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A quick silver method for senile plaques and neurofibrillary tangles in paraffin sections
Brain Research Bulk&n, Vol. 3.5, No. 3, pp. 279-284, 1994 Copyright 6 1994 Elsevier Science Ltd Printed in the USA. At1tights reserved
Pergamon
0361-9230/94$6.00 + .M)
0361-9230(94)00099-9
BRIEF COMMUNICATION
A Quick Silver Method for Senile Plaques and Neurofibrillary Tangles in Paraffin Sections JOHN
C. HEDREEN,*tP
LISABETH
S. RASKJN$
AND
DONALD
L. PRICE*Wj
Departments of *Pathology, fNeuroscience, SNeurology, and #the Neuropathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2196 Received
11 October
1993; Accepted
27 April 1994
HARES, J. C., L. S. RASKIN AND D. L. PRICE. A quick silver method for senile pl~ues and neuro~briifa~ fang/es in paraj3fn sections. BRAIN RES BULL 35(3) 279-284, 1994.-A new silver method for senile plaques and neurofib~ll~ tangles (NFT) in paraffin sections is presented. This new technique is rapid in execution (15 min) and reveals senile plaques of all morphological types as well as NFJ. The silver staining is spatially congruent with immunocytochemical staining for the pamyloid peptide (A/3) in adjacent sections. AD peptide
Alzheimer’s disease
Diagnosis of Alzheimer’s disease
Neurofibrillary tangles
Senile plaques
the ready identification of senile plaques and NFI in standard paraffin sections of the cerebral cortex, hippocampus, amygdala, and nucleus basalis in 9 cases of AD and in brainstem sections of 1 case of progressive supranuclear palsy. Twelve control brains without neurological disease were also examined (age range, 18-73 years). All solutions are used at room temperature.
SENILE plaques and NFT, the characteristic histopathoIog~c~ features of Afzheimer’s disease (AD), are usually demonstrated in autopsied brains using silver methods. A variety of methods have been used, but modifications of the Bielschowsky technique (3,24,28,38) are perhaps the closest to a standard. These methods vary in their efficacy (13,20,31,34,38). Recently, we developed a silver method that specifically stains senile plaques (14,15,17). Neurofibrillary tangles, normal axons, and other features that are usually stained darkly by Biefschowsky procedures are lightly stained by this method. Because of the need to demonstrate NPI’ for diagnostic purposes, we have devised a new method that demonstrates both senile plaques and NFI. The most prominent features of this new procedure are its rapidity in comparison with standard silver methods, its ability to stain senile plaques of all morphological types, and the clarity and delicacy of the silver staining of NFT.
1. Cut paraffin sections (IO- 12 pm), lay out on gelatinized water (about 1.5 g!l) in a water bath, mount onto subbed slides, and dry for l-3 days at 37°C. 2. Deparaffinize and bring to distilled water (DH,O). 3. Immerse in 5% AgNOl in DH20 (5 min) with occasional agitation. 4. Dip briefly in DH20. 5. Immerse in ammoniacal silver solution (2 min) with gentle, continual agitation. This solution consists of the following ingredients, added in the order listed: 18 ml 5% AgNO,; 10 ml 95% ethanol; 2 ml DH20; 1.8 ml concentrated N&OH solution; and 1.5 ml 2.5% NaOH (25). The IQ&OH and NaOH solutions may be mixed together first and added in small aliquots to the silver-ethanol solution with vigorous agitation, or these solutions may be added separately in a similar manner. The ammoniacal silver solution should be made immediately before use. Ingredients may be stored as stock solutions. Because of the potential formation of explosive compounds on standing (33), the solution should be discarded after use, and glassware should be rinsed ~o~ughly.
METHOD
This technique is a modification of our method for senile plaques (14,15,17) and of the 1954 version of the method of Nauta and Gygax (25), both of which are Bielschowsky-type procedures (18). In the new technique, deparaffinized sections are passed through solutions of silver nitrate, ammoniacal silver nitrate, and reducer. Trials were performed that varied the concentration of silver nitrate, the incubation time in silver nitrate, and the concentration of different elements of the ammoniacal silver solution. The following procedure was judged optimal for
Requests for reprints should be addressed to John C. Hedreen, M.D., at his present address: Dept. of Psychiatry #1007, New England Medical Center, 750 Washington St., Boston, MA 02111.
279
280
HE~REEN.
RASKIN
AND PRICE
Drain excess solution quickly, and transfer slides to two solutions of reducer. The reducer solution consists of: 400 ml DH20; 45 ml 95% ethanol; 13.5 ml 10% unbuffered formaldehyde USP. (40 g/IO0 ml formaldehyde with IO-15% methanol); and 13.5 ml 1% citric acid. Agitate slides immediately for S- 15 s in the first reducer solution and then transfer slides to the second solution for up to 2 min. Sections will become a gray to brown color. Rinse three times in DH,O and then immerse (with occasional agitation) in 1.0% sodium thiosulfate for 3 min. Rinse thoroughly three times in DH20, dehydrate, clear, and coverslip. The use of gelatinized water in the cutting water bath proved be essential in obtaining successful staining. It should be pointed out that sections to be stained ~mmun~ytochemically should not be mounted from gelatinized water. Short times {25 min) in the AgNO? solution provide the best contrast between senile plaques and surrounding neuropil and often produce golden-brown NFT against a gray background. At 10 min, the background is partly gray and partly brown, and NFI are more deeply stained. At 20 min, the background is brown and resembles that seen in standard Bielschowsky-type silver stains (24,28,38). However, at this incubation time, contrast is reduced, and NIT are overstained. Using 2% or 5% AgN03 in the first step produces a lighter background than 10% AgN03 and provides better contrast between NFT and normal neurons and between plaques and background. Three, 5, and 7 min trials in 5% AgN07 are recommended when the method is tried initially. The following ammoniacal silver solution, derived from Method One of Fink and Heimer (9), also produces useful results: 50 ml 2.5% AgN03; and 4 ml of a 1:l mixture of concentrated ~monium hydroxide and 2.5% NaOH. The latter is added slowly with vigorous agitation. Because this ammoniacal silver solution produces a pleasing golden-brown background, it may be preferred by some microscopists, but the contrast of plaques and NFI against this background is less than with the method given above. In some cases, adjacent sections were stained with other modern silver methods (6,38), and a Bielschowsky variant method from our laboratory that stains mainly NFT and enlarged neurites in classic senile plaques in AD as well as axonal enlargements in amyo~ophic lateral sclerosis. Some serial sections of amygdala and cerebral cortex were also stained imm~~yt~hemitally (method of Walker et al. (32)) for the A/3 peptide (23) or tau (5,26). Both the new silver method and the immunocytochemical staining procedure for A@ were performed with and without pretreatment in formic acid. RESULTS
Senile plaques were readily seen, including the diffuse type (Fig. 1A) with low amyloid density and few or no fine, abnormal neurites (12,16,35-37). In addition, the classic type of plaque (Fig. 1B) with enlarged dystrophic neurites and high amyloid content as well as perivascular and subpial plaques were readily identified. Senile plaques of the diffuse type in the lateral amygdaloid nucleus, which are difficult to demonstrate with some standard silver techniques (12), were clearly stained with the present method (Fig. 1A). Serial section analysis showed that diffuse plaques demonstrated with AP peptide i~unocytoche~st~ were all stained with the present technique and showed a similar outline (Figs. 1A Br 1C). Formic acid pretreatment was necessary for A@ immunostaining but prevented silver staining of plaques.
