- Email: [email protected]
[44] A mouse model for serum amyloid A amyloidosis
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15. Transfer the slides to the DAB solution and allow 10 min for the reaction. 16. Quench the reaction by immersing the slides in 0.1 M Tris-HCl, pH 7.4, for 15-30 min. 17. Counterstain nuclei using 1% methyl green in acetate buffer for 5 min. 18. Give three quick rinses in water (overextensive washing will easily remove the stain). Allow the slides to completely air dry for 30-60 min. 19. Transfer the slides to Histoclear (National Diagnostic Lab.), incubate for 15 min, and replace with fresh Histoclear. The slides can remain in this solution until mounting. 20. Mount the slides with DPX mounting medium (Fisher Scientific). Avoid trapping air bubbles between the slide and cover glass. Leave to dry on the bench overnight, and peel away excess mounting medium the following day.
Acknowledgements This work was supported by grants from the Wellcome Trust, the Deutsche Forschungsgemeinschaft, the European Union (BMH4 CT96 0244), the Hereditary Disease Foundation (in the form of an award to GB from Harry Lieberman), the Huntington's Disease Society of America, and the Special Trustees of Guy's Hospital.
[44] A M o u s e M o d e l f o r S e r u m A m y l o i d A A m y l o i d o s i s
By M A R K
S. KINDY a n d FREDERICK C. DE BEER
Introduction Amyloid A (AA) amyloidosis is associated with rheumatic diseases, certain chronic inflammatory diseases, chronic infections, and some malignant disorders. Crohn's disease and rheumatoid arthritis (particularly juvenile rheumatoid arthritis) are frequently complicated with amyloidosisl; whereas, in ulcerative colitis and systemic lupus erythematosis, amyloidosis is a rare confounding problem. Differences seen in these disease states are most likely due to altered regulation in the cytokine-mediated changes in amyloid precursor protein serum amyloid A (SAA) production. Renal 1 M. A. Gertz and R. A. Kyle, Medicine (Baltimore) 70, 246 (1991).
METHODS IN ENZYMOLOGY,VOL.309
Copyright© 1999by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/99 $30.00
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carcinomas and Hodgkin's disease are known to be associated with fevers and systemic inflammatory features leading to serum amyloid A induction. A A amyloid incidence has decreased considerably since the early 1980s as a result of therapeutic interventions that reduce inflammation. 2 Nevertheless, our capability to maintain individuals suffering from various types of trauma has contributed to an increase in systemic A A amyloidosis, particularly due to chronic urinary tract infections. Idiopathic inflammatory disease (rheumatoid arthritis, juvenile rheumatoid arthritis, and chronic infections) is the leading cause of A A amyloidosis in the Western Hemisphere. Generally, 50% of patients with amyloidosis succumb to the disease within the first 5 years after diagnosis and an additional 25% between 5 and 15 years. The deposition of A A amyloid involves multiple organ systems, usually without any initial symptomatic presentations. Approximately 25% of individuals with amyloidosis ultimately present with renal or glomerular involvement. 3 The typical clinical presentation is proteinurea due to the deposition of amyloid fibrils in the glomerulus. This results in a nephrotic syndrome leading to renal insufficiency or end-stage renal failure, the most common cause of death in these patients. Deposits can usually be detected in the spleen with some involvement of the liver, adrenals, and, to a lesser degree, in the heart, gut, and other tissues. Dialysis and renal transplantation can exacerbate the deposition of amyloid fibrils in other organs. 4 Therefore, there is a growing need to understand the process of systemic amyloid deposition and its complications. The mouse amyloid model provides an ideal system for understanding amyloid fibrillogenesis. 5 Serum amyloid A proteins are a group of highly homologous apoproteins associated with high-density lipoproteins (HDLs) and are the precursors of A A amyloid. 6 Serum amyloid A proteins are the most dramatically increased acute-phase proteins in mice with levels increasing up to 1000-fold (from 1-5 to 1000/xg/ml) within 24 hr of an inflammatory response. 7 In the mouse, SAA1 and SAA2 are induced in the liver by circulating cytokines [interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF)] in response to amyloid induction paradigms (Fig. 1). These two isotypes differ in only 9 of 103 amino adds; however, only SAA2 is 2 M. Pras, Scand. J. Rheumatol. 27, 92 (1998). 3 A. Montoli, E. Minola, F. Stabile, C. Grillo, L. RadaeUi, D. Spanti, E. Luccarelli, C. Spata, and L. Minetti, Am. J. Nephrol. 15, 142 (1995). 4 S.-Y. Tan, A. Irish, C. G. Winearls, E. A. Brown, P. E. Gower, E. J. Clutterbuck, S. Madhoo, J. P. Lavender, and M. B. Pepys, Kidney Int. 50, 282 (1996). 5 R. L. Meek, J. S. Hoffman, and E. P. Benditt, J. Exp. Med. 163, 499 (1986). 6 m. Husebekk, B. Skogen, and G. Husby, Scand. J. Imrnunol. 25, 375 (1987). 7 K. P. W. J. McAdam, and J. D. Sipe, J. Exp. Med. 144, 1121 (1976).
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selectively deposited into amyloid fibrils (Fig. 2). SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2 (Fig. 2). 8 Even though the CE/J protein overall has features similar to the SAA2 protein, it is not deposited into amyloid fibrils. Therefore, the 8 M. C. de Beer, F. C. de Beer, W. D. McCubbin, C. M. Kay, and M. S. Kindy, J. Biol. Chem. 268, 20606 (1993).
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CE/J mouse does not develop amyloid. 9 Differences in the CE/J protein that confer amyloid resistance appear to be at the amino acid positions 6 and 7, where it shows similarity with the nonamyloidogenic SAAa protein (Fig. 2). 8"1°Linkage analysis with amyloid-resistant mice (CE/J) and amyloid-susceptible mice (CBA/J) demonstrated that the CE/J isoform confers protection against amyloid deposition even in the presence of the SAA2 isoform, la These data indicate valuable information that can be obtained from inbred strains of mice on the structural properties and specific interactions that may occur in the process of amyloidogenesis. Our current understanding of the processes involved in AA amyloidosis is that it is the result of a two-stage mechanism (Fig. 3). 12 First, the generation of A A amyloid requires the synthesis of a precursor protein in sufficient quantities to allow for deposition. This is referred to as the preamyloid phase, which can persist for several days to months, depending on the stimulus and the levels of SAA expression. In response to a chronic or recurrent acute inflammatory condition, macrophage activation leads to the synthesis and secretion of cytokines (IL-1, IL-6, and TNF). These cytokines result in the hepatic synthesis of acute-phase proteins, specifically SAA proteins that are secreted and associated with H D L particles. 7 Second, in the amyloid phase, the continuous maintenance of high levels of the precursor is required to generate a nidus or fibrillar network (also referred to as amyloid-enhancing factor, AEF) onto which amyloid can be deposited. 13 The production of AEF results in the rapid deposition of A A fibrils, which can occur in days. Amyloid deposition first occurs in the spleen, liver, and kidney, and eventually amyloid can be detected in every organ, with the possible exception of the brain. However, studies have demonstrated the presence of S A A proteins in the brains of patients with Alzheimer's disease. 14'15 Amyloid deposition in the mouse is extremely rapid, with extensive deposits detectable following amyloid induction paradigms. Injection of a modified casein solution results in amyloid deposition in various organs in 9 j. D. Sipe, I. Carreras, W. A. Gonnerman, E. S. Cathcart, M. C. de Beer, and F. C. de Beer, Am. J. Pathol. 143, 1480 (1993). 10H. Patel, J. BramaU, H. Waters, M. C. de Beer, and P. Woo, Biochem. J. 318, 1041 (1996). 11 W. A. Gonnerman, R. Elliot-Bryant, I. Carreras, J. D. Sipe, and E. S. Cathcart, J. Exp. Med. 181, 2249 (1995). 12 I. Kushner and D. L. Rzewnicki, Clin. Rheumatol. 8, 513 (1994). 13M. A. Axelrad, R. Kisilevsky, J. Willmer, S. J. Chen, and M. Skinner, Lab. Invest. 47, 139 (1982). 14j. S. Liang, J. A. Sloane, J. M. Wells, C. R. Abraham, R. E. Fine, and J. D. Sipe, Neurosci. Lett. 225, 73 (1997) 15 M. S. Kindy, J. Yu, J. T. Gao, and H. Zhu, J. Alzheimer's Dis., in press (1999).
