Modified Bielschowsky Method Research Articles

LONG-TERM OUTCOMES OF RAT SCIATIC NERVE INJURY AND ITS GRAFTING WITH SILICON WIRES-BASED CONDUIT: DISTAL NERVE STUMP HISTOLOGY AND ELECTROMYOGRAPHY RESULTS

Relevance. The investigations on trauma epidemiology have shown that both combat- and noncombat-related extremity injuries are often accompanied by nerve injuries. These injuries disproportionately affect young healthy civilians and military officers and has a devastating impact on a patients’ quality of life.
 Severe nerve injuries, such as nerve trunk injury in continuity (Sunderland 5), that cannot be treated by neurorraphy without tension, require use of nerve gap bridging strategies with different materials and techniques.
 Objective. This study was aimed to evaluate any positive or negative impact of implanted silicon wires on the quality of nerve fibers at distal nerve stump.
 Materials and Methods. An experiment was performed on 40 male Whistar rats 2-4 month that were divided to the next groups:
 I, (n=10) sham-operated, only surgical access to sciatic nerve was performed.
 II (n=10) with 10 mm sciatic nerve gap that was bridged with autoneurografting.
 III (n=10) with 10 mm nerve gap that was bridged with allogenic decell aorta filled with 4% carboxymethylcellulose hydrogel.
 IV (n=10) with 10 mm nerve gap that was bridged with allogenic decell aorta filled with 4% carboxymethylcellulose hydrogel and aligned p-type silicon microvires.
 Decellularization of allogenic aortas was performed by freeze-thaw cycles.
 Silicon whiskers were fabricated by Vapor-Liquid-Solid (VLS) method in a cold wall Catalytic Chemical Vapor Deposition (Cat-CVD) chamber, pre-cleaned with hydrofluoric acid and sterilized via 180*C dry heat.
 12 weeks after surgery under general anesthesia all rats underwent invasive needle electroneurpmyography with proximal nerve stump stimulation and registration from gastrocnemius muscle. Myograms were recorded and compared by the shape of M-reflex and its amplitude.
 After myography rats were euthanized under thiopentone overdosage and distal stumps of injured sciatic nerves were harvested for light microscopy.
 Sciatic nerve transverse slices were stained with nitric silver by modified Bielschowsky method Nerve fiber diameter, axon diameter, myelin sheath thickness and axon-to-nerve fiber diameter ratio (g-ratio) were measured.
 Results. Performed analysis showed that rats from ІІ and IV groups demonstrated the best quality of nerve fibers in distal nerve stump. That was evidenced by bigger nerve fibers diameter in rats from autologous nerve grafting group and aorta with gel and wires grafting group in comparison with aorta with gel grafting group. Rats from IV demonstrated higher voltage and lower latency of M-reflexes during electromyography.
 Conclusions. It can be concluded about the possible pro-regenerative impact of implanted silicon wires that was evidenced by better nerve fibers quality at distal nerve stump.

CAIDE Dementia Risk Score, Alzheimer and cerebrovascular pathology: a population-based autopsy study.

CAIDE Dementia Risk Score is a tool for estimating dementia risk in the general population. Its longitudinal associations with Alzheimer or vascular neuropathology in the oldest old are not known. To explore the relationship between CAIDE Dementia Risk Score at baseline and neuritic plaques, neurofibrillary tangles, cerebral infarcts and cerebral amyloid angiopathy (CAA) after up to 10-year follow-up in the Vantaa 85+ population. Study population included 149 participants aged ≥85years, without dementia at baseline, and with available clinical and autopsy data. Methenamine silver staining was used for β-amyloid and modified Bielschowsky method for neurofibrillary tangles and neuritic plaques. Macroscopic infarcts were identified from cerebral hemispheres, brainstem and cerebellum slices. Standardized methods were used to determine microscopic infarcts, CAA and α-synuclein pathologies. The CAIDE Dementia Risk Score was calculated based on scores for age, sex, BMI, total cholesterol, systolic blood pressure, physical activity and APOEε4 carrier status (range 0-18 points). A CAIDE Dementia Risk Score above 11 points was associated with more cerebral infarctions up to 10years later: OR (95% CI) was 2.10 (1.06-4.16). No associations were found with other neuropathologies. In a population of elderly aged ≥85years, higher CAIDE Dementia Risk Score was associated with increased risk of cerebral infarcts.