Success in staining of plaques was similar to, or better than, that achieved with other modem silver methods and with immuno-
FIG. I. A) Senile plaques of the diffuse, iow amyloid type in the lateral amygdala in AD. New method. Bar, 40 pm. 8) Seniie plaques of the classic, dense amyloid type with dystrophic neurites and cores in the medial amygdala in AD. New method. Bar, 20 pm. C) Section, adjacent section to that of Fig. I A, stained immunocytochemically for AD peptide following formic acid treatment. Bar, 40 pm.
QUICK SILVER METHOD
PIG. 2. Senile plaques in cerebral neocortex in AD demonstrated with three methods. The pial surface in A-C is at upper right. Bar, 200 pm. A) New method. B) Yamamoto and Hirano method. Section adjacent to A. C) Immunocytochemisuy for the AP peptide. Section not adjacent to A or B.
cyi~hemis~ for the A@ peptide (Figs. 2A-C). Perivascular plaques were seen with both i~un~yt~he~~~ and silver methods, but vascular amyloid was not always stained well with the silver method. Reticulin and other elements of blood vessels were variably stained with silver (Fig. 4D), regardless of the presence or absence of amyloid in the blood vessel wall. r~unos~ning and silver staining of various types of senile plaques in the amygdala in the brains of individuals with AD
281
PIG. 3. Serial sections through a diffuse plaque in the lateral amygdala from the brain of a patient with AD. Bar. 20 urn. A) New method. All plaques demonstrated by immun~~o~hemis~ for ;\a were also seen with the new silver method. B) Immunocytochemistry for the Ap peptide. Plaque indicated by arrowheads. C) Immunocytochemistry for tau. Note the absence of staining for enlarged neurites containing cytoskeletal eiements. Very fine, lightly stained processes are present (arrowheads).
were performed in serial sections (16). Senile plaques in the lateral amygdala in most cases were of the type usually designated “diffuse.” Diffuse plaques identified with the new method had the following characteristics: silver stains showed a homogeneous group of fine fibrils (Fig. 3A) without huge, distorted neurites that characterize classic, dense-amyloid plaques (see periphery
FIG. 4. A> Flame-shaped NIT ia pyramida neuron of cerebral neocortex in AD. New method. Bar, 20 pm (for A-Q. B) Globose NFT in neur~rts of nucleus basalis of Meynert irk AD. Note contrast with nearby normal neurot~s. New method. C) Diffuse piaqire and NIT {~~h~ds)~ D) Extracelfular NFT derived from large tayer-El neurons of eotorhinal cortex in AD. No neurons remained in an adjacem H&E-stained section. Four of the many NFT are indicated by arrowheads. New method. Bar. 40 p,rn (for D,E). E) Same area as D; adjacent section stained with the Yamamoto and Hirano method. Note faint or absent staining of NFT.
of plaques in Fig. IB); immunocytochemistry for A/? revealed light, homogeneous staining (Fig. 38); a silver method specific for NFT and eniarged neurites of senile plaques was negative (not shown); and i~~~yt~hem~s~ for tau revealed either no staining or fine, faintty stained elements (Fig. 3C) that were much smaller than the typical club-shaped “neurites” of classic plaques.