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21 days after initiation. The use of AEF and silver nitrate has reduced this time to 3 days.13 In addition, mouse AA amyloid has serum amyloid P component (SAP) and heparan sulfate proteoglycan (HSPG) associated with the amyloid fibrils. 16J7 Studies have shown that the mouse apolipoprotein E (apoE) is associated with mouse A A amyloid.TM Kisilevsky et al. 19 showed that a series of small anionic sulfates and sulfonates interfered with the S A A - H S P G interaction and were effective in not only inhibiting amyloidogenesis but in reversing the process in vivo. These studies indicate the importance of HSPG in the events associated with amyloid formation. The generation of SAP and apoE knockout mice showed that inactivation of these genes resulted in delayed and reduced deposition of amyloid.2°'21 As suggested earlier, the involvement of accessory factors in the evolution of amyloid diseases needs to be examined as a possible mechanism in the development of therapeutic strategies for treatment of these diseases. This article describes the methods used in the generation of AA amyloid in the mouse as a model of amyloid disease. Several different protocols have been used in the deposition of AA amyloid in mice. The first requires the continuous injection of a modified casein solution over an extended period of time to initiate amyloid deposition. The second utilizes AEF and silver nitrate to give extensive amyloid deposits within 3-5 days.
Induction of AA Amyloid in Mouse Initial studies on amyloid induction in the mouse were performed using casein and azocasein with multiple injections over a several week period. 22 For induction of amyloid with casein or azocasein, 0.5 ml of a prepared 10% solution of sterile azocasein is injected subcutaneously daily for up to 21 days (or sometimes the injections are performed 5 days a week for up to 4 weeks). Injection of the azocasein results in a sterile abscess, which will cause an inflammatory reaction inducing SAA in the mouse. Using this paradigm, significant amounts of amyloid develop over the 21-day period. Initial deposition does not begin for several days after the injections 16M. B. Pepys, in "Amyloidosis" (J. Marrink and M. van Ryswijk, eds.), p. 43. Martinus Nijhoff, Boston, 1986. 17 A. D. Snow, J. Willmer, and R. Kisilevsky, Lab. Invest. 56, 170 (1987). i8 M. S. Kindy, A. R. King, G. Perry, M. C. de Beer, and F. C. de Beer, Lab. Invest. 73, 469 (1995). 19 R. Kisilevsky, L. J. Lemieux, P. E. Faser, X. Kong, P. G. Hultin, and W. A. Szarek, Nature Med. 2, 143 (1996). 20 M. Botto, P. N. Hawkins, M. C. M. Bicerstaff, J. Herbert, A. E. Bygrave, A. McBride, W. L. Hutchinson, G. A. Tennent, M. J. Walport, and M. B. Pepys, Nature Med. 3, 855 (1997). 21 M. S. Kindy and D. J. Rader, A m . J. Pathol. 152, 1387 (1998). 22 D. T. Janigan, A m . J. Pathol. 55, 379 (1969).
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have commenced, delaying the process. This is due to the synthesis and initial deposition of the amyloid fibrils onto which amyloid can form. Subsequently, amyloid is now induced in the mouse model by the injection of A E F (100/zg protein) intravenously via the tail vein followed by a subcutaneous injection of 0.5 ml of a 2% solution of silver nitrate (sterile preparation in water). 23 The AEF is injected into the tail vein as follows: the mouse is placed in a mouse holder and the tail is heated for 5 to 10 sec in a water bath or beaker of water heated to 50°. This will bring the tail vein to the surface and allow for easier injection of AEF. The tail is removed from the water bath and swabbed with alcohol to sterilize and then A E F (100 tzg in 100/zl) is injected with a 29-gauge tuberculin syringe into the tail vein. Immediately following the tail vein injection, the animals are treated with a subcutaneous injection of sterile silver nitrate (0.5 ml of a 2% solution). This allows for extensive deposition of amyloid within 3-5 days. Studies have shown that in the AEF and silver nitrate injection paradigm, amyloid deposits can be detected as early as 12 hr posttreatment. This allows for a rapid model of amyloid formation to test the effects of gene mutations on amyloid formation and the effects of inhibitors of amyloid deposition. A A amyloid deposits first develop in the spleen, possibly due to the metabolic effects of the spleen. Secondary deposition occurs in the liver, heart, kidneys, lungs, intestine, and every major organ except the brain. If the inflammatory stimulus is maintained for extended periods of time, the amyloid deposits will continue to expand, resulting in organ failure and death. Amyloid-lnducing Agents
Preparation of Casein Various experimental protocols have been utilized to induce amyloid deposition in animals over the years. It was originally assumed that amyloidosis was due to chronic infections or the presence of bacterial toxins. Subsequent to these findings, it was shown that the injection of sodium caseinate gave rise to experimental amyloidosis in mice. The hypothesis was that the presence of large quantities of protein material led to insolubility and deposition of the amyloid substance. Since that time, a modified form of casein, azocasein, has been used extensively for the generation of amyloid in the mouse model. 24 The preparation of azocasein is time23 M. Axelrad, R. Kisilevsky, and S. Beswetherick, A m . J. PathoL 78, 277 (1975). 24 D. T. Janigan and R. L. Druet, Am. J. Pathol. 48, 1013 (1966).
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consuming and there can be variability if the proper casein is not used in the preparation protocol. Hammerstein-grade casein from ICN (Costa Mesa, CA) has been used successfully in the production of azocasein that will consistently give amyloid deposits in mice. A solution of sulfanilic acid (20 g, sodium salt, Sigma Chemical Co., St. Louis, MO) is prepared in 400 ml of cold distilled-deionized water containing 12 ml of 5 N NaOH. To this solution, 4.4 g of NaNO3 is added and allowed to dissolve. Next, 32 ml of 5 N HCI, followed by 32 ml of 5 N NaOH, is added and the solution is stirred continuously in the cold. Casein (100 g) is dissolved in 2 liters of a 1% (w/v) NaHCO3 solution prepared in distilled-deionized water and cooled on ice to 4 °. The casein solution is added slowly to the sulfanilic acid to allow the casein to remain in solution. If the casein is added too rapidly, it will precipitate out of solution. The combination will turn a deep red color once the casein goes into solution and should be mixed for 2-3 hr in the cold. The azocasein is precipitated out of solution by bringing the pH to 2-3 with the addition of 0.2 N HC1, added slowly to allow for a flocculent precipitate to form. The precipitate (yellowish in color) can be filtered through cheesecloth in a large filter funnel. The azocasein is dissolved in a large volume of 0.2 N NaOH (1 liter) and is subjected to two additional rounds of precipitation and solubilization. The final precipitate is resuspended in 0.2 M NaHCO3 and dialyzed in 10- to 12-kDa molecular mass dialysis tubing for 3 days against cold distilled-deionized water, changing the water twice a day. The azocasein solution is lyophilized completely (several days) and stored at - 8 0 ° as a powder. For injection of the azocasein, a 10% solution in 0.01 M NaHCO3 (5 g in 50 ml) is prepared in 50-ml polypropylene tubes and adjusted to a final pH of 6.5. The solution is sterilized by the addition of 1% diethyl pyrocarbonate.
Preparation of Amyloid-Enhancing Factor Over time, amyloid deposition was considered to be a result of or related to an immunological response in human and animal models. The general principle has been that the subcutaneous injection of proteinaceous material would cause an immunologic reaction and result in amyloid formation. The involvement of the immune system in the amyloidogenic process suggested the presence of factors that may contribute to amyloid deposition. Transfer experiments using tissue and cells from amyloidogenic animals demonstrated the presence of an enhancing factor in the amyloid tissue. 25'26Subse25 F. Hardt and P. Ranlov, Int. Rev. Exp. Pathol. 49, 273 (1976). 26 I° Keizman, A. Rimon, E. Sohar, and J. Gafni, Acta Pathol. Microbiol. Scand. (A) 233, 172 (1970).
