The role of glia and immune cells in trigeminal nerve pain

Stacy is on vacation with her family when she suddenly has a sharp, shooting pain on the right side of her face. The pain is so intense that it stops her in her tracks, and she cannot focus on anything else. Thankfully, it lasts only a few minutes. Unfortunately for her, this would not be the last time she feels such debilitating pain. Stacy is experiencing trigeminal neuralgia (TN), a condition characterized by pain coming from the trigeminal nerve, which starts near the top of the ear and diverges toward the eye, cheek, and jaw. The trigeminal nerve transmits pain, touch, and temperature sensations from your face to your brain. Even though we have two trigeminal nerves total (one for each side of the face), TN pain usually affects only one side. TN is a common and severe type of chronic neuropathic pain, a general term that describes prolonged pain that arises due to disease or injury of the nervous system. Scientists suspect that trigeminal pain arises when blood vessels compress the trigeminal nerve (Figure 1). However, we still lack complete clarity on how TN arises.

Figure 1: Left: Image depicting normal anatomy around the trigeminal root entry zone (TREZ). Right: Apparent vascular compression in the trigeminal root entry zone (TREZ). Adapted from A. M. Kaufmann & M. Patel, Centre for Cranial Nerve Disorders

It so happens that the trigeminal root entry zone (TREZ) has glial cells. Glial cells are non-neuronal cells found in the nervous system. There are different types of glial cells, including astrocytes, microglia, Schwann cells, and oligodendrocytes. Researchers have reported that astrocytes, microglia, and macrophages in the TREZ are activated after compression injury in rats (Luo et al. 2019). We also know that microglia, astrocytes, and peripheral immune cells like macrophages play critical roles in the development and maintenance of neuropathic pain (Takeura et al. 2019; Jha et al. 2019). Macrophages constitute an early peripheral immune response, while microglia dominate the early glial response and contribute to the development of neuropathic pain. Astrocytes, on the other hand, are important for maintenance of chronic pain. However, previous studies did not distinguish between microglia and infiltrating macrophages or between different subtypes of astrocytes involved in pain.   

Lin and his colleagues, therefore, sought to further clarify the categories of microglia, infiltrating macrophages, and astrocytes activated in the TREZ in a rat model of TN using immunohistochemistry and flow cytometry. Immunohistochemistry (IHC) involves selective imaging of cell types in intact tissue (e.g. a brain slice), providing a visual representation of where those cell types  exist. On the other hand, flow cytometry is a technique used to characterize trends in populations of cells removed from the tissue, providing quantitative representations of how many cells exist and how strongly they express certain markers. The authors chose these two methods because they can be used complementarily to differentiate the cells of interest in other contexts (Gieryng et al. 2017; Badie & Schartner 2000). To model chronic compression of the trigeminal nerve in rats, the authors made an incision at the upper edge of the right eye of the rat to expose a branch of the trigeminal nerve. Then, they inserted a small plastic filament to compress the nerve in TN rats. In a separate group of control rats, the authors only surgically exposed the nerve without nerve compression injury. They confirmed development of pain using a facial mechanical stimulation test in which filaments are used to poke the face and the withdrawal response is observed. This test was used because TN can present as hypersensitivity to touch. As expected, the TN rats experienced mechanical hypersensitivity twenty-eight (28) days after surgery, while the control rats did not.

Using IHC, the authors found that, at day 28 post-injury, microglia were back to normal levels and the total number of  astrocytes increased in TN rats. Also, different types of astrocytes were activated in the TREZ in TN rats. Activated/reactive astrocytes can be divided into A1-type toxic astrocytes (labeled with GFAP and C3), and A2-type protective astrocytes (labeled with GFAP and S100A10) (Figure 2). A1 and A2 astrocytes increased twenty-eight days after compression injury. There was a more significant expansion of A1 astrocytes than A2 astrocytes after chronic compression, and the ratio of A1:A2 increased in the TN group compared to the control group. Hence, from IHC, they concluded that trigeminal nerve compression resulted in a greater increase in toxic astrocytes than protective astrocytes. Also, this greater activation of astrocytes than microglia 28 days after injury supports current knowledge that microglia are involved in pain initiation and astrocytes play a more significant role in the persistence of chronic pain.

Figure 2: IHC of A1- and A2-reactive astrocytes in the TREZ on POD 28. (a–c) and (g–i) Reactive astrocytes in the TREZ in the two groups. (d, j) show GFAP/ C3-positive A1 astrocytes in the TREZ. (c, k) show GFAP/S100A10-positive A2 astrocytes in the TREZ. (f, l) show triple positive cells (GFAP, C3 and S100A10). (d1–f1) and (j1–l1) show the higher magnification of (d–f) and (j–l). (a–l). scale bar=100 μm. (d1–f1, j1–l1), scale bar=50 μm.  Adapted from Lin, J. et al. Sci. Rep. 2021

On the contrary, flow cytometry did not show any differences in total astrocytes, A1 astrocytes, and A2 astrocytes between the two groups. Interestingly, the authors also did not observe a difference in microglial activation between TN and control rats twenty-eight days after surgery. The total numbers of microglia in the TREZ and trigeminal ganglia were also not significantly different. However, the ratios of infiltrating lymphocytes and macrophages were significantly higher in TN rats than sham rats. The discovery of an increase in infiltrating lymphocytes and macrophages provides a new perspective into the central-peripheral interactions underlying TN.

This study provided a deeper understanding of glial changes after chronic compression. The contrast between the immunohistochemistry and flow cytometry results for astrocyte activation in this study could result from the isolation techniques used to obtain astrocytes for flow cytometry. Also, it is likely that a single chronic time point was not sufficient to capture the actual behavior of these cells in TN. This highlights the importance of studious experimental design, approaching scientific questions from different angles, using reliable cell markers, and employing rigorous analyses for scientific data. Further studies could resolve the changes in microglia and macrophages and potentially target astrocytes, lymphocytes, and macrophages. In the end, we hope researchers can develop bett­­­­er treatments for TN to benefit patients like Stacy.

Edited by Tamara Chan

REFERENCES

Lin, Junjin, et al. "Flow cytometry analysis of immune and glial cells in a trigeminal neuralgia rat model." Scientific Reports 11.1 (2021): 23569.

Luo, Lin, et al.  “Glial Plasticity in the Trigeminal Root Entry Zone of a Rat Trigeminal Neuralgia Animal Model.” Neurochemical Research 8 (2019):1893-1902.

Takeura, Nakajima, et al. “Role of macrophages and activated microglia in neuropathic pain associated with chronic progressive spinal cord compression.” Scientific Reports 9 (2019): 15656.

Jha, Jo, et al. “Microglia-Astrocyte Crosstalk: An Intimate Molecular Conversation.” The Neuroscientist 25(3) (2019): 227-240.

Gieryng, Pszczolkowska, et al. “Immune microenvironment of experimental rat C6 gliomas resembles human glioblastomas.” Scientific Reports 7 (2017): 17556.

Badie & Schartner. “Flow cytometric characterization of tumor-associated macrophages in experimental gliomas.” Neurosurgery 46 (2000): 957–961.