Neurofibrilhrry tangles, whether flame shaped or globose, appeared as delicately impregnated, brown-staining cytoplasmic incfusions in neurons of the hippocampal formation, amygdaia, neocortex, and nucleus basaiis in ‘AD (Figs. 4A St B) and in various brainstem nuclei in progressive supranuckar palsy. Because of the short silver nitrate impregnation, NFT are more lightly stained with the present method than with most silver
283
QUICK SILVER METHOD
techniques, and internal structure is often apparent (Fig. 4B). Both plaques and NFT were easily visualized despite short incubations in silver nitrate and ammoniacal silver solutions (Fig. 4C). In some cases, especially with shorter incubation times, NIT were stained a different color than were other tissue elements. In such cases, the brown or golden-brown staining of NFT provided excellent color contrast against the gray staining of other argyrophilic tissue structures. Extracellular “tombstone” NFT in layer II of the entorhinal area were often better demonstrated with our new method than with an excellent modem Bielschowsky variant (Figs. 4D & E) (38). Axons in the neuropil were variably stained in different cases. As a result, the method cannot be used to identify “neuropil threads.” Neuromelanin granules were strongly stained; red blood cells and intracytoplasmic granules in occasional neurons were sometimes stained. Except for very large cells (e.g., oculomotor nucleus, mesencephalic trigeminal nucleus, Purkinje cells), most normal neuronal cell bodies were lightly stained (gray to light brown) (Figs. 3A, 4A & B), in contrast to neighboring neurons that harbor NFT, However, there was variability from case to case in staining of neuronal nuclei and perikarya (note absence of staining in Figs. 1A & B, 4C & D). Control cases showed staining of normal neurons and axons similar to that seen in cases of AD. As expected, occasional cortical senile plaques and ~pp~arnpal NFT were encountered in older individuals. Thus, the pattern and specificity of staining in controIs are similar to those found with other commonly used Bielschowsky method variants (6,24,38). DISCUSSlON The most specific and sensitive histological criterion for the diagnosis of AD at autopsy is the demonstration of numerous senile plaques in the cerebral neocortex (2,19). Neurofibrillary tangles are also important histopathological features in AD; thus, a method for histological diagnosis of AD at autopsy should have the ability to stain both senile plaques and NFT. In addition, such a method should stain all types of plaques demons~able by immun~yt~hemist~ for the A/3 peptide and should be relatively labor efficient and inexpensive. Most silver methods for nervous tissue derive either from the Bielschowsky method (silver nitrate, ammoniacal silver, reducer) or the Cajal method (silver nitrate impregnation of tissue blocks,
reducer) (18). Variants of the Bielschowsky method were used in most of the initial studies with silver methods of senile plaques and NIT ( 1,4,10,11). Staining of neurofibrillar elements, namely NFT and enlarged, often club-shaped, neurites in plaques, was emphasized with these methods. Using a modified Cajal method, Marinesco (21,22) described and illustrated diffuse plaques as we know them today and contrasted the morphology of plaques stained with the Bielschowsky and Cajal methods. Nevertheless, the acceptance of diffuse plaques in descriptions of the histopathoiogy of AD was rare until certain Bielschowsky method variations that reliably demons~at~ this type of plaque came into common use in the last decade (13,14,20,24,27,29-31,34,38) and until i~un~yt~hemis~ for the A/3 polypeptide confirmed the biochemical relationship of diffuse and classic plaques (7,8,35,36). Diffuse plaques, as defined in the Results section, were reliably stained with the present method. The principal advantages of the new technique described here over other silver methods for paraffin sections are: its speed (total staining time < 15 min); the clarity of impregnation of all types of AD peptide-containing senile plaques, including diffuse plaques; and the readiness with which NET can be differentiated from other argyrophilic tissue structures. We have completed a study comparing numbers of senile plaques stained in AD cerebral neocortex with six different silver stains and A/3 peptide immun~yt~hemis~ (13). In this study, the quick silver method described above revealed more neocortical plaques in sections of temporal neocortex from 8 AD cases than any other method. Recent experience in our laboratory indicates that this new method may prove to be useful in both diagnostic and research situations. ACKNOWLEDGEMENTS The authors thank Drs. Lary C. Walker, Juan C. Troncoso, Robert G. Struble, and Linda C. Cork for helpful discussions on the nature and methods of demonstration of plaques and NFT. in addition, the authors thank Lisa J. Sansone, Eleanor Brown, and Leila M. Rowland for testing a variety of silver methods. ~ti~ies against the Afi polypeptide and tau were generous gifts from Drs. Cohn Masters and Konrad Beyreuther and Dr. Lester Binder, respectively. This work was supported by grants from the U.S. Public Health Service (NIH AG 05146, AG 03359). Dr. Price is the recipient of a Javits Neuroscience Investigator Award (NIH NS 10580) and a Leadership and Excellence in Alzheimer’s Disease (LEAD) award (NIA AG 07914).
REFERENCES I. Alzheimer, A. ijber eine eigenartige Erkrankung der Himrinde.
2.
3.
4.
5.
6.
7.
Allg. 2. Psychiatric Psychisch-Gerichtlich Med. 64: 146 148; 1907. Berg, L.; M&eel, D. W.. Jr.: Miller. J. P.: Batv. J.: Morris. J. C. Ne&opathological indexes of Alzheimer’s &e&e in ‘demented and nondemented persons aged 80 years and older. Arch. Neurol. S&349-358; 1993. Bielschowsky, M. Die Sil~~mp~gnadon der Neurofib~llen. Einige Bemerkungen zu der von mir angegebenen Methode und den von ihr gelieferten Bildem. J. Psychol. Neurol. 3: 169- 189; 1904. Bielschowsky, M. Zur Kenntnis der Alzheimerschen Krankheit (pmsenilen Demenz mit Herdsymptomen). 3. Psychol. Neurol. 18:273292; 1911. Binder, L. I.; Frankfurter, A.; Rebhun, L. I. The distribution of tau in the mammalian central nervous system. J. Cell Biol. 101: 137 I1378; 1985. Campbell, S. K.; Switzer, R. C., III; Martin, T. L. Alzheimer’s plaques and tangles: a controlled and enhanced silver staining method. Sot. Neurosci. Abstr. 13:678; 1987. Davies, L.; Wolska, B.; Hilbich, C.; Multhaup, G.; Martins, R.; Simms, G.; Beyreuther, K.; Masters, C. L. A4 amyloid protein deposition and the diagnosis of Alzheimer’s disease: prevalence in aged
8.
9.
10.
1I. 12. 13.
brains determined by immunocytochemistry compared with conventional neuropathologic techniques. Neurology 38: 1688- 1693; 1988. Dickson, D. W.; Farlo, J.; Davies, P.; Crystal, H.; Fuld, P.; Yen, S-.H. C. Alzheimer’s disease: a double-labeling immunohistochemical study of senile plaques. Am. J. Pathol. 132:86- 101; 1988. Fink, R. P.; Heimer, L. Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4:369-374; 1967. Fischer, 0. Die presbyophrene demenz, deren anatomische grundlage und klinische abgrenzung. Zeit. f.d. ges. Neurol. Psychiat. 3:371-471; 1910. Fischer, 0. Miliare Nekrosen mit drusigen Wucherungen der Neurofibrillen, eine regelmtissige Veranderung der Himrinde bei seniler Demenz. Monatsschr. Psychiatr. Neurol. 22:361-372; 1907. Hedreen, J. C.; Broadhead, J. C.; Price, D. L. Senile plaques in the amygdala in Alzheimer’s disease. Neurology 38:227; 1988. Hedreen, J. C.; Raskin, L. S.; Applegate, M. D.; Price, D. L.; Troncoso, J. C. A quick silver method for senile plaques and a comparison of eight methods for the demons~tion of senile plaques. J. Ne~opa~ol. Exp. Neurol. 49:308; 1990.
