scholarly journals Attenuation in Proinflammatory Factors and Reduction in Neuronal Cell Apoptosis and Cerebral Vasospasm by Minocycline during Early Phase after Subarachnoid Hemorrhage in the Rat

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Chia-Li Chung ◽  
Hung-Pei Tsai ◽  
Yu-Hua Huang ◽  
Shu-Chuan Wu ◽  
Chee-Yin Chai ◽  
...  
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Western Blot ◽  
Cerebral Vasospasm ◽  
Cell Apoptosis ◽  
Early Phase ◽  
Autologous Blood ◽  
Neuronal Cell ◽  
Immunofluorescence Staining ◽  
Immunohistochemistry Staining ◽  
Tnf Α

Background. Subarachnoid hemorrhage (SAH) is an important subcategory of stroke due to its high mortality rate as well as severe complications such as neurological deficit. It has been suggested that cerebral inflammation is a major factor in advanced brain injury after SAH. Microglia and astrocytes are known supporting cells in the development and maintenance of inflammation in central nervous system. However, the role of microglia and astrocytes in the development of inflammation and neuronal cell apoptosis during the early phase after SAH has not been thoroughly investigated. Materials and Methods. Sprague-Dawley rats were divided into 4 groups ( n = 6 /group): sham group, animals subjected to SAH without treatment, SAH animals pretreated with the microglia inhibitor minocycline (50 mg/kg, ip), and SAH animals pretreated with the astrocyte inhibitor fluorocitrate (50 mg/kg, ip). SAH was induced by injecting autologous blood (1 ml/kg) into the cistern magna on day 0. Pretreatment with minocycline or fluorocitrate was given three days prior to the induction of SAH. Rats were sacrificed 6 hr after SAH, and their cerebral spinal fluids were used to measure protein levels of neuroinflammatory cytokines IL-1β, IL-6, and TNF-α by ELISA. In addition, the cerebral cortex was utilized to determine the levels of caspase-3 by western blot and to evaluate neuronal cell apoptosis by immunohistochemistry staining and detect microglia and astrocyte by immunofluorescence staining for Iba-1 and GFAP. In this study, all SAH animals were given an injection of autologous blood and SAH rats treated with minocycline or fluorocitrate received ip injections on day 1, 2, and 3 before inducing SAH. Neurological outcome was assessed by ambulation and placing/stepping reflex responses on day 7. Results. Immunofluorescence staining showed that SAH induced proliferation of microglia and astrocyte and minocycline inhibited the proliferation of both microglia and astrocyte. However, fluorocitrate inhibited only the proliferation of astrocyte. ELISA analysis showed that SAH upregulated TNF-α and IL-1β, but not IL-6 at 6 hr after SAH. Minocycline, but not fluorocitrate, attenuated the upregulation of TNF-α and IL-1β. Western blot analysis and immunohistochemistry staining showed that SAH induced neuronal cell apoptosis. Pretreatment with minocycline, but not fluorocitrate, decreased SAH-induced neuronal death and cerebral vasospasm. Furthermore, significant improvements in neurobehavioral outcome were seen in the minocycline treatment group, but not in animals treated with fluorocitrate. Conclusions. Microglia may play an important role to regulate neuronal cell apoptosis and cerebral vasospasm through inhibiting inflammation at an early phase after SAH in the rat.

Identification and Expression Analysis of CD73 Inhibitors in Cervical Cancer

2020 ◽  
Vol 16 ◽  
Author(s):  
Jamshed Iqbal ◽  
Ayesha Basharat ◽  
Sehrish Bano ◽  
Syed Mobasher Ali Abid ◽  
Julie Pelletier ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Western Blot ◽  
Hela Cell ◽  
Cell Line ◽  
Cell Apoptosis ◽  
Immunofluorescence Staining ◽  
Cell Cycle Analysis ◽  
Hela Cell Line ◽  
Cell Line Hela

