P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation.

BACKGROUND: Guillain-Barré syndrome (GBS), a post-infectious, immune-mediated, acute demyelinating disease of the peripheral nerves and nerve roots, represents the most prevalent and severe acute paralyzing neuropathy. Purinergic P2X7 receptors (P2X7R) play a crucial role in central nervous system inflammation. However, little is known about their role in the immune-inflammatory response within the peripheral nervous system. METHODS: Initially, we assessed the expression of purinergic P2X7R in the peripheral blood of patients with GBS using flow cytometry and qRT-PCR. Next, we explored the expression of P2 X7R in CD4+ T cells, CD8+ T cells, and macrophages within the sciatic nerves and spleens of rats using immunofluorescence labeling and flow cytometry. The P2X7R antagonist brilliant blue G (BBG) was employed to examine its therapeutic impact on rats with experimental autoimmune neuritis (EAN) induced by immunization with the P0180 - 199 peptide. We analyzed CD4+ T cell differentiation in splenic mononuclear cells using flow cytometry, assessed Th17 cell differentiation in the sciatic nerve through immunofluorescence staining, and examined the expression of pro-inflammatory cytokine mRNA using RT-PCR. Additionally, we performed protein blotting to assess the expression of P2X7R and NLRP3-related inflammatory proteins within the sciatic nerve. Lastly, we utilized flow cytometry and immunofluorescence labeling to examine the expression of NLRP3 on CD4+ T cells in rats with EAN. RESULTS: P2X7R expression was elevated not only in the peripheral blood of patients with GBS but also in rats with EAN. In rats with EAN, inhibiting P2X7R with BBG alleviated neurological symptoms, reduced demyelination, decreased inflammatory cell infiltration of the peripheral nerves, and improved nerve conduction. BBG also limited the production of pro-inflammatory molecules, down-regulated the expression of P2X7R and NLRP3, and suppressed the differentiation of Th1 and Th17 cells, thus protecting against EAN. These effects collectively contribute to modifying the inflammatory environment and enhancing outcomes in EAN rats. CONCLUSIONS: Suppression of P2X7R relieved EAN manifestation by regulating CD4+ T cell differentiation and NLRP3 inflammasome activation. This finding underscores the potential significance of P2X7R as a target for anti-inflammatory treatments, advancing research and management of GBS.

Microwave-assisted and methanol/acetic acid-free method for rapid staining of proteins in SDS-PAGE gels.

We describe a microwave-assisted, methanol and acetic acid-free, inexpensive method for rapid staining of SDS-PAGE proteins. Only citric acid, benzoic acid, and Coomassie brilliant blue G-250 (CBG) were used. Microwave irradiation reduced the detection duration, and proteins in a clear background were visualized within 30 min of destaining, after 2 min of fixing and 12 min of staining. By using this protocol, comparable band intensities were obtained to the conventional methanol/acetic acid method.

Long-term effects of combined brilliant blue G and xenon light induced retinal toxicity following macular hole repair surgery.

BACKGROUND: The purpose of this study was to look at the long-term effects of retinal phototoxicity after macular hole repair surgery using xenon endolight illumination and Brilliant blue G (BBG) dye. CASE PRESENTATION: An elderly man in his late seventies underwent para plana vitrectomy with BBG dye to repair an idiopathic full-thickness macular hole (MH) in his right eye. Prior to macular hole surgery, his visual acuity in the right eye was 6/60, N24 at the time of presentation. The MH closed with type 1 closure immediately after surgery, but there was extensive damage to the outer retinal layers and retinal pigment epithelium (RPE) at the macula, resulting in a reduction in visual acuity to 2/60. We presumed that the combination of BBG and xenon light, is the probable reason of retinotoxicity in the current patient. There was a progressive increase in the area of retinal and RPE layer damage and choroidal thinning over a 4-year period. CONCLUSION: Due to combined BBG-induced dye and endoilluminator toxicity, a rare case of continuously progressing RPE layer damage with choroidal thinning over a long follow-up interval was described. Such long-term effects of BBG and endolight induced retinotoxicity have not been reported in the literature, to the best of our knowledge.