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quent studies by Kisilevsky and co-workers 27 have shown that the amyloid tissue contained amyloid-enhancing activity, which facilitated the deposition of amyloid in the mouse. Extraction of A E F from amyloidotic tissue (spleens and livers) and subsequent injection into mice with an inflammatory stimulus reduce the time of amyloid deposition from 4 weeks to 3 days. The composition of A E F is still not known; however, it is thought to be composed of preformed amyloid fibrils and proteoglycans. Studies have strengthened this hypothesis. Injection of amyloidogenic peptides from SAA and transthyretin proteins, as well as from A/3 peptides, resulted in the rapid accumulation and deposition of A A amyloid. 28'29 The utilization of SAA and other amyloidogenic peptides indicates that the amyloidogenic activity of A E F is due to amyloid fibrils. A E F appears to act as a seed or nucleus onto which the amyloid can form. Amyloid-enhancing factor is prepared from the spleens and livers of animals that were injected with azocasein or AEF and silver nitrate to establish amyloid deposits, as described previously. 3° Animals injected with casein for 4 weeks or A E F and silver nitrate for 2 weeks are taken for A E F preparation. Using sterile techniques, the spleens and livers are removed from the amyloidotic mice (about 25 animals) and homogenized in 10 volumes of phosphate-buffered saline (PBS) containing protease inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF), 1/zM leupeptin; 5/xg aprotinin/ml] using a homogenizer (Polytron, Brinkmann Instruments, Westbury, NY). The tissue is homogenized on a low setting until a homogeneous suspension is obtained. The homogenate is centrifuged at 10,000 rpm for 20 min at 4 ° in a superspeed centrifuge. The pellet is resuspended in 10 volumes of PBS plus protease inhibitors (low-speed homogenization to disrupt the pellet) and centrifuged repeatedly until the optical density of the supernatant at 280 nm is less than 0.1. The final pellet is homogenized/ extracted with sterile/distilled water (10 times the volume of the pellet). The sample is centrifuged at 16,000 rpm for 1 hr at 4°. The first supernatant can be discarded. The water extractions are repeated; the second and third supernatants are saved and quantified for protein content. Samples of AEF are stored frozen at - 8 0 ° and tested for activity as described previously. Detection of AA Amyloid Several methods have been used in the detection of A A amyloid. The most frequently used method is Congo red staining in which the Congo 27 R. Kisilevsky, M. D. Benson, M. A. Axelrad, and L. Boudreau, Lab. Invest. 41, 206 (1979). 28 K. Johan, G. Westermark, U. Engstrom, A. Gustausson, P. Jultman, and P. Westermark, Proc. Natl. Acad. Sci. U.S.A. 95, 2558 (1998). 29 M. S. Kindy, unpublished results (1999). 30 R. Kisilevsky and L. Boudreau, Lab. Invest. 48, 33 (1983).
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red dye will stain amyloid structures specifically (the cross/3-pleated sheet arrangement). 31 Congo red appears to bind to linear polymers that have a similar structure to cellulose. 24 Tissue sections should be fixed in 10% (w/v) buffered formalin and sections should be 6-12/xm in thickness.
Congo Red Staining Paraffin-embedded sections are deparaffinized and hydrated to water (two changes for 5 min each in xylene, 100, 95, 80, 50, and 30% ethanol, then distilled water). The sections are stained in a 1% solution of Congo red for I hr at room temperature (1 g Congo red, color index number 22120 containing 98% dye [Sigma, St. Louis, MO], in 100 ml of distilled water). The Congo red solution should be stirred for several hours at room temperature to allow the dye to dissolve completely, then filtered through a Whatman (Clifton, N J) No. 2 filter. This solution should be used within i month, as beyond this time the dye will not bind well to the amyloid. The sections are rinsed in running water and differentiated in alkaline alcohol (0.01% sodium hydroxide in 50% ethyl alcohol) for 3-5 sec and rinsed for 5 rain in running water. It is easy to overdifferentiate the sections; if the sections are pale, repeat the Congo red staining. Counterstain with Mayer's hematoxylin for 5 min, wash for 15 rain in water, dehydrate to xylene (two changes for 5 min in 50, 80, 95, and 100% ethanol followed by two changes in xylene), and mount with Permount (Fisher, Pittsburgh, PA). Results of the staining protocol are amyloid stains red to pink, nuclei stain blue, elastic fibers stain light red, and most other structures are not stained. When examined under light microscopy with polarizing filters (light-polarizing microscope filter set 31-52-62-75) the amyloid material shows an applegreen birefringence surrounding the splenic follicles (Fig. 4A). In the spleen, amyloid deposits are detected surrounding the splenic follicles, beginning as small amounts of congophilic material that accumulate over time in the presence of a continuous inflammatory response. In Fig. 4, the amyloid present in the spleen creates a dramatic effect with large deposits around the follicles. In other organs, the amyloid is more diffuse, with vascular amyloid appearing in the veins and arteries, as well as general deposits in the tissue.
Immunocytochemical Detection A second method of staining amyloid is by immunocytochemical analysis using rabbit antimouse SAA or A A antibodies. TM After generation of amyloid, the tissue (spleen, liver, heart, etc.) is drop fixed in formalin and embedded in paraffin. Alternatively, the animal can be perfused with cold 31H. Puchtler, F. Sweat, and M. Levine,J. Histochem. Cytochem. 10, 355 (1962).
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B
FIG. 4. Detection of A A amyloid in the spleen. (A) Congo red staining of an amyloidotic spleen and visualization using bipolar filters of apple-green birefringent material. (B) Immunocytochemical staining of amyloid. An antibody against mouse SAA proteins was used as the primary antibody. Detection was performed with a peroxidase secondary antibody and diaminobenzidine hydrochloride. Dark staining indicates A A fibrils and amyloid deposits.
saline (25 ml) followed by 4% paraformaldehyde (25 ml) and the tissues harvested. Paraformaldehyde (PF, 4 g) is suspended in saline and adjusted to a pH of 8.0 and heated to 60° to allow the PF to dissolve. The solution is cooled and adjusted to pH 7.4 for perfusion. Saline and paraformaldehyde are perfused by means of a 30-cm 3 syringe and 23-gauge needle inserted into the heart while the animal is anesthetized. After perfusion, the tissues are removed and submerged in fresh 4% (w/v) paraformaldehyde for 24 hr and then in 30% sucrose (30 g sucrose in 100 ml saline). After 24 to 48 hr, the tissue can be frozen in OCT medium (Tissue-Tek, Torrance, CA) and stored at - 8 0 °. Eight- to 10-t~m sections of tissue are prepared and mounted on Superfrost Plus microscope slides (Fisher, Pittsburgh, PA) coated with 1% gelatin for immunocytochemistry. For slide preparation, 1 g of gelatin is dissolved in 100 ml water by heating and stirring. Slides are dipped in gelatin and allowed to air dry. Paraffin sections are deparaffinized and hydrated to water (as described earlier) and incubated for 3 min in 100% formic acid to help disrupt amyloid fibrils (this allows for better detection of the amyloid fibrils by exposing the epitopes on the A A for antibody binding). Sections are incubated in hydrogen peroxide (3% in methanol for 30 min) to remove any endogenous peroxidase activity in the tissue and hydrated (two changes of 5 min each) to Tris-buffered saline (TBS; 0.1 M Tris, 0.150 M NaCI, pH 7.4). Sections are incubated in 15% goat serum (in TBS) to block nonspecific sites and are incubated in the presence of the primary antibody at a dilution of 1 : 2000 (rabbit antimouse SAA antibody). After incubation at room temperature for 1 hr or overnight at 4°, the sections are washed (3 x 15 min in TBS plus 15% goat serum) and incubated with the secondary antibody (peroxidase-conjugated goat antirabbit; 1 : 5000; Sigma Chemical Co., St. Louis, MO) for 1 hr at room temperature. Sections are washed as before and are developed in enzyme
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buffer containing diaminobenzidine hydrochloride (50/~g/ml) until staining appears (2 to 10 min). Staining is stopped by rinsing in water and counterstaining for 10 sec in hematoxylin (Sigma, St. Louis, MO). Slides are dehydrated to xylene (reverse of process for hydration, as described earlier) and mounted with Permount. Figure 4B illustrates the presence of immunoreactive AA protein detected in the amyloid deposits. As with the Congo red staining, the splenic deposits are detected around the follicles. In animals with extensive amyloid, the deposits can extend from each follicle to overlap and appear to be a solid sheet of amyloid.
123[-Labeled S A P Detection o f A m y l o i d Amyloid in mouse and human can be monitored specifically by using quantitative scintigraphy with 123I-labeled human serum amyloid P (SAP) component or technetium-99m pyrophosphate. 32,33 SAP is used for the general detection of amyloid deposits, and pyrophosphate has been used in detecting amyloid in cardiac amyloidosis. We will focus on the SAP detection system because it is more relevant to these studies. Human SAP is isolated by affinity chromatography to 99% purity from sterile serum heated to 56° for 30 min. 34 Human sera (2 liters) is passed over a 5 x 85cm column of Sepharose 4B equilibrated with Tris-Ca buffer (0.01 M Trisbuffered 0.14 M NaC1, pH 8.0, containing 0.002 M CaCI2 and 0.1 g/liter NAN3) at 100 ml/hr at 4°. The column is washed in Tris buffer until the effluent is zero at A280 and the bound protein elutes Tris buffer with EDTA (0.01 M EDTA). The eluted protein is passed over Sepharose-anti-NHS (normal human serum, 10 ml) followed by Blue-Sepharose (1 ml) to remove contaminating serum proteins. The samples are concentrated to 5-10 ml and gel filtered on a 2.6 x 100-cm column of Sephacryl S-300 (Amersham Pharmacia Biotech, Piscataway, N J) eluted with Tris-EDTA at a flow rate of 16 ml/hr (5.8-ml fractions). Peak SAP as determined by Western blot analysis or electroimmunoassay is pooled, concentrated, and stored under liquid nitrogen. SAP is labeled with iodine-123 using N-bromosuccinimide and is purified by gel filtration on Sephadex G-25 (Sigma, St. Louis, MO). Approximately 100/zg of labeled SAP (550/zCi), administered by intravenous injection, will bind specifically but in a reversible fashion to amyloid fibrils in vivo. Mice injected with 123I-labeled SAP are subjected to whole body scintigraphy. Animals are anesthetized [chloral hydrate (350 mg/kg) and xylazine (4 mg/kg)] and scanned with a Toshiba gamma camera and pinhole collimator for 1-4 min at t = 0 to t = 24 hr. The labeled SAP will 32p. N. Hawkins,J. P. Lavender,and M. B. Pepys,N. Engl. J. Med. 323, 508 (1990). 33p. N. Hawkins,M. J. Myers,A. A. Epenetos,D. Caspi,and M. B. Pepys,J. Exp. Med. 167, 903 (1988). 34F. C. de Beer and M. B. Pepys,J. IrnmunoL Methods 50, 17 (1982).
