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Examples of parenchymal and vascular plaques with CR and amyloid antibodies in the brain sections of one-year old APPPS1 rat brain sections. In all instances, the middle row represents CR labeling, the top row indicates immunolabeling and lowest row represents double merged images. A) distribution of Aβ plaques that are immunolabeled with mOC64 antibody in the brain. The same section was used to label with CR as illustrated in D. While both the parenchymal and vascular plaques are comparable, the only difference was noticed in vascular staining which is limited in mOC64 (D) while CR labeled sections reveal all the plaques, i.e. both luminal and abluminal sided of the vessels (G). B represents the distribution of Aβ 1–40 in the temporal cortex of the rat brain but CR labeling showing all the parenchymal and vascular plaques as shown in E. When both B and E images were merged in H, Aβ1–40 stains only minimal amount of plaques compared to CR labeling. Lastly, we also explored Aβ40/42 distribution as shown in C and compared with CR labeling as shown in F. When compared between Aβ40/42 and CR labeling, it appears that Aβ40/42 stains more comprehensively parenchymal plaques. However, only luminal vascular plaques were stained by this antibody, albeit CR can stain all the parenchymal and vascular plaques in the brain (I). Magnification bar A-I = 100 μm
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Although there are multiple histochemical tracers available to label plaques and tangles in the brain to evaluate neuropathology in Alzheimer disease (AD), few of them are versatile in nature and compatible with immunohistochemical procedures. Congo Red (CR) is an anisotropic organic stain discovered to label amyloid beta (Aβ) plaques in the brain....
Citations
... Congo red (CR) was used to visualize amyloid and neurofibrillary pretangles in tissue sections where it binds to β-pleated sheets (Moloney et al., 2021;Sarkar et al., 2020). Brain sections were deparaffinized and rehydrated, after which stained in 0.5 % Congo Red solution for 25 min at room temperature, then rinsed in distilled water, alkaline alcohol solution (1 ml of 1 % sodium hydroxide diluted in 50 % alcohol), dehydrated, and cleared with xylene, afterward mounted with DPX as described in the literature (Manickam et al., 2018). ...
High-altitude environments present extreme conditions characterized by low barometric pressure and oxygen deficiency, which can disrupt brain functioning and cause edema formation. The objective of the present study is to investigate several biomolecule expressions and their role in the development of High Altitude Cerebral Edema in a rat model. Specifically, the study focuses on analyzing the changes in total arginase, nitric oxide, and lipid peroxidation (MDA) levels in the brain following acute hypobaric hypoxic exposure (7620 m, SO2=8.1 %, for 24 h) along with the histopathological assessment. The histological examination revealed increased TNF-α activity, and an elevated number of mast cells in the brain, mainly in the hippocampus and cerebral cortex. The research findings demonstrated that acute hypobaric hypoxic causes increased levels of apoptotic cells, shrinkage, and swelling of neurons, accompanied by the formation of protein aggregation in the brain parenchyma. Additionally, the level of nitric oxide and MDA was found to have increased (p<0.0001), however, the level of arginase decreased indicating active lipid peroxidation and redox imbalance in the brain. This study provides insights into the pathogenesis of HACE by evaluating some biomolecules that play a pivotal role in the inflammatory response and the redox landscape in the brain. The findings could have significant implications for understanding the neuronal dysfunction and the pathological mechanisms underlying HACE development.
... for 2 minutes each, then submerged in xylene 1 and 2 for 2 minutes each before drying for 30 minutes. The slides were then placed in DPX mounting medium (BDH Chemicals, UK) and coated with coverslips (HmbG Inc., Germany) (14). Finally, the slides were analysed by two blinded scientists using a light microscope (Olympus Corporation, Japan) coupled to an image analyzer at 20x objective lens power magnification. ...
