Glial activation in a mouse model of endometriosis and impact of Di(2-ethylhexyl) phthalate on glial cells, ileum, and colon

Shah, Tauseef Bashir

Permalink

https://hdl.handle.net/2142/117555 Copy

Description

Title

Glial activation in a mouse model of endometriosis and impact of Di(2-ethylhexyl) phthalate on glial cells, ileum, and colon

Author(s)

Shah, Tauseef Bashir

Issue Date

2022-11-22

Director of Research (if dissertation) or Advisor (if thesis)

Nowak, Romana A

Doctoral Committee Chair(s)

Raetzman, Lori T

Committee Member(s)

• Flaws, Jodi A
• Nelson, Erik
• Steelman, Andrew J

Department of Study

Molecular & Integrative Physl

Discipline

Molecular & Integrative Physi

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

Endometriosis, Glial activation, Microglia, Astrocytes, DEHP, Ileum, Colon

Abstract

Endometriosis is a chronic inflammatory disease where endometrial tissues grow on surfaces outside the uterus, negatively impacting the reproductive system. The ectopic lesions are commonly discovered on the pelvic organs and peritoneum, but they can also be found on the ovaries, kidneys, intestines, bladder, skin, brain, and lungs. Endometriosis affects 5- 10% of women of reproductive age. Chronic pelvic pain (CPP) and infertility are observed in 30-50% of women with endometriosis and are the two most common symptoms of endometriosis. Other symptoms of endometriosis include bowel pain, dysuria, dysmenorrhea, and menorrhagia. Due to the chronic nature of the resulting pain, women with endometriosis report a significant decrease in quality of life and other mental health concerns such as anxiety and depression. Increasing evidence suggests that endometriosis affects the central nervous system, as evidenced by animal and human studies. Most of the earlier studies have focused on the changes in the functional activity of the brain, or the effects endometriosis has on neuronal function. Moreover, recent evidence indicates that exposure to environmental toxicants such as endocrine-disrupting chemicals (EDCs) can impact the etiopathology of endometriosis. There is a significant gap in knowledge about the effect of endometriosis on the glial cells in the brain. Gaps in knowledge also exist on how EDCs may affect endometriosis-associated glial activation. In the first study, I explored the astrocytic and microglial morphology changes and the inflammatory markers interleukin-6 (IL6) and tumor necrosis factor (TNF) in different brain regions in mice with endometriosis. I observed an increase in microglial soma size in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls beginning at day 8 after tissue inoculation, indicating microglia activation. The percentage of ionized calcium binding adapter molecule-1 (IBA1) and glial fibrillary acidic protein (GFAP) positive area was increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls at day 16. The number of microglia and astrocytes did not differ between endometriosis and sham control groups. Although IL6 and TNF expression did not differ between treatment groups in specific individual regions of the brain, we saw increased TNF and IL6 expression when expression levels from all regions were combined. To my knowledge, this is the first report of central nervous system-wide glial activation in a mouse model of endometriosis. These results have significant implications for understanding chronic pain associated with endometriosis and other issues, such as anxiety and depression, in women with endometriosis. For the second experiment, I dissected the cell-type-specific transcriptional changes in the brains of mice with induced endometriosis. All the mice that received donor tissue developed endometriotic lesions. I identified 10 cell types distributed into 15 different clusters from the single-cell sequencing data, even though the number of different cell subtypes had to be reduced because of the batch effect from the day of processing. I did not observe a change in the differential expression between the endometriotic and sham mice. Based on recent studies, I determined that cell dissociation using an enzymatic method at 37°C caused microglial activation in the processed tissue. Further evaluation showed an increase in microglia activation markers in both groups. In the next study, I explored the effect of concurrent exposure to intermediate doses of DEHP on the changes in astrocytic and microglial morphology and the inflammatory markers IL6 and TNF in different brain regions in mice with endometriosis. I observed an increase in microglial soma size at specific timepoints after induction in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls. The total microglia and astrocyte area were increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls at day 16. DEHP exposure increased the expression of IBA1 in the hippocampus and hypothalamus on day 32. An increase in GFAP expression was observed in endometriotic mice exposed to DEHP on day 32 but not in mice with endometriosis only. The expression of IL6 and TNF expression did not differ between treatment groups in different regions of the brain. These results indicate that exposure to intermediate doses of DEHP variably affects the endometriosis-associated glial activation in different brain regions. Di(2-ethylhexyl) phthalate (DEHP) is a large-molecular-weight phthalate added to plastics to impart versatile properties. DEHP can be found in medical equipment and devices, food containers, building materials, and children’s toys. Although DEHP exposure occurs most commonly by ingesting contaminated foods in most populations, its effects on the gastrointestinal tract have not been well studied. In the final study, I analyzed the effects of subchronic exposure to DEHP on the ileum and colon morphology, gene expression, and immune microenvironment. Subchronic DEHP exposure in the ileum altered the expression of several immune-mediating factors (Muc1, Lyz1, Cldn1) and cell viability factors (Bcl2, and Aifm1). Similarly, DEHP exposure in the colon impacted the gene expression of factors involved in mediating immune responses (Muc3a, Zo2, Ocln, Il6, and Il17a); and also altered the expression of cell viability factors (Ki67, Bcl2, Cdk4, and Aifm1) as well as specialized epithelial cell marker (Vil1). Immunohistochemical analysis of the ileum showed increased expression of VIL1, CLDN1, and TNF and decreased the number of T-cells in the villi. Histological analysis of the colon showed altered morphology and reduced cell proliferation. Moreover, the expression of MUC2, MUC1, VIL1, CLDN1, and TNF was increased in the colon. The number of T-cells and Th2-cells was also increased in the colon. These data suggest that subchronic DEHP exposure differentially affects the ileum and colon and alters colonic morphology and the intestinal immune microenvironment. These results have important implications for understanding the effects of DEHP on the gastrointestinal system. Collectively, the data from my dissertation indicate that induced endometriosis in a mouse model causes CNS-wide glial activation. Moreover, concurrent DEHP exposure variably affected the microglial activation in different brain regions and caused delayed reactive astrocytosis in the hippocampus. Finally, subchronic DEHP exposure impacts the histomorphology and the immune microenvironment of the ileum and colon in adult female mice, with the colon being more sensitive to DEHP than the ileum.

Graduation Semester

2022-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Tauseef Bashir Shah

Owning Collections