<meta charset="utf8"></head><body>Sistema de aire de sobrepresión personalizado para inducir lesiones del SNC en modelos murinos

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Pentanol</td>
			<td>Fisher Scientific</td>
			<td>AC160600250</td>
			<td>Used to make Avertin solution&#160;</td>
		</tr>
		<tr>
			<td>2,2,2-tribomoethanol</td>
			<td>Sigma Aldrich</td>
			<td>T48402</td>
			<td>Used to make Avertin solution&#160;</td>
		</tr>
		<tr>
			<td>24-well plates with lid</td>
			<td>VWR</td>
			<td>76520-634</td>
			<td>24-well plate</td>
		</tr>
		<tr>
			<td>2-Propanol&#160;</td>
			<td>Fisher Scientific</td>
			<td>A451-1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>50 kS/s/channel Bridge Analog Input Module&#160;</td>
			<td>National Instruments</td>
			<td>NI-9237</td>
			<td>DAQ module</td>
		</tr>
		<tr>
			<td>Albumin Bovine Fraction V (BSA)&#160;</td>
			<td>Research Products International&#160;</td>
			<td>A30075</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Anti-Iba1 Primary Antibody (Goat polyclonal)&#160;</td>
			<td>Abcam&#160;</td>
			<td>ab5076</td>
			<td>Marker for microglia, Used at 1:500 concentration&#160;</td>
		</tr>
		<tr>
			<td>Anti-Synaptophysin Primary Antibody (Mouse monoclonal)&#160;</td>
			<td>Abcam&#160;</td>
			<td>ab8049</td>
			<td>Marker for photoreceptor ribbon synapses, Used at 1:20 concentration</td>
		</tr>
		<tr>
			<td>Araldite GY 502&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>10900</td>
			<td></td>
		</tr>
		<tr>
			<td>Cacodylate buffer</td>
			<td>Electron Microscopy Sciences</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>Charcoal Filter Canister</td>
			<td>E-Z Systems</td>
			<td>EZ-258</td>
			<td>Collection of anesthetic waste</td>
		</tr>
		<tr>
			<td>Clear H20 DietGel 76A</td>
			<td>Clear H2O&#160;</td>
			<td>72-07-5022</td>
			<td>Used post blast to aid animal recovery</td>
		</tr>
		<tr>
			<td>CompactDAQ Chassis</td>
			<td>National Instruments</td>
			<td>USB-9162</td>
			<td>DAQ chassis</td>
		</tr>
		<tr>
			<td>Compressed Air</td>
			<td>A-L Gas</td>
			<td>GSMCA300&#160;</td>
			<td>Used to refill pressurized air tank</td>
		</tr>
		<tr>
			<td>DAPI Fluoromount-G&#160;</td>
			<td>Southern Biotech</td>
			<td></td>
			<td>Mounting media with DAPI</td>
		</tr>
		<tr>
			<td>Diamond knife</td>
			<td>Micro Star Technologies, Group of Bruker Nano, Inc.&#160;</td>
			<td></td>
			<td>For sectioning optic nerves, 3 mm/45 degrees/Style H&#160;</td>
		</tr>
		<tr>
			<td>Donkey Anti-Goat IgG (H+L) High Cross Adsorbed Secondary Antibody, Alex Fluor 594</td>
			<td>Invitrogen (Supplier: Fisher Scientific)</td>
			<td>A-11058</td>
			<td>Secondary antibody for microglia, Used at 1:200 concentration</td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IgG (H+L) High Cross Adsorbed Secondary Antibody, Alex Fluor 594</td>
			<td>Invitrogen (Supplier: Fisher Scientific)</td>
			<td>A-21203</td>
			<td>Secondary antibody for photoreceptor ribbon synapses, Used at 1:200 concentration</td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IgG (H+L) High Cross Adsorbed Secondary Antibody, Alex Fluor 488</td>
			<td>Invitrogen (Supplier: Fisher Scientific)</td>
			<td>A-21206</td>
			<td>Secondary antibody for rod bipolar cells, Used at 1:200 concentration&#160;</td>
		</tr>
		<tr>
			<td>Donkey Serum&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D9662</td>
			<td>NDS</td>
		</tr>
		<tr>
			<td>Dumont #3 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11231-30</td>
			<td>Fine forceps for whole eye enucleation&#160;</td>
		</tr>
		<tr>
			<td>Ethanol (200 proof)&#160;</td>
			<td>KOPTEC (Supplier: VWR)&#160;</td>
			<td>89125-188</td>
			<td>Ethanol&#160;</td>
		</tr>
		<tr>
			<td>Fluoromount-G&#160;</td>
			<td>Invitrogen (Supplier: Fisher Scientific)</td>
			<td>00-4958-02</td>
			<td>Mounting media</td>
		</tr>
		<tr>
			<td>Genteal Tears Ophthalmic Gel</td>
			<td>Covetrus</td>
			<td>72359</td>
			<td>Eye lubricant to prevent eyes from drying out during/after anesthesia&#160;</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16200</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated Cylinder 1000 mL</td>
			<td>Fisher Scientific</td>
			<td>08-572G</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated Cylinder 250 mL</td>
			<td>Fisher Scientific</td>
			<td>08-572E</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated Cylinder 500 mL</td>
			<td>Fisher Scientific</td>
			<td>08-572F</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad&#160;</td>
			<td>Braintree Scientific</td>
			<td>AP-R 26E</td>