Numerous animal models have shown that minocycline has strong impacts on the architecture and functioning of the nervous system. Its neuroprotective qualities, however, after a single lipopolysaccharide (LPS) injection in an adult rat model remain unclear. This study examined the neuroprotective properties of minocycline in a rat model of LPS-induced neuroinflammation.. Methods: Five sets of fifty-year-old male Sprague Dawley rats were created: (i) LPS treated with distilled water, (ii) LPS treated with 25 mg/kg minocycline, and (iv) LPS treated with 50 mg/kg minocycline. treated LPS and (v) LPS treated with 10 mg/kg memantine. Day 5 was a single intraperitoneal injection of LPS (5 mg/kg), followed by daily injections of minocycline and memantine for a period of 14 days. Results: It was discovered that minocycline treatment significantly attenuated the effects of LPS on β-amyloid peptide buildup, neuronal damage, and memory loss. According to these findings, LPS causes neuronal damage and β-amyloid peptide buildup in the cortex and hippocampus, which subsequently affects cognitive memory. Additionally, we examined the administration of minocycline and memantine. The results indicated that, although minocycline (50 mg/kg) therapy produced superior outcomes, minocycline © 2024 IJNRD | Volume 9, Issue 5 May 2024 | ISSN: 2456-4184 | IJNRD.ORG IJNRD2405364 International Journal of Novel Research and Development (www.ijnrd.org) d586 (25 mg/kg) and memantine (10 mg/kg) treatment were comparable. It lessens memory loss, neuronal damage, and beta-amyloid peptide formation brought on by LPS.. Conclusions: These results suggest that minocycline might be a promising therapy option for neuroinflammatory conditions including Alzheimer's disease (AD).
... In brief, the free-floating sections were rinsed in distilled water for 30 s and then incubated in 0.5% Congo-red solution for 10 min. The sections were then washed three times in tap water for 30 s each and subsequently incubated in 50mM NaOH for postquenching the autofluorescence for 30 s [27]. The sections were then washed three times in tap water, air-dried on a slide warmer for 5 min, mounted with ProLong™ gold antifade reagent and imaged at 10x and 20x magnifications using a Nikon Eclipse 90i fluorescence microscope equipped with a Retiga 2000R Q imaging system (Nikon Instruments Inc., NY, USA). ...
... It is well established that Congo red dye is commonly used in the histological staining of amyloid plaques in the brains of both the animal models of AD as well as postmortem human AD patients which appear as apple-green birefringence in polarized light and bright red color fluorescence in Texas red filter (i.e., 561-590 nm) [27]. To validate that FITC-labelled PLGA detect Aβ-containing neuritic plaques, we performed Congo-red staining of brain sections of 5xFAD mice at different times following single intracerebral injection of labelled PLGA. ...
Evidence suggests that increased level/aggregation of β-amyloid (Aβ) peptide, together with enhanced phosphorylation/aggregation of tau protein, play a critical role in the development of Alzheimer’s disease (AD), the leading cause of dementia in the elderly. At present, AD diagnosis is based primarily on cognitive assessment, neuroimaging, and immunological assays to detect altered levels/deposition of Aβ peptides and tau protein. While measurement of Aβ and tau in the cerebrospinal fluid/blood can indicate disease status, neuroimaging of aggregated Aβ and tau protein in the brain using positron emission tomography (PET) enable to monitor the pathological changes in AD patients. With advancements in nanomedicine, several nanoparticles, apart from drug-delivery, have been used as a diagnostic agent to identify more accurately changes in AD patients. Recently, we reported that FDA approved native PLGA nanoparticles can interact with Aβ to inhibit its aggregation/toxicity in cellular and animal models of AD. Here, we reveal that fluorescence labelled native PLGA following acute intracerebellar injection can identify majority of the immunostained Aβ as well as Congo red labelled neuritic plaques in the cortex of 5xFAD mice. Labelling of plaques by PLGA is apparent at 1 h, peak around 3 h and then start declining by 24 h after injection. No fluorescent PLGA was detected in the cerebellum of 5xFAD mice or in any brain regions of wild-type control mice following injection. These results provide the very first evidence that native PLGA nanoparticles can be used as a novel nano-theragnostic agent in the treatment as well as diagnosis of AD pathology.
... Although these approaches have been applied for decades for amyloid visualization, there is evidence of both binding nonspecificity and lack of amyloid detection for Congo Red, Thioflavin S, which can lead to inaccuracies in tissue analysis (Clement and Truong, 2014;Yakupova et al., 2019;Guselnikova et al., 2021). However, these techniques have additional limitations such as fluorophore broad excitation/emission profile and chemical instability (Levine, 1993;Fernandez-Flores, 2011;Dapson, 2018;Sarkar et al., 2020;Setti et al., 2021;Nosova et al., 2022;Schmued, 2022). Briefly, Thioflavin S has a broader emission profile, as a result, studies assessing specificity using multiple labelling may be inherently flawed. ...
