The effects of chronic, continuous ß-funaltrexamine pre-treatment on lipopolysaccharide-induced inflammation and behavioral deficits in C57BL/6J mice.

BACKGROUND: Inflammation and neuroinflammation are integral to the progression and severity of many diseases and are strongly associated with cardiovascular disease, cancer, autoimmune disorders, neurodegenerative disease, and neuropsychiatric disorders. These diseases can be difficult to treat without addressing the underlying inflammation, and, as such, a growing need has arisen for pharmaceutical treatments that target inflammatory mediators and signaling pathways. Our lab has investigated the therapeutic potential of the irreversible µ-opioid antagonist ß-funaltrexamine (ß-FNA) and discovered that acute treatment ameliorates inflammation in astrocytes in vitro and inhibits central and peripheral inflammation and reduces anxiety- and sickness-like behavior in male C57BL/6J mice. Now, our investigation has expanded to investigate the chronic pre-treatment effects of ß-FNA on lipopolysaccharide (LPS)-induced inflammation and behavior in male C57BL/6J mice. RESULTS: Micro-osmotic drug pumps were surgically inserted into the subcutaneous intrascapular space of male C57BL/6J mice. ß-FNA or saline vehicle was continuously administered for seven days. On the sixth day, mice were given intraperitoneal injections of LPS or saline. An elevated plus maze test, followed by a forced swim test, were administered 24 h post-injection to measure sickness-, anxiety- and depressive-like behavior. Immediately after testing, frontal cortex, hippocampus, spleen, and plasma were collected. Levels of inflammatory chemokines C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 10 (CXCL10) were measured in tissues by enzyme-linked immunosorbent assay (ELISA). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to assess expression of the enzyme indoleamine 2, 3-dioxygenase 1 (IDO1) and the NLR family pyrin domain-containing protein 3 (NRLP3) inflammasome in frontal cortex and spleen tissues. Chronic pre-treatment robustly decreased inflammation in the hippocampus, frontal cortex, and spleen and reduced or abolished anxiety- and sickness-like behavior (e.g., increased time spent motionless, increased time spent in a contracted position, and reduced distance moved). However, treatment with ß-FNA alone increased both inflammation in the frontal cortex and anxiety-like behavior. CONCLUSION: These findings provide novel insights into the anti-inflammatory and behavior-modifying effects of chronic ß-FNA pre-treatment and continue to support the therapeutic potential of ß-FNA under inflammatory conditions.

Nerve Growth Factor Signaling Modulates the Expression of Glutaminase in Dorsal Root Ganglion Neurons during Peripheral Inflammation.

Glutamate functions as the major excitatory neurotransmitter for primary sensory neurons and has a crucial role in sensitizing peripheral nociceptor terminals producing sensitization. Glutaminase (GLS) is the synthetic enzyme that converts glutamine to glutamate. GLS-immunoreactivity (-ir) and enzyme activity are elevated in dorsal root ganglion (DRG) neuronal cell bodies during chronic peripheral inflammation, but the mechanism for this GLS elevation is yet to be fully characterized. It has been well established that, after nerve growth factor (NGF) binds to its high-affinity receptor tropomyosin receptor kinase A (TrkA), a retrograde signaling endosome is formed. This endosome contains the late endosomal marker Rab7GTPase and is retrogradely transported via axons to the cell soma located in the DRG. This complex is responsible for regulating the transcription of several critical nociceptive genes. Here, we show that this retrograde NGF signaling mediates the expression of GLS in DRG neurons during the process of peripheral inflammation. We disrupted the normal NGF/TrkA signaling in adjuvant-induced arthritic (AIA) Sprague Dawley rats by the pharmacological inhibition of TrkA or blockade of Rab7GTPase, which significantly attenuated the expression of GLS in DRG cell bodies. The results indicate that NGF/TrkA signaling is crucial for the production of glutamate and has a vital role in the development of neurogenic inflammation. In addition, our pain behavioral data suggest that Rab7GTPase can be a potential target for attenuating peripheral inflammatory pain.

Assessing haematological parameters and probable toxicity analysis in two coastal fish species at harbouring areas of Digha coastal belt, West Bengal, India.