284
HEDREEN,
14. Hedreen. J. C.; Raskio, L. S.; Struble, R. G.; Price, D. L. Selective silver impregnation of senile plaques: a method useful for computer imaging. J. Neurosci. Meth. 25: 15 I- 158; 1988. 15. Hedreen, J. C.; Raskin, L. S.; Struble, R. G.; Whitehouse, P. J.; price, D. L. A new h&h-contrast silver stain for senile plaques facilitating computer-assisted counting of senile plaques in Alzheimer’s disease and in aging. Sot. Neurosci. Abstr. 12: 101; 1986. 16. Hedreen, J. C.; Price. D. Morphology and classification of senile plaques in Alzheimer’s disease using a new silver method. J. Neuropathol. Exp. Neurol. 47:339; 1988. of senile plaques in 17. Hedreen, J. C.; Price, D. L. Distribution Alzheimer’s disease, as seen with a new silver method. 1. Neuropathol. Exp. Neural. 46335; 1987. of degenerating axoplasm. 18. Heimer, L. Selective silver-impregnation In: Nauta, W. J. H.: Ebbesson, S. 0. E., eds. Contempora~ research methods in neuroanatomy. New York: Springer-Verlag: 1970: 1Ofi-
27.
28. 29.
30.
31.
131. 19. Khachaturian, Z. S. Diagnosis of Alzheimer’s disease. Arch. Neural. 42:1097-l 105; 1985. 20. Lamy, C.; Duyckaerts, C.; Delaere, P.; Payan, C. H.; Fetmanian, J.; Poulain, V.; Hauw, J.-J. Comparison of seven staining methods for senile plaques and neurofibrillary tangles in a prospective series of 15 elderly patients, Neuropathol. Appl. Neurobiol. 15563-578; 1989. 21. Marinesco, G. Etude anatomique et clinique des plaques dites seniles. L’Enc6phaIe 7:105- 132; 1912. G.; Minea, J. Untersuchungen iiber die “senilen 22. Marinesco, Plaques.” Monats. Psychol. Neural. 31:79- 133; 1912. 23. Masters, C. L.; Multhaup, G.; Simms, G.; Pottgiesser, J.; Martins, R. N.; Beyreuther, K. Neuronal origin of a cerebral amyfoid: neuro~b~lla~ tangles of Atzheimer’s disease contain the same protein as the amyioid of plaque cores and blood vessels. EMBO J. 4:27572763; 198.5. 24. Mirra, S. S.; Hart, M. H.; Terry, R. D. Making the diagnosis of Alzheimer’s disease, A primer for practicing pathologists. Arch. Pathol. Lab. Med. 117:132-144; 1993. of degenerating 25. Nauta. W. J. H.: Gvnax. P. A. Silver impregnation axons’in the centraiiervous system: a modified technic. S?ain Technol. 29:91-93; 1954. 26. Pardo, C. A.; Martin, L. J.; Troncoso, J. C.; Price, D. L. The human pineal gland in aging and Alzheimer’s disease: patterns of cytoske-
32.
33. 34.
35.
36.
37.
38.
RASKIN
AND PRICE
letal antigen immunt~r~activity. Acta Neur~~p~~thol. 80:535 --540: 1990. Probst. A.; Brunnschweiler. H.; Lautenschlager. C.; Uirich. J. A special type of senile plaque, possibly an initial stage. Acta Neuropathol. 74:133-141: 1987. Sevier, A. C.; Munger. B. L. A silver method for paraftin sections of neural tissue. J. Neuropathol. Exp. Neural. 24: 130- 135; 1965. Ulrich, J. Alzheimer changes in nondemented patients younger than sixty-five: possible early stages of Alzheimer’s disease and senile dementia of Alzheimer type. Ann. Neurol. 17:273-277; 1985. Ulrich, J.; Probst, A.: Anderton. 8. H.; Kahn. J. Dementia of Alrheimer type (DAT)--a review of its morbid anatomy. Klin. Wochenschr. 64: 103- I 14; 1986. Vallet, P. G.; Guntern. R.; Hof, P. R.; Golaz. J.; Delacourte, A.; Robakis, N. K.; Bouras, C. A comparative study of histological and immunoh~st~hemical methods for neuro~b~llary tangles and senile plaques in Alzheimer’s disease. Acta Neuropathol. 83: 170- 178; 1992. Walker, L. C.; Masters, C.; Beyreuther, K.; Price, D. L. Amyloid in the brains of aged squirrel monkeys. Acta Neuropathol. 80:381387; 1990. Wallington, E. A. The explosive properties of ammoniacal-silver solutions. J. Med. Lab. Tech. 22:220-223; 1965. Wisniewski, H. M.; Wen, G. Y.; Kim, K. S. Comparison of four staining methods on the detection of neuritic plaques. Acta Neuropathol. 78:22-27; 1989. Yamaguchi. H.; Hirai, S.; Morimatsu, M.; Shoji, M.; Ihara, Y. A variety of cerebral amyloid deposits in the brains of the AIzheimertype dementia demonstrated by p protein immunostaining. Acta Neuropa~ol. 76541-549; 1988. Yamaguchi. H.; Hirai, S.; Morimatsu, M.; Shoji, M.; Harigaya, Y. Diffuse type of senile plaques in the brains of Aizheimer-type dementia. Acta Neuropathol. (Berl.) 77:113- 119; 1988. Yamaguchi, H.; Nakazato, Y.; Hirai, S.; Shoji, M.; Harigaya, Y. Electron micrograph of diffuse plaques. Initial stage of senile plaque formation in the Alzheimer brain. Am. J. Pathol. 135593-597; 1989. Yamamoto, T.; Hirano, A. A comparative study of modified Bielschowsky, Bodian and thioflavin S stains on Alzheimer’s neurofibrillary tangles. Neuropathol. Appl. Neurobiol. 12:3-9; 1986.
