GFAP Staining Research Articles

Effect of citronellol on oxidative stress, neuroinflammation and autophagy pathways in an in vivo model of Parkinson's disease

Citronellol, a monoterpene found in the essential oils of Cymbopogo plants has been reported to possess various biological properties. In the present study, we investigated the neuroprotective mechanisms of citronellol against rotenone induced neurodegeneration by using rat model of Parkinson's disease (PD). Our results demonstrated that oral administration of citronellol prevented rotenone induced reactive oxygen species production, lipid peroxidation and enhanced Nrf2 expression, catalase, glutathione peroxidase and superoxide dismutase levels in the brain. Enzyme-linked immunosorbent assays showed that citronellol reduced secretion of TNF-α, IL-1β, IL-6 and decreased MMP-9 expression levels. Further, citronellol prevented rotenone induced microglia (Iba-1 staining) and astrocyte (GFAP staining) activation. Western blot analysis showed that citronellol significantly decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase-2 that are key markers of neuroinflammation. We further evaluated the effect of citronellol on dopaminergic neurons in substantia nigra pars compacta (SNpc) and striatum (ST) which are key anatomical structures in PD. Tyrosine hydroxylase (TH) immunoreactivity showed that citronellol preserved Tyrosine hydroxylase (TH) positive dopaminergic neurons and enhanced TH striatal expression levels significantly compared to rotenone alone group. Further, to understand the effect of citronellol on apoptosis and proteotoxicity, we evaluated apoptotic markers (Bax, Bcl-2), growth regulator (mTOR) and α-synuclein expression. Citronellol attenuated rotenone induced expression of pro-apoptotic protein Bax, reduced α-synuclein expression and enhanced Bcl-2 and mTOR levels. In addition, citronellol modulated autophagy pathway by decreasing LC-3 (Microtubule-associated proteins) and p62 levels. Taken together, our results demonstrate that citronellol protected dopaminergic neurons through its antioxidant, anti-inflammatory, anti-apoptotic and autophagy modulating properties.

Structural and Functional Reorganization of the Sensorimotor Cortex During Ligation of the Common Carotid Arteries (Experimental Study)

Aim of the study. To explore the structural and functional changes of neurons, glial cells, and synaptic terminals in layers I, III, and V of the sensorimotor cortex (SMC) of the rat brain after bilateral common carotid artery ligation (CCAL).Material and methods. Incomplete cerebral ischemia was simulated by irreversible bilateral CCAL (2-vessel model of global ischemia without hypotension) on white rats (n=36). Comparative evaluation of the studied SMC structures was performed in the control group (intact rats, n=6) on days 1, 3, 7, 14, and 30 (n=30) after CCAL. Nissl, hematoxylin-eosin staining, and immunohistochemical reactions for NSE, MAP-2, p38, GFAP, and IBA1 were used. Numerical density of pyramidal neurons, astrocytes, oligodendrocytes, microglial cells, and relative area of p38-positive material (synaptic terminals) were determined. Statistical hypotheses were tested using nonparametric methods with Statistica 8.0 software.Results. After CCAL, the number of degenerative neurons in rat brain SMCs increased. The peak of numerical density of unshrunken neurons was detected after day 1. Later, the numerical density of hyperchromic unshrunken neurons decreased, while that of shrunken neurons increased. These parameters did not reach the control values. The changes in SMC neurons were accompanied by an increase in the numerical density of microglial cells after day 1 and its subsequent decrease. Immunohistochemistry for IBA1 revealed signs of microglial cell activation such as change in shape and loss of processes. Maximum increase in the SMC density of oligodendrocytes was observed on day 7, and that of astrocytes on day 14 after CCAL. The maximum number of NSE-positive neurons occurred on day 1 after CCAL. There was a significant decrease in the number of NSEpositive neurons in SMC layer III on days 3, 7, and 14, and an increase in the number of NSE-positive neurons on day 30. The number of NSE-positive neurons in layer V of the SMC progressively decreased throughout the whole study period. The evolution of changes in the proportion of p38-positive material (synaptic terminal area) differed significantly between the layers of SMC. In the layers I and III, this parameter first decreased (days 1 and 3) and then increased (days 7, 14, and 30). In layer V of SMC, the activation of the protein expression was observed in the acute phase (days 1 and 3), then it decreased on days 7 and 14, and increased again on day 30. The changes found in the numerical density of neurons, glial cells and synaptic terminals were associated with dehydration and overhydration of SMC. We found strong to medium significant associations between the relative area of terminals and neuropil swelling and edema zones.Conclusion. After CCAL, layers I, III, and V of the SMC of white rats revealed destructive and compensatory changes in neurons, glial cells, and inter-neuronal communication structures. Taken together, all these changes indicate a significant layer-by-layer variability of the neural tissue response to CCAL. Layer III (secondary projection complex) of the SMC was affected to a greater extent. Reorganization of neuronal-glial and interneuronal interrelations occurred along with a prominent neuropil overhydration.

A Mouse Model of Neurodegeneration Induced by Blade Penetrating Stab Wound to the Hippocampus.

