Bielschowsky Staining Research Articles

Genetic association of ?2-macroglobulin with Alzheimer's disease in a Finnish elderly population

Recently, two studies have reported an association between the alpha2-macroglobulin gene on chromosome 12 and late-onset Alzheimer's disease, whereas others have not been able to replicate these findings. By using a prospective population-based study, we have investigated the relation between two polymorphisms in this gene with the presence of the disease and also with the extent of pathological changes in the cerebral cortex. The Vantaa 85+ Study includes all 601 persons, at least 85 years of age, who were living in Vantaa, Finland, on April 1, 1991. The neocortical beta-amyloid protein load and the number of neurofibrillary tangles were determined on tissue sections by using methenamine silver staining and a modified Bielschowsky staining, respectively. The A/A genotype in exon 24 of the alpha2-macroglobulin gene was associated with neuropathologically defined diagnosis of Alzheimer's disease according to the CERAD (Consortium to Establish a Registry for Alzheimer's Disease) criteria and with an increase in the neocortical beta-amyloid protein load. The effect of this association was stronger in the apolipoprotein E epsilon4-negative group. Therefore, genetic variability in the alpha2-macroglobulin gene is a risk factor associated with neuropathologically defined Alzheimer's disease in our population, as well as with the extent of neocortical beta-amyloid protein deposition.

Subfield- and layer-specific changes in parvalbumin, calretinin and calbindin-D28k immunoreactivity in the entorhinal cortex in Alzheimer's disease

The entorhinal cortex, which is involved in neural systems related to memory, is selectively degenerated in early Alzheimer's disease. Here, we examined neuropathological changes in the eight entorhinal subfields in post mortem Alzheimer's disease subjects using Thionin and Bielschowsky stains and parvalbumin, calretinin and calbindin-D28k immunohistochemistry. Both histological stains revealed the most dramatic cell loss and neurofibrillary tangle formation to be in layers II and V of the lateral, intermediate and caudal subfields. In accordance, immunohistochemical staining showed that neurons and fibres that contain calcium-binding proteins were also more frequently altered in these subfields than in the rostromedial subfields. Detailed analysis further revealed that non-principal cells containing parvalbumin or calbindin-D28k showed morphological alterations early in the entorhinal pathology of Alzheimer's disease, whereas non-principal neurons containing calretinin were better preserved even in Alzheimer's disease patients with severe entorhinal pathology. The degeneration of parvalbumin-immunoreactive neurons and basket-like networks and calbindin-positive non-principal neurons was observed mainly in layer II, where the calretinin-positive non-principal neurons formed aggregates especially at late stages of the disease. The pyramidal-shaped neurons containing either calretinin or calbindin-D28k were often preserved, although morphological alterations were observed. Our findings indicate that specific subfields of the entorhinal cortex involving neurons that contain distinct calcium-binding proteins are differentially vulnerable in Alzheimer's disease. This could have an impact on the topographically organized inputs and outputs of the entorhinal cortex in Alzheimer's patients.

Early Amyloid Deposition in the Medial Temporal Lobe of Young Down Syndrome Patients: A Regional Quantitative Analysis

The presence of the neuropathological alterations of Alzheimer's disease (AD) in essentially all older Down syndrome (DS) patients suggests that the examination of younger DS patients may clarify the early pathological progression of AD. We examined the hippocampus and parahippocampal-inferior temporal gyri of 42 DS patients (ages 4 days to 38 years) for the deposition of amyloid β protein (Aβ) using both a modified Bielschowsky stain and immunohistochemistry for Aβ protein. The parahippocampal and inferior temporal gyri demonstrated Aβ staining in cases as young as 8 years of age. As age and degree of Aβ deposition increased, staining included the CA-1/subiculum and dentate molecular layer followed then by the remainder of the CA hippocampal regions. The first neuritic plaques were observed in the CA-1/subiculum, despite this being a later region of Aβ deposition. Although Aβ staining increased with age, there was substantial variability in the severity of Aβ deposition within age groups. These results suggest that within the hippocampal/parahippocampal region there is a progressive stereotypic deposition of Aβ. The variable severity of Aβ deposition within age groups suggests that other factors, besides DS, may be contributing to the timing and severity of Aβ deposition.

Age-related changes in the olfactory system of dogs.

