Limits...
High Resolution Dissection of Reactive Glial Nets in Alzheimer's Disease.

Bouvier DS, Jones EV, Quesseveur G, Davoli MA, A Ferreira T, Quirion R, Mechawar N, Murai KK - Sci Rep (2016)

Bottom Line: Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease.Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs).The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Neuroscience, Department of Neurology and Neurosurgery, The Research Institute of the McGill University Health Centre, Montreal General Hospital, Montreal, Quebec, Canada.

ABSTRACT
Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation, and tauopathy and display unique features around β-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

No MeSH data available.


Related in: MedlinePlus

A broadly accessible, systematic method to study neurons and glia in human brain samples recovered from long-term storage.(a) The staining of pan-axonal neurofilaments (SMI312) reveals the general organization of neurons in cortical sections (female, 86 years old, 25 years of fixation). (b) Calbindin expressing interneurons are enriched in layers I/II of the human cortex (male, 88 years old, 19 years of fixation) and show diversified morphologies after 3D reconstruction. (c) High-resolution imaging of synapses in cortex (female, 82 years old, 15 years of fixation) shows vGlut1-positive presynaptic boutons (magenta) juxtaposed to PSD95-positive postsynaptic structures (green) (synapses indicated by arrows). (d) Maximum projection of a large field showing 1,95 mm2 (x:1.95 mm, y:0.9 mm) of tissue with a 30 μm depth labeled for Iba1 (microglia; magenta) and GFAP (astrocytes; green). (e) High magnification image of a GFAP-positive astrocyte ‘island’ in a cortical section. (f) 3-dimensional reconstructions of cortical protoplasmic GFAP+ astrocytes and Iba1+ microglia (female, 86 years old). (g) GFAP+ astrocytes (green) and Iba1+ microglia (magenta) in cortical layers III/IV show complex interactions in a control brain (female, 86 years old), resolvable by visualizing individual frames of confocal Z-stacks (step-size: 1 μm). Scale bars: 50 μm (a); 20 μm (b); 5 μm (c); 200 μm (d); 10 μm (e–g).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835751&req=5

f1: A broadly accessible, systematic method to study neurons and glia in human brain samples recovered from long-term storage.(a) The staining of pan-axonal neurofilaments (SMI312) reveals the general organization of neurons in cortical sections (female, 86 years old, 25 years of fixation). (b) Calbindin expressing interneurons are enriched in layers I/II of the human cortex (male, 88 years old, 19 years of fixation) and show diversified morphologies after 3D reconstruction. (c) High-resolution imaging of synapses in cortex (female, 82 years old, 15 years of fixation) shows vGlut1-positive presynaptic boutons (magenta) juxtaposed to PSD95-positive postsynaptic structures (green) (synapses indicated by arrows). (d) Maximum projection of a large field showing 1,95 mm2 (x:1.95 mm, y:0.9 mm) of tissue with a 30 μm depth labeled for Iba1 (microglia; magenta) and GFAP (astrocytes; green). (e) High magnification image of a GFAP-positive astrocyte ‘island’ in a cortical section. (f) 3-dimensional reconstructions of cortical protoplasmic GFAP+ astrocytes and Iba1+ microglia (female, 86 years old). (g) GFAP+ astrocytes (green) and Iba1+ microglia (magenta) in cortical layers III/IV show complex interactions in a control brain (female, 86 years old), resolvable by visualizing individual frames of confocal Z-stacks (step-size: 1 μm). Scale bars: 50 μm (a); 20 μm (b); 5 μm (c); 200 μm (d); 10 μm (e–g).

Mentions: To demonstrate the utility of this method, we analyzed the organization and morphology of brain cells in cortical samples from healthy, aged individuals (average age, 84.2 years; average storage, 20.4 years; Table 1). Specific antibodies were used to label neurons (SMI312, calbindin), microglia (ionized calcium-binding adapter molecule 1; Iba1), and astrocytes (Glial Fibrillary Acidic Protein; GFAP). Staining of neurofilaments (SMI312) resolved the layered organization of neurons in neocortex and their general morphology (Fig. 1a). Discrete populations of pyramidal neurons including calbindin D28-positive interneurons of layer III were detected, allowing their complex morphology to be visualized (Fig. 1b). Labeling for the presynaptic protein VGLUT1 and postsynaptic scaffold PSD95 demonstrated the ability of this approach to resolve individual synapses (Fig. 1c). Thus, this method allows for simultaneous analysis of neuronal populations, single-cell anatomy, and synaptic organization in long-term fixed human brain tissue.


