fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer’s disease using an automated quantitative analysis workflow

Highet, Blake; Anekal, Praju Vikas; Ryan, Brigid; Murray, Helen C.; Coppieters, Natacha; Dieriks, Birger; Singh‐Bains, Malvindar K.; Mehrabi, Nasim F.; Faull, Richard L. M.; Dragunow, Michael; Curtis, Maurice A.

doi:10.1111/jnc.15283

Cited by 5 publications

(5 citation statements)

References 38 publications

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“…Total RNA was extracted from fresh-frozen human brain tissue sections using the RNeasy Mini Kit (QIAGEN) according to manufacturer’s instructions with pre-processing using a recently published protocol ( Highet et al, 2021 ). Tissue was placed in a pre-chilled Eppendorf tube with 300 μL of lysis buffer and disrupted using pre-chilled plastic pestles.…”

Section: Methodsmentioning

confidence: 99%

“…Six genes were chosen to examine the effects of RIN on mRNA quantification. The genes were selected based on their use in previous publications quantifying mRNA expression in human brain tissue ( Coulson et al, 2008 ; Penna et al, 2011 ; Rydbirk et al, 2016 ; Highet et al, 2021 ). Details for the primers used in this study are available in Supplementary Table 2 .…”

Section: Methodsmentioning

confidence: 99%

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RNA Quality in Post-mortem Human Brain Tissue Is Affected by Alzheimer’s Disease

Highet

Parker

Faull

et al. 2021

Front. Mol. Neurosci.

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Gene expression studies of human post-mortem brain tissue are useful for understanding the pathogenesis of neurodegenerative disease. These studies rely on the assumption that RNA quality is consistent between disease and neurologically normal cases; however, previous studies have suggested that RNA quality may be affected by neurodegenerative disease. Here, we compared RNA quality in human post-mortem brain tissue between neurologically normal cases (n = 14) and neurodegenerative disease cases (Alzheimer’s disease n = 10; Parkinson’s disease n = 11; and Huntington’s disease n = 9) in regions affected by pathology and regions that are relatively devoid of pathology. We identified a statistically significant decrease in RNA integrity number (RIN) in Alzheimer’s disease tissue relative to neurologically normal tissue (mixed effects model, p = 0.04). There were no statistically significant differences between neurologically normal cases and Parkinson’s disease or Huntington’s disease cases. Next, we investigated whether total RNA quality affected mRNA quantification, by correlating RIN with qPCR threshold cycle (CT). CT values for all six genes investigated were strongly correlated with RIN (p < 0.05, Pearson correlation); this effect was only partially mitigated by normalization to RPL30. Our results indicate that RNA quality is decreased in Alzheimer’s disease tissue. We recommend that RIN should be considered when this tissue is used in gene expression analyses.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

RNA Quality in Post-mortem Human Brain Tissue Is Affected by Alzheimer’s Disease

Highet

Parker

Faull

et al. 2021

Front. Mol. Neurosci.

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“…RNAscope and immunohistochemically labeled sections were imaged on the Zeiss Axio Imager Z2 (MetaSystems Metafer5 VSlide software) using the 10× objective (0.45 numerical aperture). As shown by Highet et al the RNAscope Fast Red chromogen is visible under both transmitted light (GLT) and the TexasRed (TxRed) filter [ 27 ]. Therefore, to minimize the effect of autofluorescence and tissue artifacts, the RNAscope ® puncta were imaged under GTL and TxRed filters.…”

Section: Methodsmentioning

confidence: 99%

“…The RNAscope ® analysis was adapted from [ 27 ]. Images were extracted using the VSviewer software (MetaSystems) from the base, the middle and the top of the cortex and the intragyral white matter of the first parasagittal gyrus for analysis in an uncompressed 8-bit TIFFs format using FIJI.…”

Section: Methodsmentioning

confidence: 99%

Persistent cortical and white matter inflammation after therapeutic hypothermia for ischemia in near-term fetal sheep

