This work was supported by funding from NIH NEI P30 EY008126, the Potocsnak Discovery Grant in Regenerative Medicine, the Ret. Maj. General Stephen L. Jones, MD Fund, and Research Prevent Blindness, Inc Unrestricted Funds (VEI).

Custom Overpressure Air System for Inducing CNS Injuries in Murine Models

The authors have no conflicts of interest to disclose.

This custom overpressure air system is a useful tool for studying closed-system CNS injuries in murine models. The representative results from the example experiment demonstrate that the focal delivery of overpressure air using this system can effectively induce ITON, resulting in significant axon loss and degeneration. This highlights the system&#39;s ability to produce precise and reproducible CNS injury.
One of the major strengths of this system is its customizability to induce a range of CNS injuries. The severity of the injury can be adjusted by modifying the overall output pressure of the system, the distance of the animal from the end of the barrel using the x-y positioning stage, the size and shape of the exposure aperture, the number of exposures to overpressure air, and the interval between exposures. Additionally, the location of the CNS injury can be adjusted by modifying the location of the exposure aperture within the animal holder. This versatility has enabled the system to produce a spectrum of closed-system CNS injuries in murine models. Initially, the system was used to model closed-globe injuries, focusing on anterior and posterior pole damage and related deficits34,36, including the impacts of immune system response37, strain-specific outcomes38, and the efficacy of neuroprotective agents39. Eventually, this application expanded to assess the sequelae of repeated eye-directed exposures to model indirect traumatic optic neuropathy (ITON)30 and explore the effect of the number and interval between repeated exposures33. Since then, the application of the system has expanded to model closed-head mild traumatic brain injury (mTBI) through head-directed exposures40,41 and closed-body spinal cord injury (SCI) through dorsum-directed exposures42, emphasizing the device&#39;s adaptability and versatility in studying varied CNS injury domains.
When using this system, it is critical to take measures to minimize variability in injury outcomes to ensure the reproducibility and reliability of experimental results. Key measures include calibrating the system&#39;s output pressure levels before and after each series of three exposures to ensure consistent pressure delivery. Although variability is low when the system is operated between 15 psi and 50 psi when using compressed air34, consistent calibration helps detect unexpected errors, such as low battery or low air. Additionally, position each animal at the same distance from the end of the barrel to ensure consistent overpressure magnitude, as the intensity of the pressure wave decreases with distance. Uniform positioning also ensures each animal is impacted by the same part of the airwave. Furthermore, securing animals uniformly within the holder ensures the tissue of interest is consistently targeted, especially in repeated exposure models when there is risk of movement. Finally, uniformity in the age, sex, and genetic background of the animals is crucial as these factors influence the response to injury. For example, previous studies using this system compared the effects of eye-directed overpressure air on different mouse strains, highlighting significant differences in injury response between C57Bl/6J36, DBA/2J37, and Balb/c38 mice. The DBA/2J and Balb/c mice exhibited more severe anterior pole pathologies, greater retinal damage, higher oxidative stress, and more pronounced neuroinflammatory responses compared to the C57Bl/6J mice with Balb/c mice showing particularly robust and lasting injury profiles38.
System troubleshooting 
If the pressure values are uncharacteristically low for a given pressure gauge setting, pull the trigger 5-10x, allowing air to pass through the system and the regulator to adjust to a new setting. There must be no leaks in the air tank. The O-ring on the air tank must not be damaged or worn, the air tank should have enough air, and the battery of the gun should not be depleted. The x-y table should not have shifted away from its usual position from the end of the barrel and the overpressure air exposure aperture should be lined up with the barrel of the gun and not occluding it. The regulator should be tightly secured to the grip of the gun. If the pressure values are too low despite using the highest setting on the pressure gauge, the pressure gauge must not be increased beyond 200 psi, and the velocity setting on the gun should be adjusted to the maximum setting. If the pressure settings are inconsistent (e.g., high then low), ensure the air tank has enough air, the regulator is tightly secured to the grip of the gun, there are no leaks in the air tank and that it is screwed on tight, and the O-ring on the air tank is not damaged or worn.