			<td>Controlled heating support</td>
		</tr>
		<tr>
			<td>High Pressure Fill Station</td>
			<td>Ninja Paintball</td>
			<td>HPFSV2</td>
			<td>Used to refill pressurized air tank</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health&#160;</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Invert Mini</td>
			<td>Empire Paintball</td>
			<td></td>
			<td>Paintball gun</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Covetrus</td>
			<td>29405</td>
			<td>Inhalation anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane Vaporizer</td>
			<td>VetEquip</td>
			<td>901806</td>
			<td>Animal anesthesia</td>
		</tr>
		<tr>
			<td>Masterflex Pump</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td>Used for animal perfusion</td>
		</tr>
		<tr>
			<td>Methanol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>322415-2L&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Globe Scientific</td>
			<td>1358W</td>
			<td>White glass microscope slides</td>
		</tr>
		<tr>
			<td>NI LabVIEW&#160;</td>
			<td>National Instruments</td>
			<td></td>
			<td>Software to acquire data from DAQ system (other examples include Matlab, Python, or other softwares provided by different DAQ hardware manufacturers)</td>
		</tr>
		<tr>
			<td>NI Measurement and Automation Explorer (NI MAX)&#160;</td>
			<td>National Instruments</td>
			<td></td>
			<td>Software to configure DAQ system settings</td>
		</tr>
		<tr>
			<td>NI-DAQmx drivers&#160;</td>
			<td>National Instruments</td>
			<td></td>
			<td>Driver for interacing with DAQ system&#160;</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ni-E microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide 2%</td>
			<td>Electron Microscopy Sciences</td>
			<td>19152</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 32%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>PFA diluted down to 4%</td>
		</tr>
		<tr>
			<td>Paraphenylenediamine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P6001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011044</td>
			<td>PBS diluted down to 1x</td>
		</tr>
		<tr>
			<td>Propylene oxide&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>20401</td>
			<td></td>
		</tr>
		<tr>
			<td>PROV3 48 L, 48 in3 Aluminum 3000 psi Rated Tank</td>
			<td>Ninja Paintball</td>
			<td></td>
			<td>Pressurized air tank</td>
		</tr>
		<tr>
			<td>Pyrex Reusable Media Storage Bottle 1000 mL</td>
			<td>Fisher Scientific</td>
			<td>06-414-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Reusable Media Storage Bottle 500 mL</td>
			<td>Fisher Scientific</td>
			<td>06-414-1C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Reusable Media Storage Bottles 250 mL</td>
			<td>Fisher Scientific</td>
			<td>06-414-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Anti-PKC-a Primary Antibody (Rabbit monoclonal)&#160;</td>
			<td>Abcam&#160;</td>
			<td>ab32376</td>
			<td>Marker for rod bipolar cells, Used at 1:500 concentration&#160;</td>
		</tr>
		<tr>
			<td>Resin 812</td>
			<td>Electron Microscopy Sciences</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>Series TJE Pressure Transducer, 100 psi&#160;</td>
			<td>Honeywell&#160;</td>
			<td>060-0708-10TJG</td>
			<td>Consider the range when selecting pressure transducer to optimize resolution of measurements&#160;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S5016</td>
			<td></td>
		</tr>
		<tr>
			<td>Super TJE Pressure Transducer, 7500 psi&#160;</td>
			<td>Honeywell&#160;</td>
			<td></td>
			<td>Consider the range when selecting pressure transducer to optimize resolution of measurements&#160;</td>
		</tr>
		<tr>
			<td>Syringe/Needle Combo</td>
			<td>Covetrus&#160;</td>
			<td>60728</td>
			<td>Syringe/Needle to perform IP injections</td>
		</tr>
		<tr>
			<td>Tissue-Plus OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>23-730-571&#160;</td>
			<td>Freezing medium&#160;</td>
		</tr>
		<tr>
			<td>Toluidine blue</td>
			<td>Fisher Scientific</td>
			<td>BP107-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>UniSlide XY Table</td>
			<td>Velmex&#160;</td>
			<td>AXY40 Series</td>
			<td>XY positioning table&#160;</td>
		</tr>
		<tr>
			<td>University Brush - Series 233- Round, Size 000</td>
			<td>Winsor and Newton&#160;</td>
			<td></td>
			<td>Paintbrush</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 2.5mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>Scissors for whole eye enucleation</td>
		</tr>
		<tr>
			<td>Virtual Instrument</td>
			<td>National Instruments&#160;</td>
			<td></td>
			<td>Digital tool for data acquisition software&#160;</td>
		</tr>
	</tbody>
</table>

