Ex Vivo Neuroimaging Tools in Rodents - 2024 PDF

Ex vivo neuroimaging tools in rodents DGCN35 – Animal models for brain function and disorders BMS30 - Animal models for psychological and neurological disorders Bram Geenen, MSc Research technician Dept. of Imaging, Anatomy, Radboudumc Visit gosocrative.com and enter room name DGCN35...

Ex vivo neuroimaging tools in rodents DGCN35 – Animal models for brain function and disorders BMS30 - Animal models for psychological and neurological disorders Bram Geenen, MSc Research technician Dept. of Imaging, Anatomy, Radboudumc Visit gosocrative.com and enter room name DGCN35  Today My career so far Your future careers? Topic: a few examples of ex vivo techniques used for brain imaging Goal: learn about the techniques often used in labs, understand their steps, be able to advise on which technique to use for a specific research question 1-4 Pagina 2  Outline Histochemistry In vivo experiments Brain sections Immunohistochemistry In situ hybridization Mouse brain 3DISCO Whole brain clearing uDISCO iDISCO Pagina 3 Pagina 4  From in vivo to ex vivo: sacrificing the animals Anesthetics, analgesia Killing: CO2, cervical dislocation, guillotine, perfusion Harvesting of organs Fixation of organs Pagina 5 Pagina 6  Mouse brain A mouse brain → ex vivo neuroimaging To section or not to section It all depends on what you want to know Pagina 7  Mouse brain Section Making brain sections: Cryostat Freezing microtome Paraffin microtome Pagina 8  Mouse brain sections Section Making brain sections: Cryostat Freezing microtome Paraffin microtome Pagina 9  Mouse brain sections What to do with brain sections: Make images GM mouse with fluorescent label Mouse injected with fluorescent substance Pagina 10  Mouse brain sections What to do with brain sections: Histochemical staining to visualize cells Hematoxylin/eosin Brain morphology Pagina 11  Mouse brain sections What to do with brain sections: Histochemical staining to visualize cells Nissl (cresyl violet) Brain morphology, neuron quantification, etc. Pagina 12  History of histochemistry 17th century: Marcello Malpighi studied the microstructure of the lungs of different animals Larger animals: capillaries and alveoli Insects: Malpighi tubule system 19th century: Camillo Golgi & Santiago Ramón y Cajal 5 Pagina 13  History of histochemistry Camillo Golgi & the Golgi staining A technique developed in 1863 to visualize neurons in brain tissue Immersion of a block or thick section of tissue in potassium dichromate Immersion in silver nitrate Deposition of silver chromate Section to make thinner sections Cover with a coverglass Investigate using a light microscope Entire neuron is stained, but only a small percentage of all neurons How it works? Nobody knows… Pagina 14  Mouse brain sections What to do with brain sections: Immunohistochemistry: detecting specific proteins in a tissue section Pagina 15  Immunohistochemistry Antibodies: Bind specifically to an antigen (mostly proteins) Have a variable and a constant region Variable region binds antigen Constant region differs between species Polyclonal antibodies are made by: Injecting an animal with the antigen Waiting for an immune reaction (B cells) Isolating the specific antibodies from the blood Testing for cross reactivity Monoclonal antibodies are made by: Injecting an animal with the antigen Waiting for an immune reaction (B cells) Isolating the specific antibody-producing B cells Fusing B cells to immortal cells in petri dish Isolating the specific antibodies from the medium Pagina 16  Immunohistochemistry General steps of the protocol (2 day protocol): 1. Blocking Incubate with BSA (bovine serum albumin) Pagina 17  Immunohistochemistry General steps of the protocol (2 day protocol): 1. Blocking 2. Incubation with primary antibody (antibody binds antigen) Pagina 18  Immunohistochemistry General steps of the protocol (2 day protocol): 1. Blocking 2. Incubation with primary antibody 3. Incubation with labeled secondary antibody Pagina 19  Immunohistochemistry General steps of the protocol (2 day protocol): 1. Blocking 2. Incubation with primary antibody 3. Incubation with labeled secondary antibody 1. Labeled with an enzyme 2. Labeled with a fluorophore