Coastal ecosystems are vital for maintaining the biodiversity and human livelihoods, but they are increasingly subjected to anthropogenic pressures, including pollution from various sources. Present work intends to assess the possible threats in coastal ecosystem as well as coastal fish species, in particular, through haematological parameters caused due to exposure of environmental contaminants like polycyclic aromatic hydrocarbons (PAHs), potentially toxic metals (PTMs), etc. This study analysed the haematological parameters and probable toxicity levels in two important coastal fish species, viz., Mystus sp. and Mugil sp. widely available in Digha coastal belt. Different haematological parameters, such as WBCs (White Blood Cells), Lym (Lymphocytes), Gran (Granulocytes), Mid (Monocytes), RBCs (Red Blood Cells), HCT (Haematocrit) value, MCV (Mean Corpuscular Volume), MCH (Mean Corpuscular Haemoglobin), MCHC (Mean Corpuscular Haemoglobin Concentration), RDW- CV (Red Cells Distribution Width-Co-efficient of Variation), RDW- SD (Red Cells Distribution Width-Standard Deviation), PLT (Total Platelet Count), MPV (Mean Platelet Volume), PDW- SD (Platelet Distribution Width-Standard Deviation), PDW- CV (Platelet Distribution Width-Co-efficient of Variation), PCT (Plateletcrit), PLCR (Platelet Large Cell Ratio), PLCC (Platelet Large Cell Count) and many others were measured directly through Erba H360 Haematology Analyser, simultaneously air dried blood smear was stained by Haematoxylin-Eosin(H-E) and Giemsa stain for assessing morphometric alterations of RBCs, WBCs, platelets as well as to determine the differential counts of WBCs by observing through Leica DM2000 microscope. Evidence of several abnormalities in the erythrocyte's nucleus (ENAs) and the abundance of abnormal celled erythrocytes (ECAs), carcinoma (lymphoproliferative disorder, polycythaemia vera, Hodgkin lymphoma and non-Hodgkin lymphoma), elevation of WBCs content, Lym %(Lymphocyte percentage), Eo(Eosinophils), monocytes, HCT and gross depletion of Ne(Neutrophils), basophils, and PLCR levels indicated a sign of major impact of contamination to two intoxicated fishes which may also affect the human being through food chain and may result into leukaemia in mammalian species, finally. However, comprehensive evaluation of the long-term impacts of the contaminants like PAHs and/or PTMs, etc., on fish populations, human health risk and coastal ecosystem is required to be addressed.

Influence of polymorphisms in TNF-α and IL1ß on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1ß mediated multi-cellular signaling in liver microenvironment.

Background and aims: Alcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p. Materials and methods: Bio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required. Results: The combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1ß/IL8/MCP1/IL6/TGFß) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1ß were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1ß was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1ß-31CT+TT than TNF-α-238GG/IL1ß-31CC. The TNF-α/IL1ß promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1ß over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFß and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1ß were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFßR2/MCP1, and ICAM1 respectively. Conclusion: Thus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1ß gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1ß and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients.

Time dependent ultrastructural alterations on the skin, eye, barbel and fins of the spawn of Clarias batrachus (Linn. 1758) exposed to UV-B radiation.

Present study highlighted the ultramicroscopic (SEM) alterations of the skin, eye, barbel, and fins of spawn of an air-breathing teleost (Clarias batrachus, Linn. 1758) induced by UV-B radiation (280-320 nm) at a dose (@4.07 × 10-20J/photon/m2) under the time-frame of 5, 10 and 15 min/d in the laboratory condition for the periods of 5 and 10 days. Limnological parameters revealed no significant changes throughout the period of experimentation which were measured by PCS Testr 35 Multi-Parameter. Morphometric analysis revealed that during the extended exposure period of 10 days the spawn size and weight were reduced as analysed through Specific Growth Rate (SGR). SGR values in terms of weight for 5 and 10 days under 3 time-frames were 17.12%, 12.52%, 11.46% and 9.09%, 6.43%, 6.09% respectively, which revealed a declined trend along with the exposure days. In the skin of C. batrachus, the compact regular orientation of the stratified epithelial cells and mucous cells became distorted and the microridges and double-ridged structures showed destruction and fragmentations. The body striations and microfolds became shrinked and swollen and finally degenerated to form a mass. The distribution of mucous cells throughout the epidermis was disorganised and releasing secretory contents on the surface through small pores. Appearance of huge quantity of biogenic semi-hexagonal plate like crystals (guanine platelets) on the skin surface of the body was the most significant observations during UV-B radiation. In the developmental phases the eyeball showed shrinkage loosing normal regular concave structure and to become a dome-shaped one. The supportive connective infoldings became loosened. The choroid coat displayed deformities and the iris deformed the pupil. The fibroblast on the epithelium and melanocytes depicted dispersed arrangement. The pairs of ventral barbels near the mouth depicted the presence of taste buds that became severely damaged exposing the sensory as well as neuroepithelial cells. Compact regular arrangement of the SECs was completely destroyed leaving long and deep channels inbetween them; the disintegrated concentric MRs also showed a mass.