Treatment with 1, 10 Phenanthroline-5-Amine Reduced Amyloid Burden in a Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most prevalent age-related dementia, and, despite numerous attempts to halt or reverse its devastating progression, no effective therapeutics have yet been confirmed clinically. However, one class of agents that has shown promise is certain metal chelators. OBJECTIVE: For the novel assessment of the effect of oral administration of 1,10-phenanthroline-5-amine (PAA) on the severity of amyloid plaque load, we used a transgenic (Tg) mouse model with inserted human autosomally dominant (familial) AD genes: amyloid-ß protein precursor (AßPP) and tau. METHODS: AßPP/Tau transgenic mice that model AD were allotted into one of two groups. The control group received no treatment while the experimental group received PAA in their drinking water starting at 4 months of age. All animals were sacrificed at 1 year of age and their brains were stained with two different markers of amyloid plaques, Amylo-Glo+ and HQ-O. RESULTS: The control animals exhibited numerous dense core plaques throughout the neo- and allo- cortical brain regions. The experimental group treated with PAA, however, showed 62% of the amyloid plaque burden seen in the control group. CONCLUSIONS: Oral daily dosing with PAA will significantly reduce the amyloid plaque burden in transgenic mice that model AD. The underlying mechanism for this protection is not fully known; however, one proposed mechanism involves inhibiting the "metal-seeding" of Aß.

Use of a stable and solvent-soluble variant of Amylo-Glo, Amylo-Glo+, for the high-contrast and resolution fluorescent localization of amyloid plaques in murine and human brain tissue sections.

BACKGROUND: Although numerous different fluorescent compounds have been used for the fluorescent localization of amyloid plaques, limitations include weak fluorescence, low contrast, low resolution, broad excitation / emission profile and chemical instability. NEW METHOD: Amylo-Glo+ was reconstituted from a powder into a stable solution for the histological staining of plaques in brain tissue sections from both patients diagnosed with Alzheimer's disease and APP/PS1 transgenic mice. RESULTS: Amylo-Glo+ was found to result in high resolution and contrast labeling of amyloid plaques. The tracer was excited exclusively by ultraviolet light illumination. The staining of amyloid plaques corresponded with the labeling pattern seen following staining with HQ-O. Counterstaining with ethidium bromide resulted in no bleed-through of the Amylo-Glo+ under blue or green light excitation. The tracer was stable in both powder and solution form. COMPARISON WITH EXISTING METHODS: Amylo-Glo+ was found to be more stable, in both a powder and solution form, than the original Amylo-Glo, brighter than Congo red, of narrower emission and greater contrast than Thioflavine S and of narrower emission than X-34 and BSB. CONCLUSION: Amylo-Glo+ is a stable and solvent soluble variant of Amylo-Glo that allows for the high contrast and resolution localization of amyloid plaques in tissue sections from the brains of transgenic mice and humans with Alzheimer's disease. The tracer is also solely excited by ultraviolet light, making it ideal for fluorescent multiple labeling studies in combination with commonly used red or green fluorophores. It is also stable in both solution and powder form, unlike the original Amylo-Glo which is only available as a basic solution that is prone to precipitation.

High Contrast and Resolution Labeling of Amyloid Plaques in Tissue Sections from APP-PS1 Mice and Humans with Alzheimer's Disease with the Zinc Chelator HQ-O: Practical and Theoretical Considerations.

BACKGROUND: Various methodologies have been employed for the localization of amyloid plaques in numerous studies on Alzheimer's disease. The majority of these stains are thought to label the plaques by virtue of their affinity for aggregated Aß. However, plaques are known to contain numerous other components, including multivalent metals such as zinc. OBJECTIVE: This investigates whether it is possible to localize the presence of zinc in parenchymal and vascular amyloid plaques in afflicted brains. To accomplish this, a novel fluorescent zinc chelator, HQO, was investigated to determine its mechanism of binding and to optimize a stain for the high contrast and resolution histological localization of amyloid plaques. METHODS: A novel zinc chelator, HQ-O, was developed for localizing zinc within amyloid plaques. The histology involves incubating tissue sections in a dilute aqueous solution of HQ-O. Its compatibility with a variety of other fluorescent methodologies is described. RESULTS: All amyloid plaques are stained in fine detail and appear bright green under blue light excitation. The staining of parenchymal plaques correlates closely with that seen following staining with antibodies to Aß, however, the HQ-O sometimes also label additional globular structures within blood vessels. In situ mechanistic studies revealed that fluorescent plaque-like structures are only observed with HQ-O when synthetic Aßx-42 is aggregated in the presence of zinc. CONCLUSION: Zinc is intimately bound to all amyloid plaques, which was demonstrated by its histological localization using a novel fluorescent zinc chelator, HQ-O. Additionally, the tracer is also capable of labeling intravascular leucocytes due to their high zinc content.

