SC-NSPC proliferation and astrogliosis were analyzed by nestin/Bromodeoxyuridine (BrdU) and Glial fibrillary acidic protein (GFAP) staining. The injured spinal cord was then examined histologically, including quantification of cavitation.ResultsThe BBB score of the SCI-curcumin group was better than that of SCI-vehicle group up to 14 days (p<0.05). The co-immunoreactivity of nestin/BrdU in the SCI-curcumin group was much higher than that of the SCI-vehicle group 1 week after surgery (p<0.05). The GFAP immunoreactivity of the SCI-curcumin group was remarkably lower than that of the SCI-vehicle group 4 weeks after surgery (p<0.05). The lesion cavity was significantly reduced in the curcumin group as compared to the control group (p<0.05).ConclusionThese results indicate that curcumin could increase the expression of SC-NSPCs, and reduce the activity of reactive astrogliosis and lesion cavity. Consequently curcumin could improve the functional recovery after SCI via SC-NSPC properties.

Roles of Cdc42 and Rac in Bergmann glia during cerebellar corticogenesis

Bergmann glia (BG) are important in the inward type of radial migration of cerebellar granule neurons (CGNs). However, details regarding the functions of Cdc42 and Rac in BG for radial migration of CGN are unknown. To examine the roles of Cdc42 and Rac in BG during cerebellar corticogenesis, mice with a single deletion of Cdc42 or Rac1 and those with double deletions of Cdc42 and Rac1 under control of the glial fibrillary acidic protein (GFAP) promoter: GFAP-Cre;Cdc42flox/flox (Cdc42-KO), GFAP–Cre;Rac1flox/flox (Rac1-KO), and GFAP-Cre; Cdc42flox/flox;Rac1flox/flox (Cdc42/Rac1-DKO) mice, were generated. Both Cdc42-KO and Rac1-KO mice, but more obviously Cdc42-KO mice, had disturbed alignment of BG in the Purkinje cell layer (PCL). We found that Cdc42-KO, but not Rac1-KO, induced impaired radial migration of CGNs in the late phase of radial migration, leading to retention of CGNs in the lower half of the molecular layer (ML). Cdc42-KO, but not Rac1-KO, mice also showed aberrantly aligned Purkinje cells (PCs). These phenotypes were exacerbated in Cdc42/Rac1-DKO mice. Alignment of BG radial fibers in the ML and BG endfeet at the pial surface of the cerebellum evaluated by GFAP staining was disturbed and weak in Cdc42/Rac1-DKO mice, respectively. Our data indicate that Cdc42 and Rac, but predominantly Cdc42, in BG play important roles during the late phase of radial migration of CGNs. We also report here that Cdc42 is involved in gliophilic migration of CGNs, in contrast to Rac, which is more closely connected to regulating neurophilic migration.

Indirubin Derivative 7-Bromoindirubin-3-Oxime (7Bio) Attenuates Aβ Oligomer-Induced Cognitive Impairments in Mice.

Indirubins are natural occurring alkaloids extracted from indigo dye-containing plants. Indirubins could inhibit various kinases, and might be used to treat chronic myelocytic leukemia, cancer and neurodegenerative disorders. 7-bromoindirubin-3-oxime (7Bio), an indirubin derivative derived from indirubin-3-oxime, possesses inhibitory effects against cyclin-dependent kinase-5 (CDK5) and glycogen synthase kinase-3β (GSK3β), two pharmacological targets of Alzheimer's disease (AD). In this study, we have discovered that 2.3–23.3 μg/kg 7Bio effectively prevented β-amyloid (Aβ) oligomer-induced impairments of spatial cognition and recognition without affecting bodyweight and motor functions in mice. Moreover, 7Bio potently inhibited Aβ oligomer-induced expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Furthermore, 7Bio significantly prevented the decreased expression of synapsin-1 and PSD-95, biomarkers of pre-synaptic and post-synaptic proteins in Aβ oligomer-treated mice. The mean optical density (OD) with hyper-phosphorylated tau (pTau), glial fibrillary acidic protein (GFAP) and CD45 positive staining in the hippocampus of 7Bio-treated mice were significantly decreased compared to those of Aβ oligomer-treated mice. In addition, Western blotting analysis showed that 7Bio attenuated Aβ oligomer-decreased expression of pSer9-GSK3β. Those results suggested that 7Bio could potently inhibit Aβ oligomer-induced neuroinflammation, synaptic impairments, tau hyper-phosphorylation, and activation of astrocytes and microglia, which may contribute to the neuroprotective effects of 7Bio. Based on these findings, we expected that 7Bio might be developed as a novel anti-AD lead compound.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions.