Simple SummaryTo date, various animal models of traumatic brain injury (TBI) have been developed to investigate the cellular and molecular networks underlying the pathogenesis of neurodegeneration, and to examine the safeness and therapeutic efficacy of drugs. However, the commonly used animal TBI models usually have the weaknesses of low reproducibility and high mortality rates. In the present study, we created a mouse model of cognitive deficits induced by a blade penetrating stab wound to the hippocampus (HBSI). This model will contribute a better understanding of neurodegeneration and accelerate the translation of preclinical research to clinical applications.Traumatic brain injury (TBI) is closely associated with the later development of neurodegenerative and psychiatric diseases which are still incurable. Although various animal TBI models have been generated, they usually have weaknesses in standardization, survivability and/or reproducibility. In the present study, we investigated whether applying a blade penetrating stab wound to the hippocampus would create an animal model of cognitive deficits. Open-field, Morris water maze and Barnes maze tests were used to evaluate the animal behaviors. The immunofluorescence staining of NeuN, GFAP, IBA1, and TUNEL was conducted to analyze the changes in neurons, astrocytes, and microglia, as well as cell death. Mice with a hippocampal blade stab injury (HBSI) displayed the activation of microglia and astrocytes, inflammation, neuronal apoptosis, and deficits in spatial learning and memory. These findings suggest that HBSI is an easy approach to generate a reliable in vivo model of TBI to capture hemorrhage, neuroinflammation, reactive gliosis, and neural death, as well as cognitive deficits observed in human patients.

4. Elucidating the potential mechanisms underlying distraction spinal cord injury: associated neuroinflammation and apoptosis

<h3>BACKGROUND CONTEXT</h3> The incidence of distraction spinal cord injury (DSCI), which results from spinal cord ischemia due to vascular compromise and spinal cord tract disturbances, remains high. Furthermore, because no ideal animal model that mimics DSCI in clinical settings is available thus far, the related molecular mechanisms underlying DSCI remain unclear. <h3>PURPOSE</h3> This study aimed to establish a porcine model of DSCI and investigate the neuroinflammation and apoptosis mechanisms in these pigs. <h3>STUDY DESIGN/SETTING</h3> Animal experiment. <h3>PATIENT SAMPLE</h3> Before surgery, all pigs were randomly divided into three groups: sham group, osteotomy surgery only; the incomplete distraction spinal cord injury (IDSCI) and complete distraction spinal cord injury (CDSCI) group, osteotomy plus DSCI surgery with a motor-evoked potential (MEP) amplitude decreased by approximately 75% and 100%, respectively. <h3>OUTCOME MEASURES</h3> 1. The neurologic function changes of hind limbs following DSCI. 2. The DSCI lesions examined by MRI in different groups. 3. The pathological changes in the spinal cord tissues of different groups following DSCI. 4. The representative images of spinal cord tissues stained with H&E (40 × and 400 ×) and LFB staining (40 ×). 5. The levels of inflammatory cytokines in spinal cord tissues and cerebrospinal fluid at 7 days after DSCI. 6. Immunofluorescence staining of GFAP, Iba-1, and NeuN in the central DSCI lesions at 7 days after DSCI in different groups. 7. Determination of the expression levels of P53/ Bcl-2/Bax/Caspase-3 signaling pathway-related proteins following DSCI. <h3>METHODS</h3> After surgery, modified Tarlov scoring and MRC muscle strength scoring were used to evaluate neurologic function in each group. We observed the distracted spinal cord using MRI, and then all pigs were sacrificed. We used immunofluorescence staining to assess the neuronal and microglial structure, and function and astrocyte hyperplasia in the central DSCI lesions (T15). Inflammatory cytokine levels in the spinal cord and cerebrospinal fluid (CSF) were also analyzed. Western blotting was used to determine the expression of apoptosis-related proteins. <h3>RESULTS</h3> Results showed that the modified Tarlov scoring and muscle strength decreased significantly in the two DSCI groups. T2-MRI showed a relative enhancement at the center of the DSCI lesions. H&E and Lxol fast blue staining revealed that spinal cord distraction destroyed the normal structure of spinal cord tissues and nerve fiber tracts, exacerbating inflammatory cell infiltration, hyperemia, and edema. The IL-1β, IL-6, and TNF-α levels increased in the spinal cord and CSF following DSCI. Immunofluorescence staining results indicated the GFAP, Iba-1 expression increased following DSCI, whereas the NeuN expression reduced. Moreover, DSCI promoted the protein expression of P53, Bax, and Caspase-3 in the spinal cord tissues, whereas it reduced the Bcl-2 expression. <h3>CONCLUSIONS</h3> This study successfully established a porcine DSCI model that closely mimics DSCI in clinical settings, and clarified the mechanisms underlying DSCI-associated neuroinflammation and apoptosis; thus, our findings highlight potential DSCI-treatment strategies for further establishing suitable drug therapies. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Astrocytes profiling in acute hepatic encephalopathy: Possible enrolling of glial fibrillary acidic protein, tumor necrosis factor-alpha, inwardly rectifying potassium channel (Kir 4.1) and aquaporin-4 in rat cerebral cortex.

