In Vivo confocal microscopy evaluation of corneal layer alterations in patients with trigeminal neuralgia

Introduction

Trigeminal neuralgia (TN) is a chronic neuropathic pain disorder characterized by sudden, severe, and recurrent episodes of facial pain affecting one or more divisions of the trigeminal nerve. ¹ Although the classical pathophysiological concept of TN has emphasized central mechanisms such as neurovascular compression and central sensitization, increasing evidence suggests that peripheral nerve degeneration and neuroinflammatory processes also contribute significantly to disease onset and persistence.^2,3

Peripheral involvement of trigeminal nerve fibers may result in altered sensory input, impaired neurotrophic support, and dysregulated immune–neural interactions, which together may perpetuate chronic pain. ⁴ The cornea is innervated primarily by the ophthalmic division (V1) of the trigeminal nerve. Trigeminal afferent fibers enter the cornea through the long ciliary nerves and form the subbasal nerve plexus, which plays an essential role in corneal sensation, epithelial trophism, wound healing, and neuroimmune regulation.^5–7 This anatomical relationship makes the cornea a useful surrogate for evaluating peripheral trigeminal small fiber integrity.

Objective assessment of peripheral nerve pathology in TN, however, remains challenging in routine clinical practice. The cornea represents a unique model for evaluating peripheral trigeminal nerve integrity, as it is one of the most densely innervated tissues in the human body and receives its sensory innervation primarily from the ophthalmic branch of the trigeminal nerve.^5–7 Corneal nerves are essential not only for sensory perception but also for epithelial maintenance, wound healing, and immune homeostasis.^8,9 Disruption of corneal innervation has been associated with epithelial instability, inflammation, and stromal remodeling. ¹⁰

In vivo confocal microscopy (IVCM) is a non-invasive imaging modality that allows real-time visualization of corneal layers at a cellular level, including corneal nerves, immune cells, and stromal components. ¹¹ Previous studies have demonstrated corneal nerve alterations in systemic neuropathies and neurodegenerative disorders.^12–14 However, comprehensive, layer-by-layer evaluation of corneal involvement in trigeminal neuralgia remains limited.

The aim of this study was to evaluate microstructural alterations across all corneal layers from epithelium to endothelium in patients with trigeminal neuralgia using IVCM and to compare these findings with healthy controls.

Materials and methods

Results

Subbasal nerve plexus

Quantitative analysis of the subbasal nerve plexus demonstrated significant reductions in all nerve parameters in trigeminal neuralgia affected eyes compared with healthy controls (Figure 1). CNFD was lower in affected eyes than in controls (13.4 ± 1.8 vs. 21.8 ± 2.2 fibers/mm², p < 0.001). Contralateral eyes showed intermediate CNFD values (18.2 ± 2.0 fibers/mm²), with borderline significance compared with controls before correction for multiple comparisons. However, these differences did not remain statistically significant after Bonferroni correction. CNFL was also lower in affected eyes than in controls (9.1 ± 1.2 vs. 15.0 ± 1.5 mm/mm², p < 0.001). Similarly, contralateral eyes demonstrated intermediate CNFL values (11.8 ± 1.3 mm/mm²), showing borderline uncorrected significance compared with controls; however, statistical significance was not maintained after Bonferroni correction.(Figure 2) Similarly, CNBD was lower in affected eyes than in controls (21.2 ± 1.7 vs. 34.1 ± 1.9 branches/mm², p < 0.001). Contralateral eyes also demonstrated intermediate CNBD values (26.5 ± 1.6 branches/mm²), although these differences did not remain statistically significant after correction for multiple comparisons. Effect size analysis demonstrated large differences between affected eyes and controls for all subbasal nerve parameters, with Cohen's d values of 1.42 for CNFD, 1.51 for CNFL, and 1.47 for CNBD (Figure 3). In addition, a significant positive association was observed between CNFL and CNFD across the pooled dataset (Figure 4). Representative in vivo confocal microscopy images illustrating subbasal nerve loss and increased dendritic cell density in trigeminal neuralgia are shown in Figure 5.

