2023 MSC Poster Contest Presentation: Prrxl1 knockout – a non-invasive model of chronic pain

The 2023 MSC Emerging Science Contest for Early-Career Investigators took place on December 13, 2023. Below is a written summary of one of the presentations from the contest. Read about other presentations from the event in our Early-Career Science Library.

Category: Pain

Runner up: Ezekiel Willerson, PhD student, Queens College, CUNY, US

Title: Prrxl1 knockout – a non-invasive model of chronic pain.

Hypothesis, methodology, findings and conclusions.

The work focuses on non-neuronal components of chronic, neuropathic orofacial pain. We examined changes in microglia and perineuronal nets in a genetic knockout mouse (Prrxl1) we are positing as a model for chronic pain. Microglia, the brain and spinal cord’s resident macrophages, are known to undergo morphological changes in disease/disorder, including in pain. Microglia shift from a “surveillant” state to an “activated” state, where they are reduced in complexity and their cell bodies become larger/rounder. This morphological change is typically accompanied by upregulation of factors related to phagocytosis (e.g., CD68) and inflammation (e.g., IL-6). Perineuronal nets (PNNs) are a form of the extracellular matrix that stabilizes synapses. As with microglia, PNNs are altered in many diseased states. In pain, they have been observed to be degraded and reduced in quantity in affected brain regions.

Prrxl1 is a homeodomain transcription factor necessary for patterning of the trigeminal nuclei during development. Prrxl1 is key to the proper formation of “Barrelettes,” somatotopic representation of a mouse’s whiskers, in the trigeminal nuclei, and knockout (KO) of Prrxl1 disrupts this patterning. Interestingly, disruption is primarily in the trigeminal’s primary sensory nucleus (PrV) but not in the spinal trigeminal nucleus (SpV), which is traditionally thought of as the “pain pathway.” Behaviorally, we have shown that KO results in chronic facial pain. Here, we hypothesize that microglia and PNNs will be affected as in other models of chronic pain. We hypothesize that microglia will appear morphologically “activated,” and that PNNs will be reduced.

To test our hypotheses, we performed immunohistochemistry to stain microglia and PNNs in both our KO mice and in wildtype (WT) controls. Using Neurolucida software, we reconstructed microglia, allowing us to analyze each cell for measures of complexity – process length, branching, and number of nodes. We further measured soma size, number of microglia per region, and the average distance between microglia in each given area. We compared across regions (PrV and SpV) and across genotypes (Prrxl1 KOs and WT). For PNNs, we used luminance to determine the density of nets in each region. We measured the amount of light able to penetrate each stained region, and we contrasted that with neighboring white matter (which contains little to no PNNs). Higher luminance scores indicated lower levels of nets.

We found that microglia trended towards an “activated” morphology in the trigeminal nuclei of Prrxl1 KO mice, when compared with WT controls. On average, they had fewer branches and nodes, as well as lower overall process length, than their WT counterparts. PNNs were also reduced in the PrV of KO mice. Interestingly, we found that few PNNs exist in the SpV across all genotypes (making comparison moot). Taken together, these findings indicate that Prrxl1 KO mice have the anatomical correlates of chronic pain, aligning with our previous findings that these mice exhibit pain behavior.

Existing models of chronic pain, particularly orofacial pain, are limited to invasive procedures such as nerve ligation. These procedures can result in many side effects (e.g., damage to surrounding tissue), as well as inconsistency between procedures. This model will allow us to study glia, PNNs, and other pain factors in a non-invasive way. This research also helps us better understand the role of non-neuronal components of chronic pain, and how they interact. By investigating these specific elements, we hope to expand the list of potential therapeutic targets for the eventual elimination of this issue.

Implications for understanding migraine disease and/or its comorbidities, or how the research holds promise as a new avenue of future migraine study.

The trigeminal system has been implicated in migraine pathology. Here, we investigate chronic orofacial pain tied to changes in trigeminal nuclei (disruption of patterning). Neuroinflammation is also thought to play a key function in migraine development. The role of microglia in inflammation has been well established, with microglial activation linked to upregulation of inflammatory cytokines (tumor necrosis factor and IL-6, among others). Our research aims to characterize changes in microglia in chronic, trigeminal-linked pain, which may help us understand their role in migraine as well.

Ultimately, the goal of this research is to better understand the role of microglia and other non-neuronal factors in chronic orofacial pain, with the objective of expanding the list of potential therapeutic targets for the eventual elimination of this issue. In doing so, it seems likely that we can gain insight into how these factors may be targeted in migraine physiopathology as well.