Elsevier

Brain Research

Volume 1035, Issue 1, 21 February 2005, Pages 24-31
Brain Research

Research report
Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons

https://doi.org/10.1016/j.brainres.2004.11.054Get rights and content

Abstract

The causes and effects of neuronal degeneration are of major interest to a wide variety of neuroscientists. Paralleling this growing interest is an increasing number of methods applicable to the detection of neuronal degeneration. The earliest methods employing aniline dyes were methodologically simple, but difficult to interpret due to a lack of staining specificity. In an attempt to circumvent this problem, numerous suppressed silver methods have been introduced. However, these methods are labor intensive, incompatible with most other histochemical procedures and notoriously capricious. In an attempt to develop a tracer with the methodological simplicity and reliability of conventional stains but with the specificity of an ideal suppressed silver preparation, the Fluoro-Jade dyes were developed. Fluoro-Jade C, like its predecessors, Fluoro-Jade and Fluoro-Jade B, was found to stain all degenerating neurons, regardless of specific insult or mechanism of cell death. Therefore, the patterns of neuronal degeneration seen following exposure to either the glutamate agonist, kainic acid, or the inhibitor of mitochondrial respiration, 3-NPA, were the same for all of the Fluoro-Jade dyes. However, there was a qualitative difference in the staining characteristics of the three fluorochromes. Specifically, Fluoro-Jade C exhibited the greatest signal to background ratio, as well as the highest resolution. This translates to a stain of maximal contrast and affinity for degenerating neurons. This makes it ideal for localizing not only degenerating nerve cell bodies, but also distal dendrites, axons and terminals. The dye is highly resistant to fading and is compatible with virtually all histological processing and staining protocols. Triple labeling was accomplished by staining degenerating neurons with Fluoro-Jade C, cell nuclei with DAPI and activated astrocytes with GFAP immunofluoresence.

Introduction

The detection of neuronal degeneration is important for a number of reasons. Unlike other cells in the adult body, dead neurons are not typically replaced by new neurons, so neuronal loss is of great consequence. It is therefore important to detect neuronal degeneration caused by chemical agents, physical insult, or disease. By doing so, the risks associated with exposure to neurotoxic chemicals including drugs, food contaminants or environmental hazards can be determined and their exposure avoided. The detection of neuronal degeneration is also important in the understanding of neurodegenerative diseases and in the evaluation of putative neuroprotective drugs.

Early studies relied on traditional detection methods such as Nissl or hematoxylin and eosin (H & E) stains in which neuronal degeneration was inferred either by morphological changes or by changes in staining intensity. Although degenerating neurons tend to be hypochromic when Nissl stained and hyperchromic when H & E stained, there is no staining specificity, as both viable and degenerating neurons are labeled. Furthermore it has been demonstrated [1], [21] that H & E hyperchromia is not a reliable marker of neuronal degeneration. A significant advance was the development of suppressed silver stains [2], [4], [6], [12] that selectively label degenerating neurons. Although superior to the more routine methods, the suppressed silver methods were less than ideal in several aspects. These limitations include a propensity to be capricious, labor intensive, time consuming and incompatible with most multiple labeling procedures. In addition, several reports [7], [11] suggest that the presence of argyrophilic dark neurons simply reflect exposure to an insult that will ultimately result in neurons either dying or recovering.

A significant advance was the development of the Fluoro-Jade dyes which are fluorescent ligands for the detection and localization of degenerating neurons. Structurally, the three Fluoro-Jade dyes are related. The most fundamental of these fluorochromes is Fluoro-Jade [19], which is disodium 5′/6′ carboxyfluorescein. Fluoro-Jade B [18] is a mixture of the following three related fluorochromes: (1) trisodium 5-(6-hydroxy-3-oxo-3H-xanthen-9-yl) benzene, 1,2,4, tricarboxylic acid; (2) disodium 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-(2,4-dihydroxybenzol) terephthalic acid; and (3) disodium 2,5-bis(6-hydroxy-3-oxo-3H-xanthen-9-yl) terephthalic acid [22]. Although the resolution of the exact chemical structure of Fluoro-Jade C is presently under investigation, preliminary mass spectroscopy data suggests that it is the sulfate ester of one of the aforementioned fluorescent components of Fluoro-Jade B.