Plasma homocysteine, Alzheimer and cerebrovascular pathology: a population-based autopsy study

Elevated plasma total homocysteine is associated with increased risk of dementia/Alzheimer's disease, but underlying pathophysiological mechanisms are not fully understood. This study investigated possible links between baseline homocysteine, and post-mortem neuropathological and magnetic resonance imaging findings up to 10 years later in the Vantaa 85+ population including people aged ≥85 years. Two hundred and sixty-five individuals had homocysteine and autopsy data, of which 103 had post-mortem brain magnetic resonance imaging scans. Methenamine silver staining was used for amyloid-β and modified Bielschowsky method for neurofibrillary tangles and neuritic plaques. Macroscopic infarcts were identified from cerebral hemispheres, brainstem and cerebellum slices. Standardized methods were used to determine microscopic infarcts, cerebral amyoloid angiopathy, and α-synuclein pathology. Magnetic resonance imaging was used for visual ratings of the degree of medial temporal lobe atrophy, and periventricular and deep white matter hyperintensities. Elevated baseline homocysteine was associated with increased neurofibrillary tangles count at the time of death: for the highest homocysteine quartile, odds ratio (95% confidence interval) was 2.60 (1.28-5.28). The association was observed particularly in people with dementia, in the presence of cerebral infarcts, and with longer time between the baseline homocysteine assessment and death. Also, elevated homocysteine tended to relate to amyloid-β accumulation, but this was seen only with longer baseline-death interval: odds ratio (95% confidence interval) was 2.52 (0.88-7.19) for the highest homocysteine quartile. On post-mortem magnetic resonance imaging, for the highest homocysteine quartile odds ratio (95% confidence interval) was 3.78 (1.12-12.79) for more severe medial temporal atrophy and 4.69 (1.14-19.33) for more severe periventricular white matter hyperintensities. All associations were independent of several potential confounders, including common vascular risk factors. No relationships between homocysteine and cerebral macro- or microinfarcts, cerebral amyoloid angiopathy or α-synuclein pathology were detected. These results suggest that elevated homocysteine in adults aged ≥85 years may contribute to increased Alzheimer-type pathology, particularly neurofibrillary tangles burden. This effect seems to be more pronounced in the presence of cerebrovascular pathology. Randomized controlled trials are needed to determine the impact of homocysteine-lowering treatments on dementia-related pathology.

Diabetes, Alzheimer disease, and vascular dementia

To investigate the relation of diabetes to dementia, Alzheimer disease (AD), and vascular dementia (VaD), through analyses of incidence, mortality, and neuropathologic outcomes in a prospective population-based study of the oldest old. The Vantaa 85+ study included 553 residents living in the city of Vantaa, Finland, and aged ≥85 years on April 1, 1991. Survivors were reexamined in 1994, 1996, 1999, and 2001. Autopsies were performed in 291 persons who died during the follow-up (48% of total population). Diabetes was assessed according to self-report, medical record of physician-diagnosed diabetes, or use of antidiabetic medication. Macroscopic infarcts were identified from 1-cm coronal slices of cerebral hemispheres, 5-mm transverse brainstem slices, and sagittal cerebellum slices. Methenamine silver staining was used for β-amyloid, methenamine silver-Bodian staining for neurofibrillary tangles, and modified Bielschowsky method for neuritic plaques. Cox proportional hazards and multiple logistic regression models were used to analyze the association of diabetes with dementia and neuropathology, respectively. Diabetes at baseline doubled the incidence of dementia, AD, and VaD, and increased mortality. Individuals with diabetes were less likely to have β-amyloid (hazard ratio [HR] [95% confidence interval (CI)] was 0.48 [0.23-0.98]) and tangles (HR [95% CI] 0.72 [0.39-1.33]) but more likely to have cerebral infarcts (HR [95% CI] 1.88 [1.06-3.34]) after all adjustments. Elderly patients with diabetes develop more extensive vascular pathology, which alone or together with AD-type pathology (particularly in APOE ε4 carriers) results in increased dementia risk.