Age-related changes in the olfactory system were examined in 22 dogs ranging in age from 10 to 19 years old. Atrophic changes with degeneration were observed in the olfactory epithelium of dogs older than 14 years; the changes were prominent in the dogs over the age of 17 years. Immunohistochemistry using an anti-carnosine antibody, which is a marker for the olfactory cells, demonstrated a decrease in the number of olfactory cells. Electron microscopy also showed a decrease in the number of cilia of olfactory cells and microvilli of supporting cells. Atrophic changes with the features of regeneration were rarely observed in the aged animals. Lipofuscin-like granules in the olfactory epithelium became prominent with age. These age-related changes were similar to those reported in the olfactory epithelium of man and rats. Dystrophic neurites were not detected by a modified Bielschowsky stain or by neurofilament, synaptophysin and tau immunohistochemistry in the olfactory mucosa. There was no beta-amyloid- and ubiquitin-immunostaining in the olfactory mucosa. Senile brain changes, including cerebrovascular amyloidosis, age-related astrocytic gliosis and ubiquitin deposits were found in the olfactory bulb, although neurofibrillary tangles and senile plaques were not detected either by a modified Bielschowsky stain or by beta-amyloid immunohistochemistry. These results indicate that dog may be a useful animal model to study the age-related changes in the olfactory system in man.

Induction of lysosomal abnormalities and tau hyperphosphorylation in human neuroblastoma cells by colchicine and okadaic acid: Evidence that microtubule disruption contributes to Alzheimer neurodegeneration

Two early pathological features of neurons in Alzheimer's disease, the intraneuronal accumulation of lysosomal hydrolases and alterations in tau antigenicity, distribution and electrophoretic mobility, have been suggested to arise in whole or in part from disruption of the microtubule system. To test this hypothesis, we subjected SH-SY-5Y human neuroblastoma cells to treatment with colchicine and the phosphatase inhibitor okadaic acid (OA), both of which have been previously demonstrated to depolymerize microtubules in these cells. We observed that these treatments induced the accumulation of lysosomal hyrolases and AD-like tau immunoreactivity. These findings support the hypothesis that microtubule breakdown may represent a contributing factor to Alzheimer neurodegeneration. Unlike the alteration in tau antigenicity, Bielschowsky staining indicated that neither treatment induced neurofibrillary pathology in these cells. This latter finding underscores the possibility that the interrelated events of microtubule disruption, tau hyperphosphorylation and lysosomal accumulation are antecedent events in AD neuropathology.

Axonal reinnervation does not influence Schwann cell proliferation after rat sciatic nerve transection

We asked whether reinnervating axons are Schwann cell mitogens in vivo as they are in vitro. Left sciatic nerves of 50 Wistar rats were transected. In one-half of the animals, axonal reinnervation from the proximal to the distal stump was allowed to take place, while in the other half, sutures were placed on the transected nerve ends to prevent reinnervation. Samples were collected from 3 days up to 8 weeks after the transection proximally and distally from the point of transection. PCNA-immunostaining was performed on paraffin sections to determine the number of proliferating cells. Axonal reinnervation was followed by Bielschowsky staining and Schwann cell number was determined by counting S-100-immunopositive cells from paraffin sections. In the distal stump Schwann cell proliferation was similar in both experimental groups. There was no statistical evidence of S-100 negative cell proliferation during the study. Proximally to the site of transetion of she number of small initial axonal sprouts and also the number of Schwann cells increased if the nerve stump had been sutured. In conclusion, although axons may be mitogenic for Schwann cells to a greater extent than other potential effects associated with nerve transection.

Demonstration of Nucleolar Organizer Region Associated Protein: Utility of the Bielschowsky Stain and Counterstains

AbstractThe demonstration of nucleolar organizer regions (NORs) has received increasing popularity in the study of basic cell biology and tumorigenesis. The most widely used method requires a 50% silver nitrate gelatin emulsion (AgNOR) and dark room manipulation. Morphologic detail is often poor. We investigated Bielschowsky (Biel) and Grimelius (Grim) silver techniques and nuclear fast red (NFR) and metanil yellow (MY) counterstains. We compared them with the AgNOR technique. Five breast carcinomas and 5 malignant melanomas were stained and counted in a single blinded fashion. Comparisons to AgNOR were made and correlation coefficients (r) calculated as follows: AgNOR vs AgNOR + NFR, r = 0.96; AgNOR vs Biel, r = 0.96; AgNOR + NFR vs Biel + NFR, r = 0.98; Biel vs Biel + NFR, r = 0.93. Grim and MY were unsatisfactory.These data demonstrated that Bielschowsky offers a comparable alternative to AgNOR that may be more suitable for smaller laboratories. NFR improved morphologic detail and did not significantly...