The first of these fluorochromes, Fluoro-Jade [19], was initially shown to label neuronal degeneration following diverse insults including exposure to kainic acid, domoic acid, ibogaine, MK-801, 3-nitropropionic acid (3-NPA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and physical trauma. Subsequent studies employed this compound to localize neuronal degeneration following exposure to MPTP [5], methamphetamine [3], [17] and d-fenfluramine [20]. An analogue of this compound, Fluoro-Jade B [18], was subsequently found to have an even greater affinity for kainic acid induced neuronal degeneration, as reflected by staining with increased contrast and resolution. Subsequent studies using Fluoro-Jade B characterized the temporal progression of kainic acid induced lesions [10], and demonstrated the patterns of degeneration resulting from exposure to aurothioglucose [15] and methylenedioxymethamphetamine (MDMA) [16]. Similarly, the present study demonstrates that the recently developed Fluoro-Jade C exhibits the greatest affinity for degenerating neurons and therefore results in staining with the highest resolution and contrast of the Fluoro-Jade dyes.

The present manuscript was actually the product of a preliminary structure–activity relationship study designed to allow inferences to be made concerning the structure of the endogenous neurodegeneration molecule. Inferences would be based on altered staining of degenerating neurons resulting from the addition of specific functional groups to the dye molecule. Thus, FJ-B variants with the addition of two 5 carbon alkane chains resulted in a fluorescent dye with no affinity for degenerating neurons, while the substitution of two additional hydroxyl groups produced a non-fluorescent compound. Increasing the dye's acidity by incorporating a sulfonate group resulted in a fluorescent compound of relatively low affinity for degenerating neurons, while the formation of a sulfate ester resulted in a fluorescent molecule with an extremely high affinity for degenerating neurons. Only this latter compound, introduced here as Fluoro-Jade C, was characterized further because of its practical histological applications.

Section snippets

Animal use

Ten adult male Sprague Dawley rats were given a single intraperitoneal injection of kainic acid (10 mg/kg), ten animals were given a single subcutaneous dose of 3-nitropropionic acid (30 mg/kg) and two animals were given a comparable volume of saline. All neurotoxicants were obtained from Sigma Chemicals (St. Louis, MO). All animals were used in accordance with institutional animal care and use guidelines. A survival interval of 2 days was used for animals receiving kainic acid, while a 4 day

Saline treated control tissue

Labeling of neurons in control tissue was nonexistent. In those brains that were incompletely perfused, stained red blood cells could be observed within vascular elements. Some tissues adjacent to the brain also stained, specifically the meninges and the choroid plexus. Although glial labeling is not common, occasionally weakly stained astrocytes could be observed within certain white matter structures such as the middle cerebellar peduncle.

Comparing FJ-B vs. FJ-C labeling

Neurons were labeled in their entirety including cell

Discussion

The findings of this study indicate that Fluoro-Jade C is the most sensitive of the fluorescent markers of neuronal degeneration in terms of producing a stain of highest resolution and contrast. This allows for the unequivocal localization of the finest nerve processes, including distal dendrites, axons and terminals. The ability to label with increased resolution implies that Fluoro-Jade C possesses a higher affinity for the endogenous neurodegeneration molecule(s) than its predecessors

Acknowledgments

This work was funded by FDA protocol # E-7111.01. The authors would like to thank Becky Divine for histological assistance, Tom Heinze for providing MS data, and Mangala Manivannan for help with digital acquisitions. The authors would also like to thank Merle Paule and Bill Slikker Jr. for thoughtful discussions.

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