Pergamon
0361-9230/94$6.00 + .M)
0361-9230(94)00099-9
BRIEF COMMUNICATION
A Quick Silver Method for Senile Plaques and Neurofibrillary Tangles in Paraffin Sections JOHN
C. HEDREEN,*tP
LISABETH
S. RASKJN$
AND
DONALD
L. PRICE*Wj
Departments of *Pathology, fNeuroscience, SNeurology, and #the Neuropathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2196 Received
11 October
1993; Accepted
27 April 1994
HARES, J. C., L. S. RASKIN AND D. L. PRICE. A quick silver method for senile pl~ues and neuro~briifa~ fang/es in paraj3fn sections. BRAIN RES BULL 35(3) 279-284, 1994.-A new silver method for senile plaques and neurofib~ll~ tangles (NFT) in paraffin sections is presented. This new technique is rapid in execution (15 min) and reveals senile plaques of all morphological types as well as NFJ. The silver staining is spatially congruent with immunocytochemical staining for the pamyloid peptide (A/3) in adjacent sections. AD peptide
Alzheimer’s disease
Diagnosis of Alzheimer’s disease
Neurofibrillary tangles
Senile plaques
the ready identification of senile plaques and NFI in standard paraffin sections of the cerebral cortex, hippocampus, amygdala, and nucleus basalis in 9 cases of AD and in brainstem sections of 1 case of progressive supranuclear palsy. Twelve control brains without neurological disease were also examined (age range, 18-73 years). All solutions are used at room temperature.
SENILE plaques and NFT, the characteristic histopathoIog~c~ features of Afzheimer’s disease (AD), are usually demonstrated in autopsied brains using silver methods. A variety of methods have been used, but modifications of the Bielschowsky technique (3,24,28,38) are perhaps the closest to a standard. These methods vary in their efficacy (13,20,31,34,38). Recently, we developed a silver method that specifically stains senile plaques (14,15,17). Neurofibrillary tangles, normal axons, and other features that are usually stained darkly by Biefschowsky procedures are lightly stained by this method. Because of the need to demonstrate NPI’ for diagnostic purposes, we have devised a new method that demonstrates both senile plaques and NFI. The most prominent features of this new procedure are its rapidity in comparison with standard silver methods, its ability to stain senile plaques of all morphological types, and the clarity and delicacy of the silver staining of NFT.
1. Cut paraffin sections (IO- 12 pm), lay out on gelatinized water (about 1.5 g!l) in a water bath, mount onto subbed slides, and dry for l-3 days at 37°C. 2. Deparaffinize and bring to distilled water (DH,O). 3. Immerse in 5% AgNOl in DH20 (5 min) with occasional agitation. 4. Dip briefly in DH20. 5. Immerse in ammoniacal silver solution (2 min) with gentle, continual agitation. This solution consists of the following ingredients, added in the order listed: 18 ml 5% AgNO,; 10 ml 95% ethanol; 2 ml DH20; 1.8 ml concentrated N&OH solution; and 1.5 ml 2.5% NaOH (25). The IQ&OH and NaOH solutions may be mixed together first and added in small aliquots to the silver-ethanol solution with vigorous agitation, or these solutions may be added separately in a similar manner. The ammoniacal silver solution should be made immediately before use. Ingredients may be stored as stock solutions. Because of the potential formation of explosive compounds on standing (33), the solution should be discarded after use, and glassware should be rinsed ~o~ughly.
METHOD
This technique is a modification of our method for senile plaques (14,15,17) and of the 1954 version of the method of Nauta and Gygax (25), both of which are Bielschowsky-type procedures (18). In the new technique, deparaffinized sections are passed through solutions of silver nitrate, ammoniacal silver nitrate, and reducer. Trials were performed that varied the concentration of silver nitrate, the incubation time in silver nitrate, and the concentration of different elements of the ammoniacal silver solution. The following procedure was judged optimal for
Requests for reprints should be addressed to John C. Hedreen, M.D., at his present address: Dept. of Psychiatry #1007, New England Medical Center, 750 Washington St., Boston, MA 02111.
279
280
HE~REEN.
RASKIN
AND PRICE
Drain excess solution quickly, and transfer slides to two solutions of reducer. The reducer solution consists of: 400 ml DH20; 45 ml 95% ethanol; 13.5 ml 10% unbuffered formaldehyde USP. (40 g/IO0 ml formaldehyde with IO-15% methanol); and 13.5 ml 1% citric acid. Agitate slides immediately for S- 15 s in the first reducer solution and then transfer slides to the second solution for up to 2 min. Sections will become a gray to brown color. Rinse three times in DH,O and then immerse (with occasional agitation) in 1.0% sodium thiosulfate for 3 min. Rinse thoroughly three times in DH20, dehydrate, clear, and coverslip. The use of gelatinized water in the cutting water bath proved be essential in obtaining successful staining. It should be pointed out that sections to be stained ~mmun~ytochemically should not be mounted from gelatinized water. Short times {25 min) in the AgNO? solution provide the best contrast between senile plaques and surrounding neuropil and often produce golden-brown NFT against a gray background. At 10 min, the background is partly gray and partly brown, and NFI are more deeply stained. At 20 min, the background is brown and resembles that seen in standard Bielschowsky-type silver stains (24,28,38). However, at this incubation time, contrast is reduced, and NIT are overstained. Using 2% or 5% AgN03 in the first step produces a lighter background than 10% AgN03 and provides better contrast between NFT and normal neurons and between plaques and background. Three, 5, and 7 min trials in 5% AgN07 are recommended when the method is tried initially. The following ammoniacal silver solution, derived from Method One of Fink and Heimer (9), also produces useful results: 50 ml 2.5% AgN03; and 4 ml of a 1:l mixture of concentrated ~monium hydroxide and 2.5% NaOH. The latter is added slowly with vigorous agitation. Because this ammoniacal silver solution produces a pleasing golden-brown background, it may be preferred by some microscopists, but the contrast of plaques and NFI against this background is less than with the method given above. In some cases, adjacent sections were stained with other modern silver methods (6,38), and a Bielschowsky variant method from our laboratory that stains mainly NFT and enlarged neurites in classic senile plaques in AD as well as axonal enlargements in amyo~ophic lateral sclerosis. Some serial sections of amygdala and cerebral cortex were also stained imm~~yt~hemitally (method of Walker et al. (32)) for the A/3 peptide (23) or tau (5,26). Both the new silver method and the immunocytochemical staining procedure for A@ were performed with and without pretreatment in formic acid. RESULTS
Senile plaques were readily seen, including the diffuse type (Fig. 1A) with low amyloid density and few or no fine, abnormal neurites (12,16,35-37). In addition, the classic type of plaque (Fig. 1B) with enlarged dystrophic neurites and high amyloid content as well as perivascular and subpial plaques were readily identified. Senile plaques of the diffuse type in the lateral amygdaloid nucleus, which are difficult to demonstrate with some standard silver techniques (12), were clearly stained with the present method (Fig. 1A). Serial section analysis showed that diffuse plaques demonstrated with AP peptide i~unocytoche~st~ were all stained with the present technique and showed a similar outline (Figs. 1A Br 1C). Formic acid pretreatment was necessary for A@ immunostaining but prevented silver staining of plaques.
