In vivo analysis of the stratum corneum of Japanese neonates and infants using confocal Raman spectroscopy: a pilot study

ABSTRACT

Background

Physiological skin properties of neonates and infants change drastically after birth and are implicated in the onset of atopic dermatitis and other diseases. Studies have measured physiological skin properties in infants; however, how these properties change over time remains unclear. No reports have measured ceramide in the stratum corneum of infants using confocal Raman spectroscopy; hence, we used it to measure the physiological properties of the skin, including ceramide, in infants.

Materials and methods

The water content and other factors in the skin of infants aged 0, 1, and 6 months were measured. All measurements were performed five times indoors at 22 ± 2°C and 50% ± 10% relative humidity in the middle of the calf at 4‐µm distances, and their mean was calculated.

Results

The water content of the area between the skin surface and superficial layers was the lowest in newborns as compared with other ages, and the deeper the skin layer, the higher the water content. The stratum corneum, evaluated using confocal Raman spectroscopy, was the thickest in newborns and gradually thinned with age. Its water content was the lowest in newborns. The levels of natural moisturizing factor, ceramide, and cholesterol were higher in newborns and tended to decrease with age.

Conclusion

This report is the first to evaluate ceramide in the stratum corneum of infants using confocal Raman spectroscopy and could help in conducting subsequent longitudinal measurements of physiological skin properties in neonates and infants.

1. INTRODUCTION

It is well known that transepidermal water loss (TEWL) and moisturization are implicated in atopic dermatitis (AD) onset. ¹ , ² The amount of ceramide in the stratum corneum (SC), which is important for skin barrier function, has also been implicated in AD. ³ , ⁴ , ⁵ However, how the physiological properties of the skin change over time remains unclear. There are a few reports on the natural moisturizing factor (NMF) and water content in the SC of neonates and infants using confocal Raman spectroscopy (CRS), but no reports on ceramide. ⁶ , ⁷ , ⁸ Identifying the specific SC components involved in AD onset may aid with early intervention in high‐risk patients and efficiently prevent AD by replenishing these components upon depletion.

Many studies have investigated the use of CRS to noninvasively measure more than one corneal component in adults. ⁹ , ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ In comparison with the findings of in vitro studies, there was no difference in the NMF measured using CRS. It has been revealed that the SC is thin, and contains low NMF and ceramide levels due to the presence of a mutation in the filaggrin gene. Furthermore, studies showed that patients with AD had less water, NMF, and ceramide content in the SC, as well as a reduced lipid‐to‐protein ratio, which was critical for the effective maintenance of the skin barrier function. ⁹ , ¹⁰ , ¹¹ , ¹² It was also reported that the SC water content was lower in infants below 2 weeks of age than in older infants; meanwhile, the NMF content, excluding lactate, was higher in newborns. ¹⁵ , ¹⁶ However, no longitudinal study has used CRS to measure physiological skin properties, including ceramide, from neonates to infants to examine their association with diseases due to the difficulty of using CRS among neonates and infants. Before conducting longitudinal evaluations of neonate skin to investigate the relationship between skin physiological properties, including ceramide, and diseases such as AD, we conducted a study to validate the results of CRS and SC hydration measurements during the neonatal and infancy periods and assess ceramide in the SC using CRS.