Elevated Zinc Transporter-6 in Mild Cognitive Impairment, Alzheimer Disease, and Pick Disease

Filamentous cytoplasmic inclusions are hallmarks of Alzheimer disease (AD) and Pick disease (PD). Although previous studies show elevated zinc (Zn) in AD brain, there has been little study of zinc transporter (ZnT) proteins that are critical in the maintenance of Zn homeostasis. Using Western blot analysis, we show significantly elevated ZnT-6, the protein responsible for sequestration of Zn in the trans-Golgi network, in the hippocampus/parahippocampal gyrus (HPG) of AD subjects compared to age-matched controls and a trend toward elevated ZnT-6 in subjects with amnestic mild cognitive impairment (MCI). Based on these data, we used immunohistochemistry to investigate the cellular distribution of ZnT-6 in the HPG of control subjects and subjects with MCI, AD, and PD. Comparison of immediately adjacent serial sections stained using the modified Bielschowsky method and immunostained for ZnT-6 showed elevated ZnT-6 in 89 +/- 7% of neurofibrillary tangle (NFT)-bearing neurons in AD and 100 +/- 19% of Pick bodies in PD specimens. Confocal microscopy of HPG from MCI subjects double labeled for ZnT-6 and MC-1, a marker of early NFT formation, showed 85 +/- 4% of MC-1-positive cells were strongly ZnT-6-positive. Increased ZnT-6 immunostaining in neurons containing cytoplasmic inclusions in MCI, AD, and PD suggests a role for ZnT-6 in the pathogenesis of these lesions.

Focally Elevated Creatine Detected in Amyloid Precursor Protein (APP) Transgenic Mice and Alzheimer Disease Brain Tissue

The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the beta-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21-89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process.

Ectopic Expression of Musashi-1 in Alzheimer Disease and Pick Disease

Abnormal accumulation of proteins in filamentous cytoplasmic inclusions is a hallmark of several neurodegenerative disorders, including Alzheimer disease (AD) and Pick disease (PD). Musashi-1 (Msi-1), an RNA-binding protein associated with neural progenitor cells, has been shown by others to increase the accumulation of tau isoforms in intracellular inclusions in frontotemporal dementia and parkinsonism linked to chromosome 17. We investigated the expression of Msi-1 in the hippocampus of AD, PD, and aged normal control subjects using immunohistochemistry. Comparison of immediately adjacent serial sections stained using the modified Bielschowsky method and immunostained for Msi-1 showed that Msi-1 was present in 83 +/- 6% of neurofibrillary tangle bearing neurons in AD and 94 +/- 14% of Pick bodies in PD specimens. Aged control hippocampus demonstrated virtually no Msi-1 immunostaining. The presence of Msi-1 in a significant percentage of neurons containing cytoplasmic inclusions in 2 different neurodegenerative diseases suggests that it may play a role in the pathogenesis of these lesions.

Discontinuous distribution of senile plaques within striate cortex hypercolumns in Alzheimer's disease