Extensive internal limiting membrane peeling for proliferative vitreoretinopathy.

PURPOSE: The aim of this study was to describe the anatomical outcomes of Brilliant Blue G (BBG)-assisted extensive internal limiting membrane peeling for proliferative vitreoretinopathy (PVR) under three-dimensional (3D) visualization. METHODS: This study constitutes a retrospective case series conducted in a private retina practice, of 14 consecutive patients (14 eyes) with rhegmatogenous retinal detachment complicated by PVR who underwent pars plana vitrectomy between January 2019 and January 2020. The internal limiting membrane (ILM) was selectively stained with BBG, and perspectives were enhanced with a 3D visualization system. We peeled off the ILM beyond the vascular arcades up to the periphery. The main outcome was anatomical success, defined as persistent retinal reattachment after removal of the silicone oil tamponade. RESULTS: Anatomic success was achieved with a single surgery in 11 of 14 (78.6%) eyes, and eventual success was achieved in all eyes. The mean patient follow-up time was 12.3 months (range, 7-16 months). The mean preoperative best-corrected visual acuity (BCVA) was 2.93 ± 0.79 logMAR which improved to 1.75 + 0.91 at the last follow-up. CONCLUSION: Extensive ILM peeling allowed the creation of a cleavage plane underlying the PVR membranes that facilitated its complete removal, thereby achieving anatomically reattached retina and reducing the risk of recurrence of retinal detachment. The long-term effects of this technique need further research.

Effects of a P2X7 receptor antagonist on myenteric neurons in the distal colon of an experimental rat model of ulcerative colitis.

Inflammatory bowel diseases (IBDs) are chronic diseases of the gastrointestinal tract that include ulcerative colitis and Crohn's disease and affect enteric neurons. Research has shown that Brilliant Blue G (BBG), a P2X7 receptor antagonist, restores enteric neurons following ischemia and reperfusion. This study aimed to evaluate the effect of BBG on myenteric neurons of the distal colon in an experimental rat model of ulcerative colitis. Colitis was induced by injection of 2,4,6-trinitrobenzene sulfonic acid (TNBS) into the large intestine. BBG was administered 1 h after colitis induction and for five consecutive days thereafter. Distal colons were collected 24 h or 7 days after TNBS injection. The animals were divided into 24-h and 7-day sham (vehicle injection rather than colitis induction), 24-h colitis, 24-h BBG, 7-day colitis and 7-day BBG groups. The disease activity index (DAI), neuronal density and profile of neuronal nitric oxide synthase (nNOS)-, choline acetyltransferase (ChAT)- and P2X7 receptor-immunoreactive enteric neurons were analyzed, and histological analysis was performed. The results showed recovery of the DAI and histological tissue integrity in the BBG groups compared to those in the colitis groups. In addition, the numbers of neurons positive for nNOS, ChAT and the P2X7 receptor per area were decreased in the colitis groups, and these measures were recovered in the BBG groups. Neuronal size was increased in the colitis groups and restored in the BBG groups. In conclusion, BBG is effective in improving experimental ulcerative colitis, and the P2X7 receptor may be a therapeutic target.

Efficacy of different doses of dye-assisted internal limiting membrane peeling in idiopathic macular hole: a systematic review and network meta-analysis.