Eetal Nerve Healing

An experimental study was performed to assess fetal nerve repair and regeneration both qualitatively and quantitatively. The posterior tibial nerves in one hindlimb were transected in 16 midgestational fetal lambs and in their mothers. The nerves were then repaired with epineurial sutures and allowed to progress to 2, 4, 6, and 8 weeks postinjury. Qualitative assessment was performed through standard nerve histologic staining, including Luxol fast and toluidine blue for myelin and Bielschowsky stain for axons, and quantitative assessment through nerve conduction velocity studies and morphometry to determine mean myelinated fiber diameter, total fiber number, and density. A frequency histogram of the distribution of myelinated nerve fibers according to fiber diameter also was generated. In our model, the subsequent fetal nerve response to injury was characterized by earlier degeneration than in the adult counterparts. Repair and regeneration proceeded with dense collagenous scar formation in both groups. Electrophysiologic studies showed nerve impulse conduction across the repair site only at 6 and 8 weeks postinjury in both fetus and adult. Action potential amplitudes at 6 and 8 weeks were measured at 3 to 5 percent of control nerves in both nerve types. No electrophysiologic differences in the recovery of the injured fetal and adult nerves could be identified. Morphometry revealed that fetal nerve regeneration appeared to occur at a rate equivalent to that of the adult, although by 8 weeks the total percentage of remyelinated nerves appeared more complete in the fetus than in the adult (87 versus 59 percent), suggesting that fetal nerves may have a more favorable regenerative capacity than their adult counterparts.

Demonstration of Nucleolar Organizer Region Associated Protein: Utility of the Bielschowsky Stain and Counterstains

Demonstration of Nucleolar Organizer Region Associated Protein: Utility of the Bielschowsky Stain and Counterstains

Morphology of cerebral plaque-like lesions in hereditary cerebral hemorrhage with amyloidosis (Dutch)

We studied the presence and morphology of plaque-like lesions in the frontal cortex of six patients, aged 40 to 76 years, with hereditary cerebral hemorrhage with amyloidosis--Dutch type (HCHWA-D), using beta/A4 immuno-, silver, Congo red and thioflavin S staining. Two types of beta/A4 immunoreactive and Congo red-negative plaques were detected. The first type was composed of argyrophilic fibrous material in periodic acid-methenamine silver (PAM) and modified Bielschowsky staining and lacked silver-stained degenerating neurites. Therefore, this type of plaque has the same staining properties as the diffuse plaque described in Alzheimer's disease, Down's syndrome and nondemented elderly. The second type of plaque, occurring only in the three oldest patients and numerically increasing with age, consisted of a spherical non-argyrophilic area of granular texture with a rim of PAM-positive material. The PAM-positive fibrous material of both types of plaques was mingled with coarser and compact, irregular-shaped argyrophilic structures in the oldest patient. The described plaques did not show bright fluorescence with thioflavin S staining. These results indicate, that the morphology of plaques, encountered in HCHWA-D, is diverse and changes with age.

Descriptive morphology of developing fetal neostriatal allografts in the rhesus monkey: A correlated light and electron microscopic golgi study