Hepatic encephalopathy (HE) is a neurological disarray manifested as a sequel to chronic and acute liver failure (ALF). A potentially fatal consequence of ALF is brain edema with concomitant astrocyte enlargement. This study aims to outline the role of astrocytes in acute HE and shed light on the most critical mechanisms driving this role. Rats were allocated into two groups. Group 1, the control group, received the vehicle. Group 2, the TAA group, received TAA (300 mg/kg) for 3 days. Serum AST, ALT, and ammonia were determined. Liver and cerebral cortical sections were processed for hematoxylin and eosin staining. Additionally, mRNA expression and immunohistochemical staining of cortical GFAP, TNFα, Kir4.1, and AQP4 were performed. Cortical sections from the TAA group demonstrated neuropil vacuolation and astrocytes enlargement with focal gliosis. GFAP, TNFα, and AQP4 revealed increased mRNA expression, positive immunoreactivity, and a positive correlation to brain water content. In contrast, Kir 4.1 showed decreased mRNA expression and immunoreactivity and a negative correlation to brain water content. In conclusion, our findings revealed altered levels of TNFα, Kir 4.1, GFAP, and AQP4 in HE-associated brain edema. A more significant dysregulation of Kir 4.1 and TNFα was observed compared to AQP4 and GFAP.

Unraveling the Mechanisms of Clinical Drugs-Induced Neural Tube Defects Based on Network Pharmacology and Molecular Docking Analysis.

Chemotherapeutic agents such as methotrexate (MTX), raltitrexed (RTX), 5-fluorouracil (5-FU), hydroxyurea (HU), and retinoic acid (RA), and valproic acid (VPA), an antiepileptic drug, all can cause malformations in the developing central nervous system (CNS), such as neural tube defects (NTDs). However, the common pathogenic mechanisms remain unclear. This study aimed to explore the mechanisms of NTDs caused by MTX, RTX, 5-FU, HU, RA, and VPA (MRFHRV), based on network pharmacology and molecular biology experiments. The MRFHRV targets were integrated with disease targets, to find the potential molecules related to MRFHRV-induced NTDs. Protein-protein interaction analysis and molecular docking were performed to analyze these common targets. Utilizing the kyoto encyclopedia of genes and genomes (KEGG) signaling pathways, we analyzed and searched the possible causative pathogenic mechanisms by crucial targets and the signaling pathway. Results showed that MRFHRV induced NTDs through several key targets (including TP53, MAPK1, HSP90AA1, ESR1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN) and multiple signaling pathways such as PI3K/Akt pathway, suggesting that abnormal proliferation and differentiation could be critical pathogenic contributors in NTDs induced by MRFHRV. These results were further validated by CCK8 assay in mouse embryonic stem cells and GFAP staining in embryonic brain tissue. This study indicated that chemotherapeutic and antiepileptic agents induced NTDs might through predicted targets TP53, MAPK1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN and multiple signaling pathways. More caution was required for the clinical administration for women with childbearing potential and pregnant.

From Molecular to Functional Effects of Different Environmental Lead Exposure Paradigms.

Simple SummaryOur comparative study brings new insights regarding the effects of environmental lead exposure on the cardiorespiratory and nervous systems. We show how various kinds of exposure can lead to different toxicities, with various degrees of nefarious effects. The developmental period is of utmost importance to the toxicity of environmental lead; however, we found that the duration of exposure is the prime reason for stronger effects, even though the dual effect of intermittent exposure causes greater molecular neuronal alterations. Lead is a heavy metal whose widespread use has resulted in environmental contamination and significant health problems, particularly if the exposure occurs during developmental stages. It is a cumulative toxicant that affects multiple systems of the body, including the cardiovascular and nervous systems. Chronic lead exposure has been defined as a cause of behavioral changes, inflammation, hypertension, and autonomic dysfunction. However, different environmental lead exposure paradigms can occur, and the different effects of these have not been described in a broad comparative study. In the present study, rats of both sexes were exposed to water containing lead acetate (0.2% w/v), from the fetal period until adulthood. Developmental Pb-exposed (DevPb) pups were exposed to lead until 12 weeks of age (n = 13); intermittent Pb exposure (IntPb) pups drank leaded water until 12 weeks of age, tap water until 20 weeks, and leaded water for a second time from 20 to 28 weeks of age (n = 14); and the permanent (PerPb) exposure group were exposed to lead until 28 weeks of age (n = 14). A control group (without exposure, Ctrl), matched in age and sex was used. After exposure protocols, at 28 weeks of age, behavioral tests were performed for assessment of anxiety (elevated plus maze test), locomotor activity (open-field test), and memory (novel object recognition test). Metabolic parameters were evaluated for 24 h, and the acute experiment was carried out. Blood pressure (BP), electrocardiogram, and heart (HR) and respiratory (RF) rates were recorded. Baroreflex gain, chemoreflex sensitivity, and sympathovagal balance were calculated. Immunohistochemistry protocol for NeuN, Syn, Iba-1, and GFAP staining was performed. All Pb-exposed groups showed hypertension, concomitant with a decrease in baroreflex gain and chemoreceptor hypersensitivity, without significant changes in HR and RF. Long-term memory impairment associated with reactive astrogliosis and microgliosis in the dentate gyrus of the hippocampus, indicating the presence of neuroinflammation, was also observed. However, these alterations seemed to reverse after lead abstinence for a certain period (DevPb) and were enhanced when a second exposure occurred (IntPb), along with a synaptic loss. These results suggest that the duration of Pb exposure is more relevant than the timing of exposure, since the PerPb group presented more pronounced effects and a significant increase in the LF and HF bands and anxiety levels. In summary, this is the first study with the characterization and comparison of physiological, autonomic, behavioral, and molecular changes caused by different low-level environmental lead exposures, from the fetal period to adulthood, where the duration of exposure was the main factor for stronger adverse effects. These kinds of studies are of immense importance, showing the importance of the surrounding environment in health from childhood until adulthood, leading to the creation of new policies for toxicant usage control.

