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A novel animal model for neuroinflammation and white matter degeneration

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Preprint: A novel animal model for neuroinflammation and white matter degeneration https://t.co/HvzwnOhuUR https://t.co/Mb05psKato
A novel animal model for neuroinflammation and white matter degeneration https://t.co/8p4H598XfJ Small interference RNA has been …
136 days ago
A novel animal model for neuroinflammation and white matter degeneration https://t.co/yQvdet3VsM
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Supplemental Information

Design of D1shRNAs and suppression of Drd1 protein in mouse striatum

(A) Three different shRNAs were designed to target different sites of mouse Drd1 gene. Immunohistochemical staining of Drd1 was performed using rabbit anti-Drd1 antibody in mouse striatum transduced with either scAAV8-hrGFP (B) or scAAV8-D1shRNA1 (C) virus. The virus was injected into the left striatum only. Reduced Drd1 protein was observed in the left striatum injected with scAAV8-D1shRNA1 virus, but not with scAAV8-hrGFP virus. High magnification of Drd1 reduction was shown in the left striatum transduced with scAAV8-D1sdhRNA1 virus (D) in comparison with Drd1 expression in the right control striatum (E). Scale bar: 50 µm

DOI: 10.7287/peerj.preprints.3117v1/supp-1

Mild neuroinflammation in striatum transduced with scAAV8-D1shRNA2 virus

(A) Immunohistochemical staining of Iba-1 in mice injected with scAAV8-D1shRNA2 virus. Microglial cells were activated to surrounding blood capillaries in the left striatum transduced with the scAAV8-D1shRNA2 virus. (B) and (C) are the higher magnifications of mild neuroinflammation around the blood capillaries. Scale bar: 50 µm.

DOI: 10.7287/peerj.preprints.3117v1/supp-2

Immunohistochemical staining of MBP

There is no difference in MBP staining in the left corpus callosum transduced with scAAV8-hrGFP virus (A) in comparison with the control right corpus callosum without virus injection (B). Scale bar: 100 µm. MBP was reduced (black arrow) on the left corpus callosum (C) of mice injected with scAAV8-D1shRNA3 in comparison with the normal right corpus callosum (D). Scale bar: 200 µm. Under a higher magnification, MBP staining was also decreased in striatal white matter tracts that were swollen and blebbed (black arrowheads) in the left striatum transduced with scAAV8-D1shRNA3 (E) in comparison with normal white matter tracts in the control right striatum (F). Scale bar: 60 µm.

DOI: 10.7287/peerj.preprints.3117v1/supp-3

Absence of neuroinflammation and white matter degeneration in the left striatum transduced with scAAV8-hrGFP virus

Two consecutive paraffin sections from mice injected with scAAV8-hrGFP virus were immunostained 7 weeks post-injection with either anti-Iba-1 (A) or anti-NF-L (B) antibody. There was no activation of microglial cells in the left striatum transduced with scAAV8-hrGFP virus in comparison with the uninjected control right striatum. There was no reduction of NF-L staining in the corpus callosum on the left side (C) transduced with the virus in comparison with the control right side without virus infection (D). Scale bar: 150 µm.

DOI: 10.7287/peerj.preprints.3117v1/supp-4

White matter degeneration and neurofilament reduction in striatum transduced with scAAV8-D1shRNA3 virus

Two consecutive brain paraffin sections from mice injected with scAAV8-D1shRNA3 virus were immunostained with either anti-Iba-1 (A) or anti-NF-L (B) antibody. Extensive microglial activation as shown by anti-Iba-1 immunostaining was observed in the left striatum transduced with the scAAV8-D1shRNA3 virus in comparison with the control right striatum. Decreased NF-L staining was observed in the left corpus callosum (C) compared with the control right corpus callosum (D). Scale bar: 200 µm. Neurofilament staining was reduced in blebbed striatal white matter tracts (black arrow) in the left striatum transduced with the virus (E) in contrast to the control right striatum (F). Scale bar: 60 µm.

DOI: 10.7287/peerj.preprints.3117v1/supp-5

Western blot raw data with mouse anti-Drd1

DOI: 10.7287/peerj.preprints.3117v1/supp-6

Western blot raw data using anti-mouse IgG

DOI: 10.7287/peerj.preprints.3117v1/supp-7

Western blot raw data mouse IgG week 4 to 6

DOI: 10.7287/peerj.preprints.3117v1/supp-8

Western blot raw data mouse IgG week 7 to 15

week 7, 8, 9, 15

DOI: 10.7287/peerj.preprints.3117v1/supp-9

Western blot raw data NR1 week 4 to 6

normalization controls

DOI: 10.7287/peerj.preprints.3117v1/supp-10

Western blot raw data NR1 week 7 to 15

Normalization controls for week 7,8,9, and 15.

DOI: 10.7287/peerj.preprints.3117v1/supp-11

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Baohu Ji performed the experiments, analyzed the data, prepared figures and/or tables.

Kerin Higa performed the experiments.

Virawudh Soontornniyomkij contributed reagents/materials/analysis tools.

Atsushi Miyanohara contributed reagents/materials/analysis tools.

Xianjin Zhou conceived and designed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The surgery and other procedures were approved by both the UCSD Animal Care and Use Committee (UCSD Animal Use Protocol: S04190M) and local Veteran’s Administration Hospital (Animal Use Protocol: 04-036 (VAH)) prior to the onset of the experiments.

Funding

This work was supported by the grant to XZ from the National Institute of Mental Health MH081037. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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