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Social stress induces neurovascular pathology_Masuma_Akter
- 1. MASUMAAKTER
- 2. ๏ Major depressive disorder (MDD), also known simply as depression, often accompanied by low self-esteem, loss of interest, and pain without a clear cause. ๏ MDD is associated with an โผ80% increased risk of cardiovascular morbidity and mortality ๏ Clinical studies report higher levels of circulating pro-inflammatory cytokines in patients with MDD, a pattern that has been replicated in preclinical animal models of depression ๏ Proinflammatory cytokines can diffuse into the brain of stressed individuals as a result of stress-induced neurovascular damage and increased BBB permeability.
- 3. ๏ The bloodโbrain barrier (BBB) is a highly selective semipermeable membrane barrier in the CNS. ๏ The BBB is formed by endothelial cells sealed by tight junction proteins pericytes and astrocytes. The bloodโbrain barrier (BBB) Tight junctions Proteins: ๏ Closely associated areas of two cells, forming a barrier impermeable to fluid ๏ Tight junctions are composed of a branching network of sealing strands ๏ Major types are the claudins and the occludins
- 4. Question: To evaluate the effect of chronic stress on BBB-related gene expression and the establishment of depression-like behaviors or MDD Methods: 1. Chronic social defeat stress (CSDS) 2. Microdefeat stress 3. Social interaction/avoidance test 4. Behavioral studies (Splash test, Sucrose preference test, Forced swim test) 5. Transcriptional profiling 6. Immunohistochemistry (IHC) and quantification of tight junction proteins 7. IHC for Evans blue (EB) 8. Capillary extraction and western blot 9. Imipramine treatment 10. Transmission electron microscopy (TEM) 11. Human post-mortem tissue collection 12. Stereotaxic surgery and viral gene transfer 13. Magnetic resonance imaging (MRI) scans 14. Isolation of brain leukocytes and flow cytometry 15. IL-6 biotinylation Conclusion: Chronic social stress alters BBB integrity through down regulation of the tight junction protein drives the development of depression-like behaviors
- 5. ๏ A male C57BL/6 J mouse is exposed daily (10min/d) to social defeat by a larger, physically aggressive CD-1 mouse for 10 days. Chronic social defeat stress (CSDS) Sam A Golden,2014
- 6. Microdefeat stress ๏ A sub threshold variation of the CSDS protocol was used to evaluate increased susceptibility to stress ๏ Without the introduction of pro-depressive manipulations, this protocol is insufficient to induce depression like behaviors Day 1 Physical stress (5 min) Physical stress (5 min) Physical stress (5 min) Rest (15 min) Rest (15 min) SI test Social interaction/avoidance test ๐๐ผ ๐ ๐๐ก๐๐ = The time spent in the interaction zone when the AGG is present The time spent in the interaction zone when the AGG is absent
- 7. Purpose: Comparison between SS and RES mice to identify individual differences in the neurovascular mechanisms potentially underlying chronic stress responses. Methods: Transcriptional Profiling of genes, Social interaction behavioral screening Supp_Fig:1 Conclusion: Cldn5 mRNA was significantly reduced in NAc of SS mice and ss mice showed high social avoidance behavior. Pecam1: an endothelial cell marker
- 8. Methods: Immunohistochemistry, Social interaction behavioral screening Purpose: Detection of a a specific reduction of Cldn5 protein in SS mice Conclusion: Cldn5 protein levels in NAc blood vessels also positively correlated with social avoidance behavior where as other tight junction protein does not. CD31: an endothelial cell marker Occludin: Plasma membrane protein at Tight junctions ZO-1: Tight junction protein-1
- 9. Purpose: CSDS induces ultrastructural abnormalities of blood vessels within the NAc of SS mice or not? Methods: Transmission Electron Microscopy Conclusion: Ultrastructural abnormalities were present in both large vessels and capillaries of the NAc where as No significant change was observed in the PFC region.
