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Journal of Histochemistry and Cytochemistry, Vol. 47, 969-972, July 1999, Copyright © 1999, The Histochemical Society, Inc.

TECHNICAL NOTE

Detection of Pathological Zinc Accumulation In Neurons: Methods for Autopsy, Biopsy, and Cultured Tissue

Sang Won Suha,c,f, Kathy Listiacka, Brent Bella, Jefferson Chena, Massoud Motamedia, David Silvab, Gorm Danscherc, William Whetselld, Richard Thompsone, and Christopher Fredericksona,f
a Center For Biomedical Engineering, University of Texas Medical Branch, Galveston, Texas
b Laboratory for Zinc Neurobiology, MicroFab Technologies, Inc., Plano, Texas
c Institute of Neurobiology, University of Aarhus, Aarhus, Denmark
d Department of Pathology, Vanderbilt School of Medicine, Nashville, Tennessee
e Department of Chemistry, University of Maryland at Baltimore, Baltimore, Maryland
f NeuroBioTex, Inc., Galveston, Texas

Correspondence to: Christopher Frederickson, NeuroBioTex, Inc., 921 Sealy & Smith Bldg., 200 University Boulevard, Galveston, TX 77550


*   Summary
*Top
 *Summary
*Introduction
 *Materials and Methods
 *Results
 *Discussion
 *Literature Cited

It has been repeatedly shown that synaptically released zinc contributes to excitotoxic neuronal injury in ischemia, epilepsy, and mechanical head trauma. Such zinc-induced injury leaves an unmistakable "footprint" in the injured neurons, allowing an easy and unambiguous postmortem diagnosis. This footprint is the presence of weakly bound, histochemically reactive zinc in the cytoplasm of the perikaryon and proximal dendrites. Such staining appears to be a necessary and sufficient marker for zinc-induced neuronal injury. Here we show how to prepare and stain tissue from biopsy, autopsy, or experimental animal sources for maximal contrast and visibility of zinc-injured neurons. (J Histochem Cytochem 47:969–972, 1999)

Key Words: zinc, neuronal injury, perikarya, dendrites, staining


*   Introduction
*Top
 *Summary
 *Introduction
*Materials and Methods
 *Results
 *Discussion
 *Literature Cited

During the excitotoxic events that accompany ischemia, seizures, and mechanical head injury, one contributing cause of neuronal injury is the translocation of weakly bound zinc (or Zn2+) from zinc-containing presynaptic boutons into the perikarya and proximal dendrites of vulnerable postsynaptic neurons. Evidence for this zinc translocation has been observed after status epilepticus (Frederickson et al. 1988 Down, Frederickson et al. 1989 Down; Suh et al. 1996 Down), ischemia (Tonder et al. 1990 Down; Koh et al. 1996 Down), and mechanical head/brain trauma (Long et al. 1998 Down). Apparently, the weakly bound zinc in presynaptic vesicles is released in pathological excess from the boutons (Howell et al. 1984 Down; Sloviter 1985 Down; Frederickson et al. 1989 Down) and enters postsynaptic neurons through the zinc-permeable channels (Yin and Weiss 1995 Down), raising intracellular Zn2+ by several orders of magnitude (Yin and Weiss 1995 Down) and thereby killing those neurons (Frederickson 1989 Down; Choi and Koh 1998 Down). The crucial role of the translocated zinc in this scenario has been verified by two approaches. (a) It has been repeatedly shown that cells showing translocated zinc are eosinophilic or acidophilic, i.e., undergoing degenerative changes (e.g., Koh et al. 1996 Down; Frederickson et al. 1989 Down; Long et al. 1998 Down). (b) It has been repeatedly shown that blocking the translocation of zinc by extracellular chelation reduced neuronal death up to 85% in ischemia (Koh et al. 1996 Down), seizures (Suh et al. 1996 Down), and mechanical head trauma (Long et al. 1998 Down).