Tangential sections of the primary visual (striate) cerebral cortex from five patients with histopathologically verified Alzheimer's disease were used to study the laminar and tangential disposition of senile plaques. These lesions were visualized with thioflavin S or the modified Bielschowsky method, and classified into four different, purely morphological types: "classical", (predominantly) "neuritic", (primarily amyloid) "core" and "diffuse", which were charted and analyzed using computer-assisted three- and two-dimensional reconstruction and mapping methods. These analyses reveal a tendency for a selective laminar disposition of the lesions (preferentially in layers II/III and V) which is generally consistent with previous reports performed at lower resolution, yet the specific pattern is highly variable among patients, and among plaque subtypes within individual patients. In addition, we observed a clustering of senile plaques in the tangential domain (i.e. parallel to the pial surface) in layers II/III, that suggests a selective involvement of iterated circuits within the "units", "modules", or "hypercolumns" that some believe compose this region of the cortex. These findings also imply an intriguing relative sparing of immediately adjacent components of the modular circuitry of the cerebral cortex, in the same cytoarchitectonic layers. Taken together, these findings indicate that: (1) senile plaques may arise in functionally and anatomically distinct subsets of iterated neuronal circuits that cannot be reduced to schemes based on traditional cytoarchitectonic layers; and (2) that individual variability in the patterns of striate cortex involvement and clinical manifestations must be taken into consideration when addressing the specific mechanisms underlying visual dysfunction in Alzheimer's disease.

Immunohistochemical, ultrastructural and immunoelectron microscopic studies of spinal cord neurofibrillary tangles in progressive supranuclear palsy.

Immunohistochemical, ultrastructural and immunoelectron microscopic studies of spinal cord neurofibrillary tangles (NFTs) in progressive supranuclear palsy (PSP) were performed. The spinal cord NFTs reacted with antibodies to tau protein (tau-2), ubiquitin and Alzheimer neurofibrillary tangles (ANTs, Ab 39). Ultrastructurally, the NFTs consisted of bundles of straight fibrils. In longitudinal sections, the individual NFT fibrils appeared as straight fibrils with a diameter of approximately 15 nm. In cross sections, circular structures approximately 15 nm in diameter were seen, and some had a central density. Electron microscopic examination of specimens stained with the antibodies and by the modified Bielschowsky method revealed the products of the tau, ubiquitin and ANTs immunoreactions and silver deposits on the NFT fibrils. This is the first demonstration of the ultrastructure of spinal cord NFTs in PSP.

Sensory nerve endings in the puborectalis and anal region of the fetus and newborn.

The study was carried out in seven fetuses (ovulation ages were 16, 18, 20, 22, 26, 28, and 31 weeks, respectively) and in five full-term newborn babies. The modified Bielschowsky method and Barker's block silver impregnation were used. It was found that the muscle spindle was arranged in the puborectalis and the external sphincter muscles from the 20th week onward; the tendon organ was located in association with the musculo-tendinous junction of the puborectalis from the 20th week onward; the Pacinian corpuscle lay in the plane of cleavage between the internal anal sphincter and the external anal sphincter from the 22nd week onward, and in the presacral space from the 28th week onward, and the later lagged behind the former in development; the globular endings lay in the presacral space from the 28th week onward; and the free nerve ending was distributed in the epithelium of the anal mucosa and the epidermis around the anus from the 31st week onward. These sensory nerve endings tend to mature gradually with increasing age. It was concluded that these endings may be responsible for the reflex of the external anal sphincter and the anal sensation.

Ultrastructural identification of neurofibrillary tangles in the spinal cords in Guamanian amyotrophic lateral sclerosis and parkinsonism-dementia complex on Guam.

The immunohistochemical and ultrastructural characteristics of spinal cord neurofibrillary tangles (NFTs) were examined in Guamanian amyotrophic lateral sclerosis and in parkinisonism-dementia complex on Guam. The spinal cord NFTs reacted with antibodies to tau protein (tau-2), ubiquitin and paired helical filaments (PHFs). Ultrastructurally, the components of the NFTs were seen as randomly arranged fibrils which were often associated with osmiophilic granules; small bundle-like arrangements were also occasionally observed. Individual NFT fibrils appeared as straight fibrils with a diameter of approximately 15 nm and constricted fibrils with a periodicity of approximately 80 nm. Ultrastructural microscopic examination of specimens stained by the modified Bielschowsky method and with the antibodies revealed silver particles and the products of the tau, ubiquitin and PHF immunoreactions on the NFT fibrils. This is the first demonstration of the fine structure of the spinal cord NFTs.