Introducing Euro-Glo, a rare earth metal chelate with numerous applications for the fluorescent localization of myelin and amyloid plaques in brain tissue sections.

The vast majority of fluorochromes are organic in nature and none of the few existing chelates have been applied as histological tracers for localizing brain anatomy and pathology. NEW METHOD: In this study we have developed and characterized a Europium chelate with the ability to fluorescently label normal and pathological myelin in control and toxicant-exposed rats, as well as the amyloid plaques in aged AD/Tg mice. RESULTS: This study demonstrates how Euro-Glo can be used for the detailed labeling of both normal myelination in the control rat as well as myelin pathology in the kainic acid exposed rat. In addition, this study demonstrates how E-G will label the shell of amyloid plaques in an AD/Tg mouse model of Alzheimer's disease a red color, while the plaque core appears blue in color. The observed E-G staining pattern is compared with that of well characterized tracers specific for the localization of myelin (Black-Gold II), degenerating neurons (Fluoro-Jade C), A-beta aggregates (Amylo-Glo) and glycolipids (PAS). COMPARISONS WITH EXISTING METHODS: This study represents the first time a rare earth metal (REM) chelate has been used as a histochemical tracer in the brain. This novel tracer, Euro-Glo (E-G), exhibits numerous advantages over conventional organic fluorophores including high intensity emission, high resistance to fading, compatibility with multiple labeling protocols, high Stoke's shift value and an absence of bleed-through of the signal through other filters. CONCLUSIONS: Euro-Glo represents the first fluorescent metal chelate to be used as a histochemical tracer, specifically to localize normal and pathological myelin as well as amyloid plaques.

Development and application of novel histochemical tracers for localizing brain connectivity and pathology.