As of this writing, the only way to diagnose prior zinc-induced neuronal injury in a biopsy or autopsy specimen is to stain the tissue for zinc and search for signs of zinc translocation. Evidence of prior zinc translocation epsiodes includes both depletion of zinc from presynaptic boutons and the anomalous appearance of weakly bound zinc in the cytoplasm of injured postsynaptic neurons. Of these two signs, the latter is by far the most reliable because normal healthy neurons in the brain never show any staining for zinc in the perikaryon or proximal dendrites. In general, therefore, the appearance of the atypical, anomalous zinc staining in the perikaryon is both a necessary and a sufficient condition for the diagnosis of a zinc translocation-mediated neuronal injury.

The present studies were undertaken in search of optimal zinc staining methods for detecting zinc translocation. Both fluorescent and silver-amplification histochemistry were explored and a number of variations in tissue preparation and staining methods were tested. Special emphasis was placed on the use of these methods to identify zinc translocation and thus to diagnose zinc-induced neuronal injury in tissue that was taken from human subjects as autopsy or biopsy specimens.


*   Materials and Methods
*Top
 *Summary
 *Introduction
 *Materials and Methods
*Results
 *Discussion
 *Literature Cited

Fluorescent probes included TSQ (Frederickson et al. 1987 Down, Frederickson et al. 1992 Down) and its congener TFlZn (Teflabs; Austin TX) and an enzyme-based system employing zinc-free human carbonic anhydrase II (apoCA) as the "detector" with the fluorochrome ABD-N (7-(N-amino ethane-2-ol)benz-2-oxa-1,3-diazole-4-sulfonamid) as the "reporter" [see Thompson et al. 1997 Down for further methods]. The silver method was the frozen-tissue, sulfide gas method recently developed by Danscher and colleagues for human autopsy/biopsy material [see Danscher et al. 1997 Down for details].

Several models of zinc-induced neuronal injury were explored, including kainic acid (KA)-induced seizures [Methods in Frederickson et al. 1988 Down, Frederickson et al. 1989 Down] and mechanical brain trauma (Long et al. 1998 Down). In addition, some brain slices [prepared and maintained as in Howell et al. 1984 Down and Easley et al. 1995 Down)] and hippocampal organotypic cultures (Whetsell and Schwarcz 1983 Down) were subjected to excitotoxic insults in vitro and stained either in vitro or after harvesting, freezing, and sectioning. For the acute slices, simply interrupting the superfusate flow for 1 hr was the insult. For the cultures, the insult was to delay supplying fresh medium and oxygen for 1–2 days [see Whetsell and Schwarcz 1983 Down for details of this ischemia/hypoglycemia method]. In the KA model of zinc-induced cell death (Frederickson et al. 1988 Down, Frederickson et al. 1989 Down), the KA was administered IP (12 mg/kg), and the rats were watched thereafter and selected if they exhibited tonic–clonic seizures (Frederickson et al. 1988 Down, Frederickson et al. 1989 Down). For the mechanical brain trauma, rats were deeply anesthetized and subjected to a calibrated acceleration of the dura by weight-drop onto the exposed dura (15 g dropped 10 cm; 3 mm maximal dura deflection), resulting in focal and some remote neuron loss (Long et al. 1998 Down). Brains were harvested 1–24 hr after insults.


*   Results
*Top
 *Summary
 *Introduction
 *Materials and Methods
 *Results
*Discussion
 *Literature Cited