Primate fetal striatal neurons were transplanted into the ibotenic acid lesioned rhesus monkey striatum. Ten weeks after transplantation the monkeys were transcardially perfused and graft tissue was histologically stained, Golgi impregnated, and processed for electron microscopy. The monkeys received magnetic resonance imaging (MRI) scans before lesioning, after lesioning, and ten weeks after transplantation to noninvasively study the striatal grafts. The study demonstrated that fetal striatal grafts, measuring up to 0.4 × 0.8 cm, can survive for extended periods of time in the non-human primate. Hematoxylin-eosin stained sections of the transplant demonstrated that neuronal, glial, vascular, and lymphocytic cells were present in the graft. The majority of the neurons had somatic diameters between 8 and 20 μm and were characterized by nuclei containing multiple nucleoli. A few neurons within the graft had somatic diameters up to 40 μm. These larger neurons exhibited more mature cytoplasm containing a moderate amount of Nissl substance. Some of the blood vessels within the graft were surrounded by a large number of plasma cells, but there was no evidence of hemorrhage or necrosis. Bielschowsky staining and Golgi impregnation of the transplanted tissue demonstrated that there were neurons at various degrees of differentiation. Some of the neurons had varicose dendrites, growth cones, and filopodia, which are all characteristics of immature neurons, while others had a much more mature appearance, including a moderate number of dendritic spines. Some of these neurons had an appearancetypical of differentiating “medium spiny” neurons of the normal striatum. Electron microscopic analysis of the transplanted tissue and individual Golgi-impregnated neurons within the transplant confirmed that there were developing neurons within the graft. These neurons had an increased nuclear-to-cytoplasmic ratio and had nuclei containing multiple nucleoli. The neuropil surrounding these neurons was loosely organized and contained large areas of extracellular space. The neuropil exhibited developing dendrites, numerous growth cones, and mature synapses. In summary, the study demonstrated that fetal striatal allografts can survive for up to three months in the rhesus monkey and undergo normal differentiation as assessed by Golgi impregnation and electron microscopy.

Topographic relationship between senile plaques and cerebrovascular amyloidosis in the brain of aged dogs.

The distributions of senile plaques (SP) and cerebrovascular amyloidosis (CA) were studied by employing thioflavin S and modified Bielschowsky stains, and beta-protein immunohistochemistry on serial sections of the brains of aged dogs older than 10 years. Mature and perivascular plaques, both of which contained compact amyloid deposits, always showed a close topographic relationship to CA. In contrast, the majority of diffuse plaques showed no topographic relationship to CA. Cell bodies of neurons and/or glia were almost always involved in the diffuse plaques. In addition, beta-protein immunohistochemistry demonstrated amyloid deposits on the periphery of occasional neurons. These findings suggest that different mechanisms may be involved in the development of the different subtypes of SP in the brains of aged dogs.

A case of progressive supranuclear palsy with widespread senile plaques

A case of progressive supranuclear palsy (PSP) with frontal lobe atrophy is reported, in which many senile plaques were widely distributed in the neocortex, the entorhinal cortex, the amygdala, and, to a lesser extent, the cerebellar cortex, but not in the hippocampus. Most of the plaques were of the diffuse and primitive types. They were well visualized by beta-protein immunostaining, modified Bielschowsky staining and methenamine silver staining, but were not seen by Bodian staining. The widespread distribution of senile plaques in the cerebral and cerebellar cortices was far beyond that seen in normal aging, and was reminiscent of concomitant Alzheimer's disease (AD). Unlike AD, however, this case had neither senile changes in the hippocampus nor neurofibrillary tangles in the amygdala and entorhinal cortex. It seems that many senile plaques may appear widely in the cerebral cortex and even, to a lesser extent, in the cerebellar cortex of some patients with PSP. Additional case studies using sensitive silver and amyloid antibody preparations are required to elucidate the presence of senile plaques in the cerebral cortex of PSP.

Modified Bielschowsky and immunohistochemical studies on senile plaques in aged dogs

Aged dogs developed senile plaques (SP) in the brain which were similar to those occurring in aged humans. These SP were studied with a modified Bielschowsky stain and immunohistochemical methods using polyclonal antibodies raised against β-protein and glial fibrillary acidic protein (GFAP). Serial sections stained by immunohistochemical and modified Bielschowsky stains showed that all areas of slight β-protein immunoreactivity were intensely stained by the modified Bielschowsky stain. Most SP were observed in the neocortex of the cerebrum. Plaque densities were highest in the cingulate and temporal cortices. There were occasional SP in the subcortical nuclei including the caudate nucleus and putamen, and hippocampus. Based on the morphological characteristics demonstrated by the modified Bielschowsky stain, SP in the brains of the dogs were grouped into 3 types: diffuse, mature and perivascular plaques, of which diffuse plaques were predominant. Various degrees of astroglial reaction were observed in all subtypes of SP except diffuse plaques. These findings indicate that dog may serve as a model for study of the pathogenesis of SP.