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localize to the amyloid deposits dependent on the amount of amyloid present in the tissue. Thus, one can determine the percentage of the injected dose that is retained in the body. In addition, the organs can be removed and counted in a gamma counter to determine the amount of labeled SAP that localizes to that organ (i.e., spleen). This number corresponds to the histological grade described later. Quantification of Amyloid Load (Amyloid Deposition) The quantification of amyloid varies dramatically from estimated semiquantitative analysis to quantitative analysis by determining the proportion of the area of the sections occupied by amyloid material. Original analysis was performed by visual examination to determine the amount of amyloid based on a crude scale of 0 to 4Y The grade is as follows: grade 0, no amyloid; grade i, trace amounts of amyloid in the perifollicular zone; grade 2, narrow ring of amyloid in the perifollicular zone; grade 3, broad bands of amyloid in the perifollicular zone; and grade 4, broad bands of amyloid with bridging between the follicles. To quantitate the amount of amyloid present in the tissue of mice, the following protocol is used. 19~I A standard set of amyloid-containing tissues is generated (0, 10, 20, 30, 40, 50% of tissue containing amyloid). These are used as reference points to determine the amount of amyloid in a given tissue. Standard sections are examined under the microscope (Nikon, Garden City, NY; using polarizing filters to generate birefringence) and recorded on videotape (Sony, New York, NY). The images are digitized and transferred to a Macintosh computer for analysis. The digitized images are analyzed (Kontron, Prism, or MCID image analysis) for color (intensity and area, operator specified area of interest) under low power (20 ×). This generates a standard for comparison. Experimental tissue sections are analyzed and compared to the standards for the quantitation of amyloid. Approximately 20 sections from each spleen are examined by image analysis and averaged for amyloid quantification. Data generated from these studies are calculated as the percentage of amyloid in the tissue (i.e., the area of amyloid present in the spleen to the total area). Alternatively, sections are stained with fluorescence-labeled anti-SAA or SAP (I/1000 dilution) antibodies and images are analyzed on a confocal laser scanning microscope (Molecular Dynamics, Sunnyvale, CA) using ImageSpace ScanControl software and a SGI UNIX workstation (Summit, NJ). In most cases the spleens are analyzed for amyloid; however, we will take other tissues to verify the levels of amyloid in the mice. In addition, some animals are injected with 1251-1abeled SAP (labeled as described for Iz31-1abeled SAP) for the quantitative assessment of amyloid. 35E. S. Cathcart, C. A. Leslie,S. N. Meydani, and K. C. Hayes,J. Immunol. 139, 1850(1987).
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Animals are injected with 0.6/zCi of SAP (0.4/zCi//zg) at t = 0 and killed at t = 24 hr, the animals are bled out, and the organs weighed and subjected to gamma counting. SAP is cleared rapidly into the amyloid tissue, and quantitation by homogenization and scintillation counting is very reproducible. Inhibition of Amyloid Fibrillogenesis The mouse A A model is ideal for studying fibrillogenesis and developing inhibitors of fbril formation. As discussed earlier, many similarities exist between the mouse model of A A amyloid and other amyloid diseases, and this model can be used as an in vivo screening technique to identify compounds that may prevent or slow the process of amyloid fibril development. Several studies have shown the utility of this model. Kisilevsky et al. 19,36 predicted that the interactions between amyloid fibrils and HSPG were important and, by disruption of these contacts, may prevent amyloid deposition. Oral administration of low molecular weight anionic sulfates or sulfonates reduced amyloid development in the mouse model. In addition, they interfered with Aft fibril formation in vitro. Many other potential compounds that may prove to be important in the inhibition of amyloid fibrillogenesis or in the regression of amyloid deposits can be tested in this model. These compounds include methyl 4 , 6 - O [ ( R ) - l - c a r b o x y e t h y l i d e n e ] f l D-galactopyranoside (MOflDG, interferes with SAP interaction with fibrils) and 4'-iodo-4'-deoxydoxorubicin (I-DOX, may interfere with fibril deposition), which have been shown to interfere with fibril formation and may prove to be effective in vivo. 37"38 Concluding Remarks Experimental approaches designed to characterize the mechanisms of amyloid deposition, components involved in the process, and inhibitors that block amyloid deposition should ideally include a systematic evaluation of the entire spectrum of amyloid diseases. The described methodology provides a strategy for evaluating amyloidogenesis in an in vivo paradigm that has most of the components present in all amyloid diseases. Amyloidogenic diseases demonstrate a high degree of similarity and one can be used to help understand the basic principles of all the disorders. The A A model demonstrates the same effect seen in APP transgenic mice. 36R. Kisilevsky,Drugs Aging 2, 75 (1996). 37G. A. Tennent, L. B. Lovat, and M. B. Pepys, Proc. Natl. Acad. Sci. U.S.A. 92, 4299 (1995). 38L. Gianni, V. Bellotti, A. M. Gianni, and G. Merlini, Blood 86, 855 (1995).
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In both models, inactivation of the apoE gene resulted in a decrease in amyloid deposition. 21'39 Also, the generation of a SAP knockout mouse may show greater similarities in the two models. 2° The unique features of the A A model are the rapid development of the disease, and the ease in studying the role of specific factors and testing of drugs.
Acknowledgments The authors thank Drs. Maria C. de Beer and Deneys van der Westhuyzen for critical reading of the manuscript, Drs. Jin Yu and Hong Wu, Mr. Darin Cecil, Ms. Amy King, Ms. Connie Gerardot, and Mr. John Cranfill for expert technical assistance during various phases of the investigations. This work was supported by National Institutes of Health Grants NS-32221 and AG-12981 (MSK), AG-10886 and the Veterans Affairs Medical Research Fund (FCD), and the Sanders-Brown Center on Aging. 39 K. R. Bales, T. Verina, R. C. Dodel et aL, Nature Genet. 17, 263 (1997).
[45] T o x i c i t y o f P r o t e i n A g g r e g a t e s i n P C 1 2 Cells: 3-(4, 5 - D i m e t h y l t h i a z o l - 2 - y l ) - 2 , 5 - d i p h e n y l t e t r a z o l i u m Bromide Assay
By M A R K
S. SHEARMAN
Introduction Senile plaques, a neuropathological feature of Alzheimer's disease (AD), consist primarily of an insoluble aggregate of amyloid-/3 peptide (A/3), a 40-43 amino acid peptide. 1 Dense core plaques of A/3 deposited in AD brain are typically surrounded by dystrophic neurites, 2 an observation that led to the proposal that .Aft itself may be neurotoxic. 3'4 Although studied intensively in the early 1990s, the biochemical mechanisms that underlie the neurotoxicity of A/3 remain uncertain. 5 One observation that remains consistent, however, is that amyloidogenic peptides such 1 C. L. Masters, G. Simms, N. A. Weinman, G. Multhaup, B. L. McDonald, and K. Beyreuther, Proc. Natl. Acad. Sci. U.S.A. 82, 4245 (1985). 2 D. M. A. Mann, P. O. Yates, and B. Marcyniuk, Neurosci. Lett. 56, 51 (1985). 3 R. E. Tanzi, P. H. St. George-Hyslop, and J. F. Gusella, Trends Neurosci. 12, 152 (1989). 4 D. J. Selkoe, Neuron 6, 487 (1991). s L. L. Iversen, R. M. Mortishire-Smith, S. J. Pollack, and M. S. Shearrnan, Biochem. J. 311, 1 (1995).
METHODS IN ENZYMOLOGY, VOL. 309
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15. Transfer the slides to the DAB solution and allow 10 min for the reaction. 16. Quench the reaction by immersing the slides in 0.1 M Tris-HCl, pH 7.4, for 15-30 min. 17. Counterstain nuclei using 1% methyl green in acetate buffer for 5 min. 18. Give three quick rinses in water (overextensive washing will easily remove the stain). Allow the slides to completely air dry for 30-60 min. 19. Transfer the slides to Histoclear (National Diagnostic Lab.), incubate for 15 min, and replace with fresh Histoclear. The slides can remain in this solution until mounting. 20. Mount the slides with DPX mounting medium (Fisher Scientific). Avoid trapping air bubbles between the slide and cover glass. Leave to dry on the bench overnight, and peel away excess mounting medium the following day.