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Figure 1.

Composite bar graph of subbasal nerve Plexus parameters. Composite bar graph comparing corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), and corneal nerve branch density (CNBD) among trigeminal neuralgia affected eyes, unaffected eyes, and healthy controls. Three group statistical comparisons were performed using ANOVA or Kruskal-Wallis tests with post-hoc multiple comparison correction. Bars represent mean values with standard error of the mean. Statistical significance between groups is indicated by brackets.

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Figure 2.

Violin plot showing distribution of corneal nerve fiber length (CNFL). Violin plot illustrating the distribution of corneal nerve fiber length (CNFL) in trigeminal neuralgia affected eyes, unaffected eyes, and healthy controls. The width of each violin represents kernel density, the central line denotes the median, and overlaid jittered points represent individual measurements, highlighting within-group variability.

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Figure 3.

Forest plot of effect sizes for subbasal nerve parameters. Forest plot displaying effect sizes (Cohen's d) with 95% confidence intervals for corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), and corneal nerve branch density (CNBD), comparing trigeminal neuralgia affected eyes with healthy controls. Negative effect sizes indicate lower parameter values in affected eyes.

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Figure 4.

Scatter plot demonstrating the association between CNFL and CNFD. Scatter plot showing the association between corneal nerve fiber length (CNFL) and corneal nerve fiber density (CNFD) across the pooled study population. Each point represents an individual eye, and the solid line represents the fitted linear regression.

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Figure 5.

In vivo confocal microscopy images of the subbasal nerve plexus obtained from study participants. Images acquired from healthy control eyes (left panels) show a dense, continuous, and well-organized subbasal nerve plexus, characterized by long, uniformly thick, and predominantly parallel-running nerve fibers with minimal tortuosity and preserved nerve continuity. In contrast, images obtained from patients with trigeminal neuralgia (right panels) demonstrate marked disruption of subbasal nerve architecture, including pronounced reduction in corneal nerve fiber density and length, increased nerve tortuosity, focal nerve fragmentation, and areas of nerve dropout. Numerous hyperreflective dendritic cells with activated morphology are also evident in the subbasal region, indicating an associated inflammatory microenvironment. These qualitative in vivo findings directly support and visually corroborate the quantitative reductions in corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), and corneal nerve branch density (CNBD) observed in Figures 1–4. Scale bar = 50 µm.

Quantitative subbasal nerve parameters for all groups are summarized in Table 1.

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Table 1.

Quantitative subbasal nerve plexus parameters in trigeminal neuralgia affected eyes, contralateral eyes, and healthy controls.

Parameter	TN affected	TN unaffected	Controls	P value
CNFD (fibers/mm2)	13.4 ± 1.8	18.2 ± 2.0	21.8 ± 2.2	<0.001
CNFL (mm/mm2)	9.1 ± 1.2	11.8 ± 1.3	15.0 ± 1.5	<0.001
CNBD (branches/mm2)	21.2 ± 1.7	26.5 ± 1.6	34.1 ± 1.9	<0.001

Data are presented as mean ± standard deviation. Group comparisons were performed using one-way ANOVA or Kruskal–Wallis tests with Bonferroni-adjusted post-hoc analyses, as appropriate. CNFD: corneal nerve fiber density; CNFL: corneal nerve fiber length; CNBD: corneal nerve branch density.

Descemet's membrane and endothelium

No significant abnormalities were detected in Descemet's membrane. Endothelial cell density, hexagonality, and pleomorphism were comparable between TN patients and controls (p > 0.05).

Table 2 provides a structured summary of the frequency of qualitative corneal alterations across different corneal layers, supporting the descriptive findings observed above and demonstrating a higher prevalence of epithelial irregularity, Bowman's layer disorganization, nerve tortuosity, nerve fragmentation, dendritic cell activation, and keratocyte activation in trigeminal neuralgia patients compared with controls.