http://www.bio-rad.com/en-nl/applications-technologies/detection-methods Pagina 20  Immunohistochemistry Detection methods Colorimetric detection (secondary antibody labeled with an enzyme) Most used: HRP enzyme and DAB/H2O2 substrate enzyme secondary primary antigen Pagina 21  Immunohistochemistry Detection methods Colorimetric detection (secondary antibody labeled with an enzyme) Most used: HRP enzyme and DAB/H2O2 substrate Pagina 22  Immunohistochemistry Detection methods Fluorescent detection (secondary antibody labeled with a fluorophore) Most used: Alexa Fluor Pagina 23  Immunohistochemistry Detection methods Fluorescent detection (secondary antibody labeled with a fluorophore) Most used: Alexa Fluor Pagina 24  Example study Effect of a multinutrient intervention after ischemic stroke in female C57Bl/6 mice Maximilian Wiesmann, Nienke M. Timmer, Bastian Zinnhardt, Dirk Reinhard, Sarah Eligehausen, Anja Königs, Hasnae Ben Jeddi, Pieter J. Dederen, Andreas H. Jacobs, Amanda J. Kiliaan Pagina 25  Example study Pagina 26  Example study Pagina 27  Example study DCX = doublecortin = marker for newly-formed neurons Pagina 28  Example study Effect of a multinutrient intervention after ischemic stroke in female C57Bl/6 mice “We induced a transient middle cerebral artery occlusion (tMCAo) in C57Bl/6 female mice and immediately after surgery switched to either Fortasyn or an isocaloric control diet.” Stroke → less new neurons Stroke + Fortasyn → more new neurons DCX = doublecortin = marker for newly-formed neurons Pagina 29  Mouse brain sections What to do with brain sections: In situ hybridization: detecting specific mRNAs in a tissue section Pagina 30  In situ hybridization General steps of the protocol: 1. Incubating the tissue sections with the DIG-labeled probe 2. Incubating with an anti-DIG antibody that is labeled with alkaline phosphatase enzyme 3. Colorimetric detection by incubating with NBT/BCIP substrate antibody enzyme RNA probe Pagina 31  In situ hybridization General steps of the protocol: 1. Incubating the tissue sections with the DIG-labeled probe 2. Incubating with an anti-DIG antibody that is labeled with alkaline phosphatase enzyme 3. Colorimetric detection by incubating with NBT/BCIP substrate Pagina 32  In situ hybridization General steps of the protocol: 1. Incubating the tissue sections with the DIG-labeled probe 2. Incubating with an anti-DIG antibody that is labeled with a fluorophore 3. Detection of the light signal with a fluorescence microscope Pagina 33  Mouse brain A mouse brain → ex vivo neuroimaging To section or not to section It all depends on what you want to know Pagina 34  Mouse brain Tissue clearing Tissue clearing: Protocol for making organs (i.e. mouse brains) transparent, to make them suitable for rapid imaging using light-sheet fluorescence microscopy (LSFM) First publication on clearing: over 100 years ago! Visualizing fluorescently labeled proteins in the brain in 3D, without sectioning the tissue Pagina 35  Mouse brain Tissue clearing Pagina 36  Tissue clearing Tissue clearing: Protocol for making organs (i.e. mouse brains) transparent, to make them suitable for rapid imaging using light-sheet fluorescence microscopy (LSFM) Three different approaches: Organic solvent-based Aqueous solvent-based Hydrogel crosslink-based Pagina 37  3DISCO General steps of the protocol: Dehydrate Dissolve lipids Refractive index matching Imaging using LSFM, in a glass box filled with the refractive index matching liquid Pagina 38  3DISCO Pagina 39 LSFM  3DISCO Clearing the brain 1. Dehydrate using tetrahydrofuran (THF) or methanol (MeOH) 2. Dissolve lipids in THF or MeOH and dichloromethane (DCM) 3. Refractive index matching in dibenzylether (DBE) Pagina 41  3DISCO Pagina 42  3DISCO Pagina 43  Break Pagina 44  uDISCO uDISCO: ultimate three-Dimensional Imaging of Solvent Cleared Organs Optimization of the 3DISCO protocol Using the tissue shrinkage of the original protocol to their advantage Able to image larger structures than before Whole body clearing Pagina 45  uDISCO 6 Pagina 46  uDISCO Pagina 47  uDISCO 3D visualization of neuronal connections throughout the intact mouse CNS (GFP-M mouse, 4 months old). The fine details of the neuronal connections are evident from head to the nerves invading the