Five results merit presentation. First, as has been shown previously for bouton zinc staining (Frederickson et al. 1987 Down) the time elapsed between death (or live tissue harvesting) and the freezing of the tissue has proved to be critical. Tissue that has remained unfrozen for more than 1–2 hr post mortem shows an artifactual decline in the intensity of fluorescent staining that declines to virtually no staining at all by 12–24 hr. Likewise, in normal tissue, the staining of boutons declines over 12–24 hr and there is no corresponding increase in the staining of neuronal perikarya (not shown). Second, the manner in which the tissue is frozen can determine whether or not neurons are found. Specifically, freezing procedures that promote morphological damage, such as freezing too slowly or too rapidly, or (worse) freezing, thawing, and refreezing, can obliterate the discernible staining of zinc-injured neurons (Figure 1). The third important finding is that a histological marker that cannot easily penetrate cell membranes is superior for highest-contrast definition of zinc-injured neurons. This is shown in Figure 2, in which the staining of CA3 pyramidal neurons (injured by KA-induced status epilepticus) is vivid, whereas the adjacent mossy fiber neuropil is virtually unstained. The failure of staining in the neuropil, coupled with the positive reaction in the perikarya, enables the latter to stand out in high contrast. The fourth noteworthy finding is that zinc-injured neurons can be reliably and densely stained in autopsy or biopsy material by a variation of the sulfide gas–silver method of Danscher et al. 1997 Down. The variation that allows the injured perikarya to be stained (in addition to the normal bouton staining) is to treat the cut tissue sections with a basic (pH 10) buffer (so called "universal buffer" (Na-barbital/Na-acetate) (Frederickson et al. 1987 Down) before exposing them to the sulfide gas. For this variant, one immerses the cut and mounted cryostat sections in the buffer for 2 min, rinses briefly in saline, then places the tissue in the GAS-AMG exposure chamber, as described by Danscher et al. 1997 Down. Whereas few zinc-killed neurons can be stained by conventional silver methods for zinc (Sloviter 1985 Down; Frederickson et al. 1989 Down), many (perhaps all) are stained by this novel variant (Figure 3). Fifth, and finally, neurons subjected to ischemic insult in vitro, as cut slices or organotypic cultures, can be readily stained by a cell-permeable fluorescent marker, in vitro or after frozen sectioning, by the standard TSQ methods (Figure 4).



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  		Figure 1. Effect of refreezing on nearby sections of rat dentate gyrus hilar region. This rat was subjected to 3 hr of kainic acid-induced convulsions before sacrifice, resulting in zinc translocation into somata. Standard TSQ methods were used to stain both sections. (a) Arrows point to the only cell-like "puffs" of "shreds" left in this section of hilar tissue after the section was cut frozen, thawed, then refrozen on the glass slide before thawing, staining, and photographing. (b) In contrast, this nearby section clearly shows 10+ distinct neuronal somata and some proximal dendrites. This section was cut frozen, thawed, stained, and photographed in one session. Bar = 100 µm.

Figure 2. Staining of neuronal somata by carbonic anhydrase-based zinc stain. After 4 hr of KA-induced convulsions, this rat was sacrificed and the brain was cut frozen, thawed, and stained. The stain was a mixture of 1 µm zinc-free human carbonic anhydrase and the fluorochrome ABDN (Thompson et al. 1997 Down), which fluoresces brightly (560-nm emission, 430-nm excitation) when the carbonic anhydrase binds any available free zinc. Many pyramidal somata in stratum pyramidale (SP) are vividly fluorescent, whereas the mossy boutons in stratum lucidum (SL) are not appreciably brighter than the subjacent stratum oriens (SO). Bar = 50 µm.

Figure 3. Silver staining for translocated zinc. This tissue was harvested from a rat (dorsal cortex) subjected to traumatic head injury, which causes translocation of zinc from boutons to somata. The silver grains were produced by the Danscher gas-AMG method, in which the fresh-frozen, unfixed sections are exposed to sulfide gas to create ZnS catalyst, then developed by the Danscher AMG development (Danscher et al. 1997 Down). This silver-stained material would be suitable for EM determination of the exact localization of the translocated zinc. Bar = 30 µm.