PURPOSE: Pars plana vitrectomy is the gold standard for the treatment of idiopathic macular hole. Several chromovitrectomy dyes have been used to improve the visualization of the internal limiting membrane (ILM), including indocyanine green, trypan blue (TB), brilliant blue G (BBG), and triamcinolone acetonide (TA). We conducted a network meta-analysis (NMA) to establish the optimum concentration of chromovitrectomy dye-assisted ILM peeling for IMH. METHODS: We searched PubMed, Embase, and Cochrane Library for relevant studies before January 2020. We performed a random-effects NMA using STATA version 15.1 to assess mean difference and odds ratios with 95% confidence intervals. RESULTS: We identified twelve retrospective trails and five randomized controlled trials (RCTs), comprising 1 492 patients of IMH on stage II-IV for ILM peeling. The results of IMH closure rate show that the effect of ILM peeling without dye was better than 0.25% ICG, the effects of ILM peeling with 0.5% ICG or TA were better than without dye, and the effects of ILM peeling with 0.05% BBG, 0.15% TB, 0.5% ICG or 0.05% ICG were better than 0.25% ICG. Ranking probability analysis shows that the rates of IMH closure after ILM peeling with 0.15% TB or 0.05% BBG were better than nine other concentrations of chromovitrectomy dyes. CONCLUSION: The 0.15% TB and 0.05% BBG were recommended as the better efficient treatment-assisted ILM peeling for IMH closure. For retina specialists who prefer to use ICG to assist ILM peeling, 0.05% ICG may be a good choice. However, high-quality large-scale RCTs are recommended to confirm the NMA results.

Infracyanine Green vs. Brilliant Blue G in Inverted Flap Surgery for Large Macular Holes: A Long-Term Swept-Source OCT Analysis.

Background and Objectives: To compare the long-term toxicity of infracyanine green (IFCG) to brilliant blue G (BBG) in inverted internal limiting membrane flap surgery (I-ILMFS) for large, full-thickness macular holes (FTMHs). Materials and Methods: Prospective randomized study including 39 eyes with ≥ 400 µm idiopathic FTMH who underwent I-ILMFS with either IFCG or BBG. Postoperative 6- and 12-month corrected distance visual acuity (CDVA), closure rate, and swept-source optical coherence tomography parameters, including ellipsoid zone (EZ) and external limiting membrane (ELM) mean defect length, central foveal thicknesses (CFT), parafoveal macular thickness (MT), ganglion cells and inner plexiform layer (GCL++) thickness, and peripapillary nerve fiber layer (pRNFL) thickness, were compared. Results: Nineteen eyes were included in the IFCG group and 20 eyes in the BBG group. In all cases a FTMH closure was found. CDVA improved at 6 and 12 months in both groups (p < 0.0005); the increase at 12 months was greater in the BBG group (p = 0.036). EZ and ELM defects did not differ between groups at either follow-up time. CFT at 12 months was greater in the BBG group (p = 0.041). A 12-months compared to 6-months MT decrease was present in both groups (p < 0.01). The GCL++ superior inner sector was thicker in the BBG group at 12 months (p = 0.036), as were the superior outer sector (p = 0.039 and p = 0.027 at 6 and 12 months, respectively) and inferior outer sector (p = 0.011 and p = 0.009 at 6 and 12 months, respectively). Conclusion: In our study BBG in I-ILMFS exhibits better long-term CDVA and retinal thickness than does IFCG, suggesting a lesser toxicity from BBG. These findings support the use of BBG over IFCG in I-ILMFS.

Long-term treatment with the P2X7 receptor antagonist Brilliant Blue G reduces liver inflammation in a humanized mouse model of graft-versus-host disease.

