fallrevP41Change 12/28/04 12:34 PM Page 21 Vol. XXXVII, No. 2 December 2004 AB Managing Editor, Gilles Lefebvre Scientific Editor, Vinnie Della Speranza, MS, HTL (ASCP) HT, MT Introduction Alzheimer’s disease is a slowly progressive cerebral degeneration characterized by dementia. Increasing age and the presence of the beta-amyloid protein in neuritic plaques are risk factors associated with AD.1 Extensive research is ongoing in an attempt to better understand AD. The following theories have been proposed for its etiology: invasion by slow viruses, acetylcholinesterase deficiency, aluminum excess, autoimmune responses, genetic predisposition, amyloid protein deposition, and Fig. 1. Single photon emission computerized tomography. A) Altered blood circulation in an AD brain. vascular changes. In patients with B) Normal brain. AD, neurons and neuritic processes are lost. The gyri narrow, the sulci A Brief Review of Alzheimer’s Disease widen, and cortical atrophy by Using Conventional Silver Staining becomes apparent (Fig. 2B).1 Procedures A common finding in the brains of Alzheimer’s disease patients is a Dr. Alicia A. Zuniga, CLS, HTL (ASCP); Patricia Gonzalez, BS, HT; loss of approximately 200 g in a Antonio Fernandez, MD span of 3 to 8 years. The atrophy is School of Natural and Health Sciences bilateral and symmetric and targets Barry University, Miami Shores, FL the frontal and hippocampal cortex [email protected] (Fig. 2). A definitive Alzheimer’s diagnosis is based on large Abstract Alzheimer’s Disease (AD) is the leading cause of dementia. As many as IN THIS ISSUE 10% of all people 65 years of age and older have AD, and as many as A Brief Review of Alzheimer’s Disease 50% of those 85 and older have the disease. The high morbidity and by Using Conventional Silver Staining mortality associated with this disorder and its increasing prevalence in Procedures …………………………………21 Combined UCHL-1/PASH aging populations require the histology laboratory to have in its arsenal Staining in the Diagnosis of reproducible staining methods to render this diagnosis in biopsy sections. Renal Transplant Rejection ………………25 We evaluated different silver stains to identify which is most useful for Cryomicrotomy of Murine Tissues making the diagnosis of AD in histological sections. We found that the for Research Immunohistochemistry and Routine Hematoxylin and Eosin Bielschowsky silver stain is the best method to demonstrate AD Staining. Part II ……………………………29 pathology in our laboratory. The senile or neuritic plaques were the most Alcian Blue-H&E-Metanil Yellow conspicuous histologic lesion observed in sections stained with the Stain for Diagnosing Barrett’s Bielschowsky technique. Esophagus …………………………………35 Double Embedding: Double the Trouble?………………………39 Mark Your Calendar! Educational Opportunities in 2005 ………41 21 fallrevP41Change 12/16/04 4:54 PM Page 22 water, collected onto precleaned, AB charged slides, and dried overnight in a 39°C oven, deparaffinized, and rehydrated to distilled water prior to staining. Staining techniques included the Bielschowsky silver stain, Bodian silver stain, hematoxylin & eosin, and a modification of the methenamine silver stain, using both microwave and conventional procedures. Slides were observed under a light microscope and cortical areas were selected at 40X, 100X, and 400X magnifications using the DP12 Olympus® Microscope Digital Camera System (Olympus Fig. 2A. Normal brain. Fig. 2B. The brain of a patient with AD shows cortical atrophy, characterized by slender gyri America, Melville, NY). and prominent sulci. The pictures recorded in the SmartMedia™ installed in the numbers of microscopic We evaluated the Bielschowsky camera system were viewed with neurofibrillary tangles, neuropil silver stain, Bodian silver stain, and the software provided with the threads, neuritic plaques, and modifications of the methenamine DP12-BSW. Saved pictures were granulovacuolar degeneration silver stain for the utility in printed using a Hewlett Packard present in several tissue samples of demonstrating microscopic changes Deskjet 660 color printer. cerebral cortex. Symptoms include in brains from patients with loss of memory, judgment and Alzheimer’s disease. Results reasoning, difficulty with day-to- The preliminary results of this day function, and changes in mood Materials and Methods overview indicate that the and behavior. The utilization of Brain samples from three patients Bielschowsky silver stain2 is the imaging studies in the diagnosis of who died from cardiorespiratory best staining method used in our AD is done mainly to exclude failure and had a history of progres- laboratory to demonstrate AD structural lesions such as subdural sive cognitive decline were fixed in pathology. The conventional hemorrhage and brain tumors. 