FLUORO-GOLD: A NEW FLUORESCENT RETROGRADE AXONAL TRACER WITH NUMEROUS UNIQUE PROPERTIES: A new fluorescent dye, Fluoro-Gold, has been demonstrated to undergo retrograde axonal transport. Its properties include (1) intense fluorescence, (2) extensive filling of dendrites, (3) high resistance to fading, (4) no uptake by intact undamaged fibers of passage, (5) no diffusion from labeled cells, (6) consistent and pure commercial source, (7) wide latitude of survival times and (8) compatibility with all other tested neuro-histochemical techniques. © 1986. Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons: The causes and effects of neuronal degeneration are of major interest to a wide variety of neuroscientists. Paralleling this growing interest is an increasing number of methods applicable to the detection of neuronal degeneration. The earliest methods employing aniline dyes were methodologically simple, but difficult to interpret due to a lack of staining specificity. In an attempt to circumvent this problem, numerous suppressed silver methods have been introduced. However, these methods are labor intensive, incompatible with most other histochemical procedures and notoriously capricious. In an attempt to develop a tracer with the methodological simplicity and reliability of conventional stains but with the specificity of an ideal suppressed silver preparation, the Fluoro-Jade dyes were developed. Fluoro-Jade C, like its predecessors, Fluoro-Jade and Fluoro-Jade B, was found to stain all degenerating neurons, regardless of specific insult or mechanism of cell death. Therefore, the patterns of neuronal degeneration seen following exposure to either the glutamate agonist, kainic acid, or the inhibitor of mitochondrial respiration, 3-NPA, were the same for all of the Fluoro-Jade dyes. However, there was a qualitative difference in the staining characteristics of the three fluorochromes. Specifically, Fluoro-Jade C exhibited the greatest signal to background ratio, as well as the highest resolution. This translates to a stain of maximal contrast and affinity for degenerating neurons. This makes it ideal for localizing not only degenerating nerve cell bodies, but also distal dendrites, axons and terminals. The dye is highly resistant to fading and is compatible with virtually all histological processing and staining protocols. Triple labeling was accomplished by staining degenerating neurons with Fluoro-Jade C, cell nuclei with DAPI and activated astrocytes with GFAP immunofluoresence. © 2005. ARTICLE ABSTRACT: The development of novel tracers and associated histochemical methods has always been need driven. One such need was the development of tracers that could be administered to discrete brain regions in vivo to subsequently reveal neuronal connectivity via axonal transport of the tracer. One such compound is Fluoro-Gold (F-G), which can be used to demonstrate retrograde axonal transport. Advantages of this fluorescent tracer include brightness, sensitivity, contrast, stability, permanence and compatibility with multiple labeling studies. It may be applied to resolve either the afferent or efferent connections of brain regions of interest. Another need addressed was for a simple and definitive way to localize degenerating neurons in brain tissue sections. This led to the development of Fluoro-Jade B (FJ-B) and Fluoro-Jade C (FJ-C). Advantages of these fluorescent histochemical tracers include high specificity, resolution, contrast, stability and suitability for use in multiple labeling studies. These methods can be applied to detect both apoptotic and necrotic neuronal degeneration following a variety of insults including physical trauma, neurodegenerative disease and a wide variety of neurotoxicants. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Oral Administration of Thioflavin T Prevents Beta Amyloid Plaque Formation in Double Transgenic AD Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the fourth leading cause of death in the United States and most common cause of adult-onset dementia. The major hallmarks of AD are the formation of senile amyloid plaques made of beta amyloid and neurofibrillary tangles (NFT) which are primarily composed of phosphorylated tau protein. Although numerous agents have been considered as providing protection against AD, identification of potential agents with neuroprotective ability is limited. Thioflavin T has been used in the past to stain amyloid beta plaques in brain. In this study, Thioflavin T (ThT) and vehicle (infant formula) were administered orally by gavage to transgenic (B6C3 APP PS1; AD-Tg) mice beginning at 4 months age and continuing until sacrifice at 9 months of age at 40 mg/kg dose. The number of amyloid plaques was reduced dramatically by ThT treatment in both male and female transgenic mice compared to those in control mice. Additionally, GFAP and Amylo-Glo labeling suggest that astrocytic hypertrophy is minimized in ThT-treated animals. Similarly, CD68 labeling, which detects activated microglia, along with Amylo-Glo labeling, suggests that microglial activation is significantly less in ThT-treated mice. Both Aß-40 and Aß-42 concentrations in blood rose significantly in the ThT-treated animals suggesting that ThT may inhibit the deposition, degradation, and/or clearance of Aß plaques in brain.

The use of MRI to assist the section selections for classical pathology assessment of neurotoxicity.

MRI was utilized to probe T2 changes in living brain following exposure of rats to one of ten classical neurotoxicants. Brains were subsequently perfused for classical neuropathology examination. This approach was predicated on the assumption that the T2 changes represent loci of neurotoxicity encompassing those seen using neuropathology techniques. The traditional neurotoxicologic approach of selecting a few arbitrary brain sections is dramatically improved by MRI targeting that can indicate the location(s) at which to collect "smart sections" for subsequent workup. MRI scans can provide the equivalent of 64 coronal sections; the number estimated for full coverage of the rat brain if only traditional neuropathology is utilized. Use of MRI allows each animal to serve as its own control as well as longitudinal observations of the life cycle of the neurotoxic lesion(s) (inception, apex and regression). Optimization of time of sacrifice and selection of an appropriate stain based on MRI-identified brain areas could be greatly enhanced should this approach prove successful. The application of full brain MRI imaging that informs neuropathology offers the potential to dramatically improve detection of neurotoxicity produced by new drugs and facilitate new drug development, review and approval processes, and to qualify an imaging biomarker of neuropathology.

Neuroprotective effect of the chemical chaperone, trehalose in a chronic MPTP-induced Parkinson's disease mouse model.

