Senescence Associated With Neurodegeneration: Simultaneous Assessment of β-gal Activity And Nissl Stain In Histological Sections of Rodent Brain

Summary

This protocol is presented as a tool to detect senescence-associated β-galactosidase activity, to perform Nissl staining for the simultaneous assessment of senescence and neuronal integrity, and to investigate links between senescence burden and neurodegeneration.

Abstract

Cellular senescence is a physiological process characterized by irreversible cell cycle arrest that impairs tissue regeneration and function. This phenomenon has emerged as a key driver of neurodegeneration, fueled by the accumulation of senescent cells within the central nervous system (CNS). Senescent cells acquire a pro-inflammatory senescence-associated secretory phenotype (SASP) that sustains chronic neuroinflammation and disrupts the neuronal microenvironment. Consequently, essential processes such as neurogenesis, synaptic plasticity, and neuronal survival are compromised. An extensive body of literature associates cellular senescence with several neurodegenerative disorders, such as Parkinson’s disease, Alzheimer’s disease, or multiple sclerosis, and acute neuronal-related damage, such as cerebral ischemia or traumatic brain injury.

The combined assessment of senescence-associated β-galactosidase (SA-β-gal) activity and Nissl staining in histological sections provides a comprehensive approach to evaluate cellular senescence and neuronal integrity simultaneously within the same tissue context. This strategy enables precise spatial correlation between the accumulation of senescent cells in specific vulnerable regions (e.g., the hippocampus or cortex) and neuronal loss or tissue damage. By integrating a functional marker of senescence with a classical indicator of neuronal morphology and density, this approach strengthens the interpretative robustness of the analysis. Moreover, it enables a more accurate characterization of the relationship between senescent burden and neurodegenerative changes, maximizing the information yield from limited tissue samples.

Moreover, this protocol can determine how senescent cell accumulation occurs in response to interventions (pharmacological, genetic manipulation, etc.) in rodent models of neurodegenerative diseases, thereby providing a powerful tool to analyze this contribution to their pathophysiology.

Introduction

Cellular senescence is defined by an essentially irreversible arrest of the cell cycle accompanied by extensive macromolecular alterations¹. Moreover, senescent cells acquire a hypersecretory phenotype (SASP), characterized by the robust secretion of a diverse array of pro-inflammatory cytokines, growth factors, and proteases². Collectively, these factors contribute to the establishment of a pro-inflammatory microenvironment that reinforces senescence and impacts tissue homeostasis. In the central CNS, senescent cells accumulate progressively with age, particularly within regions vulnerable to age-related pathology

Protocol

Perform all animal procedures in accordance with institutional and national guidelines for the care and use of laboratory animals, and obtain prior approval from the appropriate ethics committee. This protocol complies with the current Mexican Official Standard for the Care and Use of Laboratory Animals (NOM-062-ZOO-1999(Muñoz, 2001)), prior revision and approval by the Ethics and Research Committees, and the Institutional Animal Care and Use Committee (CICUAL) of the Facultad de Medicina, Universidad Nacional Autónoma de México (FM/DI/039/2023, CICUAL-009-2023). Animals were supplied by the vivarium of the Unidad de Medicina Experimental, Facultad de M....

Results

The combined application of senescence-associated β-galactosidase (SA-β-gal) staining and Nissl staining enables the simultaneous visualization of senescent cell burden and neuronal cytoarchitecture within the same brain section.

Following the SA-β-gal staining procedure (step 1), examine the slides under a bright-field microscope. Successful staining is indicated by the presence of a distinct blue precipitate localized within cells exhibiting β-galactosidase activity (Fig.......

Discussion

Currently, the limited availability of robust, specific tools to identify, isolate, and selectively remove senescent cells in the CNS remains a major barrier to mechanistic studies. This methodological gap limits our ability to disentangle the causal contributions of cellular senescence to neuropathology from mere correlation with disease progression. The workflow presented here combines SA-β-gal enzymatic detection with Nissl counterstaining to map the putative senescent cell burden within defined neuroanatomical c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-metilbutane	SIGMA	M32631-500ML
5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal)	PROMEGA	V394A	Store at -30 to -10 °C
Citric acid	MERCK	251275-5G
Cresyl violet acetate	SIGMA	C5042-10G
DPX Mountant for histology	SIGMA	06522-500ML
Filter paper	Whatman	WHA1001929
Glacial acetic acid	SIGMA	695092-500ML
Hidrophobic pen	MERCK	Z672548
Histological-grade Xylenes	SIGMA	534056-500ML
Humidity Chamber	STAINTRAY	25501
Magnesium chloride anhydrous	SIGMA	M8266-100G	Adjust molarity if hydrated magnesium chloride is used.
OCT	SAKURA FINE TEK	4583
Phosphate buffered saline (PBS)	SIGMA	P4417-50TAB	Commercial tablets are used.
Potassium ferricyanide (Potassium hexacyanoferrate(III))	SIGMA	P8131-100G
Potassium ferrocyanide (Potassium hexacyanoferrate(II) trihydrate)	SIGMA	P9387-100G
Sodium acetate anhydrous	SIGMA	S2889-250G	Adjust molarity if hydrated sodium acetate is used.
Sodium chloride	SIGMA	S9888-2.5KG
Sodium phosphate (Sodium phosphate dibasic dodecahydrate)	SIGMA	71649-500G