Mesenchymal stem cell-derived extracellular vesicles protect retina in a mouse model of retinitis pigmentosa by anti-inflammation through miR-146a-Nr4a3 axis

BackgroundRetinitis pigmentosa is a rod-cone degenerative disease that induces irreversible vision loss. This study probed the protective capacity of mesenchymal stem cell-derived small EVs (MSC-EVs) on the retinas of rd10 mice and the underlying mechanism.MethodsMSC-EVs were injected into the vitreous of rd10 mice at postnatal day 14 and P21; morphology and function were examined at P28. The mechanism of action was explored by using co-culture of photoreceptor cell line 661 W and microglia cell line BV2.ResultsTreatment with MSC-EVs increased the survival of photoreceptors and preserved their structure. Visual function, as reflected by optomotor and electroretinogram responses, was significantly enhanced in MSC-EVs-treated rd10 mice. Mechanistically, staining for Iba1, GFAP, F4/80, CD68 and CD206 showed that MSC-EVs suppressed the activation of microglial, Müller glial and macrophages. Furthermore, western blotting showed that the treatment inhibited the NF-κB pathway. RNA-seq and qPCR showed that MSC-EVs upregulated anti-inflammatory cytokines while downregulating pro-inflammatory cytokines. MSC-EVs application in vitro decreased the number of TUNEL-positive 661 W cells co-cultured with LPS-stimulated BV2, with similar impact on the cytokine expression as in vivo study. Genetic screening predicted miR-146a to be the downstream target of MSC-EVs, which was detected in MSC-EVs and upregulated in co-cultured 661 W cells and BV2 cells after MSC-EVs treatment. Upregulation of miR-146a by using its mimic decreased the expression of the transcription factor Nr4a3, and its downregulation inhibition promoted Nr4a3 expression in both 661 W and BV2 cells. Nr4a3 was further identified as the target gene of miR-146a by dual-luciferase assay. Furthermore, overexpressing miR-146a significantly decreased the expression of LPS-induced pro-inflammatory cytokines in BV2 cells.ConclusionsMSC-EVs delays retinal degeneration in rd10 mice mainly by its anti-inflammatory effect via the miR-146a-Nr4a3axis. Hence, MSC-EVs may be used in the treatment of neurodegenerative diseases.

NPC transplantation rescues sci-driven cAMP/EPAC2 alterations, leading to neuroprotection and microglial modulation

Neural progenitor cell (NPC) transplantation represents a promising treatment strategy for spinal cord injury (SCI); however, the underlying therapeutic mechanisms remain incompletely understood. We demonstrate that severe spinal contusion in adult rats causes transcriptional dysregulation, which persists from early subacute to chronic stages of SCI and affects nearly 20,000 genes in total tissue extracts. Functional analysis of this dysregulated transcriptome reveals the significant downregulation of cAMP signalling components immediately after SCI, involving genes such as EPAC2 (exchange protein directly activated by cAMP), PKA, BDNF, and CAMKK2. The ectopic transplantation of spinal cord-derived NPCs at acute or subacute stages of SCI induces a significant transcriptional impact in spinal tissue, as evidenced by the normalized expression of a large proportion of SCI-affected genes. The transcriptional modulation pattern driven by NPC transplantation includes the rescued expression of cAMP signalling genes, including EPAC2. We also explore how the sustained in vivo inhibition of EPAC2 downstream signalling via the intrathecal administration of ESI-05 for 1 week impacts therapeutic mechanisms involved in the NPC-mediated treatment of SCI. NPC transplantation in SCI rats in the presence and absence of ESI-05 administration prompts increased rostral cAMP levels; however, NPC and ESI-05 treated animals exhibit a significant reduction in EPAC2 mRNA levels compared to animals receiving only NPCs treatment. Compared with transplanted animals, NPCs + ESI-05 treatment increases the scar area (as shown by GFAP staining), polarizes microglia into an inflammatory phenotype, and increases the magnitude of the gap between NeuN + cells across the lesion. Overall, our results indicate that the NPC-associated therapeutic mechanisms in the context of SCI involve the cAMP pathway, which reduces inflammation and provides a more neuropermissive environment through an EPAC2-dependent mechanism.Graphical abstract

Abstract 334: Convection enhanced delivery of EGFR-targeting antibody drug conjugates ABBV-321 and Depatux-M