To comprehensively understand this system&#39;s full capabilities, it is important to recognize its limitations. Mimicking real-world scenarios in a laboratory setting remains challenging. Although this system generates overpressure air, it does not replicate the complex dynamics of an explosive event, such as the varying pressure and temperature gradients, the presence of debris and reflected waves, and a multiphasic nature. Additionally, it does not mimic a Friedlander waveform (&#34;primary blast wave&#34;), which is characterized by a sharp, near-instantaneous peak in pressure followed by a rapid exponential decay that drops below ambient pressure before returning to baseline43. Rather, the waveform produced by this system represents a simpler, more symmetrical profile in which there is a more gradual rise and fall in pressure with no distinct negative phase (see Figure 2C in Hines-Beard et al.34). Somewhat advantageously, this waveform combines elements of both blast and blunt injuries. The bell-shaped &#34;pressure pulse&#34; delivers a consistent overpressure impact, akin to a &#34;wall of air&#34; hitting the subject. Yet, the overpressure air delivered by the wave is also a key characteristic aspect of blast injuries. Some may argue that while this waveform includes aspects of both injury types, it does not fully capture the complexity of either one. However, this consistent and reproducible &#34;pressure pulse&#34; is ideal for controlled experiments in a laboratory setting to study focal closed-system CNS injury. We have demonstrated the focal nature of the injury previously. For example, exposure to one eye does not cause damage to the primary nasal epithelium or brain44. Also, when directed to the side of the mouse head, a small area of the brain is affected45. Finally, the energy from the overpressure air from this system at the pressure level used for ITON did not affect the mouse unless repeated with a short time interval33. Thus, the pressure is non-injurious and therefore does not replicate a jet-end force. Further, even with repeated overpressure air exposure to the eye, there was no effect on anterior eye structures33. Significant optic nerve degeneration and vision loss only occurred with repeated exposure with an inter-exposure interval of less than 1 min33.
Compared to other laboratory devices for creating closed-system CNS injuries, this system offers unique benefits. It can deliver sequential bursts of overpressure air in rapid succession (0.5 s intervals)33, mimicking conditions in high-risk occupational environments where rapid blast exposures are a common hazard. For example, military personnel, both in training and combat scenarios, use a host of automatic firearms capable of rapid repeated firing, including automatic rifles (e.g., M16, AK-47), machine guns (e.g., M2 .50 caliber), Gatling guns, and miniguns. Other slower, yet repetitive weaponry used by military personnel include artillery, mortars, grenades, and improvised explosive devices (IEDs). Demolition workers involved in controlled demolition and miners involved in blasting operations to break up rock and extract minerals also experience sequential blasts in rapid succession. Finally, construction workers using pneumatic tools, pile drivers, or other heavy equipment that generative powerful percussive forces can experience rapid repeat impacts that mimic blast exposures. Notably, rapid delivery of overpressure air is not possible with devices like shock tubes that require extensive reconfiguration or re-pressurization between each event. Shock tubes use diaphragms that burst to generate shock waves, and after each burst, the diaphragm must be replaced. This process takes time, as the shock tube must be opened, the spent diaphragm removed, a new diaphragm installed, and the system allowed time to reset and repressurize. Thus, especially for studies investigating CNS injury after rapid repeat blast exposure, a system that does not require extensive reconfiguration or re-pressurization between each event is ideal.
Future applications of this modulatory, user-friendly, cost-effective system are promising. Leveraging its adaptable and unique attributes, this system opens several promising avenues for future pre-clinical therapeutic studies. Its ability to deliver rapid, sequential bursts of overpressure air can be leveraged to study the cumulative effects of repeated blast exposures, which is relevant for understanding chronic traumatic encephalopathy and other long-term neurodegenerative conditions. Additionally, this system can be used to explore the effectiveness of various pharmacological interventions aimed at mitigating closed-system CNS injuries, including the timing and dosing of neuroprotective drugs to determine optimal treatment windows. Furthermore, the system&#39;s precision in mimicking aspects of both blunt and blast injury mechanisms allows for the development of comprehensive injury models that reflect the complex trauma experienced by individuals in real-world scenarios. This can facilitate the testing of multi-modal therapies that address common global aspects of injury, such as inflammation, oxidative stress, and neuronal death. Overall, this device offers a versatile and powerful platform for advancing our understanding of closed-system CNS injuries and developing effective therapeutic interventions.