hind limbs. Pagina 48  uDISCO A single traced axons (red) in the spinal cord of the sample is shown. The trajectories of individual axons in the entire CNS of the adult mice can be determined. Note that occasional curling of long spinal cord axons does not interfere with the tracing of their entire trajectories. The pseudo-colored traced axon is shown thicker than its actual size for easier visualization. Pagina 49  uDISCO uDISCO: ultimate three-Dimensional Imaging of Solvent Cleared Organs Only endogenous fluorescently labeled proteins Pagina 50  iDISCO Pagina 51  iDISCO iDISCO: immunolabeling-enabled three-Dimensional Imaging of Solvent Cleared Organs iDISCO+: version 2.0 of the protocol Combining immunohistochemistry and tissue clearing Visualizing protein expression in 3D In general the same steps as normal immunohistochemistry, with extra steps: 1. Permeabilization 2. Bleaching 3. Blocking 4. Incubation with primary antibody 5. Incubation with labeled (fluorescence!) secondary antibody 6. Clearing the brain (modified 3DISCO method) 1. Dehydrate using methanol 2. Dissolve lipids in methanol and dichloromethane 3. Refractive index matching in dibenzylether Pagina 52  iDISCO TrkA is the high affinity catalytic receptor for the neurotrophin Nerve Growth Factor (NGF) Pagina 53  iDISCO (A) tdTomato immunolabeling in an adult ChAT::cre; Ai14 half forebrain. The movie first shows sagittal optical cross-sections through the forebrain and then a fly over of the midbrain highlighting the fasciculus retroflexus as well as virtual dissection of the striatum to show cholinergic striatal interneurons. Then, a higher magnification shows the whole striatum and subsequently isolates an area of the cortex to highlight cholinergic neurons and their neurites. (B) iDISCO GFP immunolabeling in an adult Thy1::GFP-M half forebrain. The movie first shows a quick fly-over of the whole brain, in which the hippocampus is clearly visible along with the fornix. Then, a first zoom is made on the anterior forebrain showing the cortical pyramidal cells and their projections through the striatum. The following zoom shows a more detailed view of the hippocampus along with the axons of the fornix. (C) iDISCO β-galactosidase immunolabeling in an E14 Netrin-1lacZ/+ embryo. The movie shows the expression of the reporter gene produced from the trapped allele of Netrin-1. The expression is very high in the midline throughout the brain. Pagina 54  iDISCO+ @ Radboudumc Workflow: iDISCO+ method (immunolabeling and clearing) Pagina 55  iDISCO+ Major limitation: endogenous fluorescently labeled proteins (uDISCO) are not preserved Solution: do immunolabeling on the tags (i.e. anti-GFP antibody) Pagina 56  iDISCO+ @ Radboudumc Workflow: iDISCO+ method (immunolabeling and clearing) Imaging using Ultramicroscope (light-sheet fluorescence microscope) Making a 3D model of the immunolabeling using VOREEN software Pagina 57 Urocortin in the EWcp nucleus  iDISCO+ Automatic cFos detection using ClearMap Workflow: iDISCO+ method (immunolabeling and clearing) on a whole brain Imaging using Ultramicroscope (light-sheet fluorescence microscope) Use ClearMap scripts to detect cFos staining Pagina 58 Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes, Renier et al., 2016  iDISCO+ Pagina 59  iDISCO+ Mouse brain, 1 hr after a new environment exploration. All whiskers were clipped, except the B row. The first part shows the raw data acquired at 1.6X on the ultramicroscope, after c-Fos immunolabeling. The second part shows a detail of the barrel cortex, after manual segmentation of c-Fos signal in the cortical layer 4 (green) and 2-3 (red) layered with the autofluorescence signal (gray), showing the increased activity in the B row. Pagina 60  iDISCO+ 7-8 Pagina 61  iDISCO+ Application of iDISCO+ to visualize and quantify amyloid-β plaques in mouse and human brain samples Visualization of plaques together with inflammation and vasculature Greater 3D complexity of amyloid-β plaques in humans, when compared to mice 9 Pagina 62  iDISCO+ Application of iDISCO+ to visualize and quantify amyloid-β plaques in mouse and human brain samples Visualization of plaques together with inflammation and vasculature Greater 3D complexity of amyloid-β plaques in humans, when