Aims: The present study was conducted to examine the inhibitory effects of synthesized sulfonylhydrazones on the expression of CD73 (ecto-5′-NT). Background: CD73 (ecto-5′-NT) represents the most significant class of ecto-nucleotidases which are mainly responsible for dephosphorylation of adenosine monophosphate to adenosine. Inhibition of CD73 played an important role in the treatment of cancer, autoimmune disorders, precancerous syndromes, and some other diseases associated with CD73 activity. Objective: Keeping in view the significance of CD73 inhibitor in the treatment of cervical cancer, a series of sulfonylhydrazones (3a-3i) derivatives synthesized from 3-formylchromones were evaluated. Methods: All sulfonylhydrazones (3a-3i) were evaluated for their inhibitory activity towards CD73 (ecto-5′-NT) by the malachite green assay and their cytotoxic effect was investigated on HeLa cell line using MTT assay. Secondly, most potent compound was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis. After that, CD73 mRNA and protein expression were analyzed by real-time PCR and Western blot. Results: Among all compounds, 3h, 3e, 3b, and 3c were found the most active against rat-ecto-5′-NT (CD73) enzyme with IC50 (µM) values of 0.70 ± 0.06 µM, 0.87 ± 0.05 µM, 0.39 ± 0.02 µM and 0.33 ± 0.03 µM, respectively. These derivatives were further evaluated for their cytotoxic potential against cancer cell line (HeLa). Compound 3h and 3c showed the cytotoxicity at IC50 value of 30.20 ± 3.11 µM and 86.02 ± 7.11 µM, respectively. Furthermore, compound 3h was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis which showed promising apoptotic effect in HeLa cells. Additionally, compound 3h was further investigated for its effect on expression of CD73 using qRT-PCR and western blot. Conclusion: Among all synthesized compounds (3a-3i), Compound 3h (E)-N'-((6-ethyl-4-oxo-4H-chromen-3-yl) methylene)-4-methylbenzenesulfonohydrazide was identified as most potent compound. Additional expression studies conducted on HeLa cell line proved that this compound successfully decreased the expression level of CD73 and thus inhibiting the growth and proliferation of cancer cells.

scholarly journals Alteration of Basilar Artery Rho-Kinase and Soluble Guanylyl Cyclase Protein Expression in a Rat Model of Cerebral Vasospasm following Subarachnoid Hemorrhage

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Chih-Jen Wang ◽  
Pei-Yu Lee ◽  
Bin-Nan Wu ◽  
Shu-Chuan Wu ◽  
Joon-Khim Loh ◽  
...  
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Protein Kinase ◽  
Protein Expression ◽  
Cerebral Vasospasm ◽  
Basilar Artery ◽  
Guanylyl Cyclase ◽  
Autologous Blood ◽  
Rho Kinase ◽  
Soluble Guanylyl Cyclase ◽  
Western Blots

Background and Purpose. The vasoconstrictor endothelin-1 (ET-1) has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). Previous results showed that CGS 26303, an endothelin converting enzyme (ECE) inhibitor, effectively prevented and reversed arterial narrowing in animal models of SAH. In the present study, we assessed the effect of CGS 26303 on neurological deficits in SAH rats. The involvement of vasoactive pathways downstream of ET-1 signaling in SAH was also investigated.Methods. Sprague-Dawley rats were divided into five groups (n=6/group): (1) normal control, (2) SAH, (3) SAH+vehicle, (4) SAH+CGS 26303 (prevention), and (5) SAH+CGS 26303 (reversal). SAH was induced by injecting autologous blood into cisterna magna. CGS 26303 (10 mg/kg) was injected intravenously at 1 and 24 hr after the initiation of SAH in the prevention and reversal protocols, respectively. Behavioral changes were assessed at 48 hr after SAH. Protein expression was analyzed by Western blots.Results. Deficits in motor function were obvious in the SAH rats, and CGS 26303 significantly improved the rate of paraplegia. Expressions of rho-kinase-II and membrane-bound protein kinase C-δand rhoA were significantly increased, while those of soluble guanylyl cyclaseα1andβ1as well as protein kinase G were significantly decreased in the basilar artery of SAH rats. Treatment with CGS 26303 nearly normalized these effects.Conclusions. These results demonstrate that the rhoA/rho-kinase and sGC/cGMP/PKG pathways play pivotal roles in cerebral vasospasm after SAH. It also shows that ECE inhibition is an effective strategy for the treatment of this disease.

scholarly journals Inhibition of the NKCC1/NF-κB Signaling Pathway Decreases Inflammation and Improves Brain Edema and Nerve Cell Apoptosis in an SBI Rat Model

2021 ◽  
Vol 14 ◽  
Author(s):  
Yating Gong ◽  
Muyao Wu ◽  
Jinchao Shen ◽  
Jiafeng Tang ◽  
Jie Li ◽  
...  
Keyword(s):  
Brain Injury ◽  
Brain Edema ◽  
Nerve Cell ◽  
Cell Apoptosis ◽  
Neuronal Cell ◽  
Neurological Dysfunction ◽  
Inflammatory Factors ◽  
Inflammatory Factor ◽  
Tnf Α ◽  
Bbb Permeability