Figure 4. TSQ staining of translocated zinc in neuronal somata of hippocampal culture and (inset) acute hippocampal slice preparation. This organotypic hippocampal culture was subjected to hypoglycemia/hypoxia for 48 hr before staining with TSQ. Healthy cultures have essentially no zinc-stained perikarya, whereas these "ischemic" cultures have selective zinc staining in many pyramidal neurons (arrows) of the dentate hilus (H) and CA3 of Ammon's horn. The mossy boutons of stratum luicidum (SL) are faintly visible. (Inset) Many hilar pyramids have developed zinc staining as a result of blocking the oxygen and medium flow for 1 hr while the slice was incubating. Note the TSQ-stained fluorescent neurons. Bar = 300 µm; inset = 200 µm.


*   Discussion
*Top
 *Summary
 *Introduction
 *Materials and Methods
 *Results
 *Discussion
*Literature Cited

The present results show that the postmortem diagnosis of zinc-mediated neuronal injury can be carried out on any tissue that has been (a) harvested within 1–2 hr of death, (b) left unfixed, and (c) frozen properly and maintained frozen. Such tissue can be stained immediately with either the TSQ (Frederickson et al. 1987 Down) or the CA-ABDN method (Thompson et al. 1997 Down), photographed, then postfixed for archival staining. Alternatively, one can treat the sections with basic buffer, then use the sulfide gas method for permanent light- and electron-dense silver staining of tissue (Danscher et al. 1997 Down). In all cases, thinner (10-µm) sections give better cell definition than thicker.

The reason that a short postmortem period is critical is presumably that the Zn2+ present in the fresh tissue binds fairly rapidly with protein residues and small molecules (including S-) liberated by postmortem autolysis. Concerning the peculiar sensitivity of the zinc-positive cell staining to freeze–thaw–freeze damage (freezer burn), we speculate that this is because the Zn2+ (unlike the macromolecules of Nissl) is a mobile ion in solution and would be expected to diffuse quickly away through ruptured membranes. The "smudges" or "clouds" of fluorescence staining that remain after such diffusion are difficult to recognize as former neurons.

The advantage of CA-ABDN as a stain for zinc-filled neurons was an unexpected finding, which we tentatively interpret thus. If most of the vesicles in zinc-containing boutons are intact after single freezing and cutting of the tissue at 10–20 µm, then most of the bouton zinc (in the vesicles) would be unstained by the membrane-impermeable CA-ABDN. Most neural somata, on the other hand, will be sliced open (especially in the thinner 10-µm sections) and thus open to the CA-ABDN. Alternatively, it is possible that the biochemical microenvironment of the zinc in vesicles is sufficiently different from that of the zinc in the perikarya that the CA-ABDN staining favors the latter. This idea is also consistent with the finding that pretreatment with a basic buffer is needed to achieve consistent staining of perikaryal zinc, but not vesicular zinc, with the sulfide gas method.

We know of only three conditions in which neurons in the brain will show staining for zinc: after excitotoxic zinc translocation (as in this report), in Alzheimer's disease (Suh et al. 1998 Down), and after exposure to a pathological excess of NO (Cuajungco and Lees 1998 Down). In Alzheimer's, there is evidence that excess synaptically released zinc may be a contributing factor in the neuronal pathology. In the case of the NO-mediated injury, it is also quite plausible that the NO causes release of zinc from vesicles, thus triggering the familiar zinc translocation cell injury.

Zinc translocation in stroke, ischemia, hypoxia, seizures, and head injury may be a leading cause of neuronal injury in adults. The ability to identify and diagnose this particular pathology in any suitably collected and archived tissue sample should facilitate further research.


*   Acknowledgments

Supported in part by NIH-GM 48894, MH 56335, and NS 37658 (to CJF), by the Schriners Burn Foundation (JWC), NSF (RBT, DSS), the Danish Medical Research Council (SWS, GD), and the Office of Naval Research (RBT).

We thank Gabrielle Schneider for technical assistance.

Received for publication January 15, 1999; accepted January 29, 1999.


