Dr. Neha Shilpy, S18084 , Dr. Rohit Shetty, Dr. Rashmi
Deshmukh, Dr. Swaminathan Sethu
Introduction:
In vivo confocal microscopy (IVCM) is a useful tool to image the cornea at a cellular level both in ophthalmic clinical practice and research. IVCM is used to study corneal diseases such as ectasias, keratitis, Dry eye disease (DED) and dystrophies [1]. Corneal nerves, epithelial cells, keratocytes, endothelial cells and immune cells have been demonstrated on IVCM in different ocular and systemic diseases [2]. Changes in corneal nerve morphology have been reported in keratoconus [3] and dry eye including those associated with systemic conditions.
DED is one of the common disorders of the eye with an estimated prevalence of 5.5% – 33.7% worldwide [4]. The hallmarks of DED include discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. There is a lack of substantial understanding with relevance to the etiopathology of the symptoms and their association with other in vivo clinical findings. The source of ocular discomfort or pain in DED cannot solely be explained by tear film metrics suggesting the role of other factors in causation of symptoms. Pain associated with dry eye has been described as neuropathic pain [5] and there have been emerging reports regarding dysfunctional ocular somatosensory nerves including the sub-basal nerve plexus in ocular pain [6].
Keratoconus is characterized by stromal thinning and protrusion of the cornea. Bilateral disease is more common, however, the frequency of unilateral disease has been reported to range from 14.3% to 41% [7]. Unilateral KC provides insight into disease pathogenesis as the unaffected fellow eye acts as an ideal control for the affected one with other contributing factors constant for both.Corneal nerves are known to regulate multiple pathways, which play a role in KC disease pathogenesis [8]. In-vivo confocal microscopy (IVCM) studies have demonstrated changes in the Sub-basal nerve plexus (SBNP) of patients suffering from KC, however it’s role in Unilateral KC has not been described so far [9].
Purpose:
The purpose of our study is to analyse the changes in sub-basal nerve plexus (SBNP) using confocal microscopy in healthy and diseased cornea.
Materials and methods:
This was a cross-sectional study with two arms. First arm had 52 evaporative dry eye (EDE) patients (Group1) and 43 normal individuals (Group2). In the second arm we had 30 eyes of healthy patients (Group 3) and 33 patients with unilateral keratoconus with 33 normal eyes (Group 4) and 33 affected eyes (Group 5). The study was approved by the Ethics Committee of Narayana Nethralaya Hospital. Informed consent of study subjects was obtained at the time of enrollment. A thorough medical history was elicited to rule out any other ocular and systemic co-morbidity. Detail examination was done for all that included visual acuity, refraction, slit-lamp and fundus evaluation and DED investigations (tear film break-up time, Schirmer’s test and meibography). Patient ocular pain or discomfort was graded using ocular surface disease index (OSDI) questionnaire. Based on OSDI scores, the severity of symptoms can be grouped as normal (OSDI score 0-12), mild (OSDI score 13-22), moderate (OSDI score 23-32), or severe (OSDI score 33-100) [28]. Exclusion criteria include the use of contact lenses, the presence of drug allergy or ocular or systemic diseases with ocular manifestations such as Sjogren’s syndrome, rheumatoid arthritis and diabetes mellitus.
In vivo confocal microscopy
IVCM imaging was performed using Rostock Corneal Module/Heidelberg Retina Tomograph ll (RCM/HRT ll; Heidelberg Engineering GmBH, Dossenheim, Germany).[7] The device uses a diode laser of 670 nm wavelength. 0.5% proparacaine drops were used to anaesthetize the cornea before the procedure. The central cornea was scanned in a single area at a desired depth. A drop of 0.5% moxifloxacin was instilled after the procedure.
