Journal: bioRxiv

Article Title: Pharmacological Inhibition of BTK reduces neuroinflammation and stress induced anxiety in vivo

doi: 10.1101/2021.01.11.426241

Figure Lengend Snippet: (A) Examination of anxious behavior of stressed and non-stressed control mice using open field test (OFT). Mice exposed to predator odor and physical stress exhibited significantly higher anxiety levels when compared to the controls (Control-vivarium, and Control-arena), as depicted by their hesitation to explore or spend more time in the central area of the OFT. (B) Light-Dark test (LDT) revealed mice exposed to predator odor and restrain stress exhibited significantly higher anxiety levels (Dark-Light ratios) when compared to the controls, as explained by their reluctance to spend more time in the light chamber of the LDT, with physically stressed mice displaying greater anxious behavior. (C) Elevated plus maze (EPM) test revealed mice subjected to predator odor, and physical stress displayed significantly increased anxiety levels compared to the controls, as evidenced by their hesitation to spend more time in the open arms of the EPM. (D) Examination of Interleukin 1β (IL1β) in the hippocampus of mice exposed to predator odor and physical stress showed aberrantly higher IL1β levels compared to the controls. (E) Examination of Interleukin 6 (IL6) in the hippocampus of mice by ELISA showed that mice exposed to predator odor and physical stress showed aberrantly higher IL6 levels relative to the controls. All data are presented as Mean with 95% CI (n=20-21/group); ***p<0.001, ns (not significant); one-way ANOVA (Shapiro-Wilk’s, p >0.05) followed by Bonferroni post-hoc test.

Article Snippet: The supernatant was immediately used to determine the levels of IL1β and IL6 using Quantikine Mouse ELISA kits and following the manufacturer’s protocols (R&D Systems, Minneapolis, Mn).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Pharmacological Inhibition of BTK reduces neuroinflammation and stress induced anxiety in vivo

doi: 10.1101/2021.01.11.426241

Figure Lengend Snippet: (A) Evaluation of anxious behavior of stressed (predator odor) and non-stressed control mice using open field test (OFT). Female mice exposed to predator odor exhibited significantly higher anxiety levels when compared to stressed males and controls, as depicted by their hesitation to explore the central area of the OFT. (B) The light-dark test showed female mice exposed to predator odor exhibited significantly higher anxiety levels (higher Dark-Light ratios) when compared to the male stressed mice and control, as demonstrated by their reluctance to spend more time in the light chamber of the LDT. (C) Elevated plus maze test showed female mice subjected to predator odor displayed significantly increased anxiety levels compared to the other groups, as evidenced by their avoidance to spend more time in the open arms of the EPM. (D) Evaluation of IL1β levels by ELISA in hippocampal homogenates revealed female mice exposed to predator odor showed aberrantly higher IL1β as compared to stressed males. (E) Exposure of predator odor to female mice caused aberrantly higher IL6 levels in the hippocampus as compared to their male counterparts. All data are presented as Mean with 95% CI (n=17/group); ***p<0.001, ns (not significant); one-way ANOVA (Shapiro-Wilk’s, p >0.05) followed by Bonferroni post-hoc test.

Article Snippet: The supernatant was immediately used to determine the levels of IL1β and IL6 using Quantikine Mouse ELISA kits and following the manufacturer’s protocols (R&D Systems, Minneapolis, Mn).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Pharmacological Inhibition of BTK reduces neuroinflammation and stress induced anxiety in vivo

doi: 10.1101/2021.01.11.426241

Figure Lengend Snippet: Physical stress was induced by subjecting mice to restraint and underwater trauma. Control mice were treated with either vehicle or MCC950. Similarly, stressed mice were treated with either vehicle or MCC950. (A) Open Field Test: as compared to controls, physically stressed mice dosed with MCC950 exhibited significantly decreased anxiety levels, as illustrated by the increased time spent in the central area of the OFT, in both male and female mice. (B) Light-Dart Test: physically stressed mice dosed with MCC950 exhibited significant improvement from hyper-anxious behavior, as explained by the reduced D/L ratio, i.e. increased exploration time in the light chamber of the LDT. (C) Elevated plus maze test revealed physical stress mice dosed with MCC950 displayed significant rescue from anxiety compared to the vehicle controls, as evidenced by the increased duration of time spent in the open arms of the EPM. (D) Examination of IL1β in hippocampal homogenates by ELISA revealed physically stressed mice dosed with MCC950 showed diminished IL1β levels compared to the vehicle controls, denoting significant rescue from anxiety. (E) Caspase 1 activity in the hippocampus: physically stressed mice administered with MCC950 exhibited attenuated Caspase 1 activation relative to their vehicle controls. All values are presented as Mean with 95% CI (n=22/group); ***p<0.001, ns (not significant); two-way ANOVA (Shapiro-Wilk’s, p >0.05) followed by Bonferroni post-hoc test.