... Briefly, Thioflavin S has a broader emission profile, as a result, studies assessing specificity using multiple labelling may be inherently flawed. In addition, staining with Thioflavin S demonstrates a significantly higher level of lipofuscin autofluorescence, which is contained aplenty by the ageing brain, which leads to background staining increasing and difficulties with analysis of images (Sarkar et al., 2020;Setti et al., 2021). ...
... Some authors reported, that there are no specific antibodies that can label all types of plaques (Sarkar et al., 2020), which makes it attractive to finding out for alternative dyes. We previously proposed a novel approach using Alcian Blue for the visualization of amyloid plaques in association with astrocytes and microglial cells by light microscopy only (Guselnikova et al., 2019;Nosova et al., 2022). ...
Background:
Astrocytes play an essential role in the normal functioning of the nervous system and are active contributors to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). Therefore, to comprehend the astrocytes and amyloid plaques relationship there is a need for imaging techniques providing simultaneous visualization of astrocytes using fluorescence and amyloid plaques revealed by transmitted light microscopy. New Method The possibility of simultaneous detection of astrocytes by immunocytochemistry (fluorescent) and amyloid plaques by cytochemical Alcian Blue (transparent) using confocal microscopy in 8-month-old 5хFAD mice samples shown.
Results:
The described method supposes performing astrocytes fluorescent labelling by GFAP or S100beta and amyloid plaques staining by Alcian Blue. Comparison with Existing Methods Proposed approach circumvents some limitations of fluorescence microscopy, such as weak fluorescence, low contrast, fluorophore broad excitation/emission profile and chemical instability.
Conclusions:
The proposed technique provides high-quality resulting images of GFAP/s100beta- labelled astrocytes and Alcian Blue-stained amyloid plaques. These images are appliable for prospective qualitative and quantitative three-dimensional analysis due to the z-axis scanning. Moreover, it demonstrated the formation of stable Alcian Blue staining.
... However, cortical and hippocampal tissues of minocycline and memantine groups showed lesser β-amyloid peptide deposition formation, as shown in Figure 2. for 2 minutes each, then submerged in xylene 1 and 2 for 2 minutes each before drying for 30 minutes. The slides were then placed in DPX mounting medium (BDH Chemicals, UK) and coated with coverslips (HmbG Inc., Germany) (14). Finally, the slides were analysed by two blinded scientists using a light microscope (Olympus Corporation, Japan) coupled to an image analyzer at 20x objective lens power magnification. ...
Introduction: Minocycline has been demonstrated to have potent effects on neurologic structures and functions in several animal models. However, its neuroprotective properties following a single injection of lipopolysaccharide (LPS) in an adult rat model have not been clearly elucidated. This study investigated minocycline’s neuroprotective effects in the LPS-induced neuroinflammation rat model. Methods: Fifty adult male Sprague Dawley rats were split into five groups at random: (i) control, (ii) distilled water-treated LPS, (iii) 25 mg/kg minocycline-treated LPS, (iv) 50 mg/kg minocycline-treated LPS, and (v) 10 mg/kg memantine-treated LPS. On day 5, LPS (5 mg/kg) was given intraperitoneally once, whereas minocycline and memantine were given once daily for 14 days. Results: LPS was found to significantly induce β-amyloid peptide deposition and neuronal damage, and impair recognition memory, while administration of minocycline dose-dependently reversed these effects. These data suggest that LPS-induced recognition memory impairment by inducing β-amyloid peptide deposition and neuronal damage in the cortical and hippocampal areas. Furthermore, we compared minocycline with memantine administration, and these data suggested better effects in minocycline (50 mg/kg) and comparable effects between minocycline (25 mg/kg) and memantine (10 mg/kg) treatments in reducing β-amyloid peptide deposition, neuronal damage and recognition memory impairment induced by LPS. Conclusion: Minocycline may be a strong contender as an effective preventive-therapeutic drug for neuroinflammatory diseases such as Alzheimer’s disease (AD) based on these findings.
