Chronic dry eye induced corneal hypersensitivity, neuroinflammatory responses, and synaptic plasticity in the mouse trigeminal brainstem

Abstract

BackgroundDry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation, pain, and nerve abnormalities. We studied the peripheral and central neuroinflammatory responses that occur during persistent DED using molecular, cellular, behavioral, and electrophysiological approaches.MethodsA mouse model of DED was obtained by unilateral excision of the extraorbital lachrymal gland (ELG) and Harderian gland (HG) of adult female C57BL/6 mice. In vivo tests were conducted at 7, 14, and 21 days (d) after surgery. Tear production was measured by a phenol red test and corneal alterations and inflammation were assessed by fluorescein staining and in vivo confocal microscopy. Corneal nerve morphology was evaluated by nerve staining. Mechanical corneal sensitivity was monitored using von Frey filaments. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous corneal nerve activity. RT-qPCR and immunostaining were used to determine RNA and protein levels at d21.ResultsWe observed a marked reduction of tear production and the development of corneal inflammation at d7, d14, and d21 post-surgery in DED animals. Chronic DE induced a reduction of intraepithelial corneal nerve terminals. Behavioral and electrophysiological studies showed that the DED animals developed time-dependent mechanical corneal hypersensitivity accompanied by increased spontaneous ciliary nerve fiber electrical activity. Consistent with these findings, DED mice exhibited central presynaptic plasticity, demonstrated by a higher Piccolo immunoreactivity in the ipsilateral trigeminal brainstem sensory complex (TBSC). At d21 post-surgery, mRNA levels of pro-inflammatory (IL-6 and IL-1β), astrocyte (GFAP), and oxidative (iNOS2 and NOX4) markers increased significantly in the ipsilateral trigeminal ganglion (TG). This correlated with an increase in Iba1, GFAP, and ATF3 immunostaining in the ipsilateral TG of DED animals. Furthermore, pro-inflammatory cytokines (IL-6, TNFα, IL-1β, and CCL2), iNOS2, neuronal (ATF3 and FOS), and microglial (CD68 and Itgam) markers were also upregulated in the TBSC of DED animals at d21, along with increased immunoreactivity against GFAP and Iba1.ConclusionsOverall, these data highlight peripheral sensitization and neuroinflammatory responses that participate in the development and maintenance of dry eye-related pain. This model may be useful to identify new analgesic molecules to alleviate ocular pain.

Morphological and Functional Changes of Corneal Nerves and Their Contribution to Peripheral and Central Sensory Abnormalities

“…In line with these observations, a severe DED induced by the excision of extraorbital lachrymal and Harderian glands in mice provoked corneal mechanical allodynia and corneal inflammation associated with an increase in the ongoing ciliary nerve fiber electrical activity compared to control mice (Fakih et al, 2019) (Figure 2). In addition, during photorefractive keratectomy, another corneal injury condition, functional and morphologic alterations in mechanical, polymodal, and cold sensory nerve fibers of the cornea have been noted, which can lead to postoperative pain (Bech et al, 2018).…”

Lacrimal gland excision in male and female mice causes ocular pain and anxiety-like behaviors

“…In a previous study in rabbits palpebral opening was used to quantify eye closure after application of capsaicin 33 . A modification of this method was used recently to measure ongoing pain in female C57BL/6 mice after excision of the extraorbital lacrimal and harderian gland 8 . Similar to our findings, Fakih et al 8 reported a decrease in eye closure ratio after lacrimal and harderian gland excision.…”

“…A modification of this method was used recently to measure ongoing pain in female C57BL/6 mice after excision of the extraorbital lacrimal and harderian gland 8 . Similar to our findings, Fakih et al 8 reported a decrease in eye closure ratio after lacrimal and harderian gland excision. www.nature.com/scientificreports/ Less severe dry eye, induced in the present study by extraorbital LGE, caused a reduction in palpebral opening only in female mice, which is consistent with the greater corneal damage observed in female compared to male mice 2-weeks after single LGE 10 .…”

“…Aqueous tear deficiency, a form of dry eye caused by insufficient tear production from the lacrimal glands, has been modeled in the rodent through excision or functional disruption of the lacrimal glands [8][9][10][11][12][13][14] . Lacrimal gland excision (LGE) has been demonstrated to sensitize corneal polymodal nociceptive neurons to the TRPV1 agonist capsaicin and low pH induced by CO 2 , as well as sensitize corneal cold cells to the TRPM8 agonist menthol 11,[15][16][17] .…”

Expression Profiles of CircRNA and mRNA in Lacrimal Glands of AQP5–/– Mice With Primary Dry Eye

“…Previous studies have used animal models of dry eye by administering drugs to inhibit the secretion of tears or by removing the lacrimal or Harderian glands of mice (Gilbard et al, 1987;Viau et al, 2008;Fakih et al, 2019). However, these methods are too complex and cumbersome, the efficiency of the models is not stable, and the damage to the mice is greater.…”