High Resolution Dissection of Reactive Glial Nets in Alzheimer's Disease.

Bouvier DS, Jones EV, Quesseveur G, Davoli MA, A Ferreira T, Quirion R, Mechawar N, Murai KK - Sci Rep (2016)

A broadly accessible, systematic method to study neurons and glia in human brain samples recovered from long-term storage.(a) The staining of pan-axonal neurofilaments (SMI312) reveals the general organization of neurons in cortical sections (female, 86 years old, 25 years of fixation). (b) Calbindin expressing interneurons are enriched in layers I/II of the human cortex (male, 88 years old, 19 years of fixation) and show diversified morphologies after 3D reconstruction. (c) High-resolution imaging of synapses in cortex (female, 82 years old, 15 years of fixation) shows vGlut1-positive presynaptic boutons (magenta) juxtaposed to PSD95-positive postsynaptic structures (green) (synapses indicated by arrows). (d) Maximum projection of a large field showing 1,95 mm2 (x:1.95 mm, y:0.9 mm) of tissue with a 30 μm depth labeled for Iba1 (microglia; magenta) and GFAP (astrocytes; green). (e) High magnification image of a GFAP-positive astrocyte ‘island’ in a cortical section. (f) 3-dimensional reconstructions of cortical protoplasmic GFAP+ astrocytes and Iba1+ microglia (female, 86 years old). (g) GFAP+ astrocytes (green) and Iba1+ microglia (magenta) in cortical layers III/IV show complex interactions in a control brain (female, 86 years old), resolvable by visualizing individual frames of confocal Z-stacks (step-size: 1 μm). Scale bars: 50 μm (a); 20 μm (b); 5 μm (c); 200 μm (d); 10 μm (e–g).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835751&req=5

f1: A broadly accessible, systematic method to study neurons and glia in human brain samples recovered from long-term storage.(a) The staining of pan-axonal neurofilaments (SMI312) reveals the general organization of neurons in cortical sections (female, 86 years old, 25 years of fixation). (b) Calbindin expressing interneurons are enriched in layers I/II of the human cortex (male, 88 years old, 19 years of fixation) and show diversified morphologies after 3D reconstruction. (c) High-resolution imaging of synapses in cortex (female, 82 years old, 15 years of fixation) shows vGlut1-positive presynaptic boutons (magenta) juxtaposed to PSD95-positive postsynaptic structures (green) (synapses indicated by arrows). (d) Maximum projection of a large field showing 1,95 mm2 (x:1.95 mm, y:0.9 mm) of tissue with a 30 μm depth labeled for Iba1 (microglia; magenta) and GFAP (astrocytes; green). (e) High magnification image of a GFAP-positive astrocyte ‘island’ in a cortical section. (f) 3-dimensional reconstructions of cortical protoplasmic GFAP+ astrocytes and Iba1+ microglia (female, 86 years old). (g) GFAP+ astrocytes (green) and Iba1+ microglia (magenta) in cortical layers III/IV show complex interactions in a control brain (female, 86 years old), resolvable by visualizing individual frames of confocal Z-stacks (step-size: 1 μm). Scale bars: 50 μm (a); 20 μm (b); 5 μm (c); 200 μm (d); 10 μm (e–g).
Mentions: To demonstrate the utility of this method, we analyzed the organization and morphology of brain cells in cortical samples from healthy, aged individuals (average age, 84.2 years; average storage, 20.4 years; Table 1). Specific antibodies were used to label neurons (SMI312, calbindin), microglia (ionized calcium-binding adapter molecule 1; Iba1), and astrocytes (Glial Fibrillary Acidic Protein; GFAP). Staining of neurofilaments (SMI312) resolved the layered organization of neurons in neocortex and their general morphology (Fig. 1a). Discrete populations of pyramidal neurons including calbindin D28-positive interneurons of layer III were detected, allowing their complex morphology to be visualized (Fig. 1b). Labeling for the presynaptic protein VGLUT1 and postsynaptic scaffold PSD95 demonstrated the ability of this approach to resolve individual synapses (Fig. 1c). Thus, this method allows for simultaneous analysis of neuronal populations, single-cell anatomy, and synaptic organization in long-term fixed human brain tissue.

Bottom Line: Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease.Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs).The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Neuroscience, Department of Neurology and Neurosurgery, The Research Institute of the McGill University Health Centre, Montreal General Hospital, Montreal, Quebec, Canada.

ABSTRACT
Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation, and tauopathy and display unique features around β-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

No MeSH data available.


Related in: MedlinePlus