Acknowledgements This work was supported by grants from the Wellcome Trust, the Deutsche Forschungsgemeinschaft, the European Union (BMH4 CT96 0244), the Hereditary Disease Foundation (in the form of an award to GB from Harry Lieberman), the Huntington's Disease Society of America, and the Special Trustees of Guy's Hospital.
[44] A M o u s e M o d e l f o r S e r u m A m y l o i d A A m y l o i d o s i s
By M A R K
S. KINDY a n d FREDERICK C. DE BEER
Introduction Amyloid A (AA) amyloidosis is associated with rheumatic diseases, certain chronic inflammatory diseases, chronic infections, and some malignant disorders. Crohn's disease and rheumatoid arthritis (particularly juvenile rheumatoid arthritis) are frequently complicated with amyloidosisl; whereas, in ulcerative colitis and systemic lupus erythematosis, amyloidosis is a rare confounding problem. Differences seen in these disease states are most likely due to altered regulation in the cytokine-mediated changes in amyloid precursor protein serum amyloid A (SAA) production. Renal 1 M. A. Gertz and R. A. Kyle, Medicine (Baltimore) 70, 246 (1991).
METHODS IN ENZYMOLOGY,VOL.309
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carcinomas and Hodgkin's disease are known to be associated with fevers and systemic inflammatory features leading to serum amyloid A induction. A A amyloid incidence has decreased considerably since the early 1980s as a result of therapeutic interventions that reduce inflammation. 2 Nevertheless, our capability to maintain individuals suffering from various types of trauma has contributed to an increase in systemic A A amyloidosis, particularly due to chronic urinary tract infections. Idiopathic inflammatory disease (rheumatoid arthritis, juvenile rheumatoid arthritis, and chronic infections) is the leading cause of A A amyloidosis in the Western Hemisphere. Generally, 50% of patients with amyloidosis succumb to the disease within the first 5 years after diagnosis and an additional 25% between 5 and 15 years. The deposition of A A amyloid involves multiple organ systems, usually without any initial symptomatic presentations. Approximately 25% of individuals with amyloidosis ultimately present with renal or glomerular involvement. 3 The typical clinical presentation is proteinurea due to the deposition of amyloid fibrils in the glomerulus. This results in a nephrotic syndrome leading to renal insufficiency or end-stage renal failure, the most common cause of death in these patients. Deposits can usually be detected in the spleen with some involvement of the liver, adrenals, and, to a lesser degree, in the heart, gut, and other tissues. Dialysis and renal transplantation can exacerbate the deposition of amyloid fibrils in other organs. 4 Therefore, there is a growing need to understand the process of systemic amyloid deposition and its complications. The mouse amyloid model provides an ideal system for understanding amyloid fibrillogenesis. 5 Serum amyloid A proteins are a group of highly homologous apoproteins associated with high-density lipoproteins (HDLs) and are the precursors of A A amyloid. 6 Serum amyloid A proteins are the most dramatically increased acute-phase proteins in mice with levels increasing up to 1000-fold (from 1-5 to 1000/xg/ml) within 24 hr of an inflammatory response. 7 In the mouse, SAA1 and SAA2 are induced in the liver by circulating cytokines [interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF)] in response to amyloid induction paradigms (Fig. 1). These two isotypes differ in only 9 of 103 amino adds; however, only SAA2 is 2 M. Pras, Scand. J. Rheumatol. 27, 92 (1998). 3 A. Montoli, E. Minola, F. Stabile, C. Grillo, L. RadaeUi, D. Spanti, E. Luccarelli, C. Spata, and L. Minetti, Am. J. Nephrol. 15, 142 (1995). 4 S.-Y. Tan, A. Irish, C. G. Winearls, E. A. Brown, P. E. Gower, E. J. Clutterbuck, S. Madhoo, J. P. Lavender, and M. B. Pepys, Kidney Int. 50, 282 (1996). 5 R. L. Meek, J. S. Hoffman, and E. P. Benditt, J. Exp. Med. 163, 499 (1986). 6 m. Husebekk, B. Skogen, and G. Husby, Scand. J. Imrnunol. 25, 375 (1987). 7 K. P. W. J. McAdam, and J. D. Sipe, J. Exp. Med. 144, 1121 (1976).
[44]
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FIG. 1. Induction of SAA proteins following amyloid-enhancing factor (AEF) and silver nitrate (SN) injection. Immunocytochemical staining of apoSAA in C57BL6 mice: 10/zg of plasma electrophoresed onto a 5-20% acrylamide gel; lane 1, plasma from mice 24 hr after injected with AEF/SN; lane 2, plasma from normal mice.
selectively deposited into amyloid fibrils (Fig. 2). SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2 (Fig. 2). 8 Even though the CE/J protein overall has features similar to the SAA2 protein, it is not deposited into amyloid fibrils. Therefore, the 8 M. C. de Beer, F. C. de Beer, W. D. McCubbin, C. M. Kay, and M. S. Kindy, J. Biol. Chem. 268, 20606 (1993).
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CE/J mouse does not develop amyloid. 9 Differences in the CE/J protein that confer amyloid resistance appear to be at the amino acid positions 6 and 7, where it shows similarity with the nonamyloidogenic SAAa protein (Fig. 2). 8"1°Linkage analysis with amyloid-resistant mice (CE/J) and amyloid-susceptible mice (CBA/J) demonstrated that the CE/J isoform confers protection against amyloid deposition even in the presence of the SAA2 isoform, la These data indicate valuable information that can be obtained from inbred strains of mice on the structural properties and specific interactions that may occur in the process of amyloidogenesis. Our current understanding of the processes involved in AA amyloidosis is that it is the result of a two-stage mechanism (Fig. 3). 12 First, the generation of A A amyloid requires the synthesis of a precursor protein in sufficient quantities to allow for deposition. This is referred to as the preamyloid phase, which can persist for several days to months, depending on the stimulus and the levels of SAA expression. In response to a chronic or recurrent acute inflammatory condition, macrophage activation leads to the synthesis and secretion of cytokines (IL-1, IL-6, and TNF). These cytokines result in the hepatic synthesis of acute-phase proteins, specifically SAA proteins that are secreted and associated with H D L particles. 7 Second, in the amyloid phase, the continuous maintenance of high levels of the precursor is required to generate a nidus or fibrillar network (also referred to as amyloid-enhancing factor, AEF) onto which amyloid can be deposited. 13 The production of AEF results in the rapid deposition of A A fibrils, which can occur in days. Amyloid deposition first occurs in the spleen, liver, and kidney, and eventually amyloid can be detected in every organ, with the possible exception of the brain. However, studies have demonstrated the presence of S A A proteins in the brains of patients with Alzheimer's disease. 14'15 Amyloid deposition in the mouse is extremely rapid, with extensive deposits detectable following amyloid induction paradigms. Injection of a modified casein solution results in amyloid deposition in various organs in 9 j. D. Sipe, I. Carreras, W. A. Gonnerman, E. S. Cathcart, M. C. de Beer, and F. C. de Beer, Am. J. Pathol. 143, 1480 (1993). 10H. Patel, J. BramaU, H. Waters, M. C. de Beer, and P. Woo, Biochem. J. 318, 1041 (1996). 11 W. A. Gonnerman, R. Elliot-Bryant, I. Carreras, J. D. Sipe, and E. S. Cathcart, J. Exp. Med. 181, 2249 (1995). 12 I. Kushner and D. L. Rzewnicki, Clin. Rheumatol. 8, 513 (1994). 13M. A. Axelrad, R. Kisilevsky, J. Willmer, S. J. Chen, and M. Skinner, Lab. Invest. 47, 139 (1982). 14j. S. Liang, J. A. Sloane, J. M. Wells, C. R. Abraham, R. E. Fine, and J. D. Sipe, Neurosci. Lett. 225, 73 (1997) 15 M. S. Kindy, J. Yu, J. T. Gao, and H. Zhu, J. Alzheimer's Dis., in press (1999).
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FIG. 3. Flow diagram of the events in amyloidogenesis. Sequence of events in amyloid formation in both mouse and human. Generation of an inflammatory response through the injection of casein in the mouse or infection in human results in the activation of a pathway leading to the deposition of amyloid fibrils. The preamyloid phase culminates in the synthesis and secretion of SAA. The amyloid phase originates from the generation of amyloid-enhancing factor (AEF; preformed amyloid fibrils, proteoglycans, etc.) to the deposition of amyloid fibrils in the tissue.