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.

<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

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.

Author Spotlight: Developing Precise and Clinically Relevant Models for Studying Secondary Degeneration in Traumatic Optic Neuropathy

System for Focal, Closed-System Central Nervous System Injury

Using the overpressure air-producing system described here, indirect traumatic optic neuropathy (ITON) was elicited by exposing the left eye of adult (3-month-old) male C57Bl/6 mice (n = 4) to six consecutive bursts of 15 psi overpressure air separated by 0.5 s intervals. Sham animals (n = 8; data taken from Vest et al.33) were anesthetized, placed into the animal holder, and exposed to the sound but not the overpressure air.
The proximal optic nerves of sham animals (Figure 3A) appeared healthy with densely packed and uniformly sized axons surrounded by glial cells with normal morphology and distribution. In comparison, the proximal optic nerves of mice exposed to ITON (i.e., 6 consecutive bursts of 15 psi overpressure air separated by 0.5 s intervals) (Figure 3B) appeared to be degenerating with signs of axon loss, such as increased spacing between remaining axons, signs of axon degeneration, including swelling, irregularities in axon shape, and breakdown of the axons&#39; myelin sheath, and signs of gliosis, including the hypertrophy and hyperplasia of glial cells. Mann-Whitney U tests confirmed a significant difference in total axons (p = 0.0040) (Figure 3C) and degenerative profiles (p = 0.0028) (Figure 3D) between ITON and sham mice. These results suggest that ITON significantly decreases total axons and significantly increases degenerative profiles. Mann-Whitney U tests were performed because the data for the ITON group did not have a sample size large enough for an Independent Samples t-test.
Immunohistochemical staining of retina cross-sections with anti-Iba1 (see Table of Materials), a marker for microglia (the primary immune cells of the central nervous system), was performed on both sham (Figure 4A) and ITON (Figure 4B) mice. The staining revealed that microglia were in their resting state for all mice, characterized by small cell bodies with long, thin, and highly ramified processes. Notably, an increased number of microglia was noted in ITON mice (Figure 4B), suggesting microglial proliferation in response to injury. Additionally, in ITON mice, microglia were observed to be abnormally extending into the outer nuclear layer (ONL), where photoreceptor cell bodies reside (Figure 4B). This contrasts with the sham animals (Figure 4A), where microglia were localized to the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL) - the layers where microglia typically reside in a healthy, uninjured retina.
Subsequent immunohistochemical staining with anti-PKC-&#945; (see Table of Materials) and anti-synaptophysin (see Table of Materials), markers for rod bipolar cells and photoreceptor ribbon synapses, respectively, revealed intact synaptic connections in both sham (Figure 5A) and ITON mice (Figure 5B). Specifically, the dendrites of rod bipolar cells were observed extending and overlapping with the synaptic terminals of rod photoreceptors. This finding contrasts with an early study35, which showed a retraction of rod bipolar cell dendrites towards their cell bodies four weeks after ITON from two consecutive 15 psi bursts of overpressure air (0.5s apart) once daily for 3 days. This discrepancy may be attributed to the different tissue collection time points between the two studies. The current samples were collected 2 weeks post-ITON compared to 4 weeks post ITON in the earlier study. Although no synaptopathy was detected in the current analysis, we did note the extension of microglia processes into the ONL (Figure 4B), where photoreceptor cell bodies are located. This observation suggests that the disruption of synaptic connections between bipolar cells and photoreceptors may emerge as a secondary effect of the injury, while axon loss, axon degeneration, and gliosis constitute primary effects of damage.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66948/66948fig01.jpg" /> 
