Chronic Alcoholism and HHV-6 Infection Synergistically Promote Neuroinflammatory Microglial Phenotypes in the Substantia Nigra of the Adult Human Brain
Abstract
:1. Introduction
2. Materials and Methods
2.1. Autopsy Brain Tissue Collection and Characteristics
2.2. Immunohistochemistry (IHC), Double Immunohistochemistry, and Immunofluorescence (IF)
2.3. DNA Extraction and HHV-6 Detection Using Nested Polymerase Chain Reaction (nPCR)
2.4. HHV-6A and HHV-6B Variant Detection Using HindIII Restriction Endonuclease
2.5. Viral Load Determination Using Real-Time PCR (RT-PCR)
2.6. Statistical Analysis
3. Results
3.1. Alcoholics Showed a Significant Decrease in CD68+ Cells Than Controls in Both Gray and White Matter
3.2. Alcoholics Showed a Significant Increase in Iba1+ Cells Compared to Controls in Both Gray and White Matter
3.3. Alcoholics Showed Significantly More CD11b+ Cells Than Controls in Both Gray and White Matter
3.4. Exposure to Alcohol Induces Stronger Iba1 Expression Than CD11b in Microglial Cells
3.5. Morphological Characterization of Microglial Subpopulations
3.6. Alcoholics Showed Detectable HHV-6 Positivity and Viral Load
3.7. Alcohol and HHV-6 Infection in a Synergistic and Potentiating Relationship Cause Disruption of Homeostasis in the SN Region
4. Discussion
4.1. Abundance of CD68+/Iba1− Microglial Subpopulation in Controls Indicates a Special Physiological “Controlled Phagocytotic Activated State”
4.2. Decrease in the Number of CD68+ Microglia with Increase in Iba1+ Expression Shows Microglial Dystrophy Which Leads to Compensatory Mobility from Perivascular to Diffuse Locations
4.3. Alcohol and HHV-6 Infection Co-Induce and Accelerate Microglial Dystrophy
4.4. Dominance of Iba1+/CD11b− Microglial Subpopulation in Alcoholics Leads to Chronic Inflammation, Hyperalgesia, and Allodynia
4.5. Limitations of the Present Study
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Group A (Control Group) | Group B (Age-Matched Alcoholics) | Group C (Non-Age-Matched Alcoholics) | ||||||
---|---|---|---|---|---|---|---|---|
Individual Code | Age (Years) | Gender (Male/ Female) | Individual Code | Age (Years) | Gender (Male/ Female) | Individual Code | Age (Years) | Gender (Male/ Female) |
A1 | 34 | M | B1 | 36 | F | C1 | 48 | M |
A2 | 31 | M | B2 | 23 | M | C2 | 55 | M |
A3 | 27 | M | B3 | 31 | M | C3 | 60 | M |
A4 | 23 | M | B4 | 26 | M | C4 | 50 | F |
A5 | 32 | M | B5 | 33 | M | C5 | 63 | M |
A6 | 37 | M | B6 | 34 | F | C6 | 45 | M |
A7 | 33 | M | B7 | 25 | F | C7 | 45 | M |
A8 | 22 | M | B8 | 30 | M | C8 | 55 | M |
A9 | 36 | M | B9 | 35 | M | C9 | 45 | F |
A10 | 17 | M | B10 | 22 | M | C10 | 60 | F |
A11 | 37 | M | B11 | 35 | M | C11 | 66 | M |
A12 | 20 | M | B12 | 34 | M | C12 | 63 | M |
A13 | 26 | M | B13 | 29 | M | C13 | 44 | F |
C14 | 49 | M | ||||||
C15 | 45 | F | ||||||
C16 | 60 | M | ||||||
C17 | 38 | F | ||||||
C18 | 40 | M | ||||||
Group median age | 31 ± 6.79 | Group median age | 31 ± 4.85 | Group median age | 49.5 ± 8.66 |
Primary Antibody * | Antibody Characteristics ** | Clone | Dilution | Manufacturer |
