In-vivo corneal confocal microscopy is a powerful imaging technique which provides clinicians and researcher with the capabilities to observe microstructures at the ocular surfaces in significant detail. In this Mini Review, the optics and image analysis methods with the use of corneal confocal microscopy are discussed. While novel insights of neuroanatomy and biology of the eyes, particularly the ocular surface, have been provided by corneal confocal microscopy, some debatable elements observed using this technique remain and these are explored in this Mini Review. Potential improvements in imaging methodology and instrumentation are also suggested.

Ocular surface dysfunction is common in patients receiving anti-cancer drug treatment. The effects of paclitaxel, a neurotoxic chemotherapeutic drug, on ocular surface discomfort associated with dry eye disease was investigated. Patients with cancer who had completed paclitaxel treatment between 3 and 24 months prior to assessment (n = 29) and age- and sex-matched healthy control subjects (n = 29) were recruited and assessed with the Ocular Surface Disease Index (OSDI) to measure ocular surface discomfort. In-vivo corneal confocal microscopy was used to evaluate corneal nerve parameters in the right eye. Peripheral neurotoxicity was assessed using patient-reported outcomes and clinical grading scales. The paclitaxel group had significantly worse OSDI total scores compared with controls (Median, Md = 19.3 and Md = 0, p = 0.007, respectively). Corneal nerve fiber and inferior whorl lengths were reduced in the paclitaxel group compared with controls (14.2 ± 4.0 and 14.4 ± 4.0 mm/mm² vs. 16.4 ± 4.0 and 16.9 ± 4.9 mm/mm², respectively, p = 0.04). When analyzed by presence of peripheral neuropathy, paclitaxel-treated patients with neuropathy showed worse OSDI total scores compared to those without peripheral neuropathy post-treatment (p = 0.001) and healthy controls (p < 0.001). More severe ocular discomfort and worse visual function was associated with greater peripheral neurotoxicity symptoms (r = 0.60, p = 0.001) and neuropathy severity (r = 0.49, p = 0.008), respectively. Patients who have been treated with paclitaxel have a higher risk of ocular surface discomfort associated with dry eye disease, particularly those with peripheral neuropathy. Future longitudinal studies should investigate the clinical impact of corneal nerve reduction in dry eye disease.

Purpose: Dry eye disease (DED) is a growing global health problem with a significant impact on the quality of life of patients. While neurosensory abnormalities have been recognised as a contributor to DED pathophysiology, the potential role of in vivo corneal confocal microscopy in detecting nerve loss or damage remains unclear. This systematic review with meta‐analysis (PROSPERO registered CRD42022381861) investigated whether DED has an impact on sub‐basal corneal nerve parameters. Methods: PubMed, Embase and Web of Science Core Collection databases were searched from inception to 9 December 2022. Studies using laser scanning confocal microscopy to compare corneal nerve parameters of DED with healthy eyes were included. Study selection process and data extraction were performed by two independent members of the review team. Results: Twenty‐two studies with 916 participants with DED and 491 healthy controls were included, with 21 of these studies included in subsequent meta‐analyses. There was a decrease in total corneal nerve length (−3.85 mm/mm ² ; 95% CI −5.16, −2.55), corneal main nerve trunk density (−4.81 number/mm ² ; 95% CI −7.94, −1.68) and corneal nerve branch density (−15.52 number/mm ² ; 95% CI −27.20, −3.84) in DED eyes compared with healthy eyes, with subgroup analysis demonstrating that these differences were more evident in studies using NeuronJ software, a semi‐automated procedure. While this review found evidence of loss of corneal nerve parameters in eyes with DED compared with healthy controls, particularly with the use of a semi‐automated image analysis method, it is evident that there is substantial heterogeneity between studies in terms of corneal nerve imaging methodology. Conclusions: Standardisation is required in terms of terminology and analysis, with more research needed to potentially improve the clinical applicability and practicality of corneal nerve imaging. Further investigation is also required to confirm the diagnostic accuracy of this imaging modality and its potential for monitoring DED treatment efficacy.