Parkinson's disease (PD) is a progressive motor disease of unknown etiology in the majority of cases. The clinical features of PD emerge due to selective degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), which project to the caudate putamen (CPu) where they release DA. In the current in vivo mouse model study, we tested trehalose for its ability to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage to DA neurons. Trehalose is a naturally occurring disaccharide present in plants and animals and appears capable of protecting cells against various environmental stresses. The effect of trehalose is likely due to its action as a pharmacological chaperone which promotes protein stability. In the present study, there were four treatment groups: saline only (control); probenecid only; MPTP+probenecid; and trehalose+MPTP+probenecid. MPTP-induced losses in tyrosine hydroxylase and DA transporter immunoreactivity in the ventral midbrain SNc and CPu were significantly reduced by trehalose. Decreases in CPu dopamine levels produced by MPTP were also blocked by trehalose. Microglial activation and astrocytic hypertrophy induced by MPTP were greatly reduced by trehalose, indicating protection against neuroinflammation. These effects are commensurate with the observed trehalose sparing of motor deficits produced by MPTP in this mouse model. Two tight junctional proteins, ZO-1 and occludin, are downregulated following MPTP treatment and trehalose blocks this effect. Likewise, the glucose transporter-1 that is expressed in brain endothelial cells is also protected by trehalose from MPTP-induced down-regulation. This study is the first to demonstrate using fluoro-turoquoise FT gel perfusion techniques, the protection afforded by trehalose from MPTP-induced damage to microvessels and endothelial and suggests that trehalose therapy may have the potential to slow or ameliorate PD pathology.

The use of recently developed histochemical markers for localizing neurotoxicant induced regional brain pathologies.

Neuronal and vascular brain components are interrelated morphologically, physiologically and developmentally. Due to this close interrelationship, it is often difficult to understand the cause and effect relationship between neuronal vs. vascular dysfunction and pathology. This review will discuss four of the more promising recent developments for detecting vascular pathology, and will compare them with the labeling pattern seen with markers of glial and neuronal pathology; following exposure to well characterized neurotoxicants. To detect the vascular dysfunction in the brain, we recently developed a Fluoro-Turquoise gelatin conjugate (FT-gel), a fluorescent probe that helps to delineate between healthy vs. sclerotic vessels. Similarly, we have investigated the potential for Fluoro-Gold to label in vivo all the endothelial cells in the brain as they co-localize with RECA, an endothelial cell marker. We have also developed Amylo-Glo, a fluorescent tracer that can detect neurotoxic A-beta aggregates in the brain. In this article, we will discuss the potential use of these novel histochemical markers to study the neurotoxicant induced brain. We will also discuss neurovascular strategies that may offer novel therapeutic opportunities for neurodegenerative disorders.

K114 inhibits A-beta aggregation and inflammation in vitro and in vivo in AD/Tg mice.

Alzheimer's disease (AD) is the most common age related human neurodegenerative disorder. The major histopathological characteristics of the AD brain are extracellular amyloid-beta (Aß) peptide loaded plaques and intraneuronal neurofibrillary tangles made of phosphorylated tau proteins. Amyloid plaques consist primarily of aggregated Aß1-42 and Aß1-40 peptides. The aim of our current study was to test novel ligands/agents with the potential to disrupt or inhibit the aggregation of Aß peptide, specifically K114, (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene, which was initially developed as a potential positron emission tomography (PET) ligand for the in vivo detection of amyloid plaques. Systemic administration of K114 has been shown in the AD/transgenic (Tg) mouse model to be capable of crossing the blood-brain barrier (BBB) and be colocalized with amyloid plaques. In this study we determined whether K114 has the potential to inhibit Aß aggregation in vitro in AD/Tg mice and also tested, in vivo, whether chronic daily orally administered K114 has any therapeutic potential as evidenced by inhibition or reduction of the deposits of amyloid aggregates in the brains of AD/Tg mice. Our results demonstrated that K114 strongly blocked, in vitro, the aggregation of Aß peptide in the amyloid plaques of AD/Tg mouse brain. Systemic treatment with K114 was also effective in significantly reducing the deposits of amyloid plaques in the brains of living transgenic AD mice. Additionally, K114 significantly inhibited the typically observed plaque associated astrocytic activation, as revealed by GFAP immunohistochemistry, suggesting possible anti-inflammatory properties.

Systemic administration of fluoro-gold for the histological assessment of vascular structure, integrity and damage.