Allogeneic haematopoietic stem cell transplantation (HSCT) is a frequent curative therapy for numerous haematological malignancies. However, HSCT is limited by the occurrence of graft-versus-host disease (GVHD), with current therapies restricted to general immunosuppression. Activation of the P2X7 receptor by extracellular adenosine triphosphate (ATP) causes inflammation and tissue damage in GVHD. Short-term pharmacological blockade of P2X7 has been shown to reduce clinical disease and/or reduce inflammatory markers in allogeneic and humanized mouse models of GVHD. The current study demonstrates that long-term P2X7 blockade by intra-peritoneal injection of Brilliant Blue G (BBG) thrice weekly for up to 10 weeks did not impact human (h) peripheral blood mononuclear cell (PBMC) engraftment, predominantly T cells, in blood at 3 weeks post-hPBMC injection or in spleens at end-point in humanized mice. Histological analysis demonstrated long-term BBG treatment reduced leukocyte infiltration in the livers of humanized mice. Immunohistochemical analysis demonstrated that BBG treatment reduced liver apoptosis. Long-term BBG treatment did not alter clinical disease, mRNA expression of pro-inflammatory markers in tissues or serum human interferon (IFN)-γ concentrations. Therefore, this study demonstrates that P2X7 activation plays a role in GVHD pathogenesis in the livers of humanized mice, supporting a role for this receptor in GVHD development in HSCT recipients.

Effect of brilliant Blue-G on cellular stress response in retinal pigment epithelial cells: In vitro.

To assess the cellular stress evoked by exposure of Brilliant Blue-G (BBG), adult retinal pigment epithelial (ARPE-19) cells were treated with various dilutions of BBG in balanced salt solution plus (BSS-PLUS) with and without endoillumination (Alcon Constellation Vision System). The treatments lasted for acute periods of 2 and 5 min. MTT and presto blue assays were performed to assess the changes in cell viability; reactive oxygen species (ROS) production was quantified by DCFDA (dichlorofluorescin diacetate) assay, and the expression of inflammatory stress and endoplasmic reticulum (ER) genes were quantified by qPCR. We observed no reduction in cell viability at 2 min of dye treatment with and without endoillumination while at 5 min exposure, a reduction in cell viability at all concentrations of the dye was observed compared to control. Though there was an increase in ROS with endoillumination, it was insignificant. There was no change in the mRNA expression of TNF-α while that of GRP78, and inflammatory genes viz. IL-8, IL-1ß showed a significant increase at 0.5 mg/ml dye with endoillumination. BBG reduced cell viability with increasing concentration and time. The undiluted concentration of the dye results in inflammatory stress compared to the diluted formulations. Interestingly, increased GRP78 at undiluted concentration indicates a protective response in cells exposed to light. However, further studies are needed to evaluate the effect of cellular stress on the visual outcome. We infer that the commercially available formulation of BBG is safe for the RPE, at the recommended dose for a short duration however its toxicity to other cell types need to be addressed.

Blockage of the P2X7 Receptor Attenuates Harmful Changes Produced by Ischemia and Reperfusion in the Myenteric Plexus.

INTRODUCTION: Our work analyzed the effects of a P2X7 receptor antagonist, Brilliant Blue G (BBG), on rat ileum myenteric plexus following ischemia and reperfusion (ISR) induced by 45 min of ileal artery occlusion with an atraumatic vascular clamp with 24 h (ISR 24-h group) or 14 d of reperfusion (ISR 14-d group). MATERIAL AND METHODS: Either BBG (50 mg/kg or 100 mg/kg, BBG50 or BBG100 groups) or saline (vehicle) was administered subcutaneously 1 h after ischemia in the ISR 24-h group or once daily for the 5 d after ischemia in the ISR 14-d group (n = 5 per group). We evaluated the neuronal density and profile area by examining the number of neutrophils in the intestinal layers, protein expression levels of the P2X7 receptor, intestinal motility and immunoreactivity for the P2X7 receptor, nitric oxide synthase, neurofilament-200, and choline acetyl transferase in myenteric neurons. RESULTS: The neuronal density and profile area were restored by BBG following ISR. The ischemic groups showed alterations in P2X7 receptor protein expression and the number of neutrophils in the intestine and decreased intestinal motility, all of which were recovered by BBG treatment. CONCLUSION: We concluded that ISR morphologically and functionally affected the intestine and that its effects were reversed by BBG treatment, suggesting the P2X7 receptor as a therapeutic target.

