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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Characterization of neuronal aging in Cortical organoids
<ol>
	<li>Process cortical organoids for cryosections: 
	NOTE: The steps were performed in a Class 2 biosafety hood.
	<ol>
		<li>Prepare 2 mL tubes, each filled with 1.5 mL of 4% paraformaldehyde (PFA).</li>
		<li>Cut the end of a P1000 pipette tip (to make it a wide-bore) and gently transfer each organoid to one of the 2 mL tubes prepared above (one organoid per tube). 
		NOTE: To prevent excess Differentiation Medium (DM) media mixing with the PFA, allow the organoid to sink toward the opening of the P1000 pipette tip and rest the tip just above the top of the PFA in the tube before pipetting the organoid out. This will enable the researcher to transfer just the organoid and very minimal media.</li>
		<li>Allow the fixation process to take place at 4&#176;C for 1 h.</li>
		<li>Using an uncut P1000 pipette tip, carefully aspirate excess PFA and add 1.5 mL of cold 1x PBS (Phosphate buffered saline).</li>
		<li>Transfer the tubes to an orbital shaker set at 70 rpm for 10 min at room temperature (RT).</li>
		<li>Repeat the washing process with cold 1x PBS three times to ensure all PFA has been thoroughly removed. 
		NOTE: Do not dispose of PFA in regular waste containers; instead, prepare a specific chemical waste disposal container for this, as PFA is a hazard.</li>
		<li>Immerse the organoids in 1x PBS containing 30% sucrose and incubate at 4 &#176;C until all organoids have sunk to the bottom of the tube. 
		NOTE: The time required for the organoids to sink depends on their size and age. Three-month-old organoids may take up to 5 h.</li>
		<li>Using a cut, wide-bore P1000 pipette tip, gently transfer three to five organoids into a mounting mold containing a mounting solution made of 30% sucrose and 100% optimal cutting temperature (OCT) medium at a ratio of 3:2.</li>
		<li>Use a 10 &#181;L pipette tip with the aid of a stereomicroscope to orientate and position organoids in a grid-like pattern.</li>
		<li>Place the mold on dry ice to solidify the sucrose OCT solution before proceeding with cryo-sectioning (16-20 &#181;m) using a cryostat. 
		NOTE: For senescence-associated beta-galactosidase, all tissues must be processed for sectioning once they have sunk. For immunofluorescence, tissues can be processed for sectioning the next day. If not stained immediately, all the slides containing sections must be stored at -20 &#176;C before subsequent immunofluorescence or beta-galactosidase.</li>
	</ol>
	</li>
	<li>Process for analysis of senescence in the cortical brain organoids: 
	NOTE: The following steps can be performed on a regular lab bench.
	<ol>
		<li>Transfer the slides into a microscope slide staining container with a lid and wash the sectioned organoid tissue three times with 1x PBS for 10 min at RT to remove any excess mounting solution.</li>
		<li>Following this, incubate the washed tissue with freshly made beta-galactosidase staining solution overnight at 37 &#176;C. 
		NOTE: The beta-galactosidase staining solution is made of phosphate buffer (for 10 mL of phosphate buffer: 8.15 mL of 1 M NaH2PO4, 1.85 mL of 1M Na2HPO4) adjusted pH = 6, 100 mM of potassium hexacyanoferrate (III), 100 mM of potassium hexacyanoferrate (II) trihydrate, 5 M of NaCl, 1 M of MgCl2, 20 mg/mL of X-Gal. Avoid using a standard cell culture incubator containing CO2 as the CO2 will alter the pH of the beta-galactosidase staining solution.</li>
		<li>To remove the beta-galactosidase solution, wash the stained tissues with 1x PBS three times for 10 min each at RT.</li>
		<li>Mount the washed tissues with a glass antifade mountant and allow the mounting solution to solidify for 30 min at RT before viewing under the microscope.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% Formaldehyde (W/V) Methanol-free</td>
			<td>Thermo Fisher Scientific</td>
			<td>28908</td>
			<td>4% of PFA are diluted in 1x PBS</td>
		</tr>
		<tr>
			<td>CKX53 microscope with SC50 camera</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>D1408-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td>Used in DM media</td>
		</tr>
		<tr>
			<td>OCT Embedding Compound Sakura Clear (118 mL/Bottle)</td>
			<td>Tissue Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hexacyanoferrate (II) Trihydrate</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>CP1087</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate(III)</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>455946</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Glass Antifade Mountant</td>
			<td>Life Technologies Australia (TFS)</td>
			<td>P36980</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>PHR1001-1G</td>
			<td>30% of sucrose are diluted in 1x PBS</td>
		</tr>
	</tbody>
</table>

detection of neuronal aging in a cortical organoid section

Source: Shaker, M. R., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/63714">Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro.</a> J. Vis. Exp. (2022).
This video demonstrates the method of detecting aging neurons in cortical organoid sections by staining for beta-galactosidase activity, which is elevated in senescent cells. The enzyme cleaves a substrate to produce a blue product, followed by microscopic observation of the stained neurons.

Detection of Neuronal Aging in a Cortical Organoid Section

Source: Shaker, M. R., et al. Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro. J. ...

Begin with a slide containing a thin section of a cortical organoid that contains glial cells and neurons at various stages of aging.
The aging neurons, in a state of senescence with declining cell function, contain high levels of beta-galactosidase.
The section is pretreated with a fixative to stabilize the cell structure and a permeabilizing agent to permeabilize their membranes.
Place the slide in a staining container to wash with a buffer to remove any solution traces.
Stain the slide with a solution containing a substrate for the beta-galactosidase enzyme and incubate.
The substrate enters the cells, where beta-galactosidase cleaves the substrate, producing a blue-color product that accumulates more in the aging neurons.
Repeatedly wash the section.
Add an antifade mountant and allow it to solidify, ensuring the integrity of the stained section.
Observe the slide under a microscope; the area with an intense blue-color product indicates the aging neurons within the organoid.

detection-of-neuronal-aging-in-a-cortical-organoid-section

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

13 Görüntüleme. Source: Shaker, M. R., et al. Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro. J. Vis. Exp. (2022). This video demonstrates the method of detecting aging neurons in cortical organoid sections by staining for beta-galactosidase activity, which is elevated in senescent cells. The enzyme cleaves a substrate to produce a blue product, followed by microscopic observation of the stained neurons. 

Video: Detection of Neuronal Aging in a Cortical Organoid Section - Kavram

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Travmatik Beyin Hasarı için Kontrollü Kortikal Etki Modeli

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

JoVE Journal

Tıp

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

İnsan Embriyonik Kök Hücrelerinden Beyin Organoidleri Üretmek için Statik Kendi Kendini Yöneten Bir Yöntem

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Gelişim Biyolojisi

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Subselüler Ca Floresans ve Biyoparlaklık Görüntüleme<sup&gt; 2+</sup&gt; Yaşlı Hipokampal Nöronlar

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Detection of Neuronal Aging in a Cortical Organoid Section

TRANSKRIPT

Detection of Neuronal Aging in a Cortical Organoid Section

Travmatik Beyin Hasarı için Kontrollü Kortikal Etki Modeli

İnsan Embriyonik Kök Hücrelerinden Beyin Organoidleri Üretmek için Statik Kendi Kendini Yöneten Bir Yöntem

Subselüler Ca Floresans ve Biyoparlaklık Görüntüleme