Fluoro-Gold (F-G) has been used extensively as a fluorescent retrograde neuronal-track tracer in the past. We now report that intraperitoneal administration of 10 to 30 mg/ kg of F-G from 30 min to 7 days prior to sacrifice labels vascular endothelial cells of the brain, choroid plexus and meninges and can be used to assess vascular integrity and damage. F-G vascular labeling co-localized with rat endothelial cell antigen (RECA-1) in the membrane. F-G also intensely labeled the nuclei of the endothelial cells, and co-localized with propidium iodide staining of these nuclei. As well, the administration of F-G during neurotoxic insults produced by amphetamine, kainic acid or "penetrating" wound to the brain can detect where vascular leakage/ hemorrhage has occurred. Histological methods to detect F-G labeled brain vasculature were performed in the same manner as that used for fluorescent visualization of neuronal elements labeled with F-G after perfusion fixation and coronal sectioning (15 to 40 µm) of the brain. This in vivo F-G labeling of endothelial cells and their nuclei yields a clear picture of the integrity of the vasculature and can be used to detect changes in structure. Vascular leaks after "penetrating" wounds through the cortex and striatum, hyperthermic amphetamine exposure or excitotoxic kainate exposure were detected by F-G in the extracellular space and via parenchymal F-G subsequently labeling the terminals and neurons adjacent to the lesioned or damaged vasculature. Further studies are necessary to determine the extent of the leakage necessary to detect vasculature damage. Visualization of the F-G labeling of vasculature structure and leakage is compatible with standard fluorescent immuno-labeling methods used to detect the presence and distribution of a protein in histological sections. This method should be directly applicable to studying brain vascular damage that occurs in the progression of Alzheimer's disease, diabetes and for monitoring the brain vascular changes during development.

Neurovascular changes in acute, sub-acute and chronic mouse models of Parkinson's disease.

Although selective neurodegeneration of nigro-striatal dopaminergic neurons is widely accepted as a cause of Parkinson's disease (PD), the role of vascular components in the brain in PD pathology is not well understood. However, the neurodegeneration seen in PD is known to be associated with neuroinflammatory-like changes that can affect or be associated with brain vascular function. Thus, dysfunction of the capillary endothelial cell component of neurovascular units present in the brain may contribute to the damage to dopaminergic neurons that occurs in PD. An animal model of PD employing acute, sub-acute and chronic exposures of mice to methyl-phenyl-tetrahydropyridine (MPTP) was used to determine the extent to which brain vasculature may be damaged in PD. Fluoro-Turquoise gelatin labeling of microvessels and endothelial cells was used to determine the extent of vascular damage produced by MPTP. In addition, tyrosine hydroxylase (TH) and NeuN were employed to detect and quantify dopaminergic neuron damage in the striatum (CPu) and substantia nigra (SNc). Gliosis was evaluated through GFAP immunohistochemistry. MPTP treatment drastically reduced TH immunoreactive neurons in the SNc (20.68 ± 2.83 in acute; 22.98 ± 2.14 in sub-acute; 10.20 ± 2.24 in chronic vs 34.88 ± 2.91 in controls; p<0.001). Similarly, TH immunoreactive terminals were dramatically reduced in the CPu of MPTP treated mice. Additionally, all three MPTP exposures resulted in a decrease in the intensity, length, and number of vessels in both CPu and SNc. Degenerative vascular changes such as endothelial cell 'clusters' were also observed after MPTP suggesting that vasculature damage may be modifying the availability of nutrients and exposing blood cells and/or toxic substances to neurons and glia. In summary, vascular damage and degeneration could be an additional exacerbating factor in the progression of PD, and therapeutics that protect and insure vascular integrity may be novel treatments for PD.

In situ demonstration of Fluoro-Turquoise conjugated gelatin for visualizing brain vasculature and endothelial cells and their characterization in normal and kainic acid exposed animals.

The present study describes a new method for the visualization of the vasculature lumen and endothelial cells and characterizes their morphology in the brains of normal and kainic acid (KA) treated rats. Herein, labeling was accomplished using Fluoro-Turquoise (FT), a novel reactive blue fluorochrome conjugated to gelatin. Strong blue fluorescence was observed throughout the brain vasculature following intra-cardiac perfusion with FT-gel in normal animals. However, in the brains of KA treated rats (hippocampus, midline and ventral thalamus, piriform cortex), the vascular lumen was typically constricted, sclerotic and only faintly stained. The advantages of FT-gel over other markers can be attributed to its unique chemical and spectral properties. Specifically, Fluoro-Turquoise is a very bright blue UV excitable dye that does not bleed through when visualized using other filters, making it ideal for multiple immunofluorescent labeling studies. Its brightness at low magnification also makes it ideal for low magnification whole brain imaging. Compared to alternative techniques for visualizing blood vessels, such as India ink, fluorescent dye-conjugated dextran, the corrosion technique, endothelial cell markers and lectins, the present method results in excellent visualization of blood vessels.