Success in staining of plaques was similar to, or better than, that achieved with other modem silver methods and with immuno-
FIG. I. A) Senile plaques of the diffuse, iow amyloid type in the lateral amygdala in AD. New method. Bar, 40 pm. 8) Seniie plaques of the classic, dense amyloid type with dystrophic neurites and cores in the medial amygdala in AD. New method. Bar, 20 pm. C) Section, adjacent section to that of Fig. I A, stained immunocytochemically for AD peptide following formic acid treatment. Bar, 40 pm.
QUICK SILVER METHOD
PIG. 2. Senile plaques in cerebral neocortex in AD demonstrated with three methods. The pial surface in A-C is at upper right. Bar, 200 pm. A) New method. B) Yamamoto and Hirano method. Section adjacent to A. C) Immunocytochemisuy for the AP peptide. Section not adjacent to A or B.
cyi~hemis~ for the A@ peptide (Figs. 2A-C). Perivascular plaques were seen with both i~un~yt~he~~~ and silver methods, but vascular amyloid was not always stained well with the silver method. Reticulin and other elements of blood vessels were variably stained with silver (Fig. 4D), regardless of the presence or absence of amyloid in the blood vessel wall. r~unos~ning and silver staining of various types of senile plaques in the amygdala in the brains of individuals with AD
281
PIG. 3. Serial sections through a diffuse plaque in the lateral amygdala from the brain of a patient with AD. Bar. 20 urn. A) New method. All plaques demonstrated by immun~~o~hemis~ for ;\a were also seen with the new silver method. B) Immunocytochemistry for the Ap peptide. Plaque indicated by arrowheads. C) Immunocytochemistry for tau. Note the absence of staining for enlarged neurites containing cytoskeletal eiements. Very fine, lightly stained processes are present (arrowheads).
were performed in serial sections (16). Senile plaques in the lateral amygdala in most cases were of the type usually designated “diffuse.” Diffuse plaques identified with the new method had the following characteristics: silver stains showed a homogeneous group of fine fibrils (Fig. 3A) without huge, distorted neurites that characterize classic, dense-amyloid plaques (see periphery
FIG. 4. A> Flame-shaped NIT ia pyramida neuron of cerebral neocortex in AD. New method. Bar, 20 pm (for A-Q. B) Globose NFT in neur~rts of nucleus basalis of Meynert irk AD. Note contrast with nearby normal neurot~s. New method. C) Diffuse piaqire and NIT {~~h~ds)~ D) Extracelfular NFT derived from large tayer-El neurons of eotorhinal cortex in AD. No neurons remained in an adjacem H&E-stained section. Four of the many NFT are indicated by arrowheads. New method. Bar. 40 p,rn (for D,E). E) Same area as D; adjacent section stained with the Yamamoto and Hirano method. Note faint or absent staining of NFT.
of plaques in Fig. IB); immunocytochemistry for A/? revealed light, homogeneous staining (Fig. 38); a silver method specific for NFT and eniarged neurites of senile plaques was negative (not shown); and i~~~yt~hem~s~ for tau revealed either no staining or fine, faintty stained elements (Fig. 3C) that were much smaller than the typical club-shaped “neurites” of classic plaques.