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Table 2.

Frequency (%) of qualitative corneal morphological findings in trigeminal neuralgia and control groups.

Parameter	Controls (n = 60)	TN (n = 60)	P-value
Irregular epithelium	8 (%13.3)	24 (%40.0)	0.003
Bowman's layer disorganization	6 (%10.0)	22 (%36.7)	0.002
Increased nerve tortuosity	10 (%16.7)	42 (%70.0)	<0.001
Nerve fragmentation	5 (%8.3)	38 (%63.3)	<0.001
Activated dendritic cells	12 (%20.0)	45 (%75.0)	<0.001
Keratocyte activation	9 (%15.0)	30 (%50.0)	0.001
Posterior stromal changes	4 (%6.7)	6 (%10.0)	0.52
Endothelial changes	3 (%5.0)	5 (%8.3)	0.47

Data are presented as the number and percentage of eyes exhibiting each qualitative feature, including activated dendritic cell morphology, rather than quantitative dendritic cell density measurements. Statistical comparisons were performed using chi-square or Fisher's exact test, as appropriate.

Discussion

This study demonstrates that trigeminal neuralgia (TN) is associated with significant multilayer corneal microstructural alterations detectable by in vivo confocal corneal microscopy (IVCM), supporting the concept that TN is not solely a central neuropathic pain disorder but also involves progressive peripheral trigeminal small fiber pathology accompanied by neuroinflammatory activation. These findings are consistent with mechanistic models suggesting that demyelination and ectopic impulse generation at the trigeminal root entry zone may lead to distal axonal degeneration involving peripheral sensory terminals, including corneal nerves. ¹⁶

Our results demonstrate selective multilayer corneal involvement, predominantly affecting the epithelium, Bowman's layer, subbasal nerve plexus, and anterior stroma, while deeper corneal layers such as the posterior stroma, Descemet's membrane, and endothelium remain largely preserved. This distribution pattern strongly supports a nerve-dependent pathogenic mechanism rather than diffuse corneal degeneration. Previous histological and confocal investigations have established that corneal subbasal nerves and the limbal nerve plexus originate from trigeminal afferent fibers, providing anatomical continuity between the ophthalmic branch of the trigeminal nerve and corneal neural architecture.^5,6

Degeneration of the subbasal nerve plexus represents the most prominent structural abnormality observed in TN. Significant reductions in corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), and corneal nerve branch density (CNBD), accompanied by large effect sizes, indicate a distinct neurodegenerative signature detectable by IVCM. The magnitude of corneal nerve loss observed in this study was substantial, with approximately 38% reduction in CNFD, 39% reduction in CNFL, and 38% reduction in CNBD in affected eyes compared with controls, indicating pronounced small fiber degeneration. These findings are supported by prior studies demonstrating that corneal confocal nerve parameters are sensitive and reproducible biomarkers of peripheral small fiber neuropathy in systemic neuropathic disorders, including diabetic neuropathy.^12,13 Additionally, morphological abnormalities such as increased nerve tortuosity, fragmentation, focal thinning, and localized nerve loss further suggest an active and progressive neurodegenerative process rather than static nerve damage.