Abstract Introduction: EGFR targeted antibody-drug conjugates (ADCs) show promise as a novel treatment in a subset of glioblastoma (GBM). Two EGFR targeting ADCs include first generation Depatux-M, with an antimitotic toxin monomethyl auristatin F (MMAF), and ABBV-321, with a DNA crosslinking agent pyrrolobenzodiazepine dimer (PBD) toxin. Due to the large molecular weight, poor drug distribution across the blood-brain barrier significantly limits the efficacy in EGFR-amplified GBM. We studied whether convection enhanced delivery (CED) can be used to safely infuse these two EGFR-targeted ADCs in patient-derived xenograft (PDX) models of EGFR-amplified GBM. Methods: The efficacy of Depatux-M and ABBV-321 was evaluated in vitro and in vivo in two EGFRviii-amplified PDXs (GBM6 and GBM108). Immunofluorescence staining was used to evaluate drug distribution along with pharmacodynamics of the ADCs. CED was performed by stereotactic placement of an infusion catheter in the same location as the original tumor implantation. Immunohistochemistry was used to explore mechanisms of normal cell toxicity. Results: Despite potent activity in vitro (high ng/ml IC50), systemic administration of either ADC conferred minimal extension in survival for either GBM6 or GBM108. In contrast, CED significantly enhanced ADC delivery to tumor and peri-tumoral regions and extended survival. In a pilot study in GBM6 with n=3 mice per group, a single CED infusion of 0.002mg ABBV-321 resulted in extended survival beyond 365 days for two mice, while other doses and placebo were associated with shorter median survival (0 mg - 39 days; 0.03 mg - 105 days; 0.3 mg - 49 days). In a subsequent trial evaluating serial infusions performed every 21 days, four infusions at 0.002 mg ABBV-321 resulted in lethal toxicity. In contrast, limiting ABBV-321 to only two serial CED infusions in GBM108 was associated with extended survival of more than 300 days vs. 53 days for AB095 antibody control infusion. In contrast, serial infusion with Depatux-M was much better tolerated. In a single infusion in GBM6, a 0.06 mg dose was well tolerated and associated with a 49-day extension in median survival. Further, four serial infusions at 0.06 mg given every 21 days in GBM6 and GBM108 was associated with 100 day and more than 250-day extension in survival as compared to AB095 antibody control infusion. To investigate the mechanism of toxicity, infusion of non-tumor bearing C57Bl6 mice with ABBV-321 resulted in a marked loss in NeuN staining and elevated CD68 and GFAP staining 7 days later; Depatux-M infusion was only associated with modest elevation in GFAP without loss of NeuN staining or elevated CD68-positive cells. Conclusion: Depatux-M is well tolerated when infused into normal brain and results in extended survival in orthotopic GBM PDXs. In contrast, ABBV-321, with a distinct PBD toxin, had a much narrower therapeutic window when delivered by CED. Citation Format: Kendra A. Porath, Ann Mladek, Rachael A. Vaubel, Michael S. Regan, Sonia Jain, Danielle Burgenske, Katrina Bakken, Brett Carlson, Margaret Connors, Zeng Hu, Lihong He, Paul A. Decker, Gaspar Kitange, Shiv Gupta, Nathalie Y. Agar, Thomas M. Feldsien, Didier R. Lefebvre, Jann N. Sarkaria. Convection enhanced delivery of EGFR-targeting antibody drug conjugates ABBV-321 and Depatux-M [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 334.

Mitophagy boosting protects cells against MNU toxicity

Objective: A pharmacological model to study the human disease Retinitis Pigmentosa is N-methyl-N-nitrosourea (MNU) injection which results in retinal degeneration. Our objective is to study the autophagy and mitophagy status of the cells in this disease model. Materials and methods: The mitophagy reporter (MitoQC) and autophagy reporter mice (mCherry-GFP-LC3) have been used to determine the mitophagic and autophagic flux in the retina after MNU injection. Retinal pigment epithelium cell line (ARPE-19) MitoQC has been used to elucidate the cellular and molecular mechanisms after MNU treatment. Results: Neurodegeneration events appear one day after intraperitoneal MNU injection: reduction of photoreceptors thickness layer and increase of TUNEL and GFAP staining. Mitophagosomes and autophagosomes are less frequent in the retina of mice treated with MNU, but they are bigger and they tend to accumulate in the external limiting membrane. ARPE-19 cells are also vulnerable to MNU in a dose-dependent manner producing DNA damage, cytoplasm vacuolization, organelle alterations and cell death. Mitophagy levels depend on the MNU dose: an increase of the mitophagic flux is observed at low doses, whereas it seems to be blocked at high doses. We demonstrate this mitophagy is PINK-Parkin dependent and boosting this pathway makes cells more resistant to MNU. Finally, we achieve to protect MNU-treated retinae explants with the mitophagy inductor, DFP. Conclusion: MNU treatment induces activation of PINK-Parkin dependent mitophagy. We propose that mitophagy could act as a defense mechanism in this disease model.