Purpose: Sub-basal corneal nerves have been shown to change during neurotoxic chemotherapy treatment. This cross-sectional study investigated corneal nerve morphology in patients who have completed neurotoxic chemotherapy well after treatment cessation and its association with peripheral nerve function. Methods: Central corneal nerve fiber length (CNFL) and inferior whorl length (IWL), average nerve fiber length (ANFL), corneal nerve fiber density (CNFD) and corneal nerve branch density (CNBD), and nerve fiber area (CNFA) were examined using in vivo corneal confocal microscopy in patients with cancer who had completed treatment with either paclitaxel or oxaliplatin between 3 and 24 months prior to assessment in comparison with 2 separate groups of healthy controls. Neurological assessments were conducted including clinician- and patient-reported outcomes, and neurological grading scales. Results: Both paclitaxel- (n = 40) and oxaliplatin-treated (n = 30) groups had reduced IWL and ANFL compared to the respective healthy control groups (n = 15 in each group) (paclitaxel: IWL = P = 0.02, ANFL = P = 0.009; and oxaliplatin: IWL = P = 0.008, ANFL P = 0.02). CNFL and CNFD reduction were observed only in the paclitaxel-treated group compared with healthy controls (P = 0.008 and P = 0.02, respectively), whereas CNFA was reduced in the oxaliplatin-treated group (P = 0.04). IWL reduction correlated with worse fine hand dexterity in chemotherapy-treated patients (r = −0.33, P = 0.007). Conclusions: There is evidence of corneal nerve loss in patients with cancer who have been treated with paclitaxel and oxaliplatin well after treatment cessation associated with worse upper limb function. Translational Relevance: Sub-basal corneal nerve reduction is evident even after cessation of neurotoxic treatment. In vivo corneal confocal microscopy may be useful in the monitoring of nerve function in patients receiving chemotherapy.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy with an increased incidence of sudden death. Evidence of interictal breathing deficits in DS suggests that alterations in subcortical projections to brainstem nuclei may exist, which might be driving comorbidities in DS. The aim of this study was to determine whether a subcortical structure, the bed nucleus of the stria terminalis (BNST) in the extended amygdala, is activated by seizures, exhibits changes in excitability, and expresses any alterations in neurons projecting to a brainstem nucleus associated with respiration, stress response, and homeostasis. Experiments were conducted using F1 mice generated by breeding 129.Scn1a^+/−mice with wild-type C57BL/6J mice. Immunohistochemistry was performed to quantify neuronal c-fosactivation in DS mice after observed spontaneous seizures. Whole-cell patch-clamp and current-clamp electrophysiology recordings were conducted to evaluate changes in intrinsic and synaptic excitability in the BNST. Spontaneous seizures in DS mice significantly enhanced neuronal c-fosexpression in the BNST. Further, the BNST had altered AMPA/NMDA postsynaptic receptor composition and showed changes in spontaneous neurotransmission, with greater excitation and decreased inhibition. BNST to parabrachial nucleus (PBN) projection neurons exhibited intrinsic excitability in wild-type mice, while these projection neurons were hypoexcitable in DS mice. The findings suggest that there is altered excitability in neurons of the BNST, including BNST-to-PBN projection neurons, in DS mice. These alterations could potentially be driving comorbid aspects of DS outside of seizures, including respiratory dysfunction and sudden death.

Immune cell infiltration has been implicated in neurotoxic chemotherapy for cancer treatment. However, our understanding of immune processes is still incomplete and current methods of observing immune cells are time consuming or invasive. Corneal dendritic cells are potent antigen-presenting cells and can be imaged with in-vivo corneal confocal microscopy. Corneal dendritic cell densities and nerve parameters in patients treated with neurotoxic chemotherapy were investigated. Patients treated for cancer with oxaliplatin (n = 39) or paclitaxel (n = 48), 3 to 24 months prior to assessment were recruited along with 40 healthy controls. Immature (ImDC), mature (MDC) and total dendritic cell densities (TotalDC), and corneal nerve parameters were analyzed from in-vivo corneal confocal microscopy images. ImDC was increased in the oxaliplatin group (Median, Md = 22.7 cells/mm²) compared to healthy controls (Md = 10.1 cells/mm², p = 0.001), but not in the paclitaxel group (Md = 10.6 cells/mm²). ImDC was also associated with higher oxaliplatin cumulative dose (r = 0.33, p = 0.04) and treatment cycles (r = 0.40, p = 0.01). There was no significant difference in MDC between the three groups (p > 0.05). Corneal nerve parameters were reduced in both oxaliplatin and paclitaxel groups compared to healthy controls (p < 0.05). There is evidence of elevation of corneal ImDC in oxaliplatin-treated patients. Further investigation is required to explore this potential link through longitudinal studies and animal or laboratory-based immunohistochemical research.