- 10. Purpose: To evaluate that Chronic antidepressant treatment can reverse CSDS-induced depression-like behaviors & social avoidance or not? MDD: Major depressive disorder AD: Antidepressant Imipramine: Tricyclic antidepressant Conclusion: Chronic imipramine treatment normalized Cldn5 mRNA expression in the NAc of SS mice where as acute imipramine injection was not sufficient to alter Cldn5 mRNA. Method: Tricyclic antidepressant treatment and social interaction test?
- 11. Purpose: To confirm a causal role for Cldn5 downregulation in social- stress-induced depression-like behaviors Methods: Conditional knockdown of Cldn5 in the NAc, social stress test downregulation of Cldn5 mRNA & Protein Conclusion: Stressed AAV-shRNA-Cldn5 mice spent significantly less grooming time , displayed anhedonia, spent more time immobile, displayed greater social avoidance.
- 12. Purpose: To perform a rescue experiment to confirm the causal role of NAc Cldn5 expression in the establishment of depression like behaviors Conclusion: Cldn5 expression plays a protective role in behavioral response to social stress, possibly by maintaining BBB impermeability to stress-induced passage of peripheral immune signals. Methods: Conditional knockdown of Cldn5 in the NAc, Rescue of Cldn5 expression, social stress test
- 13. Purpose: Weather CSDS induced loss of Cldn5 and altered blood vessel morphology or BBB integrity in SS mice Conclusion: ๏ High Gd-DTPA suggesting leakiness of the BBB in these brain areas ๏ social avoidance is significantly correlated with Gd-DTPA level in the NAc Methods: MRI scan, retro-orbital injection, social interaction test
- 14. Purpose: Weather CSDS induced loss of Cldn5 and altered blood vessel morphology or BBB integrity in SS mice perivascular marker isolectin B4(Ib4) intravascular Evans blue (EB) Conclusion: NAc BBB may be more vulnerable to neurovascular damage than other brain regions, leading to greater infiltration of larger proteins following stress Methods: Assessment of BBB permeability with cadaverine Alexa Fluor-555 and Evans blue
- 15. Purpose: confirmation of BBB leakiness in the NAc Methods: Down regulation of Cldn5 expression, Injection of Alexa Fluor-555-conjugated cadaverine, Evans blue, HRP, transmission electron microscopy Conclusion: These observations indicate that BBB integrity is impaired in SS mice, with a loss of Cldn5 and abnormal blood vessel ultrastructure.
- 16. Methods: Flow Cytometry , immunohistochemistry, Conclusion: Flow cytometry revealed more Ccr2RFP+;Cx3cr1GFPโ peripheral monocytes in the brain of stressed mice Purpose: Evaluating whether peripheral monocytes can infiltrate the brain parenchyma in stress-related brain regions or not?
- 17. Purpose: Evaluation of whether circulating IL-6 is associated with stress vulnerability and MDD or not? Conclusion: ๏ Higher IL-6 protein is found in the NAc region and blood of SS mice compared to that in unstressed CTRL mice ๏ Confirm the importance of NAc IL-6 in the establishment of depression-like behaviors Methods: IL-6 i.p. injection, assessment of IL-6 levels by ELISA, Osmotic minipump surgery and IL-6 infusion
- 18. Purpose: To determine whether peripheral IL-6 is able to penetrate the brain parenchyma of Stress susceptible mice or not? Methods: IL-6 biotinylation, retro-orbital injection , IHC Conclusion: ๏ Biotinylated IL-6 was only detectable in the NAc of SS mice, and revealed passage outside of blood vessels into the parenchyma ๏ Downregulation of Cldn5 expression with an AAV-shRNA allowed the passage of biotinylated IL-6 into the NAc parenchyma
- 19. Discussion: ๏ถ Complement decades of clinical evidence ๏ถ IL-6 are involved in the establishment of MDD ๏ถ Chronic stress activates the immune system and undermines BBB integrity
Editor's Notes
- CD31: an endothelial cell marker Occludin: Plasma membrane protein at Tight junctions ZO-1: Tight junction protein-1ย
- Representative pictures of large blood vessels and capillaries in the NAc of CTRL, SS and RES mice. Mice were injected with horseradish peroxidase (HRP) that was allowed to circulate for 2 hours before 20-min perfusion and fixation. No significant difference was measured for tight junction discontinuity in the PFC of SS or RES mice when compared to CTRL mice. Mice were administered horseradish peroxidase type II through retro-orbital injection 24 h after SI test screening. HRP was allowed to circulate for 2 h
- Lack of significant medication effects may be related to suicide as cause of death for the majority of MDD patients in our cohorts.