[441
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21 days after initiation. The use of AEF and silver nitrate has reduced this time to 3 days.13 In addition, mouse AA amyloid has serum amyloid P component (SAP) and heparan sulfate proteoglycan (HSPG) associated with the amyloid fibrils. 16J7 Studies have shown that the mouse apolipoprotein E (apoE) is associated with mouse A A amyloid.TM Kisilevsky et al. 19 showed that a series of small anionic sulfates and sulfonates interfered with the S A A - H S P G interaction and were effective in not only inhibiting amyloidogenesis but in reversing the process in vivo. These studies indicate the importance of HSPG in the events associated with amyloid formation. The generation of SAP and apoE knockout mice showed that inactivation of these genes resulted in delayed and reduced deposition of amyloid.2°'21 As suggested earlier, the involvement of accessory factors in the evolution of amyloid diseases needs to be examined as a possible mechanism in the development of therapeutic strategies for treatment of these diseases. This article describes the methods used in the generation of AA amyloid in the mouse as a model of amyloid disease. Several different protocols have been used in the deposition of AA amyloid in mice. The first requires the continuous injection of a modified casein solution over an extended period of time to initiate amyloid deposition. The second utilizes AEF and silver nitrate to give extensive amyloid deposits within 3-5 days.
Induction of AA Amyloid in Mouse Initial studies on amyloid induction in the mouse were performed using casein and azocasein with multiple injections over a several week period. 22 For induction of amyloid with casein or azocasein, 0.5 ml of a prepared 10% solution of sterile azocasein is injected subcutaneously daily for up to 21 days (or sometimes the injections are performed 5 days a week for up to 4 weeks). Injection of the azocasein results in a sterile abscess, which will cause an inflammatory reaction inducing SAA in the mouse. Using this paradigm, significant amounts of amyloid develop over the 21-day period. Initial deposition does not begin for several days after the injections 16M. B. Pepys, in "Amyloidosis" (J. Marrink and M. van Ryswijk, eds.), p. 43. Martinus Nijhoff, Boston, 1986. 17 A. D. Snow, J. Willmer, and R. Kisilevsky, Lab. Invest. 56, 170 (1987). i8 M. S. Kindy, A. R. King, G. Perry, M. C. de Beer, and F. C. de Beer, Lab. Invest. 73, 469 (1995). 19 R. Kisilevsky, L. J. Lemieux, P. E. Faser, X. Kong, P. G. Hultin, and W. A. Szarek, Nature Med. 2, 143 (1996). 20 M. Botto, P. N. Hawkins, M. C. M. Bicerstaff, J. Herbert, A. E. Bygrave, A. McBride, W. L. Hutchinson, G. A. Tennent, M. J. Walport, and M. B. Pepys, Nature Med. 3, 855 (1997). 21 M. S. Kindy and D. J. Rader, A m . J. Pathol. 152, 1387 (1998). 22 D. T. Janigan, A m . J. Pathol. 55, 379 (1969).
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have commenced, delaying the process. This is due to the synthesis and initial deposition of the amyloid fibrils onto which amyloid can form. Subsequently, amyloid is now induced in the mouse model by the injection of A E F (100/zg protein) intravenously via the tail vein followed by a subcutaneous injection of 0.5 ml of a 2% solution of silver nitrate (sterile preparation in water). 23 The AEF is injected into the tail vein as follows: the mouse is placed in a mouse holder and the tail is heated for 5 to 10 sec in a water bath or beaker of water heated to 50°. This will bring the tail vein to the surface and allow for easier injection of AEF. The tail is removed from the water bath and swabbed with alcohol to sterilize and then A E F (100 tzg in 100/zl) is injected with a 29-gauge tuberculin syringe into the tail vein. Immediately following the tail vein injection, the animals are treated with a subcutaneous injection of sterile silver nitrate (0.5 ml of a 2% solution). This allows for extensive deposition of amyloid within 3-5 days. Studies have shown that in the AEF and silver nitrate injection paradigm, amyloid deposits can be detected as early as 12 hr posttreatment. This allows for a rapid model of amyloid formation to test the effects of gene mutations on amyloid formation and the effects of inhibitors of amyloid deposition. A A amyloid deposits first develop in the spleen, possibly due to the metabolic effects of the spleen. Secondary deposition occurs in the liver, heart, kidneys, lungs, intestine, and every major organ except the brain. If the inflammatory stimulus is maintained for extended periods of time, the amyloid deposits will continue to expand, resulting in organ failure and death. Amyloid-lnducing Agents
Preparation of Casein Various experimental protocols have been utilized to induce amyloid deposition in animals over the years. It was originally assumed that amyloidosis was due to chronic infections or the presence of bacterial toxins. Subsequent to these findings, it was shown that the injection of sodium caseinate gave rise to experimental amyloidosis in mice. The hypothesis was that the presence of large quantities of protein material led to insolubility and deposition of the amyloid substance. Since that time, a modified form of casein, azocasein, has been used extensively for the generation of amyloid in the mouse model. 24 The preparation of azocasein is time23 M. Axelrad, R. Kisilevsky, and S. Beswetherick, A m . J. PathoL 78, 277 (1975). 24 D. T. Janigan and R. L. Druet, Am. J. Pathol. 48, 1013 (1966).
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consuming and there can be variability if the proper casein is not used in the preparation protocol. Hammerstein-grade casein from ICN (Costa Mesa, CA) has been used successfully in the production of azocasein that will consistently give amyloid deposits in mice. A solution of sulfanilic acid (20 g, sodium salt, Sigma Chemical Co., St. Louis, MO) is prepared in 400 ml of cold distilled-deionized water containing 12 ml of 5 N NaOH. To this solution, 4.4 g of NaNO3 is added and allowed to dissolve. Next, 32 ml of 5 N HCI, followed by 32 ml of 5 N NaOH, is added and the solution is stirred continuously in the cold. Casein (100 g) is dissolved in 2 liters of a 1% (w/v) NaHCO3 solution prepared in distilled-deionized water and cooled on ice to 4 °. The casein solution is added slowly to the sulfanilic acid to allow the casein to remain in solution. If the casein is added too rapidly, it will precipitate out of solution. The combination will turn a deep red color once the casein goes into solution and should be mixed for 2-3 hr in the cold. The azocasein is precipitated out of solution by bringing the pH to 2-3 with the addition of 0.2 N HC1, added slowly to allow for a flocculent precipitate to form. The precipitate (yellowish in color) can be filtered through cheesecloth in a large filter funnel. The azocasein is dissolved in a large volume of 0.2 N NaOH (1 liter) and is subjected to two additional rounds of precipitation and solubilization. The final precipitate is resuspended in 0.2 M NaHCO3 and dialyzed in 10- to 12-kDa molecular mass dialysis tubing for 3 days against cold distilled-deionized water, changing the water twice a day. The azocasein solution is lyophilized completely (several days) and stored at - 8 0 ° as a powder. For injection of the azocasein, a 10% solution in 0.01 M NaHCO3 (5 g in 50 ml) is prepared in 50-ml polypropylene tubes and adjusted to a final pH of 6.5. The solution is sterilized by the addition of 1% diethyl pyrocarbonate.
Preparation of Amyloid-Enhancing Factor Over time, amyloid deposition was considered to be a result of or related to an immunological response in human and animal models. The general principle has been that the subcutaneous injection of proteinaceous material would cause an immunologic reaction and result in amyloid formation. The involvement of the immune system in the amyloidogenic process suggested the presence of factors that may contribute to amyloid deposition. Transfer experiments using tissue and cells from amyloidogenic animals demonstrated the presence of an enhancing factor in the amyloid tissue. 25'26Subse25 F. Hardt and P. Ranlov, Int. Rev. Exp. Pathol. 49, 273 (1976). 26 I° Keizman, A. Rimon, E. Sohar, and J. Gafni, Acta Pathol. Microbiol. Scand. (A) 233, 172 (1970).