2. METHODS

Thirty‐six infants, aged 0 (1–7days old), 1 (± 14 days), and 6 months (± 2 months), were recruited. The water content, levels of NMF (defined here as the sum of pyrrolidone carboxylic acid, arginine, ornithine, citrulline, serine, proline, glycine, histidine, and alanine), total ceramide with ceramide structure and cholesterol, and SC thickness were estimated using CRS (Model 3510; River Diagnostics BV, Rotterdam, the Netherlands). Two lasers operating at excitation wavelengths of 785 and 671 nm were used to detect spectra from 400 to 1800 cm⁻¹ (fingerprint region) and 2500 to 4000 cm⁻¹ (high wavenumber region), respectively. In addition, the open‐chamber Tewameter TM300 and the capacitive Corneometer CM825 were attached to a Multi Probe Adapter (MPA5; Courage & Khazaka) and used to measure TEWL and SC hydration. ¹⁷ Measurements were performed indoors at a temperature and humidity level of 22 ± 2°C and 50% ± 10%, respectively. In CRS, measurements were repeatedly performed on the skin around the middle of the calf of each participant at 4‐µm distances, and the average value was calculated for each depth and used as the representative value. For the 0–20 µm area under the curve (AUC), the average value of repeated measurements was also used as the representative value. If measurements could not be repeated up to five times owing to strong crying and resistance from the participants, the mean of the obtained measurements was used. TEWL and SC hydration measurements were performed on the calf, similar to CRS. Measurements using a Tewameter were performed once, and measurements using a Corneometer were performed until data were obtained five times, with the mean as a representative value. The use of moisturizers on the measurement site was prohibited on the day of the measurement. This study was approved by the Ethics Review Committee of the National Center for Child Health and Care Research (approval number: 1962) and was conducted after obtaining consent from the participant's family members.

3. RESULTS

Using CRS, five participants were measured at 0 and 1 month, five at 0, 1, and 6 months, two at 1 and 6 months, and seven at 6 months. The ages were 0 months: 3.6 ± 0.8 days, 1 month: 34.8 ± 12.1 days and 6 months: 148.5 ± 27.7 days (mean ± SD) (Table 1). All 0‐month participants had no experience with bathing which meant dry care. Figure 1 presents a graph of the SC water content in the middle of the calf, as well as the levels of NMF, ceramide, and cholesterol in the SC for each age group calculated from the CRS results. The water content of the area between the skin surface and superficial layers was low, and the deeper the skin layer, the higher the water content. In particular, the water content in the area between the skin's surface and superficial layers was the lowest at 0 months (3.6 ± 0.8 days) when compared to other ages.

TABLE 1.

Patient enrollment

Item	0 Months N = 10	1 Month N = 12	6 Months N = 14
Age (days), mean (SD)	3.6 (0.8)	34.8 (12.1)	148.5 (27.7)
Gestational age (weeks), median [IQR]	39.0 [38.0−40.8]	39.0 [38.0−40.2]	38.5 [38.0−39.0]
Sex male, n (%)	5 (50.0%)	6 (50.0%)	7 (50.0%)
Height (cm), mean (SD)	50.0 (1.6)	53.8 (3.4)	62.2 (1.9)
Body weight (g), mean (SD)	3134.8 (260.3)	4448.3 (1203.5)	6886.5 (779.9)
Status of a history of diagnosis of atopic dermatitis, n (%)	0 (0.0%)	0 (0.0%)	2 (14.3%)
A history of diagnosis of allergic diseases other than atopic dermatitis *Present, n (%)	0 (0.0%)	0 (0.0%)	0 (0.0%)
A family history of atopic dermatitis, n (%)	5 (50.0%)	5 (41.7%)	6 (42.9%)
A family history of allergic diseases other than atopic dermatitis*, n (%)	9 (90.0%)	10 (83.3%)	12 (85.7%)
Use of topical moisturizer, n (%)	6 (60.0%)	10 (83.3%)	14 (100.0%)

Note: Allergic diseases other than atopic dermatitis.

^*Bronchial asthma, allergic rhinitis, allergic conjunctivitis, pollen allergy, chronic urticaria, and food allergy.

FIGURE 1.