Modified Bielschowsky stain and immunohistochemical studies on striatal plaques in Alzheimer's disease

The nature of senile plaques (SP) in the striatum in 14 cases of Alzheimer's disease (AD) was investigated with the modified Bielschowsky stain and immunohistochemistry using antibodies to a beta amyloid synthetic peptide, ubiquitin, tau protein, and paired helical filaments (PHF). Striatal SP, composed of beta amyloid deposits with or without neuritic elements, were demonstrated in all AD cases examined. Compact and perivascular amyloid deposits were concentrated in the ventral striatum, including the nucleus accumbens. Many diffuse amyloid deposits in the ventral striatum contained ubiquitin-positive granular elements, presumably representing dystrophic neurites, whereas most of those in the dorsal striatum did not have such elements. On the other hand, most compact amyloid deposits in both ventral and dorsal striatum had ubiquitin immunoreactivity. Dystrophic neurites with tau or PHF immunoreactivity were detected particularly around compact amyloid deposits. Our results indicate that the ventral striatum, which is closely affiliated with the limbic system, is frequently affected by amyloid deposits with dystrophic neurites, and suggest that the ventral striatum is particularly vulnerable to AD. Furthermore, our results suggest that amyloid deposits, especially compact deposits, may induce dystrophic neurites.

Cytoplasmic argyrophilic inclusions in neurons of pontine nuclei in patients with olivopontocerebellar atrophy: immunohistochemical and ultrastructural studies

Patients with olivopontocerebellar atrophy (OPCA) were studied, and cytoplasmic inclusions were observed in some of the remaining neurons of the pontine nuclei, nuclei reticularis tegmenti pontis and arcuate nuclei. The cytoplasmic argyrophilic inclusions were demonstrated by silver impregnation techniques such as Bielschowsky and Bodian staining. With hematoxylin and eosin stain, the inclusions were sharply demarcated and appeared pale. The inclusions were not stained by the following routine histological methods: Klüver-Barrera, phosphotungstic acid hematoxylin, Holzer, periodic acid-Schiff, Mallory azan, alcian blue, nile blue, Masson trichrome, Congo red, thioflavine S, oil red O and Sudan black B stains. Immunohistochemistry with anti-ubiquitin antiserum showed that these inclusions were ubiquitinated. However, the inclusions did not react with any of the following antibodies (Abs) or antisera: anti-phosphorylated neurofilament (NF) Ab, anti-nonphosphorylated NF Abs (160 and 200 kDa), anti-paired helical filament antiserum, anti-tau antiserum, anti-tubulin Abs (alpha and beta), anti-microtubule-associated protein antiserum, anti-glial fibrillary acidic protein antiserum, anti-vimentin Ab, anti-desmin Ab, anti-cytokeratin Abs (low and high molecular weights), anti-actin antiserum, anti-skeletal myosin antiserum and anti-myelin basic protein Ab. Ultrastructurally, the inclusion bodies noted in OPCA were composed primarily of fibrils having a width ranging from about 24 to 40 nm, which were entirely coated with osmiophilic granular material along their whole length. They were occasionally intermingled with a few filaments about 10 nm in width. Electron microscopical examination on silver-impregnated specimens revealed that each granule-coated fibril had a great affinity for silver particles. In elucidating the pathogenesis of OPCA, it was considered to be an important neuropathological finding that some of the remaining pontine neurons affected by OPCA developed characteristic cytoplasmic argyrophilic inclusions.

Modified Bielschowsky and Immunocytochemical Studies on Cerebellar Plaques in Alzheimer's Disease

Senile plaques (SP) in the cerebellum of 23 cases of Alzheimer's disease (AD), three with widespread amyloid angiopathy, were studied with a modified Bielschowsky stain and immunocytochemical methods using antibodies to a beta-amyloid synthetic peptide (beta ASP), phosphorylated neurofilament proteins, ubiquitin, tau protein, and glial fibrillary acidic protein (GFAP). The four subtypes of SP (diffuse plaques, compact plaques, perivascular plaques, and subpial fibrillar deposits) that were observed with the modified Bielschowsky stain were also stained with antibodies to beta ASP. Many cerebellar SP contained ubiquitin-positive granular elements resembling dystrophic neurites. In contrast to neuritic elements in cerebral SP in AD, ubiquitin-positive elements in cerebellar SP were not labeled with antibodies to phosphorylated neurofilament or tau proteins. Various degrees of glial reaction were observed in all subtypes of SP except diffuse plaques. The absence of phosphorylated neurofilament and tau epitopes in neuritic elements in cerebellar SP is not surprising since paired helical filaments have not been seen in the cerebellum. Nevertheless, our results suggest that cerebellar SP are frequently associated with dystrophic neurites.