Corneal sub-basal nerve plexus and dendritic cell density assessments
An experienced masked observer selected five representative IVCM frames for corneal sub-basal nerves and dendritic cells image based analyses. Quantitative analyses of the nerve fibers were used performed using Automatic CCMetrics software, Ver. 1.0 (University of Manchester, UK). The parameters quantified include corneal nerve fiber density (CNFD), the total number of major nerves per square millimeter; nerve fiber length (CNFL), the total length of all nerve fibers and branches (millimeters per square millimeter); nerve branch density (CNBD), number of branches emanating from major nerve trunks per square millimeter, total branch density (CTBD) the total number of branch points per square millimeter; the nerve fiber area (CNFA) and the total nerve fiber area per square millimeter and the nerve fiber width (CNFW) the average nerve fiber width per square millimeter [9,10].
Results:
A significant decrease in nerve features such as fiber length, width, branch points, number of major nerves and branches was observed in EDE patients as compared to controls (Table 1). In the second arm, significant reduction in corneal nerve fiber density and length was observed in Group 5 compared to Group 3 and Group 4 (Table 2).
Conclusion:
SBNP changes play a role in the symptomatology of EDE and based on the severity of EDE, the nerve features also change. Also, IVCM can be used as an imaging marker for early diagnosis of keratoconus.
Discussion:
IVCM is a unique tool that can give us an idea of the corneal architecture. The study of sub basal nerve plexus can help in the early diagnosis and appropriate management of various corneal disorders like EDE and keratoconus.
The management of patients with dry eye poses a challenge many a times, especially when the symptoms do not correlate with the findings. Our study showed that the analysis of corneal nerve features could give an insight into the disease pathogenesis. Castillo et al. showed a significant decrease in the nerve density in patients with dry eye [11] and that is similar to what we have observed in the current study. In our current study we have also observed a significant decrease in various nerve features in EDE patients with moderate to severe symptoms. Thus suggesting the use of corneal nerve morphological features as a predictor for the presence of pain in EDE patients.
This study demonstrates that changes in the SBNP occur early in keratoconus. Quantitative changes might, therefore help in establishing a diagnosis of KC even before it is manifested clinically or topographically. SBNP changes can also help in monitoring disease progression [12].
Table 1: Sub basal nerve plexus feature differences based on severity of symptoms in EDE patients
| EDE(OSDI<23) | EDE(OSDI>23) | ||
| Sub basal nerve plexus | Mean + SEM | Mean + SEM | P-value |
| CNFL (length in mm/mm2) | 16.9 + 0.3 | 15.9 + -0.4 | 0.0165∗ |
| CNFD (major nerves/mm2) | 27.9 +-0.7 | 26.2 + -1.0 | 0.0447∗ |
| CNFW (average nerve fiber width/mm2) | 0.0210 + 0.0001 | 0.0211 + 0.0001 | 0.6107∗ |
| CTBD (branch points/mm2) | 61.5 + -3.5 | 50.8 + -3.4 | 0.0398∗ |
| CNBD (number of branches/mm2) | 42.7 + -2.3 | 34.3 + -2.6 | 0.0208∗ |
| CNFA (total nerve fiber area/mm2) | 0.0071 + -0.0002 | 0.0064 + -0.0002 | 0.4098∗ |
Table 2: Sub basal nerve plexus feature differences between normal individuals and the two eyes of patients with unilateral keratoconus
| Nerve Parameters | Group 1 (n=30) | Group 2 (n=33) | Group 3 (n=33) | Group 1 vs 2 | Group 1 vs 3 | Group 2 vs 3 |
| CNFD | 30.51 + 5.81 | 28.48 + 23.82 | 23.82 + 8.02 | 0.26 | <0.001 | 0.01 |
| CNBD | 43.45 + 15.29 | 34.7 + 16.1 | 37.61 + 19.82 | 0.05 | 0.02 | 0.51 |
| CNFL | 17.59 + 3.16 | 16.6 + 2.42 | 14.82 + 3.61 | 0.2 | 0.001 | 0.02 |
| CTBD | 59.73 + 20.75 | 49.05 + 25.3 | 54.66 + 27.15 | 0.09 | 0.41 | 0.36 |
| CNFA | 0.01 + 0.002 | 0.006 + 0.001 | 0.006 + 0.002 | 0.13 | 0.06 | 0.68 |
| CNFW | 0.02 + 0.001 | 0.021 + 0002 | 0.02 + 0.002 | 0.29 | 0.13 | 0.63 |
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