Neurofibrilhrry tangles, whether flame shaped or globose, appeared as delicately impregnated, brown-staining cytoplasmic incfusions in neurons of the hippocampal formation, amygdaia, neocortex, and nucleus basaiis in ‘AD (Figs. 4A St B) and in various brainstem nuclei in progressive supranuckar palsy. Because of the short silver nitrate impregnation, NFT are more lightly stained with the present method than with most silver
283
QUICK SILVER METHOD
techniques, and internal structure is often apparent (Fig. 4B). Both plaques and NFT were easily visualized despite short incubations in silver nitrate and ammoniacal silver solutions (Fig. 4C). In some cases, especially with shorter incubation times, NIT were stained a different color than were other tissue elements. In such cases, the brown or golden-brown staining of NFT provided excellent color contrast against the gray staining of other argyrophilic tissue structures. Extracellular “tombstone” NFT in layer II of the entorhinal area were often better demonstrated with our new method than with an excellent modem Bielschowsky variant (Figs. 4D & E) (38). Axons in the neuropil were variably stained in different cases. As a result, the method cannot be used to identify “neuropil threads.” Neuromelanin granules were strongly stained; red blood cells and intracytoplasmic granules in occasional neurons were sometimes stained. Except for very large cells (e.g., oculomotor nucleus, mesencephalic trigeminal nucleus, Purkinje cells), most normal neuronal cell bodies were lightly stained (gray to light brown) (Figs. 3A, 4A & B), in contrast to neighboring neurons that harbor NFT, However, there was variability from case to case in staining of neuronal nuclei and perikarya (note absence of staining in Figs. 1A & B, 4C & D). Control cases showed staining of normal neurons and axons similar to that seen in cases of AD. As expected, occasional cortical senile plaques and ~pp~arnpal NFT were encountered in older individuals. Thus, the pattern and specificity of staining in controIs are similar to those found with other commonly used Bielschowsky method variants (6,24,38). DISCUSSlON The most specific and sensitive histological criterion for the diagnosis of AD at autopsy is the demonstration of numerous senile plaques in the cerebral neocortex (2,19). Neurofibrillary tangles are also important histopathological features in AD; thus, a method for histological diagnosis of AD at autopsy should have the ability to stain both senile plaques and NFT. In addition, such a method should stain all types of plaques demons~able by immun~yt~hemist~ for the A/3 peptide and should be relatively labor efficient and inexpensive. Most silver methods for nervous tissue derive either from the Bielschowsky method (silver nitrate, ammoniacal silver, reducer) or the Cajal method (silver nitrate impregnation of tissue blocks,
reducer) (18). Variants of the Bielschowsky method were used in most of the initial studies with silver methods of senile plaques and NIT ( 1,4,10,11). Staining of neurofibrillar elements, namely NFT and enlarged, often club-shaped, neurites in plaques, was emphasized with these methods. Using a modified Cajal method, Marinesco (21,22) described and illustrated diffuse plaques as we know them today and contrasted the morphology of plaques stained with the Bielschowsky and Cajal methods. Nevertheless, the acceptance of diffuse plaques in descriptions of the histopathoiogy of AD was rare until certain Bielschowsky method variations that reliably demons~at~ this type of plaque came into common use in the last decade (13,14,20,24,27,29-31,34,38) and until i~un~yt~hemis~ for the A/3 polypeptide confirmed the biochemical relationship of diffuse and classic plaques (7,8,35,36). Diffuse plaques, as defined in the Results section, were reliably stained with the present method. The principal advantages of the new technique described here over other silver methods for paraffin sections are: its speed (total staining time < 15 min); the clarity of impregnation of all types of AD peptide-containing senile plaques, including diffuse plaques; and the readiness with which NET can be differentiated from other argyrophilic tissue structures. We have completed a study comparing numbers of senile plaques stained in AD cerebral neocortex with six different silver stains and A/3 peptide immun~yt~hemis~ (13). In this study, the quick silver method described above revealed more neocortical plaques in sections of temporal neocortex from 8 AD cases than any other method. Recent experience in our laboratory indicates that this new method may prove to be useful in both diagnostic and research situations. ACKNOWLEDGEMENTS The authors thank Drs. Lary C. Walker, Juan C. Troncoso, Robert G. Struble, and Linda C. Cork for helpful discussions on the nature and methods of demonstration of plaques and NFT. in addition, the authors thank Lisa J. Sansone, Eleanor Brown, and Leila M. Rowland for testing a variety of silver methods. ~ti~ies against the Afi polypeptide and tau were generous gifts from Drs. Cohn Masters and Konrad Beyreuther and Dr. Lester Binder, respectively. This work was supported by grants from the U.S. Public Health Service (NIH AG 05146, AG 03359). Dr. Price is the recipient of a Javits Neuroscience Investigator Award (NIH NS 10580) and a Leadership and Excellence in Alzheimer’s Disease (LEAD) award (NIA AG 07914).
REFERENCES I. Alzheimer, A. ijber eine eigenartige Erkrankung der Himrinde.
2.
3.
4.
5.
6.
7.
Allg. 2. Psychiatric Psychisch-Gerichtlich Med. 64: 146 148; 1907. Berg, L.; M&eel, D. W.. Jr.: Miller. J. P.: Batv. J.: Morris. J. C. Ne&opathological indexes of Alzheimer’s &e&e in ‘demented and nondemented persons aged 80 years and older. Arch. Neurol. S&349-358; 1993. Bielschowsky, M. Die Sil~~mp~gnadon der Neurofib~llen. Einige Bemerkungen zu der von mir angegebenen Methode und den von ihr gelieferten Bildem. J. Psychol. Neurol. 3: 169- 189; 1904. Bielschowsky, M. Zur Kenntnis der Alzheimerschen Krankheit (pmsenilen Demenz mit Herdsymptomen). 3. Psychol. Neurol. 18:273292; 1911. Binder, L. I.; Frankfurter, A.; Rebhun, L. I. The distribution of tau in the mammalian central nervous system. J. Cell Biol. 101: 137 I1378; 1985. Campbell, S. K.; Switzer, R. C., III; Martin, T. L. Alzheimer’s plaques and tangles: a controlled and enhanced silver staining method. Sot. Neurosci. Abstr. 13:678; 1987. Davies, L.; Wolska, B.; Hilbich, C.; Multhaup, G.; Martins, R.; Simms, G.; Beyreuther, K.; Masters, C. L. A4 amyloid protein deposition and the diagnosis of Alzheimer’s disease: prevalence in aged
8.
9.
10.
1I. 12. 13.
brains determined by immunocytochemistry compared with conventional neuropathologic techniques. Neurology 38: 1688- 1693; 1988. Dickson, D. W.; Farlo, J.; Davies, P.; Crystal, H.; Fuld, P.; Yen, S-.H. C. Alzheimer’s disease: a double-labeling immunohistochemical study of senile plaques. Am. J. Pathol. 132:86- 101; 1988. Fink, R. P.; Heimer, L. Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4:369-374; 1967. Fischer, 0. Die presbyophrene demenz, deren anatomische grundlage und klinische abgrenzung. Zeit. f.d. ges. Neurol. Psychiat. 3:371-471; 1910. Fischer, 0. Miliare Nekrosen mit drusigen Wucherungen der Neurofibrillen, eine regelmtissige Veranderung der Himrinde bei seniler Demenz. Monatsschr. Psychiatr. Neurol. 22:361-372; 1907. Hedreen, J. C.; Broadhead, J. C.; Price, D. L. Senile plaques in the amygdala in Alzheimer’s disease. Neurology 38:227; 1988. Hedreen, J. C.; Raskin, L. S.; Applegate, M. D.; Price, D. L.; Troncoso, J. C. A quick silver method for senile plaques and a comparison of eight methods for the demons~tion of senile plaques. J. Ne~opa~ol. Exp. Neurol. 49:308; 1990.