The marked lateralization of corneal nerve alterations to the eyes ipsilateral to the symptomatic trigeminal nerve further supports the functional relationship between trigeminal sensory pathways and corneal innervation. Contralateral eyes demonstrated intermediate corneal nerve parameter values between affected eyes and healthy controls. However, these differences did not remain statistically significant after Bonferroni correction, suggesting that the predominant pathological involvement remains localized to the clinically affected trigeminal side. Nevertheless, the presence of intermediate contralateral values may indicate subtle bilateral neurophysiological changes or early central sensitization mechanisms, which have previously been described in chronic neuropathic pain disorders. To further clarify the interpretation of contralateral eye findings, additional three-group statistical comparisons were performed between affected eyes, contralateral eyes, and healthy controls. While contralateral eyes demonstrated intermediate corneal nerve parameter values, these differences did not reach statistical significance after correction for multiple comparisons. Therefore, although subtle bilateral alterations may suggest early subclinical involvement or central sensitization mechanisms, our findings primarily support localized peripheral neurodegeneration in the symptomatic trigeminal distribution. This cautious interpretation strengthens the biological plausibility of our results while avoiding overestimation of bilateral disease involvement. Because corneal innervation is derived exclusively from the ophthalmic division (V1) of the trigeminal nerve, the observed corneal changes primarily reflect involvement of this branch, even in patients with clinically predominant maxillary (V2) or mandibular (V3) symptoms.Beyond structural neurodegeneration, our findings demonstrate a prominent neuroimmune response at the corneal level in TN patients. Increased dendritic cell density and activated cellular morphology indicate an inflammatory microenvironment associated with trigeminal nerve injury. Previous studies have also demonstrated corneal nerve loss in trigeminal neuralgia without significant differences in dendritic cell density in small patient cohorts, suggesting that inflammatory responses may vary depending on disease stage. ¹⁵ Dendritic cells are key mediators of neuroimmune signaling and play a central role in regulating inflammatory cascades and peripheral sensitization following nerve injury. ¹⁷ In the present study, descriptive evaluation suggested close spatial association and occasional apparent contact between dendritic cells and subbasal nerve fibers. Although not quantitatively assessed, these findings are in line with previous reports and further support the presence of active neuroimmune crosstalk at the corneal level. Increasing evidence indicates that neuroimmune interactions among neurons, immune cells, and glial cells play a critical role in the development and maintenance of neuropathic pain.^18–22 Experimental and clinical studies have demonstrated that chronic peripheral axonal injury may alter immune cell activity and stromal cell behavior through sustained neuroinflammatory signaling, potentially creating a self-perpetuating cycle of inflammation and neurodegeneration.

Anterior stromal involvement observed in patients with longer disease duration provides further evidence supporting progressive corneal tissue remodeling in TN. Increased keratocyte reflectivity and cellular activation have previously been associated with chronic inflammation, oxidative stress, and dysregulation of neurotrophic signaling pathways essential for corneal homeostasis. ²³ The preservation of posterior stromal and endothelial structures reinforces the selective nature of TN-associated corneal alterations and supports the hypothesis that peripheral trigeminal nerve pathology is the primary driver of secondary stromal changes.

A major contribution of this study is the demonstration of disease duration-dependent corneal alterations. Correlation and regression analyses revealed that prolonged disease duration is associated with worsening corneal nerve degeneration and increased inflammatory cell density, suggesting that peripheral trigeminal neuropathy in TN represents a progressive biological process. Emerging evidence indicates that delayed diagnosis and prolonged disease duration may result in irreversible peripheral nerve remodeling characterized by reduced regenerative capacity and altered neuroimmune balance. Furthermore, corneal confocal microscopy studies in chronic neuropathic pain conditions have shown that prolonged nerve injury is associated with increased immune cell infiltration and stromal remodeling.^24–26 To the best of our knowledge, this study represents one of the first comprehensive demonstrations of disease duration-dependent multilayer corneal microstructural alterations in trigeminal neuralgia using in vivo confocal microscopy.

Receiver operating characteristic analysis in this study identified CNFL as a high-performance parameter for distinguishing disease chronicity, highlighting its potential clinical utility as an objective biomarker for disease staging and longitudinal monitoring. These ROC findings should nevertheless be interpreted cautiously given the subgroup-based exploratory design and require validation in larger prospective cohorts. The identification of reliable imaging biomarkers capable of quantifying peripheral trigeminal involvement is of substantial translational importance, as current TN evaluation primarily relies on clinical symptom assessment and neuroimaging findings, which may not adequately reflect peripheral nerve integrity.

