Discussion
The primary findings in the present research are as follows: (1) ALFF signals in the supplemental somatosensory area, secondary auditory cortex, agranular insular cortex, temporal association areas, and ectorhinal cortex brain areas were enhanced in the DE group. (2) Insular cortex showed upregulated ALFF and played a dominant role in chronic corneal pain. (3) Cyclotraxin-B injection in insular cortex markedly blocked chronic corneal pain by inhibiting the upregulation of inflammatory cytokines.
Recent research suggested that some dry eye patients described different responses to the same treatment, implying different pathophysiological mechanisms between patients with and without chronic corneal pain9, 10. It is well known that persistent peripheral ocular nerve damage and inflammation might cause structures and functional changes in the CNS11. However, the evidence of the central sensitization mechanism underlying chronic corneal pain is still scarce.
In this study, we applied ALFF by rs-fMRI to map the functional brain associated with chronic corneal pain and distinguish spontaneous cerebral neuro-activities. ALFF has been used to evaluate functional abnormalities of brain in some pain-related diseases such as discogenic low-back and leg pain, migraine, phantom limb pain, and trigeminal neuralgia12-14. However, there’s only few rs-fMRI alterations research about acute corneal pain in DED. Yan. H. et al. revealed ReHo values alterations in the limbic-cortical circuits in patients with DED15. Zhi-Ming Pan et al. showed higher ALFF values in parahippocampal gyri and caudate in acute eye pain patients 16. In our present study, the results demonstrated increase ALFF in S2, GU, VISC, AUDp, AuV, AlD, AlP, TeA, PERI, Ect, OLF, HIP, RHP, CTXsp, STR brain areas. We detected increased ALFF in S2. This is different from the previous case reported which uncovered noxious acute stimulation of the cornea could produce somatotopic activation in primary somatosensory cortex17. This may be due to the difference in acute and chronic noxious stimulation to peripheral corneal nerves.
ALFF analysis also revealed some brain areas involved in anxiety. Many studies demonstrated that DE patients experienced increased anxiety, chronic pain can aggravate emotional disorders such as anxiety and depression18. In current study, ALFF showed significant changes in temporal areas in DE mice. The temporal cortex has been reported involved in anxiety and could record the physical impact of negative emotions 19,20. Moreover, we also noticed an increase ALFF in the primary auditory area, which contributes to anxiety disorders21.
C-Fos expression was also used to verify the ALFF changed brain areas and find abnormal neuronal activity brain areas. C-Fos expression has previously been found to have significant upregulation in frontal cortex, the third ventricle, fewer in hippocampal area and the spinal nucleus of the trigeminal nerve in the medulla after menthol-stimulated rabbits, cornea22,23. In line with the rs-fMRI results, the upregulated mount of c-Fos verified the abnormal neuronal activity mapping especially in S2, AID, AIP, AuV, TeA, Ect in chronic corneal pain mice.
Then, we found a significant correlation between ALFF values of the insular cortex and eye blinking times after chemical application. This result is in line with the documented role of the insular cortex in trigeminal neuropathic pain, which showed insular cortex representation of dynamic mechanical allodynia24. The insular cortex receives afferents from sensory thalamic nuclei involved in widely different functions, such as pain perception and speech production, to the processing of social emotions25.
Experimental results in the present study showed mRNA expression of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, in addition to BDNF, increased, reflecting an underlying over-active state related to altered neuron function. The present result is also consistent with previous research that pro-inflammatory cytokines, neuronal and microglial markers were upregulated in the trigeminal brainstem sensory complex of DED animals4. BDNF, crucial in synaptic transmission and neuronal plasticity, has been defined as a neurotransmitter and neuromodulator26, and the BDNF-TrkB signaling pathway could mediate inflammatory responses27. BDNF could affect central sensitization by activation of NMDA receptor28. Remarkably, it has been reported that BDNF enhances the excitability of the small-diameter TRG neurons projecting onto the Vi/Vc after trigeminal inflammatory hyperalgesia29. So, we speculated an essential role of BDNF-derived neuroinflammation changes in the central mechanism of chronic corneal pain. The local inhibition of TrkB in the insular cortex of DE mice suppressed chronic pain and increased inflammation factors significantly.
The present research is the first attempt to apply ALFF in chronic corneal pain, but a few issues need to be declared. Our study mainly concentrated on one major brain area, other brain regions associated with chronic corneal pain have not been further confirmed. Besides Trk-B, the different type of BDNF receptors, such as the p75 neurotrophin receptor, and the signaling mechanism involved may also be established in the future.