Modification of Brain Processing Techniques: Tissue Embedding in Glycol Methacrylate and Stain Modifications

Abstract Increased interest in quantitation of the histopathological changes in a variety of neurological disorders (including neurodegenerative disease such as Alzheimer's disease) continues in an attempt to develop specific clinical-histopathological correlations. Most previous efforts at quantitation have used paraffin embedded sections of brain tissue, although plastic embedded sections have recently become a preferable alternative because they provide greatly reduced tissue shrinkage and distortion during processing, and greater clarity and improved resolution to the tissue sections. We have developed techniques for glycol methacrylate embedding and sectioning of brain tissue blocks on a standard histology laboratory microtome. In addition, we have modified routine diagnostic and investigational neurohistological stains for use in glycol methacrylate embedded brain sections, including hematoxylin and eosin, modified Bielschowsky stain, Jamarri silver technique, Einarson's Nissl stain, gallocyanin-Dar...

Diffuse Lewy body disease: light and electron microscopic immunocytochemistry of senile plaques.

The nature of senile plaques (SP) in 27 cases of diffuse Lewy body disease (LBD) was investigated using immunocytochemistry and antibodies to beta amyloid protein synthetic peptides (BetaSP), ubiquitin (UBQ), paired helical filaments (PHF; Ab39) and a 68-kDa protein in Alzheimer brains (Alz50). Lewy bodies were present in widespread areas of the neocortex of all cases and were more easily detected with ubiquitin immunocytochemistry than with conventional stains. All cases had neocortical SP, but only six cases had neocortical neurofibrillary tangles (NFT). SP were very numerous in most cases and were usually "pale", "diffuse" or "very primitive" plaques with thioflavin S fluorescent microscopy. SP in diffuse LBD were immunostained with BetaSP. Several cases had extensive amyloid angiopathy that was also immunoreactive with BetaSP. SP in diffuse LBD were characterized by amyloid deposits with few or no neuritic elements that could be detected with thioflavin S, Bielschowsky's stain or double staining with BetaSP and Bodian's silver stain. They differed from plaques in Alzheimer's disease by lack of PHF-type neurites that could be stained with Ab39. In diffuse LBD, SP contained PHF-type neurites only in areas coexistent with NFT. Some SP had round, granular neurites that were immunoreactive with UBQ, but weakly argyrophilic with Bodian's stain and nonfluorescent with thioflavin S. Diffuse LBD lacked significant neuritic change in the neuropil that could be detected with UBQ, Ab39 and Alz50. The latter finding is a characteristic feature that distinguishes Alzheimer's disease from diffuse LBD.

Diffuse type of senile plaques in the cerebellum of Alzheimer-type dementia demonstrated by? protein immunostain

We studied senile plaques (SP) in the cerebella of six autopsied subjects with Alzheimer-type dementia (ATD) and ten non-ATD autopsied subjects between the ages of 78 and 90. Neither SP nor amyloid angiopathy (AA) was observed in any of the non-ATD subjects. In the four of the six ATD subjects, diffuse plaques in the molecular layer were seen as ill-defined areas of fine fibrillar materials by beta protein immunostaining with formic acid pretreatment, the modified Bielschowsky stain, and periodic acid-methenamine silver (PAM) stain. The plaques were not visible with Bodian, Congo red, or periodic acid-Schiff stains. Compact plaques in the Purkinje cell or in the granular cell layers were found in three of the six subjects. Their amyloid core was often surrounded by areolar amyloid deposits. AA was observed in three of the six subjects. The argyrophilia of the diffuse and compact plaques, demonstrated by the modified Bielschowsky and PAM stains, became undetectable when the sections were first treated with formic acid. Such treatment made the plaques immunoreactive with beta protein antiserum. The findings suggested that cerebellar diffuse plaques and compact plaques consist mainly of an amyloid component, and are characteristic of ATD.