Surgical brain injury (SBI) triggers microglia to release numerous inflammatory factors, leading to brain edema and neurological dysfunction. Reducing neuroinflammation and protecting the blood-brain barrier (BBB) are key factors to improve the neurological function and prognosis after SBI. Na+-K+-Cl– cotransporter 1 (NKCC1) and nuclear factor κB (NF-κB) have been implicated in the secretion of inflammatory cytokines by microglia in brain injury. This study aimed to establish the role of NKCC1 in inducing inflammation in SBI, as well as to determine whether NKCC1 controls the release of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) via phosphorylation of NF-κB in microglia, thus affecting BBB permeability and neuronal cell apoptosis. Male Sprague-Dawley (SD) rats were used to establish an SBI model. This study revealed that compared with the sham group, the expression levels of p-NKCC1, p-p65-NF-κB, and related inflammatory factor proteins in SBI model group significantly increased. After p-NKCC1 was inhibited, p-p65-NF-κB, IL-6, IL-1β, and TNF-α were downregulated, and nerve cell apoptosis and BBB permeability were significantly reduced. These findings suggest that the SBI-induced increase in p-NKCC1 exacerbates neuroinflammation, brain edema, and nerve function injury, which may be mediated by regulating the activity of p65-NF-κB that in turn influences the release of inflammatory factors.

scholarly journals Blocking Hepatoma-Derived Growth Factor Attenuates Vasospasm and Neuron Cell Apoptosis in Rats Subjected to Subarachnoid Hemorrhage

2021 ◽  
Author(s):  
Chia-Li Chung ◽  
Chieh-Hsin Wu ◽  
Yu-Hua Huang ◽  
Shu-Chuan Wu ◽  
Chee-Yin Chai ◽  
...  
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Growth Factor ◽  
Western Blot ◽  
Blot Analysis ◽  
Subarachnoid Space ◽  
Cell Apoptosis ◽  
Neurological Outcome ◽  
Post Treatment ◽  
Inflammatory Factors ◽  
Protein Levels

Abstract Subarachnoid hemorrhage (SAH) is an important subcategory of stroke due to its unacceptably high mortality rate as well as the severe complications it causes, such as cerebral vasospasm, neurological deficits, and cardiopulmonary abnormality. Hepatoma-derived growth factor (HDGF) is a growth factor related to normal development and is involved in liver development and regeneration. This study explored the relationship between SAH and HDGF. Sixty rats were divided into five groups (n = 12/group): (A) control group; (B) rHDGF ab only group [normal animals treated with 50 µM recombinant HDGF antibodies (rHDGF ab)]; (C) SAH group; (D) SAH + pre-rHDGF ab group (SAH animals pre-treated with 50 µM rHDGF ab into the subarachnoid space within 24 h before SAH); and (E) SAH + post-rHDGF ab group (SAH animals post-treated with 50 µM rHDGF ab into the subarachnoid space within 24 h after SAH). At 48 h after SAH, serum and cerebrospinal fluid (CSF) samples were collected to measure the levels of pro-inflammatory factors by ELISA, and rat cortex tissues were used to measure protein levels by western blot analysis. Immunofluorescence staining for Iba-1, GFAP, TUNEL, and NeuN was detected proliferation of microglia and astrocyte and apoptosis of neuron cells. Neurological outcome was assessed by ambulation and placing/stepping reflex responses. Morphology assay showed that pre-treatment and post-treatment with rHDGF ab attenuated vasospasm after SAH. SAH up-regulated the levels of TNF-α, IL-1β, and IL-6 in both the CSF and serum samples, and both pre- and post-treatment with rHDGF ab inhibited the up-regulation of these pro-inflammatory factors, except for the serum IL-6 levels. Western blot analysis demonstrated that SAH up-regulated pro-BDNF and NFκB protein levels, and both pre- and post-treatment with rHDGF ab significantly reduced the up-regulation. The result from immunofluorescence staining showed that SAH induced proliferation of microglia and astrocyte and apoptosis of neuron cells. Both pre- and post-treatment with rHDGF ab significantly attenuated proliferation of microglia and astrocyte and inhibited apoptosis of neuron cells. Furthermore, treatment with rHDGF ab significantly improved neurological outcome. Blocking HDGF attenuates neuron cell apoptosis and vasospasm through inhibiting inflammation in brain tissue at early phase after SAH.

