Vascular Pathology of the Prefrontal Cortex in Major Depressive Disorder

1. Mentor: Dr. Grazyna Rajkowska Co-mentor: Dr. Dorota Maciag Intern: Rachael Vaughan Vascular Pathology of the Prefrontal Cortex in Major Depressive Disorder

2. Why study Major Depressive Disorder? highest mortality rate of any mental disorder (suicide) over 12 million U.S. citizens affected each year (and growing) Numerous negative effects: Social withdrawal Anhedonia Agitation, restlessness, and irritability Extreme difficulty concentrating Fatigue and lack of energy Insomnia or hypersomnia Appetite change Weight gain or loss Anhedonia: : a loss of interest or pleasure in activities that were once enjoyed (including sexual activity) Hypersomnia: sleeping excessively These effects are usually accompanied by feelings of hoplessness/helplessness/worthlessness/self-hate/inappropriate guilt, etc. Each of these factors greatly impact the patient�s physical, social, economic, and overall well-being. (ex: can�t concentrate at work therefore decreased productivity and possible loss of income)Anhedonia: : a loss of interest or pleasure in activities that were once enjoyed (including sexual activity) Hypersomnia: sleeping excessively These effects are usually accompanied by feelings of hoplessness/helplessness/worthlessness/self-hate/inappropriate guilt, etc. Each of these factors greatly impact the patient�s physical, social, economic, and overall well-being. (ex: can�t concentrate at work therefore decreased productivity and possible loss of income)

3. So�what does vascular disease have to do with depression? Growing evidence suggests direct relationship Mayo Clinic lists cardiovascular disease as a main risk factor for depression and vice versa US Department of Health and Human Services has predicted that depression will be the second leading cause of death (after cardiovascular disease) worldwide by 2020. Recent studies have indicated that depression may precede cardiovascular malfunctions and cardiovascular disorders may precede depressive episodes American Heart Association suggests that all heart disease patients be screened regularly for depression. Recent studies have indicated that depression may precede cardiovascular malfunctions and cardiovascular disorders may precede depressive episodes American Heart Association suggests that all heart disease patients be screened regularly for depression.

4. Key ideas Astroglia are essential in cerebral blood flow. Reductions in neuronal and glial cell densities are found in the prefrontal cortex of depressed patients (previous studies) Coverage of blood vessels walls by astrocytic endfeet is essential to transducing synaptic activity signals that cause vasodilation and increase microcirculation in the brain. What are astroglia? Type of macroglial cells, identified usually by their radial orientation and endfeet contact with neuronal processes and blood vessels. To put it simply, they are star shaped cells found mainly in the brain and spinal cord. What are astroglia? Type of macroglial cells, identified usually by their radial orientation and endfeet contact with neuronal processes and blood vessels. To put it simply, they are star shaped cells found mainly in the brain and spinal cord.

5. Quantitative reduction in astrocytes (the star shaped cells just mentioned) means a quantitative reduction in vascular �coverage� Not due to anti depressant medications but rather the ACTUAL pathology of Major Depressive Disorder itself In depression there are alterations in vascular morphology that are associated with deficits in angiogenic and neurotrophic factors as well as pathology of neurons and glia. Quantitative reduction in astrocytes (the star shaped cells just mentioned) means a quantitative reduction in vascular �coverage� Not due to anti depressant medications but rather the ACTUAL pathology of Major Depressive Disorder itself In depression there are alterations in vascular morphology that are associated with deficits in angiogenic and neurotrophic factors as well as pathology of neurons and glia.

6. Methodology Tissue and slide preparation Tissue was obtained through the coroner�s office of Cleveland, Ohio (CNTR=8, MDD=8) Frozen brain tissue was cut in cryostat at approximately - 14�C to 20 micrometers in thickness Double Immunohistochemistry used to selectively illuminate the blood vessels and astrocytes in tissue. Imaging via confocal microscope Computational analysis using Adobe Photoshop CS4 and Image J software to isolate and quantify the ratio of coverage between astrocytic end feet and vascular tissue. How we performed our study: our three main approaches in order of procedure Confocal microscope to bring out the chemilluminescence Computer programs to calculate our numerical data About to explain what the ratio of coverage is How we performed our study: our three main approaches in order of procedure Confocal microscope to bring out the chemilluminescence Computer programs to calculate our numerical data About to explain what the ratio of coverage is

7. Chemiluminescence: Anti mouse linked to flourescein�anti rabbit linked with CY5 (gives you red color) Immunohistochemistry= method of staining using the mammalian immune system with its ability to recognize foreign proteins and to manufacture antibody molecules that strongly bind to the substances that elicit them GFAP=marker for presence of astrocytes (found in cytoskeleton of astrocytes) Collagen IV= allows us to see location of blood vessels (collagen is a structural marker of vessels because it is one of the components of a vessel wall, so called basal membrane)Chemiluminescence: Anti mouse linked to flourescein�anti rabbit linked with CY5 (gives you red color) Immunohistochemistry= method of staining using the mammalian immune system with its ability to recognize foreign proteins and to manufacture antibody molecules that strongly bind to the substances that elicit them GFAP=marker for presence of astrocytes (found in cytoskeleton of astrocytes) Collagen IV= allows us to see location of blood vessels (collagen is a structural marker of vessels because it is one of the components of a vessel wall, so called basal membrane)

8. We use double-immunohistochemical staining with luminescent colors to distinguish the collagen within the walls of vessels in the brain (GREEN) and astrocytic feet (RED) that we hope �cover� or provide �coverage� for the vessels, which would reveal a YELLOW color overlap.We use double-immunohistochemical staining with luminescent colors to distinguish the collagen within the walls of vessels in the brain (GREEN) and astrocytic feet (RED) that we hope �cover� or provide �coverage� for the vessels, which would reveal a YELLOW color overlap.

9. Using the replace color tool in Photoshop CS4 we could isolate the areas of red/green color colocalizationUsing the replace color tool in Photoshop CS4 we could isolate the areas of red/green color colocalization

10. Enlarge image to see pixels Enlarge image to see pixels

11. Results There are more blood vessels visible in gray matterThere are more blood vessels visible in gray matter

12. Conclusion: A Work In Progress Time consuming analysis: only two pairs of subjects fully analyzed. Must allow at least one slide per day for analysis (isolation of yellow pixels in Photoshop is tedious) 4 subjects (2 pairs); White Matter and Gray Matter; 3 slides per matter per subject; 10-13 images per slide; 2 versions of each image (1 green and 1 with all colors present) Multiple subjects still awaiting analysis (several more pairs including depressed subjects and their carefully matched controls) Data from gray matter more closely followed our expectations than white matter

13. Possible Considerations for Further Study Although every glial fibrillary acidic protein (GFAP)-immunopositive cell in the brain can be called an astrocyte, there are many astrocytic endfeet that are not labeled by anti-GFAP antibodies Use of another marker for astrocytic endfeet? Aquaporin 4 is a marker for water channels present specifically in astrocytic endfeet. Viability of Photoshop method of isolation: finding a more accurate way to select and extract only the desired color range versus point and click pixel method More work to be done! It�s possible to see more coverage with the use of marker Aquaporin 4It�s possible to see more coverage with the use of marker Aquaporin 4

14. Acknowledgements Neuroscience Summer Scholars Program Dr. Grazyna Rajkowska Dr. Dorota Maciag Dr. Ian Paul CPN Imaging Core