Delayed Xenograft Transplantation of Mouse Olfactory Ensheathing Cells in Adult Rats

Regeneration of the central nervous system after injury using appropriate cells for transplantation is a controversial issue. Accessibility of allograft olfactory ensheathing cells to transplant in the spinal cord of patients is not applicable. Therefore, in this study, an attempt has been made to xenotransplant cells from mouse into a corticospinal tract lesion in a rat in order to achieve a plausible preclinical approach for future application to a clinical study. Adult rats were trained to use their forepaws for retrieving. The dorsal corticospinal tract was lesioned by a stereotactic radio-frequency lesion maker at the level of the first/second cervical segments. Rats that had shown no forepaw retrieval by 8 weeks were xenotransplanted with a suspension of cultured olfactory ensheathing cells derived from the mouse olfactory bulb. Starting between 1 and 3 weeks, 10 rats with transplants bridging the lesion site resumed ipsilateral forepaw reaching. After transplanting cells into the lesion side, the cross and horizontal sections of GFAP and NF staining of 10 animals that have the Directed Forepaw Reaching (DFR) function returned showed the regenerated CST fibers in the lesion area after 8 weeks postoperative. Xenotrasplant of olfactory ensheathing cells from the mouse olfactory bulb into a rat corticospinal tract lesion was promising and positive. Animals that had difficulty in Directed Forepaw Reaching had returned the function 8 weeks postoperatively. Doi: 10.28991/SciMedJ-2022-04-02-01 Full Text: PDF

Corydalis decumbens Can Exert Analgesic Effects in a Mouse Neuropathic Pain Model by Modulating MAPK Signaling.

Objectives This study is aimed at investigating the analgesic effect of the administration of Corydalis decumbens (CD) in a mouse model of postherpetic neuralgia (PHN) and at elucidating its mechanism of analgesic action. Methods Adult Kunming (KM) mice were randomly divided into control, CD, and vehicle-treated groups. Neuropathic pain was induced with a single intraperitoneal injection of resiniferatoxin (RTX). Thermal hyperalgesia was assessed with a hot/cold plate test, and mechanical allodynia was evaluated using von Frey filaments. The activation states of astrocytes, microglia, and the mitogen-activated protein kinase (MAPK) pathway in the spinal cord were determined by immunofluorescence staining and Western blot analysis of Iba-1, GFAP, phospho-p38, and phospho-Jun N-terminal kinase (JNK). Results RTX diminished thermal sensitivity and gradually increased sensitivity to tactile stimulation. The expression of Iba-1, GFAP, phospho-p38 MAPK, and phospho-JNK was upregulated in the RTX-induced postherpetic neuralgia mouse model. Systemic treatment with CD significantly ameliorated thermal sensitivity and mechanical hyperalgesia and was accompanied by a reduction in the expression of Iba-1 and GFAP and reduced phosphorylation of p38 and JNK. Conclusions This study suggests that CD is effective at ameliorating mechanical hyperalgesia in PHN mice and that its mechanism of action may involve modulation of MAPK phosphorylation and glial cell activation. Thus, CD may be a promising alternative therapy for PHN.

Potential Protective Role of Curcumin on the Toxic Effect of Food Azo Dye Tartrazine on the Brain of Young Albino Rats

The use of coloring agents in food industry has dramatically increased. Children are the main consumers of colored food products and beverages and are more vulnerable to the effects of synthetic colorants. Curcumin, an active constituent of turmeric plant, has been the subject of extensive studies assessing its antioxidant properties. The present study was conducted to evaluate the protective potentials of curcumin against neurotoxicity induced by the synthetic food colorant; tartrazine. Thirty-six young male albino rats were divided into six groups; negative control (Group I), distilled water (Group II), corn oil (Group III), curcumin (Group IV), tartrazine (Group V) and tartrazine+curcumin (Group VI). At the end of the study, the rats were subjected to biochemical, histological and immunohistochemical staining for GFAP. Our results showed that tartrazine induced a significant decrease in GSH, serotonin and GABA levels and a significant increase in MDA levels in the brain as compared to control groups. Tartrazine induced apoptosis, vacuolations, congestion and cellular infiltration in the brain tissue. An immunohistochemistry study showed astrogliosis with increased expression of GFAP. Curcumin ameliorated all these effects. In conclusion, tartrazine is neurotoxic. Increased brain oxidative stress and cellular damage are contributing factors. Curcumin can prevent tartrazine-induced neurotoxicity.

Continuous administration of mirabegron has advantages in inhibition of central sensitization compared with short-term treatment cessation in a mouse model of overactive bladder.

