Analysis of the Effects of Light Emitting Diode (LED) Phototherapy on the Hematological and Biochemical Parameters of Rabbits Using Repeated Measures Longitudinal ANOVA

Dita Amelia; Suliyanto Suliyanto; Anisah Nabilah Ghasani; Nike Meliana Rahmawati; Dwi Syarifatun Nisya’; Dinda Rahma Alya

doi:10.20956/j.v22i3.49375

Authors

Dita Amelia Airlangga University
Suliyanto Suliyanto
Anisah Nabilah Ghasani
Nike Meliana Rahmawati
Dwi Syarifatun Nisya’
Dinda Rahma Alya

DOI:

https://doi.org/10.20956/j.v22i3.49375

Keywords:

LED Photography, Hematological, Biochemical, Repeated Measures ANOVA, Rabbit Model

Abstract

This study examined the longitudinal effects of Light Emitting Diode (LED) phototherapy on hematological (hemoglobin) and biochemical (creatinine) parameters in rabbits (Oryctolagus cuniculus). Although LED phototherapy is widely applied as a non-invasive treatment, its systemic effects in repeated-measure settings remain limited. Fourteen rabbits were randomly assigned to a control group (n = 7) and a treatment group (n = 7). The treatment group received two 12-hour LED phototherapy sessions on consecutive days, while the control group underwent identical conditions without LED activation. Hemoglobin and creatinine levels were measured at three time points and analyzed using Repeated Measures ANOVA, with assumption testing for normality, homogeneity, and sphericity; Greenhouse–Geisser correction was applied when necessary. The results showed that observation time significantly affected hemoglobin (p = 0.004967) and creatinine levels (p = 0.03577), indicating temporal physiological changes that occurred regardless of treatment exposure. However, no significant differences were observed between the control and treatment groups for hemoglobin (p = 0.936) or creatinine (p = 0.357), and no significant group–time interaction was detected, suggesting that the observed changes were independent of LED phototherapy. Post-hoc pairwise comparisons based on estimated marginal means (EMMs) with Tukey adjustment revealed a significant increase in hemoglobin from time 0 to time 1 and a significant difference in creatinine between time 1 and time 2, further supporting that these variations reflect natural physiological processes rather than treatment-induced effects. These findings indicate a lack of evidence of treatment effect under the studied conditions, although the relatively small sample size warrants cautious interpretation. Future studies with larger samples are recommended.

References

[1] Adewale, A. O., Alagbe, J. O., & Adeoye, A. G., 2021. Dietary Supplementation of Rauvolfia Vomitoria Root extract as a Phytogenic Feed additive in Growing Rabbit diets : Haematology and Serum Biochemical Indices. International Journal on Orange Technologies, 3(3), 31–42.

[2] Bhutani, V. K., Wong, R. J., Turkewitz, D., Rauch, D. A., Mowitz, M. E., & Barfield, W. D., 2024. Phototherapy to prevent severe neonatal hyperbilirubinemia in the newborn infant 35 or more weeks of gestation: technical report. Pediatrics, 154(3).

[3] Blanca, M. J., Arnau, J., García-Castro, F. J., Alarcón, R., & Bono, R., 2023. Repeated measures ANOVA and adjusted F-tests when sphericity is violated: Which procedure is best? Frontiers in Psychology, 14, 1–11.

[4] Fan, J., Chen, Y., Yan, H., Niimi, M., Wang, Y., & Liang, J., 2018. Principles and applications of rabbit models for atherosclerosis research. Journal of Atherosclerosis and Thrombosis, 25(3), 213–220.

[5] Glass, G. E., 2021. Photobiomodulation: The clinical applications of low-level light therapy. Aesthetic Surgery Journal, 705–714.

[6] Hamblin, M. R., 2016. Photobiomodulation or low-level laser therapy. Journal of Biophotonics, 9(11-12), 1122-1124.

[7] Hassan, K. M., 2021. Applied repeated measures design in medical field. Journal of Kurdistani for Strategic Studies, 11.

[8] Hernández-Bule, M. L., Naharro-Rodríguez, J., Bacci, S., & Fernández-Guarino, M., 2024. Unlocking the power of light on the skin: a comprehensive review on photobiomodulation. International Journal of Molecular Sciences, 25(8), 4483.

[9] Kassie, A., Berta, D. M., Yalew, A., Sitotaw, C., Workineh, M., & Woldu, B., 2025. Hematological parameter changes and associated factors in neonates with hyperbilirubinemia treated with phototherapy. BMC Pediatrics, 25(1), 115.

[10] Langenberg, B., Helm, J. L., Günther, T., & Mayer, A., 2023. Understanding, testing, and relaxing sphericity of repeated measures ANOVA with manifest and latent variables using SEM. Methodology, 19(1), 60–95.

[11] Maghfour, J., 2024. Photobiomodulation CME part I: Overview and mechanism. Journal of the American Academy of Dermatology, 90(3), 565–572.

[12] Midway, S. R., & White, J. W., 2025. Testing for normality in regression models: mistakes abound (but may not matter). Royal Society Open Science.

[13] Moskvin, S. V., & Kozlov, V. A., 2021. The main mechanisms of action of photobiomodulation. Lasers in Medical Science, 36(10), 1967–1975.

[14] Muhammad, L. N., 2023. Guidelines for repeated measures statistical analysis approaches with basic science research considerations. The Journal of Clinical Investigation, 133(11).

[15] Pan, W. T., Liu, P. M., Ma, D., & Yang, J. J. (2023). Advances in photobiomodulation for cognitive improvement by near-infrared derived multiple strategies. Journal of Translational Medicine, 21(1), 135.

[16] Park, S.-R., Han, Y., Lee, S. J., & Lee, K.-I., 2023. Efficacy of Low-Level Laser Therapy in a Rabbit Model of Rhinosinusitis. International Journal of Molecular Sciences, 24(1), 760.

[17] Pessini, P. G. D. S., Knox de Souza, P. R., Chagas, C. D. S., Sampaio, E. G., Neves, D. S., Petri, G., Fonseca, F. L. A., & da Silva, E. B., 2020. Hematological reference values and animal welfare parameters of BALB/C‐FMABC (Mus musculus) inoculated with Ehrlich tumor kept in the vivarium at ABC Medical School. Animal Models and Experimental Medicine, 3(1), 32–39.

[18] Zhou, Y., Zhu, Y., & Wong, W. K., 2023. Statistical tests for homogeneity of variance for clinical trials and recommendations. Contemporary Clinical Trials Communications, 33.