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Evidence of Carotenoid in Surgically Removed Lamellar Hole-Associated Epiretinal Proliferation.

To determine the constituents and origin of the yellow pigment in surgically removed lamellar hole-associated epiretinal proliferation (LHEP) in patients with lamellar macular hole (LMH). This prospective case series comprised nine eyes with LMH in patients aged 41 to 83 years. The presence of LHEP was confirmed by preoperative optical coherence tomography; the distribution of macular pigment was observed by two-wavelength fundus autofluorescence technique before and after surgery. The subjects underwent a 25-gauge vitrectomy, and the surgically removed epiretinal membranous tissue was fixed with formalin. The specimens were examined using resonance Raman microscopy, and paraffin sections were stained with antiglial fibrillary acidic protein. Seven cases presented with LHEP, and the presence of yellow pigment was confirmed using an operating microscope. Carotenoid-specific Raman signals with three major Raman peaks could be identified in the specimens with LHEP. These specimens were positive for glial fibrillary acidic protein staining. Using the fundus autofluorescence technique, a central defect in the distribution of the macular pigment was noted in the exact area of the lamellar hole. This type of defect was no longer visible after surgical repair of the lamellar hole. The constituents of the yellow pigment in the removed LHEP were carotenoids that typically originate from the macular xanthophyll pigments at the fovea. Since LHEP is reported to be composed of Müller cells, we hypothesize that xanthophyll carotenoids at the fovea are contained in the Müller cells.

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

Inflammation, oxidative stress, and glial cell activation characterize stellate ganglia from humans with electrical storm.

Neuronal remodeling in human heart disease is not well understood. Stellate ganglia from patients with cardiomyopathy (CMY) and refractory ventricular arrhythmias undergoing cardiac sympathetic denervation (n = 8), and from organ donors with normal hearts (n = 8) collected at the time of organ procurement were compared. Clinical data on all subjects were reviewed. Electron microscopy (EM), histologic, and immunohistochemical assessments of neurotransmitter profiles, glial activation and distribution, and lipofuscin deposition, a marker of oxidative stress, were quantified. In CMY specimens, lipofuscin deposits were larger, and present in more neurons (26.3% ± 6.3% vs. 16.7% ± 7.6%, P < 0.043), than age-matched controls. EM analysis revealed extensive mitochondrial degeneration in CMY specimens. T cell (CD3+) infiltration was identified in 60% of the CMY samples, with one case having large inflammatory nodules, while none were identified in controls. Myeloperoxidase-immunoreactive neutrophils were also identified at parenchymal sites distinct from inflammatory foci in CMY ganglia, but not in controls. The adrenergic phenotype of pathologic samples revealed a decrease in tyrosine hydroxylase staining intensity compared with controls. Evaluation of cholinergic phenotype by staining for the vesicular acetylcholine transporter revealed a low but comparable number of cholinergic neurons in ganglia from both groups and demonstrated that preganglionic cholinergic innervation was maintained in CMY ganglia. S100 staining (a glial cell marker) demonstrated no differences in glial distribution and relationship to neurons; however, glial activation demonstrated by glial fibrillary acidic protein (GFAP) staining was substantially increased in pathologic specimens compared with controls. Stellate ganglia from patients with CMY and arrhythmias demonstrate inflammation, neurochemical remodeling, oxidative stress, and satellite glial cell activation. These changes likely contribute to excessive and dysfunctional efferent sympathetic tone, and provide a rationale for sympathectomy as a treatment for arrhythmias in this population. This work was made possible by support from NIH grants HL125730 to OAA, GM107949 to DBH, and HL084261 and OT2OD023848 to KS.