Demonstration of β amyloid protein-containing neurofibrillary tangles in parkinsonism-dementia complex on Guam

This report concerns the immunohistochemical demonstration of beta amyloid protein-containing neurofibrillary tangles (beta-NFTs) from patients with parkinsonism-dementia complex on Guam (PDC). Formalin-fixed paraffin-embedded tissue sections from the temporal lobe of nine PDC cases and the spinal cord of three were examined. An affinity-purified antibody to the N-terminal peptide sequence of beta amyloid protein was used. beta-NFTs were demonstrated in the temporal lobes of the three oldest individuals. Some senile plaques, probably a manifestation of ageing, were also seen. The beta-NFTs were most frequent in the entorhinal cortex. Senile plaques were invariably present in the vicinity of the beta-NFTs. Conversely, in the regions without senile plaques, even though numerous neurofibrillary tangles were identifiable with the modified Bielschowsky method, no beta-NFTs were seen. The density of beta-NFTs appeared to be proportional to that of senile plaques, none was seen in cases lacking senile plaques. No beta-NFTs were detected in the spinal cords. Our data suggest that beta-NFTs result from secondary beta amyloid deposition on certain neurofibrillary tangles.

Immunohistochemical Study of the Hippocampus in Parkinsonism-Dementia Complex on Guam

This investigation concerns the expression of paired helical filaments, tau protein, ubiquitin, beta-amyloid protein, and synaptophysin in the hippocampus of patients with parkinsonism-dementia complex on Guam (PDC) and Alzheimer's disease. Alzheimer's neurofibrillary tangles (NFTs) were identified in all cases of PDC and Alzheimer's disease by the modified Bielschowsky method, with which they were readily detected, and by immunohistochemical procedures using antibodies to paired helical filaments, tau protein, and ubiquitin. Observations regarding the different morphological stages indicated that NFTs were similar in PDC and Alzheimer's disease. The same markers were also useful for detecting neuropil threads, abundant in the CA1 field and the subiculum in both diseases. In the CA4 region of some PDC cases, prominent threads were noted. No senile plaques or amyloid angiopathies were seen in the hippocampus of the PDC cases examined. There was a significant decrease in synaptophysin immunoreactivity, most pronounced in the subfield CA1 and the subiculum, as well as in the outer molecular layer of the dentate gyrus, in both disorders.

Presenile appearance of abundant Alzheimer's neurofibrillary tangles without senile plaques in the brain in myotonic dystrophy

In 7 myotonic dystrophy (MyD) cases of 35- to 56-year old and 18 non-neurological age-matched controls paraffin-embedded temporal lobe sections were stained by the modified Bielschowsky method to count neurofibrillary tangles (NFTs) and senile plaques (SPs). In the parahippocampal gyrus, NFTs were observed in all the MyD cases; a few in the youngest, with an increase in number with age to the abundant appearance in the 4 cases in their 50s. The eldest also had many NFTs in the hippocampus. By contrast, the control subjects had, if any, only a few NFTs in the hippocampus and the parahippocampal gyrus. No SPs were observed in any MyD or control case. Thus, it was found that in MyD NFTs appear, unaccompanied by SPs, at an abnormally early age in the parahippocampal gyrus, with a rapid age-related increase in their number. This neuronal change may belong to the progeric features observed in this condition.

Sensory nerve endings in puborectalis and anal region: Normal findings in the newborn and changes in anorectal anomalies

This study was carried out in five full-term newborn babies and 11 patients with anorectal anomalies (five of whom had high deformity, five had intermediate, and one had low), using the modified Bielschowsky method. The following four kinds of sensory nerve ending were found: muscle spindle in the puborectalis and the external anal sphincter, Pacinian corpuscle in the plane between the internal anal sphincter and the external anal sphincter and in the presacral space, globular ending in the presacral space, and free nerve ending in the epithelium of the anal canal and in the perianal skin. It was found that there were significantly fewer muscle spindles in the puborectalis and many fewer Pacinian corpuscles and globular endings in the presacral space of high and intermediate deformities than normal. No spindle was found in the external sphincteric muscles of the deformities. On the other hand, the Pacinian corpuscles and the globular endings of high and intermediate deformities lagged behind normal in development. The densities of the nerve bundles in the subepidermis and the nerve fibers in the dermis of perineal region in high and intermediate deformities were much lower than normal, and the decrease in density corresponded to the levels of the rectal pouch. The observation showed that the agenesis of sensory nerve endings in the puborectalis and the anal region was one of the most significant pathological features in high and intermediate types of anorectal anomalies, and might be responsible for the abnormalities of the reflex of the anal sphincter and the anal sensation in such patients. The muscle spindles in the puborectalis may be only potential receptors for the reflex and sensation after surgery.