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[44]
quent studies by Kisilevsky and co-workers 27 have shown that the amyloid tissue contained amyloid-enhancing activity, which facilitated the deposition of amyloid in the mouse. Extraction of A E F from amyloidotic tissue (spleens and livers) and subsequent injection into mice with an inflammatory stimulus reduce the time of amyloid deposition from 4 weeks to 3 days. The composition of A E F is still not known; however, it is thought to be composed of preformed amyloid fibrils and proteoglycans. Studies have strengthened this hypothesis. Injection of amyloidogenic peptides from SAA and transthyretin proteins, as well as from A/3 peptides, resulted in the rapid accumulation and deposition of A A amyloid. 28'29 The utilization of SAA and other amyloidogenic peptides indicates that the amyloidogenic activity of A E F is due to amyloid fibrils. A E F appears to act as a seed or nucleus onto which the amyloid can form. Amyloid-enhancing factor is prepared from the spleens and livers of animals that were injected with azocasein or AEF and silver nitrate to establish amyloid deposits, as described previously. 3° Animals injected with casein for 4 weeks or A E F and silver nitrate for 2 weeks are taken for A E F preparation. Using sterile techniques, the spleens and livers are removed from the amyloidotic mice (about 25 animals) and homogenized in 10 volumes of phosphate-buffered saline (PBS) containing protease inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF), 1/zM leupeptin; 5/xg aprotinin/ml] using a homogenizer (Polytron, Brinkmann Instruments, Westbury, NY). The tissue is homogenized on a low setting until a homogeneous suspension is obtained. The homogenate is centrifuged at 10,000 rpm for 20 min at 4 ° in a superspeed centrifuge. The pellet is resuspended in 10 volumes of PBS plus protease inhibitors (low-speed homogenization to disrupt the pellet) and centrifuged repeatedly until the optical density of the supernatant at 280 nm is less than 0.1. The final pellet is homogenized/ extracted with sterile/distilled water (10 times the volume of the pellet). The sample is centrifuged at 16,000 rpm for 1 hr at 4°. The first supernatant can be discarded. The water extractions are repeated; the second and third supernatants are saved and quantified for protein content. Samples of AEF are stored frozen at - 8 0 ° and tested for activity as described previously. Detection of AA Amyloid Several methods have been used in the detection of A A amyloid. The most frequently used method is Congo red staining in which the Congo 27 R. Kisilevsky, M. D. Benson, M. A. Axelrad, and L. Boudreau, Lab. Invest. 41, 206 (1979). 28 K. Johan, G. Westermark, U. Engstrom, A. Gustausson, P. Jultman, and P. Westermark, Proc. Natl. Acad. Sci. U.S.A. 95, 2558 (1998). 29 M. S. Kindy, unpublished results (1999). 30 R. Kisilevsky and L. Boudreau, Lab. Invest. 48, 33 (1983).
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711
red dye will stain amyloid structures specifically (the cross/3-pleated sheet arrangement). 31 Congo red appears to bind to linear polymers that have a similar structure to cellulose. 24 Tissue sections should be fixed in 10% (w/v) buffered formalin and sections should be 6-12/xm in thickness.
Congo Red Staining Paraffin-embedded sections are deparaffinized and hydrated to water (two changes for 5 min each in xylene, 100, 95, 80, 50, and 30% ethanol, then distilled water). The sections are stained in a 1% solution of Congo red for I hr at room temperature (1 g Congo red, color index number 22120 containing 98% dye [Sigma, St. Louis, MO], in 100 ml of distilled water). The Congo red solution should be stirred for several hours at room temperature to allow the dye to dissolve completely, then filtered through a Whatman (Clifton, N J) No. 2 filter. This solution should be used within i month, as beyond this time the dye will not bind well to the amyloid. The sections are rinsed in running water and differentiated in alkaline alcohol (0.01% sodium hydroxide in 50% ethyl alcohol) for 3-5 sec and rinsed for 5 rain in running water. It is easy to overdifferentiate the sections; if the sections are pale, repeat the Congo red staining. Counterstain with Mayer's hematoxylin for 5 min, wash for 15 rain in water, dehydrate to xylene (two changes for 5 min in 50, 80, 95, and 100% ethanol followed by two changes in xylene), and mount with Permount (Fisher, Pittsburgh, PA). Results of the staining protocol are amyloid stains red to pink, nuclei stain blue, elastic fibers stain light red, and most other structures are not stained. When examined under light microscopy with polarizing filters (light-polarizing microscope filter set 31-52-62-75) the amyloid material shows an applegreen birefringence surrounding the splenic follicles (Fig. 4A). In the spleen, amyloid deposits are detected surrounding the splenic follicles, beginning as small amounts of congophilic material that accumulate over time in the presence of a continuous inflammatory response. In Fig. 4, the amyloid present in the spleen creates a dramatic effect with large deposits around the follicles. In other organs, the amyloid is more diffuse, with vascular amyloid appearing in the veins and arteries, as well as general deposits in the tissue.
Immunocytochemical Detection A second method of staining amyloid is by immunocytochemical analysis using rabbit antimouse SAA or A A antibodies. TM After generation of amyloid, the tissue (spleen, liver, heart, etc.) is drop fixed in formalin and embedded in paraffin. Alternatively, the animal can be perfused with cold 31H. Puchtler, F. Sweat, and M. Levine,J. Histochem. Cytochem. 10, 355 (1962).
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B
FIG. 4. Detection of A A amyloid in the spleen. (A) Congo red staining of an amyloidotic spleen and visualization using bipolar filters of apple-green birefringent material. (B) Immunocytochemical staining of amyloid. An antibody against mouse SAA proteins was used as the primary antibody. Detection was performed with a peroxidase secondary antibody and diaminobenzidine hydrochloride. Dark staining indicates A A fibrils and amyloid deposits.
saline (25 ml) followed by 4% paraformaldehyde (25 ml) and the tissues harvested. Paraformaldehyde (PF, 4 g) is suspended in saline and adjusted to a pH of 8.0 and heated to 60° to allow the PF to dissolve. The solution is cooled and adjusted to pH 7.4 for perfusion. Saline and paraformaldehyde are perfused by means of a 30-cm 3 syringe and 23-gauge needle inserted into the heart while the animal is anesthetized. After perfusion, the tissues are removed and submerged in fresh 4% (w/v) paraformaldehyde for 24 hr and then in 30% sucrose (30 g sucrose in 100 ml saline). After 24 to 48 hr, the tissue can be frozen in OCT medium (Tissue-Tek, Torrance, CA) and stored at - 8 0 °. Eight- to 10-t~m sections of tissue are prepared and mounted on Superfrost Plus microscope slides (Fisher, Pittsburgh, PA) coated with 1% gelatin for immunocytochemistry. For slide preparation, 1 g of gelatin is dissolved in 100 ml water by heating and stirring. Slides are dipped in gelatin and allowed to air dry. Paraffin sections are deparaffinized and hydrated to water (as described earlier) and incubated for 3 min in 100% formic acid to help disrupt amyloid fibrils (this allows for better detection of the amyloid fibrils by exposing the epitopes on the A A for antibody binding). Sections are incubated in hydrogen peroxide (3% in methanol for 30 min) to remove any endogenous peroxidase activity in the tissue and hydrated (two changes of 5 min each) to Tris-buffered saline (TBS; 0.1 M Tris, 0.150 M NaCI, pH 7.4). Sections are incubated in 15% goat serum (in TBS) to block nonspecific sites and are incubated in the presence of the primary antibody at a dilution of 1 : 2000 (rabbit antimouse SAA antibody). After incubation at room temperature for 1 hr or overnight at 4°, the sections are washed (3 x 15 min in TBS plus 15% goat serum) and incubated with the secondary antibody (peroxidase-conjugated goat antirabbit; 1 : 5000; Sigma Chemical Co., St. Louis, MO) for 1 hr at room temperature. Sections are washed as before and are developed in enzyme
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buffer containing diaminobenzidine hydrochloride (50/~g/ml) until staining appears (2 to 10 min). Staining is stopped by rinsing in water and counterstaining for 10 sec in hematoxylin (Sigma, St. Louis, MO). Slides are dehydrated to xylene (reverse of process for hydration, as described earlier) and mounted with Permount. Figure 4B illustrates the presence of immunoreactive AA protein detected in the amyloid deposits. As with the Congo red staining, the splenic deposits are detected around the follicles. In animals with extensive amyloid, the deposits can extend from each follicle to overlap and appear to be a solid sheet of amyloid.
