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Corneal nerve loss in children with type 1 diabetes mellitus without retinopathy or microalbuminuria

Abstract Aims/Introduction Corneal confocal microscopy is a rapid, non‐invasive ophthalmic technique to identify subclinical neuropathy. The aim of this study was to quantify corneal nerve morphology in children with type 1 diabetes mellitus compared with age‐matched healthy controls using corneal confocal microscopy. Materials and Methods A total of 20 participants with type 1 diabetes mellitus (age 14 ± 2 years, diabetes duration 4.08 ± 2.91 years, glycated hemoglobin 9.3 ± 2.1%) without retinopathy or microalbuminuria and 20 healthy controls were recruited from outpatient clinics. Corneal confocal microscopy was undertaken, and corneal nerve fiber density (n/mm2), corneal nerve branch density (n/mm2), corneal nerve fiber length (mm/mm2), corneal nerve fiber tortuosity and inferior whorl length (mm/mm2) were quantified manually. Results Corneal nerve fiber density (22.73 ± 8.84 vs 32.92 ± 8.59; P < 0.001), corneal nerve branch density (26.19 ± 14.64 vs 47.34 ± 20.01; P < 0.001), corneal nerve fiber length (13.26 ± 4.06 vs 19.52 ± 4.54; P < 0.001) and inferior whorl length (15.50 ± 5.48 vs 23.42 ± 3.94; P < 0.0001) were significantly lower, whereas corneal nerve fiber tortuosity (14.88 ± 5.28 vs 13.52 ± 3.01; P = 0.323) did not differ between children with type 1 diabetes mellitus and controls. Glycated hemoglobin correlated with corneal nerve fiber tortuosity (P < 0.006) and aspartate aminotransferase correlated with corneal nerve fiber density (P = 0.039), corneal nerve branch density (P = 0.003) and corneal nerve fiber length (P = 0.037). Conclusion Corneal confocal microscopy identifies significant subclinical corneal nerve loss, especially in the inferior whorl of children with type 1 diabetes mellitus without retinopathy or microalbuminuria.


INTRODUCTION
Type 1 diabetes mellitus affects over half a million children worldwide 1,2 . Diabetes is associated with chronic microvascular complications in adults, which increase morbidity and all-cause mortality 3 . Diabetes is the main cause of distal symmetric polyneuropathy (DSPN) [4][5][6] . Adults with DSPN present with a combination of symptoms, such as numbness, pain and tingling in the feet 7 . The American Diabetes Association endorses screening for DSPN at diagnosis of type 2 diabetes, 5 years after the diagnosis of type 1 diabetes and annually thereafter 8 . Children and adolescents with type 1 diabetes rarely complain of neuropathic symptoms. However, a study of children with type 1 diabetes showed reduced motor and sensory nerve conduction velocities (24%), and at least one neuropathic symptom (60%) or sign (58%) 9 . In another study, symptomatic neuropathy was present in 13.5% of patients, whereas 22.5% of patients had neurophysiological evidence of neuropathy 10 and 18% had impaired vibrotactile sense 11 . Furthermore, in one study, 36% of patients had more than two abnormal autonomic function tests, and 18.8% had severe autonomic neuropathy 12 . In a prospective study, abnormal nerve conduction velocity was found in 31.6% at baseline, which increased to 63.2% after 5 years 13 . In another study, over a period of 10 years, the prevalence of clinical neuropathy increased from 6.5% to 16.1%, whereas nerve conduction velocity abnormalities increased from 17.7% to 46.8% 14 . Although neurophysiological assessments are highly sensitive, they are not easily carried out in children 15 . Vibration perception threshold and tactile perception threshold tests are easy to carry out, but lack sensitivity for the early detection of DSPN 16 . There is a need for non-invasive sensitive screening tools for the early detection of neuropathy in children with diabetes.
Corneal confocal microscopy (CCM) is a rapid, non-invasive and well-tolerated technique to detect and quantify neuropathy in adults with type 1 diabetes [17][18][19][20][21][22][23][24] . An early study found no significant changes in CCM parameters among children with   Raynaud's phenomenon), previous corneal trauma or systemic disease that affects the cornea, surgery and a history of or current contact lens wear were excluded from the study. All participants provided assent and parental informed consent. The research adhered to the tenets of the Declaration of Helsinki, and was approved by Sidra Medicine and the Weill Cornell Medicine Research Ethics Committee.

Image selection and quantification
Six central sub-basal nerve plexus images were selected from the central cornea and corneal nerve fiber density (CNFD; n/mm 2 ), corneal nerve branch density (CNBD; n/mm 2 ), corneal nerve fiber length (CNFL; mm/mm 2 ) and corneal nerve fiber tortuosity (CNFT) were quantified using manual CCMetrics (The University of Manchester, Manchester, UK). Six images centered on the inferior whorl and adjacent areas (upper right/left corner and lower right/left corners) were selected, and the inferior whorl length (IWL) (mm/mm 2 ) was quantified utilizing the manual CNFL mode in CCMetrics (Figure 1) 26 . The investigator was blind to the study group when carrying out CCM and analyzing CCM images.

