Effects of different lighting regimens on the ultrastructure of rat pinealocytes

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Abstract

BACKGROUND: The morphofunctional state of the pinealocytes is closely related to the lighting regimen. Light deprivation is known to lead to increased metabolic activity of these cells and to ultrastructural changes. In contrast, darkness deprivation is associated with decreased pinealocyte activity and reduced numbers of mitochondria and lipid inclusions. Studying the effect of various lighting regimens on the ultrastructure of rat pinealocytes provides deeper insight into the mechanisms underlying pineal gland adaptation to environmental changes and the regulation of circadian rhythms and may be relevant for developing approaches to correcting biorhythm disorders.

AIM: To study the ultrastructure of the pineal gland in Wistar rats under constant light and under a light regimen that models a "one day on, two days off" shift work schedule.

METHODS: The study was conducted on 120 male Wistar rats aged 5 months. The animals were divided into three equal groups (n = 40 per group) based on the light regimen: control group was kept under a fixed light/dark (LD) regimen (10 h light / 14 h dark); experimental group I under constant light (LL); and experimental group II under a light regimen simulating a shift work schedule (1 day of LL followed by 2 days of LD). The experiment lasted 21 days; euthanasia for pineal gland harvesting was performed on day 22 at four time points (9:00 AM, 3:00 PM, 9:00 PM, 3:00 AM). Structural and ultrastructural evaluation of pinealocytes was performed on pineal gland sections using light and transmission electron microscopy. Micromorphometric analysis included measuring the size of pinealocytes and their ultrastructural components. Statistical analysis was performed using GraphPad Prism v8.41 (GraphPad Software, USA).

RESULTS: Constant light leads to vacuolization of pinealocytes, destructive changes in lipid inclusions, the Golgi complex, and the endoplasmic reticulum, the formation of autophagosomes of various sizes, mitochondrial polymorphism, and changes in nuclear structure, including the nuclear envelope. The presence of necrotic cells, perivascular edema, and leukocyte infiltration was also observed. The most pronounced effect of the shift regimen on pinealocytes is changes in mitochondrial ultrastructure, accompanied by relative structural and functional preservation of the endoplasmic reticulum and Golgi complex.

CONCLUSION: Thus, both constant light and the "one day on, two days off" shift regimen are associated with pronounced ultrastructural rearrangements in rat pinealocytes. Constant light causes deep disintegration of the pinealocyte ultrastructure, characterized by oxidative stress, suppression of transcriptional and metabolic activity, mitochondrial dysfunction, and cell death. The alternating lighting regimen causes profound disturbances in the nuclear and mitochondrial apparatus of pinealocytes, likely due to desynchronization of circadian rhythms and dysregulated apoptosis.

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About the authors

Anna I. Anurkina

Petrovsky National Research Centre of Surgery

Author for correspondence.
Email: anyaaai1925@gmail.com
ORCID iD: 0009-0003-0011-1114
SPIN-code: 9812-3412
Russian Federation, Moscow

Maria A. Kozlova

Petrovsky National Research Centre of Surgery

Email: kma-morph@mail.ru
ORCID iD: 0000-0001-6251-2560
SPIN-code: 5647-1372

Cand. Sci. (Biology)

Russian Federation, Moscow

Valery P. Chernikov

Petrovsky National Research Centre of Surgery

Email: 1200555@mail.ru
ORCID iD: 0000-0002-3253-6729
SPIN-code: 3125-7837

MD, Cand. Sci. (Medicine)

Russian Federation, Moscow

David A. Areshidze

Petrovsky National Research Centre of Surgery

Email: labcelpat@mail.ru
ORCID iD: 0000-0003-3006-6281
SPIN-code: 4348-6781

Cand. Sci. (Biology)

Russian Federation, Moscow

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2. Fig. 1. The sequence of sample selection and the stages of the study.

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3. Fig. 2. Histological sections of the pineal gland of rats kept under different light regimes: a — control group, fixed light regime (light/dark — 10/14 hours); b — Experimental group I, dark deprivation (constant illumination); c — Experimental group II, light regime simulating shift work (1 day — round-the-clock illumination, 2 days — light regime similar to that of the control group); В — vacuoles in pinealocytes; arrows — necrotic cells; stained with haematoxylin and eosin; magnification ×400.

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4. Fig. 3. Ultrastructure of pineal cells from rats in the control group — transmission electron microscopy: Я — nucleus, ГЭР — granular endoplasmic reticulum, КГ — Golgi complex, ПС — polysomes, М — mitochondria, К — cristae, Мт and arrows — matrix; magnification a — ×8,000, b — ×14,000, c — ×30,000, d — ×55,000.

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5. Fig. 4. Ultrastructure of pineal cells from rats kept under conditions of constant illumination (dark deprivation) — transmission electron microscopy: АФС — autophagosome, Я — nucleus, М — mitochondria, ГЭР — granular endoplasmic reticulum, СЛ — synaptic ribbon; magnification a, b — ×8,000, c — ×12,000, d — ×28,000, e — ×16,000.

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6. Fig. 5. The effect of continuous illumination on the structure of the pineal gland — transmission electron microscopy: a — swelling of the mitochondria (М), dilation and fragmentation of the cisternae of the Golgi apparatus (КГ); b — perivascular leukocyte infiltrate (ЛЦ); magnification: a — ×55,000; b — ×12,000.

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7. Fig. 6. Ultrastructure of pineal cells from rats kept under an alternating lighting regime (1 day of continuous illumination, followed by 2 days of a lighting regime identical to that of the control group) — transmission electron microscopy: АФС — autophagosome, Я — nucleus, М — mitochondrion, Мт — matrix, К — cristae, ГЭР — granular endoplasmic reticulum, ЭР — smooth endoplasmic reticulum, КГ — Golgi complex, СЛ — synaptic ribbon; magnification: a — ×14,000, b — ×4,500, c — ×20,000, d — ×40,000, e — ×55,000.

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8. Fig. 7. The pineal gland of a rat kept under an alternating lighting regime (1 day of continuous illumination, followed by 2 days of a lighting regime similar to that of the control group) — transmission electron microscopy: ПО — perivascular oedema; magnification ×2300.

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