... However, cortical and hippocampal tissues of minocycline and memantine groups showed lesser β-amyloid peptide deposition formation, as shown in Figure 2. for 2 minutes each, then submerged in xylene 1 and 2 for 2 minutes each before drying for 30 minutes. The slides were then placed in DPX mounting medium (BDH Chemicals, UK) and coated with coverslips (HmbG Inc., Germany) (14). Finally, the slides were analysed by two blinded scientists using a light microscope (Olympus Corporation, Japan) coupled to an image analyzer at 20x objective lens power magnification. ...
Introduction: Minocycline has been demonstrated to have potent effects on neurologic structures and functions in several animal models. However, its neuroprotective properties following a single injection of lipopolysaccharide (LPS) in an adult rat model have not been clearly elucidated. This study investigated minocycline's neuroprotective effects in the LPS-induced neuroinflammation rat model. Methods: Fifty adult male Sprague Dawley rats were split into five groups at random: (i) control, (ii) distilled water-treated LPS, (iii) 25 mg/kg minocycline-treated LPS, (iv) 50 mg/kg minocycline-treated LPS, and (v) 10 mg/kg memantine-treated LPS. On day 5, LPS (5 mg/kg) was given intra-peritoneally once, whereas minocycline and memantine were given once daily for 14 days. Results: LPS was found to significantly induce β-amyloid peptide deposition and neuronal damage, and impair recognition memory, while administration of minocycline dose-dependently reversed these effects. These data suggest that LPS-induced recognition memory impairment by inducing β-amyloid peptide deposition and neuronal damage in the cortical and hip-pocampal areas. Furthermore, we compared minocycline with memantine administration, and these data suggested better effects in minocycline (50 mg/kg) and comparable effects between minocycline (25 mg/kg) and memantine (10 mg/kg) treatments in reducing β-amyloid peptide deposition, neuronal damage and recognition memory impairment induced by LPS. Conclusion: Minocycline may be a strong contender as an effective preventive-therapeutic drug for neuroinflammatory diseases such as Alzheimer's disease (AD) based on these findings.
... Finally, the slices were dehydrated through immersion in 95% alcohol (3 min) and 100% alcohol (3 min). The glass slides were cleared in xylene and mount with a mounting medium (Sarkar et al. 2020). ...
Insulin signaling disruption and caspase-3 cleavage play a pathologic role in Alzheimer’s disease (AD). Evidence suggested that cinnamaldehyde (Cin), the major component of cinnamon, has the ability to act as a neuroprotective agent. However, little evidence is available to demonstrate its effectiveness in regulating the insulin and caspase-3 signaling pathways and underlying molecular mechanisms. Therefore, the present study was conducted to correlate the molecular mechanisms of these signaling pathways and Cin treatment on animal behavioral performance in an intracerebroventricular (ICV)-streptozotocin (STZ, 3 mg/kg) model. The sporadic AD rat model was treated with Cin (10 and 100 mg/kg; intraperitoneal, i.p) daily for 2 weeks. Novel object recognition (NOR), Morris water maze (MWM), and elevated plus maze (EPM) tests were performed to assess recognition/spatial memory and anxiety-like behavior, respectively. Hippocampal Aβ aggregation was assessed using Congo red staining. The activity of hippocampal caspase-3 and IRS-1/Akt/GSK-3β signaling pathways were analyzed using the Western blot technique. The results revealed that Cin (100 mg/kg, effective dose) improved recognition/spatial memory deficits and anxiety-like behavior. In addition, Cin negated the effects of STZ on Aβ aggregation and caspase-3 cleavage in the hippocampus. Furthermore, the Western blot method showed that hippocampal IRS-1/AKT/GSK-3β phosphorylation was altered in ICV-STZ animal model, while Cin modulated this signaling pathway through decreasing Phospho.IRS-1Ser307/Total.IRS-1 ratio and also increasing Phospho.AktSer473/Total.Akt and Phospho.GSK-3βSer9/Total.GSK-3β ratios. These findings suggest that Cin is involved in the regulation of hippocampal IRS-1/AKT/GSK-3β and caspase-3 pathways in a sporadic AD model, and modulation of these signaling pathways also influences the animal behavioral performance.