Early P2X7R-dependent activation of microglia during the asymptomatic phase of autoimmune encephalomyelitis.

Microglia-mediated neuroinflammation accompanies many central nervous system (CNS) diseases, including multiple sclerosis (MS), and is strongly dependent on the purinergic P2X7 receptor. The nature of the inflammatory response in MS is studied for decades indicating, that proinflammatory microgliosis is involved in advanced stages of MS and is associated with active tissue damage and neurological dysfunctions. Evidence on the role of microgliosis in initial stages of the disease is scarce. Thus, in the present study, we investigated the time course of microglial activation in rat brain subjected to experimental autoimmune encephalomyelitis (EAE) which is the animal model of MS. We show that activation of microglia occurs in brains of immunized rats at a very early stage of EAE, well before the development of neurological symptoms of the disease. Enhanced immunoreactivity of microglia/macrophage-specific protein Iba-1, together with morphological features of microgliosis, was identified beginning at day 4 post immunization. Concomitantly, microglial expression of P2X7R was also examined. Moreover, our results reveal that administration of Brilliant Blue G, an antagonist of P2X7R, delays the onset of the disease and partially inhibits development of neurological symptoms in EAE rats. Blockage of P2X7R significantly reduces activation of microglia as confirmed by decreased Iba-1 immunoreactivity and suppresses neuroinflammation in EAE rat brains, as indicated by decreased protein levels of investigated proinflammatory cytokines: IL-1ß, IL-6 and TNF-α. Our results indicate that microglia are involved in inducing neuroinflammation at a very early stage of MS/EAE via a P2X7R-dependent mechanism.

Comparison of trypan blue and Brilliant Blue G for staining of the anterior lens capsule during cataract surgery: short-term results.

PURPOSE: The present study aimed to evaluate the potential corneal endothelial cell toxicity of trypan blue (TB) and Brilliant Blue G (BBG), two dyes used to stain the anterior capsule in cataract surgery. METHODS: We conducted a single-center, prospective, randomized study in which 150 eyes of 117 patients were randomly divided into control (CT), TB, and BBG groups. Preoperative and postoperative (1, 3, and 6 months) values for corrected distance visual acuity (CDVA), corneal endothelial cell count, and central corneal thickness were compared among the three groups. RESULTS: A total of 111 eyes from 88 patients were completely analyzed. The CDVA (logarithm of the minimal angle of resolution) values in the CT, TB, and BBG groups 1 month after surgery were 0.001, 0.023, and 0.019, respectively. The corneal endothelial cell counts 6 months after surgery were 2711 ± 225, 2748 ± 251, and 2680 ± 284 cells/mm2, respectively. The central corneal thicknesses 6 months after surgery were 524.3 ± 35.5, 532.2 ± 36.1, and 531.4 ± 33.0 µm, respectively. There were no significant differences in CDVA, endothelial cell count, or central corneal thickness among the three groups during the follow-up period. CONCLUSIONS: Our findings indicate that neither TB nor BBG was associated with detectable toxicity to corneal endothelial cells during cataract surgery, even when injected directly into the anterior chamber. Additionally, BBG exhibited equivalent staining efficiency to TB.

Specific glycine to alanine mutation eliminates dynamic interaction of polymeric GlyT1a N-terminus with Coomassie Brilliant Blue G-250.

We previously found that multimeric GlyT1aN16 protein exhibits increased diffusion in a polyacrylamide gel and shows an unusual time-dependent absorbance rearrangement, as revealed by the Bradford assay. Here, we find that glycine to alanine mutation eliminates the absorbance shift, but not the altered diffusion properties of GlyT1aN16, indicating that these two phenomena are not interconnected. The absorbance shift is apparent with both native and urea-denatured GlyT1aN16, suggesting that the effect is either not dependent on protein structure, or the required structure is restored very quickly following denaturant removal. In the far-UV spectra, circular dichroism (CD) curves for both wild-type and mutated GlyT1aN16 are under the zero line, indicating largely unstructured character. However, a significant shift of the mutant CD curve suggests possible microstructural heterogeneity. Deconvolution of the CD data indicates a potential 3-fold increase in isolated helical content, which would inhibit an absorbance shift, as we demonstrated previously. These results suggest that, in addition to protein quantification, Coomassie Brilliant Blue G-250 can be used to reveal certain properties of the secondary structure of proteins.

