Association between age-related changes in rat choroid plexus and lipofuscin accumulation in epithelial cells

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BACKGROUND

The choroid plexus of the brain is the primary source of cerebrospinal fluid (CSF). It consists of a stromal component containing connective tissue elements and blood vessels, and a covering epithelium that is sometimes considered together with the ventricular ependyma as a special glioependymal tissue, which is associated with their common origin [1, 2]. Some studies demonstrate the proliferative capacity of choroid plexus epithelium in explants [3]. At the same time, there is evidence suggesting that these cells should be classified as a slowly renewing population, and their regenerative capacity in adult animals and humans remains questionable. Consequently, similar to neurons and cardiomyocytes, choroid plexus epithelial cells should exhibit structural and cytochemical features characteristic of responses to oxidative stress and other pathological events. With aging, such reactions may manifest through the accumulation of lipofuscin granules and melanin, as well as the occurrence of intranuclear and cytoplasmic inclusions [4]. It has been established that in humans, both lipofuscin and special cytoplasmic inclusions containing lipofuscin-like granular structures and amyloid fibrils (Biondi bodies) accumulate in the choroid plexus epithelium during natural aging and more frequently in age-related abnormalities (Alzheimer disease and others) [5]. In laboratory animals with significantly shorter lifespans than humans, such manifestations of age-related changes have not been previously described. Rabbits are an exception, as lipofuscin accumulation has been detected in the choroid plexus of the lateral cerebral ventricles when cholesterol is introduced into their diet [6]. However, the choroid plexus of naturally aging rabbits has not been studied for signs of lipofuscin accumulation.

This study aimed to test the hypothesis that epithelial cells of rat choroid plexus accumulate lipofuscin as they age.

METHODS

Study Design

A single-center, cross-sectional study was conducted to investigate the choroid plexuses of lateral ventricles in the brains of Wistar rats of different ages. Frontal brain sections were prepared from paraffin blocks, mounted on glass slides, deparaffinized, and embedded in permanent mounting medium.

Study Setting

The work was performed at the Institute of Experimental Medicine. Study material was collected from male Wistar rats under deep ether anesthesia. Tissue dehydration and paraffin embedding preparation were performed using a Microm STP 120 histoprocessor (Microm, Germany). Images were digitized using Axio Observer Z1 fluorescence (Zeiss, Germany) and LSM 800 confocal (Zeiss, Germany) microscopes.

Eligibility Criteria

The study used brain tissue samples from male Wistar rats aged: 4–5 months (n = 3), 18 months (n = 3), and 28 months (n = 3).

Intervention

The animal brain was fixed in a combined zinc-ethanol-formaldehyde fixative for 24 hours at room temperature, dehydrated, and embedded in paraffin. Frontal 5-μm-thick sections of the brain were prepared using a Rotary 3003 PFM Medical rotary microtome (PFM Medical, Germany) and mounted on slides with an adhesive coating HistoBond+ (Paul Marienfeld, Germany). After deparaffinization and washing, the sections were embedded in the permanent mounting medium Cytoseal 60 (Richard-Allan Scientific, USA) without prior staining. Some of the specimens were stained with hematoxylin and eosin, aniline blue, toluidine blue, and by the Nissl stain method. The specimens were analyzed using transmitted light microscopy in the visible range (Leica DM 750 microscope; Leica Microsystems, Germany). Fluorescence microscopy was performed using an Axio Observer Z1 microscope (Zeiss, Germany). For confocal microscopy, an LSM 800 microscope (Zeiss, Germany) controlled by ZEN Blue 2012 software (Zeiss, Germany) and lasers with wavelengths of 405, 488, and 561 nm were used. Fluorescence was detected in the ranges of 405–510 nm, 510–570 nm, and 570–700 nm, respectively.

RESULTS

In the study of unstained specimens under transmitted light, no pigment granules were detected in neurons or choroid plexus cells. The application of various overview staining methods failed to reveal granules in the choroid plexus with tinctorial properties similar to typical lipofuscin inclusions. Concurrently, an expanded space occupied by the fibrous component of the choroid plexus stroma was observed in rats aged 18 and 28 months.

Fluorescence microscopy using a low-magnification EC Plan-Neofluar 10×/0.30 M27 objective also failed to detect autofluorescent granules, likely because of their small size and low fluorescence intensity.

Confocal laser microscopy with an immersion Plan-Apochromat 63×/1.4 Oil DIC M27 objective revealed fluorescent structures in choroid plexus epithelial cells of 18- and 28-month-old rat brain specimens, appearing as cytoplasmic granules measuring 0.2–0.5 µm (Fig. 1). The emission spectrum of detected granules corresponded to 570–700 nm when excited by a 561 nm laser and 510–570 nm when excited by a 488 nm laser. Excitation with 405 nm light did not induce fluorescence in the detected granules in the 405–510 nm range.