*   Literature Cited
*Top
 *Summary
 *Introduction
 *Materials and Methods
 *Results
 *Discussion
 *Literature Cited

Choi DW, Koh JY (1998) Zinc and brain injury. Annu Rev Neurocsi 21:347-375

Cuajungco MP, Lees GJ (1998) Nitric oxide generators produce accumulation of chelatable zinc in hippcampal neuronal perikarya. Brain Res 799:118-129[Medline]

Danscher G, Juhl S, Stoltenberg M, Krunderup B, Schroder HD, Andreasen A (1997) Autometallographic silver enhancement of zinc sulfide crystals created in cryostat sections from human brain biopsies: a new technique that makes it feasible to demonstrate zinc ions in tissue sections from biopsies and early autopsy material. J Histochem Cytochem 45:1503-1510[Abstract/Free Full Text]

Easley J, Lee A, Fleming E, Frederickson CJ (1995) A selective role for vesicular zinc during fast neuronal firing. Soc Neurosci Abst 21:1062

Frederickson CJ, Kasarskis EJ, Ringo D, Frederickson RE (1987) A quinoline fluorescence method for visualizing and assaying the histochemically reactive zinc (bouton zinc) in the brain. J Neurosci Methods 20:91-103[Medline]

Frederickson CJ, Hernandez MD, Goik SA, Morton JD, McGinty JF (1988) Loss of zinc staining from hippocampal mossy fibers during kainic acid induced seizures: a histofluorescence study. Brain Res 446:383-386[Medline]

Frederickson CJ (1989) Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol 31:145-238[Medline]

Frederickson CJ, Hernandez MD, McGinty JF (1989) Translocation of zinc may contribute to seizure-induced death of neurons. Brain Res 480:317-321[Medline]

Frederickson CJ, Rampy BA, Reamy–Rampy S, Howell GA (1992) Distribution of histochemically reactive zince in the forebrain of the rat. J Chem Neuroanat 5:521-530[Medline]

Howell GA, Welch MG, Frederickson CJ (1984) Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308:736-738[Medline]

Koh JY, Suh SW, Gwag BJ, He YY, Hsu CY, Choi DW (1996) The role of zinc in selective neuronal death following transient global cerebral ischemia. Science 272:1013-1016[Abstract]

Long YY, Suh SW, Pons NF, Jensen MS, Chen JW, Motamedi M, Danscher G, Frederickson CJ (1998) Chelation of extracellular Zn2+ reduces neuronal death after head trauma. Soc Neurosci Abst V 24:1727

Sloviter RS (1985) A selective loss of hippocampal mossy fiber Timm stain accompanies granule cell seizure activity induced by perforant path stimulation. Brain Res 330:150-153[Medline]

Suh SW, Jensen KB, Jensen MS, Silva DS, Kesslak P, Danscher G, Frederickson CJ (1998) Histologic evidence of a zinc role in Alzheimer's disease. Soc Neurosci Abst 24:1217

Suh SW, Koh JY, Choi DW (1996) Extracellular zinc mediates selective neuronal death in hippocampus and amygdala following kainate-induced seizure. Soc Neurosci Abst 22:2101

Thompson RB, Lin HJ, Ge Z, Johnson K, Fierke CA (1997) Fluorescence lifetime-based determination of anions using a site-directed mutant enzyme transducer. Proc SPIE Conf Adv Fluorescence Sensing Tech III 2980:247-256

Tonder N, Johansen FF, Frederickson CJ, Zimmer J, Diemer NH (1990) Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat. Neurosci Lett 109:247-252[Medline]

Whetsell W, Schwarcz R (1983) The organotypic tissue culture model of corticostriatal system used for examining amino acid neurotoxicity and its antagonism: studies on kainic acid, quinolinic acid and (-) 2-amino-7-phosphonoheptanoic acid. J Neural Transm Suppl 19:53-63[Medline]

Yin HZ, Weiss JH (1995) Zn2+ permates Ca2+ permeable AMPA/kainate channels and triggers selective neuronal injury. Neuroreport 6:2553-2556[Medline]


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