Figure 1: System for focal, closed-system central nervous system injury. Dashed rectangles in the top image are enlarged and shown as B, C, and A in the two images below (as denoted with white arrows). (A)&#160;Custom 1.5-inch, non-fenestrated barrel (I) at the end of the paintball gun. (B)&#160;Pressure regulator with guide cap removed to expose adjustment screw (II). (C)&#160;Feed neck with the gravity feed loader removed and a feed neck cover installed (III). (D)&#160;Base platform consisting of a 1.5 ft x 1.5 ft piece of fiberboard elevated above a larger 2.5 ft x 1.5 ft piece of fiberboard. (E)&#160;Compressed air tank connected to the pressure regulator of the paintball gun and secured to the fiberboard platform using a durable strap. (F)x-y animal positioning table. <a href="https://www.jove.com/files/ftp_upload/66948/66948fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66948/66948fig02.jpg" /> 
Figure 2: Custom animal holder for focal delivery of overpressure air. (A)&#160;Inside of the animal holder consisting of a narrow PVC tube with a rectangular-shaped hole (3 x 5 cm) to expose the animal&#39;s head and upper hind shoulders. (B)&#160;Outside of the animal holder consisting of a wider PVC tube that the narrower PVC tube slides into, shielding the entirety of the animal&#39;s body apart from the exposed tissue within the exposure aperture. (C)&#160;Exposure aperture for focal delivery of overpressure air to the CNS injury site of interest. (D)&#160;Pressure transducer to calibrate the system&#39;s output pressure. <a href="https://www.jove.com/files/ftp_upload/66948/66948fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66948/66948fig03.jpg" /> 
Figure 3: ITON due to focal delivery of overpressure air. (A,B) Representative brightfield micrographs of proximal optic nerve cross-sections from (A) sham and (B) ITON. (C) Quantification of total axon counts.(D) Quantification of degenerative axon profiles. n = 4 for ITON. n = 9 for sham. Axon count data for the sham group was taken from Vest et al.33. **p &#60; 0.005.&#160;Error bars represent standard deviation. Scale bars = 20 &#956;m. Abbreviation: ITON = indirect traumatic optic neuropathy. <a href="https://www.jove.com/files/ftp_upload/66948/66948fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66948/66948fig04.jpg" /> 
Figure 4: Abnormal microglia proliferation and migration into the ONL due to system-induced ITON. (A,B) Representative fluorescence micrographs of retina cross-sections with anti-Iba1 labeling of microglia (red) from (A) sham and (B) ITON animals. Scale bars = 100 &#956;m. Abbreviations: ITON = indirect traumatic optic neuropathy; GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer. <a href="https://www.jove.com/files/ftp_upload/66948/66948fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66948/66948fig05.jpg" /> 
Figure 5: Early perseveration of synaptic connections between rod bipolar cells and photoreceptors due to system-induced ITON, despite the potential for delayed synaptopathy. (A,B) Representative fluorescence micrographs of retina cross-sections with anti-synaptophysin labeling of photoreceptor ribbon synapses (red) and anti-PKC-&#945; labeling of rod bipolar cells (green) from (A) sham and (B) ITON animals. Scale bars = 100 &#956;m. Abbreviations: ITON = indirect traumatic optic neuropathy; PKC = protein kinase C. <a href="https://www.jove.com/files/ftp_upload/66948/66948fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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

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JoVE Journal

Neuroscience

562 Views.  Vanderbilt University. 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. 