---|---|---|---|---|
CD68 | Monoclonal mouse AB against human AG | Kp-1 | 1:200 | Cell Marque (USA) |
CD11b | Monoclonal rabbit AB against human AG | EP45 | 1:100 | Epitomics (USA) |
Iba1 | Monoclonal rabbit AB against human AG | EP289 | 1:150 | Epitomics (USA) |
HHV-6 (20) | Monoclonal mouse AB against viral lysate | - | 1:200 | Santa Cruz (USA) |
Primer | Primer Sequence |
---|---|
Outer primer O1 | 5′-AGTCATCACGATCGGCGTGCTATC-3′ |
Outer primer O2 | 5′-TATCTAGCGCAATCGCTATGTCG-3′ |
Inner primer I3 | 5′-TCGACTCTCACCCTACTGAACGAG-3′ |
Inner primer I4 | 5′-TGACTAGAGAGCGACAAATTGGAG-3′ |
Region | Group A | Group B | Group C | p Value † |
---|---|---|---|---|
Pars Compacta (SNpc) | ||||
Gray matter | 6.50 ± 0.27 | 6.32 ± 0.20 | 6.22 ± 0.15 | 0.908 |
White matter | 6.55 ± 0.25 | 6.07 ± 0.18 | 5.97 ± 0.20 | 0.360 |
p value ‡ | 0.749 | 0.145 | 0.519 | - |
Pars Reticulata (SNpr) | ||||
Gray matter | 6.50 ± 0.25 | 6.34 ± 0.22 | 5.61 ± 0.25 | 0.031 ** |
White matter | 7.40 ± 0.29 | 6.75 ± 0.25 | 5.86 ± 0.19 | <0.001 ** |
p value ‡ | 0.001 ** | 0.002 ** | 0.020 ** | - |
Region | Group A | Group B | Group C | p Value † |
---|---|---|---|---|
Pars Compacta (SNpc) | ||||
Gray matter | 2.97 ± 0.15 | 4.41 ± 0.16 | 4.72 ± 0.17 | <0.001 ** |
White matter | 3.50 ± 0.17 | 4.57 ± 0.14 | 4.80 ± 0.17 | <0.001 ** |
p value ‡ | <0.001 ** | <0.001 ** | 0.232 | - |
Pars Reticulata (SNpr) | ||||
Gray matter | 1.97 ± 0.13 | 2.20 ± 0.15 | 3.27 ± 0.14 | <0.001 ** |
White matter | 2.30 ± 0.16 | 2.45 ± 0.16 | 3.31 ± 0.13 | <0.001 ** |
p value ‡ | <0.001 ** | 0.342 | 0.752 | - |
Region | Group A | Group B | Group C | p Value † |
---|---|---|---|---|
Pars Compacta (SNpc) | ||||
Gray matter | 1.02 ± 0.07 | 1.47 ± 0.07 | 1.58 ± 0.09 | <0.001 ** |
White matter | 1.34 ± 0.09 | 1.60 ± 0.07 | 1.73 ± 0.09 | 0.005 ** |
p value ‡ | 0.002 ** | 0.191 | 0.165 | - |
Pars Reticulata (SNpr) | ||||
Gray matter | 0.72 ± 0.08 | 0.83 ± 0.08 | 0.88 ± 0.07 | 0.143 |
White matter | 0.79 ± 0.09 | 0.83 ± 0.07 | 0.95 ± 0.09 | 0.724 |
p value ‡ | 0.815 | 0.270 | 0.603 | - |
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Jain, N.; Smirnovs, M.; Strojeva, S.; Murovska, M.; Skuja, S. Chronic Alcoholism and HHV-6 Infection Synergistically Promote Neuroinflammatory Microglial Phenotypes in the Substantia Nigra of the Adult Human Brain. Biomedicines 2021, 9, 1216. https://doi.org/10.3390/biomedicines9091216
Jain N, Smirnovs M, Strojeva S, Murovska M, Skuja S. Chronic Alcoholism and HHV-6 Infection Synergistically Promote Neuroinflammatory Microglial Phenotypes in the Substantia Nigra of the Adult Human Brain. Biomedicines. 2021; 9(9):1216. https://doi.org/10.3390/biomedicines9091216
Chicago/Turabian StyleJain, Nityanand, Marks Smirnovs, Samanta Strojeva, Modra Murovska, and Sandra Skuja. 2021. "Chronic Alcoholism and HHV-6 Infection Synergistically Promote Neuroinflammatory Microglial Phenotypes in the Substantia Nigra of the Adult Human Brain" Biomedicines 9, no. 9: 1216. https://doi.org/10.3390/biomedicines9091216