Statistical analysis
All statistical analyses were carried out using IBM SPSS Statistics software version 26 (IBM Corporation, Armonk, NY, USA), and P < 0.05 was considered statistically significant. Normally distributed data were expressed as the meanstandard deviation, and the means were compared using an independent sample t-test. Pearson's correlation was undertaken to investigate the association between clinical parameters and corneal nerve fiber parameters. GraphPad Prism version 8 (La Jolla, CA, USA) was used to build the plots.

RESULTS
A total of 20 participants with type 1 diabetes and 20 healthy controls underwent CCM. Participants with type 1 diabetes were slightly older (P < 0.02) and taller (P < 0.02), but had comparable weight and body mass index (BMI). They also had a lower aspartate aminotransferase (AST; P < 0.02), but comparable bilirubin and alanine aminotransferase (Table 1). Just four (20%) of the patients met the American Diabetes Association criteria (aged >10 years and >5 years of diabetes) to undergo screening for microvascular complications. Eight (40.0%) underwent assessment for retinopathy, and 11 (55.0%) underwent assessment for microalbuminuria, of whom none had retinopathy or microalbuminuria.

DISCUSSION
In the present study, there was evidence of significant corneal nerve loss in children with type 1 diabetes without retinopathy or microalbuminuria. It is critical to detect and prevent nerve damage at the earliest stage of diabetic neuropathy, as improvement in glycemic control and other risk factors, such as obesity, hypertension and dyslipidemia, might prevent nerve degeneration and promote nerve regeneration 27,28 .
Previous studies of adults with type 1 diabetes have found a significant reduction in central CNFD, CNBD and CNFL compared with healthy controls 18,[29][30][31][32][33][34][35] and in type 1 diabetes patients without retinopathy or microalbuminuria 34 . Corneal nerve loss has good diagnostic utility for both diabetic somatic and autonomic neuropathy 22 . Furthermore, a lower CNFL is associated with the development of clinical diabetic neuropathy 31,36,37 , and a more rapid reduction in CNFL predicts the development and progression of diabetic neuropathy 38 . Significant improvements in CNFD, CNBD and CNFL have been observed in type 1 diabetes patients after simultaneous pancreas and kidney transplantation 39,40 , omega-3 supplementation 41 , and an improvement in multiple risk factors for diabetic neuropathy 42 .
In the present study, a significant reduction in central corneal nerve fiber parameters in young children with type 1 diabetes has been shown, which is comparable to a previous study of children and young adolescents with type 1 diabetes 25 . Established risk factors for diabetic neuropathy, such as age, height, glycated hemoglobin and BMI, were not associated with the reduction in corneal nerve parameters, consistent with previous findings in adults with type 1 diabetes 35,43 . CNFT was not altered, in contrast with a study of adults with diabetes where nerve tortuosity was higher 44 . A reduction in corneal nerves occurs, regardless of diabetes duration, in young patients with type 1 diabetes 35 and adults with type 2 diabetes 45 .
The American Diabetes Association has recommended initial screening for albuminuria and retinopathy in patients with type 1 diabetes aged >10 years, after 3-5 years of diabetes 46 . Although 20% of this cohort fulfilled the criteria for screening, none had microalbuminuria or retinopathy. Indeed, the significant corneal nerve loss in these children with type 1 diabetes without retinopathy or microalbuminuria agrees with previous findings in adults with type 1 diabetes 25,34 , and supports the thesis that neuropathy might precede retinopathy 47 . It also argues for earlier screening of diabetic neuropathy in children with type 1 diabetes using CCM. AST was lower in the present cohort with type 1 diabetes, and correlated with CNFD, CNBD and CNFL. No relationship between AST and CCM has been observed in studies in adults with diabetes 21,22,30,48 . Although the association between BMI and elevated AST is well established as a marker for liver injury in obese adults [49][50][51][52] , in the present study, AST was inversely correlated with BMI.
The inferior whorl is distal to the central nerves, and might allow the identification of earlier nerve damage 26,53 . Studies of adults with type 1 diabetes and type 2 diabetes have shown a greater reduction in IWL 48,54 , especially in those with painful diabetic neuropathy 55,56 . This is the first study of children with type 1 diabetes showing a marked reduction in IWL, with 50%  having a reduction greater than two standard deviations lower than the mean in controls.
A limitation of the current study was the cross-sectional design, relatively small number of participants studied and the lack of additional measures of diabetic neuropathy. Prospective studies are required to assess progression of corneal nerve abnormalities in relation to other complications and risk factors for diabetic neuropathy.
Significant corneal nerve loss has been shown in the central cornea and inferior whorl indicative of neuropathy in children with type 1 diabetes without microalbuminuria or retinopathy. This suggests that CCM could be used to screen for early subclinical neuropathy and to assess disease progression in children with type 1 diabetes.

DISCLOSURE
The authors declare no conflict of interest.