compared to mice Permeabilization: getting the antibodies inside the tissue Detergents and chemicals to break down cell membranes Bleaching: getting rid of autofluorescence Hydrogen peroxide and methanol to destroy autofluorescent molecules Pagina 63  iDISCO+ (A) The first video shows a region of the cortex labeled for plaques and cell nuclei with anti-beta amyloid antibodies (PAb #10) and TO-PRO-3, respectively. Surface rendering isolates the beta amyloid deposits in plaques and along blood vessels. (B) The second video shows plaque labeling with Congo red and the surrounding microglia labeled with anti-Iba1 antibodies. Surface rendering shows the enveloping of plaques by microglia. Pagina 64  iDISCO+ (A) The first video shows an entire hemisphere labeled for beta amyloid plaques (Congo red), microglia (anti-Iba1), and vasculature (anti-mouse secondary antibody) using iDISCO. The video zooms into the region of the cortex and hippocampus featured in the following video. (B) The second video shows a magnified view of optical slices through the hippocampus and cortex from the sample previously described, followed by a volume reconstruction and surface rendering of an isolated volume. Pagina 65  iDISCO+ iDISCO for entire mouse brain vasculature Automatic analysis (TubeMap) Investigate effects of ischemic stroke and sensory loss Pagina 66  iDISCO+ Pagina 67  iDISCO+ Pagina 68  iDISCO+ Video S3. Reconstruction of a Stroked Brain 3 Weeks after an Electrocauterization of the Middle Cerebral Artery, Related to Figure 6 Comparison of a control (left panel) and ischemic (right panel) brains, 3 weeks after an ischemic stroke. The brains were not perfused, and stained for Podocalyxin, mouse immunoglobulins (blue) and Acta2 (red). The modifications to the middle cerebral artery arbor are visible at the surface, with a missing ventral branch, and remodeled anastomosis with dorsal arteries. In the cortex, reorientation of the vessels is visible, with a change of directions showing a convergence toward the stroked site. Pagina 69  EyeDISCO EyeDISCO protocol for whole eye clearing Investigate role of Dcc in optic nerve guidance during development Pagina 70  EyeDISCO Frontal view of 3D-rendered brains using after anterograde axon tracing of visual projections with AlexaFluor-555 and/or AlexaFluor-647-conjugated cholera toxin β subunit (CTB). RE: right eye, LE: left eye, OC: optic chiasm, SCN: supra chiasmatic nucleus, MTN: medial terminal nucleus, LGN: lateral geniculate nucleus, NOT: nucleus of the optic tract, OPT: olivary pretectal nucleus, SC: superior colliculus. (A) Control brain, and (B) a Dkk3:cre;Dccfl/fl brain. Pagina 71  SHANEL SHANEL clearing of intact human organs Pagina 72  SHANEL Pagina 73 Clearing protocols Pagina 74  All the DISCO protocols 3DISCO: the original tissue clearing protocol, for transgenic animals (strong endogenous fluorescent labels) uDISCO: modified 3DISCO protocol, shrinkage used to image larger specimens, for transgenic animals (strong endogenous fluorescent labels) iDISCO: using immunohistochemistry (fluorescence) to label proteins, then use 3DISCO protocol to clear tissue iDISCO+: improved iDISCO protocol, substitute THF for DCM to eliminate (uneven) tissue shrinkage EyeDISCO: iDISCO+ adapted for eye tissue (de-pigmentation steps) 10 Pagina 75  Tissue clearing Hydrogel crosslink-based: CLARITY Pagina 76  Tissue clearing Aqueous solvent-based: CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) Pagina 77  Translational research From mouse to man iDISCO of human AD brain tissue (Liebmann paper) Small blocks Our tries so far with “fresh” brains Fixation iDISCO+ stainings CSVD AD Pagina 78 High blood pressure & SVD What have I talked about Histochemistry Immunohistochemistry In situ hybridization 3DISCO/uDISCO/iDISCO & clearing techniques I didn’t talk about: ELISA, PLI, ex vivo MRI, etc.  Important concepts The underlined, important concepts of this class: Histology, histochemistry, immunohistochemistry Antigen, antibody Blocking, primary antibody, secondary antibody Colorimetric detection (enzyme), fluorescent detection (fluorophore) In situ hybridization, DIG-labeled probe, target mRNA Light-sheet fluorescence microscope, tissue clearing General steps 3DISCO, iDISCO+ Pagina 82