Attenuation of experimental subarachnoid hemorrhage–induced cerebral vasospasm by the adenosine A2A receptor agonist CGS 21680

2007 ◽  
Vol 106 (3) ◽  
pp. 436-441 ◽  
Author(s):  
Chih-Lung Lin ◽  
Huei-Chuan Shih ◽  
Ann-Shung Lieu ◽  
Kung-Shing Lee ◽  
Aaron S. Dumont ◽  
...  
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Cerebral Vasospasm ◽  
Receptor Agonist ◽  
Autologous Blood ◽  
Physiological Parameter ◽  
Inos Mrna ◽  
Sprague Dawley ◽  
Cross Sectional ◽  
Cgs 21680 ◽  
Enos Mrna

Object Impaired endothelium-dependent relaxation is present in vasospastic cerebral vessels after subarachnoid hemorrhage (SAH) and may result from deficient production of endothelial nitric oxide synthase (eNOS) or increased production and/or activity of inducible NOS (iNOS). Accumulating evidence demonstrates that adenosine A2A receptors increase the production of NO by human and porcine arterial endothelial cells, which in turn leads to vasodilation. This study was designed to examine the effects of an adenosine A2A receptor agonist, (2(4-[2-carboxyethyl]phenyl)ethylamino)-5′-N-ethylcarboxamidoadenosine (CGS 21680), in the prevention of SAH-induced vasospasm. Methods Experimental SAH was induced in Sprague–Dawley rats by injecting 0.3 ml of autologous blood into the cisterna magna of each animal. Intraperitoneal injections of CGS 21680 or vehicle were administered 5 minutes and 24 hours after induction of SAH. The degree of vasospasm was determined by averaging measurements of cross-sectional areas of the basilar artery (BA) 48 hours after SAH. Expression of eNOS and iNOS in the BA was also evaluated. Prior to perfusion–fixation, there were no significant differences among animals in the control and treated groups in any physiological parameter that was recorded. The CGS 21680 treatment significantly attenuated SAH-induced vasospasm. Induction of iNOS mRNA and protein in the BA by the SAH was significantly diminished by administration of CGS 21680. The SAH-induced suppression of eNOS mRNA and protein was also relieved by the CGS 21680 treatment. Conclusions This is the first evidence that adenosine A2A receptor agonism is effective in preventing SAH-induced vasospasm without significant complications. The beneficial effect of adenosine A2A receptor agonists may be, at least in part, related to the prevention of augmented expression of iNOS and the preservation of normal eNOS expression following SAH. Adenosine A2A receptor agonism holds promise in the treatment of cerebral vasospasm following SAH and merits further investigation.

scholarly journals Effect of thrombin injection on cerebral vascular in rats with subarachnoid hemorrhage

2019 ◽  
Vol 47 (7) ◽  
pp. 2819-2831
Author(s):  
Gang Li ◽  
Qingsong Wang ◽  
Tingting Lin ◽  
Chengye Liu
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Cerebral Vasospasm ◽  
Subarachnoid Space ◽  
Physiological Saline ◽  
Pathological Examination ◽  
Cross Sectional ◽  
Arterial Lumen ◽  
Tnf Α ◽  
Significant Difference ◽  
Group 3

Objective To evaluate the effect of thrombin (TM) injection via the cerebellomedullary cistern on cerebral vessels in rats with subarachnoid hemorrhage (SAH). Methods Eighteen rats were randomly divided into three groups. In the A1 group, physiological saline was injected via the cerebellomedullary cistern; in the A2 group, 3 U of TM was injected into the subarachnoid space; and in the A3 group, SAH models were established and 3 U of TM was injected with the first injection of whole blood. Three days later, basilar artery specimens were collected for pathological examination. Results The basilar arterial lumen cross-sectional area was significantly smaller in the A2 versus the A1 group, and proteinase-activated receptor (PAR)-1 and tumor necrosis factor (TNF)-α average optical densities were significantly higher (all P < 0.05). Basilar arterial lumen cross-sectional areas were significantly smaller in the A3 than the A2 group and average TNF-α optical densities were significantly lower (both P < 0.05), while those of PAR-1 did not differ significantly. Conclusions There was no significant difference in the extent of cerebral vasospasm between SAH and non-SAH model groups following TM injection into the subarachnoid space, so TM was considered to be an independent factor affecting cerebral vasospasm.