10% buffered formalin for 14 days. procedure was compared to the Figure 1 shows a single photon Several tissue blocks from each case microwave modification.3 The two emission computerized tomography were cut on a rotary microtome. techniques differed in the amount indicating how blood is circulating Sections of 8-10 µ were floated on of time employed to do the in normal (B) and AD brain (A). a water bath at 41°-43°C in distilled procedures but results were AB Fig. 3A. Section of cerebral cortex revealing the presence of neuritic plaques. Fig. 3B. At higher magnification, the uniform size and spherical shape of the neuritic H&E, 40X plaques are observed. Silver stain, 100X 22 fallrevP41Change 12/16/04 4:54 PM Page 23 AB Fig. 4A. The cytoplasm of a neuron is distended by neurofibrillary tangles. Fig. 4B. A Bielschowsky silver stain demonstrates the fibrillary character H&E, 100X of the cytoplasmic inclusions. 100X conclusive in both. The major microscopic features observed in the sections of cortex from patients with Alzheimer’s disease were neurofibrillary tangles and neuritic plaques (Figs. 3, 4). The neurofibrillary tangles found within the cytoplasm of abnormal neurons consist of fibrous proteins that are wound around each other as pairs of helical filaments. The main component of tangles1 (Fig. 4A, 4B) is an abnormal phosphorylated form of a normally occurring microtubule-associated protein (MAP), termed tau, which is necessary to stabilize neuronal microtubules for proper axonal transport. The abnormally Fig. 5. Section of cortex from a patient with AD, demonstrating senile plaques. Bielschowsky stain, phosphorylated tau is less able microwave method. 40X to bind microtubules, probably causing microtubule depolymerization, disrupted axonal transport, compromised synaptic neurotransmission, and finally, neuronal death. These tangles are resistant to chemical and enzymatic breakdown, and they persist in brain tissue long after the neuron in which they arose has died and disappeared. The observed neurofibrillary tangles are composed of argentaphilic fibers arranged in irregular bundles, knots, and curves. The senile or neuritic plaques were the most conspicuous histologic lesions observed in sections stained with the Bielschowsky technique. Fig. 6. Section of cortex. Numerous senile plaques are observed. Bielschowsky stain, microwave method. 100X 23 fallrevP41Change 12/16/04 4:54 PM Page 24 that accumulates in neuritic plaques as amyloid fibrils5 (Fig. 9). The core of these plaques contains a distinct form of BAP, which is 40 amino acids in length. BAP is derived from proteolysis of a much larger (695 amino acids) membrane-spanning amyloid protein.5 Under the microscope, different developmental stages of neuritic plaques were observed. In Figs. 3A, 5, 6, 7, diffuse plaques are observed; primitive plaques without a ß-amyloid core are observed in Figs. 3A, 6, 7. Classic neuritic plaques are observed at low magnification in Fig. 3B (100X) and at higher magnification in Fig. 10 (400X). In our laboratory, Fig. 7. Three different types of neuritic plaques are observed. Diffuse plaques, primitive plaques the quantitative studies of different without a ß-amyloid core, and classic plaques are evident. 100X plaques and comparison of staining methods in serial sections are underway, as well as the electron microscopy of such cases. Discussion The incidence of the progressive neurological disorder known as Alzheimer’s disease is characterized by loss of memory, carelessness about personal appearance, emotional disturbances that progress to complete disorientation, severe deterioration in speech, incontinence, and stereotypical repetitive movements. Pathologic changes include cortical degeneration that is more marked in frontal, temporal, and parietal lobes. Characteristic degeneration Fig. 8. Senile plaques. Bielschowsky stain, microwave method. 400X includes a decrease in neurons in regions of the brain that are responsible for cognition, memory, and other thought processes. The The neuritic plaques (Figs. 3, 5, 8, membrane-spanning amyloid cause of AD has not been fully 9, 10) are patches or flat areas precursor protein (APP). The elucidated, but there have been composed of clusters of function of APP is unclear, but it significant advances in our degenerating nerve terminals appears to be associated with the understanding of the origin of both 4 arranged around a central core of cytoskeleton of nerve fibers. AD-associated amyloid in the ß-amyloid peptide (BAP). Neuritic Normally, the degradation of APP neuritic plaques and the plaques are argentaphilic and involves cleavage in the middle neurofibrillary tangles in the contain abundant glial processes as of the BAP portion of the cytoplasm