Foetal bovine serum can reduce toxicity of indocyanine green, brilliant blue G and trypan blue in ARPE-19 cellular model that suggests new surgical staining protocols for internal limiting membrane peeling procedure.

BACKGROUND: To investigate and compare the cytotoxicity of indocyanine green (ICG), brilliant blue G (BBG) and trypan blue (TB) using ARPE-19 cells that have been pre-treated/post-treated with balanced salt solution (BSS) or foetal bovine serum (FBS). METHODS: The cultured human retina pigment epithelium ARPE-19 cells were pre-treated/post-treated with BSS or FBS (represent the autologous serum in clinic) in parallel with cells being soaked with various concentrations of ICG, BBG and TB. The cells were then assessed for viability, growth rate, reactive oxygen species (ROS) level, mitochondrial membrane potential (Δψ) and mitochondrial mass as cytotoxic indices. For the FBS pre-treated cells, only ROS was examined. RESULTS: Using the MTT assay, cytotoxicity seemed to appear when the dye concentration was above 2.5 mg/mL for ICG but no cytotoxicity for BBG and TB at the concentrations used. Cell growth was arrested at a concentration 1 mg/mL when ICG or BBG were present but no arrest at any of the tested concentrations was found for TB with the cell-growth curve was slowest for ICG. Cellular ROS levels increased at all concentrations of all dyes, but the increasing slopes were decreased after FBS post-treatment washout. CONCLUSIONS: As a rinse buffer FBS performs much better than BSS in terms of cell rescue, which agrees with a clinical report when autologous whole blood was applied to macular hole surgery. However, FBS pre-treatment seems to be much better than FBS use as washout buffer in post-treatment.

The P2X7 receptor antagonist Brilliant Blue G reduces serum human interferon-γ in a humanized mouse model of graft-versus-host disease.

Graft-versus-host disease (GVHD) remains a major problem after allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. Previous studies have demonstrated a role for the adenosine triphosphate (ATP)-gated P2X7 receptor channel in allogeneic mouse models of GVHD. In this study, injection of human peripheral blood mononuclear cells (PBMCs) into immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (NOD-SCID-IL)-2Rγnull (NSG) mice established a humanized mouse model of GVHD. This model was used to study the effect of P2X7 blockade in this disease. From five weeks post-PBMC injection, humanized mice exhibited clinical signs and histopathology characteristic of GVHD. The P2X7 antagonist, Brilliant Blue G (BBG), blocked ATP-induced cation uptake into both murine and human cells in vitro. Injection of BBG (50 mg/kg) into NSG mice did not affect engraftment of human leucocytes (predominantly T cells), or the clinical score and survival of mice. In contrast, BBG injection reduced circulating human interferon (IFN)-γ significantly, which was produced by human CD4+ and CD8+ T cells. BBG also reduced human T cell infiltration and apoptosis in target organs of GVHD. In conclusion, the P2X7 antagonist BBG reduced circulating IFN-γ in a humanized mouse model of GVHD supporting a potential role for P2X7 to alter the pathology of this disease in humans.

Evaluation of the Anticonvulsant Effect of Brilliant Blue G, a Selective P2X7 Receptor Antagonist, in the iv PTZ-, Maximal Electroshock-, and 6 Hz-Induced Seizure Tests in Mice.