system for focal, closed-system central nervous system injury

Closed-system central nervous system (CNS) injuries are injuries that are caused by damage to the brain or spinal cord without causing a break in the skull or spinal column. These injuries include traumatic brain injury (TBI) and spinal cord injury (SCI) and can occur from a variety of incidents, including blunt force injuries (e.g., falls, sports injuries, motor vehicle accidents) and explosive blasts. Closed-system CNS injuries are generally considered less severe compared to penetrating CNS injuries, yet they occur more often. However, similar to penetrating injuries, closed-system CNS injuries can result in long-term and progressive health issues, especially after repeated occurrences1,2,3,4,5,6. Concerningly, emerging evidence suggests that even subclinical closed-system CNS injuries, which fall below the diagnostic criteria for a TBI or SCI after a single occurrence7,8,9,10,11,12,13, may evolve into chronic neurodegenerative diseases after repeated injury6,14,15,16. This underscores the urgent need for a better understanding of the mechanisms and consequences of single and repeated closed-system CNS injuries.&#160; Such knowledge is imperative for improved protective and therapeutic approaches. Crucial to this endeavor are animal models that replicate closed-system CNS injuries.
Current animal models of closed-system CNS injuries have been instrumental in advancing our understanding of the pathophysiology and potential protective and therapeutic interventions for these traumas. Rodents are particularly popular due to their low cost, availability, genetic manipulability, ease of handling, well-established behavioral and physiological assays, and more favorable ethical considerations17. Common methods for inducing closed-system TBI in rodents include weight-drop devices18,19, controlled cortical impact (CCI) devices20, and compression-air-driven shock tubes21. For SCI, blunt trauma models typically require laminectomy22,23 or other surgical techniques24 to access the spinal cord or epidural space directly. However, closed-body SCI blast injury models have been developed using compression-air-driven shock tubes 25. Despite providing valuable insights, each of these models has unique limitations. Weight-drop models can have high variability and limited control of injury location and severity, producing experimental and ethical concerns for causing severe, uncontrolled injury26. CCI devices offer precision but require training to operate, may involve a craniotomy, and can suffer from mechanical variability impacting reproducibility27. Shock tubes are generally less invasive but can be difficult to acquire, complex to set up and operate, and can create unrealistic and highly variable injury conditions due to environmental factors, wave reflections, and complex pressure interactions28.
To better study the mechanisms and effects of single and repeat closed-system CNS injuries and their treatments, this paper presents a modular, user-friendly, cost-effective, and non-invasive method. The primary objective of this approach is to enable precise control and flexible modification of injury parameters, including location, severity, and timing. To support this objective, this manuscript provides a detailed protocol for constructing, calibrating, and troubleshooting an overpressure air system, which addresses some of the limitations of existing closed-system CNS injury devices. This system not only offers cost-effectiveness and minimal setup time, but it highly versatile, providing consistent and reproducible results while minimizing ethical concerns and maximizing clinical relevance. Additionally, the system&#8217;s ability to produce a range of closed-system CNS injuries in murine models is described, along with its potential applications in future studies. Notably, the goal of this manuscript is to provide a framework that enables investigators to easily acquire, adapt, and expand this system for their specific needs, thereby furthering ongoing research in CNS trauma. Representative results demonstrating the system&#8217;s efficacy in inducing axonal trauma are also presented.