284
HEDREEN,
14. Hedreen. J. C.; Raskio, L. S.; Struble, R. G.; Price, D. L. Selective silver impregnation of senile plaques: a method useful for computer imaging. J. Neurosci. Meth. 25: 15 I- 158; 1988. 15. Hedreen, J. C.; Raskin, L. S.; Struble, R. G.; Whitehouse, P. J.; price, D. L. A new h&h-contrast silver stain for senile plaques facilitating computer-assisted counting of senile plaques in Alzheimer’s disease and in aging. Sot. Neurosci. Abstr. 12: 101; 1986. 16. Hedreen, J. C.; Price. D. Morphology and classification of senile plaques in Alzheimer’s disease using a new silver method. J. Neuropathol. Exp. Neurol. 47:339; 1988. of senile plaques in 17. Hedreen, J. C.; Price, D. L. Distribution Alzheimer’s disease, as seen with a new silver method. 1. Neuropathol. Exp. Neural. 46335; 1987. of degenerating axoplasm. 18. Heimer, L. Selective silver-impregnation In: Nauta, W. J. H.: Ebbesson, S. 0. E., eds. Contempora~ research methods in neuroanatomy. New York: Springer-Verlag: 1970: 1Ofi-
27.
28. 29.
30.
31.
131. 19. Khachaturian, Z. S. Diagnosis of Alzheimer’s disease. Arch. Neural. 42:1097-l 105; 1985. 20. Lamy, C.; Duyckaerts, C.; Delaere, P.; Payan, C. H.; Fetmanian, J.; Poulain, V.; Hauw, J.-J. Comparison of seven staining methods for senile plaques and neurofibrillary tangles in a prospective series of 15 elderly patients, Neuropathol. Appl. Neurobiol. 15563-578; 1989. 21. Marinesco, G. Etude anatomique et clinique des plaques dites seniles. L’Enc6phaIe 7:105- 132; 1912. G.; Minea, J. Untersuchungen iiber die “senilen 22. Marinesco, Plaques.” Monats. Psychol. Neural. 31:79- 133; 1912. 23. Masters, C. L.; Multhaup, G.; Simms, G.; Pottgiesser, J.; Martins, R. N.; Beyreuther, K. Neuronal origin of a cerebral amyfoid: neuro~b~lla~ tangles of Atzheimer’s disease contain the same protein as the amyioid of plaque cores and blood vessels. EMBO J. 4:27572763; 198.5. 24. Mirra, S. S.; Hart, M. H.; Terry, R. D. Making the diagnosis of Alzheimer’s disease, A primer for practicing pathologists. Arch. Pathol. Lab. Med. 117:132-144; 1993. of degenerating 25. Nauta. W. J. H.: Gvnax. P. A. Silver impregnation axons’in the centraiiervous system: a modified technic. S?ain Technol. 29:91-93; 1954. 26. Pardo, C. A.; Martin, L. J.; Troncoso, J. C.; Price, D. L. The human pineal gland in aging and Alzheimer’s disease: patterns of cytoske-
32.
33. 34.
35.
36.
37.
38.
RASKIN
AND PRICE
letal antigen immunt~r~activity. Acta Neur~~p~~thol. 80:535 --540: 1990. Probst. A.; Brunnschweiler. H.; Lautenschlager. C.; Uirich. J. A special type of senile plaque, possibly an initial stage. Acta Neuropathol. 74:133-141: 1987. Sevier, A. C.; Munger. B. L. A silver method for paraftin sections of neural tissue. J. Neuropathol. Exp. Neural. 24: 130- 135; 1965. Ulrich, J. Alzheimer changes in nondemented patients younger than sixty-five: possible early stages of Alzheimer’s disease and senile dementia of Alzheimer type. Ann. Neurol. 17:273-277; 1985. Ulrich, J.; Probst, A.: Anderton. 8. H.; Kahn. J. Dementia of Alrheimer type (DAT)--a review of its morbid anatomy. Klin. Wochenschr. 64: 103- I 14; 1986. Vallet, P. G.; Guntern. R.; Hof, P. R.; Golaz. J.; Delacourte, A.; Robakis, N. K.; Bouras, C. A comparative study of histological and immunoh~st~hemical methods for neuro~b~llary tangles and senile plaques in Alzheimer’s disease. Acta Neuropathol. 83: 170- 178; 1992. Walker, L. C.; Masters, C.; Beyreuther, K.; Price, D. L. Amyloid in the brains of aged squirrel monkeys. Acta Neuropathol. 80:381387; 1990. Wallington, E. A. The explosive properties of ammoniacal-silver solutions. J. Med. Lab. Tech. 22:220-223; 1965. Wisniewski, H. M.; Wen, G. Y.; Kim, K. S. Comparison of four staining methods on the detection of neuritic plaques. Acta Neuropathol. 78:22-27; 1989. Yamaguchi. H.; Hirai, S.; Morimatsu, M.; Shoji, M.; Ihara, Y. A variety of cerebral amyloid deposits in the brains of the AIzheimertype dementia demonstrated by p protein immunostaining. Acta Neuropa~ol. 76541-549; 1988. Yamaguchi. H.; Hirai, S.; Morimatsu, M.; Shoji, M.; Harigaya, Y. Diffuse type of senile plaques in the brains of Aizheimer-type dementia. Acta Neuropathol. (Berl.) 77:113- 119; 1988. Yamaguchi, H.; Nakazato, Y.; Hirai, S.; Shoji, M.; Harigaya, Y. Electron micrograph of diffuse plaques. Initial stage of senile plaque formation in the Alzheimer brain. Am. J. Pathol. 135593-597; 1989. Yamamoto, T.; Hirano, A. A comparative study of modified Bielschowsky, Bodian and thioflavin S stains on Alzheimer’s neurofibrillary tangles. Neuropathol. Appl. Neurobiol. 12:3-9; 1986.