Graphical Abstract
... However, the contrast and resolution are of an intermediate level and the tracer exhibits a very broad excitation and emission profile, making it unsuitable for most multiple labeling studies. In contrast, CR has been observed to label both SP and filamentous tau inclusions (Sarkar et al. 2020), can be combined with other traditional immunohistochemical staining (Sarkar et al. 2020;Setti et al. 2021), and is capable of staining SP in vivo (Setti et al. 2021). However, research has focused on developing CR derivatives that have better brain bioavailability and increased relative fluorescence intensity as opposed to the original CR compound. ...
... However, the contrast and resolution are of an intermediate level and the tracer exhibits a very broad excitation and emission profile, making it unsuitable for most multiple labeling studies. In contrast, CR has been observed to label both SP and filamentous tau inclusions (Sarkar et al. 2020), can be combined with other traditional immunohistochemical staining (Sarkar et al. 2020;Setti et al. 2021), and is capable of staining SP in vivo (Setti et al. 2021). However, research has focused on developing CR derivatives that have better brain bioavailability and increased relative fluorescence intensity as opposed to the original CR compound. ...
Although several histochemical markers for senile plaques (SP) and neurofibrillary tangles (NFTs) have been synthesized since the discovery of plaques in Alzheimer’s disease (AD), only a handful of these markers stain both lesions in the human brain. Despite discovery of its ability to stain both SP and NFT over 13 years ago, the styrylbenzene derivative, (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), has only recently gained attention, primarily due to its ability to function as a contrasting agent for MRI imaging of AD pathology in vivo. The structure of the compound is a nuclide with quantized angular momentum, which explains its value as a contrast agent. In the current study, modification of the established staining procedure produced meaningful improvement in the labeling of plaques and tangles in the human brain. We utilized two rodent models of AD to show FSB’s value in labeling both Aβ and tau lesions. Furthermore, our current modification allows us to detect SP in rodent brains in 15 min and both SP and NFT in human brains within 20 min. The study presents new evidence regarding potential binding targets for FSB as well as optimization protocols in which various parameters have been manipulated to show how section thickness, use of frozen versus paraffin-embedded sections, and selection of staining media can affect the intensity of the plaque and tangle staining in the brain. To determine the target FSB potentially binds, we performed double immunolabeling of FSB with mOC64 (a conformational antibody that label Aβ1-42). Results indicated that all plaques in the brain colocalized with mOC64, suggesting that FSB has the potential to bind all Aβ containing plaques, making it a very sensitive detector of multiple forms of SP... All antibodies were assessed for the degree of colocalization with FSB in order to better understand potential binding targets. We found more than 90% hyperphosphorylated Tau against AT8, AT180 and S214 colocalized with FSB labeled tangles. On the other hand, more than 90% of the mOC64 containing plaques colocalized with FSB stained plaques. Our results indicate that FSB is a valuable marker that can be used to detect AD pathologies in human and rodent brains with greater fluorescence intensity relative to other conventional fluorescence markers.
... 67 For over 50 years, this dye was used to visualize AD neuropathology, including mature tangles and ghost tangles. 68 Congo red does not stain pretangles likely owing to the lack of fibrillar structure. ...
Neurofibrillary tangles, one of the neuropathologic hallmarks of Alzheimer's disease, have a dynamic lifespan of maturity that associates with progressive neuronal dysfunction and cognitive deficits. As neurofibrillary tangles mature, the biology of the neuron undergoes extensive changes that may impact biomarker recognition and therapeutic targeting. Neurofibrillary tangle maturity encompasses three levels: pretangles, mature tangles, and ghost tangles. In this review, we detail distinct and overlapping characteristics observed in the human brain regarding morphologic changes the neuron undergoes, conversion from intracellular to extracellular space, tau immunostaining patterns, and tau isoform expression changes across the lifespan of the neurofibrillary tangle. Post‐translational modifications of tau such as phosphorylation, ubiquitination, conformational events, and truncations are discussed to contextualize tau immunostaining patterns. We summarize accumulated and emerging knowledge of neurofibrillary tangle maturity, discuss the current tools used to interpret the dynamic nature in the postmortem brain, and consider implications for cognitive dysfunction and tau biomarkers.