<meta charset="utf8"></head><body>Autor destacado: Desarrollo de modelos precisos y clínicamente relevantes para el estudio de la degeneración secundaria en la neuropatía óptica traumática

Sistema para la lesión focal y cerrada del sistema nervioso central

Our laboratory explores the mechanisms controlling secondary degeneration after ITON, indirect traumatic optic neuropathy, with the goal of developing rational therapies for patients. Currently, the devices to induce CNS injury in the field include controlled cortical impact devices, weight drop models, and compression air-driven shock tubes. We have found that the duration of the interblast exposure interval contributes significantly to the amount of axon degeneration, and that injury to the optic nerve, similar to injury to the brain, significantly elevates the amount of reactive oxygen species.And that contributes to the secondary axon degeneration. This protocol addresses the need for a device that offers precise control over injury location and severity, while maintaining clinical relevance for closed-system injuries. It also offers a way to study the short interblast injury intervals of certain occupational environments.It's also cost effective, easy to set up and use. So while other devices also offer customization, this closed-system injury device stands out for its ease of set up and use. And this protocol provides a detailed framework simplifying this process, enabling other researchers to quickly adapt and set up the device for their own specific experimental goals and needs.

The prevalence of closed-system central nervous system (CNS) injuries underscores the need for an enhanced understanding of these traumas to improve ...

system-for-focal-closed-system-central-nervous-system-injury

Research

JoVE Journal

Neuroscience

System for Focal, Closed-System Central Nervous System Injury

387 vistas.  Vanderbilt University. Nuestro laboratorio explora los mecanismos que controlan la degeneración secundaria después de la ITON, neuropatía óptica traumática indirecta, con el objetivo de desarrollar terapias racionales para los pacientes. Actualmente, los dispositivos para inducir lesiones del SNC en el campo incluyen dispositivos de impacto cortical controlado, modelos de caída de peso y tubos de choque de compresión impulsados por aire. Hemos encontrado que la duración del...

Video: Autor destacado: Desarrollo de modelos precisos y clínicamente relevantes para el estudio de la degeneración secundaria en la neuropatía óptica traumática

The prevalence of closed-system central nervous system (CNS) injuries underscores the need for an enhanced understanding of these traumas to improve protective and therapeutic interventions. Crucial to this research are animal models that replicate closed-system CNS injuries. In this context, a custom overpressure air system was engineered to reproduce a range of closed-system CNS injuries in murine models, including ocular, brain, and spinal cord trauma. To date, the system has been used to administer eye-, head-, or spine-directed overpressure air to model anteroposterior pole injury in the eye, indirect traumatic optic neuropathy (ITON), focal traumatic brain injury, and spinal cord injury. This paper provides a detailed protocol outlining the system&#39;s design and operation and shares representative results demonstrating its effectiveness. The robust framework presented here provides a strong foundation for ongoing research in CNS trauma. By leveraging the system&#8217;s flexible attributes, investigators can modify and carefully control the location, severity, and timing of injuries. This allows for comprehensive comparisons of molecular mechanisms and therapeutic efficacy across multiple closed-system CNS injuries.

This protocol describes a custom overpressure air system designed to induce closed-system central nervous system (CNS) injuries in mice, including ocular, brain, and spinal cord traumas. The goal of this protocol is to provide a framework for researchers to easily adapt and expand the system for their unique CNS trauma studies.

Investigación

Neurociencias

Overpressure Air Delivery for the Induction of Indirect Traumatic Optic Neuropathy (ITON) in Mice

To begin, confirm the anesthetization of the mouse by assessing the absence of a response to a toe pinch. Secure the mouse into the inner animal holder with its head and upper hind shoulders exposed through the rectangular opening while the dorsum and lower hind limbs remain shielded. Apply surgical tape across the upper hind shoulders to secure the mouse.Insert the inner animal holder into the outer holder. After opening the secondary anesthesia line inside the animal holder, seal off the end of the holder to prevent the anesthetic from leaking out of the tube and the mouse from moving during the experiment. Align the outer animal holder's four millimeter circular aperture directly over the mouse's left eye.Position the animal holder on the XY table using the control knobs, so that its aperture aligns with the barrel of the paintball gun, and the external surface is five millimeters from the end of the barrel. After calibration, initiate the over-pressure air sequence to deliver two bursts of 15 PSI at an interval of 0.5 seconds. For the sham group mouse, turn the animal holder away from the barrel, block the air with a cardboard shield and expose it to the noise of the over-pressure air.Healthy densely packed axons were observed in the sham group. In contrast, the ITON group exhibited signs of axon degeneration, including irregularities in axon shape and myelin sheath breakdown. The total number of axons significantly decreased in the ITON group compared to the sham group.A significant increase in degenerative profiles was detected in the ITON group compared to the sham group. Increased microglia proliferation was observed in the retina of ITON mice, with microglia extending into the outer nuclear layer. Compared to the sham group, where microglia remained in their typical retinal layers.Intact synaptic connections between rod bipolar cells and photoreceptors were noted in both ITON and sham mice, with no detected synaptopathy.