Traumatic brain injury (TBI) poses a significant global health concern with substantial health and economic consequences. Patients often face significant consequences after injury, notably persistent cognitive changes and an increased risk of developing neurodegenerative disease later in life. Apart from the immediate insult, the resulting inflammatory response can lead to neuroinflammation, oxidative stress, tissue death, and long-term neurodegeneration. Microglia and astrocytes play critical roles in these inflammatory processes, emphasizing the unmet need for targeted therapies. Vaccine formulations consisting of poly (a-ketoglutarate) (paKG) microparticles (MPs) encapsulating PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one) and myelin proteolipid protein (PLP) were developed for prior studies and have demonstrated the production of antigen-specific adaptive T-cell responses in the brain, spleen, and lymph nodes of mice, suggesting that these formulations may be able to prevent neuronal inflammation in mice after TBI. The vaccine efficacy was further evaluated through the image analysis of immunohistochemically stained brain tissue sections from naive, saline, and paKG(PFK15+PLP) MPs or paKG(PFK15) MPs treated mice. Though microglia (Iba1), astrocytes (GFAP) and CD86 were visualized in this method, only Iba1 was found to be significantly reduced in the contralateral hemisphere for paKG(PFK15+PLP) MPs and paKG(PFK15) MPs groups when compared to naive (p=0.0373 and p=0.0186, respectively). However, the naive group also showed an unexpectedly high level of CD86 after thresholding (compared to the TBI groups), indicating flaws were present in the analysis pipeline. Challenges of the image analysis process included thresholding setting optimization, folded tissues, bubbles, and saturated punctate signal. These issues may have impacted data accuracy, underscoring the need for rigorous optimization of experimental techniques and imaging methodologies when evaluating the therapeutic potential of the vaccines in mitigating TBI-induced neuroinflammation. Thus, future analyses should consider microglial morphology and employ more accurate thresholding in FIJI/ImageJ to better measure cellular activation and the overall positive signal.