Article Snippet: The supernatant was immediately used to determine the levels of IL1β and IL6 using Quantikine Mouse ELISA kits and following the manufacturer’s protocols (R&D Systems, Minneapolis, Mn).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Pharmacological Inhibition of BTK reduces neuroinflammation and stress induced anxiety in vivo

doi: 10.1101/2021.01.11.426241

Figure Lengend Snippet: Physical stress was induced by subjecting mice to restraint and underwater trauma. Control mice were treated with either vehicle or MCC950. Similarly, stressed mice were treated with either vehicle or MCC950. (A) Analysis of IL1β in amygdala homogenates by ELISA revealed mice exposed to physical stress showed aberrantly higher IL1β compared to control mice. Treatment of stressed mice with MCC950 showed a significant reduction of IL1β. (B) Caspase 1 activity in amygdala: intraperitoneal administration of MCC950 in physically stressed mice showed reduced Caspase 1 activation, elucidating key role of NLRP3 in anxiogenic Capsase 1 - IL1β pathway. Caspase 1 activity in amygdala homogenates was measured by Caspase 1 activity assay kit. All values are presented as Mean with 95% CI (n=19-22/group); ***p<0.001, ns (not significant); two-way ANOVA (Shapiro-Wilk’s, p >0.05) followed by Bonferroni post-hoc test.

Article Snippet: The supernatant was immediately used to determine the levels of IL1β and IL6 using Quantikine Mouse ELISA kits and following the manufacturer’s protocols (R&D Systems, Minneapolis, Mn).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Pharmacological Inhibition of BTK reduces neuroinflammation and stress induced anxiety in vivo

doi: 10.1101/2021.01.11.426241

Figure Lengend Snippet: Mice subjected to restraint stress and underwater trauma were injected with Ibrutinib (3 mg/Kg, i.p.) or vehicle. (A) Immunoblot analysis of NLRP3 in hippocampus homogenates. (B) The relative densitometry of NLRP3 immunoblots from hippocampal samples showing Ibrutinib treatment significantly reduced the NLRP3 inflammasome levels in physically stressed mice as compared to vehicle-treated stressed mice. (C) Open field test: physically stressed mice dosed with Ibrutinib exhibited significantly decreased anxiety levels when compared to the vehicle controls, as illustrated by increased exploration time in the central area of the OFT. (D) Light-Dark test: physically stressed mice dosed with Ibrutinib exhibited significant rescue from hyper-anxiety as explained by the significant increase in their time spent in the light chamber of the LDT. (E) Elevated plus maze test: physically stress mice injected with Ibrutinib displayed significantly reduced anxiety, as evidenced by the increased duration of time spent in the open arms of the EPM. (F) Analysis of IL1β in the hippocampus by ELISA revealed physically stressed mice dosed with Ibrutinib showed diminished proinflammatory IL1β. (G) Caspase 1 activity in the hippocampus: administration of Ibrutinib in physically stressed mice showed reduced Caspase 1 activation, elucidating significant rescue from the anxiogenic proinflammatory pathway. All values are presented as Mean with 95% CI (n=22/group); ***p<0.001, ns (not significant); two-way ANOVA (Shapiro-Wilk’s, p >0.05) followed by Bonferroni post-hoc test.

Article Snippet: The supernatant was immediately used to determine the levels of IL1β and IL6 using Quantikine Mouse ELISA kits and following the manufacturer’s protocols (R&D Systems, Minneapolis, Mn).

Techniques: Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Pharmacological Inhibition of BTK reduces neuroinflammation and stress induced anxiety in vivo

doi: 10.1101/2021.01.11.426241

Figure Lengend Snippet: (A) Analysis of the open field test, following the administration of LMF-A13 in physically stressed mice, there was evidence of significantly decreased anxiety level, as illustrated by the increase in the time of exploration in the central area of the OFT. (B) Light-Dark test: physically stressed mice dosed with LMF-A13 showed significant rescue from anxiety, as illustrated by the significant increase in time to spend in the light chamber of the LDT. (C) Results of an elevated plus maze also revealed physically stressed mice dosed with LMF-A13 displayed a significant reduction in anxiety, as evidenced by the increase in the duration of time spent in the open arms of the EPM. (D) Examination of IL1β levels by ELISA in the hippocampus revealed physically stressed mice dosed with LMF-A13 showed diminished IL1β levels. (E) Caspase 1 activity in the hippocampus: physically stressed mice administered with LMF-A13 showed reduced Caspase 1 activation, elucidating significant rescue from anxiogenic proinflammatory pathway. All values are presented as Mean with 95% CI (n=20/group); ***p<0.001, ns (not significant); two-way ANOVA (Shapiro-Wilk’s, p >0.05) followed by Bonferroni post-hoc test.

Article Snippet: The supernatant was immediately used to determine the levels of IL1β and IL6 using Quantikine Mouse ELISA kits and following the manufacturer’s protocols (R&D Systems, Minneapolis, Mn).

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Activation Assay