Epithelial alterations observed in TN patients likely reflect impaired neurotrophic support resulting from trigeminal sensory dysfunction. Corneal sensory nerves play a critical role in epithelial maintenance through the release of neuropeptides and growth factors essential for epithelial stability and regeneration. ⁸ Reduced innervation may therefore predispose to epithelial instability and altered cellular turnover. Similarly, structural disorganization of Bowman's layer may represent secondary changes related to chronic epithelial stress and altered corneal biomechanics. ²⁷

From a clinical and translational perspective, the high interobserver agreement values obtained for corneal nerve measurements confirm the reliability and reproducibility of IVCM as a diagnostic modality. The non-invasive nature of IVCM, combined with its ability to directly visualize peripheral trigeminal nerve terminals, makes it a promising tool for evaluating peripheral nerve involvement, monitoring disease progression, and assessing treatment response. Integration of corneal confocal microscopy into clinical practice may facilitate earlier detection of peripheral neuropathy and improve patient stratification for targeted therapeutic interventions.

The strengths of this study include comprehensive layer-by-layer corneal evaluation, combined quantitative and qualitative morphological analysis, and inclusion of a well-defined cohort of unilateral TN patients with matched controls, which enhances the internal validity and biological plausibility of our findings.

This study has several limitations. First, the cross-sectional design precludes causal inference. Nevertheless, the strong correlations between disease duration and corneal nerve parameters as well as dendritic cell density support the presence of a progressive peripheral neuropathic process in trigeminal neuralgia. Second, the relatively small sample size may limit generalizability; however, the large effect sizes and high measurement reproducibility indicate robust and methodologically sound findings. In particular, the subgroup distribution for ROC analysis was not fully balanced, which may have affected the stability of AUC estimates. Additionally, specific neuropathic features such as microneuromas and bead-like axonal swellings were not systematically quantified, which may have provided further insight into neuropathic structural alterations. Although dendritic cells were descriptively characterized based on morphology and their relationship with nerve fibers, quantitative subclassification was not performed, which may have provided additional insight into neuroimmune interactions. Furthermore, clinical pain severity scores and corneal sensitivity measurements were not included, limiting correlation between structural corneal changes and functional sensory impairment. Neurovascular conflict status was not systematically analyzed, and therefore potential differences in corneal findings according to neurovascular compression could not be evaluated. In addition, medication status was not systematically analyzed in the present study. Because many patients with trigeminal neuralgia receive antineuropathic treatments such as carbamazepine or oxcarbazepine, potential treatment-related effects on corneal nerve morphology cannot be completely excluded. Third, the lack of pre- and post-treatment comparisons restricts evaluation of the potential role of in vivo confocal microscopy in monitoring treatment response. Future longitudinal studies are warranted to assess the effects of medical and surgical interventions on corneal nerve regeneration and neuroinflammatory changes. Finally, the analysis was limited to the ophthalmic branch of the trigeminal nerve, and peripheral extensions of the maxillary and mandibular branches were not directly assessed. Nonetheless, the cornea and limbal region provide a unique and accessible window for evaluating peripheral trigeminal small fiber integrity.

In conclusion, trigeminal neuralgia is associated with significant corneal neurodegeneration and neuroinflammatory activation, primarily affecting anterior corneal layers and the subbasal nerve plexus. These findings support the concept that TN involves progressive peripheral trigeminal small fiber neuropathy. In vivo confocal corneal microscopy emerges as a sensitive, reproducible, and clinically valuable imaging modality capable of detecting these alterations. The strong association between disease duration and corneal microstructural deterioration highlights the importance of early diagnosis and timely therapeutic intervention. Future prospective and interventional studies are warranted to determine whether medical or surgical treatments can reverse or stabilize peripheral nerve alterations and to evaluate the role of IVCM-derived biomarkers in guiding individualized therapeutic strategies and improving long-term clinical outcomes.

Clinical implications