scholarly journals GPR18 Agonist Resolvin D2 Reduces the Early Brain Injury in A Rat Model of Subarachnoid Hemorrhage by Multi-Mechanisms of Protection

2021 ◽  
Author(s):  
Tongyu Zhang ◽  
Gang Zuo ◽  
Hongqi Zhang
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Brain Injury ◽  
White Matter ◽  
Western Blot ◽  
Rat Model ◽  
Time Course ◽  
Early Brain Injury ◽  
Brain Regions ◽  
White Matter Injury ◽  
Tnf Α

Abstract Background Early brain injury (EBI) is the early phase of secondary complications resulted in poor prognosis of subarachnoid hemorrhage (SAH). GPR18 is a G protein-coupled receptor which has been reported for neuroprotection in ischemia. In this study, we aimed to use resolvin D2 (RvD2) as an agonist to investigate the roles of GPR18 in different brain regions during EBI. Methods Location and time course of GPR18 after SAH were measured with immunofluorescence and western blot in endovascular perforation rat model. RvD2 was given one hour intranasally post-SAH, and SAH grades, neurobehavior and brain water content tests were performed after 24 hours. TUNEL and DHE staining were measured 24 hours post-SAH in cortex. Immunofluorescence, western blot and immunohistochemistry of proteins related to EBI in different brain regions were also performed. Results We found GPR18 mainly located in meninges, hypothalamus, cortex and white matter. And GPR18 expression increased in meninges and hypothalamus after EBI, however, it decreased in cortex and white matter. RvD2 could improve neurological scores and brain edema. Mast cell degranulation was attenuated, Chymase and Typtase expression decreased after RvD2 administration in meninges. RvD2 attenuated inflammation with increase of POMC, IL-10 and decrease of NPY, TNF-α in hypothalamus. In cortex, RvD2 alleviated oxidative stress and apoptosis, protected blood-brain barrier. RvD2 also ameliorated white matter injury by MBP elevation and APP depression. Conclusions Current results emphasized the importance of GPR18 in the whole brain during EBI. Upregulation of GPR18 with RvD2 may improve neurological functions with multi-mechanisms in different brain regions.

scholarly journals Potential Contribution of Nuclear Factor-κB to Cerebral Vasospasm after Experimental Subarachnoid Hemorrhage in Rabbits

2007 ◽  
Vol 27 (9) ◽  
pp. 1583-1592 ◽  
Author(s):  
Meng-Liang Zhou ◽  
Ji-Xin Shi ◽  
Chun-Hua Hang ◽  
Hui-Lin Cheng ◽  
Xiao-Ping Qi ◽  
...  
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Dna Binding ◽  
Cerebral Vasospasm ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Expression Levels ◽  
Dna Binding Activity ◽  
Tnf Α ◽  
Gene Expression Levels

Nuclear factor-κB (NF-κB) plays a key role in inflammation, which is involved in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). In the present study, we assessed the potential role of NF-κB in regulation of cerebral vasospasm. Nuclear factor-κB DNA-binding activity was measured in cultured vascular smooth muscle cells (VSMCs) treated with hemolysate and pyrrolidine dithiocarbamate (PDTC, 80 μmol/L), an inhibitor of NF-κB. Forty-two rabbits were divided into three groups: control, SAH, and PDTC groups ( n = 14 for each group). The caliber of the basilar artery was evaluated. Nuclear factor-κB DNA-binding activity and the gene expression levels of cytokines and adhesion molecules in the basilar artery were measured. Immunohistochemical study was performed to assess the expression and localization of tumor necrosis factor (TNF)-α, intercellular adhesion molecule (ICAM)-1, and myeloperoxidase (MPO). It was observed that NF-κB DNA-binding activity was significantly increased by treatment with hemolysate in cultured VSCMs, but this increase was suppressed by pretreatment with PDTC. Severe vasospasm was observed in the SAH group, which was attenuated in the PDTC group. Subarachnoid hemorrhage could induce increases of NF-κB DNA-binding activity and the gene expression levels of TNF-α, interleukin (IL)-1β, ICAM-1, and vascular cell adhesion molecule (VCAM)-1, which were reduced in the PDTC group. Immunohistochemical study demonstrated that the expression levels of TNF-α, ICAM-1, and MPO were all increased in the SAH group, but these increases were attenuated in the PDTC group. Our results suggest that NF-κB is activated in the arterial wall after SAH, which potentially leads to vasospasm development through induction of inflammatory response.

Sign in / Sign up

Export Citation Format

Share Document