Zhou

Wassink

McDouall

et al. 2022

J Neuroinflammation

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Background Therapeutic hypothermia significantly improves outcomes after moderate–severe hypoxic-ischemic encephalopathy (HIE), but it is partially effective. Although hypothermia is consistently associated with reduced microgliosis, it is still unclear whether it normalizes microglial morphology and phenotype. Methods Near-term fetal sheep (n = 24) were randomized to sham control, ischemia-normothermia, or ischemia-hypothermia. Brain sections were immunohistochemically labeled to assess neurons, microglia and their interactions with neurons, astrocytes, myelination, and gitter cells (microglia with cytoplasmic lipid granules) 7 days after cerebral ischemia. Lesions were defined as areas with complete loss of cells. RNAscope® was used to assess microglial phenotype markers CD86 and CD206. Results Ischemia-normothermia was associated with severe loss of neurons and myelin (p < 0.05), with extensive lesions, astrogliosis and microgliosis with a high proportion of gitter cells (p < 0.05). Microglial wrapping of neurons was present in both the ischemia groups. Hypothermia improved neuronal survival, suppressed lesions, gitter cells and gliosis (p < 0.05), and attenuated the reduction of myelin area fraction. The “M1” marker CD86 and “M2” marker CD206 were upregulated after ischemia. Hypothermia partially suppressed CD86 in the cortex only (p < 0.05), but did not affect CD206. Conclusions Hypothermia prevented lesions after cerebral ischemia, but only partially suppressed microglial wrapping and M1 marker expression. These data support the hypothesis that persistent upregulation of injurious microglial activity may contribute to partial neuroprotection after hypothermia, and that immunomodulation after rewarming may be an important therapeutic target.

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“…A32849; respectively) and donkey anti-mouse IgG Secondary Antibody Alexa Fluor™ 647 (Thermofisher, Cat No. A-31571) was added for 1 h and the nuclei stained with DAPI (1:1000, KPL, Gaithersburg, MD, USA) [91,92].…”

Section: Rnascopementioning

confidence: 99%

Consistency and Variation in Doublecortin and Ki67 Antigen Detection in the Brain Tissue of Different Mammals, including Humans

Ghibaudi

Amenta

Agosti

et al. 2023

IJMS

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Recently, a population of “immature” neurons generated prenatally, retaining immaturity for long periods and finally integrating in adult circuits has been described in the cerebral cortex. Moreover, comparative studies revealed differences in occurrence/rate of different forms of neurogenic plasticity across mammals, the “immature” neurons prevailing in gyrencephalic species. To extend experimentation from laboratory mice to large-brained mammals, including humans, it is important to detect cell markers of neurogenic plasticity in brain tissues obtained from different procedures (e.g., post-mortem/intraoperative specimens vs. intracardiac perfusion). This variability overlaps with species-specific differences in antigen distribution or antibody species specificity, making it difficult for proper comparison. In this work, we detect the presence of doublecortin and Ki67 antigen, markers for neuronal immaturity and cell division, in six mammals characterized by widely different brain size. We tested seven commercial antibodies in four selected brain regions known to host immature neurons (paleocortex, neocortex) and newly born neurons (hippocampus, subventricular zone). In selected human brains, we confirmed the specificity of DCX antibody by performing co-staining with fluorescent probe for DCX mRNA. Our results indicate that, in spite of various types of fixations, most differences were due to the use of different antibodies and the existence of real interspecies variation.

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fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer’s disease using an automated quantitative analysis workflow

Cited by 5 publications

References 38 publications

RNA Quality in Post-mortem Human Brain Tissue Is Affected by Alzheimer’s Disease

RNA Quality in Post-mortem Human Brain Tissue Is Affected by Alzheimer’s Disease

Persistent cortical and white matter inflammation after therapeutic hypothermia for ischemia in near-term fetal sheep

Consistency and Variation in Doublecortin and Ki67 Antigen Detection in the Brain Tissue of Different Mammals, including Humans

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