Systemic infusions of anti-interleukin-1β neutralizing antibodies reduce short-term brain injury after cerebral ischemia in the ovine fetus

Perinatal hypoxic-ischemic reperfusion (I/R)-related brain injury is a leading cause of neurologic morbidity and life-long disability in children. Infants exposed to I/R brain injury develop long-term cognitive and behavioral deficits, placing a large burden on parents and society. Therapeutic strategies are currently not available for infants with I/R brain damage, except for hypothermia, which can only be used in full term infants with hypoxic-ischemic encephalopathy (HIE). Moreover, hypothermia is only partially protective. Pro-inflammatory cytokines are key contributors to the pathogenesis of perinatal I/R brain injury. Interleukin-1β (IL-1β) is a critical pro-inflammatory cytokine, which has been shown to predict the severity of HIE in infants. We have previously shown that systemic infusions of mouse anti-ovine IL-1β monoclonal antibody (mAb) into fetal sheep resulted in anti-IL-1β mAb penetration into brain, reduced I/R-related increases in IL-1β expression and blood-brain barrier (BBB) dysfunction in fetal brain. The purpose of the current study was to examine the effects of systemic infusions of anti-IL-1β mAb on short-term I/R-related parenchymal brain injury in the fetus by examining: 1) histopathological changes, 2) apoptosis and caspase-3 activity, 3) neuronal degeneration 4) reactive gliosis and 5) myelin basic protein (MBP) immunohistochemical staining. The study groups included non-ischemic controls, placebo-treated ischemic, and anti-IL-1β mAb treated ischemic fetal sheep at 127days of gestation. The systemic intravenous infusions of anti-IL-1β mAb were administered at fifteen minutes and four hours after in utero brain ischemia. The duration of each infusion was two hours. Parenchymal brain injury was evaluated by determining pathological injury scores, ApopTag® positive cells/mm2, caspase-3 activity, Fluoro-Jade B positive cells/mm2, glial fibrillary acidic protein (GFAP) and MBP staining in the brains of fetal sheep 24h after 30min of ischemia. Treatment with anti-IL-1β mAb reduced (P<0.05) the global pathological injury scores, number of apoptotic positive cells/mm2, and caspase-3 activity after ischemia in fetal sheep. The regional pathological scores and Fluoro-Jade B positive cells/mm2 did not differ between the placebo- and anti-IL-1β mAb treated ischemic fetal sheep. The percent of the cortical area stained for GFAP was lower (P<0.05) in the placebo ischemic treated than in the non-ischemic group, but did not differ between the placebo- and anti-IL-1β mAb treated ischemic groups. MBP immunohistochemical expression did not differ among the groups. In conclusion, infusions of anti-IL-1β mAb attenuate short-term I/R-related histopathological tissue injury, apoptosis, and reduce I/R-related increases in caspase-3 activity in ovine fetal brain. Therefore, systemic infusions of anti-IL-1β mAb attenuate short-term I/R-related parenchymal brain injury in the fetus.

Effect of Brain Tumor Presence During Radiation on Tissue Toxicity: Transcriptomic and Metabolic Changes