Epilepsy is one of the most common neurological disorders which is diagnosed in around 65 million people worldwide. Clinically available antiepileptic drugs fail to control epileptic activity in about 30% of patients and they are merely symptomatic treatments and cannot cure or prevent epilepsy. There remains a need for searching new therapeutic strategies for epileptic disorders. The P2X7 receptor has been recently investigated as a new target in epilepsy treatment. Preclinical studies revealed that P2X7 receptor antagonists have anticonvulsant properties in some models of epilepsy. We aimed to investigate whether P2X7 receptor antagonist-brilliant blue G (BBG)-is able to change seizure threshold in three acute seizure models in mice, i.e., in the intravenous pentylenetetrazole seizure threshold, maximal electroshock seizure threshold and 6 Hz psychomotor seizure threshold tests. BBG was administered acutely (50-200 mg/kg, 30 min before the tests) and sub-chronically (25-100 mg/kg, once daily for seven consecutive days). Moreover, the chimney and grip strength tests were used to estimate the influence of BBG on the motor coordination and muscular strength in mice, respectively. Our results revealed only a week anticonvulsant potential of the studied P2X7 receptor antagonist because it showed anticonvulsant action only in the 6 Hz seizure test, both after acute and sub-chronic administration. BBG did not significantly influence seizure thresholds in the remaining tests. Motor coordination and muscular strength were not affected by the studied P2X7 receptor antagonist. In summary, BBG does not possess any remarkable anticonvulsant potential in acute seizure models in mice.

Purinergic Antagonism Prevents Mitochondrial Dysfunction and Behavioral Deficits Associated with Dopaminergic Toxicity Induced by 6-OHDA in Rats.

Purinoceptors are present in neurons, microglia and oligodendrocytes and regulate dopamine (DA) release, striatal-related function and striatal neuronal and DA cells damage. Therefore, purinoceptors may be involved in the pathology of Parkinson's disease (PD) and purinergic antagonism may show neuroprotective effect. The study investigated the role of the non-selective purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS) and a selective purinergic receptor P2X7 receptor antagonist Brilliant Blue G (BBG) against 6-OHDA induced dopaminergic neurotoxicity in rats; while adenosine triphosphate (ATP) was used as a P2X receptor agonist. Behavioral parameters like spontaneous motor activity, narrow beam walk, footprint, bar catalepsy, grip strength and rotarod tests were performed to evaluate motor deficits in PD. Striatal DA contents were estimated as neurochemical measures of PD. Mitochondrial studies and oxidative status were assessed to investigate the mechanism of purinergic system antagonists. Involvement of purinergic receptors in apoptosis was assessed by expressing cytochrome-C, caspase-9 and caspase-3. Both the antagonists not only attenuated 6-OHDA induced motor deficits but also protected against 6-OHDA induced DA depletion in the striatum. Oxidative stress, mitochondrial integrity and dysfunction were attenuated by purinergic antagonists. Further, they attenuated mitochondrial-linked apoptosis as observed by a decrease in expression of cytochrome-C, caspase-9 and caspase-3. Therefore, purinoceptor antagonism shows neuroprotective effect in 6-OHDA induced dopamine toxicity through preservation of mitochondrial bioenergetics and anti-apoptotic activities.

Early P2X7R-related astrogliosis in autoimmune encephalomyelitis.

Astrocytes are the main cells responsible for maintenance of brain homeostasis. Undisturbed action and signaling with other cells are crucial for proper functioning of the central nervous system (CNS). Dysfunctional astrocytes may determine the degree of neuronal injury and are associated with several brain pathologies, among which are multiple sclerosis (MS) and the animal model of this disease which is known as experimental autoimmune encephalomyelitis (EAE). One of the many functions of astrocytes is their response to CNS damage when they undergo reactive gliosis. Our data reveal that activation of astrocytes occurs in forebrains of immunized rats at a very early stage of EAE, well before the symptomatic phase of the disease. We have noted enhanced expression of GFAP and S100ß starting from day 4 post-immunization. Temporal coincidence between the expression of astrocyte activation markers and the expression of connexin 43 and purinergic P2X7 receptor (P2X7R) was also observed. Administration of Brilliant blue G, an antagonist of P2X7R, significantly decreases astrogliosis as confirmed by immunohistochemical analysis and observation of decreased levels of GFAP and S100ß. The condition of the treated animals was improved and the neurological symptoms of the disease were alleviated. With the knowledge that cerebral astroglia represent the main source of ATP and glutamate which are potentially neurotoxic substances released through P2X7R and connexin hemichannels, we suggest that astroglia may be involved in pathogenesis of MS/EAE at a very early stage through the purinergic/glutamatergic mechanisms.