Comparison of three silver stains for demonstrating neurofibrillary tangles and neuritic plaques in brain tissue stored for long periods

Three methods were compared to find a reliable method for demonstrating neurofibrillary tangles (NFTs) and neuritic plaques (NPs) in brain tissue stored for long periods in formalin or as paraffin blocks. The short-term fixation of tissue, e.g. up to 6 months in formalin does not usually present a problem using any of the three methods tried, e.g. Gallyas, modified Palmgren, or modified Bielschowsky, but once the time lengthens to 6 years or more the demonstration of NFTs and NPs is not so reliable using the first two methods. The modified Bielschowsky method, however, demonstrates well both NFTs and NPs in material stored in formalin or as paraffin blocks for long periods, e.g. 7 years, and also compares favourably with the other methods on freshly processed material and fixed tissue, stored for shorter periods. We also noted as a consistent trend, irrespective of the staining technique employed, the detection of fewer plaques and tangles in material stored in formalin, as opposed to that stored as blocks embeded in paraffin wax.

COMPARISON OF SEVEN STAINING METHODS FOR SENILE PLAQUES AND NEUROFIBRILLARY TANGLES IN A PROSPECTIVE SERIES OF 15 ELDERLY PATIENTS

Samples of brain from 15 prospectively tested women over 75 years of age, living in the same institution and with a Blessed test score between 0 and 28 were studied to evaluate seven different staining techniques for counting senile plaques and neurofibrillary tangles. The methods included Bielschowsky (modified by Yamamoto & Hirano, 1986), Cross, Gallyas, Naoumenko-Feigin, silver methenamine, Bodian coupled with luxol fast blue, and thioflavine S; these techniques were performed on contiguous slides of the first temporal gyrus (Brodmann's area 22). The cost, difficulty and variability of the methods were evaluated. Modified Bielschowsky method revealed both amyloid and neurites. Cross, Bodian and Gallyas stained neurites preferentially and were more sensitive for neurofibrillary tangles than for senile plaques. Silver methenamine revealed amyloid in much the same way as thioflavine S. The highest count of senile plaques and neurofibrillary tangles was obtained with the modified Bielschowsky method. Best correlations between density of senile plaques and Blessed test score were obtained with modified Bielschowsky impregnation, silver methenamine, and Bodian and those for neurofibrillary tangles, with modified Bielschowsky, Naoumenko-Feigin, and Gallyas methods. Naoumenko-Feigin impregnation, even if fitted to the linear model, revealed very few changes (four times less than modified Bielschowsky) and appeared to be insensitive. All the techniques except Naoumenko-Feigin, were equally able to distinguish two groups of cases, i.e. those affected by Alzheimer's disease and those not affected. A multiple regression analysis indicated that the statistical relationship between the density of changes, evaluated after the modified Bielschowsky method, and the Blessed test score could not be improved by the additional use of other staining techniques. These data have direct implications for the evaluation of diagnostic criteria in Alzheimer's dementia. In this study, modified Bielschowsky impregnation was found to give the most complete picture of the lesions and, as such, could be considered a reference method. This technique is, however, expensive and difficult to perform. It stains a large number of normal structures and the recognition of the lesions is more subjective than with other selective stains. It is therefore difficult to recommend this technique for routine use. Other techniques alone, or in combination, can be recommended but the diagnostic criteria should be adapted for each of them.