 

Fig. 1. Fragments of the choroid plexus in the lateral ventricle of the rat brain: a–d, 28 months; e, 18 months; f, 4–5 months. Autofluorescence of lipofuscin under laser excitation at wavelengths of 561 nm (a), 488 nm (b), and 405 nm (c). Separate (a–c) and merged (d) channel views; arrows indicate lipofuscin granules. Confocal laser microscopy; scale bar, 5 μm.

 

The detected granules were consistently located near the apical portion of epithelial cells, forming small clusters of 5–20 granules that occasionally fused. Precise sizing of these structures was often unattainable because of their sub-resolution dimensions relative to the confocal capacity of the microscope. In isolated instances, larger ring-shaped granules (0.5–0.9 µm) were visualized. Fluorescence channel merging demonstrated heterogeneous spectral composition and intensity within individual clusters. Granules exhibiting fluorescence predominantly in the green and red spectral regions were most frequently observed (Fig. 1).

DISCUSSION

The presented results demonstrate that aging rats exhibit accumulation of autofluorescent granules in the green and red ranges of the visible spectrum within the epithelial lining of the cerebral choroid plexus. Confocal microscopy represents the most appropriate method for detecting such granules. It is known that aging laboratory animals (mice and rats) show lipofuscin accumulation in the brain [7], associated with lipid peroxidation and increased levels of lysosomal protein degradation products [8]. In this context, the granules identified in the choroid plexus demonstrating broad-spectrum autofluorescence most likely represent lipofuscin and lipofuscin-like inclusions. The validity of this assumption is supported by the similarity of biophysical properties and biochemical composition of lipofuscins between humans and rats [9]. Notably, the potential for lipofuscin accumulation in the rat choroid plexus has not been previously investigated, despite the recognized importance of choroid plexus epithelial cells as a key component of the blood-CSF barrier [10]. Since lipofuscin predominantly accumulates in slowly renewing cell populations such as neurons and cardiomyocytes, the detection of lipofuscin granules in choroid plexus cells may serve as an indirect indicator of their relatively slow turnover rate.

CONCLUSION

Thus, the obtained results confirm the hypothesis regarding the possibility of lipofuscin accumulation in the choroid plexus epithelium of the rat brain during aging. The presence of lipofuscin in the choroid plexus can be used as an additional criterion indicating the abnormal nature of experimental interventions.

ADDITIONAL INFORMATION

Author contributions: O.V. Kirik: investigation, formal analysis, visualization, writing—original draft; O.S. Alekseeva: investigation, writing—original draft; M.S. Faizov: investigation, formal analysis; E.A. Fedorova: investigation; A.A. Beketova: investigation, formal analysis; D.E. Korzhevsky: conceptualization, investigation, writing—review & editing. All the authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval: All procedures involving animals (housing and euthanasia) complied with the principles of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986) and with the Rules of Good Laboratory Practice (Order of the Ministry of Health of Russia No. 199n of April 1, 2016). The study was approved by the Local Ethics Committee of the Institute of Experimental Medicine (Minutes No. 1/25 of January 27, 2025).

Funding sources: This work was carried out as part of the state assignment of the Institute of Experimental Medicine (FGWG-2025-003) and the state assignment of the I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences (No. 075-00263-25-00).

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: The authors confirm that this is an original study. No previously obtained or published material was used in this study or article.

Data availability statement: The authors provide limited access to the data upon reasonable request (after the embargo period has expired).

Generative AI: No generative artificial intelligence technologies were used to prepare this article.

Provenance and peer-review: This paper was submitted unsolicited and reviewed following the standard procedure. The peer review process involved two external reviewers, a member of the Editorial Board, and the in-house science editor.

About the authors

Olga V. Kirik

Institute of Experimental Medicine

Author for correspondence.
Email: olga_kirik@mail.ru
ORCID iD: 0000-0001-6113-3948
SPIN-code: 5725-8742

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Olga S. Alekseeva

Institute of Experimental Medicine; Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: osa72@inbox.ru
ORCID iD: 0000-0001-5688-347X
SPIN-code: 4281-3091

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg; Saint Petersburg

Murodali S. Fayzov

Institute of Experimental Medicine

Email: fayzov-1994@mail.ru
ORCID iD: 0009-0001-3411-3412
Russian Federation, Saint Petersburg

Elena A. Fedorova

Institute of Experimental Medicine

Email: el-fedorova2014@ya.ru
ORCID iD: 0000-0002-0190-885X
SPIN-code: 5414-4122

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Anastasiya A. Beketova

Institute of Experimental Medicine

Email: beketova.anastasiya@yandex.ru
ORCID iD: 0009-0002-8659-733X
SPIN-code: 6780-2677
Russian Federation, Saint Petersburg

Dmitrii E. Korzhevskii

Institute of Experimental Medicine

Email: dek2@yandex.ru
ORCID iD: 0000-0002-2456-8165
SPIN-code: 3252-3029

Dr. Sci. (Medicine), professor

Russian Federation, Saint Petersburg

References

Supplementary files