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Effect of systemic inflammation on the repair of damaged brains.

초록/요약

In this study, I examined how systemic inflammation affects the repair of brain injury. To this end, I created a brain-injury model by stereotaxic injection of ATP, a component of damage-associated molecular patterns (DAMPs), into the striatum of mice. Systemic inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS-ip). The first part of my study aimed to evaluate the impact of LPS-ip treatment on brain cells, as well as on circulating blood monocytes that infiltrate into the injured brain, and their role in the repair process. An analysis of magnetic resonance images showed that LPS-ip reduced the initial brain injury but slowed injury repair. An immunostaining analysis using the neuronal marker, NeuN, showed that LPS-ip delayed removal of dead/dying neurons, despite the fact that LPS-ip enhanced infiltration of monocytes, which serve to phagocytize dead cells/debris. Notably, infiltrating monocytes showed a widely scattered distribution. Bulk RNAseq analyses showed that LPS-ip decreased expression of genes associated with phagocytosis, with PCR and immunostaining of injured brains confirming reduced levels of Cd68 and Clec7a, markers of phagocytic activity, in monocytes. Collectively, these results suggest that systemic inflammation affects properties of blood monocytes as well as brain cells, resulting in delay in clearing damaged cells and activating repair processes. In the second part of the study, I explored the impact of systemic inflammation on initial brain injury and repair processes, including neurite extension and synapse formation. Bulk RNA-sequencing (RNA-seq) analyses and immunostaining for microtubule-associated protein 2 (MAP2) and tyrosine hydroxylase (TH) showed that LPS-ip led to a reduction in initial brain injury, but inhibited neurite extension into the damaged brain. LPS-ip upregulated expression of defense response genes and anti-apoptotic genes, but decreased expression of genes associated with repair and regeneration. In addition, LPS-ip reduced levels of vGlut1 and PSD95 (markers for excitatory pre and post synapses, respectively), but had little effect on vGAT and gephyrin (markers for inhibitory pre and post synapses, respectively). Taken together, these findings suggest that systemic inflammation reduces initial damage but impedes subsequent repair process.

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목차

I. INTRODUCTION 1
A. Overview of the neuronal and non-neuronal cells in the injured brain 1
1. Neurons 1
2. Astrocytes 2
3. Microglia 4
4. Monocytes 5
B. Major events in the injured brain 6
1. Cell death 6
2. Clearance of dead cells/Phagocytosis 7
3. Repair and inflammation 8


C. Effect of systemic inflammation on injured brains 9
1. Effect of systemic inflammation on blood and brain cells 9
2. Systemic inflammation and brain injury 11
3. Systemic inflammation and repair of injured brain 12

D. Aims of the study 14

II. MATERIALS AND METHODS 16

1. Animals and LPS-ip injection 16
2. Stereotaxic injection 16
3. Magnetic Resonance Imaging (MRI) 17
4. Tissue Preparation 18
5. Immunostaining 18
6. Isolation of peritoneal macrophages and peripheral blood mononuclear cells (PBMCs) 19
7. Total protein extraction and Western blotting 20
8. Quantitative real-time polymerase chain reaction (QPCR) and Reverse Transcriptase PCR (RT-PCR) 21
9. RNA sequencing and data analysis 22
10. Statistical analysis 23
III. RESULTS 26
A. Systemic inflammation attenuates the repair of damaged brains through reduced phagocytic activity of monocytes infiltrating brain. 26
1. Transcriptomic analysis of injured brains of mice with and without systemic inflammation 27
2. LPS-ip reduces initial brain damage but delays removal of dead neurons and repair of injured brains. 33
3. Systemic inflammation increases monocytes infiltration with scattered distribution in the injured brain 39
4. Systemic inflammation decreases phagocytic efficiency of monocytes in the injured brain 47

B. Systemic inflammation decreases initial brain injury but attenuates neurite extension and synapse formation in injured brains 53

1. Systemic inflammation reduces initial brain injury and increases the expression of defense and anti-apoptosis genes 53
2. LPS-ip significantly altered gene expression related to the repair and attenuates neurites regeneration 60
3. LPS-ip attenuated synapse formation in the injured brain. 62


IV. DISCUSSION 71
A. Systemic inflammation attenuates the repair of damaged brains through reduced phagocytic activity of monocytes infiltrating brain. 71

B. Systemic inflammation decreases initial brain injury but attenuates neurite extension and synapse formation in injured brains. 74


V. SUMMARY AND CONCLUSION 79
REFERENCES 81

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