- The microdefeat allows the investigation of the prosusceptibility effects of a molecular manipulation. Specifically, in the microdefeat protocol, a C57BL/6J mouse is subjected to subthreshold levels of social defeat that consist of three 5-min defeat sessions given consecutively on a single day with 15 min of rest between each session
- Gadolinium contrast agent Gd-DTPA was then administered through retro-orbital injection retro-orbital injection is an acceptable methods for intravenous route of administration of any compounds. The retro orbital sinus is the site located behind the eye at the medial or lateral canthus.
- cadaverine : Alexa Fluor-555, A dye with a molecular weight of 0.95 kDa EB has a high affinity for serum albumin (โผ69kDa) ,which cannot readily cross the BBB in the absence of neurovascular damage
- C-C chemokine receptor 2โexpressing (Ccr2+), Ccr2RFP::Cx3cr1GFP double heterozygous mice in which adult resident microglia exhibit a Ccr2RFPโ;Cx3cr1GFP+ pattern, whereas peripheral monocytes, which are recruited in response to inflammation, are characterized by a Ccr2RFP+;Cx3cr1GFPโ pattern. Ccr2RFP::Cx3cr1GFP mice were subjected to 10-d CSDS, followed by an SI test. Half of the brain was collected for flow cytometry analyses and the other half fixed for immunohistochemistry (IHC) (Fig.ย 4a).
- vulnerability and MDD7, can infiltrate the NAc of defeated mice although this does not rule out the possibility that stress induces local production of IL-6. suggesting a region-specific accumulation of IL-6 in the NAc of SS mice following CSDS Our studies also revealed that the NAc is largely impermeable to circulating IL-6 under normal conditions To confirm the importance of NAc IL-6 in the establishment of depression-like behaviors, we implanted cannulas in the NAc of stress-naive mice and infused either saline or IL-6 before subthreshold microdefeat (Fig.ย 4f). Stressed mice that received intra-NAc IL-6 displayed social avoidance compared to stressed mice receiving intra-NAc saline or unstressed mice receiving either intra-NAc IL-6 or saline
- Biotinylated IL-6 (20 ng diluted in saline solution) was administered through retro-orbital injection in CTRL, SS or RES mice 24 h after SI test screening and allowed to circulate for 2 h. IL-6โbiotin conjugate was detected with Oregon Greenโ488 conjugate of NeutrAvidin biotin-binding protein Our studies also revealed that the NAc is largely impermeable to circulating IL-6 under normal conditions, as we were unable to detect biotinylated IL-6 in the NAc of unstressed CTRL mice These observations indicate that social stress causes direct passage of the pro-inflammatory cytokine IL-6, which then acts directly within the NAc to induce depression-like behaviors
- 10-day chronic social defeat stress (CSDS) induces loss of Cldn5 expression at mRNA and protein levels, leading to abnormalities in blood vessel morphology and increased BBB permeability in stress-susceptible (SS) mice. These molecular and cellular changes are associated with depression-like behavior such as social avoidance, anhedonia, despair and lack of self-care. Conversely, mice resilient (RES) to CSDS display normal social and stress coping behaviors and neurovascular features similar to unstressed controls (CTRL).