There is no clear pathophysiologic evidence determining how long overactive bladder (OAB) medication should be continued. We, therefore, investigated the effect of mirabegron using cessation (CES) or continuation (CON) treatment in an OAB animal model. Female C57BL/6 mice were divided into four groups (N = 8 each): Sham, OAB, CES, and CON groups. The OAB-like condition was induced by three times weekly intravesical instillations of KCl mixture with hyaluronidase. After the last intravesical instillation for inducing OAB, mirabegron (2 mg/kg/day) was administered in CES and CON groups for 10 and 20 days, respectively. Final experiments were carried out on 20 days from the last intravesical instillation in all groups. After cystometry, mRNA levels of bladder muscarinic, β-adrenergic, and P2X purinergic receptors were measured to investigate bladder efferent and afferent activity. In addition, mRNA levels of CCL2 and CCR2 in L6-S1 dorsal root ganglia (DRG) were measured to assess afferent sensitization. Immunofluorescent staining of CX3CR1, GFAP, and CCR2 in the L6 spinal cord was also conducted to investigate glial activation and central sensitization. OAB mice showed bladder overactivity evidenced by decreased intercontraction interval (3.56 ± 0.51vs. 5.76 ± 0.95 min in sham mice), increased non-voiding contractions (0.39 ± 0.11vs. 0.13 ± 0.07/min in sham mice), and inefficient voiding (72.1 ± 8.6% vs. 87.1 ± 9.5% in sham mice). Increased M2, M3, β2, β3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder and increased CCL2 and CCR2 in DRG indicate bladder efferent and afferent hyperexcitability. In addition, CX3CR1, GFAP, and CCR2 in the L6 spinal cord were upregulated in OAB mice. However, theCON group exhibited reduced β2, β3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder, reduced CCL2 and CCR2 in DRG, which are markers of afferent hyperexcitability, and reduced immunoreactivities of CX3CR1, GFAP, and CCR2 in the L6 spinal cord, which are markers of the central sensitization. Moreover, theCON group showed better improvements in nonvoiding contractions (0.16 ± 0.09vs. 0.44 ± 0.17/min) and voiding efficiency (93.9 ± 7.4% vs. 76.5 ± 13.1%) and reductions in bladder β3 receptors and CCL2 of L6-S1 DRG, and immunoreactivities of CX3CR1 and GFAP in the L6 spinal cord compared to theCES group. Continuous mirabegron treatment seems to prevent central sensitization and, thus, might be desirable for long-term disease control of OAB.

Saffron extract attenuates Sofosbuvir-induced retinal neurodegeneration in albino rat.

Sofosbuvir is a novel drug candidate for the treatment of hepatitis C viral infection; however, vision loss is one of its growing adverse effects. Saffron is a natural biomolecule with a high antioxidant potential that has been efficiently used in some diseases caused by oxidative stress. This study evaluated Sofosbuvir's neurodegenerative effect on the retina of albino rat and examined the potential protective role of saffron aqueous extract. Twenty-one adult male albino rats were randomly divided into three groups: Control, Sofosbuvir-treated (41.1 mg/kg /day for 6 weeks), and Sofosbuvir + Saffron co-treated groups. Retinal specimens were biochemically analyzed for malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels. In addition, light and transmission electron microscopic examination, as well as immunohistochemical staining for Caspase-3, COX-2, and GFAP were performed. Sofosbuvir treatment caused a significant increase in retinal MDA, IL-6, and TNF-α levels coupling with a significant decrease in retinal total antioxidant capacity level. Histopathological findings revealed disturbed retinal architecture, detached pigment epithelium, vacuolated photoreceptors, in addition to a significant decrease in the thicknesses of both outer and inner nuclear layers, and the number of ganglionic cells. Ultrastructural examination revealed extensive degenerative changes in all retinal layers. Caspase-3, COX-2, and GFAP immunohistochemical expressions were significantly increased. Meanwhile, concomitant treatment with Saffron significantly improved retinal redox status, inflammation, histological, and ultrastructural parameters. Saffron may protect the retina from the hazardous effects of Sofosbuvir. Saffron could be used as an adjuvant therapy to protect patients receiving Sofosbuvir from retinal damage.

Angiotensin II related glial cell activation and necroptosis of retinal ganglion cells after systemic hypotension in glaucoma

The purpose of this study was to design an animal model mimicking glaucoma with hemodynamic instability and to identify involvement of angiotensin II (AngII) and associated changes of the retina. Systemic hypotension was induced in Sprague–Dawley rats by oral hydrochlorothiazide administration. Rats were sacrificed at 4, 8, and 12-week time points. AngII and receptor levels were examined in the serum and retina. To examine the relationship between glia activation and associated RGC death, biochemical analysis of GFAP, Iba-1, and necroptosis associated factors such as TNFα, receptor-interacting protein (RIP) 1, 3, and inactive caspase 8 were explored. To investigate the difference in RGC death mechanism, JNK inhibitor or RIP3 inhibitor were given intraperitoneally to rats with ocular hypertension and systemic hypotension both to identify the pathway mainly involved. AngII and receptors were increased in the serum and retina of systemic hypotensive rat. At 4, 8, and 12 weeks after hypotension induction, glial activation was increased as indicated by GFAP and Iba-1 staining. TNFα, RIP3 were elevated. and downregulation of inactive caspase 8 was apparent in the retina of hypotensive rat. Electron microscopy revealed that necroptosis of RGC was gradually increased after systemic hypotension. Following intraperitoneal JNK inhibitor or RIP3 inhibitor administration, RGC loss was attenuated in systemic hypotensive rats but not in ocular hypertensive rats. In conclusion, AngII is involved in glial activation and associated RGC necroptosis following systemic hypotension. This pathway represents a novel and distinct cell death mechanism when compared to that involved in elevated intraocular pressure.