123[-Labeled S A P Detection o f A m y l o i d Amyloid in mouse and human can be monitored specifically by using quantitative scintigraphy with 123I-labeled human serum amyloid P (SAP) component or technetium-99m pyrophosphate. 32,33 SAP is used for the general detection of amyloid deposits, and pyrophosphate has been used in detecting amyloid in cardiac amyloidosis. We will focus on the SAP detection system because it is more relevant to these studies. Human SAP is isolated by affinity chromatography to 99% purity from sterile serum heated to 56° for 30 min. 34 Human sera (2 liters) is passed over a 5 x 85cm column of Sepharose 4B equilibrated with Tris-Ca buffer (0.01 M Trisbuffered 0.14 M NaC1, pH 8.0, containing 0.002 M CaCI2 and 0.1 g/liter NAN3) at 100 ml/hr at 4°. The column is washed in Tris buffer until the effluent is zero at A280 and the bound protein elutes Tris buffer with EDTA (0.01 M EDTA). The eluted protein is passed over Sepharose-anti-NHS (normal human serum, 10 ml) followed by Blue-Sepharose (1 ml) to remove contaminating serum proteins. The samples are concentrated to 5-10 ml and gel filtered on a 2.6 x 100-cm column of Sephacryl S-300 (Amersham Pharmacia Biotech, Piscataway, N J) eluted with Tris-EDTA at a flow rate of 16 ml/hr (5.8-ml fractions). Peak SAP as determined by Western blot analysis or electroimmunoassay is pooled, concentrated, and stored under liquid nitrogen. SAP is labeled with iodine-123 using N-bromosuccinimide and is purified by gel filtration on Sephadex G-25 (Sigma, St. Louis, MO). Approximately 100/zg of labeled SAP (550/zCi), administered by intravenous injection, will bind specifically but in a reversible fashion to amyloid fibrils in vivo. Mice injected with 123I-labeled SAP are subjected to whole body scintigraphy. Animals are anesthetized [chloral hydrate (350 mg/kg) and xylazine (4 mg/kg)] and scanned with a Toshiba gamma camera and pinhole collimator for 1-4 min at t = 0 to t = 24 hr. The labeled SAP will 32p. N. Hawkins,J. P. Lavender,and M. B. Pepys,N. Engl. J. Med. 323, 508 (1990). 33p. N. Hawkins,M. J. Myers,A. A. Epenetos,D. Caspi,and M. B. Pepys,J. Exp. Med. 167, 903 (1988). 34F. C. de Beer and M. B. Pepys,J. IrnmunoL Methods 50, 17 (1982).
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localize to the amyloid deposits dependent on the amount of amyloid present in the tissue. Thus, one can determine the percentage of the injected dose that is retained in the body. In addition, the organs can be removed and counted in a gamma counter to determine the amount of labeled SAP that localizes to that organ (i.e., spleen). This number corresponds to the histological grade described later. Quantification of Amyloid Load (Amyloid Deposition) The quantification of amyloid varies dramatically from estimated semiquantitative analysis to quantitative analysis by determining the proportion of the area of the sections occupied by amyloid material. Original analysis was performed by visual examination to determine the amount of amyloid based on a crude scale of 0 to 4Y The grade is as follows: grade 0, no amyloid; grade i, trace amounts of amyloid in the perifollicular zone; grade 2, narrow ring of amyloid in the perifollicular zone; grade 3, broad bands of amyloid in the perifollicular zone; and grade 4, broad bands of amyloid with bridging between the follicles. To quantitate the amount of amyloid present in the tissue of mice, the following protocol is used. 19~I A standard set of amyloid-containing tissues is generated (0, 10, 20, 30, 40, 50% of tissue containing amyloid). These are used as reference points to determine the amount of amyloid in a given tissue. Standard sections are examined under the microscope (Nikon, Garden City, NY; using polarizing filters to generate birefringence) and recorded on videotape (Sony, New York, NY). The images are digitized and transferred to a Macintosh computer for analysis. The digitized images are analyzed (Kontron, Prism, or MCID image analysis) for color (intensity and area, operator specified area of interest) under low power (20 ×). This generates a standard for comparison. Experimental tissue sections are analyzed and compared to the standards for the quantitation of amyloid. Approximately 20 sections from each spleen are examined by image analysis and averaged for amyloid quantification. Data generated from these studies are calculated as the percentage of amyloid in the tissue (i.e., the area of amyloid present in the spleen to the total area). Alternatively, sections are stained with fluorescence-labeled anti-SAA or SAP (I/1000 dilution) antibodies and images are analyzed on a confocal laser scanning microscope (Molecular Dynamics, Sunnyvale, CA) using ImageSpace ScanControl software and a SGI UNIX workstation (Summit, NJ). In most cases the spleens are analyzed for amyloid; however, we will take other tissues to verify the levels of amyloid in the mice. In addition, some animals are injected with 1251-1abeled SAP (labeled as described for Iz31-1abeled SAP) for the quantitative assessment of amyloid. 35E. S. Cathcart, C. A. Leslie,S. N. Meydani, and K. C. Hayes,J. Immunol. 139, 1850(1987).
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Animals are injected with 0.6/zCi of SAP (0.4/zCi//zg) at t = 0 and killed at t = 24 hr, the animals are bled out, and the organs weighed and subjected to gamma counting. SAP is cleared rapidly into the amyloid tissue, and quantitation by homogenization and scintillation counting is very reproducible. Inhibition of Amyloid Fibrillogenesis The mouse A A model is ideal for studying fibrillogenesis and developing inhibitors of fbril formation. As discussed earlier, many similarities exist between the mouse model of A A amyloid and other amyloid diseases, and this model can be used as an in vivo screening technique to identify compounds that may prevent or slow the process of amyloid fibril development. Several studies have shown the utility of this model. Kisilevsky et al. 19,36 predicted that the interactions between amyloid fibrils and HSPG were important and, by disruption of these contacts, may prevent amyloid deposition. Oral administration of low molecular weight anionic sulfates or sulfonates reduced amyloid development in the mouse model. In addition, they interfered with Aft fibril formation in vitro. Many other potential compounds that may prove to be important in the inhibition of amyloid fibrillogenesis or in the regression of amyloid deposits can be tested in this model. These compounds include methyl 4 , 6 - O [ ( R ) - l - c a r b o x y e t h y l i d e n e ] f l D-galactopyranoside (MOflDG, interferes with SAP interaction with fibrils) and 4'-iodo-4'-deoxydoxorubicin (I-DOX, may interfere with fibril deposition), which have been shown to interfere with fibril formation and may prove to be effective in vivo. 37"38 Concluding Remarks Experimental approaches designed to characterize the mechanisms of amyloid deposition, components involved in the process, and inhibitors that block amyloid deposition should ideally include a systematic evaluation of the entire spectrum of amyloid diseases. The described methodology provides a strategy for evaluating amyloidogenesis in an in vivo paradigm that has most of the components present in all amyloid diseases. Amyloidogenic diseases demonstrate a high degree of similarity and one can be used to help understand the basic principles of all the disorders. The A A model demonstrates the same effect seen in APP transgenic mice. 36R. Kisilevsky,Drugs Aging 2, 75 (1996). 37G. A. Tennent, L. B. Lovat, and M. B. Pepys, Proc. Natl. Acad. Sci. U.S.A. 92, 4299 (1995). 38L. Gianni, V. Bellotti, A. M. Gianni, and G. Merlini, Blood 86, 855 (1995).
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In both models, inactivation of the apoE gene resulted in a decrease in amyloid deposition. 21'39 Also, the generation of a SAP knockout mouse may show greater similarities in the two models. 2° The unique features of the A A model are the rapid development of the disease, and the ease in studying the role of specific factors and testing of drugs.
Acknowledgments The authors thank Drs. Maria C. de Beer and Deneys van der Westhuyzen for critical reading of the manuscript, Drs. Jin Yu and Hong Wu, Mr. Darin Cecil, Ms. Amy King, Ms. Connie Gerardot, and Mr. John Cranfill for expert technical assistance during various phases of the investigations. This work was supported by National Institutes of Health Grants NS-32221 and AG-12981 (MSK), AG-10886 and the Veterans Affairs Medical Research Fund (FCD), and the Sanders-Brown Center on Aging. 39 K. R. Bales, T. Verina, R. C. Dodel et aL, Nature Genet. 17, 263 (1997).
[45] T o x i c i t y o f P r o t e i n A g g r e g a t e s i n P C 1 2 Cells: 3-(4, 5 - D i m e t h y l t h i a z o l - 2 - y l ) - 2 , 5 - d i p h e n y l t e t r a z o l i u m Bromide Assay
By M A R K
S. SHEARMAN
Introduction Senile plaques, a neuropathological feature of Alzheimer's disease (AD), consist primarily of an insoluble aggregate of amyloid-/3 peptide (A/3), a 40-43 amino acid peptide. 1 Dense core plaques of A/3 deposited in AD brain are typically surrounded by dystrophic neurites, 2 an observation that led to the proposal that .Aft itself may be neurotoxic. 3'4 Although studied intensively in the early 1990s, the biochemical mechanisms that underlie the neurotoxicity of A/3 remain uncertain. 5 One observation that remains consistent, however, is that amyloidogenic peptides such 1 C. L. Masters, G. Simms, N. A. Weinman, G. Multhaup, B. L. McDonald, and K. Beyreuther, Proc. Natl. Acad. Sci. U.S.A. 82, 4245 (1985). 2 D. M. A. Mann, P. O. Yates, and B. Marcyniuk, Neurosci. Lett. 56, 51 (1985). 3 R. E. Tanzi, P. H. St. George-Hyslop, and J. F. Gusella, Trends Neurosci. 12, 152 (1989). 4 D. J. Selkoe, Neuron 6, 487 (1991). s L. L. Iversen, R. M. Mortishire-Smith, S. J. Pollack, and M. S. Shearrnan, Biochem. J. 311, 1 (1995).
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