Efficacy and toxicity of clioquinol treatment and A-beta42 inoculation in the APP/PSI mouse model of Alzheimer's disease.

Alzheimer's disease (AD), the most common human neurodegenerative disease, is characterized pathologically by numerous deposits of amyloid plaques in the brain. Systemic administration of clioquinol (CQ) and inoculation with amyloid-beta42 (Aß42) vaccines have been demonstrated to significantly inhibit deposits of amyloid in AD brains. However, each of these treatments has also been reported to be neurotoxic. The generation of transgenic mice models of AD has made it possible to study aspects of this disease employing experimental animals. In the present study, we investigated the efficacy and toxicity of CQ and Aß42 vaccine in a transgenic AD (APP/PS1) mouse model. Our results confirmed that both CQ and Aß42 vaccine were effective in significantly reducing the deposits of amyloid in the brains of transgenic AD mice. We also report here that systemic CQ induces myelinopathies in the dorsal lateral geniculate nucleus (DLG), which was almost devoid of amyloid plaques and is the primary site of retinal efferent projections via the optic nerve. This is the first report that systemic administration of CQ causes myelinopathies in the central nervous system (CNS) of a transgenic AD mouse model as well as wild-type mice. Inoculation with an Aß42 vaccine was also found, for the first time, to result in a significant increase in plaque-independent astrocytic hyperplasia in the dorsal part of the lateral septal nucleus (LSD) which was also devoid of plaques, reflecting potential brain inflammatory processes.

Longitudinal behavioral changes in the APP/PS1 transgenic Alzheimer's disease model.

The APP/PS1 double transgenic mouse is an Alzheimer's Disease-like model. However, cognitive deficits measured at one age do not necessarily indicate age-related progressions. Further, results of the most widely used behavioral assessment, water maze performance, are generally limited to 1-2 endpoints. Here, male APP/PS1 and noncarrier wildtypes (n=11/group) were assessed at 7-15 months of age for water maze, open field, and motor coordination performance. Body weights and motor coordination were comparable for both groups throughout. Beginning at approximately 9 months of age, the transgenic group exhibited hypoactivity in the open field which continued throughout. Latency to locate the platform and swim path length were longer in the transgenic group; however, these appeared to be more related to increased floating and thigmotactic behavior and only partially related to a cognitive impairment. Age-related decrements in performance were not substantial; however, substantial plaque numbers were measured in six representative 16-month-old transgenic mice. The stability of water maze performance may be related to the longitudinal testing and repetitive experience, which previous research has demonstrated can confer beneficial effects on behavior and plaque deposition in transgenic Alzheimer's Disease models [1]. These results emphasize the importance of measuring multiple water maze endpoints and demonstrate the feasibility of longitudinal assessments in this model.

Characterization of myelin pathology in the hippocampal complex of a transgenic mouse model of Alzheimer's disease.

We have characterized the myelin changes observed within the hippocampal complex (HC) of a transgenic (Tg) mouse model of Alzheimer's disease (AD). Individual myelinated fibers were labeled with Black-Gold II while amyloid plaques were labeled with either Congo Red or Pan-A-beta immunofluoresence. Myelinated fibers were never seen passing through amyloid plaques in any region, while conspicuous myelin pathology was seen within, and immediately adjacent to, the amyloid plaques in the HC of the AD-Tg mouse. This pathology consisted of a complete disruption of myelinated fibers passing through the plaque and the region immediately adjacent to the plaques exhibited an edematous swelling of the fibers. This pathology was most frequently observed within the molecular and polymorph layers of the dentate gyrus and the molecular layer of Ammon's horn. The remaining layers of Ammon's horn exhibited minimal myelin pathology, while moderate myelinopathy was observed in the subiculum. Since the HC is integral for memory function, these findings may help account for the memory problems so characteristic of the disease process. Because the molecular layers of the dentate gyrus and Ammon's horn are the sites of inputs to the HC, the extensive myelin pathology observed in these regions would imply functional deafferentation of the HC. The appearance of some Black-Gold II positive debris within the plaques may reflect a possible cascade mechanism whereby the presence of plaques results in myelin degeneration, some of which is incorporated within the plaque, causing it to further expand in a self-perpetuating fashion.

Introducing Amylo-Glo, a novel fluorescent amyloid specific histochemical tracer especially suited for multiple labeling and large scale quantification studies.