The innate immune system stimulating cytokine GM-CSF improves learning/memory and interneuron and astrocyte brain pathology in Dp16 Down syndrome mice and improves learning/memory in wild-type mice

Down syndrome (DS) is characterized by chronic neuroinflammation, peripheral inflammation, astrogliosis, imbalanced excitatory/inhibitory neuronal function, and cognitive deficits in both humans and mouse models. Suppression of inflammation has been proposed as a therapeutic approach to treating DS co-morbidities, including intellectual disability (DS/ID). Conversely, we discovered previously that treatment with the innate immune system stimulating cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which has both pro- and anti-inflammatory activities, improved cognition and reduced brain pathology in a mouse model of Alzheimer's disease (AD), another inflammatory disorder, and improved cognition and reduced biomarkers of brain pathology in a phase II trial of humans with mild-to-moderate AD. To investigate the effects of GM-CSF treatment on DS/ID in the absence of AD, we assessed behavior and brain pathology in 12–14 month-old DS mice (Dp[16]1Yey) and their wild-type (WT) littermates, neither of which develop amyloid, and found that subcutaneous GM-CSF treatment (5 μg/day, five days/week, for five weeks) improved performance in the radial arm water maze in both Dp16 and WT mice compared to placebo. Dp16 mice also showed abnormal astrocyte morphology, increased percent area of GFAP staining in the hippocampus, clustering of astrocytes in the hippocampus, and reduced numbers of calretinin-positive interneurons in the entorhinal cortex and subiculum, and all of these brain pathologies were improved by GM-CSF treatment. These findings suggest that stimulating and/or modulating inflammation and the innate immune system with GM-CSF treatment may enhance cognition in both people with DS/ID and in the typical aging population.

Topical Ocular Administration of Progesterone Decreases Photoreceptor Cell Death in Retinal Degeneration Slow (rds) Mice.

Retinitis pigmentosa (RP) is an inherited eye disorder which triggers a cascade of retinal disorders leading to photoreceptor cell death and for which there is currently no effective treatment. The purpose of this research was to study whether ocular administration of a solution of progesterone (PG) in β-cyclodextrins (CD) could delay photoreceptor cell death and counteract the gliosis process in an animal model of RP (rds mice). The possible effect of PG reaching the contralateral eye through the circulatory system was also evaluated. Finally, this research discusses and evaluates the diffusion of the drug from possible topical formulations for ocular administration of PG. A group of rds mice received one drop of a solution of PG in CD every 12 h for 10 days to the left eye, while the right eye was left untreated. Another group of rds mice (control) received the drug vehicle (PBS) on the left eye and, again, the right eye was left untreated. Once the treatment was finished on postnatal day 21, the animals were euthanized and histological immunofluorescence studies (TUNEL, GFAP, and DAPI staining) were carried out. Our results showed that the administration of a solution of PG in CD (CD-PG) as drops significantly decreased cell death and inflammation in the retina of the PG-treated eyes of rds mice. No effect was seen in the contralateral eye from PG that may have entered systemic circulation. In conclusion, CD-PG applied topically as drops to the eye decreases photoreceptor cell death in the early stages of RP, delaying vision loss and decreasing gliosis.

Methicillin resistance in Staphylococcus aureus modulates the transcriptome and disease pathology in a murine model of endophthalmitis

The ever-increasing incidence of methicillin-resistant strains of Staphylococcus aureus (MRSA) endophthalmitis is of particular concern as they are associated with poor outcomes. To compare the histology and whole transcriptome of Methicillin resistant (MRSA) and Methicillin-susceptible (MSSA) Staphylococcus aureus endophthalmitis in an experimental murine model. MRSA and MSSA endophthalmitis was induced in C57BL/6 mice and disease progression was scored clinically and histologically at 24 h p.i. Retinal changes were monitored by H&E, CD45, MPO and GFAP staining followed by retinal cell death evaluation. Whole Transcriptome was analysed using the SuperPrint G3 Mouse Gene Expression v2 chip. Differential gene expression analysis (Limma package, R) was done followed by enrichment of pathways (KEGG database). Increased corneal haze, diminished vitreous clarity and red reflex was observed in MRSA infected mice eye compared to MSSA (p = 0.04). Histological assessment also corroborated with increased disease severity in MRSA (p = 0.02). Although MRSA infected eye displayed higher CD45+ cells and greater GFAP intensity, the difference was not statistically significant. However, higher retinal cell death was found to be associated with the MRSA infection (p = 0.007). Our study also revealed that MRSA infection induces changes in host transcriptome (FC = 1.5, p = 0.05), revealing the involvement of several interleukins (IL-11,15,10,1ra), chemokines (CCL-11, CXCL-1), Interferon receptors, GM-CSF, M-CSF, MMPs, Neruopilin2 (NRP-2), Ubiquitin associated peptidase and apoptotic ligands. ErbB signalling, JAK-STAT, adipocytokine and Ras signalling were the top divergently enriched pathways. Our study confirms the differential host immune response triggered by MRSA infection in the eye. Our study may help to elucidate the mechanisms of pathogenesis and to identify additional candidate drug targets for the treatment of MRSA endophthalmitis.