Radiation therapy (RT) causes functional and transcriptomic changes in the brain; however, most studies have been carried out in normal rodent brains. Here, the long-term effect of irradiation and tumor presence during radiation was investigated. Male Wistar rats ∼7weeks old were divided into 3 groups: sham implant, RT+sham implant, and RT+tumor implant (C6 glioma). Hypofractionated irradiation (8 or 6Gy/day for 5days) was localized to a 1-cm strip of cranium starting 5days after implantation, resulting in complete tumor regression and prolonged survival. Biopsy of tissue was performed in the implant area 65days after implantation. RNA was hybridized to GeneChip Rat Exon 1.0 ST array. Data were analyzed using significant analysis of microarrays and ingenuity pathway analysis. 1H magnetic resonance spectroscopy (1H-MRS) imaging was performed in the implantation site 65 to 70days after implantation using a 9.4T Biospec magnetic resonance imaging scanner with a quadrature rat brain array. Immunohistochemical staining for astrogliosis, HMG-CoA synthase 2, γ-aminobutyric acid (GABA) and taurine was performed at ∼65days after implantation. Eighty-four genes had a false discovery rate <3.5%. We compared RT+tumor implant with RT+sham implant animals. The tumor presence affected networks associated with cancer/cell morphology/tissue morphology. 1H-MRS showed significant reduction in taurine levels (P<.04) at the implantation site in both groups. However, the RT+tumor group also showed significant increase in levels of neurotransmitter GABA (P=.02). Hippocampal taurine levels were only significantly reduced in the RT+tumor group (P=.03). HMG-CoA synthase 2, GABA and taurine levels were confirmed using staining. Glial fibrillary acidic protein staining demonstrated a significant increase in inflammation that was heightened in the RT+tumor group. Our data indicate that tumor presence during radiation significantly affects long-term functional transcriptomics landscape and neurotransmitter levels at the tumor implantation site/normal tissue, accompanied by increased inflammation (astrogliosis).

Histological and cognitive alterations in adult diabetic rats following an episode of juvenile diabetic ketoacidosis: Evidence of permanent cerebral injury

Evidence suggests that diabetic ketoacidosis (DKA) may cause subtle cognitive alterations in children but the mechanisms are poorly understood. Acute DKA is associated with reactive astrogliosis and microglial activation in a rat model. Whether these inflammatory changes permanently alter brain histology is unknown. We aimed to determine whether DKA results in permanent alterations in brain histology and whether these changes are associated with cognitive deficits in a rat model. We induced diabetes in juvenile rats with streptozotocin at 4 weeks of age. We induced DKA in one group (n=21) at 5 weeks of age and compared this group to rats with diabetes without DKA episodes (n=13). Beginning at 7 weeks, rats underwent a series of cognitive tests to evaluate memory. At 15 weeks, rat brains were harvested and examined using immunohistochemistry (IHC). In tests of novel object recognition and social recognition, both groups performed similarly, however, the DKA group performed more poorly in object-place recognition tests, suggesting alterations in hippocampal function. IHC studies demonstrated increased glial fibrillary acidic protein staining intensity in the hippocampus of DKA rats suggesting astrogliosis, and decreased NeuN positive cell counts in the cortex suggesting neuron loss. These studies demonstrate that DKA results in permanent alterations in brain microstructure in a rat diabetes model. These structural changes are associated with deficits in hippocampal function.

Expression of peptidylarginine deiminase 4 in an alkali injury model of retinal gliosis

Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to identify the PADs that mediate citrullination in a model of alkali-induced retinal gliosis. Mouse corneas were exposed to 1.0 N NaOH and posterior eye tissue from injured and control uninjured eyes was evaluated for transcript levels of various PADs by reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qPCR). Retinas were also subjected to immunohistochemistry (IHC) for glial fibrillary acidic protein (GFAP), citrullinated species, PAD2, and PAD4 and tissue levels of GFAP, citrullinated species, and PAD4 were measured by western blots. In other experiments, the PAD4 inhibitor streptonigrin was injected intravitreally into injured eyes ex vivo to test inhibitory activity in an organ culture system. We found that uninjured retina and choroid expressed Pad2 and Pad4 transcripts. Pad4 transcript levels increased by day 7 post-injury (p < 0.05), whereas Pad2 levels did not change significantly (p > 0.05) by qPCR. By IHC, PAD2 was expressed in uninjured eyes along ganglion cell astrocytes, but in injured retina PAD2 was downregulated at 7 days. On the other hand, PAD4 showed increased staining in the retina upon injury revealing a pattern that overlapped with filamentous GFAP staining in Müller glial processes by 7 days. Injury-induced citrullination and soluble GFAP protein levels were reduced by PAD4 inhibition in western blot experiments of organ cultures. Together, our findings for the first time identify PAD4 as a novel injury-inducible druggable target for retinal gliosis.