One of the hallmark pathologies associated with Alzheimer's disease (AD) is the conspicuous deposition of extracellular amyloid plaques within the forebrain. These plaques are primarily composed of fibrular aggregates of the A-beta peptide. Traditional methods for the histological localization of these plaques typically rely on the use of the tracers Congo Red or Thioflavin S. This study describes the characterization of a novel fluorescent histochemical probe, Amylo-Glo, for the high resolution and contrast localization of amyloid plaques in brain tissue sections. Potential advantages over conventional amyloid plaque stains such as Congo Red or Thioflavin S can be attributed to its unique chemical and spectral properties. Specifically, it results in a very bright blue UV excitable stain under physiological conditions that will not bleed through when illuminated with other filters. Its brightness makes it ideal for low magnification quantification studies, while its unique excitation/emission profile and mild staining conditions makes it ideal for combination with multiple immunofluorescent labeling studies.

One-step labeling of degenerative neurons in unfixed brain tissue samples using Fluoro-Jade C.

Neurodegeneration is the underlying cause of a vast majority of neurological disorders and often a result of brain trauma, stroke, or neurotoxic insult. Here we describe a simple method for labeling degenerating neurons in unfixed brain tissue samples. This method could provide a new avenue for identifying and harvesting degenerative neurons from unfixed brain tissues for subsequent molecular analyses.

Temporal progression of kainic acid induced changes in vascular laminin expression in rat brain with neuronal and glial correlates.

We recently demonstrated a dramatic up regulation of laminin expression within the blood vessels of brain regions vulnerable to excitotoxin mediated neuronal degeneration. Although this effect was clearly demonstrable at 2 days post kainic acid exposure, its expression at shorter and longer post-dosing intervals has not been reported. Therefore, a primary goal of the present study was to characterize the laminin labeling at intervals ranging from 4 hours to 2 months following i.p. injection of kainic acid. To better characterize the nature and possible underlying mechanism of action of the changes in laminin expression, both Fluoro-Jade C and GFAP immunohistochemistry were employed respectively at all survival intervals. At the shortest intervals examined (4hr, 8hr), Fluoro-Jade C positive cells could be detected in the hippocampus, thalamus, and piriform cortex. In these same regions, both vascular laminin and astrocytic GFAP expression were up regulated. At intermediate survival intervals (2, 5, 14, and 21 days), the respective labeling of degenerating neurons, astrocytes and capillaries were all maximal. Morphologically, Fluoro-Jade C labeled degenerating neurons were labeled in their entirety, GFAP positive astrocytes appeared hypertrophic and blood vessels took on a fragmented appearance. Hypertrophied GFAP positive astrocytes were conspicuous around periphery of the lesion but absent within the core of the lesion at these times. At longer survival intervals (1-2 months), the number of FJ-C labeled degenerating neurons was greatly reduced, while GFAP staining essentially returned to base line and laminin expression remained noticeably elevated, although the vessels appeared to be intact morphologically. These data allow for speculation on possible mechanisms underlying these events.

In vivo administration of fluorescent dextrans for the specific and sensitive localization of brain vascular pericytes and their characterization in normal and neurotoxin exposed brains.

We have aimed to develop novel histochemical markers for the labeling of brain pericytes and characterize their morphology in the normal and the excitotoxin-exposed brain, as this class of cells has received little attention until recently. Pericyte labeling was accomplished by the intracerebroventricular injection of certain fluorescent dextran conjugates, such as Fluoro-Gold-dextran, FR-dextran, FITC-dextran and Fluoro-Turquoise (FT)-dextran. 1-7 days after the tracer injection, extensive labeling of vascular pericytes was seen throughout the entire brain. These cells were found distal to the endothelial cells and exhibited large dye containing vacuoles. The morphology of the pericytes was somewhat variable, exhibiting round or amoeboid shapes within larger intracellular vesicles, while those wrapping around capillaries exhibited a more elongated appearance with finger-like projections. The use of FG-dextran resulted in bluish yellow fluorescently labeled pericytes, while FR-dextran resulted in red fluorescent labeled pericytes, FITC-dextran exhibited green fluorescent pericytes and FT-dextran showed fluorescent blue pericytes in the brain. We have used these tracers to study possible changes in morphology and pericyte number following kainic acid insult, observing that the number of pericytes in the injured or lesioned areas of the brain is dramatically reduced compared to the non-injured areas. These novel fluorochromes should be of use for studies involving the detection and localization of pericytes in both normal and pathological brain tissues.