The depth profile (mean ± SD) of water (A), natural moisturizing factor (NMF) (B), ceramide (C), and cholesterol (D) for each age group was obtained by confocal Raman spectroscopy (CRS) (0 months: 3.6 ± 0.8 days, 1 month: 34.8 ± 12.1 days, 6 months: 148.5 ± 27.7 days)

Figure 2 presents the estimated SC thickness, AUC values for water content, and levels of NMF, ceramide, and cholesterol. The SC, which was evaluated using CRS, was the thickest at 0 months (3.6 ± 0.8 days) and gradually thinned with age; meanwhile, the SC water content was lowest at 0 months (3.6 ± 0.8 days). The NMF, ceramide, and cholesterol levels were higher at 0 months (3.6 ± 0.8 days) and tended to decrease with age. The TEWL and SC hydration are shown in Figure 3. They were lowest at 0 months (3.6 ± 0.8 days), and both tended to gradually increase with age. The means and 95% confidence interval (CI) for each age group are shown in Tables S1 and S2.

FIGURE 2.

The area under the curve (AUC) (0–20 µm) of water (A), natural moisturizing factor (NMF) (B), ceramide (C), cholesterol (D), and stratum corneum (SC) thickness (E) for each age group, were obtained by confocal Raman microspectroscopy (0 months: 3.6 ± 0.8 days, 1 month: 34.8 ± 12.1 days, 6 months: 148.5 ± 27.7 days)

FIGURE 3.

The results of transepidermal water loss (TEWL)(A) and stratum corneum (SC) hydration (B) for each age group (0 months: 3.6 ± 0.8 days, 1 month: 34.8 ± 12.1 days, 6 months: 148.5 ± 27.7 days)

4. DISCUSSION

Based on CRS measurements, SC tended to be the thickest in newborns and thinned gradually over the first 6 months of life, which was similar to that reported by Miyauchi et al. ¹⁸ The thickness of the SC in newborns was 21.8 µm, which differs from the 9.3 µm of Holbrook et al. and the 6.25 µm of Fluhr et al. who also measured neonates. ¹⁹ , ²⁰ The reason for this difference is unknown but may depend on differences in the sites of the body, methods for measurement, and bathing experience. The levels of NMF, ceramide, and cholesterol in the SC tended to be higher in newborns, and the NMF observed in this study were similar to those reported by Fluhr et al. ¹⁶ TEWL was lowest in neonates as suggested by Stamatas et al. ²¹ These results indicated that NMF, ceramide, and cholesterol levels were related to the skin barrier function. Since all neonates received dry care, the SC that is removed by bathing may have remained, resulting in a thicker SC and increased NMF, ceramide, and cholesterol. In addition, there may have been more ceramide and cholesterol in the newborns’ skin due to the presence of vernix caseosa. ²² Furthermore, since activation of filaggrin degradation is dependent on a decrease in external water activity, ²³ it is possible that low SC water content in newborns is related to high NMF. Figure 1A suggests that the SC of neonates is less moist than that of other age groups, and Figure 3B SC hydration reflects this result.

It was reported that the total level of SC ceramide in lesional AD skin was significantly lower than that in normal skin of healthy individuals, which was sampled using the tape‐stripping method. ³ While tape stripping is useful and minimally invasive for sampling SC components, CRS is a non‐invasive and highly sensitive optical means that can measure SC components to depth. Therefore, CRS is more suitable for measuring SC components in neonates, who are more sensitive than adults. ²⁴

There are limitations to our study. Filaggrin mutation testing was not performed in this study but should be performed in the future. In adults, an association between filaggrin and NMF has been reported. ²⁵ This study did not perform sample size or power calculations because the number of cases was determined by feasibility. In addition, the data from neonate participants might be affected by vernix caseosa due to dry care, and CRS cannot measure each ceramide class, which is reported to be associated with barrier functions. However, to the best of our knowledge, this is the first report of CRS, which is a non‐invasive tool to measure the level of ceramide in the SC of infants and can potentially help in investigating changes in the physiological properties of the skin of infants.

FUNDING

Grants‐in‐Aid for Scientific Research, Grant number: JP18K16073 from Japan Society for the Promotion of Science; research funds from Pigeon Home Products Corporation

ETHICS STATEMENT

This study was approved by the Ethics Review Committee of the National Center for Child Health and Care Research (approval number: 1962) and was conducted after obtaining consent from the participant's family members.

Supporting information

Supporting Information

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.