Inner retinal thinning after Brilliant Blue G-assisted internal limiting membrane peeling for vitreoretinal interface disorders.

The aim of this study was to evaluate ganglion cell layer and nerve fiber layer thickness after Brilliant Blue G (BBG)-assisted internal limiting membrane (ILM) peeling for vitreomacular disorders. Retrospective analysis of spectral domain optical coherence tomography (SD-OCT) of 42 eyes of 42 patients, who underwent pars plana vitrectomy with BBG-assisted ILM peeling, was performed. Inclusion criteria were idiopathic macular hole, idiopathic vitreomacular traction, and idiopathic epiretinal membrane. Key exclusion criteria were vitreoretinal interface abnormalities secondary to any other diseases, follow-up period of less than 3 months, and any other associated retinal pathology. Average, minimum, and sectoral ganglion cell, and inner plexiform layers (GCIPL) and retinal nerve fiber layer (RNFL) parameters were collected. Changes in these parameters from baseline to 3- and 6-month visits after surgery were analyzed. At 3 months after surgery, we found a statistically significant reduction in the average GCIPL thickness (P = 0.031) and also in the superior sectors (P < 0.05) compared to the baseline values. A similar reduction was observed in the minimum RNFL thickness (P = 0.028) as well as in the superior sectoral RNFL thickness (P < 0.05). In 14 eyes with 6 months of follow-up, a similar statistically significant thinning of the GCIPL and RNFL was observed. However, the difference between the 3-month and 6-month values was not statistically significant (P = 0.679). BBG-assisted ILM peeling for vitreomacular interface disorders leads to thinning of the inner retina including GCIPL and RNFL. These structural changes should be correlated with retinal function tests to explore the pros and cons of this surgical step.

Visual and anatomical outcome of macular hole surgery at a tertiary healthcare facility.

OBJECTIVE: To assess visual and anatomical outcome of full thickness macular hole (FTMH) surgery with ILM peeling using brilliant blue G dye. METHODS: Thirty patients who had clinically evident macular hole were selected. Pre-operative Optical Coherence Tomography (OCT) was done. In all cases vitrectomy was performed via 23guage 3 ports pars plana (3PPV) vitrectomy system and Brilliant blue G dye, 0.5ml dye was injected over macula which resulted in light blue stain of ILM and peeling was performed around hole in circular motion and after gas fluid exchange gas tamponade with SF6 was done. Final visual and anatomical outcome was measured as postoperative BCVA and postoperative OCT at three months respectively. Descriptive statistics were computed. Paired t-test was applied. P value≤0.05 were considered as significant. RESULTS: There were 12 male and 18 female patients. The mean age was 57.40±4.76 years. The mean size of macular hole was 452.20±242.33µm. The mean duration of symptoms was 16.73±13.49 weeks. Mean pre operative BCVA was 1.30±0.73 log MAR and post operative was 0.51±0.23 log MAR. Mean increased BCVA was found to be 0.22±0.13 log MAR. Primary closure of hole was achieved in 29(96.7%). Significant mean difference was found in pre operative and post operative BCVA. CONCLUSION: Brilliant blue G exhibits sufficient staining qualities and safety profile to peel ILM in the management of full thickness macular hole with significant visual and anatomical improvement.